Characterising the Role of Pa2g4 and Its Interaction with Mycn in Neuroblastoma Progression

CHARACTERISING THE ROLE OF PA2G4 AND ITS INTERACTION WITH MYCN IN NEUROBLASTOMA PROGRESSION

Jessica A Koach B.Sc., M.Sc. (Hons)

Children’s Cancer Institute School of Women’s and Children’s Health, Faculty of Medicine, University of New South Wales, Australia

This thesis is submitted to the University of New South Wales in fulfilment of the requirements for the degree of Doctorate of Philosophy

February 2016

i THESIS/DISSERTATION SHEET

PLEASE TYPE THE UNIVERSITY OF NEW SOUTH WALES Thesis/Dissertation Sheet

Surname or Family name: Koach

First name: Jessica Other name/s: AuNguyet

Abbreviation for degree as given in the University calendar: PhD

School: School of Women’s and Children’s Health Faculty: Faculty of Medicine

Title: Characterising the role of PA2G4 and its interaction with MYCN in neuroblastoma progression

Abstract 350 words maximum: (PLEASE TYPE)

Neuroblastoma, an embryonal tumour of the sympathetic nervous system, is the most common solid tumour in childhood. MYCN oncogene amplification is found in one third of primary neuroblastoma at diagnosis, and correlates with poor prognosis. We identified Proliferation-associated protein 2G4 (PA2G4) as a binding partner for MYCN using co-immunoprecipitation and mass spectrometry. The long isoform of PA2G4 (p48) has a known oncogenic function, whereas the short isoform (p42) acts as a tumour suppressor. However, the role of PA2G4 in neuroblastoma and its link with MYCN is currently unknown.

Using a panel of human neuroblastoma cell lines and patient tumour samples, we analysed the expression of PA2G4 by real-time PCR and Western blotting. We found that high expression of PA2G4 was a strong independent clinical predictor of poor survival and positively correlated with MYCN expression. Analysis of pre-cancerous ganglia cells from TH-MYCN neuroblastoma mouse model showed PA2G4 mRNA was expressed 8-fold higher in ganglia from TH-MYCN transgenic homozygous, compared to wild-type, mice.

Chromatin immunoprecipitation showed MYCN binds to PA2G4 promoter to activate its transcription. Surprisingly, suppression of PA2G4 in neuroblastoma cell lines markedly reduced MYCN protein level. Cycloheximide-chase assay confirmed that PA2G4 increased MYCN protein stability, thus creating a positive forward feedback loop essential for maintaining mutual high expression. Suppression of PA2G4 down-regulated pMDM2 and pAKT, leading to an increase in p53 expression. Furthermore, PA2G4 knockdown reduced cell migration and colony formation; importantly expression of MYCN was required for the effect of PA2G4 on colony formation.

Most significantly, overexpression of PA2G4 induced tumorigenesis in a non-tumorigenic cell line. Furthermore, a small molecule inhibitor of PA2G4, WS6, significantly decreased neuroblastoma cell growth and PA2G4 and MYCN protein levels in vitro, and delayed tumour growth in vivo. This research highlights the importance and underlying mechanisms of PA2G4 as an onco-factor in MYCN driven neuroblastoma. It identifies PA2G4 as a novel MYCN-binding protein which increases MYCN protein stability and acts as an oncogenic co-factor in a forward feedback expression loop with MYCN. It provides strong evidence that PA2G4 plays a critical role in tumorigenesis and a novel molecular target for the treatment of neuroblastoma.

Declaration relating to disposition of project thesis/dissertation

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

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

……………………………………………………… ……………………………………..………… ……….……………………...… Signature Witness Date

The University recognises that there may be exceptional circumstances requiring restrictions on copying or conditions on use. Requests for restriction for a period of up to 2 years must be made in writing. Requests for a longer period of restriction may be considered in exceptional circumstances and require the approval of the Dean of Graduate Research.

FOR OFFICE USE ONLY Date of completion of requirements for Award:

ORIGINALITY STATEMENT

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

Signed:

Date:

iii COPYRIGHT STATEMENT

‘I hereby grant the University of New South Wales or its agents the right to archive and to make available my thesis or dissertation in whole or part in the University libraries in all forms of media, now or here after known, subject to the provisions of the Copyright Act 1968. I retain all proprietary rights, such as patent rights. I also retain the right to use in future works (such as articles or books) all or part of this thesis or dissertation. I also authorise University Microfilms to use the 350 word abstract of my thesis in Dissertation Abstract International (this is applicable to doctoral theses only). I have either used no substantial portions of copyright material in my thesis or I have obtained permission to use copyright material; where permission has not been granted I have applied/will apply for a partial restriction of the digital copy of my thesis or dissertation.'

Signed:

Date:

AUTHENTICITY STATEMENT

‘I certify that the Library deposit digital copy is a direct equivalent of the final officially approved version of my thesis. No emendation of content has occurred and if there are any minor variations in formatting, they are the result of the conversion to digital format.’

Signed:

Date:

iv ACKNOWLEDGEMENTS

I would like to acknowledge my supervisors Dr Belamy Cheung and Dr Jamie Fletcher. I wish to thank Belamy for her constant support, guidance, motivation and belief in me and the project during the course of my PhD study, it has made the whole PhD a wonderful experience. I would like to thank Jamie for providing valuable suggestions and insightful critiques to this work, I have enjoyed our conversations and banter about science and research. I would also like to acknowledge Professor Glenn Marshall for his guidance and support and providing funding to this project.

I started this PhD thinking that the destination (completion of the PhD) was most important but I have learned so much along the way that I have come to realise that the journey to obtaining my PhD is more valuable than the degree itself. Because of this, I would like to give thanks to the following people who have either helped me with the project or have helped me to develop into a better scientist: Dr Jessica Bell and Dr Stefan Hüttelmaier for giving me the opportunity to work in their lab at the Martin Luther University (Halle, Germany). Jayne Murray, Kathleen Kimpton and Dr Joshua McCarroll for their help and guidance with the mouse models and animal ethics application. Dr Bing Liu for his help with the bioinformatics analysis, Dr Cheng Xue, Dr Sharon Sagnella, Dr Daniel Carter and Dr Emanuele Valli for their scientific input and technical assistance. Claudia Flemming, Tanya Dwarte and Joanna Keating for always making the time to hear me practise my presentations and providing valuable suggestions. I would specially like to express my gratitude to Claudia Flemming for editing my thesis so thoroughly, I am extremely lucky to have such a dedicated friend.

I would like to thank my colleagues in the Molecular Carcinogenesis Program, past and present members, for their help and support throughout the years, especially Owen Tan for his assistance with the flow cytometry work. I would also like to thank Aldona Konopka and Anna Tomczak for their hard work in looking after the researchers, it is always a pleasure to see their happy faces early in the morning.

Finally, thank you to my parents and my partner, David, for their constant support and love. I would especially like to acknowledge David’s patience and understanding for all my late nights and weekend work in the lab.

v PUBLICATIONS, PRESENTATIONS AND AWARDS

Publications in preparation from this thesis

1. Jessica Koach, Giorgio Milazzo, Giovanni Perini, Jayne E Murray, Joshua McCarroll, Jessica L Bell, Stefan Hüttelmaier, Bing Liu, Daniel R Carter, Tao Liu, Michelle Haber, Murray D Norris, Jamie I Fletcher, Belamy B Cheung, Glenn M Marshall. PA2G4 predicts poor prognosis in neuroblastoma patients and promotes neuroblastoma progression by enhancing MYCN protein stability. Manuscript in preparation.

2. Jessica Koach, Jessica K Holien, Michael W Parker, Michelle Haber, Murray D Norris, Jamie I Fletcher, Glenn M Marshall, Belamy B Cheung. PA2G4 inhibitor, WS6, disrupts PA2G4 and MYCN binding. Manuscript in preparation.

Publications in collaboration during this thesis

1. Xue CY, Yu D, Gherardi S, Koach J, Milazzo G, Gamble L, Liu B, Russell A, Liu T, Cheung BB, Marshall M, Perini G, Haber M, Norris MD. MYCN promotes neuroblastoma malignancy by establishing a regulatory circuit with transcription factor AP4. Oncotarget (2016) In press

2. Carter DR, Murray J, Cheung BB, Gamble L, Koach J, Tsang J, Sutton S, Kalla H, Syed S, Gifford AJ, Issaeva N, Biktasova A, Atmadibrata B, Sun Y, Sokolowski N, Ling D, Kim PY, Webber H, Clark A, Ruhle M, Liu B, Oberthuer A, Fischer M, Byrne J, Saletta F, Thwe LM, Purmal A, Haderski G, Burkhart C, Speleman F, De Preter K, Beckers A, Ziegler D, Liu T, Gurova KV, Gudkov AV, Norris MD, Haber M, Marshall GM. Therapeutic targeting of the MYC signal by inhibition of histone chaperone FACT in neuroblastoma. Science Translational Medicine (2015) Nov 4;7 (312)

vi 3. Cheung BB, Tan O, Koach J, Liu B, Shum MS, Carter DR, Sutton S, Po'uha ST, Chesler L, Haber M, Norris MD, Kavallaris M, Liu T, O'Neill GM, Marshall GM. Thymosin-β4 is a determinant of drug sensitivity for Fenretinide and Vorinostat combination therapy in neuroblastoma. Molecular Oncology (2015) Aug 9(7):1484-500

4. Sutton SK, Koach J, Tan O, Liu B, Carter DR, Wilmott JS, Yosufi B, Haydu LE, Mann GJ, Thompson JF, Long GV, Liu T, McArthur G, Zhang XD, Scolyer RA, Cheung BB, Marshall GM. TRIM16 inhibits proliferation and migration through regulation of interferon beta 1 in melanoma cells. Oncotarget (2014) Oct 30;5(20):10127-39.

5. Liu PY, Xu N, Malyukova A, Scarlett CJ, Sun YT, Zhang XD, Ling D, Su SP, Nelson C, Chang DK, Koach J, Tee AE, Haber M, Norris MD, Toon C, Rooman I, Xue C, Cheung BB, Kumar S, Marshall GM, Biankin AV, Liu T. The histone deacetylase SIRT2 stabilizes Myc oncoproteins. Cell Death and Differentiation (2013) March 20(3):503-14.

6. Gardner CR, Cheung BB, Koach J, Black DS, Marshall GM, Kumar N. Synthesis of retinoid enhancers based on 2-aminobenzothiazoles for anti-cancer therapy. Bioorg Med Chem. (2012) Dec 1;20(23):6877-84

7. Bell JL, Malyukova A, Holien JK, Koach J, Parker MW, Kavallaris M, Marshall GM, Cheung BB. TRIM16 acts as an E3 ubiquitin ligase and can heterodimerize with other TRIM family members. PLoS ONE (2012) May 21; e37470

Presentations arising from and during this thesis

1. Jessica Koach, Jayne E Murray, Joshua McCarroll, Giorgio Milazzo, Giovanni Perini, Michelle Haber, Murray D Norris, Jamie I Fletcher, Belamy B Cheung, Glenn M Marshall. Targeting PA2G4, a novel MYCN co-factor, for the treatment of Neuroblastoma. European Association for Cancer Research, Manchester, England, 2016. Oral presentation.

vii 2. Jessica Koach, Jayne E Murray, Joshua McCarroll, Giorgio Milazzo, Giovanni Perini, Michelle Haber, Murray D Norris, Jamie I Fletcher, Belamy B. Cheung, Glenn M. Marshall. Targeting a novel MYCN onco-factor, PA2G4, for the treatment of Neuroblastoma. Advances in Neuroblastoma Research, Cairns, Australia, 2016. Oral presentation.

3. Jessica Koach, Bing Liu, Jessica L Bell, Stefan Hüttelmaier, Tao Liu, Daniel R Carter, Michelle Haber, Murray D Norris, Jamie I Fletcher, Belamy B Cheung, Glenn M Marshall. PA2G4 predicts poor prognosis in neuroblastoma patients and promotes neuroblastoma progression by enhancing MYCN protein stability. American Association for Cancer Research, Philadelphia, USA, 2015. Oral presentation.

4. Jessica Koach, Jayne E Murray, Joshua McCarroll, Michelle Haber, Murray D Norris, Jamie I Fletcher, Glenn M. Marshall, Belamy B. Cheung. Targeting a novel oncogenic protein, PA2G4, for the treatment of Neuroblastoma. 43rd Annual TOW Research Meeting, Sydney, Australia, 2015. Oral presentation winner.

5. Jessica Koach, Bing Liu, Jayne E Murray, Tao Liu, Daniel R Carter, Michelle Haber, Murray D Norris, Jamie I Fletcher, Glenn M Marshall, Belamy B Cheung. PA2G4 predicts poor prognosis in neuroblastoma patients and promotes neuroblastoma progression by enhancing MYCN protein stability. Australian Society for Medical Research, Sydney, Australia, 2015. Oral presentation.

6. Jessica Koach, Bing Liu, Jessica L Bell, Stefan Hüttelmaier, Tao Liu, Daniel R Carter, Michelle Haber, Murray D Norris, Jamie I Fletcher, Glenn M Marshall, Belamy B Cheung. High PA2G4 predicts poor prognosis in NB patients and promotes neuroblastoma progression by controlling MYCN stability. 42nd Annual TOW Research Meeting, Sydney, Australia, 2014. Oral presentation.

7. Jessica Koach, Bing Liu, Tao Liu, Jessica L Bell, Stefan Hüttelmaier, Daniel R Carter, Michelle Haber, Murray D Norris, Jamie I Fletcher, Glenn M Marshall, Belamy B Cheung. High PA2G4 predicts poor prognosis in neuroblastoma patients and promotes cell growth by controlling stability of MYCN oncoprotein. Australian

viii Association of Chinese Biomedical Scientist (AACBS) meeting, Sydney, Australia, 2014. Oral presentation winner.

8. Jessica Koach, Jamie I Fletcher, Bing Liu, Michelle Haber, Murray D Norris, Glenn M Marshall, Belamy B Cheung. PA2G4 promotes neuroblastoma oncogenesis through direct binding and modulation of MYCN protein levels. Advances in Neuroblastoma Research, Cologne, Germany, 2014.

9. Jessica Koach, Owen Tan, Maria Kavallaris, Michelle Haber, Murray D Norris, Tao Liu, Glenn M Marshall and Belamy B Cheung. Increasing the effectiveness of retinoid anticancer therapy for neuroblastoma. The Lowy Cancer Symposium, Sydney, Australia, 2013. Oral presentation.

Awards

 Best oral presentation, Senior Division, 43rd Annual TOW Research Meeting, Sydney, Australia, 2015.

 Best oral presentation, Australian Association of Chinese Biomedical Scientists (AACBS) meeting, Sydney, Australia, 2014

 Kids Cancer Alliance (KCA) PhD Top-up Award, 2014

 UNSW Post Graduate Research Travel Grant, 2014

 Australian Post Graduate Award, 2012

ix TABLE OF CONTENTS THESIS/DISSERTATION SHEET ...... ii ORIGINALITY STATEMENT ...... ii COPYRIGHT STATEMENT ...... iv AUTHENTICITY STATEMENT ...... iv ACKNOWLEDGEMENTS ...... v PUBLICATIONS, PRESENTATIONS AND AWARDS ...... vi LIST OF FIGURES ...... xvi LIST OF TABLES ...... xix ABBREVIATIONS AND ACRONYMS ...... xx ABSTRACT ...... xxiii Chapter 1 Introduction ...... 1 1.1 Neuroblastoma ...... 2 1.1.1 Epidemiology ...... 2 1.1.2 Predisposition ...... 3 1.1.3 Histopathology ...... 4 1.1.4 Molecular abnormalities...... 6 1.1.5 Staging and risk stratification...... 7 1.1.6 Neuroblastoma chemotherapy ...... 12 1.1.7 Treatment of low- and intermediate-risk neuroblastoma ...... 12 1.1.8 Treatment of high-risk neuroblastoma ...... 13 1.1.9 Retinoid and immunotherapy ...... 14 1.1.10 Molecular targeted therapy ...... 15 1.2 MYCN ...... 19 1.2.1 The MYC family ...... 19 1.2.2 MYCN in cancer ...... 22 1.2.3 Function of MYCN ...... 23 1.2.4 Regulation of MYCN ...... 27 1.2.5 Therapeutic targeting of MYCN ...... 29 1.3 PA2G4 ...... 34 1.3.1 Structure of PA2G4 ...... 34 1.3.2 Two distinctive isoforms of PA2G4 ...... 37 1.3.3 Cellular localisation of PA2G4 ...... 41 1.3.4 PA2G4 is an ErbB-3 binding protein ...... 41 1.3.5 Phosphorylation of PA2G4 ...... 42 x 1.3.6 PA2G4 binds DNA ...... 43 1.3.7 The role of PA2G4 in RNA processing and translation ...... 44 1.3.8 The role of PA2G4 in cancer ...... 47 1.3.9 Pa2g4 knockout mouse ...... 49 1.4 Research perspectives ...... 50 Chapter 2 Materials and Methods ...... 52 2.1 Cell culture ...... 53 2.1.1 Reagents and equipment ...... 53 2.1.2 Cell lines ...... 53 2.1.3 Inducible cell line: SH-EP-MYCN3 ...... 54 2.1.4 Patient tumour samples ...... 54 2.1.5 Cell viability counts ...... 54 2.1.6 Transient transfection with plasmid DNA ...... 57 2.1.7 Transient transfection with siRNA ...... 57 2.1.8 Stable transfection with PA2G4 plasmid DNA ...... 61 2.1.9 Stable transfection with PA2G4 shRNA ...... 61 2.2 Cell phenotype assays ...... 63 2.2.1 Reagents and equipment ...... 63 2.2.2 Cell viability assay ...... 63 2.2.3 Cell proliferation assay ...... 64 2.2.4 Cytotoxicity assay ...... 64 2.2.5 Colony formation assay...... 65 2.2.6 Neurite growth assay with retinoic acid treatment ...... 65 2.2.7 Flow Cytometry ...... 65 2.2.8 Immunofluorescent staining of endogenous protein ...... 66 2.2.9 Transwell cell migration assay ...... 67 2.2.10 3D cell formation and outgrowth assay ...... 67 2.2.11 3D cell invasion assay ...... 68 2.3 Protein Analysis ...... 70 2.3.1 Reagents and equipment ...... 70 2.3.2 Whole cell lysate protein extraction ...... 70 2.3.3 Cytoplasmic and nuclear protein extraction ...... 71 2.3.4 Protein quantification ...... 71 2.3.5 Co-immunoprecipitation ...... 71 2.3.6 Western blotting ...... 72 xi 2.3.7 Inhibition of protein synthesis and degradation ...... 75 2.3.8 Chromatin Immunoprecipitation (ChIP) Assays ...... 75 2.4 Gene expression analysis ...... 77 2.4.1 Reagents and equipment ...... 77 2.4.2 RNA isolation ...... 77 2.4.3 cDNA synthesis ...... 78 2.4.4 Real-time quantitative PCR...... 78 2.4.5 RNA amplification ...... 80 2.4.6 Microarray gene expression analysis ...... 80 2.5 Additional molecular biology techniques ...... 81 2.5.1 Reagents and equipment ...... 81 2.5.2 Bacterial transformation ...... 81 2.5.3 DNA isolation and analysis ...... 81 2.5.4 Restriction enzyme digestion ...... 82 2.6 In vivo models of neuroblastoma ...... 82 2.6.1 Reagents and equipment ...... 82 2.6.2 Assessing the ability of PA2G4 to initiate tumour development ...... 83 2.6.3 Silencing PA2G4 expression to reduce tumour growth in mice ...... 83 2.6.4 Treating neuroblastoma tumours with PA2G4 inhibitor, WS6 ...... 84 2.6.5 Histological tissue preparation ...... 84 2.7 Statistical analysis ...... 84 2.8 Solutions and Reagents ...... 85 2.8.1 Resazurin solution ...... 85 2.8.2 Crystal violet solution ...... 85 2.8.3 T.E. buffer pH 8.0 ...... 85 2.8.4 50x TAE buffer ...... 85 2.8.5 Hypotonic lysis buffer ...... 86 2.8.6 Cytoplasmic lysis buffer ...... 86 2.8.7 Nuclear lysis buffer ...... 86 2.8.8 Ponceau S staining solution ...... 87 2.8.9 10x TGS running buffer ...... 87 2.8.10 10x Transfer buffer ...... 87 2.8.11 10x Tris-buffered saline (TBS) ...... 87 2.8.12 Stripping buffer for 1o antibody ...... 87 2.8.13 5x stripping buffer for 2o antibody ...... 87

xii 2.8.14 Elution buffer for ChIP assay ...... 88 Chapter 3 Identification of MYCN binding partner PA2G4, and its interaction with MYCN ...... 89 3.1 Introduction ...... 90 3.2 Results ...... 92 3.2.1 Identification of MYCN binding proteins ...... 92 3.2.2 PA2G4 is a novel MYCN binding protein ...... 97 3.2.3 High PA2G4 expression predicts poor patient prognosis ...... 100 3.2.4 PA2G4 is an independent prognostic marker for poor outcome ...... 103 3.2.5 PA2G4 expression increases postnatally ...... 109 3.2.6 MYCN positively regulates PA2G4 expression ...... 110 3.2.7 MYCN protein binds to the PA2G4 promoter ...... 116 3.2.8 PA2G4 stabilises MYCN protein ...... 121 3.2.9 PA2G4 controls MYCN stability through phosphorylation of Serine 62 . 128 3.2.10 Common genes in the PA2G4 and MYCN signalling pathways ...... 133 3.3 Discussion ...... 136 Chapter 4 PA2G4: A potential oncogenic protein in neuroblastoma ... 140 4.1 Introduction ...... 141 4.2 Results ...... 142 4.2.1 Overexpression of PA2G4 increases cell growth...... 142 4.2.2 Suppression of PA2G4 decreases cell growth ...... 145 4.2.3 PA2G4-mediated increase in cell growth is independent of MYCN expression ...... 148 4.2.4 The PA2G4 long isoform, p48, plays a role in colony formation ...... 151 4.2.5 Overexpression of MYCN reverses the decrease in colony formation caused by suppression of PA2G4 ...... 153 4.2.6 PA2G4 regulates cell growth through MDM2 and AKT signalling ...... 155 4.2.7 The protein half-life of PA2G4 ...... 157 4.2.8 PA2G4 degradation pathway ...... 159 4.2.9 Suppression of PA2G4 does not increase apoptosis ...... 160 4.2.10 PA2G4 increases cell migration ...... 164 4.2.11 Up-regulation of MYCN increases TFAP4 expression ...... 167 4.2.12 TFAP4 expression is able to rescue the decrease in cell migration caused by PA2G4 knockdown ...... 171 4.2.13 TFAP4 is part of a PA2G4 signalling pathway ...... 173 xiii 4.2.14 PA2G4 suppresses neurite formation ...... 175 4.2.15 13-cis-RA reduces MYCN, c-MYC and PA2G4 expression ...... 177 4.2.16 Combination of PA2G4 siRNA knockdown and 13-cis-RA treatment increases differentiation ...... 180 4.2.17 Establishment of PA2G4 shRNA stable cell lines ...... 181 4.2.18 PA2G4 knockdown with shRNA reduces cell viability and colony formation ...... 185 4.2.19 PA2G4-shRNA decreases TFAP4 mRNA expression ...... 187 4.2.20 Stable knockdown of PA2G4 causes senescence...... 188 4.2.21 Generation of PA2G4 overexpressing cell lines ...... 190 4.2.22 Expression of PA2G4 is lost over time ...... 194 4.2.23 3D spheroids of SH-EP cells overexpressing PA2G4...... 196 4.2.24 3D spheroids of SH-EP cells stably overexpressing PA2G4 show increased invasion ...... 199 4.2.25 PA2G4 induces tumorigenicity ...... 201 4.2.26 In vitro analysis of SH-EP PA2G4 overexpressing tumours ...... 203 4.3 Discussion ...... 205 Chapter 5 Targeting PA2G4 for the treatment of neuroblastoma ...... 212 5.1 Introduction ...... 213 5.2 Results ...... 214 5.2.1 Investigating WS6 as a potential inhibitor of PA2G4 ...... 214 5.2.2 WS6 decreases PA2G4 and MYC expression ...... 216 5.2.3 WS6 reduces cell growth ...... 218 5.2.4 WS6 is specifically inhibiting PA2G4 ...... 221 5.2.5 WS6 induces apoptosis in neuroblastoma cells ...... 222 5.2.6 WS6 reduces colony formation ...... 224 5.2.7 WS6 reduces tumour growth in TH-MYCN+/+ mice ...... 225 5.2.8 WS6 reduces PA2G4 expression in TH-MYCN+/+ mouse tumours ...... 227 5.2.9 PA2G4 siRNA delivered by nanoparticles delays tumour growth ...... 229 5.2.10 Analysis of tumours with PA2G4 siRNA + nanoparticle ...... 231 5.2.11 WS6 and vincristine combination treatment has synergistic effects against neuroblastoma cells ...... 233 5.3 Discussion ...... 238 Chapter 6 Concluding remarks and future directions ...... 244 REFERENCES ...... 250 xiv APPENDIX A: Log rank test ...... 287

xv LIST OF FIGURES

Figure 1.1 Neuroblastoma risk stratification and treatments ...... 11 Figure 1.2 Current treatment strategy for high-risk neuroblastoma ...... 13 Figure 1.3 Current clinical approaches in targeting neuroblastoma ...... 16 Figure 1.4 Conserved domains of MYC ...... 21 Figure 1.5 MYC–MAX and MYC–MIZ1 heterodimer ...... 26 Figure 1.6 Structure of PA2G4 ...... 36 Figure 1.7 PA2G4 has two isoforms ...... 38 Figure 1.8 Molecular interactions and cellular processes of PA2G4 ...... 46 Figure 2.1 Structure of the PA2G4, MYCN and PA2G4 shRNA plasmid constructs .... 59 Figure 3.1 Identification of MYCN binding proteins ...... 93 Figure 3.2 Confirmation of PA2G4 and MYCN protein binding ...... 98 Figure 3.3 Co-localisation of PA2G4 and MYCN protein in the nucleus ...... 99 Figure 3.4 High expression of PA2G4 predicts poor survival in neuroblastoma patients ...... 101 Figure 3.5 Elevated PA2G4 expression in INSS stage 3 and 4 tumours...... 103 Figure 3.6 mRNA expression analysis of 40 neuroblastoma tumour samples ...... 106 Figure 3.7 Protein expression analysis of 30 neuroblastoma tumour samples ...... 107 Figure 3.8 PA2G4 expression is elevated in ganglia of TH-MYCN transgenic mice .. 109 Figure 3.9 Higher PA2G4 protein expression in MYCN-amplified neuroblastoma cell lines ...... 110 Figure 3.10 MYCN positively regulates PA2G4 protein and mRNA expression ...... 112 Figure 3.11 c-MYC positively regulates PA2G4 protein and mRNA expression ...... 113 Figure 3.12 Overexpression of MYCN increases PA2G4 protein and mRNA levels .. 115 Figure 3.13 MYCN binds to PA2G4 DNA ...... 118 Figure 3.14 Suppression of MYCN reduces PA2G4 transcription ...... 120 Figure 3.15 PA2G4 regulates MYCN protein expression but not mRNA expression . 122 Figure 3.16 PA2G4 regulates c-MYC protein expression but not mRNA expression . 123 Figure 3.17 Knockdown of PA2G4 destabilises MYCN protein ...... 125 Figure 3.18 Overexpression of PA2G4 stabilises MYCN protein ...... 127 Figure 3.19 PA2G4 controls MYCN stability through phosphorylation of Serine 62 . 129 Figure 3.20 PA2G4 requires Serine 62 for the regulation of MYCN protein ...... 131 Figure 3.21 Common genes and pathways regulated by MYCN and PA2G4 ...... 134 Figure 4.1 Overexpression of PA2G4 increases cell growth ...... 143

xvi Figure 4.2 Knockdown of PA2G4 decreases cell growth ...... 147 Figure 4.3 The decrease in cell growth caused by PA2G4 knockdown cannot be rescued by overexpression of MYCN ...... 150 Figure 4.4 The long isoform of PA2G4, p48, is required for colony formation ...... 152 Figure 4.5 Overexpression of MYCN reverses the decrease in colony formation caused by down-regulation of PA2G4 ...... 154 Figure 4.6 PA2G4 mediates the phosphorylation of MDM2 and AKT ...... 156 Figure 4.7 PA2G4 has a long protein half-life ...... 158 Figure 4.8 PA2G4 is not degraded through the proteasomal pathway ...... 159 Figure 4.9 Knockdown of PA2G4 increases G2/M arrest and induces PARP cleavage but does not increase apoptosis ...... 161 Figure 4.10 Overexpression of PA2G4 increases cell migration ...... 165 Figure 4.11 Knockdown of PA2G4 decreases cell migration ...... 166 Figure 4.12 Up-regulation of MYCN increases PA2G4 and TFAP4 expression ...... 168 Figure 4.13 Knockdown of PA2G4-p48 decreases MYCN, c-MYC and TFAP4 expression ...... 170 Figure 4.14 TFAP4 can partially reverse the effect on migration of knocking down PA2G4 ...... 172 Figure 4.15 PA2G4 positively regulates TFAP4 ...... 174 Figure 4.16 Down-regulation of PA2G4 increases neurite formation ...... 176 Figure 4.17 13-cis-RA reduces MYCN and PA2G4 expression...... 178 Figure 4.18 13-cis-RA reduces c-MYC and PA2G4 expression ...... 179 Figure 4.19 Treatment with 13-cis-RA further increases neurite formation caused by knockdown with PA2G4 ...... 180 Figure 4.20 Establishment of PA2G4 shRNA stable cell lines ...... 182 Figure 4.21 Transfection of PA2G4-shRNA constructs into BE(2)-C cells ...... 183 Figure 4.22 PA2G4-shRNA reduces cell viability and colony formation ...... 186 Figure 4.23 PA2G4-shRNA decreases PA2G4 and TFAP4 mRNA expression ...... 187 Figure 4.24 Stable expression of PA2G4-shRNA in BE(2)-C cells causes senescence189 Figure 4.25 SH-EP cells stably overexpressing PA2G4, pool of clones ...... 191 Figure 4.26 SH-EP clones stably overexpressing PA2G4 ...... 193 Figure 4.27 Expression of PA2G4 is lost over time in SH-EP PA2G4 stable cells ...... 195 Figure 4.28 3D spheroids of SH-EP cells overexpressing PA2G4 show increased migration ...... 197

xvii Figure 4.29 3D spheroids of SH-EP cells stably overexpressing PA2G4 show increased invasion ...... 200 Figure 4.30 Overexpression of PA2G4 increases neuroblastoma tumorigenicity ...... 202 Figure 4.31 Higher PA2G4 protein and mRNA expression in mouse tumours ...... 204 Figure 5.1 Putative WS6 binding sites on PA2G4 ...... 215 Figure 5.2 WS6 decreases PA2G4, MYCN and c-MYC mRNA and protein expression ...... 217 Figure 5.3 WS6 reduces cell growth ...... 219 Figure 5.4 Cells expressing higher levels of PA2G4 are more sensitive to WS6 treatment ...... 220 Figure 5.5 WS6 was not effective in PA2G4 suppressed cells ...... 221 Figure 5.6 WS6 induces apoptosis in neuroblastoma cells, but not in normal fibroblast cells...... 223 Figure 5.7 WS6 reduces colony formation ...... 224 Figure 5.8 WS6 reduces tumour growth in TH-MYCN+/+ mice ...... 226 Figure 5.9 WS6 reduces PA2G4 protein and mRNA expression in TH-MYCN+/+ mouse tumours ...... 228 Figure 5.10 PA2G4 siRNA delivered by nanoparticles decreases tumour growth ...... 230 Figure 5.11 PA2G4 siRNA coupled to Star-nanoparticles reduces PA2G4 and MYCN protein and mRNA expression in SK-N-BE(2) xenograft mouse tumours ...... 232 Figure 5.12 WS6 + vincristine combination treatment is synergistic in BE(2)-C cells 234 Figure 5.13 WS6 + JQ1 and WS6 + vincristine combination treatments are synergistic in Kelly cells ...... 235 Figure 5.14 WS6 + Vincristine and WS6 + Cisplatin combination treatments are synergistic in SH-SY5Y ...... 236 Figure 5.15 WS6 + JQ1 combination treatment is synergistic in WI-38 cells ...... 237

xviii LIST OF TABLES

Table 1.1 International Neuroblastoma Pathology Classification and Shimada Classification of neuroblastic tumours ...... 5 Table 1.2 Neuroblastoma Staging: International Neuroblastoma Staging System (INSS) ...... 8 Table 1.3 International Neuroblastoma Risk Group Staging System (INRGSS) ...... 9 Table 1.4 Physical and functional differences between p48 and p42 ...... 40 Table 2.1 Neuroblastoma and normal cell lines ...... 55 Table 2.2 Cell seeding densities ...... 56 Table 2.3 Amount of plasmid DNA, siRNA, and Lipofectamine 2000 used in transfections ...... 58 Table 2.4 siRNA and shRNA sequences ...... 62 Table 2.5 Optimisation of Collagen I concentration for invasion assay ...... 69 Table 2.6 Antibody dilutions ...... 74 Table 2.7 Real-time PCR primer sequences ...... 79 Table 3.1 MYCN binding candidates isolated by co-immunoprecipitation and identified by mass spectrometry ...... 95 Table 3.2 Selection criteria for determining the protein to study ...... 96 Table 3.3 PA2G4 expression is an independent prognostic factor in neuroblastoma ... 104 Table 3.4 Top 57 common oncogenic pathways shared between MYCN and PA2G4 knockdown ...... 135

Table 5.1 IC50 concentration from treatment with WS6 for 24 hours to 72 hours in neuroblastoma cells and normal lung fibroblast cells...... 220

xix ABBREVIATIONS AND ACRONYMS

2D two-dimensional 3D three-dimensional 7-AAD 7-aminoactinomycin D α-MEM alpha-minimum essential media µg microgram µL microlitre µM micromolar Ab antibody ALLN N-acetyl-leucyl-leucyl-norleucinal (calpain inhibitor) BCL-2 B-cell lymphoma 2 BM bone marrow BSA Bovine Serum Albumin cDNA complementary DNA CI combination index CNS central nervous system COG Children’s Oncology Group C-terminal carboxy-terminal Da dalton DMEM Dulbecco's Modified Eagle Medium DMSO dimethyl sulfoxide DNA deoxyribonucleic acid dsRNA double-stranded RNA dsRBD double-stranded RNA binding domain Ebp1 ErbB3-binding protein 1 EFS event free survival EMT epithelial mesenchymal transition EV empty vector FCS foetal calf serum g gravity/g-force G418 Geneticin, aminoglycoside antibiotic GFP green fluorescent protein GM-CSF granulocyte macrophage colony-stimulating factor GSEA gene set enrichment analysis

xx hr hour HDAC histone deacetylase INRG International Neuroblastoma Risk Group INSS International Neuroblastoma Staging System IP intraperitoneal kDa kilo dalton kg kilogram M molar mAB monoclonal antibody mBMBC mouse bone marrow B cells min minute mL millilitre mM millimolar MQ-H2O Milli-Q water MS mass spectroscopy MTD maximum tolerated dose NB neuroblastoma nm nanometre nM nanomolar N-terminal amino-terminal PA2G4 proliferation-associated 2G4 PARP poly (ADP-ribose) polymerase PBS phosphate-buffered saline PCR polymerase chain reaction qRT-PCR quantitative reverse transcriptase-polymerase chain reaction RA retinoic acid RNA ribonucleic acid rpm revolutions per minute RPMI-1640 Roswell Park Memorial Institute-1640 S62 Serine 62 SD standard deviation SDS sodium dodecyl sulphate SEM standard error of the mean siRNA short interfering RNA shRNA small hairpin RNA

xxi T58 Threonine 58 TBS tris buffered saline TBST TBS Tween-20 TFAP4 transcription factor activating enhancer binding protein 4 VCL vinculin VCR vincristine

xxii ABSTRACT

Neuroblastoma, an embryonal tumour of the sympathetic nervous system, is the most common extracranial solid tumour in childhood. MYCN oncogene amplification is found in one third of primary neuroblastomas at diagnosis, it is a key driver of the disease and correlates with poor prognosis. Proliferation-associated protein 2G4 (PA2G4) was identified as a binding partner for MYCN using co-immunoprecipitation and mass spectrometry. The long isoform of PA2G4 (p48) has a known oncogenic function, whereas the short isoform (p42) acts as a tumour suppressor. However, the role of PA2G4 in neuroblastoma and its relationship with MYCN is currently unknown.

Using publicly available microarray data from 477 patient tumour samples and a panel of 11 human neuroblastoma cell lines, PA2G4 expression was analysed by bioinformatics, real-time PCR and Western blotting. High expression of PA2G4 was a strong independent clinical predictor of poor survival and positively correlated with MYCN expression. Pa2g4 mRNA was expressed 8-fold higher in pre-cancerous ganglia cells from the homozygous TH-MYCN neuroblastoma mouse than in ganglia from wild- type mice.

Chromatin immunoprecipitation showed MYCN binds to PA2G4 promoter and MYCN siRNA decreases PA2G4 expression; together these observations suggest PA2G4 is a transactivational target of MYCN. Surprisingly, suppression of PA2G4 in neuroblastoma cell lines markedly reduced MYCN protein level. Cycloheximide-chase assay confirmed that PA2G4 expression increased MYCN protein stability, thus creating a positive forward feedback loop which resulted from increases in ERK phosphorylation. Suppression of PA2G4 down-regulated pMDM2 and pAKT, leading to an increase in p53 expression. Furthermore, PA2G4 knockdown by siRNAs reduced cell migration and colony formation; importantly the expression of MYCN was required for the effect of PA2G4 on colony formation.

xxiii MYCN in MYCN-driven neuroblastoma. It identifies PA2G4 as a novel MYCN- binding protein which increases MYCN protein stability and acts as an oncogenic cofactor in a forward feedback expression loop with MYCN. It provides strong evidence PA2G4 is a driver of tumorigenicity and a possible therapeutic target for the treatment of neuroblastoma.

xxiv

Chapter 1 INTRODUCTION

CHAPTER 1

INTRODUCTION

Chapter 1- Introduction 1 1.1 Neuroblastoma

Neuroblastoma (NB) is an embryonal tumour of the sympathetic nervous system. It arises during the early stages of development from sympathetic cells derived from the neural crest (Brodeur, 2003; Goldsby and Matthay, 2004), which arises from the dorsal region of the closing neural tube beneath the ectoderm (Le Dourin and Kalcheim, 1999). These neural crest cells undergo an epithelial to mesenchymal transition (EMT), resulting in enhanced migratory abilities and decreased requirements for intercellular contact, which allows the neural crest cells to leave the dorsal neural tube and migrate to other sites within the body (Le Dourin and Kalcheim, 1999). Primary neuroblastoma tumours develop in adrenal medullary tissue or paraspinal ganglia, both of which are part of the sympathoadrenal lineage. This lineage produces multipotential progenitor cells that give rise to the peripheral nervous system, the enteric nervous system, pigment cells, Schwann cells, adrenal medullary cells, and cells of the craniofacial skeleton. The most common presenting feature in neuroblastoma patients is an asymptomatic abdominal mass. The tumour may be localised or widely metastatic, and metastases can include lesions in the skull, ribs, pelvis and long bones (Geatti et al., 1985; Maris et al., 2007; Papathanasiou et al., 2011). The origin and initiation of neuroblastoma are extensively reviewed by Jiang et al. 2011 and Marshall et al. 2014 (Jiang et al., 2011; Marshall et al., 2014).

1.1.1 Epidemiology

Neuroblastoma accounts for 7–8% of malignant disease in children (Young et al., 1986). It is the most common solid extracranial malignancy of childhood and the most common malignant tumour in infants (Goldsby and Matthay, 2004). Approximately 50% of patients with neuroblastoma have a clinically aggressive form of the disease, with overall survival rates of less than 40% (De Bernardi et al., 2003; Maris, 2010; Matthay et al., 1999a). Although high-risk neuroblastoma accounts for only 4% of all paediatric cancer diagnoses, it is responsible for 12% of paediatric cancer deaths (Smith et al., 2010). Furthermore, neuroblastoma is associated with one of the highest proportions of spontaneous and complete regression of all human cancers (Yamamoto et al., 1998).

Chapter 1- Introduction 2 1.1.2 Predisposition

Hereditary neuroblastoma is identified in 1–2% of cases, and the tumours that arise in patients with familial disease are heterogeneous (Maris et al., 2002; Shojaei-Brosseau et al., 2004). Genetic analyses have mapped hereditary neuroblastoma predisposition loci to chromosomes 16p12–13 and 4p16, however no genes have been shown to be inactivated or mutated in these regions (Maris et al., 2002; Perri et al., 2002). Disorders involving the autonomic nervous system are occasionally seen in patients with neuroblastoma. These disorders include: Hirschsprung disease, congenital central hypoventilation syndrome, and neurofibromatosis type 1 (Bower and Adkins, 1980; Knudson and Amromin, 1966; Maris et al., 1997). The genes implicated in these disorders have been studied for their role in tumorigenesis.

The first predisposition mutations identified in neuroblastoma were germline mutations in the paired-like homeobox 2B (PHOX2B) gene on chromosome 4p13 (Mosse et al., 2004; Trochet et al., 2004). PHOX2B is a key regulator of normal development of the autonomic nervous system; mutations that result in loss of function of this gene are associated with congenital central hypoventilation syndrome (Weese-Mayer et al., 2003). Recent findings suggest that Phox2b may be a tumour suppressor. It is necessary for the differentiation of autonomic neurons, and overexpression of Phox2b inhibits proliferation in neuron progenitors and cell lines (Raabe et al., 2008; Trochet et al., 2009).

Heritable mutations in the anaplastic lymphoma kinase (ALK) gene, a receptor tyrosine kinase, are an important cause of familial neuroblastoma, along with PHOX2B (Janoueix-Lerosey et al., 2008; Mosse et al., 2004; Mosse et al., 2008a). ALK plays a key role in normal development of the central and peripheral nervous system and it protects neuroblast growth in utero against nutrient deprivation (Cheng et al., 2011; Degoutin et al., 2009; Reiff et al., 2011). Activating mutations or amplification of ALK were found to occur in about 10–15% of high-risk neuroblastoma cases (Janoueix- Lerosey et al., 2008; Mosse et al., 2008a), however a more recent study has reported the mutation rate of ALK to be 9.2% (Pugh et al., 2013). These mutations, such as p.Arg1275Gln and p.Phe1174Val/Leu, result in constitutive activation of this receptor tyrosine kinase that contributes to the malignant phenotype of neuroblastoma (de Pontual et al., 2011; Greengard and Park, 1993; Mosse et al., 2008a). ALK translocation

Chapter 1- Introduction 3 has been implicated in inflammatory myofibroblastic tumour and non-small cell lung cancer (NSCLC), but not in neuroblastoma (Palmer et al., 2009). Overexpression of wild-type ALK has also been observed in several cancers, including neuroblastoma, thyroid carcinoma, breast cancer, melanoma, small-cell lung carcinoma, glioblastoma, retinoblastoma, and Ewing sarcoma (Cheng and Ott, 2010; Mosse et al., 2009; Palmer et al., 2009).

1.1.3 Histopathology

Neuroblastoma is an extremely heterogeneous disease (Brodeur and Nakagawara 1992); tumours can spontaneously regress or mature, even without therapy, or display a very aggressive, malignant phenotype that is poorly responsive to intensive, multimodal therapy. A number of factors responsible for this heterogeneity have been identified, and the biological and molecular features of neuroblastoma are highly predictive of clinical behaviour (Maris et al., 2007). The most important clinical variables for children with neuroblastoma appear to be age and stage at diagnosis (Cohn et al., 2009; Evans et al., 1987). Other factors including histologic category and grade of tumour differentiation were found to be statistically significantly associated with event free survival (EFS) (Cohn et al., 2009; Shimada et al., 1999; Shimada et al., 2001).

The classification of disease based on age-linked tumour morphology was first described in 1984 by Shimada and colleagues, and became known as the Shimada Classification (Shimada et al., 1984). This system distinguishes good and poor prognosis tumours based on the degree of differentiation, the Schwannian stromal content, the mitosis-karyorrhexis index (MKI), and the age at diagnosis. This classification system was later redefined by the International Neuroblastoma Pathology Classification (INPC), set up by the International Neuroblastoma Pathology Committee in 1999, comprising six pathologists with the objective of creating a prognostically significant and biologically relevant classification based on morphologic features of neuroblastic tumours (Shimada et al., 1999) (Table 1.1). Neuroblastic tumours were divided into 4 histologic subtypes on the basis of the degree of surrounding Schwannian stroma: neuroblastoma (Schwannian stroma-poor), ganglioneuroblastoma-intermixed (Schwannian stroma-rich), ganglioneuroblastoma-nodular (composite, Schwannian stroma-rich/stroma-dominant and stroma-poor), and ganglioneuroma (Schwannian

Chapter 1- Introduction 4 stroma-dominant). These tumours are assigned to favourable or unfavourable subgroups, on the basis of histologic characteristics, including the degree of neuroblast differentiation, the nuclear morphology of neuroblastic cells, and the patient’s age (Chatten et al., 1988).

Table 1.1 International Neuroblastoma Pathology Classification and Shimada Classification of neuroblastic tumours

Adapted from Shimada A. et al. (Cancer 1999).

Chapter 1- Introduction 5 1.1.4 Molecular abnormalities

The status of the MYCN oncogene is one of the most highly prognostic indicators of aggressive tumour behaviour. Genomic amplification of MYCN is detected in approximately 20% of primary tumours, and is strongly correlated with advanced-stage disease and treatment failure (Brodeur et al., 1984; Seeger et al., 1985). MYCN amplification is present in 40% of patients with advanced disease and 5% to 10% of patients with low-stage disease. Amplification of MYCN, defined as greater than 10 copies of the gene per cell, is associated with rapid tumour progression, and poor outcome in patients with otherwise favourable disease patterns.

Alterations involving gain of chromosomes 1p, 11q and/or 17q also have prognostic significance (Schleiermacher et al., 2007; Vandesompele et al., 2005). Approximately 30% of neuroblastomas have 1p deletions, with the smallest common region of loss located within region 1p36.24 (Fong et al., 1989). Approximately 70% of advanced- stage neuroblastomas have 1p deletions (Gilbert et al., 1984). Deletion of the long arm of chromosome 11 (11q) also appears to be common in neuroblastoma, being present in approximately 40% of cases. A gain of 1–3 additional 17q copies, often through unbalanced translocation with chromosome 1 or 11, can also correlate with a more aggressive phenotype (Bown et al., 1999). The 17q breakpoints vary, but gain of a region from 17q22-qter suggests that a dosage effect of one or more genes provides a selective advantage (Schleiermacher et al., 2004). Several studies have linked 17q gain with known adverse prognostic factors: patient age >1 year, advanced stage disease, deletion of chromosome arm 1p, and amplification of the MYCN oncogene. It is believed that gain of chromosome segment 17q21-qter is of great biological and clinical importance in the diagnosis of neuroblastoma (Bown et al., 2001).

Another molecular abnormality found in neuroblastoma is a change in DNA content (ploidy). Normal human cells contain 2 copies of 23 chromosomes; thus, a normal diploid cell has 46 chromosomes. The majority (55%) of primary neuroblastomas are triploid or “near-triploid,” containing between 58 and 80 chromosomes; the remainder (45%) are either “near-diploid” (35–57 chromosomes) or “near-tetraploid” (81–103 chromosomes) (Kaneko et al., 1987). Patients with near-triploid, also known as hyperdiploid, tumours typically have favourable clinical and biological prognostic factors and excellent survival rates, compared with those patients who have near-diploid

Chapter 1- Introduction 6 or near-tetraploid tumours (Look et al., 1984). Currently, ploidy only impacts the risk group assessment of infants aged 12–18 months with metastatic disease, and infants with 4S disease according to the COG risk stratification schema (see Section 1.1.5).

1.1.5 Staging and risk stratification

Biological features of neuroblastoma tumours are of critical importance for risk assessment, they contribute to many groups’, including the Children’s Oncology Group (COG), risk stratification and therapeutic approach. International criteria for a common neuroblastoma staging system were first described in 1988 and subsequently revised in 1993 (Brodeur et al., 1993). The International Neuroblastoma Staging System (INSS) considered the extent of surgical resection of the primary tumour, the presence of lymph node involvement, and metastatic disease to determine disease stage (Table 1.2). However, there proved to be inconsistency with this staging system, as the expertise and aggressiveness of the surgeon influenced tumour staging, and patients who were simply observed without surgery could not be appropriately staged. As a result, an International Neuroblastoma Risk Group (INRG) task force was established in 2004 with the aim of developing a consensus approach to pre-treatment risk stratification (Cohn et al., 2009).

In 2009, the INRG proposed a new staging system based on tumour imaging rather than the extent of surgical resection (Monclair et al., 2009). In this staging system (INGRSS), localised tumours are staged based on the absence (L1) or presence (L2) of one or more of 20 image-defined risk factors. Metastatic tumours are defined as stage M, while stage MS, similar to INSS stage 4S pattern of disease (see Table 1.2), refers to disease with metastases limited to skin, liver, and bone marrow in children younger than 18 months of age at diagnosis (Table 1.3). Although the previous staging scheme (INSS) used a cut-off point of 12 months of age in designating higher or lower risk, data indicate that a higher age cut-off of 18 months has greater prognostic significance (Monclair et al., 2009).

Chapter 1- Introduction 7 Table 1.2 Neuroblastoma Staging: International Neuroblastoma Staging System (INSS)

From Brodeur, Pritchard et al. 1993.

Chapter 1- Introduction 8 Table 1.3 International Neuroblastoma Risk Group Staging System (INRGSS)

Adapted from Monclair, Brodeur et al. 2009.

Chapter 1- Introduction 9 To predict each patient's individual risk at the time of diagnosis and to allow for the most appropriate form of treatment, patients are stratified into groups of low-, intermediate- and high-risk based on the combination of the four markers: tumour stage, patient age at diagnosis, status of MYCN and status of chromosome 1p (Brodeur et al., 1988; London et al., 2005; Maris, 2005). Figure 1.1 shows the risk stratification according to the German Neuroblastoma Trial-NB2004, based on the criteria stated above (Oberthuer et al., 2009), and includes the treatment given for each risk group (Maris, 2010), which will be covered in more detail in Sections 1.1.7 and 1.1.8.

Risk stratification provides effective treatment for patients, by identifying patients in low to intermediate risk groups and reducing treatment, as reported in a study conducted by the International Society of Paediatric Oncology European Neuroblastoma Research Network (SIOPEN). This study showed that reduced treatment of infants with unresectable tumours or disseminated neuroblastoma without MYCN amplification produced excellent survival rates (De Bernardi et al., 2009; Rubie et al., 2011). They also evaluated the efficacy of decreased chemotherapy without radiotherapy in children aged < 1 year with unresectable neuroblastoma lacking MYCN amplification, and found with the reduced treatment approach that the overall survival was excellent.

Chapter 1- Introduction 10

Adapted from Oberthuer A. et al. (Future Oncology 2009) and Maris J.M. et al (New England Journal of Medicine 2010)

Figure 1.1 Neuroblastoma risk stratification and treatments Risk stratification for neuroblastoma patients based on the combination of four markers: tumour stage, patient age at diagnosis, status of MYCN and status of chromosome 1p, and the treatments for each group.

Chapter 1- Introduction 11 1.1.6 Neuroblastoma chemotherapy

Chemotherapy has an important role in the treatment of neuroblastoma, because the majority of patients have metastatic or locally advanced disease at diagnosis and require systemic treatment. There are many different classes of cytotoxic agents targeting different cellular pathways and mechanisms. Alkylating agents (cyclophosphamide, iphosphamide, busulfan, and melphalan), platinum analogues (cis-platinum and carboplatinum), vinca-alkaloids (vincristine), epipodophyllotoxins (VP16 and VM26), and anthracyclines (doxorubicin) have well-established activities and efficacies against neuroblastoma, and are considered standard options. Over the last few years, a number of other agents, such as topotecan, irinotecan, and temozolomide have also proven effective, and combinations including these drugs have been tested in phase II studies (Bagatell et al., 2011; Kushner et al., 2010). The choice of type and dose of treatment given to patients depends on whether they have intermediate-risk or high-risk disease, and can vary between different collaborative groups. High-risk patients are usually treated with combinations of the same chemotherapeutic drugs used in patients with intermediate-risk disease, but given at higher doses and with the addition of vincristine.

1.1.7 Treatment of low- and intermediate-risk neuroblastoma

Neuroblastoma patients are segregated into two groups for treatment: low- or intermediate-risk neuroblastoma and high-risk neuroblastoma. Patients with low- or intermediate-risk neuroblastoma tend to have better outcomes according to the COG P9641 study (Children’s Oncology Group P9641 clinical trial), where for low-risk patients with INSS stage 1 or 4S neuroblastoma, the 5-year OS rates were 99% ± 1% SD and 91% ± 1% SD, respectively (Strother et al., 2012). The intermediate-risk patients with INSS stage 2A or 2B tumours had a 5-year overall survival (OS) rate of 96% ± 1% SD post-surgery. Treatment with chemotherapeutic agents can improve the OS of intermediate-risk patients (96% ± 1% SD) as reported from the COG A3961 study (Baker et al., 2010). Using the risk-based approach has led to decreased therapy- related toxicities and improved outcome for neuroblastoma patients. Patients with intermediate-risk neuroblastoma undergo surgery for the resecting of the primary tumour, followed by multi-agent chemotherapy with cyclophosphamide, cisplatin, carboplatin, etoposide, or doxorubicin, which constitutes the backbone of treatment.

Chapter 1- Introduction 12 1.1.8 Treatment of high-risk neuroblastoma

Current treatment for high-risk neuroblastoma is divided into three distinct phases: intensive induction chemotherapy to achieve disease remission, myeloablative consolidation therapy with stem cell rescue, and finally a maintenance phase focused on the eradication of minimal residual disease (Figure 1.2). The backbone of the most commonly used induction chemotherapeutic regimen includes dose-intensive cycles of cisplatin and etoposide alternating with vincristine, doxorubicin, and cyclophosphamide (Kushner et al., 1994). Recently, the COG added topotecan to this induction regimen, based on data from their clinical trials showing anti-neuroblastoma activity in cases of relapse (Garaventa et al., 2003; London et al., 2010).

Adapted from Pinto N.R. et al. (Journal of Clinical Oncology 2015)

Figure 1.2 Current treatment strategy for high-risk neuroblastoma Standard high-risk neuroblastoma treatment consists of three phases of therapy: induction (chemotherapy and primary tumour resection); consolidation (high-dose chemotherapy with autologous stem-cell rescue and external-beam radiotherapy [XRT]); and post-consolidation (anti-ganglioside 2 immunotherapy with cytokines and 13-cis-retinoic acid). Reproduced with permission from American Society of Clinical Oncology.

Chapter 1- Introduction 13 Consolidation therapy has also been intensified for high-risk patients, with the introduction of high-dose therapy regimens with stem-cell transplantation in the 1980s. Stem cell harvest is typically performed after the first 2 cycles of induction therapy, and resection of the primary tumour and bulky metastatic sites is attempted after the fifth cycle. The Children’s Cancer Group (CCG) study, which enrolled patients with high- risk neuroblastoma between 1991 and 1996, was designed to assess whether myeloablative therapy in conjunction with autologous bone marrow transplantation (BMT) improved event-free survival (EFS), compared with chemotherapy alone, and whether subsequent treatment with 13-cis-retinoic acid (isotretinoin), a differentiating agent, would further improve EFS (Matthay et al., 1999a). This study demonstrated that the 3-year EFS was significantly better in patients who underwent BMT compared to those who did not. It also showed that patients who received 13-cis-retinoic acid after BMT experienced significantly better 3-year EFS and reduced risk of relapse compared to those who did not. Thus 13-cis-retinoic acid is now part of standard therapy during the first remission in patients with high-risk neuroblastoma.

In a phase III trial conducted by SIOPEN to test the efficacy of a busulfan plus melphalan regimen, compared to the standard treatment of cisplatin-etoposide- melphalan (CEM), better EFS and OS were observed with busulfan plus melphalan, compared with CEM (Ladenstein et al., 2011; Veal et al., 2012). The busulfan plus melphalan regimen also caused less toxicity (Hartmann et al., 1999). A COG pilot study (ANBL12P1; ClinicalTrials.gov identifier NCT01798004) is ongoing to evaluate the feasibility of administering busulfan plus melphalan and stem-cell rescue after an induction chemotherapy used in previous COG studies (Pinto et al., 2015). A pilot study (ANBL09P1; ClinicalTrials.gov identifier NCT01175356) is also being conducted to test the combination of iodine-131 (131I) plus metaiodobenzylguanidine (MIBG) with stem-cell rescue followed by busulfan plus melphalan and stem-cell support (Pinto et al., 2015).

1.1.9 Retinoid and immunotherapy

The majority (50–60%) of patients with high-risk neuroblastoma experience relapse, indicating the persistence of minimal residual disease at completion of consolidation therapy. The aim of maintenance therapy is to eradicate any residual tumour cells using

Chapter 1- Introduction 14 non-cytotoxic agents that are active against chemo-resistant tumour cells that survived the intensive induction and consolidation regimens. Early experiments showed that neuroblastoma cell lines can often be induced to terminally differentiate on exposure to retinoid compounds (Sidell, 1982; Thiele et al., 1985). The use of 13-cis-retinoic acid to treat residual disease after consolidation therapy was first evaluated in the 1990s, and a randomized COG trial demonstrated that treatment with this differentiating agent improved EFS, compared with no treatment (Matthay and Reynolds, 2000; Matthay et al., 1999a). In a subsequent COG study, a post-consolidation regimen of immunotherapy consisting of anti-ganglioside 2 (GD2) chimeric 14.18 antibody and cytokines followed by granulocyte macrophage colony stimulating factor (GM-CSF), interleukin-2 (IL2) and 13-cis-retinoic acid treatment, demonstrated significant improvement in both EFS and OS compared with 13-cis-retinoic acid treatment alone (Yu et al., 2010). GD2 antibody targets the disialoganglioside GD2 expressed on the surface of most neuroblastoma cells, making this approach both disease- and patient- specific. The addition of the cytokines IL-2 and GM-CSF reduces antibody-dependent cytotoxicity (Kushner and Cheung, 1989; Yu et al., 2010). Post-consolidation treatment with immunotherapy and cytokines plus 13-cis-retinoic acid is now considered part of standard-of-care treatment. However, there are still many unanswered questions about optimal dosing of 13-cis-retinoic acid, and the frequency of potential long-term toxicities.

1.1.10 Molecular targeted therapy

The identification of somatic alterations in high-risk tumours and an increased understanding of how these mutations drive tumour growth have led to the evaluation of specific molecular targeted therapies. These molecular targets include signalling pathways such as: anaplastic lymphoma kinase (ALK), mitogen-activated protein kinase (MAPK)/extracellular regulated kinase (ERK), polo-like kinase 1 (PLK1), aurora kinase A (AURKA), phosphatidylinositol 3-kinase (PI3K), and mechanistic target of rapamycin (mTOR). Identification of small-molecular inhibitors of these activated signalling pathways may aid in improved treatments for neuroblastoma, and is reviewed by Pinto et al. 2015 (Figure 1.3).

Chapter 1- Introduction 15

Figure 1.3 Current clinical approaches in targeting neuroblastoma Small-molecule inhibitors of ALK selectively bind constitutively phosphorylated mutant ALK receptors, leading to decreased signalling through PI3K/AKT, MAPK/ERK, and JAK/STAT pathways. BET bromodomain inhibitors (BDI) bind BRD4 protein, preventing DNA binding and transcription of MYCN. MYCN directly targets ODC, inhibiting the production of polyamines, which is required for cell replication and survival. Aurora kinase inhibitors reduce the ability of aurora kinase to bind to and stabilize MYCN protein, leading to its degradation. Iodine-131 (131I)- conjugated metaiodobenzylguanidine (MIBG) is carried through the norepinephrine transporter, allowing for targeted radiotherapy. Immunotherapeutic approaches include use of chimeric antigen receptor (CAR) T cells to promote host antitumor response. Anti-GD2 antibodies bind GD2 and cause cell death. Reproduced with permission from American Society of Clinical Oncology.

Chapter 1- Introduction 16 Amplification of MYCN is the most common somatic alteration in neuroblastoma. MYCN has proven to be a difficult gene to target therapeutically, however preclinical data have shown that indirect targeting of MYCN is possible by regulating MYCN transcription through bromodomain and extraterminal (BET) inhibition (Mertz et al., 2011; Puissant et al., 2013). BET inhibitors, such as JQ-1, bind BRD4 protein, preventing DNA binding and transcription of MYCN (Filippakopoulos et al., 2010). Another way of targeting MYCN indirectly is to inhibit key downstream targets of MYCN such as ornithine decarboxylase (ODC), the rate-limiting enzyme in production of polyamines. Polyamines are important for cell replication, translation, growth, and survival. The antihelmenthic agent difluoromethylornithine (DFMO) is an ODC inhibitor which reduces polyamine synthesis, it currently has been tested in a Phase I trial in patients with relapsed/refractory neuroblastoma. Initial results have been promising, with a subset of patients responding better to therapy containing DFMO (Saulnier Sholler et al., 2015). Inhibition of MYCN is discussed more thoroughly in Section 1.2.5.

The aurora kinase A (AURKA) gene provides another promising therapeutic target in neuroblastoma. Its inhibition can destabilise the MYCN protein (Brockmann et al., 2013). AURKA is the key regulator of the cell-cycle G2–M checkpoint (Carmena and Earnshaw, 2003; Glover et al., 1995; Marumoto et al., 2003). AURKA is highly expressed in high-risk tumours and, in addition to its growth-promoting role, stabilises the MYCN protein by direct physical association, preventing its degradation (Otto et al., 2009). The Pediatric Preclinical Testing Program, which tests early-phase agents in xenograft models of paediatric cancers, identified the AURKA inhibitor MLN8237 as a potent inhibitor of neuroblastoma (Maris et al., 2010), and it is currently being evaluated in combination with irinotecan and temozolomide in a phase I study by the New Approaches to Neuroblastoma Therapy (NANT) consortium (Pinto et al., 2015).

Inherited mutations in ALK have been identified in a number of familial neuroblastoma cases (Mosse et al., 2008b). Anaplastic lymphoma kinase (ALK) somatic mutation or gene amplification occurs in up to 15% of newly diagnosed neuroblastomas (Caren et al., 2008; Chen et al., 2008; George et al., 2008; Mosse et al., 2008a), and acquired ALK mutations may arise in relapsed neuroblastoma, as suggested by the higher frequency of ALK mutation (30%) in neuroblastoma-derived cell lines from patients that have relapsed (Schleiermacher et al., 2014). These mutations result in constitutive

Chapter 1- Introduction 17 activation of this receptor tyrosine kinase, providing an oncogenic driver analogous to activation via translocation events such as NPM-ALK in anaplastic large cell lymphoma, and EML4-ALK in non-small cell lung cancer (Morris et al., 1995; Touriol et al., 2000). Recent data have shown that targeted inhibition of ALK in cell models that harbour ALK mutation or amplification is highly effective, and is a good basis for early- phase clinical trials (Mosse et al., 2009). However, in a COG phase I study evaluating single-agent crizotinib in advanced paediatric solid tumours, the response rates in patients with neuroblastoma were disappointing. Only 9% of patients with known ALK- aberrant tumours, and 6% of those with unknown ALK status, achieved greater than partial response (Mosse et al., 2013).

ALK mutations that are common in neuroblastoma, such as F1174L, appear to be relatively resistant to standard crizotinib doses, which might be overcome by increased doses (Bresler et al., 2011). Oncologists are now investigating the effectiveness of ALK inhibitors in combination therapy. A COG phase I paediatric study testing crizotinib in combination with topotecan and cyclophosphamide is ongoing, and second-generation ALK inhibitors such as entrectinib (RXDX-1) and ceritinib (LDK378) are in early clinical development or early-phase trials in North America and Europe (Pinto et al., 2015). Ceritinib is a potent and selective small molecule tyrosine kinase inhibitor of ALK (Galkin et al., 2007). Ceritinib is active in NSCLC that is resistant to crizotinib (Friboulet et al., 2014). A thorough review of ALK inhibitors can be found in (Carpenter and Mosse, 2012; Iragavarapu et al., 2015).

Chapter 1- Introduction 18 1.2 MYCN

MYCN belongs to the MYC family of proto-oncogenes. These transcription factors regulate gene expression associated with critical cellular processes, such as proliferation, apoptosis, differentiation and metabolism (Henriksson and Luscher, 1996). They all possess the basic-helix-loop-helix-zipper (bHLHZ) domain structure. MYCN was identified in 1983 as an amplified gene region in human neuroblastoma cell lines that was homologous to the c-MYC oncogene. This region was designated N-myc (MYCN), and was found to be distinct from c-MYC in human neuroblastoma (Kohl et al., 1983; Schwab et al., 1983). Like c-MYC, MYCN was able to promote transformation in rat embryo fibroblasts, and induce cellular proliferation of quiescent fibroblasts (Schwab et al., 1985; Yancopoulos et al., 1985).

1.2.1 The MYC family The discovery of the MYC gene arose from an early study of the Rous sarcoma virus (RSV), a transforming retrovirus able to cause sarcomas in infected chicken cells (Rous, 1911). With the advancement of biological tools, the gene responsible for the transforming ability of RSV was identified and termed src. Using the information gathered from the isolation of src, the transforming sequence of MC29 avian tumour virus, which is responsible for inducing myeloid leukaemia, was identified and named v-g-myc or v-myc, for myelocytomatosis (Sheiness et al., 1978). This sequence was shown to be acquired from the highly conserved cellular gene, c-MYC (Roussel et al., 1979; Sheiness and Bishop, 1979; Varmus, 1984). Thus c-MYC belongs to a large group of proto-oncogenes, identified by the homology of their nucleotide sequences to retroviral oncogenes, and which were originally acquired by retroviruses through genetic transduction (Varmus, 1984).

Genes of the MYC family contribute to the genesis of many human tumours. In mammals, there are five related genes in the family: c-MYC, MYCN, MYCL, s-Myc and the less studied B-Myc, which encodes a protein that shows significant homology to the N terminus, but lacks essential domains in the C terminus of the other MYC proteins (Ingvarsson et al., 1988). S-Myc was identified from a rat genomic library. It has nucleotide sequences similar to the mouse MYCN protein, but lacks the acidic domain.

Chapter 1- Introduction 19 Due to the suppressive effect of this gene on the tumorigenicity of rat RT4-AC tumour cells in nude mice, this myc-related gene has been designated s-Myc (Sugiyama et al., 1989). Currently, only c-MYC, MYCN and MYCL have been implicated in tumorigenesis. Studies of human tumours show elevated expression of these MYC proteins in tumour tissues relative to the surrounding normal tissues, indicating that the overexpression of MYC contributes to tumorigenesis (Nesbit et al., 1999; Pelengaris and Khan, 2003). MYCL was found to be amplified in lung cancers (Kumimoto et al., 2002) including small cell lung cancer (SCLC) (Nau et al., 1985), and is also associated with thyroid cancer (Yaylim-Eraltan et al., 2008). Gene mapping studies mapped the location of MYCL to human chromosome region 1p32 (Nau et al., 1985; Zelinski et al., 1988), a location distinct from that of either c-MYC (region 8q24) (Watt et al., 1983) or MYCN (2p24). The MYCL protein is smaller than c-MYC and MYCN (Legouy et al., 1987), as it lacks a MYC box domain found in c-MYC and MYCN (Figure 1.4).

Of all the MYC-related genes, c-MYC has been the most widely studied, as it is one of the most frequently amplified oncogenes in many different human cancers (Beroukhim et al., 2010; Lin et al., 2012). c-MYC is instrumental in the progression of Burkitt’s lymphoma (Spencer and Groudine, 1991), and is frequently translocated in multiple myeloma (Shou et al., 2000). Unlike c-MYC, which is ubiquitously expressed in most proliferating cells during development and in dividing cells of adult tissues (Zimmerman et al., 1986), MYCN’s expression is tissue-specific and occurs during embryogenesis in pre-B cells, kidney, forebrain and hindbrain, where it plays an essential role in normal brain development (Knoepfler et al., 2002); thus constitutive deletion of MYCN is embryonic lethal (Charron et al., 1992; Stanton et al., 1992). MYCN is not significantly expressed in adult tissues, being down-regulated after embryonic development (Zimmerman et al., 1986). Although MYCN and c-MYC have different expression patterns, their coding regions are structurally similar and highly homologous. They both have long 5’ and 3’ untranslated regions (UTRs), and code for similarly sized proteins (~ 55 kDa) (Kohl et al., 1986).

MYC proteins have many conserved functional domains (Figure 1.4). The N-terminus of MYC proteins contains three highly conserved regions called Myc homology boxes (MB) I, II and IIIa. MYCN and c-MYC both contain an additional MBIIIb domain that MYCL lacks. MBI and MBII make up the transcriptional activation domain and can bind to co-factors to regulate gene transcription (Cowling and Cole, 2006). These two

Chapter 1- Introduction 20 regions were found to be necessary for Myc to co-operate with H-Ras to induce transformation of primary rat embryo fibroblasts (Kato et al., 1990). The central portion contains MBIIIb and MBIV, which are necessary for cellular transformation (Bannasch et al., 2001; Cowling et al., 2006). The DNA binding/dimerisation domain of MYC is composed of three different elements: the basic region, the helix-loop-helix, and the leucine zipper region (bHLHZ). The bHLHZ domain is characteristic of a class of transcription factors that bind to specific DNA sequences called E-boxes with the core motif 5'-CANNTG. The function of this domain is to homo- or heterodimerise through the HLHZ region, and to interact with DNA through the basic region (Luscher and Larsson, 1999).

Figure 1.4 Conserved domains of MYC Schematic diagram of the conserved domains found in MYCL, c-MYC and MYCN proteins. Myc Box domains are denoted by the Roman numerals I to IV, BR is the basic region domain, and HLHZ is the helix-loop-helix-zipper domain.

Chapter 1- Introduction 21 1.2.2 MYCN in cancer

In the neural crest, MYCN expression is enhanced by Sonic Hedgehog signalling to facilitate proliferation of immature neuronal precursor cells (Swartling et al., 2012). In mice with conditional deletion of MYCN in neural progenitor cells, there is a decrease in brain size and increase in neuronal differentiation (Knoepfler et al., 2002). MYCN was originally implicated in neuroblastoma following reports of its amplification in approximately 20% of neuroblastoma cases, in addition, its amplification correlated with advance disease stage and poor prognosis in patients (Brodeur et al., 1984; Seeger et al., 1985). This strong correlation of MYCN amplification with poor outcome has led to its status as a prognostic genomic biomarker, and it is currently used to stratify risk in neuroblastoma patients.

Mice with expression of MYCN targeted to the peripheral neural crest and driven by the rat tyrosine hydroxylase (TH) promoter, all developed neuroblastoma (Weiss et al., 1997). This TH-MYCN mouse model showed that amplification of MYCN in migrating neural crest cells can initiate neuroblastoma. Additionally, tumours from these mice showed chromosomal copy number abnormalities, suggesting that genetic mutation may also be required to promote neuroblast transformation. This suggested requirement for additional mutations was supported by data showing that the loss of tumour suppressor retinoblastoma (Rb), combined with the amplification of MYCN, resulted in increased penetrance of the tumour (Huang and Weiss, 2013; Weiss et al., 1997). Other observations include mutations in the RAS gene, which led to enhancement of the oncogenic role of MYCN, due to Ras-mediated stabilisation of MYCN and its translation (Yaari et al., 2005; Yancopoulos et al., 1985).

Aberrant amplification or overexpression of MYCN leading to poor prognosis has been reported in other cancers, such as: medulloblastoma (Aldosari et al., 2002; Swartling et al., 2010), glioblastoma (Bjerke et al., 2013), retinoblastoma (Lee et al., 1984), small- cell lung cancer (Funa et al., 1987; Nau et al., 1986), prostate cancer (Beltran et al., 2011; Mosquera et al., 2013) and breast cancer (Mizukami et al., 1995). This alteration in MYCN expression is associated with tumour aggressiveness, indicating that it has a driving role in MYCN-amplified or overexpressed tumours derived from primitive cell precursors. Most of these adult cancers are driven by c-MYC, however the similar structures of MYCN and c-MYC endow them with functional and biochemical

Chapter 1- Introduction 22 properties that are so closely related, that MYCN and c-MYC can replace each other in biological processes (Malynn et al., 2000). Differential expression of MYCN and c- MYC was reported in normal developing human renal tissues and in Wilms' tumours, a neoplasm which derives from primitive kidney cells. These tumours exhibited high levels of MYCN expression in the absence of gene amplification, and high-level expression of MYCN was associated with markedly diminished levels of c-MYC, suggesting that enhanced expression of the MYCN gene might lead to down-regulation of c-MYC (Nisen et al., 1986).

1.2.3 Function of MYCN

Functionally, MYCN is a transcriptional oncogene that regulates diverse cellular processes and can induce tumorigenesis. Like c-MYC, MYCN’s primary functions are to promote cell proliferation and to arrest cell differentiation. The MYC family has been shown to regulate the expression of about 15% of all human genes, and to be involved in many physiological functions, such as cell cycle control, metabolism, protein biosynthesis, and microRNA regulation (Beltran, 2014; Dang, 2012). In addition, MYCN has a direct role in blocking differentiation pathways and maintaining pluripotency (Nakagawa et al., 2010; Wakamatsu et al., 1997; Westermark et al., 2011). MYCN is not only an inducer of cell proliferation, but also has the ability to regulate apoptosis by interacting with anti-apoptotic proteins such as BCL-2 (Strasser et al., 1990), or negatively regulating tumour suppressor p53 (Fulda et al., 2000).

MYCN controls gene expression through heterodimerisation with MAX (gene activation) or MIZ-1 (Myc-interacting Zn finger protein-1) (gene repression), via its bHLHZ domain (Blackwood and Eisenman, 1991; Iraci et al., 2011; Wenzel et al., 1991). In transcriptional activation, the MYCN–MAX complex binds to gene promoters by recognising the consensus E-box sequences (CANNTG). MYCN preferentially binds the E-box motifs CATGTG and CACGTG, however under MYCN-amplified conditions, MYCN becomes less specific and can bind to CATTTG, CATCTG, and CAACTG (Murphy et al., 2009). Once bound to the E-box, MYCN–MAX complex can recruit co-factors such as positive transcription elongation factor b (P-TEFb) and Transcription factor II Human (TFIIH), which triggers transcriptional elongation (Eberhardy and Farnham, 2002), or transforming/transcription-associated protein

Chapter 1- Introduction 23 (TRRAP), which recruits GCN5 and TIP60 (Frank et al., 2003), forming a complex that acetylates histones, and allowing the transcription of target genes (McMahon et al., 2000) (Figure 1.5A). TRRAP binds to MYCN at the Myc Box II domain, while Myc Box I is the site where MYCN recruits TATA box binding protein (TBP) (McEwan et al., 1996).

MYC protein also plays a role as a transcriptional repressor. One of the first indicators that MYC functions as a transcriptional repressor came from studies published in the 1980s that suggested that MYC participates in a negative feedback loop (Meyer and Penn, 2008). Several groups observed that the products of the v-myc gene, c-MYC and MYCN were able to downregulate endogenous c-MYC expression in rat fibroblast cells (Cleveland et al., 1988; Penn et al., 1990a). The MYC region required for this negative autoregulation was demonstrated to be the same region required for transformation (Penn et al., 1990b), suggesting that repression of targeted genes by MYC protein could contribute to transformation (Grignani et al., 1990).

In its role as a transcriptional repressor, MYCN binds to MIZ-1 and is recruited to target gene promoters; this process is independent of E-box binding (Peukert et al., 1997; Schneider et al., 1997). By binding to MIZ-1, MYCN is able to repress CDKN2B (p15INK4b) (Seoane et al., 2001) by displacing MIZ-1 cofactors, and transglutaminase- 2 (TG2) by interacting with Specificity Protein 1 (SP1) (Liu et al., 2007). The MYCN– MIZ-1 complex can recruit other co-repressors such as DNA methyltransferases DNMT3a and EZH2, to silence or repress genes through DNA methylation (Brenner et al., 2005) (Figure 1.5B). MIZ-1 is a transcription factor with 13 zinc fingers and a POZ/BTB (poxvirus and zinc finger/bric-a-brac, tramtrack and broad complex) domain at its amino terminus (Bardwell and Treisman, 1994). MIZ-1 binds to the core promoter genes involved in cell cycle regulation and activates transcription. Upon binding to MYC, transcriptional activation by MIZ-1 is abolished, and the MYC–MIZ-1 complex acts as a transcriptional repressor; this is in part due to competition between p300 and MYC for binding to MIZ-1 (Staller et al., 2001). MIZ-1 binds to MYCN at the carboxyl terminus HLH region, the region where MYCN and MAX heterodimerise. The binding of MIZ-1 to MYC/MAX dimer disrupts the interaction between MIZ-1 and p300, leading to the transcriptional repression of tumour suppressor genes (Wanzel et al., 2003). In neuroblastoma, high MIZ-1 expression is associated with good patient prognosis (Ikegaki et al., 2007).

Chapter 1- Introduction 24

MYCN, in addition to regulating protein coding genes, is also an important regulator of non-coding genes. In neuroblastoma, MYCN has been shown to activate microRNAs (miR-17-92 cluster, miR-221, miR-9, and miR-421) (Chang et al., 2008; O'Donnell et al., 2005) and long non-coding RNAs (T-UCRs, ncRAN, and lncUSMycN) (Liu et al., 2014; Mercer et al., 2009; Mestdagh et al., 2010a; Yu et al., 2009a). It can also act as a repressor of several microRNAs including miR-184 and miR-542-5p (Bray et al., 2011; Foley et al., 2010). MYCN’s regulation of microRNAs and non-coding RNAs contributes to the oncogenic phenotype through regulation of downstream target pathways involved in proliferation, apoptosis and differentiation (Buechner and Einvik, 2012).

Chapter 1- Introduction 25

Figure 1.5 MYC–MAX and MYC–MIZ1 heterodimer (A) MYC–MAX heterodimers activate transcription by binding to E-box elements. Activation involves the recruitment of multiple coactivators and protein complexes such as Transcription factor II Human (TFIIH), part of the RNA polymerase II preinitiation complex that triggers transcriptional elongation; and transforming/transcription- associated protein (TRRAP) that recruits GCN5 and TIP60 which acetylates histones and chromatin regulator, allowing the transcription of target genes. (B) Myc represses transcriptional activation on core promoters that are activated by the MIZ-1 protein. The MYC–MAX heterodimer blocks transactivation by MIZ-1, partly through disruption of the interaction between MIZ-1 and p300 and by recruiting the DNA methyltransferase DNMT3a and EZH2.

Chapter 1- Introduction 26 1.2.4 Regulation of MYCN

Tight control of MYCN expression is an essential cellular mechanism for modulating MYCN function. There are a number of mechanisms that regulate MYCN expression, one highly studied mechanism is control of MYCN turnover by protein degradation. MYCN protein turnover is cell-cycle dependent, with degradation occurring during mitosis. MYCN protein is post-translationally modified, ubiquitinated and degraded with a relatively short half-life of approximately 15–20 minutes (Gregory and Hann, 2000). However, the extremely high steady-state levels (approximately 100-times normal) of MYCN protein in MYCN-amplified tumour cells ensure that the cells stay in cycle and do not enter G0 (Dang, 2012). MYCN transcriptional activity is regulated by phosphorylation of its two amino acid residues Serine 62 (S62) and Threonine 58 (T58) by the cell-cycle kinase cyclin B/CDK1 and glycogen synthase kinase-3β (GSK3β), respectively, during the mitotic phase of the cell cycle (Pulverer et al., 1994; Sjostrom et al., 2005). However there are emerging data that indicate ERK, an extracellular signal- regulated kinase that phosphorylates S62 in c-MYC, is also able to phosphorylate MYCN at the same amino acid residue (Marshall et al., 2011; Yaari et al., 2005).

The ubiquitin ligase FBXW7 normally targets MYCN for degradation via the ubiquitin– proteasome system (Sjostrom et al., 2005). This usually occurs after phosphorylation of S62, which primes T58 for phosphorylation. Proteins that prevent dephosphorylation at T58, such as AURKA, can stabilise MYCN. AURKA is a mitotic kinase normally expressed during G2 and mitosis, and blocks FBXW7-mediated degradation of the MYCN protein (Otto et al., 2009). AURKA is also frequently overexpressed or amplified in MYCN-amplified tumours such as neuroblastoma and neuroendocrine prostate cancer. MYCN-amplified neuroblastoma cells depend on high levels of AURKA expression for maintaining MYCN function (Beltran et al., 2011; Otto et al., 2009).

Another proposed mechanism for MYCN regulation includes accelerated MYCN translation by activation of the Harvey rat sarcoma viral oncogene homologue (H-Ras) through an oncogenic Ras mutation, and other oncoproteins that hyperactivate the mitogen-activated protein kinase (MAPK) pathway (Kapeli and Hurlin, 2011). This mechanism is distinct from H-Ras/MAPK regulation of c-MYC, which is primarily through protein stabilisation. It was found that oncogenic Ras signalling triggered

Chapter 1- Introduction 27 MYCN and c-MYC accumulation, with the accumulation of c-MYC a result of enhanced protein stability, whereas Ras increased MYCN turnover, and its accumulation was associated with increases in MYCN translation and not stability (Kapeli and Hurlin, 2011). H-Ras is an oncogene that is able to transform normal embryonic fibroblasts into tumorigenic cells with MYCN’s cooperation (Yancopoulos et al., 1985). Ras is regulated by the let-7 microRNA family (Johnson et al., 2005). The 3′-UTRs of the human Ras genes contain multiple let-7 complementary sites, allowing let-7 to regulate Ras expression.

This introduces an additional mechanism for regulating MYCN involving microRNAs. MicroRNAs (miRNAs) are a class of small non-coding RNA molecules, about 21–25 nucleotides in length. They are involved in the regulation of many biological processes, and their expression pattern is deregulated in cancer (Bartel, 2004; Lagos-Quintana et al., 2001). A number of miRNAs have been shown to act either as oncogenes or as tumour suppressors and to be involved in initiation and progression of cancer. miRNAs such as miR-34a and the let-7 family, have been demonstrated to regulate MYCN by targeting its mRNA, resulting in reduced MYCN expression and decreased proliferation of MYCN-amplified neuroblastoma cells (Buechner et al., 2011; Xu et al., 2009). The MYCN 3′-UTR harbours binding sites for several miRNAs, including the let-7 family and miR-34a. The most well-known miRNAs which act downstream of MYCN are the miRNAs of the miR-17-92 cluster and miR-9 (Mestdagh et al., 2010b; Schulte et al., 2008). These miRNAs are induced by MYCN, and act as oncogenes by downregulating tumour-suppressor genes. In neuroblastoma, the miR-17-92 polycistron or cluster is a direct target of MYCN and is overexpressed in tumours with MYCN gene amplification (Bray et al., 2009; Mestdagh et al., 2010b). miR-9 has been implicated in tumour cell invasiveness and metastasis, with high miR-9 expression correlating with the presence of MYCN amplification in neuroblastoma, which in turn is associated with metastatic disease and poor prognosis (Bray et al., 2009; Ma et al., 2010). The discovery of the pathogenic role of miRNAs acting upstream and downstream of MYCN in neuroblastoma suggests they could be utilised as biomarkers or even therapeutic drug targets.

More recently, an antisense transcript of MYCN, called MYCNOS, has been suggested to negatively regulate MYCN transcription (Jacobs et al., 2009). Natural antisense RNA can inhibit gene expression at the DNA level by transcriptional interference or at the

Chapter 1- Introduction 28 mRNA level by RNA interference or RNA editing, or regulate splicing by RNA masking (Lavorgna et al., 2004). MYCNOS is the antisense transcript of MYCN (Armstrong and Krystal, 1992), and shows overlap with the first exon of MYCN. The expression of MYCNOS might be relevant to the progression of neuroblastoma, potentially by directly inhibiting MYCN transcription by transcriptional interference at the DNA level (Jacobs et al., 2009).

1.2.5 Therapeutic targeting of MYCN

The established role of MYC in tumorigenesis and the ongoing requirement for MYC signalling to maintain tumour growth and proliferation make MYC family proteins prime therapeutic targets. It is frequently activated in human tumours, several of which appear to be addicted to MYC. This addiction was clearly demonstrated in several mouse models, where tumour regression was observed upon MYC inactivation (Felsher, 2010; Felsher and Bishop, 1999; Pelengaris et al., 1999). Sustained regression after brief inactivation of MYC was reported in osteosarcoma and lymphoma models (Giuriato et al., 2006; Jain et al., 2002). However, in other models tumours reappeared when MYC was reactivated (Boxer et al., 2004; Pelengaris et al., 2004). Depending on the malignancy studied, tumour regression was associated with apoptosis, cell cycle arrest, differentiation, and/or senescence.

To address the question of whether MYC inhibition is a viable cancer therapeutic strategy, the Omo-myc transgenic mouse model was engineered by Soucek et al. Omo- myc mice carry a mutant version of the bHLHZ domain of MYC, designed to dimerise with wild type MYC and also with MAX, which can be conditionally activated to inhibit the function of endogenous MYC (Soucek et al., 1998). Induction of Omo-myc in a Ras-driven lung cancer model with endogenous MYC expression induced apoptosis and senescence, leading to tumour regression (Soucek et al., 2008). In normal proliferative tissues such as skin and intestines, Omo-myc expression had profound effects, but these effects were completely reversible and tolerated (Soucek et al., 2008). Development of therapeutic strategies to selectively target MYC only in tumour cells will likely be of great importance.

Chapter 1- Introduction 29 Many different approaches have been taken to therapeutically target MYC in cancer. They include strategies aimed at either interfering with the tumour-promoting functions of MYC, such as proliferation and metabolism, by exploiting alterations in cancer cell metabolism (Dang et al., 2009), or stimulating tumour-suppressive functions induced by MYC, such as apoptosis and oncogene-induced senescence (Larsson and Henriksson, 2010). Approaches that target MYCN directly include various methods of silencing MYCN expression, either by small molecule inhibitors or even microRNAs (Frenzel et al., 2010).

Screening for small molecules that either directly inhibit MYC, or indirectly inhibit its function, has good therapeutic potential. Many studies have been conducted to identify small molecules that disrupt MYC–Max interaction (Berg et al., 2002; Yin et al., 2003), or that inhibit their interaction with DNA complexes to block transcription (Mo and Henriksson, 2006). Furthermore, the identification of drugs that target the upstream signal transduction pathways that regulate MYC protein expression and stability, or drugs acting on downstream MYC targets, is an alternative strategy to counteract MYC. The approach of targeting MYCN by modulating its protein stability is well documented (Gustafson and Weiss, 2010; Vita and Henriksson, 2006). Phosphorylation of MYCN at amino acid T58 is controlled by GSK3β, which is downstream of the PI3K/AKT/mTOR pathway. GSK3β is inactivated by AKT, thus activation of the PI3K/AKT pathway results in stabilisation of the MYCN protein (Chesler et al., 2006), and inactivation of the pathway using a PI3K inhibitor has been shown to reduce levels of MYCN (Segerstrom et al., 2011). AKT is commonly activated in MYCN-driven cancers including neuroblastoma, and the PI3K/mTOR inhibitor LY294002 has been shown to block progression of neuroblastoma (Chesler et al., 2006; Fulda, 2009). Another PI3K inhibitor, NVP-BEZ235, was shown to decrease angiogenesis and improve survival in a neuroblastoma mouse model (Chanthery et al., 2012). Thus, targeting of the PI3K/AKT/mTOR pathway may be therapeutically relevant in MYCN- driven tumours.

Another way to destabilise MYCN protein is to disrupt or inhibit interaction of Aurora kinase A and MYCN. Aurora A is a MYCN cofactor that stabilises MYCN in a kinase- independent fashion involving protein–protein interaction (Otto et al., 2009). Increased Aurora A expression is a negative prognostic factor in neuroblastoma (Shang et al., 2009), and pre-clinical testing with Aurora A inhibitor, MLN8237, showed significant

Chapter 1- Introduction 30 promise in cell line xenograft experiments (Maris et al., 2010). CD532, the lead compound from a screen of conformation-disrupting compounds, was identified as an allosteric inhibitor of Aurora A. CD532 does not alter the abundance of Aurora A, but works by inhibiting its kinase activity and preventing its stabilising interaction with MYCN, leading to decreased MYCN protein levels (Gustafson et al., 2014).

MYCN-amplified neuroblastomas have deregulated polyamine enzymes (including ODC, SRM, SMS, AMD1, OAZ2, and SMOX), which increases polyamine biosynthesis. Polyamines are organic cations that enhance transcription, translation, and replication, and support many cellular processes governed by MYC genes (Gerner and Meyskens, 2004). A study of genetic variability affecting ODC expression provided evidence that increased synthesis and cellular retention of polyamines has a causative role in human colon cancer (Martinez et al., 2003). ODC is a known oncogene that encodes the rate-limiting enzyme in polyamine synthesis (Auvinen et al., 1992; Bello- Fernandez et al., 1993). ODC gene expression is directly activated by MYCN, and it is overexpressed in high-risk neuroblastoma tumours, independent of MYCN amplification, and is associated with reduced survival (Hogarty et al., 2008). Because polyamines are essential for cell survival and are linked to cancer progression, the use of polyamine inhibitors may be beneficial in the treatment of neuroblastoma (Pegg, 1988). The ODC inhibitor α-difluoromethylornithine (DFMO) can inhibit neuroblast proliferation in vitro, and was able to suppress oncogenesis in vivo (Hogarty et al., 2008). Due to the effectiveness of DMFO in pre-clinical trials, it is currently in Phase I clinical trials to test its safety alone, and in combination with a chemotherapeutic drug, in paediatric patients with refractory or recurrent neuroblastoma (Saulnier Sholler et al., 2015).

An indirect approach to targeting MYCN is to inhibit its interaction with coactivators containing acetyl-lysine recognition domains that are required for histone acetylation and transcriptional activation. These coactivators include the bromodomain and extraterminal (BET) members of the bromodomain proteins (BRD2, BRD3, and BRD4). These BET inhibitors target MYCN transcription factors by interfering with chromatin-dependent signal transduction and inhibiting MYCN transcription (Dey et al., 2003; Mertz et al., 2011; Puissant et al., 2013). A small molecule BET inhibitor, JQ1, can inhibit MYCN-dependent transcription, reducing neuroblastoma cell growth, and inducing apoptosis (Puissant et al., 2013). JQ1 also has anti-proliferative effects on

Chapter 1- Introduction 31 acute lymphoblastic leukaemia (ALL) (Ott et al., 2012), acute myeloid leukaemia (AML) (Roe et al., 2015) and other c-MYC driven cancers (Asangani et al., 2014; Delmore et al., 2011). JQ1 targets BRD4, which plays a critical role in transcription involving RNA polymerase II (RNA Pol II) by facilitating recruitment of the positive transcription elongation factor P-TEFb (Jang et al., 2005; Yang et al., 2005), by competitively binding the conserved bromodomain pocket which results in the displacement of BRD4 from active chromatin and the subsequent removal of RNA Pol II from target genes (Dawson et al., 2011; Filippakopoulos et al., 2010). Due to overwhelming evidence of their effectiveness in mice, JQ1 and other inhibitors of BRD4 have moved into clinical trials to investigate their effectiveness in treating relapsed or refractory neuroblastoma, as well as AML (Beltran, 2014; Roe et al., 2015).

More recently, using a MYC target gene expression signature, the H2A/H2B histone chaperone FACT (facilitates chromatin transcription) was identified as a key driver and treatment target in MYCN-driven neuroblastoma (Carter et al., 2015). FACT is a chromatin remodelling complex composed of two subunits; Structure Specific Recognition Protein (SSRP1) and suppressor of Ty 16 (SPT16), which are involved in the transcription of genes with highly ordered chromatin structure, involved in replication and mitosis (Orphanides et al., 1999; Zeng et al., 2010). In neuroblastoma, FACT and MYCN act in a positive feedback regulatory loop, with FACT enhancing MYCN transcription and protein stability. Their expression is strongly correlated during tumour initiation, resulting in susceptibility of neuroblastoma cells to the FACT inhibitor CBL0137, a member of a new class of small molecule anti-cancer agents named curaxins.

Curaxins can modulate several important signalling pathways involved in the pathogenesis of pancreatic ductal adenocarcinoma (Gasparian et al., 2011). They can simultaneously activate p53 and inhibit cancer-associated stress response pathways, such as NF-kB and HSF-1 (Gasparian et al., 2011; Neznanov et al., 2009). CBL0137 can bind to DNA without causing DNA damage, an important consideration given that DNA damage is a significant drawback of most current cancer treatments. CBL0137 causes FACT to bind with high affinity to DNA, rendering FACT unable to bind histones and so perform its normal chromatin remodelling function. CBL0137 was shown to be highly effective against established primary tumours and pulmonary metastases in TH-MYCN+/+ mice, depleting MYCN expression in tumours after a single

Chapter 1- Introduction 32 intravenous dose. Moreover, CBL0137 was particularly effective in this mouse model as a combination therapy with DNA-damaging chemotherapeutic agents such as cisplatin, cyclophosphamide, and etoposide (Carter et al., 2015). Due to the effectiveness of CBL0137 in pre-clinical studies, the Children’s Oncology Group (COG) will begin a clinical trial of CBL0137 in 2016.

Despite the diverse potential therapeutic approaches to the treatment of neuroblastoma, and the fact that it is one of the most comprehensively characterized genomes among paediatric cancers, the knowledge acquired has not yet led to more effective treatment. Neuroblastoma tumours are extremely heterogeneous and cancer cells in general are remarkably adaptable. They develop resistance to targeted therapeutics by activating functionally redundant or degenerate pathways to compensate for the pathway being targeted, as is often the case with apical kinases such as ALK. However, MYC proteins are unique in that their actions are largely non-redundant, and the only gene products that can substitute for MYC are other MYC homologues. This makes MYC, especially MYCN, an important and relevant therapeutic target for neuroblastoma and many other cancers. Unfortunately, MYCN is not easy to target and has long been considered “undruggable”, because inactivating a highly abundant nuclear transcription factor that operates through a network of protein–protein interactions is pharmacologically challenging. Even with the advances in the field of drug design, and with the elucidation of the mechanisms underlying MYC overexpression in tumour cells, it is still difficult to obtain highly specific and active anti-cancer drugs. Through the identification of various small molecule compounds that interfere with c-MYC and MYCN, it may be possible to develop novel and effective therapeutic agents. Although BET inhibitors look like promising therapeutic molecules, and despite the many clinical trials, evidence is emerging of primary and acquired resistance to BET inhibition (Rathert et al., 2015). Therefore there is a need to identify other MYCN coactivators that can be targeted to regulate or inhibit MYCN oncogenic functions.

Chapter 1- Introduction 33 1.3 PA2G4

Proliferation-associated protein 2G4 (PA2G4) was initially identified from a cDNA expression library screen as a human gene with strong homology to the mouse Pa2g4 cell cycle gene (Lamartine et al., 1997). PA2G4 was mapped by in situ hybridisation to six autosomal locations (ch 3q24–25, 6q22, 9q21, 12q13, 18q12, 20p12) and to chromosome Xq25, with the strongest signals being found on chromosomes 9, 12, and X. Radiation mapping and fluorescent in situ hybridisation (FISH) were used to identify a functional copy of PA2G4 on chromosome 12q13. The PA2G4 gene on chromosome Xq25 lacks introns and has multiple stop codons in all reading frames, and is thus a pseudogene of PA2G4.

Two major mRNA species for PA2G4, 1.6 and 2.4 kb in size, were detected by Northern blotting (Lamartine et al., 1997; Yoo et al., 2000), and are the result of alternate splicing. These two transcripts were detected in all tissues, but with varying levels of expression. The 1.6 kb transcript was found to be highly expressed in the testis (Lamartine et al., 1997) and kidneys (Yoo et al., 2000), whereas expression of the 2.4 kb transcript was higher in the ovary and heart. An open reading frame encoding a 394 amino acid protein with a predicted molecular weight of 43 kDa was identified and showed 99% homology to the mouse p38-2G4 protein, with five amino acid differences (Lamartine et al., 1997). However the actual protein detected by anti-PA2G4 antibody on western blots is slightly larger, approximately 48 kDa (Yoo et al., 2000). PA2G4 protein has important domains including phosphorylation sites for kinase C, casein kinase II and c-AMP-dependent kinase A. Thus, the difference from the predicted electrophoretic mobility may be due to post-translational phosphorylation (Yoo et al., 2000). In addition, PA2G4 protein contains nuclear targeting signal domains, and a putative amphipathic helical domain (Lamartine et al., 1997), all of which suggest that PA2G4 is able to interact with DNA and other proteins.

1.3.1 Structure of PA2G4

The structure of PA2G4 has been solved using X-ray crystallography, which provides insights into the multiple functions of this protein (Kowalinski et al., 2007; Monie et al., 2007). PA2G4 contains a core domain that is homologous to type II methionine

Chapter 1- Introduction 34 aminopeptidases (MAPs). It adopts a ‘pita-bread’ fold formed from two anti-parallel β- sheets, each flanked by a pair of α-helices, with the active site contained within a deep pocket on its concave surface (Figure 1.6). However, unlike MAPs, it has a novel C- terminal extension that contains motifs important for binding proteins and RNA (Kowalinski et al., 2007; Monie et al., 2007). Despite the high degree of structural conservation, PA2G4 shows a rather low sequence identity (less than 25%) with members of the MAP family. Notably, a PA2G4-specific insertion is found at the entrance of the central cavity (helix α5). This cavity is polar and contains positively charged residues (Lys), as often found in RNA interaction sites, and might be employed in substrate recognition (Kowalinski et al., 2007). The crystal structure of PA2G4 revealed two putative RNA binding sites located on the C-terminal extension and loop insertion. Deletion of the C-terminal Lys-rich sequence severely impaired binding of PA2G4 to RNA and DNA targets (Monie et al., 2007), confirming that this is the binding motif for both RNA and DNA.

Attached to the C-terminal extension is a short amphipathic α-helix (α10). Helix α10 contains the LXXLL motif, a binding motif of co-activators of nuclear receptors (Heery et al., 1997; Plevin et al., 2005). Mutation in this motif to LXXAA inhibits the interaction between PA2G4 and the transcription factor Sin3A (Zhang et al., 2005b). Furthermore, the PKC phosphorylation site at S360 (Ahn et al., 2006; Lessor and Hamburger, 2001) occurs beyond helix α10 and marks the end of the globular fold of PA2G4. S360 strongly binds nuclear AKT and inhibits apoptosis (Ahn et al., 2006). Due to the close proximity of S360 to the LXXLL motif, it is likely to come into contact with binding partners that interact with this motif, suggesting that such interactions may be regulated by phosphorylation.

Chapter 1- Introduction 35

Figure 1.6 Structure of PA2G4 Ribbon representation of PA2G4 structure showing the beta barrel forming the hydrophobic cavity in the centre. The pita-bread fold is coloured in blue, the insertion domain in orange, the β-sheet connecting these domains in green, the PA2G4-specific helix α5 at the entrance of the cavity in cyan, and the C-terminal extension in red with helix α10 and the LXXLL motif indicated. Reproduced with permission from Elsevier.

Chapter 1- Introduction 36 1.3.2 Two distinctive isoforms of PA2G4

PA2G4 is ubiquitously expressed in all tissues and cells. The PA2G4 gene encodes two alternatively spliced isoforms, even though the gene has three in frame ATG codons (Figure 1.7A). Immunoblotting analysis following subcellular fractionation revealed two bands with molecular masses of 48 and 42 kDa in the cytoplasmic fraction, whereas only a 48 kDa band appeared in the nuclear fraction (Liu et al., 2006). Initiation of p48 translation starts at the first ATG codon, making p48 54 amino acids longer than p42. p42 translation begins at the third ATG codon, thus the protein has a truncated N- terminus (Figure 1.7B and C). In mammalian cells, p48 is the predominant form, whereas the p42 isoform is barely detectable because it is polyubiquitinated and subsequently degraded (Liu et al., 2009). In addition, the crystal structure of p42 suggests that this protein is unstable (Monie et al., 2007).

P48 and p42 have distinct functions. Overexpression of p42 suppresses cell growth and initiates cell differentiation, and also causes accumulation of cells in the G1 phase of the cell cycle. In contrast, overexpression of p48 stimulates cell growth and prevents neurite outgrowth, and enriches cells in S phase (Liu et al., 2006; Liu et al., 2009). P48 has been shown to prevent apoptosis by interacting with AKT and HDM2 (Ahn et al., 2006; Kim et al., 2012) to regulate p53. It also binds to BCL-2 mRNA and contributes to BCL-2 overexpression (Bose et al., 2006). Collectively, these observations suggest that p42 may act as a potential tumour suppressor, while p48 may function as an oncogene. Table 1.4 summarises the differential expression profiles of the two PA2G4 isoforms.

Chapter 1- Introduction 37

Figure 1.7 PA2G4 has two isoforms (A) Nucleotide sequence of PA2G4 showing the locations of the three ATG codons, highlighted in red and underlined. Exon 2 sequences are highlighted in blue and Exon 3 sequences are highlighted in green.

Chapter 1- Introduction 38

Figure 1.7 (cont’d) PA2G4 has two isoforms (B) Amino acid sequence of PA2G4; p42 lacks the first 54 amino acids highlighted in red and underlined. (C) Schematic diagram of the two alternatively spliced PA2G4 isoforms; p48 is translated from the first ATG codon while p42 is the truncated isoform and translated from the third ATG codon.

Chapter 1- Introduction 39 Table 1.4 Physical and functional differences between p48 and p42

Chapter 1- Introduction 40 1.3.3 Cellular localisation of PA2G4

PA2G4 is localised to the cytoplasm and accumulates in nucleoli of HeLa and AU565 breast cancer cells, and is almost undetectable in the nucleoplasm (Squatrito et al., 2004; Xia et al., 2001). PA2G4 was identified as a component of human nucleoli using mass spectrometry to analyse 350 different nucleolar proteins (Andersen et al., 2002; Scherl et al., 2002). Two regions were identified as necessary for proper PA2G4 localisation: the 48 N-terminal amino acids and the C-terminal region 301–394. Deletion of the N- terminal region or generation of point mutants at amino acid residues K20A–K22A completely abolishes the nucleolar localisation of PA2G4, while mutation in the C- terminal region R364A–K365A reduces PA2G4 accumulation in the nucleolus (Squatrito et al., 2004). This observation suggests that the C-terminal region of the protein contains both a nucleolar retention signal (NoRS), which allows the protein to be kept within the nucleolus, and a nuclear export signal (NES), which exports the protein from the nucleus. Not surprisingly, the loss or lack of the 54 N-terminal amino acids, required for nuclear translocation, results in confinement of the PA2G4 splice isoform to the cytoplasm (Liu et al., 2006).

1.3.4 PA2G4 is an ErbB-3 binding protein PA2G4 has been identified as an epidermal growth factor receptor (EGFR) binding protein using yeast two-hybrid system screening to characterise proteins that interact with EGFR binding protein-3 (ErbB-3) (Yoo et al., 2000). Consequently, PA2G4 is also known as ErbB-3 binding protein (Ebp1). EGFR family members, including EGFR, ErbB-2, ErbB-3, and ErbB-4, are glycosylated transmembrane proteins with tyrosine kinase domains (Kraus et al., 1989; Plowman et al., 1990). Ligands, such as EGF or heregulin (HRG), activate ErbB family members by inducing their homo- or heterodimerisation, resulting in stimulation of tyrosine kinase activity and initiation of signalling pathways, leading to cellular proliferation and differentiation (Goldman et al., 1990; Kim et al., 1994; Olayioye et al., 2000). ErbB-3 is a functional receptor for HRG, with HRG having been shown to bind to the extracellular domain of ErbB-3 and stimulate its tyrosine phosphorylation (Carraway et al., 1995). To better understand HRG-mediated ErbB-3 signalling pathways, Yoo et al. conducted yeast two-hybrid screening with the cytoplasmic domain of ErbB-3, to isolate proteins that interact with

Chapter 1- Introduction 41 ErbB-3. PA2G4 was found to bind to the first 15 amino acids of the juxtamembrane domain of ErbB-3. Treatment of cells with heregulin resulted in dissociation of PA2G4 from ErbB-3, and the translocation of PA2G4 from the cytoplasm into the nucleus (Yoo et al., 2000), suggesting that PA2G4 may be a downstream member of an ErbB-3- regulated signal transduction pathway.

1.3.5 Phosphorylation of PA2G4

Phosphorylation of proteins is an important regulatory mechanism in cellular processes. It is a reversible procedure that alters the structural conformation of a protein, causing it to become activated, deactivated, or modifying its function. Phosphorylation status is regulated by enzymes called kinases and phosphatases. PA2G4 is reported to be phosphorylated by many different kinases, the first to be documented was Protein Kinase C (PKC) (Lessor and Hamburger, 2001). PA2G4 contains 11 putative serine/threonine phosphorylation sites, six of which are putative Protein Kinase C (PKC) phosphorylation sites as determined by sequence analysis (Yoo et al., 2000). In breast cancer cells, PA2G4 is basally phosphorylated at Threonine 366, and this phosphorylation is enhanced upon HRG treatment (Lessor and Hamburger, 2001). However, in vitro data showed phosphorylation at both Serine 363 and Threonine 366 (Lessor and Hamburger, 2001). Basal phosphorylation of PA2G4 is thought to be PKC- dependent, whereas HRG-induced phosphorylation is independent of PKC. It is unlikely that phosphorylation of PA2G4 causes it to dissociate from ErbB-3, because PA2G4 protein has already dissociated from ErbB-3 and translocated to the nucleus in the majority of cells before phosphorylation is detected (Lessor and Hamburger, 2001; Yoo et al., 2000). It is possible that HRG-mediated receptor activation results in a conformational change in PA2G4 protein, causing it to dissociate from ErbB-3.

PA2G4 is phosphorylated by p21-activated kinase (PAK1). PAK1 is one of many signalling kinases involved in cancer progression, and is up-regulated in several human cancers such as breast, prostate and hepatocellular carcinoma (Ching et al., 2007; Goc et al., 2013; Kumar et al., 2006). PA2G4 has four putative PAK1 phosphorylation sites, at S252, T261, S335 and S375, which possess the PAK1 motif (K/R-K/X-X-S/T). An in vitro kinase assay demonstrated that one of these sites, T261, and S363, were phosphorylated by PAK1, with the phosphorylation of T261 potentially repressing the

Chapter 1- Introduction 42 activity of Cyclin D1 and E2F1 promoters in breast cancer cells (Akinmade et al., 2008).

PA2G4 is phosphorylated by dsRNA-activated protein kinase (PKR). PKR is one of four kinases that are involved in the phosphorylation of the eukaryotic initiation factor 2α (eIF2α) (Clemens, 1997; Srivastava et al., 1998). PA2G4 binds PKR through its carboxyl-terminal region, and in a kinase assay, involving the incubation of GST-PKR fusion protein in the presence of the GST-PA2G4 recombinant protein and [γ-32P] ATP, PKR was able to phosphorylate the GST-PA2G4 fusion protein (Squatrito et al., 2006). Other PA2G4 phosphorylation sites are specific to other cellular processes. Phosphorylation at S363 is required for PA2G4 to bind Sin3A and HDAC2, resulting in repression of Cyclin D1- and Cyclin E-regulated promoters (Akinmade et al., 2007). Phosphorylation of PA2G4 at S360 is needed for efficient nuclear localisation, and for binding ErbB-3 and nuclear AKT (Ahn et al., 2006).

1.3.6 PA2G4 binds DNA

The ability of PA2G4 to bind nucleic acid is well documented (Ahn et al., 2006; Bose et al., 2006; Pilipenko et al., 2000; Squatrito et al., 2004; Zhang et al., 2005b). PA2G4 protein has a putative amphipathic helix containing repetitive, positively charged amino acids, suggesting that it may directly interact with DNA and/or proteins. PA2G4 has been shown to interact indirectly with E2F1 at E2F1 promoter elements by binding to a complex of nuclear proteins, including retinoblastoma (Rb) (Xia et al., 2001) and Sin3A (Zhang et al., 2005b). The activity of Rb and PA2G4 on the E2F1 promoter is regulated by the ErbB-3 ligand heregulin (Zhang and Hamburger, 2004).

Chromatin immunoprecipitation (ChIP) and DNA affinity precipitation assays demonstrated that PA2G4 directly binds to the PSA and E2F1 promoters of Sin3A (Zhang et al., 2005b). Sin3A has been demonstrated to be associated with both androgen receptor- (AR) (Sharma and Sun, 2001) and Rb- (Lai et al., 2001) regulated promoters, and to contribute to repression of the activities of these promoters. The C- terminal domain of PA2G4, which enables it to repress transcription and arrest cell growth, was necessary and sufficient for binding Sin3A and conversely, the C-terminal

Chapter 1- Introduction 43 domain of Sin3A, containing the HDAC-interacting domain, bound PA2G4 (Zhang et al., 2005b).

The C-terminal domain of PA2G4 is an important DNA binding domain. Xia et al. showed that this domain (residues 300–372) was necessary for Rb binding, and that the binding of PA2G4 to Rb is important in transcriptional repression in Rb+ cells, as PA2G4 mutants lacking the Rb binding domain could no longer repress transcription of cyclin E (Xia et al., 2001). PA2G4 has also been shown to bind to HDAC2 both in vitro and in vivo, with the ability of PA2G4 to recruit HDAC related to its ability to repress transcription. HDAC inhibitors such as trichostatin A can significantly reduce PA2G4- mediated repression (Zhang et al., 2003).

PA2G4 strongly binds AKT and suppresses apoptosis. Phosphorylated nuclear but not cytoplasmic AKT interacts with PA2G4 and enhances its anti-apoptotic action independent of AKT kinase activity (Ahn et al., 2006). Kim et al. determined that PA2G4 also binds to the p53 E3 ligase HDM2, enhancing HDM2–p53 association, and thereby promoting p53 polyubiquitination and degradation to reduce p53 levels and activity (Kim et al., 2010). Furthermore, Kim et al. proposed that PA2G4 modulates p53 levels by stabilisation of the HDM2 protein, and by fostering HDM2 interaction with AKT (Kim et al., 2012).

1.3.7 The role of PA2G4 in RNA processing and translation

PA2G4 is not only involved in transcriptional regulation, but also seems to participate in the control of protein translation (Pilipenko et al., 2000; Squatrito et al., 2006). Analysis of the crystal structure of PA2G4 revealed two putative RNA binding sites: the helix α5 (residues 205–213) which contains positively-charged lysine residues often found in RNA interaction sites (Kowalinski et al., 2007), and an extended positively- charged surface patch also with a lysine-rich sequence (364RKTQKKKKKK373), situated within the C-terminal extension (residues 338–394) (Kowalinski et al., 2007; Monie et al., 2007). These findings contradicted sequence analyses of RNA binding. It had been suggested that the s70-like domain (Squatrito et al., 2004), and a double-stranded RNA- binding domain (Squatrito et al., 2006), were the RNA binding sites of PA2G4, because deletion of these putative domains abrogated RNA binding. However, the crystal

Chapter 1- Introduction 44 structure showed that neither of these domains was present in PA2G4. Nevertheless, deletion of the residues predicted to form them would cause loss of function by severely disrupting protein folding (Monie et al., 2007), explaining the result of the deletion study. Furthermore, mutations within the lysine-rich C-terminal extension (residues 364–373) partially inhibit nucleolar localisation (Squatrito et al., 2004). As the nucleolus is the main site of ribosome biosynthesis, this suggests that the RNA-binding activity of PA2G4 contributes to this function.

PA2G4 protein binds to a number of RNAs including mRNA (Bose et al., 2006), rRNA, and ribonucleoprotein (RNP) complexes (Pilipenko et al., 2000; Squatrito et al., 2006). PA2G4 seems to bind more strongly to ribosomal 5S RNA when compared to a mixture of t-RNAs or the S-domain RNA of the signal recognition particle, suggesting a direct interaction of PA2G4 with mature ribosomes via the 5S RNA (Kowalinski et al., 2007). As a member of the ribonucleoprotein complex, which is the association of RNA and protein, PA2G4 contains a double-stranded RNA binding domain (dsRBD) that mediates its interaction with dsRNA (Squatrito et al., 2004; Squatrito et al., 2006). Deletion of this domain impairs its localisation to the nucleolus and its ability to form RNP complexes (Squatrito et al., 2006). In the cytoplasm, PA2G4 associates with mature ribosomes and acts as a cellular inhibitor of eukaryotic initiation factor 2 alpha (eIF2α) phosphorylation by binding the double-stranded RNA-activated kinase, PKR, which renders it incapable of phosphorylating eIF2α (Squatrito et al., 2006). Interestingly, PA2G4 has also been described as an Internal Ribosomal Entry Site (IRES)-specific cellular trans-acting factor that mediates translation of specific viral IRESs (Pilipenko et al., 2000). Translation initiation is mediated by specific IRES structures, a process that requires eukaryotic initiation factors (eIFs).

PA2G4 may also play a role in the processing of rRNA precursors and intermediates. It associates with nucleophosmin (B23) in the nucleolus, and ablation of either B23 or PA2G4 reduces ribosome biosynthesis by inhibiting the processing of the 32S intermediate for subsequent maturation of the 28S rRNA (Okada et al., 2007). This means PA2G4 acts as an endonuclease that cleaves the 38S pre-rRNA to generate the mature 28S rRNA. Figure 1.8 summarises the molecular interactions of PA2G4 in the cytoplasm and nucleus, and its key role in cellular processes.

Chapter 1- Introduction 45

Figure 1.8 Molecular interactions and cellular processes of PA2G4 PA2G4 binds to ErbB-3 in the cytoplasm, however upon stimulation with heregulin (HRG), PA2G4 dissociates from ErbB-3 and translocates to the nucleus. PA2G4 is phosphorylated by protein kinase C and p21-activated kinase (PAK1). Phosphorylation of PA2G4 at Serine 360 is needed for proper nuclear localisation, and for binding to ErbB-3 and nuclear AKT. PA2G4 binds to many proteins including Sin3A and retinoblastoma protein (RB) to enable transcription of E2F1, a cell cycle regulator. PA2G4 regulates cell survival by interacting with AKT and MDM2 to form a nucleoprotein complex to deregulate p53. PA2G4 also plays a role in the processing of rRNA precursors and intermediates by associating with nucleophosmin in the nucleolus and acting as an endonuclease that cleaves the 38S pre-rRNA to generate the mature 28S rRNA. In the cytoplasm, PA2G4 associates with mature ribosomes and acts as a cellular inhibitor of eIF2α (eukaryotic initiation factor) phosphorylation by binding the double-stranded RNA activated kinase, PKR, which renders it incapable of phosphorylating eIF2α, and is involved in protein translation.

Chapter 1- Introduction 46 1.3.8 The role of PA2G4 in cancer

ErbB family members are frequently overexpressed in human carcinomas including those of the breast, stomach, lung, ovary and pancreas (Gullick, 1991; Lemoine et al., 1992; Ullrich et al., 1984). Initial reports showed that ectopic expression of PA2G4 in ErbB-3-expressing breast carcinoma cell lines resulted in inhibition of colony formation and decreased proliferation (Lessor et al., 2000; Zhang et al., 2008a), suggesting that PA2G4 acts as a tumour suppressor. However, since the discovery of the two PA2G4 isoforms with their opposing cellular functions, data are emerging showing that high expression of PA2G4-p48 in tumour cells of breast cancer patients is correlated with poor clinical outcome. This is further supported by the observation that p48 expression is decreased in MCF-7 breast cancer cells with tamoxifen treatment (Ou et al., 2006). It is believed that the tumour suppressor isoform of PA2G4, p42, is polyubiquinated by Bre1 E3 ligase, resulting in its degradation in human cancer (Liu et al., 2009).

PA2G4 has also been implicated in glioblastoma and p48 is highly expressed in glioblastoma cell lines and human malignant gliomas (Kim et al., 2010). Overexpression of p48 promotes cancer cell growth and migration, and facilitates tumorigenesis in glioblastoma mouse xenograft models (Kim et al., 2010). Regulation of cell growth by p48 is through the degradation of p53, by fostering its interaction with HDM2. Overexpression of p48 reduces p53 protein levels but does not alter p53 mRNA levels, thus p48 regulation of p53 is posttranslational (Kim et al., 2010). Furthermore, treatment of glioblastoma cells with HIV-1 viral protein R (Vpr) to induce apoptosis down-regulates PA2G4 and up-regulates p53 (Zhang et al., 2014).

Reports of the role of PA2G4 in carcinomas have been contradictory. In salivary adenoid cystic carcinoma (ACC) and oral squamous cell carcinoma (OSCC), high PA2G4 expression was found to be associated with good patient prognosis, and overexpression of PA2G4 by gene transfer decreased cell proliferation and reduced tumour metastasis in mouse models (Yu et al., 2007; Zhou et al., 2010b). Inhibition of cell migration and invasion by PA2G4 was proposed to be mediated by up-regulation of E-cadherin and down regulation of MMP9 (Sun et al., 2012). In a recent article, Mei et al. reported that in 81 patients with oral premalignant lesions, PA2G4 expression is strongly related to OSCC development (Mei et al., 2014b). They showed that PA2G4 promotes oral tumorigenesis by binding to the podoplanin promoter and increasing its

Chapter 1- Introduction 47 mRNA and protein, however this study did not distinguish between the p48 and p42 isoforms. Podoplanin has been shown to be expressed at high levels in various types of cancers and associated with tumour progression (Raica et al., 2008). In OSCC, podoplanin is expressed in ~90% of primary tumours with a high rate of lymph node metastasis (Yuan et al., 2006). While the investigators did not specify whether they were discussing p48 or p42, based on their immunoblots of nuclear PA2G4, it appears to be the p48 isoform that is binding and regulating podoplanin.

Ahn et al. reported that p48 interacts with AKT by mediating its phosphorylation to suppress apoptosis in PC12 cells (Ahn et al., 2006). Further evidence for the interaction between p48 and AKT came from work carried out in breast cancer cells (Kim et al., 2012). Recently, it was reported that p42 also interacts with AKT by suppressing its activation in non-small cell lung cancer (NSCLC) (Ko et al., 2015). Low p42 expression was associated with high tumorigenicity, and restoring p42 in NSCLC cells suppressed their growth and motility, thus providing further evidence that p42 acts as a tumour suppressor.

Following the discovery that PA2G4 can interact with and regulate the androgen receptor (AR), a critical molecule in the aetiology of prostate cancer, studies were conducted to investigate whether PA2G4 plays a role in the progression of prostate cancer (Zhang et al., 2005b; Zhang et al., 2002). PA2G4 expression was found to be significantly reduced in preclinical models of hormone-refractory prostate cancer (Zhang et al., 2005a), and in advanced stages of clinical prostate cancer (Zhang et al., 2008b). PA2G4 regulated AR by promoting the decay of AR mRNA through physical interaction with a conserved UC-rich motif within the 3’-UTR of AR. PA2G4 also bound to the CAG polyglutamine repeat region of AR mRNA, and was able to inhibit AR translation (Zhou et al., 2010a). The CAG regions in AR mRNA have been postulated to be involved in the control of translation (Yeap et al., 2004). Ectopic expression of PA2G4 inhibited prostate cancer cell growth both in vitro and in xenograft mouse models of prostate cancer (Zhang et al., 2002), and decreased expression of androgen-regulated genes such as prostate specific antigen (PSA) (Zhang et al., 2005a). Conversely, suppression of PA2G4 resulted in increased cell growth in the absence of androgen, increased PSA production, and activated AKT signalling (Zhang et al., 2008b).

Chapter 1- Introduction 48 Contrary to the results above, in one study PA2G4 expression in prostate cancer patient tissues correlated with progression from normal to hormone-sensitive then to hormone- refractory prostate cancer (Gannon et al., 2008). In this study, high expression of PA2G4 in patient tissues correlated with high nuclear AR expression, an observation inconsistent with in vitro data in LNCaP cells (Zhang et al., 2005a). Gannon et al. acknowledged that PA2G4 has two distinct isoforms, however, they only used an antibody that recognised the p48 isoform. They also noted that the two PA2G4 isoforms have not yet been examined in prostate cancer, and the differential expression of p42 and p48 isoforms may explain the contrasting results reported in the different prostate cancer studies. The finding that PA2G4 is associated with disease progression is also supported by data demonstrating overexpression of PA2G4 in colorectal cancer (Santegoets et al., 2007).

1.3.9 Pa2g4 knockout mouse

To understand the physiological role of PA2G4 in a whole organism, a PA2G4 deficient mouse carrying a gene trap insertion in intron 2 of the PA2G4 gene was generated (Zhang et al., 2008c). This insertion led to the expression of a PA2G4–neomycin fusion protein containing only the first 20 amino acids encoded by Exon 1 of the PA2G4 gene, which is expected to be non-functional. These Pa2g4 -/- mice were fertile, however their litter size was less than half that of the wild type mice, 4.2 ± 1.3 and 9.1 ± 0.8 respectively. Pa2g4 -/- mice were also of smaller body size, with 1/3 having kinky tails, and they all had approximately 30% reduction in weight at day 19 compared to the wild type mice. However by day 30 the Pa2g4 -/- mice began to increase in size, and by day 60 there were no differences in weight between the Pa2g4 -/- and wild type mice (Zhang et al., 2008c).

Analysis of mouse embryonic fibroblast (MEF) cells showed that Pa2g4 -/- MEFs proliferated much more slowly than wild type MEFs, and had a shorter propagation cycle of only 3 passages prior to senescent morphology, compared to wild type MEFs which could be propagated for 5 passages. Expression profiling of MEFs identified significant changes in expression of members of the insulin (IGF-1, IGFBP-3 & 4), cyclin (cyclin D1 & Cdkn2a), and MAPK (SOS-1, Grb-14 & Mdm2) families (Zhang et al., 2008c). Zhang et al. concluded that PA2G4 may not be required for prenatal

Chapter 1- Introduction 49 development, but loss of PA2G4 affects post-natal growth, which may be related to changes in levels of IGF-1 and IGFBP, key components of cellular proliferation pathways.

1.4 Research perspectives

MYCN plays an important role in neuroblastoma biology and its gene amplification and aberrant expression have the ability to predict patient outcome. The MYC protein family is extensively studied and pharmaceutical companies and researchers have attempted to develop chemical inhibitors against MYC and MYCN. However, despite substantial efforts to identify MYCN inhibitors, there has been limited success, and no MYCN inhibitors are in clinical use or currently in clinical trials. MYCN regulates the transcription of many important genes involved in cell growth and death, including both oncogenes and tumour suppressors. Rather than directly targeting MYCN, it may be more advantageous to identify proteins that interact with or bind to MYCN as potential therapeutic targets.

This study begins with the identification of PA2G4 as a MYCN binding partner in neuroblastoma. The hypotheses for this thesis are that PA2G4 acts as an oncogene by binding directly to MYCN to facilitate cell growth in neuroblastoma, and that PA2G4 is a viable therapeutic target for cancer treatment. Three aims have been formulated to test these hypotheses. The first aim is to determine whether PA2G4 directly interacts with MYCN and plays a role in MYC oncogenesis in neuroblastoma. By addressing this first aim, the studies reported in this thesis will elucidate MYCN’s role in neuroblastoma tumorigenesis, to provide insight on how to effectively target MYCN’s oncogenic effect by exploiting its interaction with PA2G4.

PA2G4 is a relatively newly discovered protein that has not been comprehensively studied. It has been described as either an oncogene or a tumour suppressor in various cancers other than neuroblastoma. These conflicting data on PA2G4’s activity led to the second aim of this thesis, which is to determine the functional role of PA2G4 in neuroblastoma cell growth, and to identify key genes and pathways involved in its signalling. The rationale for aim two is to investigate whether PA2G4 is a potential molecular target for the treatment of neuroblastoma.

Chapter 1- Introduction 50

If PA2G4 is involved in neuroblastoma growth and progression, it has the potential to be a therapeutic target. The third aim of this thesis is to determine the role of PA2G4 in neuroblastoma tumorigenesis using in vivo models, and to characterise potential PA2G4 inhibitors for the treatment of neuroblastoma. The outcome of aim three will have important implications for the development of novel compounds with clinical potential for more effective therapy.

In summary, the objective of this thesis is to investigate the role of PA2G4 in MYCN- driven neuroblastoma and to research novel therapeutic approaches to treating this disease, through targeting the interaction of MYCN and PA2G4 with the aim of improving treatment of neuroblastoma.

Chapter 1- Introduction 51

Chapter 2 MATERIALS AND METHODS

CHAPTER 2

MATERIALS AND METHODS

Chapter 2- Materials and Methods 52 2.1 Cell culture

2.1.1 Reagents and equipment

Foetal calf serum (FCS) was purchased from Thermo Trace (Noble Park, VIC, Australia). Dulbecco’s Minimum Essential Media (DMEM), Alpha-Minimum Essential Media (α-MEM), Roswell Park Memorial Institute 1640 media (RPMI-1640), trypsin, phosphate-buffered saline (PBS), and Trypan Blue (0.4% solution) were purchased from Life Technologies (Carlsbad, CA, USA). Dimethyl sulfoxide (DMSO) was purchased from Sigma-Aldrich (St Louis, MO, USA). The MycoAlert mycoplasma detection kit was purchased from Lonza (Mt Waverley, VIC, Australia). Cell culture flasks, plates and cryo-vials were supplied by Greiner Bio-One (Frickenhausen, Germany). “Complete media” refers to either DMEM or RPMI-1640 media supplemented with 10% FCS.

Lipofectamine 2000 and Opti-MEM were purchased from Life Technologies (Carlsbad, CA, USA). All siRNAs were supplied by Dharmacon (Lafayette, CO, USA) except MYCN, c-MYC, and TFAP4 siRNAs, which were purchased from Qiagen (Hilden, Germany). pCMV6-myc-flag-PA2G4 and pGFP-C-shLenti-PA2G4 plasmid DNA were supplied by Origene (Rockville, MD, USA), and pShuttle-MYCN plasmid DNA was purchased from Addgene (Cambridge, MA, USA).

All cell culture experiments were performed in a Biological Safety Cabinet Class II (AES Environment Pty Ltd, NSW, Australia), and cells were maintained at 37oC with

5% CO2 using humidified incubators from Binder (Tuttlingen, Germany). Cell counts were performed using a Neubauer haemocytometer purchased from Dutec Diagnostics (Sydney, NSW, Australia) and visualised using an Olympus bright-field, phase contrast microscope with fluorescence capabilities (Notting Hill, VIC, Australia).

2.1.2 Cell lines

Neuroblastoma cell lines were kindly provided by Prof. Susan L. Cohn (Northwestern University, Chicago, IL, USA) and Prof. June Biedler (Memorial Sloan Kettering Cancer Centre, New York, NY, USA), or purchased from ATCC (Manassas, VA, USA). The two normal lung fibroblast cell lines, MRC-5 and WI-38, were purchased

Chapter 2- Materials and Methods 53 from ATCC. Cell line identities were validated by CellBank Australia (Westmead, NSW, Australia). Table 2.1 provides information on the anatomical origin of each cell line, its disease stage or cell type, the culture media used, and where the cells were obtained. Table 2.2 summarises the seeding density of each cell line used throughout the thesis in all experiments except colony formation assays.

2.1.3 Inducible cell line: SH-EP-MYCN3

To induce the expression of MYCN in the SH-EP-MYCN3 (Tet-ON system) cell line, 1 µg/ml doxycycline (Life Technologies, USA) was added to cells growing in complete DMEM medium. To maintain MYCN expression, doxycycline was added to the complete medium every 3 days.

2.1.4 Patient tumour samples

Neuroblastoma tissues from the Kocak dataset (477 diverse patient samples) and the Versteeg dataset (88 diverse patient samples) were analysed from publicly available gene expression databases (http://r2.amc.nl). 40 primary neuroblastoma cDNA samples were synthesised from RNA isolated from untreated patient tumours, obtained from Children’s Cancer Institute Australia. The study was approved by Sydney Children’s Hospitals Network Human Research Ethics Committee, and the requirement for written informed consent was waived as samples were collected before 2003. 30 primary neuroblastoma protein samples were provided by Dr Jessica Bell (Martin Luther University, Halle, Germany).

2.1.5 Cell viability counts To determine the number of viable cells for experimental seeding, a 50 µl aliquot of suspended cells was mixed with 50 µl Trypan Blue solution. The cells/Trypan Blue solution was pipetted onto a haemocytometer, and viable and dead cells (stained with blue) were visualised using a phase contrast microscope. The number of viable cells was calculated using the formula: Cell number per mL = dilution factor (2) x average number of cells per square x 104.

Chapter 2- Materials and Methods 54 Table 2.1 Neuroblastoma and normal cell lines

Chapter 2- Materials and Methods 55

Table 2.2 Cell seeding densities

Chapter 2- Materials and Methods 56 2.1.6 Transient transfection with plasmid DNA

Cells were seeded into cell culture wells or flasks in complete media and incubated at 37oC overnight to allow them to attach. Plasmid DNA was combined with Lipofectamine 2000 in a 1:3 ratio (weight:volume) with a small volume of cold Opti- MEM (see Table 2.3). The mixture was left at room temperature for 20 minutes to allow the transfection complex to form, before warmed Opti-MEM was added. The complete media was removed from the cells and the serum-free transfection complex solution was o added. Cells were incubated at 37 C / 5% CO2 for 7–8 hours in the serum-free media before an equal volume of complete media containing 20% FCS was added, to produce o a final FCS concentration of 10%. Cells were incubated at 37 C / 5% CO2 for 24–96 hours, depending on the time frame of the experiment. The vector constructs for PA2G4 and MYCN plasmid DNA are depicted in Figure 2.1.

2.1.7 Transient transfection with siRNA

Reverse transfection, in which cells are transfected prior to being seeded into wells or flasks, was performed with siRNAs at a final concentration of 20 nM. Lipofectamine 2000 was combined with siRNA (20 M stock concentration) and a small volume of cold Opti-MEM (see Table 2.3). The mixture was left at room temperature for 20 minutes to allow the transfection complex to form, before warmed Opti-MEM was added. Cells were detached from their flasks and rinsed with warmed PBS to remove traces of serum, then counted for transfection and seeding. Serum-free transfection complex solution was added to the cells, which were then seeded into wells or flasks. o Cells were incubated at 37 C / 5% CO2 for 5–6 hours in the serum-free media before an equal volume of complete media containing 20% FCS was added, to produce a final o FCS concentration of 10%. Cells were incubated at 37 C / 5% CO2 for 24–96 hours, depending on the time frame of the experiment. The sequences of the siRNA used are listed in Table 2.4.

Chapter 2- Materials and Methods 57 Table 2.3 Amount of plasmid DNA, siRNA, and Lipofectamine 2000 used in transfections

Chapter 2- Materials and Methods 58

Figure 2.1 Structure of the PA2G4, MYCN and PA2G4 shRNA plasmid constructs (A) PA2G4 was cloned into the pCMV6-myc-flag vector for use in PA2G4 overexpression experiments. (B) MYCN was cloned into the pShuttle-IRES-hrGFP vector for use in MYCN overexpression experiments.

Chapter 2- Materials and Methods 59

Figure 2.1 (cont’d) Structure of the PA2G4, MYCN and PA2G4 shRNA plasmid constructs (C) PA2G4 shRNA was cloned into the pGFP-C-shLenti vector for use in PA2G4 knockdown experiments.

Chapter 2- Materials and Methods 60 2.1.8 Stable transfection with PA2G4 plasmid DNA

Stable PA2G4-overexpressing cell lines were created using SH-EP neuroblastoma cells and pCMV6-PA2G4-myc-flag vector or empty vector. Cells were transfected using Lipofectamine 2000 as per the transient transfection protocol, in a 100 mm cell culture dish. Selection of clones began 48 hours post transfection with 500 g/ml G418 (Life Technologies, USA). After 30 days, visible clones were picked, expanded and screened for PA2G4 protein expression. After further expansion, selected clones were cryopreserved in liquid nitrogen. As well as isolation of individual clones, a pool of clones was generated by combining individual clones together. This process took up to 1 month to complete. Cells were grown in G418 selection medium from 48 hours post thawing, with selection medium being removed during experiments.

2.1.9 Stable transfection with PA2G4 shRNA

Stable PA2G4 knockdown cell lines were created using BE(2)-C and CHP-134 neuroblastoma cells and pGFP-C-shLenti-PA2G4 vectors or empty vector. Cells were transfected using Lipofectamine 2000 as per the transient transfection protocol, in a 100 mm cell culture dish. Selection of clones began 48 hr post transfection with 1 µg/ml Puromycin (Sigma-Aldrich, USA). Success of transfection was confirmed by expression of GFP when cells were visualised with a fluorescent microscope. After 30 days, visible clones were picked, expanded and screened for loss of PA2G4 protein expression. However, clones from both cell lines could not be expanded for long-term use or storage because the cells began to increase in size, ceased proliferating and appeared to undergo senescence.

Chapter 2- Materials and Methods 61 Table 2.4 siRNA and shRNA sequences

Chapter 2- Materials and Methods 62 2.2 Cell phenotype assays

2.2.1 Reagents and equipment

Resazurin solution and crystal violet solution were made according to the protocols in the Solutions and Reagents section (2.8). 13-cis-Retinoic acid, May-Grunwald, Giemsa stain, propidium iodine, Costar Ultra-Low Cluster round bottom 96-well plates for 3D spheroid assays, Tween 20, Paraformaldehyde, Triton X and BSA were purchased from Sigma-Aldrich (St Louis, MO, USA). RNase A was supplied by Roche (Victoria, Australia). Annexin V apoptosis detection kit, mouse type IV collagen, 24-well migration inserts and companion plates were purchased from BD Biosciences (Franklin Lakes, NJ, USA). Gibco rat-tail Collagen I for the 3D invasion assay, Alexafluor 488 anti-mouse and Alexafluor 555 anti-rabbit, and ProLong Gold anti-fade reagent were purchased from Life Technologies (Carlsbad, CA, USA). Lab Tek II 8-well chamber slides were supplied by Thermo Scientific (Waltham, MA, USA). Vincristine and etoposide were purchased from Sigma-Aldrich (St Louis, MO, USA), cisplatin was supplied by Hospira Australia (VIC, Australia), and (+)- JQ1 was purchased from Sapphire Biosciences (NSW, Australia).

Fluorescence was measured using a Victor 3 microplate reader (Perkin Elmer, USA), while absorbance was measured on a Benchmark Plus microplate reader (Bio-Rad, USA). 3D spheroids were imaged using an Axiovert 200M microscope purchased from Carl Zeiss (Oberkochen, Germany). Evaluation of cell images was performed using Image J software (National Institute of Health, USA). Cell cycle and cell death analysis were done using the BD FACSCalibur Flow Cytometer (Franklin Lakes, NJ, USA). Confocal images were captured using a Leica TCS SP5 WLL (white light laser) confocal microscopy system and analysed using the Leica LAS Lite software (Mannheim, Germany), at the Biomedical Imaging Facility (BMIF) within the Mark Wainwright Analytical Centre (NSW, Australia).

2.2.2 Cell viability assay

Measurement of cell metabolism, which is an indirect way of assessing cell viability and growth, was carried out using the indicator resazurin. Resazurin, a compound whose reduction is associated with mitochondrial activity, is non-toxic, and able to

Chapter 2- Materials and Methods 63 penetrate cell membranes. Resazurin solution was made in-house (see Solutions and Reagents) at 10x working stock, it was added to cells in culture media in 96-well plates o and incubated at 37 C / 5% CO2 for 5–8 hours. The change in media colour from blue to purple to pink indicates the reduction of resazurin to fluorescent resorufin in metabolically active cells. This change was quantified colorimetrically on a Victor 3 fluorescence microplate reader at an excitation wavelength of 570 nm and an emission wavelength of 595 nm. Cell growth was expressed as a percentage of control. For the quantification of synergy and/or additivity between two drug treatments, dose effect and combination index (CI) were analysed with CalcuSyn software (Biosoft, UK). CI < 0.9 indicates synergy; 0.9 < CI < 1.1 means an additive effect; and CI > 1.1 indicates antagonism.

2.2.3 Cell proliferation assay

DNA synthesis, used as a measure of cell proliferation, was assayed using a BrdU ELISA kit (Roche, Australia), which incorporates 5-bromo-2’-deoxyuridine (BrdU) into the DNA of proliferating cells. BrdU solution (10 µM) was added to cells in 96-well o plates and incubated at 37 C / 5% CO2 for 2 hours. Media was carefully removed by inversion of the plates, and cells were fixed with the supplied fixing solution for 20 minutes, followed by incubation in 5% FCS / PBS solution for blocking non-specific antibody binding, before the addition of the primary antibody, Peroxidase-conjugated anti-BrdU. After 1 hour of primary antibody incubation at room temperature, cells were washed with PBS three times before the peroxidase substrate was added. Changes in cell proliferation were calculated from the absorbance readings at 370 nm (490 nm reference wavelength) on the Benchmark Plus microplate reader. An absorbance range of 0.2–0.8 was used, to ensure accurate readings.

2.2.4 Cytotoxicity assay

Cells were seeded into either a 96-well plate (100 µl final volume) or a 384-well plate o (50 µl final volume) in complete media and incubated at 37 C / 5% CO2 overnight to allow cells to attach. Cytotoxic drugs were diluted to 2x the final concentration (for 96- well plate) or 10x the final concentration (for 384-well plate) in complete media, before 100 µl was added to cells in a 96-well plate or 5 µl was added to cells in a 384-well

Chapter 2- Materials and Methods 64 o plate. Cells were incubated at 37 C / 5% CO2 over a period of 24 to 96 hours before the addition of resazurin solution to measure cell viability.

2.2.5 Colony formation assay

The following cell lines were used in colony formation assays: BE(2)-C, Kelly, and SH- EP-PA2G4 stable cells. 500 cells were seeded into each well of a 6-well plate 24 hours post transfection with plasmid DNA, siRNA or shRNA. Cells were incubated at 37oC /

5% CO2 for 10 days (BE(2)-C) or 15 days (Kelly and SH-EP-PA2G4 stable cells) to allow colonies to form. Colonies were fixed and stained with crystal violet solution (see Solutions and Reagents) then washed with water to remove unincorporated stain. Cells were photographed and colony numbers were counted with Image J software. Results were expressed as a percentage of the control.

2.2.6 Neurite growth assay with retinoic acid treatment

Neurite formation was used as an indicator of neuronal differentiation with retinoic acid (RA) treatment. A stock concentration of 50 mM 13-cis-RA was prepared by dissolving 100 mg of 13-cis-RA in 33.3ml of 100% ethanol. This solution was protected from direct light and stored at -80oC for up to 2 weeks or until the RA precipitated out of solution. To assess the formation of neurites with suppression of PA2G4, BE(2)-C (4x104 cells) and SH-SY5Y (4x104 cells) were transfected with PA2G4 siRNA for 24 hours in 6-well plates, before 2 µM 13-cis-RA was added to each well. Cells were incubated with RA for 6 days then photographed for neurite counts using Image J software. Cells were also treated with 10 µM 13-cis-RA for 0–96 hours, and PA2G4 mRNA and protein expression were measured.

2.2.7 Flow Cytometry

Distribution of cell populations into different cell cycle phases was determined by measuring cellular DNA content by flow cytometry using propidium iodide (PI). Attached viable cells and floating dead cells were harvested together, and 1x106 cells were resuspended in complete media. Cells were pelleted by centrifugation at 800xg for 5 minutes followed by one wash in cold PBS, then resuspended thoroughly in 200 µl cold PBS by pipetting up and down to break up clumps. Cells were fixed by slowly

Chapter 2- Materials and Methods 65 adding 80% cold ethanol while vortexing gently at an angle of 45o followed by incubation on ice for 30 minutes then centrifugation at 1000xg for 5 minutes at 4oC. Supernatant was removed and the cells washed in 1 ml PBS + 1% Tween solution followed by centrifugation at 1000xg for 5 minutes at 4oC. Cells were resuspended in 500 µl PI/RNase A staining solution (10 µl of 1 mg/ml PI; 8 µl of 0.5 mg/ml RNase A; 982 µl of PBS + 1% Tween solution) and incubated at 37oC for 1 hour. Samples were run on a BD FACS Calibur Flow Cytometer, and for cell cycle analysis the cells were divided into three subsets that represented G0+G1 phase, S phase, and G2+M phase.

Cell death by apoptosis was analysed by staining cells with Annexin V FitC, to detect apoptotic or necrotic processes, and 7-AAD, which stains membranes of dead or damaged cells as an indicator of early stage apoptosis, according to the manufacturer’s instructions. Stained cells were run on a BD FACS Calibur Flow Cytometer to measure the proportion of apoptotic cells versus live cells.

2.2.8 Immunofluorescent staining of endogenous protein

4x103 cells in 200 µl of medium were seeded into 8-well chamber slides for 24 hours to allow time for cells to attach. Cells were washed with PBS, fixed with 300 µl of 4% paraformaldehyde for 15 minutes at room temperature, then washed with 500 µl of PBS for 5 minutes. Cells were permeabilised with 300ul of 0.4% Triton X for 5 minutes, followed by two washes with 500 µl of PBST (PBS + 0.1% Tween 20) for 5 minutes each. Primary antibodies, PA2G4 (Sigma Aldrich) and MYCN (Santa Cruz) were diluted with 2% BSA + PBST at a ratio of 1:200, and applied to each well in a final volume of 200 µl for 1 hour. Rabbit and mouse IgG antibodies were used as negative isotype controls and diluted with PBST at a ratio of 1:2500. Primary antibodies were removed with 3 washes of PBST for 5 minutes each before applying secondary antibodies: Alexafluor 488 anti-mouse (to detect MYCN) and Alexafluor 555 anti- rabbit (to detect PA2G4), diluted with PBST at a ratio of 1:500 and incubated for 30 minutes at room temperature, protected from light. Cells were washed three times with PBST before staining with ProLong Gold mounting medium, and carefully applying coverslips that were sealed with clear nail polish. Confocal images were captured using a Leica TCS SP5 WLL (white light laser) confocal microscopy system and analysed using the Leica LAS Lite software.

Chapter 2- Materials and Methods 66 2.2.9 Transwell cell migration assay

Cells were transfected with PA2G4 plasmid DNA or siRNA in T25 flasks. The complete media was removed 24 hours post transfection and replaced with 2 ml of o serum-free media. Cells were incubated at 37 C / 5% CO2 for 2 hours. The underside of 8 µm polyethylene terephthalate migration inserts for 24-well plates were coated with 70 µl of 10 µg/mL collagen IV (in 10 mM acetic acid) and incubated at room temperature for 1 hour to dry. Collagen stock was prepared as follows: 250 µg collagen

(100 µl) mixed with 10 mM acetic acid (5.7 µl acetic acid (17.47 M) + 10 ml H2O). The inserts were placed in 24-well companion plates with serum-free media at 37oC / 5%

CO2 for 30 minutes to pre-wet them. Cells were harvested and resuspended in serum- free DMEM, and 5x104 cells (in 250 µl of media) were seeded into the insert, with the companion plate beneath containing DMEM + 10% FCS. The plate was returned to the incubator for 24 hours.

The media was carefully decanted from the inserts, and cells were fixed with 100% methanol for 20 minutes then air-dried at room temperature for 1–2 hours, before being stained with May-Grunwald (1:4 dilution) for 5 minutes, followed by 20 minutes in Giemsa (1:50 dilution). After three washes for 1 minute in water, the completely dried insert membranes were carefully excised and mounted on glass slides. 20 random images were taken of each membrane (top and bottom surfaces) using a 20x objective. Results were expressed as migration index (%) = number of migrated cells (cells on under surface of membrane) divided by total number of cells on both surfaces of the membrane x 100.

2.2.10 3D cell formation and outgrowth assay SH-EP-PA2G4 overexpressing stable cells were used to assess PA2G4’s ability to induce cell migration in a 3D system. This assay indirectly assesses whether overexpression of PA2G4 increases the tumorigenicity of SH-EP cells, on the basis that increased cell migration in vitro is an indicator of increased tumorigenicity. To form 3D spheroids, 5000 SH-EP-PA2G4 clone pool cells, or SH-EP empty vector clone pool cells were seeded in 100 µl of complete media into Ultra-Low attachment 96-well o plates. The cells were incubated at 37 C / 5% CO2 for 5 days, then photographs were taken of each newly formed spheroid using an Axiovert 200M microscope, to enable measurement of spheroid size. Six spheroids from each cell line were transferred to

Chapter 2- Materials and Methods 67 individual wells of a 6-well plate containing 3 ml of complete media, then incubated at o 37 C / 5% CO2 for 16 hours to allow them to attach to the plate and migrate, followed by measurement of cell outgrowth. The remaining spheroids were harvested for protein and RNA analysis.

2.2.11 3D cell invasion assay

This assay assesses the ability of SH-EP-PA2G4-overexpressing stable cells to invade through a collagen matrix in a 3D system. It indirectly assesses whether overexpression of PA2G4 increases the tumorigenicity of SH-EP cells, on the basis that increased invasion in vitro is an indicator of increased tumorigenicity. 3D spheroids were formed as described in Section 2.2.10. To optimise the concentration of collagen I (rat tail) for the invasion matrix, 30 µl of cold collagen solution at different concentrations (Table 2.5) were added to each well of a 96-well flat bottom plate and incubated at 37oC for 1 hour to allow the gel to set. In preparation for embedding the spheroids into the collagen, excess media was carefully removed from the wells containing the spheroids by aspiration (leaving spheroids in approximately 25 µl media), then each spheroid was transferred by pipette to the 96-well plates containing polymerised collagen. Each spheroid was covered with 30 µl of cold collagen solution and incubated at 37oC for 1 hour to allow the gel to set. 30 µl of complete media was added to the top of each gel o then plates were incubated at 37 C / 5% CO2 for 2 days, with spheroids imaged at 0, 24, and 48 hours to assess cell invasion using an Axiovert 200M microscope, focusing on the centre of each spheroid to obtain the best image.

Chapter 2- Materials and Methods 68 Table 2.5 Optimisation of Collagen I concentration for invasion assay

Chapter 2- Materials and Methods 69 2.3 Protein Analysis

2.3.1 Reagents and equipment

Tween 20, RIPA buffer, protease inhibitor cocktail, Ponceau S, Crystal violet, 37% formaldehyde, and NaHCO3 were purchased from Sigma-Aldrich (St Louis, MO, USA). PhosphoStop™ phosphatase inhibitor tablets and RNase A were purchased from Roche (Victoria, Australia). Criterion 10% 18-well and 10.5-14% 12-well gels, XT sample buffer, DTT reducing agent, 0.45 µm nitrocellulose membrane, and Clarity Western ECL substrate were supplied by Bio-Rad (Hercules, CA, USA). Pierce BCA protein assay kit, SuperSignal West Dura chemiluminescent substrate, and Restore Western Blot stripping buffer were purchased from Thermo Scientific (Waltham, MA, USA). GammaBind G Sepharose beads were purchased from GE Healthcare Life Sciences (United Kingdom). Super HR-T X-ray film was supplied by Fujifilm (Tokyo, Japan). Mouse and rabbit IgG isotype controls were purchased from Vector Laboratories (Burlingame, CA, USA). ChIP Assay Kit was purchased from Merck Millipore (Billerica, MA, USA) and control IgG antibody for ChIP assay was supplied by Santa Cruz Biotechnology (Dallas, TX, USA). Proteinase K was purchased from Life Technologies (Carlsbad, CA, USA). 6x loading dye was purchased from Promega (Madison, WI, USA).

Absorbance readings used to determine protein concentrations were measured on a Benchmark Plus microplate reader (Bio-Rad, USA). Criterion™ gel running tanks and transfer tanks were used for western blotting. X-ray film was developed using an SRX- 101A X-ray developer from Konica Minolta (NSW, Australia), scanned on a Gel Doc XR imager, and analysed with Quantity One software from Bio-Rad (Hercules, CA, USA). Sonication of cells for chromatin immunoprecipitation was performed on the Bioruptor Pico from Diagenode (Liège, Belgium).

2.3.2 Whole cell lysate protein extraction

Cells were washed with cold PBS then lysed with an appropriate volume (100 µl to 500 µl, depending on the size of the cell pellet) of cold RIPA buffer containing protease inhibitors (and phosphatase inhibitors if required for phosphorylation studies). Cells were lysed using a cold water bath sonicator for 10–15 minutes then incubated on ice

Chapter 2- Materials and Methods 70 for 10 minutes. If studying phosphorylated proteins, sonication was not performed, instead, cells were lysed by incubation on ice for 30 minutes with 5 seconds vortexing every 10 minutes. Lysed cells were centrifuged at 12000 to 16000xg for 20 minutes at 4oC. The supernatant containing the extracted protein was transferred to 1.5 ml tubes and stored at -80oC.

2.3.3 Cytoplasmic and nuclear protein extraction

Cells were harvested then resuspended in 500 µl of ice-cold PBS and transferred to 1.5 ml tubes. Following centrifugation at 300xg for 5 minutes, the supernatant was discarded and cells were resuspended in 250 µl of ice-cold cytoplasmic lysis buffer (for recipe see Solutions and Reagents), mixed gently by pipetting then incubated on ice for 15 minutes. 10% NP-40 was added and mixed by pipetting, then samples were centrifuged at 14,000xg for 1 minute. The supernatant (cytosolic protein) was transferred to a new tube on ice. The pellet was washed with 100 µl of cytoplasmic lysis buffer to remove any traces of cytosolic protein, then resuspended in 100 µl of ice-cold nuclear lysis buffer (see Section 2.8.7), vortexed for 15 seconds and incubated on ice for 15 minutes. The sample was centrifuged at 14,000xg for 10 minutes, then the supernatant (nuclear protein) was transferred to a new tube and stored at -80oC.

2.3.4 Protein quantification

Quantification of proteins was conducted using a Pierce BCA protein assay kit according to manufacturer’s instructions. Briefly, protein extracts (5 µl) were diluted 1 in 5 in MQ H2O (20 µl) in a 96-well plate, before 200 µl of working reagent A and B (pre-mixed at a 50:1 ratio) was added. Dilutions of a bovine serum albumin (BSA) solution (2 mg/ml to 0.03 mg/ml) were used to produce a standard curve. Samples were incubated at 37oC for 30 minutes, then absorbance was measured on a Benchmark Plus spectrophotometer at a wavelength of 570 nm.

2.3.5 Co-immunoprecipitation

Cells were harvested from T75 flasks and lysed with 300–500 µl (depending on pellet size) of cold hypotonic lysis buffer (for recipe see Section 2.8.5) containing protease inhibitors. 500 µg to 1 mg of whole cell protein lysate was incubated with 10 µg of

Chapter 2- Materials and Methods 71 primary antibodies or 10 µg of control IgG isotype antibody overnight at 4oC with gentle rotation. GammaBind G Sepharose beads were prewashed with cold Hypotonic lysis buffer, and 100ul (50% solution of sepharose in hypotonic buffer) was added to each sample tube and incubated for 2 hours at 4oC with gentle rotation. Conjugated beads were centrifuged at 12,000xg for 2 minutes and the supernatant was transferred to a new 1.5 ml tube for Western blot analysis of unbound protein. The beads were washed 3 times with 1 ml of cold hypotonic lysis buffer and 12,000xg centrifugation between each wash. 50 µl of 4x XT sample buffer containing 100 mM DTT was added to the beads followed by heating at 95oC for 5 minutes and centrifugation at 12,000xg for 5 minutes to elute the proteins. 30 µl of protein supernatant was carefully transferred to new 1.5 ml tubes and placed on ice ready for loading on a Criterion™ polyacrylamide gel for Western blotting.

2.3.6 Western blotting

20–30 µg of protein samples were mixed with 4x XT sample buffer containing 100 mM DTT, and the appropriate lysis buffer, to a final volume of 24 µl, and heated at 95oC for 5 minutes then chilled on ice for two minutes, before centrifugation at 12,000xg for 1 minute. The samples were loaded onto either Criterion™ 10% 18-well or 10.5-14% 12- well polyacrylamide gels. 5 µl of protein size markers were also loaded onto the gels. Gels were placed in tanks containing Tris-Glycine-SDS (TGS) running buffer, and 80 volts were applied for 20 minutes, then the voltage was increased to 150 for 1 hour, or until the dye front reached the bottom of the gel. Proteins were transferred onto a 0.45 µm supported nitrocellulose membrane, sandwiched between 3MM Whatman filter paper at 4oC, at 50 volts for 2 hours (or 20 volts overnight) in 20% methanol Tris- Glycine (Transfer buffer) solution. Nitrocellulose membrane was stained with Ponceau S stain to check for air bubbles that may have interfered with the protein transfer, then trimmed for particular protein sizes.

Membranes were blocked in 10% skim milk powder in TBST for at least 1 hour, then washed 3 times in TBST, before incubation with primary antibodies diluted in 0.5% skim milk in TBST, at 4oC overnight on an orbital shaker. See Table 2.6 for information on antibodies and dilutions. Membranes were washed 3 times in TBST before incubation with secondary antibodies diluted in 0.5% skim milk powder in TBST, at room temperature for 2–4 hours on an orbital shaker. Membranes were washed 3 times

Chapter 2- Materials and Methods 72 in TBST. Chemiluminescence detection was performed using either SuperSignal™ West Dura or Clarity™ Western ECL solution. Membranes were encased in two pieces of plastic film sealed with a heat sealer before exposure to X-ray films for a range of exposure times. Films were developed using an X-ray film developer, and quantification of protein bands was carried out using Quantity One software (Bio-Rad).

Primary antibodies were stripped off membranes using Restore™ Western Blot stripping buffer for 20 minutes at room temperature. Membranes were then washed 3 times with TBST and re-blocked with 10% skim milk powder in TBST, ready for re- probing with another primary antibody. Alternatively, primary stripping buffer was also made and used (see Solutions and Reagents). If stripping off only the secondary antibodies, 50 ml of Secondary stripping buffer (see Solutions and Reagents) was used for 10 minutes, followed by 3 washes with TBST.

Chapter 2- Materials and Methods 73 Table 2.6 Antibody dilutions

Chapter 2- Materials and Methods 74 2.3.7 Inhibition of protein synthesis and degradation

Cells were treated with 30 µM MG-132 (Biomol, USA) to inhibit proteasomal protein degradation. Four hours post MG-132 treatment, cells were harvested and the PA2G4 and MYCN proteins were analysed by western blotting.

Cycloheximide (Biomol, USA) was used to inhibit protein synthesis and thus enable determination of protein half-life. Cells were treated with cycloheximide (CHX) at a final concentration of 100 µg/ml for different time periods, depending on the degradation rate of the target protein. For example, MYCN has a short protein half-life of approximately 20 minutes, therefore when examining MYCN degradation, cells were treated with CHX for 10, 20, 30, 40 and 60 minutes followed by protein isolation and analysis by western blotting. Conversely PA2G4 has a long half-life of approximately 16 hours, which required longer CHX treatment of 4, 8, 16, and 24 hours.

2.3.8 Chromatin Immunoprecipitation (ChIP) Assays

Four T75 flasks of confluent BE(2)-C or Kelly cells were used for ChIP assays (approximately 6 x107 or more cells), using a ChIP Assay Kit from Merck Millipore and following the manufacturer’s instructions as summarised here. Cells were harvested and resuspended in 40 ml of complete media, then 1.5 ml of 37% formaldehyde was added. Solutions were mixed on a rotator for 10 minutes at room temperature. 5 ml of cold 1.25 M glycine was added and each tube was mixed for an additional 5 minutes at room temperature then centrifuged at 1000 rpm, 4oC for 10 minutes. The supernatant was discarded and the cell pellet was washed with 40 ml of cold PBS before centrifugation at 1000 rpm, 4oC for 10 minutes. The cell pellet was resuspended in 1ml of cold PBS and transferred to a 1.5 ml tubes before centrifugation at 8,000 rpm, 4oC for 10 minutes. Each pellet was resuspended in 500 µl of SDS lysis buffer containing protease inhibitors then incubated on ice for 10 minutes.

The DNA was sheared using a Bioruptor Pico sonicator set at the following parameters: 30 seconds ON/30 seconds OFF for 45 cycles, before centrifugation at 13,000 rpm for 10 minutes at 4oC. The supernatant was transferred to a 1.5 ml tube, and 15 µl was set aside for loading onto an agarose gel to check the size of the DNA fragments. Sheared DNA was diluted with ChIP Dilution Buffer at a 3:1 ratio (1.5 ml ChIP Dilution Buffer: 500 µl sheared DNA). 60 µl of this mixture was transferred to a new 1.5 ml tube for use

Chapter 2- Materials and Methods 75 as a positive control and stored at -20oC. To pre-clear the samples and reduce non- specific background, 38 µl of Protein A Agarose/Salmon Sperm DNA was added to every ml of diluted supernatant then mixed on a rotating wheel at 4oC for 30 minutes. After centrifugation at 2,000 rpm for 2 minutes at 4oC, supernatants were divided into two new 1.5 ml tubes. 10 µg of MYCN antibody (Santa Cruz) was added to one tube and 10 µg of isotype control IgG antibody (Santa Cruz) was added to the other tube, and both tubes were incubated at 4oC overnight.

Protein A Agarose/Salmon Sperm DNA (50 µl per ml) was added to each tube and incubated at 4oC for 2 hours then centrifuged at 1000 rpm for 1 minute at 4oC. Supernatant was removed and the beads were washed with the supplied buffers in the following order: Low Salt Wash Buffer, High Salt Wash Buffer, LiCl Immune Complex Wash Buffer, and TE Buffer. Supernatant was removed after centrifugation at 1000 rpm for 1 minute at 4oC. The protein–DNA complex was eluted from the beads by addition of 250 µl of Elution Buffer, vortexing for 30 minutes and centrifugation at 10,000 rpm for 1 minute. 190 µl of Elution Buffer was also added to 60 µl of the cell lysate set aside on the previous day. 10 µl of 5M NaCl was added to each tube, and tubes were incubated at 65oC overnight.

RNase A (2.5 µl) was added to each tube and incubated at 37oC for 30 minutes, followed by the addition of 5 µl of 0.5M EDTA, 10 µl of 1 M Tris-HCl, and 1µl of Proteinase K (20 mg/ml). Samples were mixed by vortexing and incubated at 45oC for 90 minutes. DNA from each sample was purified using a MiniElute PCR Purification Kit (Qiagen). In brief, 1250 µl of PB buffer was added to each 250 µl sample before passing the samples through the supplied columns. Columns were then washed with 750

µl of PE buffer, and DNA was eluted with 40 µl of DNase/RNase-free H2O. Real time PCR was performed on the eluted DNA using primers targeting the PA2G4 gene promoter and intron 1 regions, as well as a control region upstream of the PA2G4 gene promoter (Figure 3.13).

To check the size of the sheared DNA fragments, 10 µl of the lysed and sonicated proteins that had been set aside for the purpose were mixed with 2 µl of 6x loading dye and loaded onto a 1% agarose gel. Samples were run at 100 volts for 40 minutes, then stained with SYBR Safe DNA stain, and DNA fragments were visualised using a Gel Doc (Bio-Rad).

Chapter 2- Materials and Methods 76 2.4 Gene expression analysis

2.4.1 Reagents and equipment

Ambion RNA isolation kit, SuperScript III reverse transcriptase, RNaseOUT,

DNase/RNase free H2O, Power SYBR Green Mix for real-time PCR, and Illumina Total RNA Amplification Kit were purchased from Life Technologies (Carlsbad, CA, USA). Absolute ethanol, β-Mercaptoethanol and PCR primers were purchased from Sigma Aldrich (St Louis, MO, USA). RNeasy mini elution kit was purchased from Qiagen

(Hilden, Germany). Tetro cDNA Synthesis Kit, oligo (dT)18, and RiboSafe RNA inhibitor were supplied by Bioline (Cincinnati, OH, USA). Human Gene Array 2.0 was purchased from Affymetrix (Santa Clara, CA, USA).

Determination of RNA concentration was performed on a NanoDrop ND-1000 spectrophotometer purchased from Thermo Fisher (Waltham, MA, USA). Applied Biosystems 7500 real-time PCR machine and a GeneAmp PCR 9700 machine were purchased from Life Technologies (Carlsbad, CA, USA). RNA quality (RNA integrity number; RIN) was assessed using an Agilent Bioanalyzer (Santa Clara, CA, USA), hybridisation of cRNA and scanning of the array chips were performed by the Ramaciotti Centre for Genomics (UNSW, Australia). Gene expression was analysed using GenePattern software (Broad Institute).

2.4.2 RNA isolation

Isolation of RNA from cell cultures was performed using an Ambion RNA isolation kit according to manufacturer’s instructions. In brief, cell pellets were washed with cold PBS then lysed with 100–300 µl of lysis buffer (depending on the size of the cell pellet) containing β-Mercaptoethanol, before the addition of 70% ethanol. The lysed cells were passed through an RNA column followed by washes with the supplied wash buffers. It is very important not to overload the columns with cell lysates. RNA was eluted from the column with 30 µl of RNase-free H2O and centrifugation at 13,000xg for 2 minutes.

Isolation of RNA from mouse tumour or tissues (ganglia) samples was performed using a Qiagen RNeasy mini elution kit. Mouse ganglia samples are extremely small and it can be difficult to isolate RNA from them. 150 µl of RLT buffer containing β- Mercaptoethanol was used to homogenise ganglia by repetitive pipetting followed by

Chapter 2- Materials and Methods 77 vortexing. When the ganglia samples had been thoroughly broken up, 200 l of RLT buffer containing β-Mercaptoethanol was added to each tube, followed by 350 µl of 70% ethanol. The RNA was passed through the supplied columns and washed according to the protocol outlined in the kit before being eluted with 30 µl of RNase-free H2O.

The concentration and purity of the RNA samples were assessed by spectrophotometry, using the Optical Density (OD) ratios of 260/280 nm and 260/230 nm. The RNA is considered relatively free of protein contaminants if the 260/280 nm ratio is 2.0, and free of solvent contaminants if the 260/230 nm ratio is ≥ 2.0. For RNA samples that were used for microarray analysis, the RNA integrity number (RIN) was analysed using the Bioanalyzer to ensure that there was no degradation. RNA samples with RIN values of 8–10 were considered good quality and suitable for microarray studies.

2.4.3 cDNA synthesis

4 µg of RNA was incubated with 1 µl of each of the following reagents: Oligo (dT)18, 10 mM dNTP mix, RiboSafe RNase inhibitor or RNaseOUT, Tetro Reverse transcriptase or SuperScript III; and 4 µl of 5x RT Buffer or 5x First Strand Buffer. o RNase-free H2O was added to a final volume of 20 µl. Samples were incubated at 45 C for 30 minutes, the reaction was stopped by heating at 85oC for 5 minutes, then samples were chilled on ice for 2 minutes. 80 µl of H2O was added to each sample to give a final cDNA concentration of 40 ng/µl.

2.4.4 Real-time quantitative PCR

Quantitative gene expression was analysed by real-time PCR using 40 ng of cDNA per reaction. In brief, 1 µl of cDNA (40 ng/µl) was mixed with 12.5 µl of 2x Power SYBR Green mix, 1 µl of forward primer (100 µg/ml) and 1 µl of reverse primer (100 µg/ml), followed by 9.5 µl of RNase-free H2O. Each RNA sample was added in duplicate to Optical 96-well plates and amplified using an Applied Biosystems 7500 real-time PCR machine. Analysis of gene expression was performed using Applied Biosystems 7500 system software. Table 2.7 contains a list of primers used and their sequences.

Chapter 2- Materials and Methods 78 Table 2.7 Real-time PCR primer sequences

Chapter 2- Materials and Methods 79 2.4.5 RNA amplification

In preparation for hybridisation onto array slides, RNA samples were converted to biotinylated cRNA using an Illumina Total Prep RNA Amplification kit, according to manufacturer’s instructions. Hybridisation of the cRNA onto Affymetrix Human Gene arrays was performed by the Ramaciotti Centre for Genomics.

2.4.6 Microarray gene expression analysis

Microarray gene expression analysis was performed on BE(2)-C cells transfected with control siRNA, PA2G4 siRNA or MYCN siRNA for 24 hours. Transfection was carried out in duplicate. The data files generated by the Ramaciotti Centre for Genomics were analysed on GenePattern Version 3.9.0 software (Broad Institute, Cambridge, MA, USA). Results were normalised and analysis for differentially expressed genes was performed using the LimmaGP (Linear model for microarray analysis) module in the GenePattern software, to identify differential gene expression based on a false discovery rate (FDR) (using Benjamini-Hochberg for multiple test correction) of <0.1, and applying a p-value of <0.05 and a fold change (FC) > = |1.5|.

Gene set enrichment analysis (GSEA – http://www.broad.mit.edu/gsea/) was conducted on the differentially expressed genes using the gene sets C1 (positional gene sets), C2 (curated gene sets), C3 (motif gene sets), C4 (computational gene sets), C5 (GO gene sets), C6 (oncogenic signatures), and C7 (immunologic signatures). GSEA analysis examines groups of genes that share common biological function and pathways, or are associated with certain types of cancers.

Chapter 2- Materials and Methods 80 2.5 Additional molecular biology techniques

2.5.1 Reagents and equipment

Wizard SV Miniprep DNA purification System, restriction enzymes, and 6x loading dye were purchased from Promega (Madison, WI, USA). HiPure Plasmid Filter Maxiprep kit, SYBR Safe stain, and 1 kb ladder were supplied by Life Technologies (Carlsbad, CA, USA). Alpha-select Gold competent cells and SOC media were purchased from Bioline (Cincinnati, OH, USA). LB agar and broth, ampicillin and kanamycin were purchased from Sigma Aldrich (St Louis, MO, USA).

DNA concentration was measured on a NanoDrop ND-1000 spectrophotometer purchased from Thermo Fisher (Waltham, MA, USA). Stained DNA gels were visualised on a Gel Doc purchased from Bio-Rad (Hercules, CA, USA).

2.5.2 Bacterial transformation

Chemically competent DH5α bacterial cells were thawed on wet ice before 100 µl of cells were mixed with 1 µg of plasmid DNA and incubated on ice for 30 minutes. Cells were then incubated for 45 seconds in a 42oC water bath then quickly chilled on ice for 2 minutes. The cells were diluted with 1 ml of SOC media and incubated at 37oC, shaking at 200 rpm for 1 hour. The transformed cells were plated using a plastic hockey stick spreader at 1:100 and 1:2 dilutions onto LB agar plates containing either 100 µg/ml ampicillin or 50 µg/ml kanamycin. pUC19 plasmid DNA was used as a positive control for transformation, and plated at a 1:100 dilution only. Single colonies were expanded in 5 ml (for small mini preps) or 200 ml (for larger Maxi preps) of LB broth containing the appropriate antibiotic.

2.5.3 DNA isolation and analysis

Small quantities of DNA were extracted using an SV Miniprep DNA purification kit, while larger quantities were extracted using a HiPure Plasmid Filter Maxiprep kit, both according to manufacturer’s instructions. The concentration of the eluted DNA was measured using a NanoDrop spectrophotometer. DNA purity was assessed using the Optical Density (OD) ratios of 260/280 nm and 260/230 nm. DNA is considered

Chapter 2- Materials and Methods 81 relatively free of protein contaminants if the 260/280 nm ratio is between 1.8 and 2.0, and free of solvent contaminants if the 260/230 nm ratio is ≥ 2.0.

2.5.4 Restriction enzyme digestion

To confirm that the DNA inserted into the vector was the correct size, restriction enzymes were used to excise the DNA from the vector, and the products were subjected to gel electrophoresis. For the CMV6-Entry vector plasmid DNA, restriction enzymes MluI and SgfI were used, while SpeI and EcoRV were used for pShuttle-IRES-hrGFP-1 vector plasmid DNA (see Figure 2.1 for maps of the vectors). In brief, 1 µg of plasmid DNA was incubated with 1 µl of each restriction enzyme, 2.5 µl of the appropriate 10x o buffer, and sterile H2O to a final volume of 25 µl, for 4 hours or overnight at 37 C.

The digested DNA fragments were analysed using DNA gel electrophoresis. Depending on the expected size of the DNA (smaller DNA fragments require a higher percentage of agarose), a 1–2% agarose solution was made up in Tris-Acetate-EDTA (TAE) solution and heated in a microwave until the agarose dissolved. Once the solution cooled, SYBR Safe stain was diluted (1:10,000) into the agarose solution, which was then left to set in an agarose gel electrophoresis mould. DNA samples were mixed with 6x loading dye then loaded into the wells of the agarose gel. 1 kb marker was used for calculation of DNA fragment sizes. Electrophoresis was conducted at 100 volts for 40 minutes, and gels were visualised under UV light using a Gel Doc.

2.6 In vivo models of neuroblastoma

2.6.1 Reagents and equipment

Matrigel™ basement membrane matrix was purchased from BD Biosciences (Franklin Lakes, NJ, USA). BALB/c Fox1nu/Ausb (BALB/c nude) mice were purchased from Animal Resource Centre (Perth, Australia). TH-MYCN+/+ mice were a gift from Dr William Weiss. Mice were housed at CCIA (Lowy Cancer Research Centre, NSW, Australia), and the TH-MYCN+/+ mice were maintained according to the ACEC UNSW Ethical Approval 03/89. All in vivo mouse experiments were carried out according to the ACEC UNSW Ethical Approval 14/152B.

Chapter 2- Materials and Methods 82 2.6.2 Assessing the ability of PA2G4 to initiate tumour development

SH-EP PA2G4 stable overexpressing cells were xenografted into nude mice to assess the ability of PA2G4 to induce tumorigenesis. SH-EP PA2G4 stable overexpressing and control cells were harvested from multiple T75 flasks and rinsed twice in media without FCS. Cells were then resuspended in PBS at a concentration of 6x106 cells per 100 µl. Matrigel™ basement membrane matrix, which had been thawed the day before at 4oC, was mixed with the harvested cells at a ratio of 1:1. The cell/Matrigel solution was kept cold on ice and 200 µl was injected subcutaneously into the dorsal flank of BALB/c nude mice (6 weeks of age). Mice were monitored for signs of tumour formation for a period of 12 weeks post injection, then humanely killed by CO2 asphyxiation. Tumour tissues were excised, measured and collected for morphological and biochemical analysis.

2.6.3 Silencing PA2G4 expression to reduce tumour growth in mice

To assess whether PA2G4 is required for tumour growth and progression, PA2G4 expression was reduced with highly specific siRNAs delivered by non-viral nanoparticles synthesised by chemists at the Australian Centre for NanoMedicine, UNSW Australia. These nanoparticles are non-toxic to normal cells and have been designed to rapidly self-assemble with siRNA. Moreover, their physicochemical properties (size and surface charge) are ideal for delivering siRNA to tumour cells both in vitro and in vivo. In brief, SK-N-BE(2) cells (4x106 in 100 µl PBS) were subcutaneously injected into the dorsal flank of BALB/c nude mice (6 weeks of age). Once tumours reached approximately 100–200 mm3 (4–5 weeks), mice were randomised into treatment groups to receive a local injection of nanoparticle–siRNA complexes directly into the tumour using a 0.5ml syringe containing a 29G needle. The solution injected into the tumour contained sterile saline with 200 µg of nanoparticles complexed to PA2G4 siRNA (40 µg) or control siRNA (40 µg) (total volume 50 µl). Mice were injected once every 3 days for up to 8 injections or until the tumour reached 3 1000 mm , then humanely killed by CO2 asphyxiation. The rate of tumour growth was measured twice weekly using digital callipers and recorded. Tumour tissues were excised, measured and collected for morphological, histological and biochemical analysis.

Chapter 2- Materials and Methods 83

2.6.4 Treating neuroblastoma tumours with PA2G4 inhibitor, WS6

To assess the capability of the PA2G4 inhibitor, WS6, to delay the growth of neuroblastoma tumours, TH-MYCN+/+ mice (3 weeks of age) were treated with 50 mg/kg of WS6 (100–200 µl) dissolved in PBS, or PBS only as control, for 5 consecutive days per week for a maximum of 6 weeks i.p. with a 27–29G needle. Treatment was discontinued when tumours reached 1000 mm3 or 6 weeks post treatment, then the mice were humanely killed by CO2 asphyxiation. Tumours were excised, measured and collected for morphological, histological and biochemical analysis.

2.6.5 Histological tissue preparation

Mouse tumour samples were infused with 4% formalin in PBS for at least 24 hours before being fixed with 70% ethanol. Tumours were placed in histological cassettes for processing at the Histopathology Facility, The Kinghorn Cancer Centre (NSW, Australia), where they were embedded and sectioned, and two slides were stained with Hematoxylin and Eosin (H&E).

2.7 Statistical analysis

All experiments included a minimum of three independent replicates. Statistical calculations were performed using GraphPad Prism 6 software. Results were compared using ANOVA among groups or two-sided unpaired t-test for two groups, and expressed as mean values with 95% confidence intervals. Graphical error bars for in vitro data represent the standard error of the mean, while error bars for in vivo data were calculated as standard deviation. A p-value of less than 0.05 was considered statistically significant.

Chapter 2- Materials and Methods 84 2.8 Solutions and Reagents

All chemicals were either purchased from Sigma Aldrich (St Louis, MO, USA) or from Ajax Finechem, Thermo Scientific (Waltham, MA, USA).

2.8.1 Resazurin solution

75 mg Resazurin 12.5 mg Methylene Blue 164.5 mg Potassium hexacyanoferrate (III) 211 mg Potassium hexacyanoferrate (II) trihyrate

Dissolve each compound in 50 ml of sterile PBS then combine them and add PBS to a final volume of 500 ml. Filter sterilise and store at 4oC for short term or at -20oC, protected from light.

2.8.2 Crystal violet solution

Dissolve 0.5g crystal violet in 50 ml methanol and 50 ml MQ-H2O.

2.8.3 T.E. buffer pH 8.0

10 mM Tris-HCl pH 8 1 mM EDTA

2.8.4 50x TAE buffer 242 g Tris (40 mM) 57 ml Glacial acetic acid (20 mM) 18.6 g EDTA (1 mM)

Make up to 1L with MQ-H2O

Chapter 2- Materials and Methods 85 2.8.5 Hypotonic lysis buffer

125 g HEPES pH7.5 (10 mM) 0.3 g NaCl (10 mM)

0.07 g KH2PO4 (1 mM)

0.4 g CaCl2 (5 mM)

0.05 g MgCl2 (0.5 mM) 1 g EDTA (5 mM) 2.23 g Sodium pyrophosphate (10 mM)

1% 200 mM Na3VO4 (2 mM)

2.8.6 Cytoplasmic lysis buffer

1 ml 100 mM HEPES pH 7.9 (10 mM)

100 µl 150 mM MgCl2 (1.5 mM) 100 µl 10 mM EGTA (0.1 mM) 1 ml 1 M KCl (100 mM)

7.8 ml MQ-H2O Then to 1 ml of the above solution add: 0.5 µl 1 M DTT (0.5 mM) 10 µl 50 mM PMSF (0.5 mM) 10 µl Protease inhibitor (1x)

2.8.7 Nuclear lysis buffer

1 ml 100 mM HEPES pH 7.9 (10 mM)

100 µl 150 mM MgCl2 (1.5 mM) 840 µl 5 M NaCl (420 mM) 100 µl 10 mM EGTA (0.1 mM) 1 ml 50% solution Glycerol (5%)

6.8 ml MQ-H2O Then to 500 µl of the above solution add: 0.25 µl 1 M DTT (0.5 mM) 5 µl 50 mM PMSF (0.5 mM) 5 µl Protease inhibitors (1x)

Chapter 2- Materials and Methods 86 2.8.8 Ponceau S staining solution

Dissolve 0.2 g Ponceau S in 5 ml glacial acetic acid and 94.8 ml MQ-H2O

2.8.9 10x TGS running buffer

30 g Tris (250 mM) 150 g Glycine (2 M) 10 g SDS (1%)

1 L MQ-H2O

2.8.10 10x Transfer buffer 30 g Tris 112 g Glycine

1 L MQ-H2O

2.8.11 10x Tris-buffered saline (TBS)

24 g Tris (200 mM) 88 g NaCl (1.5 M)

1 L MQ-H2O

2.8.12 Stripping buffer for 1o antibody

2 ml 1.5 M Tris-HCl pH 6.7 5 ml 20% SDS 350 µl β-mercaptoethanol

42.65 ml MQ-H2O Apply to the nitrocellulose membrane to be stripped, and heat at 50oC for 30 minutes.

2.8.13 5x stripping buffer for 2o antibody

15 g Glycine (0.2 M) 29 g NaCl (0.5 M)

1 L MQ-H2O

Chapter 2- Materials and Methods 87

2.8.14 Elution buffer for ChIP assay

100 µl 1 M NaHCO3 (0.1 M) 100 µl 10% SDS

800 µl MQ-H2O

Chapter 2- Materials and Methods 88 Chapter 3 IDENTIFICATION OF MYCN BINDING PARTNER PA2G4, AND ITS INTERACTION WITH MYCN

CHAPTER 3

IDENTIFICATION OF MYCN BINDING PARTNER, PA2G4, AND ITS INTERACTION WITH MYCN

Chapter 3- Identification of PA2G4 89 3.1 Introduction

It is well established that MYCN gene amplification is detrimental to neuroblastoma patient outcome and aberrant MYCN expression is a major clinical determinant of patient relapse (Brodeur et al., 1984; Buechner et al., 2011; Liu et al., 2007; Negroni et al., 1991; Seeger et al., 1985). It would be ideal to target MYCN directly, however c- MYC and MYCN control many normal biological functions such as gene regulation, cell proliferation, apoptosis and differentiation (Cole, 1986; Dang et al., 1999; Thiele et al., 1985). They have been considered undruggable or difficult targets as they encode transcription factors and carry out essential functions in proliferative tissues, and their inhibition could cause severe side effects to the homeostasis of normal proliferating tissues (Soucek and Evan, 2010). Furthermore, the structure of MYCN protein has not been crystallised, making it difficult to design inhibitors to target the protein, and currently there are no direct MYCN inhibitors in clinical trials. If MYCN cannot be targeted directly, an alternative approach may be to target its oncogenic effects indirectly by targeting proteins with which MYCN interacts, and which are required for its oncogenic activity.

This chapter will describe the identification and confirmation of PA2G4 as a MYCN binding protein and the investigation of its role in MYCN-mediated oncogenesis in neuroblastoma. PA2G4 has two isoforms, p48 and p42, however it is only possible to generate siRNA targeting both isoforms or specifically targeting the long isoform, as p42 has no unique sequence elements. Therefore throughout this thesis, siRNA used to specifically knockdown the p48 isoform will be described as PA2G4-p48, while siRNA used to knockdown both p48 and p42 at the same time will be described as PA2G4 siRNA. The plasmid DNA used to overexpress PA2G4 contained the full length DNA sequence, encoding the long isoform, p48.

The rationale for using mouse bone marrow B-cells (mBMBC) arose from another project that focused on studying tumour initiation (Calao et al., 2013). Calao et al. showed that MYCN overexpression caused resistance to death from trophic factor withdrawal in normal perinatal precursor ganglia cells, thus allowing neuroblastoma to develop. Ideally mouse ganglia cells would be used to study tumour initiation because they are neuroblastoma precursor cells, however it is technically very difficult to isolate ganglia from mice, and hundreds of mice are required to provide enough ganglia cells

Chapter 3- Identification of PA2G4 90 for a co-immunoprecipitation study. Therefore, mBMBC were the closest substitute for ganglia cells. mBMBC are primary murine Pre-B lymphocytes, purified from the bone marrow of 2-week-old normal mice infected with a retrovirus expressing MYCN or empty vector. The advantage of this system is the ability to also transfect deletion mutants of MYCN to identify specific domains that target proteins binding to MYCN. However, these Pre-B cells will only be used as a tool to identify MYCN binding partners. Once a target protein has been identified, all subsequent experiments will be carried out in human neuroblastoma cells.

Chapter 3- Identification of PA2G4 91 3.2 Results

3.2.1 Identification of MYCN binding proteins

MYCN, a member of the myc protein family, contains highly conserved regions called Myc boxes (MB) that are essential for its biological activities (Figure 3.1 A). MBI and MBII are located within the transactivation domain, between amino acids 45 and 65 and amino acids 128 and 144, respectively. MBI functions as a phosphorylation-dependent binding site for the ubiquitin ligase FBXW7 (Welcker et al., 2004). It contains the Thr- 58 and Ser-62 phosphorylation sites, which have been reported to play a role in transactivation and transformation (Gupta et al., 1993; Henriksson et al., 1993). MBII is the site for the recruitment of coactivator complexes containing histone acetyltransferases (HATs) (Frank et al., 2001). The C terminus of Myc harbors nuclear localisation signals and the bHLHZ motif that mediates dimerisation with Max and DNA binding (Blackwood and Eisenman, 1991).

To identify proteins which bind to the MBI and MBII domains of MYCN, mouse bone marrow B cells (mBMBC) with stable overexpression of FLAG-tagged full-length MYCN, and truncated constructs lacking MBI, or both MBI and MBII, were used (Figure 3.1 A, showing the truncations). Full-length or truncated MYCN and their interacting proteins were co-immunoprecipitated using the FLAG tag (Figure 3.1 B). The eluted proteins from the co-immunoprecipitation were resolved by SDS-PAGE and stained with Coomassie Blue to confirm protein enrichment (Figure 3.1 C). The enriched proteins were sent to the Australian Proteomic Analysis Facility (APAF) at Macquarie University for analysis on their QSTAR mass spectrometry and Mascot software to identify the pull-down proteins, thus generating a list of possible MYCN binding partners and identifying the MYCN domain that is required for the interaction (Table 3.1) and (Appendix A). The table ranks proteins based on the required binding domains; the two proteins at the top on the list: KV201 and PA2G4, both require MYC box I domain to bind to MYCN protein, whereas proteins such as TBB3 and DDX17 require MYC box II for binding. In order to choose which possible MYCN binding proteins to study, literature searches were carried out on the top 15 protein candidates to see if they met the following criteria:

Chapter 3- Identification of PA2G4 92 1. Has a known role in cancer 2. Correlates with disease outcome in neuroblastoma patients 3. Good antibody for experimental validations 4. Has a validated small molecule inhibitor 5. Not a highly abundant protein found in mass spectrometry e.g. Tubulin, Actin, keratin and ribosomal proteins.

Through this systematic process of elimination, PA2G4 was chosen because it meets all the criteria listed above (Table 3.2). For the correlation with neuroblastoma patient outcome, each protein was analysed on the R2 microarray Versteeg database with the statistical analysis performed at the median cut-off and P-values determined by log rank test (Appendix A). The fact that two proteins from the list, HNRFP and ENPL, are known to interact with MYCN (David, Chen et al. 2010; Regan, Jacobs et al. 2011), validates this approach.

Figure 3.1 Identification of MYCN binding proteins (A) Diagram of the MYCN domains showing the two MYC Boxes (MB) in the N terminus and the bHLHZ motif in the C terminus regions, as well as the two MYCN deletion plasmids.

Chapter 3- Identification of PA2G4 93

Figure 3.1 (cont’d) Identification of MYCN binding proteins (B) Experimental outline for pulling down MYCN and its binding partners. (C) Protein samples resolved on SDS-PAGE and stained with Coomassie Blue. Transfection of empty vector (EV), MYCN full length (FL), MYCN deletion MBI (DMI), and MYCN deletion MBII (DMII) plasmid DNA for co-immunoprecipitation.

Chapter 3- Identification of PA2G4 94 Table 3.1 MYCN binding candidates isolated by co-immunoprecipitation and identified by mass spectrometry Table indicates the MYCN domain each protein requires for binding.

Chapter 3- Identification of PA2G4 95 Table 3.2 Selection criteria for determining the protein to study Each protein was analysed on the R2 microarray Versteeg database for correlation with neuroblastoma disease outcome. Statistical analysis performed with the median cut-off and P-values determined by log rank test.

Chapter 3- Identification of PA2G4 96 3.2.2 PA2G4 is a novel MYCN binding protein

Having identified PA2G4 as a possible MYCN binding partner, the next step is to confirm that PA2G4 does in fact bind to MYCN. This was firstly done in the original mBMBC with the full length MYCN-flag protein stably overexpressing cells, using Flag antibody to pull down MYCN, followed by western blotting with PA2G4 antibody to detect the PA2G4 protein (Figure 3.2 A). Additional co-immunoprecipitation assays were performed in two MYCN-amplified neuroblastoma cell lines, BE(2)-C and CHP- 134. Endogenous MYCN protein was immunoprecipitated with a MYCN antibody, then western blotting performed with a PA2G4 antibody. PA2G4 was confirmed to associate with MYCN in neuroblastoma cells (Figure 3.2 B).

For PA2G4 and MYCN to associate, the proteins need to be co-localised. BE(2)-C and Kelly cells were separated into cytoplasmic and nuclear fractions, and western blotting was used to analyse PA2G4 and MYCN levels in each fraction. For BE(2)-C cells MYCN was found to be predominantly located in the nucleus, whereas PA2G4 was distributed between the cytoplasmic and nuclear fractions (Figure 3.3 A). In Kelly cells MYCN was present in both cytoplasmic and nuclear fractions, with the majority of the protein located in the nucleus (Figure 3.3 A), while PA2G4 distribution was the same as seen in BE(2)-C. To further validate that PA2G4 co-localised with MYCN in the nucleus in BE(2)-C and Kelly cells, both proteins were probed with fluorescent conjugated dyes and the cells analysed by confocal microscopy (Figure 3.3 B). This technique revealed that PA2G4 protein is concentrated in the nucleus and is also diffused in the cytoplasm, whereas MYCN protein is predominantly nuclear.

Chapter 3- Identification of PA2G4 97

Figure 3.2 Confirmation of PA2G4 and MYCN protein binding (A) Co-immunoprecipitation with Flag antibody and western blot with PA2G4 antibody in mBMBC confirmed the binding of PA2G4 protein to MYCN protein. (B) Co- immunoprecipitation confirmed the binding of PA2G4 protein to MYCN protein in two neuroblastoma cell lines: BE(2)-C and CHP-134.

Chapter 3- Identification of PA2G4 98

Figure 3.3 Co-localisation of PA2G4 and MYCN protein in the nucleus (A) Western blots of cytoplasmic (C) and nuclear (N) protein fractions from BE(2)-C and Kelly cells. (B) Confocal microscopy of BE(2)-C and Kelly cells showing co- localisation of PA2G4 and MYCN proteins.

Chapter 3- Identification of PA2G4 99 3.2.3 High PA2G4 expression predicts poor patient prognosis

The results above suggest that PA2G4 interacts with MYCN and this interaction may play a prognostic role in neuroblastoma patient outcome. Kaplan-Meier analysis of publicly available microarray gene expression data sets showed that high PA2G4 expression was strongly predictive of poor survival (Figure 3.4 A, B, D). Analysis of event-free survival and overall survival from the Kocak microarray data set containing mRNA expression data for 477 neuroblastoma tumours, from the R2 microarray database (http://r2.amc.nl), showed high expression of PA2G4 associated with poor outcome (Figure 3.4 A). Stratification based on low and high expression of PA2G4 and MYCN showed the worst event-free survival for patients with both high PA2G4 and MYCN amplification (Figure 3.4 B). Further analysis revealed that PA2G4 expression positively correlates with MYCN expression, r =0.41 and P =6.76 x10-7. (Figure 3.4 C). Furthermore, analysis of an independent data set (Versteeg-88) containing mRNA from 88 neuroblastoma patients, also from the R2 microarray database (http://r2.amc.nl), indicates that high PA2G4 expression is associated with poor outcome (Figure 3.4 D).

Chapter 3- Identification of PA2G4 100

Figure 3.4 High expression of PA2G4 predicts poor survival in neuroblastoma patients (A) Event-free and overall survival plots from the Kocak microarray data set containing mRNA from 477 neuroblastoma patients showing the probability of survival. (B) Event- free survival plot with stratification of low and high expression of PA2G4 and MYCN.

Chapter 3- Identification of PA2G4 101

Figure 3.4 (cont’d) High expression of PA2G4 predicts poor survival in neuroblastoma patients (C) Correlation plot between PA2G4 and MYCN mRNA expression. (D) Overall survival plot from Versteeg microarray data set containing mRNA from 88 neuroblastoma patients.

Chapter 3- Identification of PA2G4 102 3.2.4 PA2G4 is an independent prognostic marker for poor outcome

Continuing with the analysis of the Kocak data set, further stratification of the data by MYCN amplification (Figure 3.5 A) or INSS stage revealed that PA2G4 is more highly expressed in MYCN-amplified tumours and in INSS stages 3 and 4 (Figure 3.5 B). Multivariable analysis using cox regression indicated that PA2G4 expression retained prognostic significance independent of age, disease stage and MYCN amplification (Table 3.3). P-value was obtained from two-sided log-rank test.

Figure 3.5 Elevated PA2G4 expression in INSS stage 3 and 4 tumours Box and whiskers plots of PA2G4 expression showing higher PA2G4 expression in MYCN-amplified tumours (A) as well as in INSS stage 3/4 tumours (B).

Chapter 3- Identification of PA2G4 103 Table 3.3 PA2G4 expression is an independent prognostic factor in neuroblastoma

Chapter 3- Identification of PA2G4 104 To further validate the correlation between expression of PA2G4 and MYCN in neuroblastoma, 40 neuroblastoma tumours obtained from the Children’s Cancer Institute tumour bank were analysed for PA2G4 and MYCN expression by real-time PCR. The 8 tumours with MYCN amplification had extremely high levels of MYCN mRNA expression, as expected compared to MYCN-non amplified patients (Figure 3.6 A), with P <0.0001. These 8 patients also had higher levels of PA2G4 expression (Figure 3.6 B), P=0.014, for primers detecting both p42 and p48 isoforms, and P =0.005 for primers detecting only the p48 isoform. Linear regression analysis further confirmed that PA2G4 expression positively correlated with MYCN expression (Figure 3.6 C); correlation coefficient r =0.81 and P <0.0001.

Chapter 3- Identification of PA2G4 105

Figure 3.6 mRNA expression analysis of 40 neuroblastoma tumour samples Real-time PCR mRNA expression of MYCN (A) and PA2G4 (B) in 40 neuroblastoma patient tumours, separated by MYCN amplification status. (C) Linear regression analysis of PA2G4 and MYCN mRNA expression.

Chapter 3- Identification of PA2G4 106 To determine if PA2G4 and MYCN expression also correlate at the protein level, protein samples from 30 neuroblastoma tumours obtained from the University of Cologne Tumour Bank were analysed by western blotting. Higher PA2G4 expression was observed in INSS stage 4 (metastatic) tumours and in tumours with MYCN gene amplification (Figure 3.7 A and B). These results were consistent across the three different loading controls used: beta-actin (ACTB), alpha4A tubulin (TUBA4A), and vinculin (VCL). Analysis across all three control proteins showed statistical differences with P-values <0.05 between INSS Stages 1–3 and Stage 4 tumours and between tumours with and without MYCN amplification. There were no statistical differences between Stages 1–3 and Stage 4S. Kaplan-Meier analysis of event-free survival stratified by protein expression indicated that high PA2G4 expression was associated with poorer outcome (Figure 3.7 C, P =0.025, Log-rank test). RNA Deep Sequencing was performed on the 28 patient samples to analyse PA2G4 mRNA expression. The results showed that PA2G4 positively correlates with MYCN expression (Figure 3.7 D, correlation coefficient r =0.64 and P <0.001).

Figure 3.7 Protein expression analysis of 30 neuroblastoma tumour samples (A) Western blots showing PA2G4 expression across 30 neuroblastoma patient tumours. Three different loading controls were used: beta actin, alpha tubulin 4A, and vinculin.

Chapter 3- Identification of PA2G4 107

Figure 3.7 Protein expression analysis of 30 neuroblastoma tumour samples (B) Normalisation of PA2G4 protein expression to each of the three control proteins showed that PA2G4 expression is elevated in Stage 4 and MYCN amplified neuroblastoma patient tumours (Statistical analysis: t-test). (C) Kaplan-Meier analysis of event-free survival based on PA2G4 protein expression. (D) RNA Deep Sequencing of neuroblastoma tumour samples shows PA2G4 positively correlates with MYCN mRNA expression in neuroblastoma patient tumours (Statistical analysis: Spearman correlation). Chapter 3- Identification of PA2G4 108 3.2.5 PA2G4 expression increases postnatally

To assess whether PA2G4 is involved in tumour initiation, PA2G4 and MYCN mRNA expression were analysed at time intervals relevant to tumorigenesis in the TH-MYCN mouse model, a well-established system to study development and progression of neuroblastoma (Weiss et al., 1997). Ganglia from TH-MYCN +/+ mice and wild-type controls were collected at day 1 after birth, and at 2, 4 and 6 weeks of age. Real-time PCR analysis of MYCN and PA2G4 mRNA showed that MYCN expression is high at day 1 after birth in TH-MYCN +/+ mice but low in wild type mice (Figure 3.8 A). PA2G4 expression is low in both wild type and TH-MYCN +/+ mice, but there is a steady increase in PA2G4 expression in the TH-MYCN +/+ mice as the ganglia mature into tumours (Figure 3.8 B).

Figure 3.8 PA2G4 expression is elevated in ganglia of TH-MYCN transgenic mice Real-time PCR mRNA expression of MYCN (A) and PA2G4 (B) in ganglia of TH- MYCN mice compared to wild type mice collected at different ages.

Chapter 3- Identification of PA2G4 109 3.2.6 MYCN positively regulates PA2G4 expression

Having established the correlation between PA2G4 and MYCN expression in patient tumour samples, the relationship between PA2G4 and MYCN was investigated at the molecular level. A panel of neuroblastoma cell lines and normal lung fibroblast cells was screened for PA2G4 protein expression. Western blotting and subsequent quantitation revealed significantly higher PA2G4 expression in neuroblastoma cell lines with MYCN gene amplification compared to both the neuroblastoma cell lines without MYCN amplification and normal fibroblast cell lines (Figure 3.9 A and B), P =0.008. High PA2G4 expression was also observed in neuroblastoma cell lines lacking MYCN- amplification but having elevated c-MYC expression (SH-SY5Y and SK-N-AS) (Figure 3.9 A–C).

Figure 3.9 Higher PA2G4 protein expression in MYCN-amplified neuroblastoma cell lines (A) Western blot analysis of PA2G4, MYCN and C-MYC protein levels in a panel of MYCN-amplified and non-amplified neuroblastoma cell lines and normal lung fibroblast cell lines.

Chapter 3- Identification of PA2G4 110

Figure 3.9 (cont’d) Higher PA2G4 protein expression in MYCN amplified neuroblastoma cell lines (B) Protein densitometry showing higher PA2G4 protein expression in MYCN- amplified neuroblastoma cell lines. (C) Protein densitometry showing high c-MYC protein expression correlates with high PA2G4 expression in neuroblastoma cell lines. P-values determined by t-test, bars represent the mean and SEM from 4 independent experiments.

Chapter 3- Identification of PA2G4 111 To determine whether altering MYCN expression subsequently alters PA2G4 levels, MYCN expression was suppressed using two different siRNA duplexes in BE(2)-C and Kelly cells. At 48h post-transfection, MYCN expression was largely abolished in both cell lines at the protein level (Figure 3.10 A), and substantially reduced at mRNA level (Figure 3.10 B). PA2G4 protein and mRNA levels were reduced in both cell lines following MYCN suppression. Similar results were obtained in the c-MYC-expressing neuroblastoma cell lines SH-SY5Y and SK-N-AS following siRNA-mediated knockdown of c-MYC (Figure 3.11 A and B), indicating that PA2G4 may be MYC regulated as well as MYCN regulated.

Figure 3.10 MYCN positively regulates PA2G4 protein and mRNA expression (A) Western blots of BE(2)-C and Kelly cells with MYCN siRNA knockdown for 48 hours. (B) Real-time PCR analysis of MYCN and PA2G4 mRNA expression in BE(2)- C and Kelly cells with MYCN siRNA knockdown for 48 hours. P-values determined by t-test, bars represent the mean and SEM from 3 independent experiments. Chapter 3- Identification of PA2G4 112

Figure 3.11 c-MYC positively regulates PA2G4 protein and mRNA expression (A) Western blots of SH-SY5Y and SK-N-AS cells with c-MYC siRNA knockdown for 48 hours. (B) Real-time PCR analysis of MYCN and PA2G4 mRNA expression in SH- SY5Y and SK-N-AS cells with c-MYC siRNA knockdown for 48 hours. P-values determined by t-test, bars represent the mean and SEM from 3 independent experiments.

Chapter 3- Identification of PA2G4 113

To determine whether elevated MYCN leads to higher PA2G4 expression, SH-EP MYCN3 cells with doxycycline-inducible MYCN expression were used. The parental SH-EP cell line has very low to undetectable levels of MYCN (Slack et al., 2005), and very low levels of PA2G4 (Figure 3.9A). Following doxycycline exposure over a period of 24 to 96 hours, MYCN expression was substantially increased at both protein and mRNA level (Figure 3.12 A and B), with a greater than 20-fold increase in mRNA expression after 72 hours. PA2G4 levels increased in a time frame similar to that of MYCN (Figure 3.12 A and C), with a more than 3-fold increase in mRNA expression after 72 hours.

Chapter 3- Identification of PA2G4 114

Figure 3.12 Overexpression of MYCN increases PA2G4 protein and mRNA levels SH-EP MYCN cells treated with 1µg/mL doxycycline for up to 96 hours to induce MYCN expression. (A) Western blot analysis of MYCN and PA2G4 after doxycycline treatment. (B) Real-time PCR analysis of MYCN mRNA and (C) PA2G4 mRNA expression following doxycycline treatment. P-values determined by t-test, bars represent the mean and SEM from 3 independent experiments. Chapter 3- Identification of PA2G4 115 3.2.7 MYCN protein binds to the PA2G4 promoter

To determine whether MYCN regulates PA2G4 expression by binding in the vicinity of its promoter to activate transcription, analysis of the PA2G4 DNA sequence was performed to identify E-boxes (consensus MYC family binding sites). DNA sequence analysis revealed two canonical E-boxes located 523 bp and 615 bp downstream of the PA2G4 promoter, within Intron region 1, as depicted in the schematic diagram of Figure 3.13 A. Primers for chromatin immunoprecipitation were designed around these two E- boxes and also at 1200 bp upstream of the promoter, which served as a negative binding control region (Figure 3.13 A and D). Chromatin immunoprecipitation was carried out using two neuroblastoma cells lines, BE(2)-C and Kelly. DNA from each cell line was sheared by sonication then cross-linked to capture bound proteins. An anti-MYCN antibody or isotype control was used to isolate the fragmented DNA that bound to MYCN protein (Figure 3.13 B). Once isolated, proteins were removed from the DNA, which was subsequently amplified using primers specific for the upstream, promoter and intron regions of PA2G4, and for the promoter region of ODC as a positive control (Bello-Fernandez et al., 1993). PCR products from the promoter and intron regions of PA2G4 and the positive control were found to be enriched, while the PCR product from the upstream control region was not (Figure 3.13 C), indicating that MYCN binds to the PA2G4 gene in close proximity to the transcriptional start site, consistent with transcriptional regulation of PA2G4.

Chapter 3- Identification of PA2G4 116

Chapter 3- Identification of PA2G4 117

Figure 3.13 MYCN binds to PA2G4 DNA (A) Schematic showing the two canonical E-box sequences located within Intron 1 of the PA2G4 gene. Also marked is the negative control region. (B) Schematic outlining the chromatin immunoprecipitation procedure. (C) Chromatin immunoprecipitation and real-time PCR indicated enrichment of MYCN binding at the Intron a & b regions as well as the PA2G4 promoter region. ODC was used as a positive control for MYCN binding (D) PA2G4 DNA sequence showing location of the real-time PCR primers and the two E-box sequences. Highlighted sequences indicate forward and reverse primers. P-values determined by t-test, bars represent the mean and SEM from 3 independent experiments.

Chapter 3- Identification of PA2G4 118 To further investigate whether MYCN activates PA2G4 transcription, MYCN was knocked down using siRNA in both BE(2)-C and Kelly cells (Figure 3.14 A), and chromatin immunoprecipitation was performed as previously described. Real-time PCR analysis indicated enrichment of the PA2G4 promoter and intron region PCR products and the ODC product in the siRNA control samples, however, each of these products was significantly decreased with MYCN knockdown (Figure 3.14 B), consistent with the transcriptional regulation of PA2G4 by MYCN.

Chapter 3- Identification of PA2G4 119

Figure 3.14 Suppression of MYCN reduces PA2G4 transcription (A) Western blot confirming knockdown of MYCN protein and correspondingly, PA2G4 protein. (B) Knockdown with MYCN siRNA reduces the enrichment for chromatin immunoprecipitation of MYCN at the promoter region and Intron 1 a & b regions and also ODC promoter. P-values determined by t-test, bars represent the mean and SEM from 3 independent experiments.

Chapter 3- Identification of PA2G4 120 3.2.8 PA2G4 stabilises MYCN protein

Since PA2G4 appears to associate with MYCN, the influence of PA2G4 expression on MYCN levels was investigated. PA2G4 expression was suppressed in the BE(2)-C and Kelly cell lines using two independent siRNA duplexes, both of which target both the long and short isoform of PA2G4. MYCN protein levels were substantially reduced in the Kelly cell line 48h after transfection with PA2G4 siRNA (Figure 3.15 A), while MYCN mRNA levels were unaltered in either line at this time point when analysed by real-time PCR (Figure 3.15 B). Similarly, when PA2G4 was suppressed in the c-Myc- expressing cell lines SK-N-AS and SH-SY5Y, c-MYC protein, but not mRNA, was substantially reduced at the 48h time point (Figure 3.16 A and B). Similar results were obtained at 24h, 72h and 96h time points (not shown).

Chapter 3- Identification of PA2G4 121

Figure 3.15 PA2G4 regulates MYCN protein expression but not mRNA expression (A) Western blots of PA2G4 and MYCN expression in BE(2)-C and Kelly cells following siRNA-mediated PA2G4 knockdown for 48 hours. GAPDH is included as a loading control. (B) Real-time PCR analysis of PA2G4 and MYCN mRNA expression in BE(2)-C and Kelly cells with PA2G4 siRNA knockdown for 48 hours. P-values determined by t-test, bars represent the mean and SEM from 3 independent experiments.

Chapter 3- Identification of PA2G4 122

Figure 3.16 PA2G4 regulates c-MYC protein expression but not mRNA expression (A) Western blots of PA2G4 and MYCN expression in SK-N-AS and SH-SY5Y cells with PA2G4 siRNA knockdown for 48 hours. (B) Real-time PCR analysis of PA2G4 and c-MYC mRNA expression in SK-N-AS and SH-SY5Y cells with c-MYC siRNA knockdown for 48 hours. P-values determined by t-test, bars represent the mean and SEM from 3 independent experiments.

Chapter 3- Identification of PA2G4 123 The effect of PA2G4 levels on MYCN protein levels suggests that PA2G4 regulates MYCN post-transcriptionally. To determine whether PA2G4 expression influences MYCN protein stability, cycloheximide (CHX) chase assays were performed to measure MYCN protein degradation. BE(2)-C and Kelly cells were treated with 100µg/μL CHX for 10 to 60 minutes, to block protein synthesis and allow the measurement of MYCN protein half-life. Before treatment with CHX, cells were transfected with PA2G4-p48 siRNA, to knock down the long PA2G4 isoform, or control siRNA for 48 hours. Faster MYCN protein degradation was observed with knockdown of PA2G4 in both cell lines (Figure 13.7 A and B). Protein densitometry analysis revealed that the half-life of MYCN in BE(2)-C cells decreased from 13.6 minutes to 8.6 minutes (Figure 3.17 B; P=0.01), when PA2G4 was suppressed. Similarly in Kelly cells, the half-life of MYCN degradation decreased from 18.8 minutes to 9.5 minutes with suppression of PA2G4 (Figure 3.17 B; P = 0.03).

Chapter 3- Identification of PA2G4 124

Figure 3.17 Knockdown of PA2G4 destabilises MYCN protein Cycloheximide chase assay measuring the half-life of MYCN protein with knockdown of PA2G4. Knockdown with PA2G4-p48 siRNA in (A) BE(2)-C and (B) Kelly cells for 48 hours then treatment with 100µg/µl CHX for up to 60 minutes followed by western blot analysis to measure MYCN protein half-life. (C) Protein half-life of MYCN in BE(2)-C and Kelly cells. P-values determined by t-test, bars represent the mean and SEM from 3 independent experiments.

Chapter 3- Identification of PA2G4 125 Overexpression of PA2G4 was also performed in BE(2)-C and Kelly cells to determine whether higher PA2G4 levels stabilise MYCN. PA2G4 plasmid DNA or empty vector DNA was transfected into cells for 48 hours, then cells were treated with 100µg/µl CHX to block protein synthesis, and samples were taken at time intervals to measure MYCN protein half-life. MYCN half-life was longer in cells with PA2G4 overexpression than in control cells (Figure 3.18 A and B). Protein densitometry analysis showed the half-life of MYCN in BE(2)-C cells increased from 11.9 minutes to 27.4 minutes (Figure 3.18 B; P =0.006). In Kelly cells MYCN half-life increased from 12.4 minutes to 24.9 minutes with overexpression of PA2G4 (Figure 3.18 B; P =0.027).

Chapter 3- Identification of PA2G4 126

Figure 3.18 Overexpression of PA2G4 stabilises MYCN protein Cycloheximide chase assay measuring the half-life of MYCN protein with overexpression of PA2G4. Overexpression of PA2G4 in (A) BE(2)-C and (B) Kelly cells for 48 hours then treatment with 100µg/µl CHX for up to 60 minutes followed by western blot analysis to measure MYCN protein half-life. (C) Protein half-life of MYCN in BE(2)-C and Kelly cells. P-values determined by t-test, bars represent the mean and SEM from 3 independent experiments.

Chapter 3- Identification of PA2G4 127 3.2.9 PA2G4 controls MYCN stability through phosphorylation of Serine 62 To elucidate the mechanism by which PA2G4 regulates MYCN protein stability, one needs to understand how MYCN is degraded. MYCN stability is regulated by phosphorylation of Serine 62 (S62) and Threonine 58 (T58), which leads to its ubiquitination and proteasomal degradation as depicted in Figure 3.19 A. To determine whether Serine 62 and Threonine 58 phosphorylation is affected by PA2G4 expression, PA2G4 was knocked down in BE(2)-C and Kelly cells using two different PA2G4-p48 siRNA for 48 hours. The phosphorylation status of S62 and T58 was determined by western blotting. Decreases in both Serine 62 and Threonine 58 phosphorylation were observed in both cell lines (Figure 3.19 B). Also evident was the decrease in phosphorylated ERK, a tyrosine kinase that is well known to phosphorylate MYC protein at S62. Protein densitometry quantifying the relative expression of T58/MYCN, and S62/MYCN, showed that S62 expression was much lower in BE(2)-C and Kelly cells (Figure 3.19 C; P-value <0.05 for all tested data).

Chapter 3- Identification of PA2G4 128

Figure 3.19 PA2G4 controls MYCN stability through phosphorylation of Serine 62 (A) Schematic diagram of the classical MYCN degradation pathway. (B) Protein analysis of BE(2)-C and Kelly cells with PA2G4-p48 siRNA knockdown to analyse the levels of Serine 62, Threonine 58, and ERK phosphorylation. (C) Protein densitometry quantifying the relative expression of S62/MYCN, and T58/MYCN.

Chapter 3- Identification of PA2G4 129 To investigate whether Serine 62 is required for PA2G4 to stabilise MYCN protein, deletion mutants of Serine 62 and Threonine 58 along with PA2G4 were used to transfect the SH-EP cell line, a line with very low endogenous PA2G4 and MYCN expression (Figure 3.9A). Double transfection was performed using combinations of the following plasmids: MYCN + empty vector; MYCN + PA2G4; MYCN deletion mutant S62 + PA2G4; MYCN deletion mutant T58 + PA2G4. Transfection was carried out for 24 hours followed by western blotting to analyse protein expression levels (Figure 3.20 A). As expected, elevated PA2G4 expression increases MYCN expression, P <0.0001, however, when S62 is mutated, MYCN levels decrease to the same as control (no PA2G4 overexpression). In contrast, mutant T58 transfection with PA2G4 increased MYCN expression similarly to the non-mutated MYCN samples (Figure 3.20 A and B; P =0.0022).

To analyse whether the phosphorylation of ERK is affected by PA2G4, SH-EP cells were again transfected with empty vector DNA or PA2G4 DNA for 24 hours. Proteins were harvested for western blot analysis, and the results clearly showed an increase in phosphorylated ERK expression but no change in total ERK protein (Figure 3.20 C and D; P =0.0095).

Chapter 3- Identification of PA2G4 130

Figure 3.20 PA2G4 requires Serine 62 for the regulation of MYCN protein (A) Protein analysis of SH-EP cells transfected with MYCN, or mutated S62, or mutated T58 and PA2G4 for 24 hours. (B) Protein densitometry quantifying the level of MYCN expression normalised to EV/MYCN.

Chapter 3- Identification of PA2G4 131

Figure 3.20 (cont’d) PA2G4 requires Serine 62 for the regulation of MYCN protein (C) Protein analysis of SH-EP cells transfected with either empty vector or PA2G4 for 24 hours. (D) Protein densitometry quantifying the level of pERK expression.

Chapter 3- Identification of PA2G4 132 3.2.10 Common genes in the PA2G4 and MYCN signalling pathways

To assess the signalling pathways common to PA2G4 and MYCN in order to better understand their interactions, PA2G4 or MYCN were knocked down by siRNA in BE(2)-C cells for 24 hours. RNA was harvested and analysed by microarray profiling using the Affymetrix Human Gene Array 2.0, to generate a list of common genes that are down-regulated or up-regulated when PA2G4 or MYCN are suppressed (Figure 3.21 A). 174 genes from the MYCN knockdown and 129 genes from the PA2G4 knockdown data sets, of which 14 genes are common, were found to be differentially expressed after a statistical cut-off value of 2-fold was applied. Using Gene Set Enrichment Analysis (GSEA) to analyse the oncogenic signalling pathways, the top ten pathways for each dataset were identified based on their ranked normalised enrichment score (NES) and P-values (Figure 3.21 B), showing that the only two shared pathways between the MYCN and PA2G4 knockdown datasets in the top ten list were MYCN and KRAS. However, there are 57 common pathways that overlap between PA2G4 and MYCN suppression (Table 3.4).

Chapter 3- Identification of PA2G4 133

Figure 3.21 Common genes and pathways regulated by MYCN and PA2G4 (A) Microarray analysis of common genes down- and up-regulated by MYCN and PA2G4. Analysis cut-off at fold change >2. (B) Top 10 pathways from Oncogenic GSEA database for MYCN knockdown and PA2G4 knockdown.

Chapter 3- Identification of PA2G4 134 Table 3.4 Top 57 common oncogenic pathways shared between MYCN and PA2G4 knockdown

Chapter 3- Identification of PA2G4 135 3.3 Discussion

The identification of MYCN binding proteins has the potential to provide vital knowledge and new avenues to target the oncogenic effects of MYCN. Here, PA2G4 is identified as a new MYCN binding partner. Having established this, it was more difficult to provide evidence that PA2G4 directly binds to MCYN. A co- immunoprecipitation protocol was optimised using lysis buffer containing very little to no detergent, enabling detection of PA2G4 binding to MYCN with MYCN pull-down. As stated previously, PA2G4 and MYCN need to be located in the same cellular compartment for there to be any interaction. MYCN is known to be predominantly nuclear (Bannasch et al., 1999; Smith et al., 2004). When proteins from BE(2)-C cells were separated into cytoplasmic and nuclear fractions, the majority of MYCN was detected in the nucleus. However in Kelly cells, MYCN protein was present in both compartments. This would suggest that after translation, MYCN translocates to the nucleus. This is aided by its nuclear localisation signals located within the C terminus. Proteins are synthesised by ribosomes found embedded in the intracellular membranes which make up the rough endoplasmic reticulum. The endoplasmic reticulum and ribosomes are located between the membranes of the nuclear envelope. In Kelly cells, MYCN is highly amplified, with a lot of MYCN being produced by ribosomes. This could explain why there is an abundance of MYCN in the cytoplasmic fraction.

Results from the protein fractionation and confocal microscopy reveal the existence of PA2G4 in cytoplasmic and nuclear compartments, however each method shows a difference in the relative distribution of PA2G4. In the subcellular fractionation experiments, PA2G4 is predominantly expressed in the cytoplasm, while confocal microscopy analysis shows more PA2G4 in the nucleus. Published data report that PA2G4 is localised in the cytoplasm and translocates to the nucleus where it interacts with nuclear proteins such as E2F-1 and Retinoblastoma protein (Rb) (Liu et al., 2006; Yoo et al., 2000; Zhang et al., 2003). PA2G4 is believed to be part of the pre-ribosomal ribonucleoprotein (RNP) complex (Squatrito et al., 2004). RNP is a nucleoprotein formed by the association of RNA and protein, which can form complexes such as ribosomes. As mentioned above, ribosomes are located on the membranes of the nuclear envelope. It is possible that high levels of PA2G4 protein seen in the nucleus in the confocal microscopy images are actually PA2G4 ribosomal proteins located outside of the nuclear membrane, which protein fractionation has identified.

Chapter 3- Identification of PA2G4 136

Having established that PA2G4 binds or associates with MYCN protein, the next point of discussion will focus on the relevance of PA2G4 expression for neuroblastoma patient prognosis. The results from two microarray databases, as well as the analysis of patient tumour samples obtained from multiple tumour banks, clearly indicated high PA2G4 expression is associated with poorer patient outcome. One significant finding was that high PA2G4 expression maintained independent prognostic significance in multivariable analysis with other well-established prognostic factors including age at diagnosis, INSS stage and MYCN amplification status.

Insight into MYCN and PA2G4 expression associated with tumour initiation came from the TH-MYCN ganglia mRNA expression data. These homozygous mice were generated to highly express MYCN; even in the 1-day-old TH-MYCN +/+ mice, MYCN is already highly expressed compared to the wild type mice, which show extremely low levels of MYCN. An interesting observation was the low expression of PA2G4 in 1-day-old TH- MYCN +/+ and wild type mice. It appears that PA2G4 mRNA expression remains low in wild type mice throughout the developmental ganglia stages, however in TH-MYCN +/+ mice PA2G4 expression in ganglia becomes elevated at 2 weeks and continues to rise with the maturation of these ganglia into tumours. This indictaes that PA2G4 may be involved in tumour initiation.

MYCN gene amplification is associated with aggressive and metastatic disease (DuBois et al., 1999; Minard et al., 2000). The fact that normal cells do not have high levels of PA2G4 protein expression is very promising in the context of targeting PA2G4 for the treatment of neuroblastoma. Elucidating the mechanisms of interaction between MYCN and PA2G4 will lead to better understanding of how PA2G4 can be targeted. Experiments with MYCN and c-MYC siRNA knockdown clearly indicated that both MYCN and c-MYC regulate PA2G4 expression, therefore PA2G4 is also likely to bind to c-MYC. Both MYCN and c-MYC regulate PA2G4 expression at both the transcriptional and the post-translational level. The overexpression of MYCN in the SH- EP inducible system provided further evidence that MYCN positively regulate PA2G4 expression.

It is well published that MYCN is a transcription factor that binds to the DNA of its target genes to activate or suppress transcription (Beltran, 2014; Evans et al., 2015).

Chapter 3- Identification of PA2G4 137 MYCN binds to conserved sequences known as E-boxes, which are located within or surrounding the promoter region of a target gene (Murphy et al., 2009). PA2G4 has two E-box sequences located within Intron I, near enough to the promoter to allow activation of transcription. Binding of MYCN to the E-boxes on PA2G4 DNA was detected due to enrichment for this region in a chromatin immunoprecipitation (ChIP) assay followed by real-time PCR analysis. Conversely, decreasing MYCN causes a loss of enrichment for the E-box region in a ChIP assay.

Some transcription factors can target genes that are involved in their regulation. MYCN is known to activate genes such as SIRT2 (Liu et al., 2013), which indirectly stabilises MYC protein and promotes cancer cell proliferation, by repressing transcription of the HECT-domain E3 ubiquitin ligase NEDD4, described as an E3 ligase for MYC. It can also regulate genes such as Skp2 (Bretones et al., 2011; Evans et al., 2015), which recognises MYC through both MBII and HLH-LZ motifs and promotes MYC polyubiquitination and degradation (Kim et al., 2003; von der Lehr et al., 2003). Therefore, it was not unexpected to find PA2G4 expression had an effect on MYCN protein stability. Based on the data from this chapter, it is proposed that MYCN activates PA2G4 transcription, leading to an increase in PA2G4 protein production which allows it to bind and interact with MYCN protein to stabilise it, thus delaying its degradation.

PA2G4 is able to positively regulate MYCN protein expression but has no effect on MYCN mRNA expression. The mechanism by which this occurs is the phosphorylation of MYCN amino acid residue, Serine 62. The MYCN degradation pathway is well studied and is reviewed by many groups (Farrell and Sears, 2014; Gustafson and Weiss, 2010). Studies into the degradation of MYCN identified phosphorylation of two MYCN amino acids, Threonine 58 and Serine 62, governing the stability of the protein. Threonine 58 and Serine 62 are located within MBI domain (Flinn et al., 1998), the same domain that PA2G4 was identified to bind to MYCN. It is plausible to speculate that PA2G4 may act as a regulator for kinase access. Changes in PA2G4 expression were able to affect the levels of phosphorylation in ERK, which in turn phosphorylates Serine 62, thus initiating the MYCN degradation signalling cascade. PA2G4 has been shown to regulate eIF2α phosphorylation by binding to PKR and inhibiting its dsRNA- induced kinase activity on eIF2α (Squatrito et al., 2006).

Chapter 3- Identification of PA2G4 138 While elucidating the molecular interactions between PA2G4 and MYCN is an important first step, the next step is to understand how PA2G4 and MYCN interact with other genes and pathways that drive neuroblastoma. Microarray analysis can identify common genes regulated by PA2G4 and MYCN and the pathways affected by their signalling. The data generated from independent suppression of MYCN and PA2G4 identified a significant number of non-coding RNA gene targets. Non-coding RNA plays a very important role in the regulation of oncogenes and tumour suppressor genes in neuroblastoma (Cheng et al., 2014; Mei et al., 2014a; Watters et al., 2013). PA2G4 is believed to be part of ribonucleoprotein complexes, it also contains a dsRBD (double- strand RNA binding domain) that mediates its interaction with dsRNA (Squatrito et al., 2004; Squatrito et al., 2006). It is speculated that its ability to inhibit growth could be linked to a negative effect on normal rRNA processing and/or ribosome assembly (Okada et al., 2007).

Using the microarray data to analyse the oncogenic pathways, it was evident that PA2G4 and MYCN regulate several common pathways. Most notable and highly ranked on the list were MYCN and KRAS. The presence of MYCN is expected in the MYCN-suppressed RNA microarray samples. Its presence at the top of the PA2G4- suppressed RNA microarray samples is further validation that there is a strong interaction between these two genes. KRAS plays an important role in signal transduction pathways and is a well-studied oncogene known to have implications in colorectal and lung cancer (Fakih, 2015; Skoulidis et al., 2015; Zuber et al., 2000). Mutations in KRAS have also been described in neuroblastoma (Schramm et al., 2015), however the link between KRAS and PA2G4 has not been described, making it an important and relevant pathway to study, since very little is known about the mechanism of PA2G4 signalling in cancer.

In conclusion, the data generated from this thesis show for the first time that PA2G4 interacts with MYCN through protein binding, thus addressing the first aim of this thesis, which was to determine whether PA2G4 directly interacts with MYCN. It also identifies PA2G4 as an independent prognostic marker for neuroblastoma patient outcome. The strong relationship between PA2G4 and MYCN makes PA2G4 a potential target for inhibiting MYCN expression. However, little is known about the role of PA2G4 in neuroblastoma, whether it truly has an oncogenic role in neuroblastoma progression, and if its mechanism of action is dependent on MYCN.

Chapter 3- Identification of PA2G4 139

Chapter 4 PA2G4: A POTENTIAL ONCOGENIC PROTEIN IN NEUROBLASTOMA

CHAPTER 4

PA2G4: A POTENTIAL ONCOGENIC PROTEIN IN NEUROBLASTOMA

Chapter 4- Characterisation of PA2G4 140 4.1 Introduction

Oncogenic proteins are proteins encoded by oncogenes (dysregulated or activated genes), and have a potential to cause cancer. Transcription factors, kinases and growth factors are considered oncogenic proteins as they are generically involved in signalling systems leading to cell growth, survival, differentiation and programmed cell death (Croce, 2008). Classic oncogenes such as RAS (Bos, 1989; Pylayeva-Gupta et al., 2011), BCL-2 (Cory et al., 2003; Yip and Reed, 2008), and MYC (Dang, 2012; Meyer and Penn, 2008) are well studied and reviewed for their role in cancer.

PA2G4 may have oncogenic potential; the p48 isoform has been reported to promote cell growth and invasion in human glioblastoma cell line U87, and overexpression of p48 facilitated tumorigenesis in a xenograft mouse model of brain cancer (Kim et al., 2010). It also interacts with nucleophosmin, a pre-ribosomal ribonucleoprotein complex that controls ribosome biosynthesis and cell growth, and disruptions to this complex lead to decreased cell growth and increased apoptosis (Okada et al., 2007). PA2G4 binds to the promoter of E2F-1 to regulate the cell cycle (Zhang et al., 2002), whereas overexpression of p42 represses E2F-1 activity; by contrast, p48 increases E2F-1 transcription (Liu et al., 2009). Although PA2G4 has been implicated in a range of cancers, including breast, prostate and brain, its role in neuroblastoma has not been investigated.

This chapter will address the second aim of this thesis, which is to determine the functional role of PA2G4 in neuroblastoma, and to identify key genes and pathways involved in its signalling. It will examine the phenotypic changes caused by expression of PA2G4 in neuroblastoma cells, and its effects on biological processes including cell growth, tumorigenicity, cell migration and invasion.

Chapter 4- Characterisation of PA2G4 141 4.2 Results

4.2.1 Overexpression of PA2G4 increases cell growth

The neuroblastoma cell line SK-N-FI, previously demonstrated to have low endogenous levels of PA2G4 (Figure 3.9), was transfected with PA2G4 plasmid DNA, and the affect on cell growth was assessed. Overexpression of PA2G4 increased cell growth over a 96 hour period, as measured by the cells’ ability to metabolise resazurin (Figure 4.1 A). The most significant increase in cell viability (approximately 20 %, P=0.0063) occurred 72 hours post transfection. The optimal time point for transfection was identified as 72 hours. This time point was used when transfecting PA2G4 plasmid DNA into five neuroblastoma cell lines: BE(2)-C, Kelly, SK-N-AS, SH-SY5Y and SK- N-FI, and one normal fibroblast cell line, WI-38. Cell proliferation was measured by the incorporation of BrdU (Figure 4.1 B), and cell viability was measured by the metabolism of resazurin (Figure 4.1 C). Overexpression of PA2G4 increased cell viability and cell proliferation in all the neuroblastoma cell lines, however, no significant changes were observed in the WI-38 fibroblast cell line. Western blot analysis confirmed transfection of the PA2G4 plasmid DNA into all cell lines (Figure 4.1 D). GAPDH protein expression was used as a loading control.

Chapter 4- Characterisation of PA2G4 142

Figure 4.1 Overexpression of PA2G4 increases cell growth (A) PA2G4 plasmid DNA transfected into SK-N-FI cells with measurement of cell viability over time. (B) PA2G4 overexpressed in neuroblastoma cells and normal lung fibroblast cell lines, WI-38, followed by measurement of cell proliferation by BrdU incorporation and (C) cell viability at 72 hours post transfection.

Chapter 4- Characterisation of PA2G4 143

Figure 4.1 (cont’d) Overexpression of PA2G4 increases cell growth (D) Western blots confirming transfection of PA2G4 plasmid DNA into cell lines, EV= empty vector control and PA= PA2G4 plasmid DNA. P-values determined by t-test, bars represent the mean and SEM from 3 independent experiments.

Chapter 4- Characterisation of PA2G4 144 4.2.2 Suppression of PA2G4 decreases cell growth

To investigate the differential effects of the two PA2G4 isoforms, p42 and p48, on cell growth, siRNAs targeting both isoforms (henceforth referred to as PA2G4 #1 and PA2G4 #2), or siRNAs targeting only the long isoform (henceforth referred to as PA2G4-p48 #1 and PA2G4-p48 #2), were transfected into neuroblastoma cells, followed by measurement of conversion of resazurin for the quantification of cell viability. Suppression of PA2G4 with siRNA targeting both p42 and p48 isoforms caused a slight (25%), but statistically significant decrease in cell viability in BE(2)-C cells (Figure 4.2 A), and a less than 15% decrease in Kelly cells with PA2G4 #2 siRNA (Figure 4.2 C). No significant decrease in cell viability was observed in CHP-134 cells (Figure 4.2 D). Knocking down PA2G4 with siRNA specifically targeting the p48 isoform greatly and significantly decreased cell viability in BE(2)-C (Figure 4.2 B), Kelly (Figure 4.2 D) and CHP-134 (Figure 4.2 F). Western blots confirmed knockdown of PA2G4 with PA2G4-p48 siRNAs (Figure 4.2 G), and showed the changes in MYCN protein expression resulting from PA2G4 suppression in BE(2)-C cells.

Chapter 4- Characterisation of PA2G4 145

Chapter 4- Characterisation of PA2G4 146

Figure 4.2 Knockdown of PA2G4 decreases cell growth BE(2)-C cells transfected with PA2G4 siRNA (A) and PA2G4-p48 siRNA (B) followed by cell viability measurements over time. Knockdown of PA2G4 and PA2G4-p48 in Kelly (C and D) and CHP-134 cells (E and F) followed by cell viability analysis over time. (G) Western blots showing that knockdown of PA2G4 with two PA2G4-p48 siRNAs decreases PA2G4 and MYCN protein in BE(2)-C. P-values determined by t- test, bars represent the mean and SEM from 3 independent experiments.

Chapter 4- Characterisation of PA2G4 147 4.2.3 PA2G4-mediated increase in cell growth is independent of MYCN expression

To investigate whether MYCN plays a role in the PA2G4-mediated increase in cell growth, neuroblastoma cell lines, BE(2)-C, Kelly, SH-SY5Y and SK-N-AS were knocked down with PA2G4-p48 siRNA for 24 hours, then transfected with MYCN plasmid DNA for a further 48 or 72 hours, giving final experimental time points of 72 or 96 hours. Suppression of PA2G4 with PA2G4-p48 siRNA significantly reduced cell growth, however, overexpression of MYCN did not significantly increase cell growth, nor was it able to rescue cells from the effect of PA2G4 suppression (Figure 4.3 A). Western blots confirming the transfection of MYCN plasmid DNA and PA2G4-p48 siRNA are shown in Figure 4.3 B.

Chapter 4- Characterisation of PA2G4 148

Chapter 4- Characterisation of PA2G4 149

Figure 4.3 The decrease in cell growth caused by PA2G4 knockdown cannot be rescued by overexpression of MYCN (A) Knockdown of PA2G4-p48 and over expression of MYCN plasmid DNA in BE(2)- C, Kelly, SH-SY5Y and SK-N-AS cells for 72 hours and 96 hours followed by cell viability measurement. (B) Western blots confirming transfection of PA2G4-p48 siRNA and MYCN plasmid DNA. Differences in cell growth were compared to the control siRNA and empty vector sample. P-values determined by t-test, bars represent the mean and SEM from 3 independent experiments.

Chapter 4- Characterisation of PA2G4 150 4.2.4 The PA2G4 long isoform, p48, plays a role in colony formation

To characterise the effects of PA2G4’s two isoforms on cells’ ability to form colonies, siRNAs were used to knock down both the long and short isoforms, or the long isoform only, in BE(2)-C (Figure 4.4 A) and Kelly cells (Figure 4.4 B) for 24 hours. 500 transfected cells were then seeded into 6-well plates and incubated for 10 days to assess colony formation. For each cell line, siRNA targeting both the p42 and p48 isoforms decreased colony formation to approximately 60% of control values. siRNA specifically targeting the p48 isoform was more effective, with colony formation for BE(2)-C and Kelly cells being 20–40% and ~20% of their respective controls.

Chapter 4- Characterisation of PA2G4 151

Figure 4.4 The long isoform of PA2G4, p48, is required for colony formation (A) BE(2)-C and (B) Kelly cells were transfected with PA2G4 siRNA targeting both the long and short isoforms (siPA2G4) or only the long isoform (siP48) for 24 hours, before being plated for colony assays. Colonies were counted after 10 days for BE(2)-C and 14 days for Kelly cells. P-values determined by t-test, bars represent the mean and SEM from 3 independent experiments. Chapter 4- Characterisation of PA2G4 152 4.2.5 Overexpression of MYCN reverses the decrease in colony formation caused by suppression of PA2G4

To determine whether MYCN over expression can rescue the decrease in colony formation seen with suppression of PA2G4, BE(2)-C and Kelly cells were transfected with siRNA targeting PA2G4-p48 for 48 hours, followed by transfection with either MYCN plasmid DNA or empty vector DNA for 24 hours. 500 transfected cells were then plated onto 6-well plates and incubated for 10 days to allow colonies to form. Overexpression of MYCN increased colony formation in BE(2)-C and Kelly cells (Figure 4.5 A; P=0.024 and P=0.0042 respectively), while suppression of PA2G4 significantly decreased colony formation in both cell lines (P<0.0001). Suppression of PA2G4 followed by overexpression of MYCN increased colony formation compared to suppression of PA2G4 alone (P=0.003 for BE(2)-C and P=0.019 for Kelly). Furthermore, overexpression of MYCN in the PA2G4 suppressed cells increased colony formation to a level that was not significantly different to that of the siRNA and empty vector control in both cell lines. Western blots were performed to confirm transfection of siRNAs (Figure 4.5 B).

Chapter 4- Characterisation of PA2G4 153

Figure 4.5 Overexpression of MYCN reverses the decrease in colony formation caused by down-regulation of PA2G4 (A) BE(2)-C and Kelly cells with PA2G4-p48 siRNA knockdown for 48 hours then transfection with MYCN plasmid DNA for 24 hours were seeded for colony formation assay. (B) Western blots confirming transfection of PA2G4-p48 siRNA and MYCN plasmid DNA. P-values determined by t-test, bars represent the mean and SEM from 3 independent experiments.

Chapter 4- Characterisation of PA2G4 154 4.2.6 PA2G4 regulates cell growth through MDM2 and AKT signalling

Having established that overexpression of PA2G4 causes an increase in cell proliferation and suppression of PA2G4 decreases cell viability, the next step is to elucidate the mechanism by which PA2G4 is controlling cell growth. Kim et al. (Kim et al., 2012) proposed that PA2G4 is involved in the regulation of cell growth through interaction with AKT and MDM2, to facilitate the degradation of p53 (Figure 4.6 A), therefore, western blots were performed to determine the protein levels of these candidate molecules with PA2G4-p48 siRNA knockdown. SH-SY5Y cells with PA2G4-p48 siRNA knockdown had decreased levels of phosphorylated AKT and MDM2, but increased p53 and p21 expression (Figure 4.6 B). SH-SY5Y cells were used because they have wild-type p53 whereas in BE(2)-C cells the p53 gene is mutated.

Chapter 4- Characterisation of PA2G4 155

Figure 4.6 PA2G4 mediates the phosphorylation of MDM2 and AKT (A) Schematic diagram of PA2G4 interaction with MDM2 and AKT for the regulation of p53 degradation. (B) Western blots showing that knockdown with PA2G4-p48 siRNA decreases phosphorylated MDM2 and AKT but increases p53 and p21 protein expression in SH-SY5Y cells, 48 hours post transfection.

Chapter 4- Characterisation of PA2G4 156 4.2.7 The protein half-life of PA2G4

In the previous chapter, the half-life of MYCN protein was shown to be very short, less than 30 minutes. The half-life of PA2G4 protein was determined here as follows. Four neuroblastoma cell lines, BE(2)-C, Kelly, SH-SY5Y and CHP-134 were treated with 100 µg/µl of cycloheximide to prevent the synthesis of new proteins and thus measure the rate of protein degradation. PA2G4 protein levels were determined after different periods of cycloheximide treatment (Figure 4.7 A). Cyclin E2 has a half-life of less than 4 hours (Zariwala et al., 1998) and was used as a positive control. GAPDH was used as a loading control for quantitation by densitometry (Figure 4.7 B). PA2G4 protein half- life was approximately 24 hours in BE(2)-C, Kelly, and CHP-134 cells, and substantially shorter (approximately 8h) in the SH-SY5Y cell line.

Chapter 4- Characterisation of PA2G4 157

Figure 4.7 PA2G4 has a long protein half-life (A) Neuroblastoma cells treated with 100 µg/µl cycloheximide to measure the rate of PA2G4 protein degradation. Cyclin E2 protein probed as positive control for cycloheximide treatment. (B) Quantification of PA2G4 protein half-life by densitometry.

Chapter 4- Characterisation of PA2G4 158 4.2.8 PA2G4 degradation pathway

MYCN protein and many other proteins are degraded through the proteasomal pathway. To assess whether PA2G4 protein is degraded through the same pathway, BE(2)-C and Kelly cells were treated with PA2G4-p48 siRNAs, followed by 30µM MG-132 for 3 hours. MG-132 is a proteasome inhibitor that reduces the degradation of ubiquitin- conjugated proteins. PA2G4-p48 siRNAs decreased PA2G4 and MYCN expression. Treatment with MG-132 increased MYCN protein, even in the presence of PA2G4-p48 siRNA knockdown. However, MG-132 did not increase PA2G4 expression in comparison with the untreated control in both BE(2)-C and Kelly cells, which suggests that PA2G4 is not degraded through the proteasomal pathway (Figure 4.8).

Figure 4.8 PA2G4 is not degraded through the proteasomal pathway BE(2)-C and Kelly cells with PA2G4-p48 siRNA knockdown for 48 hours followed by treatment with 30 µM MG-132 for 3 hours.

Chapter 4- Characterisation of PA2G4 159 4.2.9 Suppression of PA2G4 does not increase apoptosis

To understand how overexpression or knockdown of PA2G4 is affecting cell growth, specifically whether suppression of PA2G4 causes cell cycle arrest or changes in cell cycle distribution, BE(2)-C and Kelly cells were transfected with siRNA specific to PA2G4-p48 for 48 hours then stained with propidium iodide (PI). Samples were analysed by flow cytometry and the cells were divided into three subsets that represented G0+G1 phase, S phase, and G2+M phase. A decrease in G2+M phase was observed when PA2G4-p48 was suppressed with siRNAs (siP48#1 and siP48#2) in BE(2)-C (Figure 4.9 A, P=0.0148 & P=0.0031) and Kelly (Figure 4.9 B, P=0.0257 & P=0.0097). However, there were no significant changes to S-phase for both cell lines and only a slight decrease in G0+G1 for BE(2)-C with siP48#2 siRNA knockdown (Figure 4.9 A, P=0.0119).

To assess whether the cell arrest seen in the G2+M phase leads to cell death, western blotting was performed on BE(2)-C and Kelly cells following PA2G4-p48 siRNA knockdown for 48 hours, to examine changes in the key proteins involved in apoptosis, such as level of PARP and caspase 3 cleavage. Suppression of PA2G4 increased PARP cleavage but not caspase 3 cleavage (Figure 4.9 C). To further assess apoptosis, cells were transfected with PA2G4-p48 siRNAs for 48 hours then treated with Annexin V and 7-AAD, to measure the proportion of cells in early and late stages of apoptosis using flow cytometry. Overall, the suppression of PA2G4 did not significantly increase the percentage of cells in early or late apoptosis (Figure 4.9 D).

Chapter 4- Characterisation of PA2G4 160

Figure 4.9 Knockdown of PA2G4 increases G2/M arrest and induces PARP cleavage but does not increase apoptosis (A) FACS analysis of cell cycle distribution in BE(2)-C cells knockdown with PA2G4- p48 siRNAs for 48 hours followed by treatment with PI. P-values determined by t-test comparing siRNA control against siP48#1 or siP48#2. Bars represent the mean and SEM from 3 independent experiments.

Chapter 4- Characterisation of PA2G4 161

Figure 4.9 (cont’d) Knockdown of PA2G4 increases G2/M arrest and induces PARP cleavage but does not increase apoptosis (B) FACS analysis of cell cycle distribution in Kelly cells knockdown with PA2G4-p48 siRNAs for 48 hours followed by treatment with PI. P-values determined by t-test comparing siRNA control against siP48#1 or siP48#2. Bars represent the mean and SEM from 3 independent experiments.

Chapter 4- Characterisation of PA2G4 162

Figure 4.9 (cont’d) Knockdown of PA2G4 induces PARP cleavage but does not increase apoptosis (C) Western blots showing that knockdown of PA2G4-p48 in BE(2)-C and SH-SY5Y cells induces PARP cleavage but not caspase 3 cleavage. (D) FACs analysis of apoptosis in BE(2)-C and Kelly cells with PA2G4-p48 siRNA knockdown for 48 hours followed by treatment with AnnexinV and 7AAD. P-values determined by t-test comparing the total apoptosis (early + late) for siRNA control against siP48#1 or siP48#2 siRNAs, n.s. = non-significant for P>0.05, bars represent the mean and SEM from 3 independent experiments.

Chapter 4- Characterisation of PA2G4 163 4.2.10 PA2G4 increases cell migration

PA2G4 has been reported to inhibit cell migration in adenoid cystic carcinoma (ACC) cells (Yu et al., 2007), and the expression of PA2G4 was significantly higher in non- cancerous adjacent tissues from ACC patient tumours compared with corresponding cancer tissues (Sun et al., 2012). Yu et al. also reported that overexpression of PA2G4 by gene transfer into human salivary adenoid cystic carcinoma cells inhibited cell proliferation and reduced tumour metastasis in their mouse models (Yu et al., 2007). To assess the role of PA2G4 in neuroblastoma cell migration, BE(2)-C, SK-N-AS and SH- SY5Y cells were transfected with either PA2G4 plasmid DNA or empty vector DNA to overexpress PA2G4, or PA2G4-p48 siRNAs to suppress PA2G4-p48. After 48 hours of transfection, cells were seeded in serum-free media into transwell inserts and incubated for 24 hours to allow for migration towards the media in the bottom chambers, which contained FCS as the chemo-attractant. After staining with May-Grunwald and Giemsa stains, images were taken under 10x magnification. For each field, an image was taken focused on the non-migrated cells on the top surface of the transwell insert, then a second image taken focused on the migrated cells on the underside of the insert (Figure 4.10 A and Figure 4.11 A). Overexpression of PA2G4 increased cell migration in each of the three cell lines (Figure 4.10 B), whereas suppression of PA2G4 with two different PA2G4-p48 siRNAs decreased cell migration in all three cell lines (Figure 4.11 B). Western blot analysis confirming knockdown of PA2G4 with siRNA is shown in Figure 4.11 C.

Chapter 4- Characterisation of PA2G4 164

Figure 4.10 Overexpression of PA2G4 increases cell migration (A) Representative transwell migration assay images for SK-N-AS cells transfected with either empty vector control or PA2G4 plasmid DNA, showing non-migrated cells (top) and migrated (bottom) cells. (B) Migration index from transwell migration assays in three neuroblastoma cell lines with overexpression of PA2G4. (C) Western blot showing overexpression of PA2G4 by transfection with PA2G4-flag tagged plasmid DNA. PA2G4 antibody detects the PA2G4-Flag protein (upper band) and endogenous PA2G4 protein (lower band). P-values determined by t-test, bars represent the mean and SEM from 3 independent experiments.

Chapter 4- Characterisation of PA2G4 165

Figure 4.11 Knockdown of PA2G4 decreases cell migration (A) Representative transwell migration assay images for SK-N-AS cells transfected with either control siRNA or PA2G4-p48 siRNA, showing non-migrated cells (top) and migrated (bottom) cells. (B) Migration index from transwell migration assays in three neuroblastoma cell lines with knockdown of PA2G4. (C) Western blot showing knockdown of PA2G4 with PA2G4-p48 specific siRNAs. P-values determined by t-test, bars represent the mean and SEM from 3 independent experiments. Chapter 4- Characterisation of PA2G4 166 4.2.11 Up-regulation of MYCN increases TFAP4 expression

The mechanism and signalling pathways through which PA2G4 exerts effects on cell migration are unknown. Transcription factor AP4 (TFAP4) is known to be a target of c- MYC (Jung et al., 2008), and involved in cellular migration (Jackstadt et al., 2013). More recently, studies within our group have indicated that TFAP4 may also be a target of MYCN. To show TFAP4 is regulated by MYCN in neuroblastoma cells, SH-EP MYCN3 inducible cells were treated with 1ng/µl of doxycycline, to activate the MYCN transgene over a period of 24 to 96 hours. Western blot analysis showed an increase in MYCN protein expression after 24 hours treatment with doxycycline. PA2G4 and TFAP4 protein levels also increased when MYCN was induced (Figure 4.12 A). mRNA expression was analysed by real-time PCR, and increases in MYCN, PA2G4 and TFAP4 expression were observed when MYCN was induced with doxycycline (Figure 4.12 B).

Chapter 4- Characterisation of PA2G4 167

Figure 4.12 Up-regulation of MYCN increases PA2G4 and TFAP4 expression (A) Western blots showing induction of MYCN expression in SH-EP MYCN3 inducible cells with 1 µg/ml doxycycline, also showing expression of PA2G4 and TFAP4 protein. (B) Real-time PCR on SH-EP MYCN3 inducible cells with 1 µg/ml doxycycline. P-values determined by t-test, bars represent the mean and SEM from 3 independent experiments.

Chapter 4- Characterisation of PA2G4 168 Previous preliminary experiments analysing MYCN downstream target genes showed TFAP4 expression correlating with PA2G4 expression. Therefore, to assess whether PA2G4 has an effect on TFAP4 expression, three neuroblastoma cell lines were transfected with PA2G4-p48 siRNAs for 48 hours. BE(2)-C cells were chosen because they express MYCN, whereas SH-SY5Y and SK-N-AS cells were used because they express c-MYC. BE(2)-C cells with PA2G4-p48 siRNA knockdown displayed a marked decrease in MYCN and TFAP4 protein expression (Figure 4.13 A). Suppression of PA2G4 also significantly decreased TFAP4 mRNA expression (Figure 4.13 B; P<0.01). In the two c-MYC overexpressing cell lines, suppression of PA2G4 decreased c-MYC and TFAP4 protein and mRNA expression in SH-SY5Y (Figure 4.13 C and D), and SK- N-AS (Figure 4.13 E & F).

Chapter 4- Characterisation of PA2G4 169

Figure 4.13 Knockdown of PA2G4-p48 decreases MYCN, c-MYC and TFAP4 expression (A) Protein analysis and (B) real-time PCR mRNA analysis of BE(2)-C cells with PA2G4-p48 siRNA knockdown for 48 hours. (C) Protein analysis and (D) real-time PCR mRNA analysis of SH-SY5Y. (E) Protein analysis and (F) real-time PCR mRNA analysis of SK-N-AS cells with PA2G4-p48 siRNA knockdown for 48 hours. P-values determined by t-test, bars represent the mean and SEM from 3 independent experiments.

Chapter 4- Characterisation of PA2G4 170 4.2.12 TFAP4 expression is able to rescue the decrease in cell migration caused by PA2G4 knockdown

As mentioned previously, TFAP4 is known to be involved in cell migration (Jackstadt et al., 2013). To investigate whether TFAP4 plays a role in neuroblastoma cell migration, TFAP4 plasmid DNA was overexpressed in two cell lines with relatively low levels of TFAP4 expression, SH-SY5Y and SK-N-FI. Conversely, TFAP4 siRNA knockdown was carried out in BE(2)-C cells because they have higher levels of TFAP4. After 48 hours of transfection with either the plasmid DNAs or siRNAs, cells were plated onto transwell inserts with 8 µm diameter pores and incubated for 24 hours, allowing cells to migrate to the FCS chemo-attractant in the bottom chamber. After 24 hours, cells were fixed, stained and imaged for counting. Overexpression of TFAP4 significantly increased cell migration in SH-SY5Y cells (P=0.001), and SK-N-FI cells (P=0.0005), whereas suppression of TFAP4 decreased cell migration in BE(2)-C cells (P=0.001 and 0.005) (Figure 4.14 A). Western blotting confirmed transfection of TFAP4 plasmid DNA and siRNAs (Figure 4.14 B).

To assess whether TFAP4 can compensate for the decrease in cell migration caused by PA2G4 knockdown, BE(2)-C and SH-SY5Y cells were treated with PA2G4-p48 siRNA for 24 hours, then transfected with either TFAP4 plasmid DNA or empty vector DNA for a further 24 hours, before plating onto transwell inserts. Overexpression of TFAP4 restored the cells’ ability to migrate in PA2G4 knocked down BE(2)-C (P=0.021) and SH-SY5Y (P=0.002) cells (Figure 4.14 C). Western blotting confirmed transfection of TFAP4 plasmid DNA and PA2G4-p48 siRNAs (Figure 4.14 D).

Chapter 4- Characterisation of PA2G4 171

Figure 4.14 TFAP4 can partially reverse the effect on migration of knocking down PA2G4 (A) Migration index from transwell migration assays of SH-SY5Y and SK-N-FI transfected with TFAP4 plasmid DNA, and BE(2)-C knockdown with TFAP4 siRNA for 48 hours. (B) Western blots confirming transfection of TFAP4 plasmid DNA and TFAP4 siRNA. (C) Migration index from transwell migration assays of BE(2)-C and SH-SY5Y transfected with PA2G4-p48 siRNA and TFAP4 plasmid DNA for 48 hours. (D) Western blots confirming transfection of PA2G4-p48 siRNA and TFAP4. P-values determined by t-test, bars represent the mean and SEM from 3 independent experiments.

Chapter 4- Characterisation of PA2G4 172 4.2.13 TFAP4 is part of a PA2G4 signalling pathway

The p21 gene has been characterised as a target for direct repression by TFAP4 (Jung and Hermeking, 2009; Jung et al., 2008), and previous data presented in this thesis have shown that PA2G4 can affect p21 expression (Figure 4.4 B). To investigate whether PA2G4 can regulate TFAP4 and p21 expression, SH-SY5Y cells were transfected with PA2G4-p48 siRNA for 48 hours, followed by western blot analysis. Suppression of PA2G4 caused a decrease in TFAP4 protein expression, and an increase in p53 and p21 (Figure 4.15 A). These results reveal a possible signalling cascade regulated by PA2G4, which has an effect on cell growth and cell migration (Figure 4.15 B).

Chapter 4- Characterisation of PA2G4 173

Figure 4.15 PA2G4 positively regulates TFAP4 (A) Western blot analysis on SH-SY5Y with PA2G4-p48 siRNA knockdown showing the effect on TFAP4 protein expression. (B) Schematic diagram of PA2G4 signalling cascade. N.B. the samples used in this western blot are the same samples used for Figure 4.6.

Chapter 4- Characterisation of PA2G4 174 4.2.14 PA2G4 suppresses neurite formation

One characteristic of neuroblastoma tumours is their inability to differentiate in the absence of differentiation agents such as retinoid. These tumours are highly proliferative due to the over expression of genes such as MYCN, which is able to inhibit or block cellular differentiation (Westermark et al., 2011). During the process of neuronal differentiation, cells form neurites which are projections from the cell body of at least twice the length of the cell body (Yu et al., 2009b). To assess whether suppression of PA2G4 induces neurite formation, based on PA2G4 ability to regulate MYCN, BE(2)-C and SH-SY5Y cells were transfected with PA2G4 siRNAs and incubated for 6 days before images were taken to quantitate neurites (Figure 4.16 A). Significant increases in neurite numbers were observed in each cell line with two independent PA2G4 siRNAs (Figure 4.16 B; P<0.0001).

Chapter 4- Characterisation of PA2G4 175

Figure 4.16 Down-regulation of PA2G4 increases neurite formation (A) Images of neurite formation in BE(2)-C cells with PA2G4 siRNA knockdown, 6 days post transfection. Red arrows indicate examples of neurites. (B) Quantification of neurite formation in BE(2)-C and SH-SY5Y cells with PA2G4 siRNA knockdown for 6 days. P-values determined by t-test, bars represent the mean and SEM from 3 independent experiments.

Chapter 4- Characterisation of PA2G4 176 4.2.15 13-cis-RA reduces MYCN, c-MYC and PA2G4 expression

Retinoic acid (RA) induces differentiation in neuroblastoma cells. To investigate whether PA2G4 has a role in retinoic acid-induced differentiation, BE(2)-C and SH- SY5Y cells were treated with 10 µM 13-cis-RA from 16 to 96 hours, followed by protein and mRNA analysis of MYCN or c-MYC and PA2G4 expression. In BE(2)-C cells, treatment with 13-cis-RA for 24 hours or longer decreased MYCN and PA2G4 protein expression (Figure 4.17 A and B) and mRNA expression (Figure 4.17 C). Similarly, in SH-SY5Y cells, 13-cis-RA treatment decreased c-MYC and PA2G4 protein (Figure 4.18 A and B) and mRNA expression (Figure 4.18 C).

Chapter 4- Characterisation of PA2G4 177

Figure 4.17 13-cis-RA reduces MYCN and PA2G4 expression (A) Western blots of BE(2)-C cells treated with 10 µM 13-cis-RA over time. Quantification of MYCN and PA2G4 protein expression (B) and mRNA expression by real-time PCR (C) after treatment with 10 M 13-cis-RA. P-values determined by t-test, bars represent the mean and SEM from 3 independent experiments.

Chapter 4- Characterisation of PA2G4 178

Figure 4.18 13-cis-RA reduces c-MYC and PA2G4 expression (A) Western blots of SH-SY5Y cells treated with 10 M 13-cis-RA over time. Quantification of c-MYC and PA2G4 protein expression (B) and mRNA expression by real-time PCR (C) after treatment with 10 M 13-cis-RA. P-values determined by t-test, bars represent the mean and SEM from 3 independent experiments.

Chapter 4- Characterisation of PA2G4 179 4.2.16 Combination of PA2G4 siRNA knockdown and 13-cis- RA treatment increases differentiation

As suppression of PA2G4, and treatment with 13-cis-RA, both induce neurite formation, will combining these two factors further increase neurite outgrowth in neuroblastoma cells as an indirect measurement of loss of proliferation and differentiation? To address this question, BE(2)-C and SH-SY5Y cells were transfected with two different PA2G4 siRNAs for 24 hours, then treated with 2 M 13-cis-RA for 6 days, before images were taken of the cells for neurite counting. In BE(2)-C and SH- SY5Y, addition of 13-cis-RA to cells with PA2G4 suppression caused asignificant increase in neurite formation, compared to PA2G4 suppression alone (Figure 4.19, P<0.0001 for all samples).

Figure 4.19 Treatment with 13-cis-RA further increases neurite formation caused by knockdown with PA2G4 Neurite formation in BE(2)-C and SH-SY5Y cells with PA2G4 siRNA knockdown, or treatment with 2 M 13-cis-RA for 6 days. P-values determined by t-test, bars represent the mean and SEM from 3 independent experiments.

Chapter 4- Characterisation of PA2G4 180 4.2.17 Establishment of PA2G4 shRNA stable cell lines

To analyse the long term effects of suppression or overexpression of PA2G4 on cell growth in culture and in vivo, stable clones were generated. To establish stable neuroblastoma cells with stable PA2G4 knockdown, pGFP-PA2G4-shLenti plasmid (Figure 4.20 A) was transfected into BE(2)-C cells. The plasmid DNA expresses a GFP gene, which allows visualisation and detection of transfection under a fluorescent microscope (Figure 4.20 B). Four different shRNA sequences of PA2G4: shPA2G4 A, B, C and D, were cloned into the pGFP-shLenti vector to generate four different PA2G4 DNA plasmids. These four DNA plasmids were then transfected into BE(2)-C cells for 24 to 96 hours, and their ability to suppress PA2G4 expression was analysed by western blot (Figure 4.21 A) and real-time PCR (Figure 4.21 B). shPA2G4 B, C and D all decreased PA2G4 protein and mRNA expression. shPA2G4 A did not reduce PA2G4 protein expression, and only slightly reduced its mRNA.

Chapter 4- Characterisation of PA2G4 181

Figure 4.20 Establishment of PA2G4 shRNA stable cell lines (A) Schematic diagram of the PA2G4 shLenti plasmid vector containing a GFP tag. (B) Bright field and fluorescent images of BE(2)-C cells transfected with PA2G4- shLenti-GFP plasmid DNA (shPA2G4-C) for 24 hours.

Chapter 4- Characterisation of PA2G4 182

Figure 4.21 Transfection of PA2G4-shRNA constructs into BE(2)-C cells (A) Western blotting of BE(2)-C cells transfected with four different PA2G4-shLenti- GFP constructs over a period of 96 hours.

Chapter 4- Characterisation of PA2G4 183

Figure 4.21 (cont’d) Transfection of PA2G4-shRNA constructs into BE(2)-C cells (B) Confirmation of shPA2G4 knockdown with real-time PCR. P-values determined by t-test, bars represent the mean and SEM from 3 independent experiments.

Chapter 4- Characterisation of PA2G4 184 4.2.18 PA2G4 knockdown with shRNA reduces cell viability and colony formation

To test whether the shPA2G4 DNA plasmids are able to confer the same phenotypic changes as PA2G4 siRNAs, BE(2)-C cells were transfected with shPA2G4 A, B, C and D for 48 to 96 hours. The effect of shPA2G4 on cell growth was determined by the measurement of cell viability using both short and long term assays. shPA2G4 B, C, and D were able to decrease cell viability in short term assays, while shPA2G4 A had no effect (Figure 4.22 A). This result correlated with PA2G4 protein expression (Figure 4.21A). Another phenotypic change caused by suppression of PA2G4 with siRNAs is a decrease in colony formation. Transfection of BE(2)-C cells with shPA2G4 B, C and D resulted in significant decreases in colony formation, which were comparable to those observed with PA2G4 siRNAs (Figure 4.22 B). shPA2G4 A DNA plasmid was not used in the colony formation assay because it did not significantly decrease PA2G4 protein expression and caused no change in cell growth (Figures 4.21 A and 4.22 A).

Chapter 4- Characterisation of PA2G4 185

Figure 4.22 PA2G4-shRNA reduces cell viability and colony formation (A) Cell viability measurement of BE(2)-C cells transfected with PA2G4-shLenti-GFP constructs for 48 to 96 hours. (B) Colony formation in BE(2)-C cells was analysed 10 days post transfection with either PA2G4-shLenti-GFP constructs or PA2G4 siRNA. P- values determined by t-test, bars represent the mean and SEM from 3 independent experiments.

Chapter 4- Characterisation of PA2G4 186 4.2.19 PA2G4-shRNA decreases TFAP4 mRNA expression

To assess the effect of PA2G4 knockdown with shRNA on TFAP4, a possible target of PA2G4, shPA2G4 B, C, and D were transfected into BE(2)-C cells for 48 and 72 hours. Real-time PCR was used to analyse mRNA expression of PA2G4 (Figure 4.23 A) and TFAP4 (Figure 4.23 B). As previously shown (Figure 4.21), all three shPA2G4 DNA plasmids decreased PA2G4 expression. In addition, they all decreased expression of TFAP4, as was observed with siRNAs targeting PA2G4 (Figure 4.13), further confirming the efficiency and specificity of the shRNA transfection.

Figure 4.23 PA2G4-shRNA decreases PA2G4 and TFAP4 mRNA expression BE(2)-C cells transfected with PA2G4-shLenti-GFP constructs for 48 and 72 hours. Analysis of PA2G4 (A) and TFAP4 (B) mRNA expression using real-time PCR. P- values determined by t-test, bars represent the mean and SEM from 3 independent experiments.

Chapter 4- Characterisation of PA2G4 187 4.2.20 Stable knockdown of PA2G4 causes senescence

Having confirmed the efficacy of 3 of the 4 shRNA plasmids, shPA2G4 C was chosen to generate cells with stable PA2G4 suppression. BE(2)-C cells were transfected with shPA2G4 C plasmid DNA or sh-control DNA and grown in puromycin selection media. From the few shPA2G4 colonies that grew in the selection media, two clones were isolated. However, when these cell lines were subsequently propagated, the rate of cell division began to slow down after 2 weeks, and by 4 weeks these cells had increased in size, developed a flattened morphology and had stopped dividing, consistent with senescence. Unfortunately the cells became non-viable before they could be stained with beta-galactosidase to confirm their senescence state. The delay in growth and increases in cell size were not observed in the sh-control clones (Figure 4.24 A). Because the shPA2G4 clones ceased to grow, there were insufficient cells to isolate protein; only RNA could be isolated to analyse PA2G4 mRNA expression. shPA2G4 clone 1 and shPA2G4 clone 2 showed significant decreases in PA2G4 expression (Figure 4.24 B; P=0.0037 and P=0.0005 respectively).

Chapter 4- Characterisation of PA2G4 188

Figure 4.24 Stable expression of PA2G4-shRNA in BE(2)-C cells causes senescence (A) Images of BE(2)-C stably expressing PA2G4-shRNA and control empty vector cells. (B) Real-time PCR mRNA analysis of stable clones. P-values determined by t- test, bars represent the mean and SEM from 3 independent experiments.

Chapter 4- Characterisation of PA2G4 189 4.2.21 Generation of PA2G4 overexpressing cell lines Having observed that stable knockdown of PA2G4 rendered cells non-viable, the effect on cells of stable overexpression of PA2G4 was then investigated. To generate stable PA2G4 overexpressing cells, pCMV6-PA2G4-myc/flag plasmid DNA was transfected into SH-EP cells, these cells having been chosen due to their very low levels of PA2G4 expression (Figure 3.9 A and B). Transfected cells were grown in Geneticin (G418) selection media for up to 2 months, to select individual clones and generate clone pools (individual clones combined together to eliminate clonal effects). Firstly, the clone pools were analysed by comparing cell proliferation rate between the control pools and the PA2G4-overexpressing pools. Cells from each pool were seeded into either 6-well plates for cell counting, or into 96-well plates for cell viability measurement. There were no significant differences in cell counts (Figure 4.25 A) or cell viability (Figure 4.25 B) between the control and the PA2G4-overexpressing pools. Western blotting confirmed the overexpression of PA2G4 in the clone pool (Figure 4.25 C). Flag antibody was used to detect the pCMV6-PA2G4-myc/flag plasmid DNA, and GAPDH was used as a loading control.

Chapter 4- Characterisation of PA2G4 190

Figure 4.25 SH-EP cells stably overexpressing PA2G4, pool of clones (A) Growth rate of SH-EP empty vector and PA2G4 overexpressing stable cells, pool of clones, with a starting seeding density of 1x105 cells. (B) Cell viability of SH-EP empty vector and PA2G4 overexpressing stable cells grown over 72 hours as measured by resazurin uptake. (C) Western blot analysis of stable clone pool showing overexpression of PA2G4-Flag tagged protein in the PA2G4 clones and not in the empty vector control clones.

Chapter 4- Characterisation of PA2G4 191 Individual empty vector control and PA2G4 overexpressing clones were also picked and analysed by western blots for PA2G4-myc/flag protein expression. All 7 PA2G4 overexpressing clones probed positive (Figure 4.26 A), however substantial variability was observed in the levels of PA2G4 expression. Clones EV 1–3 and PA2G4 1–3 were chosen for cell growth analysis, however no significant differences in proliferation rate were observed between clones (Figure 4.26 B). Cell proliferation rate was also measured for PA2G4 overexpressed clone 5 because it had the highest levels of PA2G4 protein expression. Again, no changes in cell numbers were observed between PA2G4- clone 5 and the empty vector control clone 1 (Figure 4.26 C).

Chapter 4- Characterisation of PA2G4 192

Figure 4.26 SH-EP clones stably overexpressing PA2G4 (A) Western blot analysis showing overexpression of PA2G4-flag tagged protein in PA2G4 overexpressing stable clones but not in SH-EP empty vector clones. (B) Cell proliferation rate of three empty vectors and three PA2G4 overexpressing stable clones, with a starting seeding density of 1x105 cells grown over 72 hours. (C) Cell proliferation rate of SH-EP empty vector clone 1 and PA2G4 overexpressing clone 5 grown over 72 hours.

Chapter 4- Characterisation of PA2G4 193 4.2.22 Expression of PA2G4 is lost over time

Before these SH-EP PA2G4-overexpressing stable cells were used for in vivo experiments, the integrity of the PA2G4 plasmid DNA needed to be assessed. It is not uncommon for cells to stop expressing plasmid DNA due to selection pressure. To determine whether PA2G4 expression is stable over time, PA2G4-clone 5 and empty vector clone 1 were passaged 6 times and the PA2G4-myc/flag protein levels were analysed by western blotting. A notable decrease in PA2G4-flag expression at passage 4 (p4) was observed, and the PA2G4-flag band was no longer visible in passage 5 and 6 cells (Figure 4.27 A). Empty vector clone 1 remained negative for the PA2G4-flag band as expected. The durability of the pCMV6-PA2G4-myc/flag plasmid DNA expression was also analysed in the pool of cells. Empty vector pool and PA2G4-overexpressing pools were passaged 6 times, and protein lysates were prepared from successive cell passages. PA2G4-flag protein expression decreased at passage 5 and 6 in the PA2G4- overexpressing pools (Figure 4.27 B). The empty vector pool remained negative for the PA2G4-flag band as expected.

To test the properties of these stable cells and to access cell growth, clonogenicity assays were carried out with empty vector clone 1 and PA2G4-clone 5, and with empty vector clone pool and PA2G4 clone pool. 500 cells were seeded in 6-well plates and incubated for 14 days before counting the numbers of colonies. There was a small but significant increase in colony formation for both PA2G4-clone 5 and PA2G4 clone pool (Figure 4.27 C; P=0.0209 and P=0.0255 respectively).

Chapter 4- Characterisation of PA2G4 194

Figure 4.27 Expression of PA2G4 is lost over time in SH-EP PA2G4 stable cells (A) Western blots of individual clones and (B) clone pools showing overexpression of PA2G4-flag tagged protein over different cell passages. (C) Colony formation (long term cell growth) of SH-EP empty vector and PA2G4 overexpressing stable clones and cell pools. P-values determined by t-test, bars represent the mean and SEM from 3 independent experiments.

Chapter 4- Characterisation of PA2G4 195 4.2.23 3D spheroids of SH-EP cells overexpressing PA2G4

In the in vivo environment, cells do not grow in two-dimensional (2D) monolayers. Therefore, generating three-dimensional (3D) spheroids in vitro may better simulate the behaviour of cells in vivo (Vinci et al., 2012). Empty vector clone pools and PA2G4 clone pools were used in all the spheroid assays. 500 cells were seeded into low adhesion U-bottom 96-well plates and incubated for 4 days to allow cells to cluster together to form spheroids. A spheroid is comprised of three different layers of cells (Figure 4.28 A) and it is the outer proliferating layer of cells that can migrate. The diameter of control and PA2G4 overexpressing spheroids was measured and found not to be significantly different (Figure 4.28 B). To assess whether there are differences in cell migration, each spheroid was re-seeded onto flat-bottom 6-well plates and given 16 hours to attach to the plate and migrate. Spheroids with PA2G4 overexpression had more cells migrating on the plate, and over a longer distance, compared to the empty vector control spheroids (Figure 4.28 C; P=0.001).

Chapter 4- Characterisation of PA2G4 196

Figure 4.28 3D spheroids of SH-EP cells overexpressing PA2G4 show increased migration (A) Schematic diagram of the structure of a spheroid. (B) Comparing spheroid size between SH-EP empty vector and PA2G4 overexpressing clone pools after 4 days of growth in a low adhesion U-bottom plate.

Chapter 4- Characterisation of PA2G4 197

Figure 4.28 (cont’d) 3D spheroids of SH-EP cells overexpressing PA2G4 show increased migration (C) Measuring the distance of migration between the SH-EP empty vector and PA2G4 overexpressing clone pools 16 hours post relocation onto a flat cell culture dish. P- values determined by t-test, bars represent the mean and SEM from 3 independent experiments.

Chapter 4- Characterisation of PA2G4 198 4.2.24 3D spheroids of SH-EP cells stably overexpressing PA2G4 show increased invasion

Having established from the 3D spheroid assays that SH-EP cells stably overexpressing PA2G4 have increased cell migration, their ability to invade through various concentrations of basement membrane matrix was assessed. The amount of cellular invasion is influenced by the concentration of the matrix that the cells have to move through. The lower the concentration of the collagen, the more easily cells can move through, and in these instances cells are not invading, but merely moving between gaps. With true invasion, cells digest collagen to move through the matrix. Figure 4.29 A & B show cell migration or invasion from spheroids embedded in 3 different concentrations of collagen – 0.25mg/ml, 0.5mg/ml, or 0.75mg/ml. At the two lowest concentrations, there was no difference in the average distance that cells moved from either spheroid. It seems likely that cells were able to migrate between gaps in the collagen, without having to digest it. At the higher concentration of 0.75mg/ml however, the distance moved by empty vector cells was greatly decreased compared to the distance moved at 0.25mg/ml or 0.5mg/ml, suggesting that cells were no longer able to migrate between gaps in the collagen. Under these conditions, the distance moved by cells in the PA2G4- overexpressing spheroids was significantly greater than the distance moved by empty vector cells. This indicates that cells in PA2G4-overexpressing spheroids had an increased ability to digest collagen, and thus invade. (Figure 4.29 C; P=0.01 and P=0.02).

Chapter 4- Characterisation of PA2G4 199

Figure 4.29 3D spheroids of SH-EP cells stably overexpressing PA2G4 show increased invasion (A) Different concentrations of collagen type I were used to optimise the invasion assay. (B) Invasion assay with SH-EP empty vector and PA2G4 overexpressing clone pools. Cells were initially grown in a low adhesion U-bottom plate for 4 days then transferred and embedded in the optimal concentration, 0.75 mg/ml of collagen type I. Images of cellular invasion were taken at time of embedding then at 24 hours and 48 hours. (C) Measurement of the average distance of cell movement into the collagen matrix. P- values determined by t-test, bars represent the mean and SEM from 3 independent experiments.

Chapter 4- Characterisation of PA2G4 200 4.2.25 PA2G4 induces tumorigenicity

PA2G4 has been described as a tumour promoter, and data presented in the thesis thus far support this claim. A conclusive way to test this hypothesis would be to determine whether overexpression of PA2G4 in a non-tumorigenic cell line induced tumour growth in an in vivo mouse model. PA2G4 plasmid DNA was overexpressed in SH-EP cells, and stable clones or clone pools were isolated and characterised in Result Chapter 3. They are ideal cells to xenograft into nude mice to test whether overexpression of PA2G4 is tumorigenic. 5x106 SH-EP PA2G4 overexpressing cells or empty vector control cells were mixed with matrigel and injected subcutaneously into the flank of nude mice. Eight mice were used in each cohort and were monitored weekly for signs of tumour development and growth. At 10 weeks post engraftment, all of the eight mice in the PA2G4 overexpressing cohort had distinct tumour formation and growth compared to the control cohort, which showed very few visible signs of tumour formation (Figure 4.30 A). All mice were culled 12 weeks post engraftment, and any tumours were excised for analysis (Figure 4.30 B). Tissues were also removed from the site of injection from the control mice that appeared to be residual matrigel, dead cells or lymph nodes. Tumour size was measured weekly from week 6 to week 12. Significant differences (P<0.0001) in tumour growth were observed between PA2G4 overexpressing cells, which were able to form tumours, compared to empty vector control cells, which were not (Figure 4.30 C).

Chapter 4- Characterisation of PA2G4 201

Figure 4.30 Overexpression of PA2G4 increases neuroblastoma tumorigenicity Stable SH-EP PA2G4 overexpressing cells and SH-EP control cells were xenografted into nude mice to analyse the ability of PA2G4 to induce tumour formation. (A) Representative images of mice from each cohort 10 weeks post engraftment. (B) Images of tumour formation after mice were culled 12 weeks post engraftment. (C) Tumour measurements taken from each mouse every week starting from 6 weeks post engraftment to time of cull, 12 weeks post engraftment. P-value obtained from 2- way ANOVA multiple comparison test.

Chapter 4- Characterisation of PA2G4 202 4.2.26 In vitro analysis of SH-EP PA2G4 overexpressing tumours

Having established that overexpression of PA2G4 could induce tumorigenesis in a non- tumorigenic cell line, it was critical to assess whether the tumours that formed still retained high levels of PA2G4, and to validate that this increase in PA2G4 caused tumour formation and growth. Tumours which developed from the SH-EP PA2G4 overexpressing cells and any from the empty vector cells were excised from the mice. Overall there was no visible tumour growth in the empty vector control cohort, however when the mice were examined post-mortem there were a few small nodules, which may have been lymph nodes rather than tumour. These small nodules were isolated for in vitro analysis. RNA and protein from the tumours and nodules were isolated for analysis of PA2G4 and MYCN expression. Higher levels of PA2G4 and MYCN were observed in the SH-EP PA2G4 overexpressing tumours compared to SH-EP empty vector control (Figure 4.31 A). Vinculin was used as a loading control, and each sample was normalised to SH-EP EV control mouse #1. Protein densitometry analysis quantitated the increase in PA2G4 expression in SH-EP PA2G4 overexpressing tumours compared to the SH-EP empty vector control (Figure 4.31 B; P=0.0053), however there were no significant changes in MYCN expression. Real-time PCR analysis supported the protein expression data, showing an increase in PA2G4 mRNA expression in SH-EP PA2G4 overexpressing tumours compared to the SH-EP empty vector control (P=0.0062), and no significant changes in MYCN expression (Figure 4.31 C).

Chapter 4- Characterisation of PA2G4 203

Figure 4.31 Higher PA2G4 protein and mRNA expression in mouse tumours (A) Protein extracted from SH-EP PA2G4 overexpressing tumour and SH-EP control tumour samples was used to analyse PA2G4 and MYCN expression. (B) Protein densitometry analysis from SH-EP PA2G4 overexpressing tumour and SH-EP control tumour samples. (C) Real-time PCR analysis of PA2G4 and MYCN mRNA expression in mouse tumours. Data show means and SD derived from four mice, P-values calculated by t-test.

Chapter 4- Characterisation of PA2G4 204 4.3 Discussion

Initially, experiments suppressing PA2G4 were performed with siRNA targeting both p42 and p48 isoforms. Although statistically significant, the decrease in cell viability was minimal. Previous reports indicate that the two isoforms of PA2G4 have opposing functions (Kim et al., 2012; Monie et al., 2007), raising the possibility that simultaneous suppression of both isoforms may cancel out phenotypic changes caused by suppression of individual isoforms. Cell viability and colony forming assays support this possibility, as siRNA targeting only the p48 isoform resulted in a substantially stronger growth suppression phenotype that siRNA targeting both the p42 and p48 isoforms, despite comparable knockdown of the p48 isoform. In addition, siRNA targeting only p48 decreased MYCN protein expression to a greater degree than siRNA targeting both p48 and p42 isoforms.

Although PA2G4 and MYCN have a close positive interaction, overexpression of MYCN could not rescue the decrease in cell growth caused by suppression of PA2G4. There are several possible explanations for this. One explanation is that the period of time for which MYCN was overexpressed, 72 hours, was too short. Increasing it further may give cells more time to increase their proliferation. A second explanation might be that suppression of PA2G4 has the greater effect on cells, and inhibits any effect that overexpression of MYCN may have. As PA2G4 levels influence MYCN protein stability (Chapter 3), suppression of PA2G4 would be expected to reduce MYCN protein half-life. The protein half-life of MYCN is already short, generally 30 minutes (Ikegaki et al., 1986), but with PA2G4 suppression, it was reduced to less than 15 minutes, which may not be enough time to adequately regulate cell growth. A third explanation might be that PA2G4 regulation of cell growth occurs through a MYCN independent process.

Kim et al. have described how the long isoform of PA2G4, p48, interacts with AKT in order to stabilise MDM2 from self-ubiquitination (Kim et al., 2012). The maintained phosphorylation state of MDM2 mediates the poly-ubiquitination of p53 resulting in cellular differentiation or cell death (Tao and Levine, 1999). The data from this thesis support the hypothesis that PA2G4 is involved in the phosphorylation of AKT and MDM2, leading to decreases in p53 and its downstream target, p21. MDM2 is also a direct target of MYCN (Slack et al., 2005), and has been shown to be necessary for

Chapter 4- Characterisation of PA2G4 205 MYCN to overcome p53-mediated apoptosis and MYCN-driven neuroblastoma tumorigenesis (Chen et al., 2010). Perhaps MYCN activates MDM2 transcription and PA2G4 is required to phosphorylate MDM2, allowing it to stay localised in the nucleus (Kim et al., 2012), where it can poly-ubiquitinate p53. Therefore overexpression of MYCN can increase MDM2 expression, but suppression of PA2G4 may renders MDM2 incapable of poly-ubiquitinating p53, thus accumulation of p53 leads to apoptosis.

Protein degradation determines the outcome of many cellular physiological processes. The ubiquitin-proteasome system is the main machinery responsible for eliminating unfolded proteins and regulatory proteins that are selected for destruction. Degradation of proteins by proteasomes occurs via various pathways, the most studied one being the ubiquitin-26S proteasome pathway (Hershko and Ciechanover, 1998; Smalle and Vierstra, 2004). Substrates are normally targeted to the 26S proteasome via polyubiquitination; these chains mediate the binding of targeted proteins to the proteasome and assist in their unfolding, but are removed from the substrate prior to proteasomal degradation. Treatment of neuroblastoma cells with MG-132, a potent and reversible proteasome inhibitor that reduces the degradation of ubiquitin-conjugated proteins, resulted in the accumulation of MYCN protein consistent with previous reports that MYCN is degraded by the proteasome (Brockmann et al., 2013; Muller and Eilers, 2008).

In contrast, there was no accumulation of PA2G4 protein following MG-132 treatment, indicating that PA2G4 is not degraded by the proteasome. Similar results have been described for endoplasmic reticulum proteins (Shenkman et al., 2007), whereby the inhibition of protein folding in the endoplasmic reticulum (ER) causes ER stress, which triggers the unfolded protein response (UPR). UPR can activate an alternative non- proteasomal pathway of degradation, which is resistant to proteasome inhibitors such as MG-132 (Shenkman et al., 2007). The authors tested 18 protease inhibitors and found that only the metal chelator o-phenanthroline could block this non-proteasomal degradation. In the case of PA2G4, it may be that prolonged incubation (greater than 3 hours) with MG-132 is required, or further incubation with other proteasomal inhibitors such as N-acetyl-leucyl-leucyl-norleucinal (ALLN) is needed to exclude the possibility that PA2G4 degradation is non-proteasomal.

Chapter 4- Characterisation of PA2G4 206 PA2G4 suppression decreased cell growth and colony formation, however these changes do not appear to be due to apoptosis. While cleavage of PARP—a widely used marker of caspase-3 activation and apoptosis—was observed, it was not accompanied by caspase-3 activation. PARP is a zinc-finger DNA-binding protein which catalyses the synthesis of poly (ADP-ribose) polymerase from its substrate beta-NAD+, and is implicated in the maintenance of genomic stability and the DNA damage-triggered signalling cascade. PARP can be cleaved by caspase-3 (Nicholson et al., 1995; Salvesen and Dixit, 1997), or caspase-7 (Germain et al., 1999) during apoptosis and become incapable of responding to DNA damage. However, discrepant evidence has also been reported showing PARP degradation can be detected in the absence of procaspase-3 or - 7 cleavage, and thus could be independent of activation of these caspases. For instance, there is also evidence that PARP may participate as an initial antiapoptotic factor via an increased cellular ability to repair DNA single-strand breaks and thus to avoid the activation of apoptotic death mechanisms (Masdehors et al., 2000). Yang et al. reported that caspase-3 is not a major caspase involved in TGF-beta1-induced apoptosis in AML-12 cells, and is not required for apoptosis-associated DNA fragmentation, suggesting that PARP cleavage may occur as an independent event that has no effect on apoptosis (Yang et al., 2004). The absence of an effect on apoptosis indicates that other forms of cell growth arrest may account for the reduction in cell numbers. One possibility is differentiation. PARP has been described to be involved in the early stages of differentiation-linked DNA replication (Smulson et al., 1995). In osteosarcoma cells, a transient increase in PARP expression occurs prior to DNA cleavage before commitment to apoptosis, as well as at the end of DNA replication prior to the onset of terminal differentiation (Simbulan-Rosenthal et al., 1999). Therefore the suppression of PA2G4 may induce PARP cleavage prior to cellular differentiation.

The observation that neuroblastoma has a very high rate of spontaneous regression has led to multiple studies of neuroblastoma differentiation in vitro. One key agent, retinoic acid, has been shown to inhibit cell proliferation and decrease anchorage-independent growth followed by neurite outgrowth in neuroblastoma cells (Reynolds et al., 1991; Sidell et al., 1983; Wada et al., 1992). Retinoic acid (RA), a biologically active retinoid and a derivative of vitamin A, is a potent differentiation inducer for neuroblastoma. 13- cis RA is currently used as a maintenance regimen for the treatment of minimal residual disease (MRD) in neuroblastoma patients, and has resulted in improved event-free survival in clinical trials (Park et al., 2011). However, many neuroblastoma patients are

Chapter 4- Characterisation of PA2G4 207 resistant to 13-cis RA or develop resistance during therapy (Matthay et al., 2009; Matthay et al., 1999b), and there are multiple neuroblastoma cell lines resistant to all- trans RA and 13-cis RA (Reynolds et al., 2000).

MYCN has a central role in neuronal differentiation and neurite formation, and down- regulation of MYCN precedes retinoic acid induced differentiation (Amatruda et al., 1985; Kang et al., 2006; Thiele et al., 1985), suggesting that retinoic acid can directly regulate MYCN expression at the transcriptional level (Wada et al., 1992). Suppression of PA2G4 in neuroblastoma cells promoted neurite formation, an early morphological marker of neuronal cell differentiation. In addition, treatment of neuroblastoma cells with 13-cis RA dramatically reduced PA2G4 and MYCN expression coincident with neurite formation, suggesting that PA2G4 may play a role in 13-cis RA signalling. Consistent with this hypothesis, combined treatment with 13-cis RA and suppression of PA2G4 resulted in an additional increase in neurite formation, raising the possibility of combined retinoid treatment and PA2G4 inhibition as an approach to neuroblastoma treatment.

PA2G4 is required for colony formation and cell growth, as evidenced by the inability of stable PA2G4 knockdown cells to proliferate, and while the effect of PA2G4 on cell growth is not dependent on MYCN, clonogenicity is MYCN-dependent. This was clearly demonstrated in the colony forming assay, where exogenous MYCN expression rescues the loss of colony forming ability caused by PA2G4 suppression. The ability of MYCN to rescue colony formation, but not short-term cell proliferation, presents the possibility of a MYCN-dependant and MYCN-independent function for PA2G4 in neuroblastoma cells. However, elucidating these mechanisms is beyond the scope of this thesis.

In addition to its apparent role in neuroblastoma proliferation, PA2G4 promotes neuroblastoma cell migration and invasion, two important processes required for metastasis. Metastasis is the dissemination of cancer cells from the primary tumour to a distant organ. It is the most frequent cause of death for patients with cancer (Chambers et al., 2002), and as previously mentioned, is associated with high risk neuroblastoma. Cancer cells that migrate and invade adjacent tissues have acquired a migratory phenotype associated with increased expression of several genes involved in cell motility. The identification of these genes may provide targets for cancer therapy.

Chapter 4- Characterisation of PA2G4 208 PA2G4 is a possible cell motility gene, as its overexpression in three neuroblastoma cell lines increased the capacity of these cells to migrate through transwell chambers. Conversely, suppression of PA2G4 decreased cell migration.

Most studies on cell motility have been performed in two-dimensional (2D) flat dishes, thus limiting our understanding of mechanisms involved in cell migration, as cells use different migration strategies in physiological 3D culture systems (Fraley et al., 2012; Sahai and Marshall, 2003; Zaman et al., 2006). Cell migration in vivo often requires cells to remodel and move through a 3D collagen-rich matrix, which depends on the expression of metalloproteinases (MMPs) and the physical properties of the 3D matrix, such as pore size, all of which are not required in 2D migration (Wirtz et al., 2011). The restricted information obtained from 2D cell migration systems highlights the importance of conducting migration and invasion assay in 3D systems. For this reason, 3D spheroids of SH-EP cells stably overexpressing PA2G4 were produced and used to analyse the cells’ ability to migrate and invade through collagen matrices. Collagen pore size was found to be a major determinant of the cells’ ability to invade: larger pores enabled the cells to migrate or move through, but smaller pores forced cells to digest the collagen to be able to invade through the matrix. The ability of PA2G4 to promote cell migration and invasion further supports its status as a tumour promoter gene.

PA2G4 most likely requires other gene co-factors to regulate cell migration. One such gene is Transcription Factor Activating enhancer binding Protein 4 (TFAP4). TFAP4 is a direct transcriptional target of c-MYC (Jung et al., 2008). It is a ubiquitously expressed basic helix-loop-helix leucine-zipper (bHLH-LZ) transcription factor, which binds to the consensus E-box sequence 5’-CAGCTG-3’. This motif is located near the p21 promoter and mediates transcriptional repression of p21 (Hu et al., 1990; Jung et al., 2008). TFAP4 controls the expression of numerous genes regulating cell proliferation, such as CDK and p21 (Jung et al., 2008); stemness, such as LGR5 and CD44; and epithelial–mesenchymal transition (EMT), such as SNAIL and E-cadherin (Jackstadt et al., 2013). These data were generated from a genome-wide characterisation of TFAP4 DNA binding and mRNA expression performed using a combination of microarray, genome-wide chromatin immunoprecipitation, next-generation sequencing, and bioinformatics analyses (Jackstadt et al., 2013). Similar to the data from this thesis, Jackstadt et al. showed that activation of TFAP4 enhanced migration and induced EMT and invasion of colorectal cancer cells, and that down-regulation of TFAP4 inhibited

Chapter 4- Characterisation of PA2G4 209 migration, suggesting that TFAP4 may be an important mediator of c-MYC-induced EMT.

MYCN can also transcriptionally regulate TFAP4 (Cheng Xue, personal communication). The finding that PA2G4 is a transcriptional target of MYCN and is involved in cell migration raises the possibility that TFAP4 may also be involved in a PA2G4 signalling pathway. We propose that TFAP4 is downstream in the MYCN– PA2G4–TFAP4 signalling cascade, as TFAP4 overexpression rescues the decreased cell migration caused by PA2G4 suppression, and altering PA2G4 levels causes a corresponding change in TFAP4 expression. However, it has not been established that PA2G4 directly activates TFAP4 transcription in this thesis.

Stable suppression of PA2G4 resulted in cellular senescence, consistent with the decrease in cell growth and colony formation in the absence of apoptosis observed with transient PA2G4 knockdown. In order to generate and expand cells to study stable PA2G4 knockdown, an inducible expression system would be beneficial. An alternative approach adopted here was to conduct the converse experiment by generating stable overexpression of PA2G4 in a neuroblastoma cell line with low endogenous PA2G4 expression. Notably however, these cells lost PA2G4 expression after relatively few passages, implying a negative selection pressure arising from PA2G4 expression and necessitating the use of very early cell passages for all experiments.

Because stably overexpressing PA2G4 in SH-EP cells caused increased colony formation, cell migration and invasion, these cells were deemed an ideal tool to study the role of PA2G4 in tumour initiation/progression in vivo. Engraftment of SH-EP cells stably overexpressing PA2G4 into nude mice resulted in one of the most significant findings of this thesis. As mentioned previously, SH-EP cells are not tumorigenic, meaning they do not form tumours when xenografted into mice. The discovery that PA2G4 overexpression caused these cells to form tumours is highly significant, providing further evidence that PA2G4 is a tumour promoter and a good molecular target for neuroblastoma therapy.

In conclusion, PA2G4 displays many characteristics of a tumour promoter gene. The second aim of this thesis was to determine the functional role of PA2G4 in neuroblastoma. Its actions were found to be diverse: it affects cell growth, colony

Chapter 4- Characterisation of PA2G4 210 formation and cell migration. Although PA2G4 interacts with and is regulated by MYCN, its effect on cell growth appears to be independent of MCYN. The identification of TFAP4, MDM2, AKT, p21, and p53 as key genes and pathways involved in PA2G4 signalling, fulfilled the second part of aim two. Further studies into these signalling pathways and protein interactions involving PA2G4 will provide a better understanding of the role of PA2G4 in neuroblastoma disease progression and will determine whether PA2G4 is a possible therapeutic target in this disease.

Chapter 4- Characterisation of PA2G4 211 Chapter 5 TARGETING PA2G4 FOR THE TREATMENT OF NEUROBLASTOMA

CHAPTER 5

TARGETING PA2G4 FOR THE TREATMENT OF NEUROBLASTOMA

Chapter 5- Targeting PA2G4 212 5.1 Introduction

Molecular phenotypic and in vivo studies conducted in Chapter 4 indicate that PA2G4 plays an important role in neuroblastoma growth and progression, suggesting that PA2G4 is a potential therapeutic target in cancer. Identification of molecular targets that promote cancer progression, and agents that inhibit these targets, is critical to the treatment of cancers. This chapter will focus on the possibility of targeting PA2G4 for the treatment of neuroblastoma. It will address the third aim of this thesis, which is to determine the role of PA2G4 in neuroblastoma tumorigenesis using in vivo models, and to characterise potential PA2G4 inhibitors and combination chemotherapy for the treatment of neuroblastoma.

Chapter 5- Targeting PA2G4 213 5.2 Results

5.2.1 Investigating WS6 as a potential inhibitor of PA2G4

There are many ways of inhibiting molecular targets, one of which is to use small molecule chemical inhibitors. Small molecules are low molecular weight (<900 Da) organic compounds with the potential to regulate biological processes. Most drugs are small molecules because these are able to rapidly diffuse across cell membranes in order to reach intracellular sites of action, and the small molecular weight is necessary for oral bioavailability. Small molecule inhibitors can inhibit a specific function of a protein, disrupt protein–protein interactions (Arkin and Wells, 2004), and interfere with transcriptional activation (Koehler et al., 2003; Leung et al., 2013), making them ideal for targeting the inhibition of PA2G4 activity.

WS6 was identified by high-throughput screening as a novel small molecule capable of inducing proliferation of pancreatic β cells (Wang, Walker et al. 2009; Shen, Tremblay et al. 2013). Affinity pull-down and kinase profiling studies identified PA2G4 and the inhibitor of kappa B (IκB) kinase pathway respectively as targets of WS6 (Shen, Tremblay et al. 2013). The piperazine ring moiety of WS6 (Figure 5.1 A) makes it water-soluble, which is an ideal property for drug administration. To model whether WS6 could bind directly to the PA2G4 protein, rather than having an indirect secondary interaction, virtual docking of WS6 to the crystal structure of PA2G4 was performed at St. Vincent's Institute of Medical Research (Structural Biology Laboratory) using Fast Rigid Exhaustive Docking (FRED; Openeye Scientific Software Inc., Santa Fe, NM, USA), a program that rigidly docks molecules from a multiconformer database into a receptor site. Two possible binding sites for WS6 were identified (Figure 5.1 B). The first site is located in the large central cavity of the PA2G4 protein, while the second site is located on a surface pocket of PA2G4. To give a more precise dock, the compound was then docked separately into both sites using Surflex-dock Geom in Sybylx2.1 (Cetera, USA). Docking into the central cavity gave a single prominent cluster of solutions. This cluster had over 70% of the solutions fitting into it, with the highest ranked solution obtaining a score of 6.093 (N.B. higher scores correlate with “better” docking in terms of energetics, compound protein interactions and/or clashes). Unlike the central cavity, docking into the surface exposed group yielded only 30–40% of the solutions filling this region.

Chapter 5- Targeting PA2G4 214

Figure 5.1 Putative WS6 binding sites on PA2G4 (A) Chemical structure of WS6. (B) Crystal structure of PA2G4 protein showing two possible WS6 binding sites. The first is in the large central cavity (magenta carbons), the second (peach carbons) is on a surface pocket with no known function. PA2G4 protein is shown as grey ribbons.

Chapter 5- Targeting PA2G4 215 5.2.2 WS6 decreases PA2G4 and MYC expression

WS6 was reported to inhibit PA2G4, therefore to investigate whether WS6 can alter PA2G4 expression at the mRNA or protein level, BE(2)-C, Kelly and SH-SY5Y neuroblastoma cell lines were treated with different concentrations of WS6: 0.5 µM, 0.75 µM and 1 µM for 48 hours. RNA and protein were isolated from the cells and analysed for changes in PA2G4 and MYCN or c-MYC expression. Real-time PCR analysis showed that treatment with 1 µM WS6 decreased PA2G4 mRNA expression 2- fold in all three cell lines (P<0.005 for each). MYCN expression in BE(2)-C and Kelly cells and c-MYC expression in SH-SY5Y cells were also decreased by more than 2-fold (Figure 5.2 A; BE(2)-C P=0.006, Kelly P<0.0001, and SH-SY5Y P<0.0001). At the protein level, 0.75 µM WS6 was needed to decrease PA2G4 and MYCN protein expression in BE(2)-C cells, but a lower concentration of 0.5 µM was sufficient to decrease PA2G4 and MYCN in Kelly, or PA2G4 and c-MYC in SH-SY5Y cells (Figure 5.2 B).

Chapter 5- Targeting PA2G4 216

Figure 5.2 WS6 decreases PA2G4, MYCN and c-MYC mRNA and protein expression (A) Real-time PCR analysis of PA2G4, MYCN and c-MYC mRNA expression in BE(2)-C, Kelly and SH-SY5Y cells treated with 1 µM WS6 for 48 hours. (B) Protein analysis of PA2G4, MYCN and c-MYC expression in BE(2)-C, Kelly, and SH-SY5Y cells treated with increasing concentrations of WS6 for 48 hours. P values determined by t-test, bars represent the mean and SEM from 3 independent experiments.

Chapter 5- Targeting PA2G4 217 5.2.3 WS6 reduces cell growth

In Chapter 4 PA2G4 was shown to be involved in cell growth. To assess the effect of WS6 on neuroblastoma cell growth, cell viability assays were conducted on several cell lines: three neuroblastoma lines with high PA2G4 and MYCN or c-MYC expression (BE(2)-C, Kelly and SH-SY5Y); one line with low PA2G4 and MYCN expression (SK- N-FI); and two normal fibroblast cell lines (MRC-5 and WI-38). Cells were treated with increasing concentrations of WS6 for 24, 48 and 72 hours. Cell viability was measured by metabolism of resazurin (Figure 5.3).

Table 5.1 summarises the IC50 concentrations from WS6 treatment for each cell line over the different incubation times. Similar IC50 values were observed for BE(2)-C,

Kelly and SH-SY5Y cells, with an IC50 of 1.2 µM after 24 hours and 0.8 µM after 48 hours treatment with WS6 (Table 5.1). IC50 values for SK-N-FI, MRC-5 and WI-38 were higher (approximately 2-fold) than those for the three neuroblastoma cell lines mentioned previously. The data also highlights the increase in WS6 IC50 concentration in cell lines with lower PA2G4 and MCYN expression. Grouping the cell lines as high or low expresser of PA2G4 (see Figure 3.9) showed that cells with higher levels of PA2G4 are more sensitive to WS6 treatment at 48 hours (Figure 5.4; P=0.007). Data represent the mean and SEM from 3 independent experiments. IC50 was calculated using nonlinear regression and dose-response-inhibition equation.

Chapter 5- Targeting PA2G4 218

Figure 5.3 WS6 reduces cell growth Cell viability was measured in four neuroblastoma cell lines and two normal lung fibroblast cell lines treated with serial dilutions of WS6 starting at 8 µM over a period of 24 hours to 72 hours.

Chapter 5- Targeting PA2G4 219 Table 5.1 IC50 concentration from treatment with WS6 for 24 hours to 72 hours in neuroblastoma cells and normal lung fibroblast cells.

Figure 5.4 Cells expressing higher levels of PA2G4 are more sensitive to WS6 treatment IC50 values from cells treated with WS6 for 48 hours grouped by either High or Low PA2G4 expression. P values determined by t-test of average IC50 value of high PA2G4 expressing cell lines compared to average IC50 value of low PA2G4 expressing cell lines, bars represent the mean and SEM from 3 independent experiments.

Chapter 5- Targeting PA2G4 220 5.2.4 WS6 is specifically inhibiting PA2G4

To assess whether WS6 is specifically targeting PA2G4, BE(2)-C and Kelly cells were transfected with P48#2 siRNA for 24 hours followed by treatment with 0.5 µM WS6 for 48 hours, before measuring cell viability. The suppression of PA2G4-p48 decreased cell viability by 60% in BE(2)-C cells (Figure 5.5 A, P=0.0001) and approximately 40% in Kelly cells (Figure 5.5 B, P=0.0044). Treatment of control cells with WS6 also decreased cell viability by 30% in both cell lines (P=0.0054 for BE(2)-C, and P=0.0056 for Kelly). However WS6 treatment of cells with reduced PA2G4 expression did not further decrease the viability of either cell line.

Figure 5.5 WS6 was not effective in PA2G4 suppressed cells Suppression of PA2G4 with siRNA followed by treatment with WS6 (0.5 µM) in (A) BE(2)-C cells and (B) Kelly cells.

Chapter 5- Targeting PA2G4 221 5.2.5 WS6 induces apoptosis in neuroblastoma cells

There are many mechanisms/signalling pathways and cell phenotypic changes responsible for decreases in cell growth, one main phenotypic change is cell death induced by apoptosis. To assess whether the decrease in cell growth caused by WS6 is due to apoptosis, three neuroblastoma cell lines: BE(2)-C, Kelly and SH-SY5Y; and two normal fibroblast cell lines: MRC-5 and WI-38, were treated with three concentrations of WS6: 0.2 µM, 0.4 µM and 0.8 µM for 48 hours. After WS6 treatment, cells were stained with Annexin V and 7AAD to measure the early and late stages of apoptosis by flow cytometry. Treatment with low concentration of WS6 (0.4 µM) induced apoptosis in the three neuroblastoma cell lines but not in the two normal fibroblast cell lines (Figure 5.6). However, treatment with 0.8 µM WS6 induced apoptosis in all cell lines.

Chapter 5- Targeting PA2G4 222

Figure 5.6 WS6 induces apoptosis in neuroblastoma cells, but not in normal fibroblast cells. Three neuroblastoma cell lines and two normal lung fibroblast cell lines (MRC-5 and WI-38) were treated with increasing concentrations of WS6 for 48 hours. Cells were then stained with Annexin V and 7AAD to measure the early and late stages of apoptosis by flow cytometry. P-values determined by t-test, bars represent the mean and SEM from 3 independent experiments.

Chapter 5- Targeting PA2G4 223 5.2.6 WS6 reduces colony formation

In Chapter 4 PA2G4 was shown to promote colony formation in neuroblastoma cells. To investigate whether inhibition of PA2G4 with WS6 can affect colony formation, colony forming assays were performed on BE(2)-C and Kelly cells treated with 0.2 µM and 0.4 µM WS6. There were fewer colonies in the wells with WS6 treatment (Figure 5.7 A), compared to untreated wells. In BE(2)-C cells, there was a 40% decrease in colony formation with 0.2 µM WS6 treatment (Figure 5.7 B; P=0.0084), and a greater than 90% decrease with 0.4 µM WS6 treatment (P<0.0001). Similar results were seen in Kelly cells: 0.2 µM WS6 treatment decreased colony formation by greater than 60% (Figure 5.7 B; P=0.0003), and 0.4µM WS6 treatment caused a greater than 80% decrease in colony formation (P<0.0001).

Figure 5.7 WS6 reduces colony formation (A) BE(2)-C and Kelly cells treated with 0.2µM or 0.4µM WS6 followed by 10-14 days of incubation for colony formation. (B) Percentage of colony formation in BE(2)-C and Kelly cells treated with WS6 compared to untreated cells. P-values determined by t-test, bars represent the mean and SEM from 3 independent experiments.

Chapter 5- Targeting PA2G4 224 5.2.7 WS6 reduces tumour growth in TH-MYCN+/+ mice

Previous data from this thesis have shown that WS6 is able to decrease PA2G4 and MYCN expression and reduce neuroblastoma cell growth and colony formation. Because WS6 produces these phenotypic changes, and is water-soluble, it is an ideal drug candidate for the treatment of neuroblastoma. To investigate whether WS6 can decrease neuroblastoma tumour growth, the TH-MYCN+/+ mouse model was used. A Maximum Tolerated Dose (MTD) study was conducted on these mice at 3 weeks of age; the starting concentration of WS6 was 5 mg/kg and was increased up to a final concentration of 50 mg/kg. The MTD was not reached, however, due to the expensive cost of the WS6 compound, so it was decided that the concentration used would be 50 mg/kg.

Three-week-old TH-MYCN+/+ mice were randomised into two treatment cohorts of 10 mice. The control group was injected with PBS, while the other group was given 50 mg/kg WS6 in PBS. Injection was given by i.p. every day for 5 days, followed by a 2- day break, until tumours reached 1000 mm3 and mice were culled. Mice were palpated twice a week to monitor tumour growth. TH-MYCN+/+ mice treated with WS6 showed a significant delay in tumour growth compared to the control PBS treated mice (Figure 5.8 A; P=0.0001), with a median survival difference of 10 days between treatments (Figure 5.8 B).

Chapter 5- Targeting PA2G4 225

Figure 5.8 WS6 reduces tumour growth in TH-MYCN+/+ mice TH-MYCN +/+ mice were treated with 50 mg/kg WS6 at 3 weeks of age. Treatment was carried out for 5 consecutive days followed by a 2-day break until tumour reached 1cm3. (A) Survival data from control mice treated with PBS and mice treated with WS6. Survival curves were analysed by log-rank test. (B) Days taken to reach medium palpable tumour (1cm3) in each of the mice in each cohort, days were taken from birth.

Chapter 5- Targeting PA2G4 226 5.2.8 WS6 reduces PA2G4 expression in TH-MYCN+/+ mouse tumours

To investigate whether WS6 decreased expression of PA2G4 and MYCN in the mouse tumours, protein and RNA were isolated from the tumours. Western blot analysis showed decreased expression of PA2G4, but not MYCN, in tumours of mice treated with WS6, compared to PBS control treated mice (Figure 5.9 A). Vinculin was used as a loading control. Protein densitometry analysis was used to quantitate the decrease in PA2G4 expression in tumours from mice treated with WS6, compared to the control PBS treated mice; each sample was normalised to control siRNA mouse #1 (Figure 5.9 B; P=0.0002). A significant decrease in PA2G4 mRNA expression was observed in tumours from mice treated with WS6, compared to the control PBS treated mice (Figure 5.9 C; P=0.0003). There were no significant differences in MCYN mRNA expression between tumours from the two cohorts.

Chapter 5- Targeting PA2G4 227

Figure 5.9 WS6 reduces PA2G4 protein and mRNA expression in TH-MYCN+/+ mouse tumours (A) Protein extracted from tumours of TH-MYCN+/+ mice treated with PBS control or WS6, was used to analyse PA2G4 and MYCN expression. (B) Protein densitometry analysis from tumours of TH-MYCN+/+ mice treated with PBS control or WS6, normalised to PBS control mouse #1. (C) Real-time PCR analysis of PA2G4 and MYCN mRNA expression from mouse tumours, normalised to PBS control mouse #1. Data show means and SD derived from 6 mice, P values calculated by t-test.

Chapter 5- Targeting PA2G4 228 5.2.9 PA2G4 siRNA delivered by nanoparticles delays tumour growth

Short-interfering RNA (siRNA) has the potential to be a therapeutic tool to silence disease-causing genes. However siRNA cannot enter cells freely on its own and requires a delivery vehicle in order to penetrate cells and silence its target gene. Cationic star polymers are novel delivery vehicles for siRNA; they have been designed to self- assemble with siRNA and form small uniform nanoparticle complexes that are stable and non-toxic to cells (Figure 5.10 A). To test the effectiveness of these nanoparticles in delivering PA2G4 siRNA into cells, a comparison was carried out with Lipofectamine 2000 transfection reagent. 50 nM of each siRNA: control, PA2G4 and PA2G4-p48, was coupled to either nanoparticles at a ratio of 18:1 (nanoparticles:siRNA), or Lipofectamine 2000 at a ratio of 3:1 (Lipofectamine:siRNA), before transfection into SK-N-BE(2) cells for 48 hours, followed by protein isolation. Western blot analysis showed that PA2G4 siRNAs coupled to nanoparticles were able to reduce PA2G4 and MYCN expression just as effectively as PA2G4 siRNAs coupled to Lipofectamine 2000 (Figure 5.10 B).

As the in vitro data demonstrated the effectiveness of nanoparticles in delivering siRNA into cells, the effect of PA2G4 siRNA coupled to Star-nanoparticles on neuroblastoma tumour growth was investigated in vivo. Neuroblastoma cell line SK-N-BE(2) was chosen because it is tumorigenic, has high levels of PA2G4, and does not form very vascular tumours, thus enabling delivery of the siRNA-nanoparticles by injection directly into the tumour. SK-N-BE(2) (4x106) cells were subcutaneously injected into the flank of nude mice. Three cohorts of mice were established to receive injections of either control siRNA, PA2G4 siRNA or PA2G4-p48 siRNA, all delivered by nanoparticles. Injection started when the tumours reached 200mm3, with 40ug siRNA given intratumorally to each mouse every 3 days, for a total of 8 injections, or until tumour reached 1000mm3, whichever came first. A significant delay in tumour latency was observed in mice injected with PA2G4 siRNA and PA2G4-p48 siRNA, compared to those injected with control siRNA (Figure 5.10 C; P=0.004 for both isoforms of PA2G4).

Chapter 5- Targeting PA2G4 229

Figure 5.10 PA2G4 siRNA delivered by nanoparticles decreases tumour growth (A) Schematic diagram of the Star-nanoparticle. (B) SK-N-BE(2) cells with PA2G4 and PA2G4-p48 siRNA knockdown using either Lipofectamine 2000 or Star-nanoparticles, to compare transfection efficiency. (C) Tumour growth in mice xenografted with SK-N- BE(2) cells then injected with PA2G4 siRNA (40 µg) coupled to nanoparticles every 3 days for a total of 8 injections, or until tumour reached 1000mm3. Survival curves were analysed by log-rank test.

Chapter 5- Targeting PA2G4 230 5.2.10 Analysis of tumours with PA2G4 siRNA + nanoparticle

To investigate whether the nanoparticle-delivered PA2G4 siRNAs had successfully reduced expression of PA2G4 in the tumour cells, molecular analysis was performed on tumours from each cohort. Mice were culled when their tumour reached 1000mm3, and then RNA and protein were isolated from the tumours for analysis of PA2G4 and MYCN expression. Decreased expression of PA2G4 and MYCN protein was observed in the PA2G4 siRNA and PA2G4-p48 siRNA tumours, compared to control siRNA (Figure 5.11 A). Vinculin was used as a loading control, and each sample was normalised to control siRNA mouse #1. Protein densitometry analysis was used to quantitate the decrease in PA2G4 and MYCN expression for both PA2G4 and PA2G4- p48 siRNA tumours, compared to the control siRNA tumours (Figure 5.11 B). Significant decreases in PA2G4 and MYCN mRNA expression were also observed for both PA2G4 and PA2G4-p48 siRNA tumours, compared to the control siRNA tumours (Figure 5.11 C).

Chapter 5- Targeting PA2G4 231

Figure 5.11 PA2G4 siRNA coupled to Star-nanoparticles reduces PA2G4 and MYCN protein and mRNA expression in SK-N-BE(2) xenograft mouse tumours Tumours from SK-N-BE(2) xenografted mice injected with control siRNA, PA2G4 siRNA or PA2G4-p48 siRNA coupled to nanoparticles were processed. (A) Western blots of three tumour samples from each cohort showing the levels of PA2G4 and MYCN expression. (B) Protein densitometry analysis. (C) Real-time PCR analysis of PA2G4 and MYCN mRNA expression from tumour samples. Data show means and SD derived from 5-6 mice per group, P-values calculated by t-test.

Chapter 5- Targeting PA2G4 232 5.2.11 WS6 and vincristine combination treatment has synergistic effects against neuroblastoma cells

The use of a single molecule or drug is not the standard for treatment of most cancers. Usually a combination of 3–4 drugs is administered to patients, therefore it was necessary to investigate the effect of WS6 in combination with other inhibitors and chemotherapy drugs. JQ1, a potent inhibitor of the Bromodomain and Extra-Terminal motif (BET) family of proteins, which can reduce MYCN activity, was used to investigate whether inhibiting MYCN and PA2G4 simultaneously could further decrease neuroblastoma cell growth. Three chemotherapeutic drugs: vincristine, etoposide and cisplatin, were chosen to be used in combination with WS6 because they are currently used chemotherapeutic drugs and have different mechanisms of action on cells. These combination treatments were conducted in the neuroblastoma cell lines

BE(2)-C, Kelly and SH-SY5Y and in WI-38 fibroblast cells. The IC50 concentration for each agent was determined, so that concentrations higher and lower than the IC50 could be used in synergy assays. Treatments were performed for 48 hours before cell viability was measured. A Combination Index (C.I.) which is a theorem of Chou-Talalay, offers a quantitative definition for additive effect (C.I. = 1), synergism (C.I. < 1), and antagonism (C.I. > 1) in drug combinations (Chou, 2010).

Treatment with WS6 and vincristine was synergistic at low concentrations but antagonistic at high concentrations. In BE(2)-C cells, combination treatment with WS6 (0.6µM) + vincristine (10nM) showed synergy (Figure 5.12; C.I.= 0.57). Figure 5.13 shows two drug combinations with WS6 having a synergistic effect in Kelly cells: combinations of WS6 (0.6µM) + JQ1 (10µM) were synergistic (C.I. = 0.62), and WS6 (0.6µM) + vincristine (0.1nM) also showed synergy (C.I. = 0.71). However, there was no synergy between WS6 and etoposide, nor between WS6 and cisplatin in BE(2)-C and Kelly cells. In SH-SY5Y cells, combinations of WS6 (1µM) + vincristine (5nM), and WS6 (1µM) + cisplatin (2.5µM), were synergistic (Figure 5.14; C.I. = 0.77 and C.I. = 0.73 respectively), but not a combination of WS6 and JQ-1 or etoposide. For WI-38 cells, only WS6 (1.4µM) + JQ1 (10µM) combination treatment showed synergy (Figure 5.15; C.I. =0.58).

Chapter 5- Targeting PA2G4 233

Figure 5.12 WS6 + vincristine combination treatment is synergistic in BE(2)-C cells BE(2)-C cells treated with WS6 in combination with JQ1, vincristine, etoposide, or cisplatin for 48 hours. Cell viability was measured, and synergy with combination treatment was calculated with CalcuSyn software.

Chapter 5- Targeting PA2G4 234

Figure 5.13 WS6 + JQ1 and WS6 + vincristine combination treatments are synergistic in Kelly cells Kelly cells treated with WS6 in combination with JQ1, vincristine, etoposide, or cisplatin for 48 hours. Cell viability was measured, and synergy with combination treatment was calculated with CalcuSyn.

Chapter 5- Targeting PA2G4 235

Figure 5.14 WS6 + Vincristine and WS6 + Cisplatin combination treatments are synergistic in SH-SY5Y SH-SY5Y cells treated with WS6 in combination with JQ1, vincristine, etoposide, or cisplatin for 48 hours. Cell viability was measured, and synergy with combination treatment was calculated with CalcuSyn.

Chapter 5- Targeting PA2G4 236

Figure 5.15 WS6 + JQ1 combination treatment is synergistic in WI-38 cells WI-38 cells treated with WS6 in combination with JQ1, vincristine, etoposide, or cisplatin for 48 hours. Cell viability was measured, and synergy with combination treatment was calculated with CalcuSyn.

Chapter 5- Targeting PA2G4 237 5.3 Discussion

The advent of molecular-targeted cancer drug development has resulted in an increasing number of successful therapies, which have impacted the lives of a large number of cancer patients (Hoelder et al., 2012). Small molecule inhibitors have been successfully used to inhibit critical cancer targets: the epidermal growth factor receptor (EGFR) kinase inhibitor gefitinib potently inhibits EGFR in patients with non-small cell lung cancer (NSCLC) (Sordella et al., 2004); inhibition of the protein kinase ALK by crizotinib (Kwak et al., 2010) is used to treat NSCLC patients with a pathogenic rearrangement of the ALK gene; and vemurafenib (Chapman et al., 2011) has been approved to treat metastatic melanoma in patients with the BRAF V600E mutation.

WS6 was first identified from high-throughput small molecule screening and later as an inhibitor of PA2G4 (Shen et al., 2013; Wang et al., 2009). Affinity pull-down and kinase profiling studies identified PA2G4 and the kappa B (IκB) kinase pathway as targets of WS6 (Shen et al., 2013), and WS6 appears to be effectively targeting PA2G4 in neuroblastoma cells. However, whether WS6 also targets the kappa B (IκB) kinase pathway in neuroblastoma has not yet been assessed. Treatment with WS6 significantly reduced PA2G4 mRNA and protein expression. WS6 appears to be selectively affecting cells with higher PA2G4 expression, as cells with higher levels of PA2G4 expression had lower IC50 values for WS6 than cells with lower PA2G4 expression, and it may also be selective towards cells with higher MYCN expression. However, cell viability assays indicate that WS6 is not strongly selective for cancer cells, with toxicity also observed on normal cells at only slightly higher doses. The mode of action of WS6 appears to be cell killing. Unlike suppression of PA2G4 with siRNA, which causes cells to undergo differentiation or senescence, WS6 treatment induces apoptosis. This might be attributable to an effect of WS6 on the IκB kinase pathway (IKK). IKK plays a role in activating and regulating the NFκB pathway, which is involved in the pro-inflammatory stress response, and has anti-apoptotic activity (Baeuerle and Baltimore, 1996; Karin and Delhase, 2000). WS6 has been reported to inhibit IKK expression, which is essential for NF-kB activation and prevention of apoptosis (Li et al., 1999); therefore inactivating NFκB allows cells to undergo apoptosis.

Chapter 5- Targeting PA2G4 238 Having established that WS6 could target PA2G4 and MYCN in in vitro systems, it was necessary to evaluate WS6’s effectiveness in an in vivo mouse model. TH-MYCN+/+ mice were used, because they have 100% penetrance of the MYCN transgene, meaning that all mice will develop tumours. Mice carrying this transgene are predisposed to develop spontaneous neuroblastoma (Weiss et al., 1997), and tumours arising in this model accurately reproduce the typical features of human neuroblastoma at the pathological level, and harbour comparable genetic modifications (Moore et al., 2008; Weiss et al., 2000). Daily treatment with 50 mg/kg of WS6 delayed tumour growth in TH-MYCN+/+ mice. This delay was comparable to that caused by treatment with 0.2 mg/kg vincristine (Burkhart et al., 2009), one of the chemotherapy drugs used to treat neuroblastoma. Mice were treated at 3 weeks of age, the time at which a tumour was palpable. However treatment with WS6 at an earlier time, for example at 1 week or 2 weeks of age, may have further delayed tumour growth. This hypothesis was not tested, because when WS6 was administered at a concentration of 50 mg/kg to 1- and 2- week- old mice, they developed signs of toxicity, in the form of diarrhoea and weight loss, so treatment was ceased. Thus, to investigate whether earlier treatment with WS6 can more effectively delay or inhibit tumour initiation and growth, the MTD would first need to be determined for 1- and 2- week-old mice.

Analysis of the tumour samples supports the hypothesis that WS6 targets PA2G4. The levels of PA2G4 protein and RNA expression in tumours from mice treated with WS6 were significantly lower than in tumours from control mice. However, the MYCN levels remained unchanged between the two cohorts. This discrepancy between the in vitro and in vivo data could be due to species and genetic differences, including differences in promoter (mouse has a TH promoter rather than a MYCN promoter) between human cell lines and mouse tumour samples. Another possible explanation is that WS6 does not directly target MYCN, but only inhibits PA2G4 expression. The decrease in PA2G4 expression caused by WS6 may only have a weak effect on suppressing MYCN protein, and not affect its mRNA expression, as previously shown in Chapter 3, in in vitro experiments with PA2G4 siRNAs.

As mentioned previously, WS6 is not a specific inhibitor of PA2G4 as it also targets other proteins in the kappa B (IκB) kinase pathway. This is a limitation in the use of WS6 as tool compound to study inhibition of PA2G4, therefore as a proof of principle that PA2G4 can be targeted for the treatment of neuroblastoma, and to provide further

Chapter 5- Targeting PA2G4 239 evidence that suppression of PA2G4 can delay tumour growth, siRNA specifically targeting PA2G4 was tested in a xenograft mouse model. siRNA is an emerging class of therapeutics for the treatment of cancer (Petrocca and Lieberman, 2011; Shen et al., 2012). It has the potential to inhibit genes that are not normally accessible to, or effectively targeted by chemical inhibitors (Rana, 2007). However, there are a number of biological challenges that need to be addressed before siRNA can be used clinically, including a non-toxic, site-specific delivery vehicle for packaging and delivering the siRNA. siRNA oligonucleotides are commonly delivered to tumour tissues in nano- scale delivery vehicles (nanoparticles). Properties of a good nanoparticle include the ability to protect the siRNA from degradation, to enrich siRNA in the target organ, and to facilitate the cellular uptake of siRNA (Peer and Lieberman, 2011; Shen et al., 2012). The decision to use cationic star polymers was based on their ability to self-assemble with siRNA and to form small uniform nanoparticle complexes that are non-toxic to cells (Boyer et al., 2013; Cho et al., 2011). The star polymers possess unique properties, originating from their multi-armed structures, leading to enhanced cellular uptake (Cho et al., 2010). In vitro testing showed that the star polymer-siRNA complex was effective in suppressing PA2G4 in neuroblastoma cells.

To assess whether star polymer could deliver siRNA to tumour cells and induce specific gene silencing in vivo, star polymer-siRNA complex was delivered intratumorally to xenografted neuroblastoma cells. Treatment with a relatively small amount of PA2G4 siRNA (40 ug) every 3 days for up to 8 treatments, delayed tumour growth, compared to the control siRNA. Hence, targeting PA2G4 has therapeutic potential for the treatment of neuroblastoma. Furthermore, targeted delivery of siRNA to neuroblastoma cells could be achieved by exploiting selective cell surface markers, such as GD2, on tumour cells (Di Paolo et al., 2011), or peptide ligands specific to either epithelial or stromal components of neuroblastoma tumours (Loi et al., 2013).

Analysis of the mouse tumour tissues confirmed the activity of the PA2G4 siRNA. Mouse tumours treated with PA2G4 siRNAs showed decreases in PA2G4 mRNA and protein expression compared to the control mouse tumours. Furthermore, treatment with PA2G4 siRNAs also decreased MYCN mRNA and protein expression. The decrease in MYCN mRNA was unexpected, since in the in vitro studies in Chapter 3, PA2G4 siRNA was only able to decrease MYCN protein expression, and not its mRNA levels.

Chapter 5- Targeting PA2G4 240 This could imply that the suppression of PA2G4 in this in vivo system may be affecting other signalling pathways that deregulate MYCN mRNA expression.

Chemotherapy remains the first-line treatment for most cancers, however the anti- tumour efficacies of current cytotoxic agents are limited, most likely because of the high degree of genetic heterogeneity in cancer cells, the complexity of their cell signalling pathways, and their side-effects. The inhibition of a single target does not necessarily eradicate the cancer, therefore the use of combination therapy, including molecular- targeted agents that inhibit multiple targets or pathways simultaneously, may be the most effective way to improve treatment efficacy and conquer resistance in chemotherapy (Li et al., 2014).

Combining WS6 with conventional cytotoxic drugs or other molecular-targeted agents may improve the treatment of neuroblastoma. Because PA2G4 regulates MYCN by stabilising its protein, using WS6 to inhibit PA2G4, in combination with a MYCN inhibitor, is a rational approach to improve neuroblastoma treatment. Currently there are no direct MYCN inhibitors, however there are agents that can target MYC (c-MYC and MYCN) indirectly by disrupting the chromatin-dependent signal transduction that regulates MYC transcription (Delmore et al., 2011). Members of the bromodomain and extraterminal (BET) subfamily of human bromodomain proteins (BRD2, BRD3, and BRD4) associate with acetylated chromatin and facilitate transcriptional activation (Rahman et al., 2011). BRD4 is involved in the control of transcriptional elongation by RNA polymerase II through its recruitment of the positive transcription elongation factor P-TEFb (Jang et al., 2005; Yang et al., 2005). Inhibitors of BRD4 have been shown to selectively inhibit transcription of key oncogenic drivers such as c-MYC in multiple tumour types (Dawson et al., 2011; Delmore et al., 2011; Mertz et al., 2011). One very promising compound that inhibits BRD4 is JQ1. JQ1 is a thieno-triazolo-1,4- diazepine that displaces bromodomain BRD4 from chromatin by competitively binding to the acetyl-lysine recognition pocket (Filippakopoulos et al., 2010). Combination treatment of WS6 and JQ1 only showed synergy in one out of three neuroblastoma cell lines. Moreover, this combination treatment also showed synergy in a non-cancerous cell line without abnormal MYC expression. It would be ideal to perform gene analysis studies, such as microarray analysis, on the two cell lines showing synergy with the combination treatment of WS6 and JQ1, to determine which gene expression or

Chapter 5- Targeting PA2G4 241 signalling pathways they have in common. This data could provide valuable insight into the profile of these cell lines and aid in developing drugs for targeted therapy.

Current conventional chemotherapeutic treatment for neuroblastoma includes the use of vincristine, etoposide, cisplatin or carboplatin, doxorubicin, and cyclophosphamide (Kohler et al., 2013; Ladenstein et al., 2010; Pearson et al., 2008). The cytotoxic agents used in this thesis in combination with WS6 were vincristine, etoposide and cisplatin. These agents were chosen for their diverse mechanisms of action. Vincristine is a vinca alkaloid and a microtubule depolymerization agent. It works by binding to tubulin dimers, inhibiting assembly of microtubule structures and arresting mitosis in metaphase, causing cells to undergo apoptosis (el Alaoui et al., 1997; Harmon et al., 1992). Because cancer cells divide more rapidly than most normal cells, they are typically more sensitive to this drug.

Etoposide (also known as VP-16) forms a complex with DNA and the topoisomerase II enzyme; this ternary complex prevents re-ligation of DNA strands, causing them to break (Loike and Horwitz, 1976). Cancer cells rely on this enzyme more than healthy cells, since they divide more rapidly. When these permanent DNA breaks are present in sufficient quantities, causing errors in DNA synthesis, they trigger a series of events that ultimately lead to cell death by apoptosis (Hande, 1998). Cisplatin belongs to a class of platinum-containing anti-cancer drugs. These platinum complexes react in vivo, binding to and causing crosslinking of DNA, which ultimately triggers apoptosis. Cisplatin can also cause neuronal differentiation of neuroblastoma cells (Parodi et al., 1989).

Vincristine was the only cytotoxic agent tested that, in combination with WS6, showed synergy in all three neuroblastoma cell lines, and had no effect on the normal cells at concentrations that killed the cancer cells. Combination treatment of WS6 and cisplatin only showed synergy in one out of three neuroblastoma cell lines, and treatment with WS6 and etoposide showed no synergy in any of the cell lines tested. These results suggest that the heterogeneity between cell lines affects their sensitivity to cytotoxic drugs as well as to molecular-targeting agents. Encouragingly, vincristine appears to be a good candidate for in vivo combination studies, and elucidating the mechanism of its synergy with WS6 would be advantageous for the design of future drug combinations.

Chapter 5- Targeting PA2G4 242 In conclusion, the small molecule WS6, previously identified as a PA2G4 inhibitor in pancreatic β cells, was confirmed as a PA2G4 inhibitor in neuroblastoma cells, and found to be a good tool compound for studying the mechanism of PA2G4 activity. This chapter has demonstrated that the expression and oncogenic functions of PA2G4 can be targeted chemically with WS6, and genetically using PA2G4-specific siRNA delivered by nanoparticles. Combining PA2G4 inhibitors with chemotherapeutic drugs may lead to more effective treatment of neuroblastoma, however more specific PA2G4 inhibitors will need to be developed.

Chapter 5- Targeting PA2G4 243 Chapter 6 CONCLUDING REMARKS AND FUTURE DIRECTIONS

CHAPTER 6

CONCLUDING REMARKS AND FUTURE DIRECTIONS

Chapter 6- Conclusions 244 The overall cure rates for paediatric cancers have improved considerably in the last 20 years. More than 85% of patients with acute lymphoblastic leukaemia (ALL) now experience long-term survival (Hunger et al., 2012; Pui et al., 2011). This increase in survival rate is due to the improvement of multi-agent chemotherapy and advances in supportive care. In contrast, the survival rate of patients with high-risk neuroblastoma is still very poor, in spite of intensive, multimodal therapy (De Bernardi et al., 2003; Maris, 2010; Matthay et al., 1999a). A key factor is that patients respond to primary therapy but then often relapse due to acquired drug resistance (London et al., 2011; Siddik, 2003). While the exact mechanisms responsible for this remain unknown, MYCN amplification has proven to be the most robust predictor of poor prognosis. However, attempts to develop direct inhibitors of MYCN have thus far been largely unsuccessful. In contrast, indirect inhibitors may prove to be the most beneficial and tractable therapeutic strategy and several examples have recently demonstrated very promising preclinical activity (Brockmann et al., 2013; Carter et al., 2015; Puissant et al., 2013).

This thesis explores indirect inhibition of MYCN by identifying and exploiting novel MYCN binding partners as molecular targets for treating neuroblastoma. Proliferation associated protein 2G4 (PA2G4) was identified as a novel MYCN binding protein that is able to regulate MYCN expression by increasing MYCN protein stability, as discussed in Chapter 3. The site of interaction between MYCN and PA2G4 was not explored in this thesis, however, it would be extremely valuable to identify this site; therefore work is under way to accomplish this using MYCN domain deletion mutants and co-immunoprecipitation. Once the peptide region in the MYCN protein is identified, surface plasmon resonance (Biacore) studies will be used to map the candidate oligopeptides to the known crystal structure of PA2G4. Confirmation of the binding region can then be mapped to individual amino acids by performing functional studies using MYCN and PA2G4 point mutants.

The discovery of PA2G4 as a transcriptional target of MYCN, coupled with the observation that high PA2G4 expression is a strong marker of poor prognosis, independent of factors such as MYCN amplification, disease stage and patient’s age at diagnosis, highlighted the potential of PA2G4 as a therapeutic target in neuroblastoma. PA2G4 and MYCN activate many shared RNA-related genes and signalling pathways, as identified in the microarray study, which is in agreement with the documented role of

Chapter 6- Conclusions 245 PA2G4 in RNA processing and ribosomal assembly. The molecular mechanism through which MYCN and PA2G4 affect these processes will need to be addressed in future studies by analysing each differentially expressed gene and the associated signalling pathways.

Chapter 4 discussed the role of PA2G4 in neuroblastoma progression and biology. It focused on the long PA2G4 isoform, p48, as a possible tumour promoter gene in neuroblastoma, with diverse mechanisms of action including effects on cell growth and cell migration. However, a limitation of this work was the lack of analysis of the short isoform, p42. It was not possible to perform knockdown studies with siRNA specifically targeting p42, because it shares its sequence with p48, however it is possible to generate plasmid DNA to overexpress p42, believed to be a tumour suppressor gene, and so to assess in future studies whether overexpression of this isoform decreases cell growth and migration.

To understand the molecular mechanism of PA2G4 as an oncogenic protein, key signalling proteins affecting cell growth and migration were analysed, and the data obtained are consistent with these proteins being PA2G4 targets. These proteins include AKT, MDM2, p53, p21, and TFAP4. In the case of TFAP4, a demonstrated transcriptional target of MYCN (Cheng et al. manuscript in submission), the question remains as to how PA2G4 interacts with or regulates TFAP4. Co-immunoprecipitation studies would confirm either a direct interaction through protein-protein binding or the possibility of an indirect interaction as part of a protein complex, while chromatin immunoprecipitation would show an interaction via transcriptional activation through protein-DNA binding. Because TFAP4 is known to be involved in cell migration, further analysis of other cell migratory genes or EMT-related genes should elucidate the way in which PA2G4 influences cell migration and invasion.

Chapter 4 also presented data showing that the decrease in cell growth caused by suppression of PA2G4 was not the result of apoptosis, but possibly of differentiation or senescence. However, cellular senescence was not confirmed, due to the inability to generate viable PA2G4 knockdown stable cell lines. A possible way of generating these clones would be to use an inducible system with short-hairpin RNAs targeting PA2G4 and can be regulated by a Tetracycline Repressor Protein (Gossen et al., 1995; Yu et al., 2002). This would allow the stably transfected cells to be selected and expanded

Chapter 6- Conclusions 246 without depletion of PA2G4. To address the question of differentiation, neurite formation may not be the most definitive measurement, therefore using differentiation markers such as tyrosine hydroxylase (TH), β-III tubulin (TUBB3), growth associated protein 43 (GAP34), neurofilament 68 (NF68), and maternally expressed 3 (MEG3) to analyse differentiation by either immunofluorescence (IF) or western blot may prove to be more specific and quantitative.

One of the most significant findings from this study was that overexpression of PA2G4 in a non-tumorigenic cell line was able to induce tumour formation and growth in the xenograft mouse model. It is worth noting that MYCN protein and mRNA expression were not significantly elevated in the SH-EP PA2G4-overexpressing tumours compared to the SH-EP control tumours/tissue samples, ruling out an increase in MYCN expression as the explanation for this tumorigenesis. To further investigate the role of PA2G4 as a tumour promoter, the next step would be to sequence these tumours to confirm that the cells are the original SH-EP cells, and any changes in their tumorigenic state are due to PA2G4 expression. Definitive evidence demonstrating that PA2G4 is a tumour promoter and is required for tumorigenesis could be gained by generating PA2G4 knockout (KO) mice in the future study. These KO mice could be assessed for viability, and crossed with TH-MYCN+/+ mice to investigate whether depletion of PA2G4 (in the case of PA2G4-/- homozygous KO mice) or reduced expression of PA2G4 (in the case of PA2G4+/- heterozygous mice) can delay or prevent tumour growth in TH-MYCN+/+ mice.

Chapter 5 examined the potential of PA2G4 as a molecular target for treating neuroblastoma. The small molecule PA2G4 inhibitor WS6 provided the platform to test whether inhibition of PA2G4 can reduce neuroblastoma growth. WS6 was found to decrease PA2G4 mRNA and protein expression in neuroblastoma cell lines and data from this study also showed WS6 had specificity to PA2G4 inhibition. However, WS6 showed some toxicity to normal cells, indicating that it is not an ideal drug for cancer treatment, but better used as a tool compound to study the mechanism of targeting PA2G4. Moreover, future work needs to be carried out to investigate the effects of WS6 on its other target(s) in the IκB kinase pathway, which was not addressed in this study. Whether IκB kinases are affected by WS6 in neuroblastoma cells is still unknown. Nevertheless, WS6 was able to significantly delay tumour growth in the TH-MYCN mouse model and reduce PA2G4 expression in the tumours. This implies that there is an

Chapter 6- Conclusions 247 opportunity to search for molecules with structural analogy to WS6, which are more specific to cancer cells. Alternatively, high throughput screening of small molecules from a diverse library could potentially identify novel and more effective PA2G4 inhibitors.

As well as chemically targeting PA2G4, this study also investigated the possibility of silencing PA2G4 expression using a genetic approach. siRNA specifically targeting PA2G4, packaged in specially designed, robust non-viral nanoparticles and delivered directly into the tumours of xenografted mice was able to significantly delay tumour growth by suppressing PA2G4 mRNA and protein expression. This study provided a proof of principle for silencing PA2G4 gene expression in vivo, it showed siRNAs can be encapsulated in nanoparticles and delivered to tumour cells. Notably, the mice did not display any signs of toxicity to the nanoparticle-siRNA treatment. These results are highly encouraging. The next phase would be to test the effectiveness of PA2G4 siRNA delivered by nanoparticles in more clinically relevant neuroblastoma mouse models such as the patient-derived xenograft (PDX) models, which can retain the genetic characteristics and recapitulate the drug-sensitivity patterns of their donor tumour, thus allowing prediction of clinical outcomes (Braekeveldt et al., 2015; Hidalgo et al., 2014), or orthotopic tumour models which can more accurately reflect the tumour’s microenvironment and are considered better predictive models of drug efficacy than standard subcutaneous models (Khanna et al., 2002; Killion et al., 1998). Furthermore, nanoparticles used in this thesis can be readily modified to have targeting moieties attached to their surface to target specific cell types. Indeed, future studies could attach GD2 antibodies to the surface of nanoparticles to target GD2 which is highly expressed on the surface of neuroblastoma cells. Another possible use and future direction for siRNA-nanoparticles in the treatment of neuroblastoma would be to combine it with chemotherapeutic drugs.

Single-agent treatments are usually not effective and are less likely to be included in clinical trials. Therefore, in this study WS6 was tested in combination with three conventional chemotherapeutic drugs; vincristine, etoposide and cisplatin, all having different modes of action; and one MYCN inhibitor, JQ-1. WS6 synergised with vincristine in a panel of neuroblastoma cell lines, causing decreases in cell viability, but was not synergistic on normal cells. Vincristine was the only drug to show consistent synergy with WS6 across the panel of neuroblastoma cells. However, as mentioned

Chapter 6- Conclusions 248 above, WS6 is not an ideal compound for treatment of neuroblastoma patients, and steps have already been taken by our group to derive structural analogues of WS6, with the aim of improving on its specificity and effectiveness on neuroblastoma cells. Furthermore, any effective analogues of WS6 should be tested in xenograft models in combination with clinical drugs, to investigate their potential to overcome acquired drug resistance in neuroblastoma patients.

In summary, the work conducted in this thesis shows for the first time the direct interaction between MYCN and PA2G4, and reveals the underlying mechanisms of PA2G4 as an onco-factor in MYCN-driven neuroblastoma: PA2G4 increases MYCN protein stability and acts as an oncogenic co-factor in a forward feedback expression loop with MYCN. It also highlights the important role of PA2G4 as a tumour promoter in neuroblastoma development and progression. This thesis therefore provides strong evidence that PA2G4 is a driver of tumorigenicity, and for the first time identifies PA2G4 as a novel therapeutic target, which can be chemically or genetically inhibited, for the treatment of neuroblastoma.

Chapter 6- Conclusions 249 REFERENCES

Ahn, J.Y., Liu, X., Liu, Z., Pereira, L., Cheng, D., Peng, J., Wade, P.A., Hamburger, A.W., and Ye, K. (2006). Nuclear Akt associates with PKC-phosphorylated Ebp1, preventing DNA fragmentation by inhibition of caspase-activated DNase. The EMBO journal 25, 2083-2095.

Akinmade, D., Lee, M., Zhang, Y.X., and Hamburger, A.W. (2007). Ebp1-mediated inhibition of cell growth requires serine 363 phosphorylation. Int J Oncol 31, 851-858.

Akinmade, D., Talukder, A.H., Zhang, Y., Luo, W.M., Kumar, R., and Hamburger, A.W. (2008). Phosphorylation of the ErbB3 binding protein Ebp1 by p21-activated kinase 1 in breast cancer cells. Br J Cancer 98, 1132-1140.

Aldosari, N., Bigner, S.H., Burger, P.C., Becker, L., Kepner, J.L., Friedman, H.S., and McLendon, R.E. (2002). MYCC and MYCN oncogene amplification in medulloblastoma. A fluorescence in situ hybridization study on paraffin sections from the Children's Oncology Group. Archives of pathology & laboratory medicine 126, 540- 544.

Amatruda, T.T., 3rd, Sidell, N., Ranyard, J., and Koeffler, H.P. (1985). Retinoic acid treatment of human neuroblastoma cells is associated with decreased N-myc expression. Biochem Biophys Res Commun 126, 1189-1195.

Andersen, J.S., Lyon, C.E., Fox, A.H., Leung, A.K., Lam, Y.W., Steen, H., Mann, M., and Lamond, A.I. (2002). Directed proteomic analysis of the human nucleolus. Current biology : CB 12, 1-11.

Arkin, M.R., and Wells, J.A. (2004). Small-molecule inhibitors of protein-protein interactions: progressing towards the dream. Nature reviews Drug discovery 3, 301-317.

Armstrong, B.C., and Krystal, G.W. (1992). Isolation and characterization of complementary DNA for N-cym, a gene encoded by the DNA strand opposite to N- myc. Cell growth & differentiation : the molecular biology journal of the American Association for Cancer Research 3, 385-390.

Asangani, I.A., Dommeti, V.L., Wang, X., Malik, R., Cieslik, M., Yang, R., Escara- Wilke, J., Wilder-Romans, K., Dhanireddy, S., Engelke, C., et al. (2014). Therapeutic targeting of BET bromodomain proteins in castration-resistant prostate cancer. Nature 510, 278-282.

Auvinen, M., Paasinen, A., Andersson, L.C., and Holtta, E. (1992). Ornithine decarboxylase activity is critical for cell transformation. Nature 360, 355-358.

Baeuerle, P.A., and Baltimore, D. (1996). NF-kappa B: ten years after. Cell 87, 13-20.

References 250 Bagatell, R., London, W.B., Wagner, L.M., Voss, S.D., Stewart, C.F., Maris, J.M., Kretschmar, C., and Cohn, S.L. (2011). Phase II study of irinotecan and temozolomide in children with relapsed or refractory neuroblastoma: a Children's Oncology Group study. Journal of clinical oncology : official journal of the American Society of Clinical Oncology 29, 208-213.

Baker, D.L., Schmidt, M.L., Cohn, S.L., Maris, J.M., London, W.B., Buxton, A., Stram, D., Castleberry, R.P., Shimada, H., Sandler, A., et al. (2010). Outcome after reduced chemotherapy for intermediate-risk neuroblastoma. The New England journal of medicine 363, 1313-1323.

Bannasch, D., Madge, B., and Schwab, M. (2001). Functional interaction of Yaf2 with the central region of MycN. Oncogene 20, 5913-5919.

Bannasch, D., Weis, I., and Schwab, M. (1999). Nmi protein interacts with regions that differ between MycN and Myc and is localized in the cytoplasm of neuroblastoma cells in contrast to nuclear MycN. Oncogene 18, 6810-6817.

Bardwell, V.J., and Treisman, R. (1994). The POZ domain: a conserved protein-protein interaction motif. Genes & development 8, 1664-1677.

Bartel, D.P. (2004). MicroRNAs: genomics, biogenesis, mechanism, and function. Cell 116, 281-297.

Bello-Fernandez, C., Packham, G., and Cleveland, J.L. (1993). The ornithine decarboxylase gene is a transcriptional target of c-Myc. Proc Natl Acad Sci U S A 90, 7804-7808.

Beltran, H. (2014). The N-myc Oncogene: Maximizing its Targets, Regulation, and Therapeutic Potential. Molecular Cancer Research 12, 815-822.

Beltran, H., Rickman, D.S., Park, K., Chae, S.S., Sboner, A., MacDonald, T.Y., Wang, Y., Sheikh, K.L., Terry, S., Tagawa, S.T., et al. (2011). Molecular characterization of neuroendocrine prostate cancer and identification of new drug targets. Cancer Discov 1, 487-495.

Berg, T., Cohen, S.B., Desharnais, J., Sonderegger, C., Maslyar, D.J., Goldberg, J., Boger, D.L., and Vogt, P.K. (2002). Small-molecule antagonists of Myc/Max dimerization inhibit Myc-induced transformation of chicken embryo fibroblasts. Proc Natl Acad Sci U S A 99, 3830-3835.

Beroukhim, R., Mermel, C.H., Porter, D., Wei, G., Raychaudhuri, S., Donovan, J., Barretina, J., Boehm, J.S., Dobson, J., Urashima, M., et al. (2010). The landscape of somatic copy-number alteration across human cancers. Nature 463, 899-905.

Bjerke, L., Mackay, A., Nandhabalan, M., Burford, A., Jury, A., Popov, S., Bax, D.A., Carvalho, D., Taylor, K.R., Vinci, M., et al. (2013). Histone H3.3. mutations drive pediatric glioblastoma through upregulation of MYCN. Cancer Discov 3, 512-519.

References 251 Blackwood, E.M., and Eisenman, R.N. (1991). Max: a helix-loop-helix zipper protein that forms a sequence-specific DNA-binding complex with Myc. Science 251, 1211- 1217.

Bos, J.L. (1989). ras oncogenes in human cancer: a review. Cancer Res 49, 4682-4689.

Bose, S.K., Sengupta, T.K., Bandyopadhyay, S., and Spicer, E.K. (2006). Identification of Ebp1 as a component of cytoplasmic bcl-2 mRNP (messenger ribonucleoprotein particle) complexes. The Biochemical journal 396, 99-107.

Bower, R.J., and Adkins, J.C. (1980). Ondine's curse and neurocristopathy. Clinical pediatrics 19, 665-668.

Bown, N., Cotterill, S., Lastowska, M., O'Neill, S., Pearson, A.D., Plantaz, D., Meddeb, M., Danglot, G., Brinkschmidt, C., Christiansen, H., et al. (1999). Gain of chromosome arm 17q and adverse outcome in patients with neuroblastoma. The New England journal of medicine 340, 1954-1961.

Bown, N., Lastowska, M., Cotterill, S., O'Neill, S., Ellershaw, C., Roberts, P., Lewis, I., and Pearson, A.D. (2001). 17q gain in neuroblastoma predicts adverse clinical outcome. U.K. Cancer Cytogenetics Group and the U.K. Children's Cancer Study Group. Med Pediatr Oncol 36, 14-19.

Boxer, R.B., Jang, J.W., Sintasath, L., and Chodosh, L.A. (2004). Lack of sustained regression of c-MYC-induced mammary adenocarcinomas following brief or prolonged MYC inactivation. Cancer Cell 6, 577-586.

Boyer, C., Teo, J., Phillips, P., Erlich, R.B., Sagnella, S., Sharbeen, G., Dwarte, T., Duong, H.T., Goldstein, D., Davis, T.P., et al. (2013). Effective delivery of siRNA into cancer cells and tumors using well-defined biodegradable cationic star polymers. Molecular pharmaceutics 10, 2435-2444.

Braekeveldt, N., Wigerup, C., Gisselsson, D., Mohlin, S., Merselius, M., Beckman, S., Jonson, T., Borjesson, A., Backman, T., Tadeo, I., et al. (2015). Neuroblastoma patient- derived orthotopic xenografts retain metastatic patterns and geno- and phenotypes of patient tumours. International journal of cancer Journal international du cancer 136, E252-261.

Bray, I., Bryan, K., Prenter, S., Buckley, P.G., Foley, N.H., Murphy, D.M., Alcock, L., Mestdagh, P., Vandesompele, J., Speleman, F., et al. (2009). Widespread dysregulation of MiRNAs by MYCN amplification and chromosomal imbalances in neuroblastoma: association of miRNA expression with survival. PloS one 4, e7850.

Bray, I., Tivnan, A., Bryan, K., Foley, N.H., Watters, K.M., Tracey, L., Davidoff, A.M., and Stallings, R.L. (2011). MicroRNA-542-5p as a novel tumor suppressor in neuroblastoma. Cancer Lett 303, 56-64.

References 252 Brenner, C., Deplus, R., Didelot, C., Loriot, A., Vire, E., De Smet, C., Gutierrez, A., Danovi, D., Bernard, D., Boon, T., et al. (2005). Myc represses transcription through recruitment of DNA methyltransferase corepressor. The EMBO journal 24, 336-346.

Bresler, S.C., Wood, A.C., Haglund, E.A., Courtright, J., Belcastro, L.T., Plegaria, J.S., Cole, K., Toporovskaya, Y., Zhao, H., Carpenter, E.L., et al. (2011). Differential inhibitor sensitivity of anaplastic lymphoma kinase variants found in neuroblastoma. Science translational medicine 3, 108ra114.

Bretones, G., Acosta, J.C., Caraballo, J.M., Ferrandiz, N., Gomez-Casares, M.T., Albajar, M., Blanco, R., Ruiz, P., Hung, W.C., Albero, M.P., et al. (2011). SKP2 oncogene is a direct MYC target gene and MYC down-regulates p27(KIP1) through SKP2 in human leukemia cells. The Journal of biological chemistry 286, 9815-9825.

Brockmann, M., Poon, E., Berry, T., Carstensen, A., Deubzer, H.E., Rycak, L., Jamin, Y., Thway, K., Robinson, S.P., Roels, F., et al. (2013). Small molecule inhibitors of aurora-a induce proteasomal degradation of N-myc in childhood neuroblastoma. Cancer Cell 24, 75-89.

Brodeur, G.M. (2003). Neuroblastoma: Biological insights into a clinical enigma. Nature Reviews Cancer 3, 203-216.

Brodeur, G.M., Pritchard, J., Berthold, F., Carlsen, N.L.T., Castel, V., Castleberry, R.P., Debernardi, B., Evans, A.E., Favrot, M., Hedborg, F., et al. (1993). REVISIONS OF THE INTERNATIONAL CRITERIA FOR NEUROBLASTOMA DIAGNOSIS, STAGING, AND RESPONSE TO TREATMENT. Journal of Clinical Oncology 11, 1466-1477.

Brodeur, G.M., Seeger, R.C., Barrett, A., Berthold, F., Castleberry, R.P., Dangio, G., Debernardi, B., Evans, A.E., Favrot, M., Freeman, A.I., et al. (1988). INTERNATIONAL CRITERIA FOR DIAGNOSIS, STAGING, AND RESPONSE TO TREATMENT IN PATIENTS WITH NEURO-BLASTOMA. Journal of Clinical Oncology 6, 1874-1881.

Brodeur, G.M., Seeger, R.C., Schwab, M., Varmus, H.E., and Bishop, J.M. (1984). AMPLIFICATION OF N-MYC IN UNTREATED HUMAN NEUROBLASTOMAS CORRELATES WITH ADVANCED DISEASE STAGE. Science 224, 1121-1124.

Buechner, J., and Einvik, C. (2012). N-myc and noncoding RNAs in neuroblastoma. Molecular cancer research : MCR 10, 1243-1253.

Buechner, J., Tomte, E., Haug, B.H., Henriksen, J.R., Lokke, C., Flaegstad, T., and Einvik, C. (2011). Tumour-suppressor microRNAs let-7 and mir-101 target the proto- oncogene MYCN and inhibit cell proliferation in MYCN-amplified neuroblastoma. Br J Cancer 105, 296-303.

Burkhart, C.A., Watt, F., Murray, J., Pajic, M., Prokvolit, A., Xue, C., Flemming, C., Smith, J., Purmal, A., Isachenko, N., et al. (2009). Small-molecule multidrug resistance-

References 253 associated protein 1 inhibitor reversan increases the therapeutic index of chemotherapy in mouse models of neuroblastoma. Cancer Res 69, 6573-6580.

Calao, M., Sekyere, E.O., Cui, H.J., Cheung, B.B., Thomas, W.D., Keating, J., Chen, J.B., Raif, A., Jankowski, K., Davies, N.P., et al. (2013). Direct effects of Bmi1 on p53 protein stability inactivates oncoprotein stress responses in embryonal cancer precursor cells at tumor initiation. Oncogene 32, 3616-3626.

Caren, H., Abel, F., Kogner, P., and Martinsson, T. (2008). High incidence of DNA mutations and gene amplifications of the ALK gene in advanced sporadic neuroblastoma tumours. The Biochemical journal 416, 153-159.

Carmena, M., and Earnshaw, W.C. (2003). The cellular geography of aurora kinases. Nature reviews Molecular cell biology 4, 842-854.

Carpenter, E.L., and Mosse, Y.P. (2012). Targeting ALK in neuroblastoma--preclinical and clinical advancements. Nature reviews Clinical oncology 9, 391-399.

Carraway, K.L., 3rd, Soltoff, S.P., Diamonti, A.J., and Cantley, L.C. (1995). Heregulin stimulates mitogenesis and phosphatidylinositol 3-kinase in mouse fibroblasts transfected with erbB2/neu and erbB3. The Journal of biological chemistry 270, 7111- 7116.

Carter, D.R., Murray, J., Cheung, B.B., Gamble, L., Koach, J., Tsang, J., Sutton, S., Kalla, H., Syed, S., Gifford, A.J., et al. (2015). Therapeutic targeting of the MYC signal by inhibition of histone chaperone FACT in neuroblastoma. Science translational medicine 7, 312ra176.

Chambers, A.F., Groom, A.C., and MacDonald, I.C. (2002). Dissemination and growth of cancer cells in metastatic sites. Nature reviews Cancer 2, 563-572.

Chang, T.C., Yu, D., Lee, Y.S., Wentzel, E.A., Arking, D.E., West, K.M., Dang, C.V., Thomas-Tikhonenko, A., and Mendell, J.T. (2008). Widespread microRNA repression by Myc contributes to tumorigenesis. Nature genetics 40, 43-50.

Chanthery, Y.H., Gustafson, W.C., Itsara, M., Persson, A., Hackett, C.S., Grimmer, M., Charron, E., Yakovenko, S., Kim, G., Matthay, K.K., et al. (2012). Paracrine signaling through MYCN enhances tumor-vascular interactions in neuroblastoma. Science translational medicine 4, 115ra113.

Chapman, P.B., Hauschild, A., Robert, C., Haanen, J.B., Ascierto, P., Larkin, J., Dummer, R., Garbe, C., Testori, A., Maio, M., et al. (2011). Improved survival with vemurafenib in melanoma with BRAF V600E mutation. The New England journal of medicine 364, 2507-2516.

Charron, J., Malynn, B.A., Fisher, P., Stewart, V., Jeannotte, L., Goff, S.P., Robertson, E.J., and Alt, F.W. (1992). Embryonic lethality in mice homozygous for a targeted disruption of the N-myc gene. Genes & development 6, 2248-2257.

References 254 Chatten, J., Shimada, H., Sather, H.N., Wong, K.Y., Siegel, S.E., and Hammond, G.D. (1988). Prognostic value of histopathology in advanced neuroblastoma: a report from the Childrens Cancer Study Group. Human pathology 19, 1187-1198.

Chen, L., Iraci, N., Gherardi, S., Gamble, L.D., Wood, K.M., Perini, G., Lunec, J., and Tweddle, D.A. (2010). p53 is a direct transcriptional target of MYCN in neuroblastoma. Cancer Res 70, 1377-1388.

Chen, Y., Takita, J., Choi, Y.L., Kato, M., Ohira, M., Sanada, M., Wang, L., Soda, M., Kikuchi, A., Igarashi, T., et al. (2008). Oncogenic mutations of ALK kinase in neuroblastoma. Nature 455, 971-974.

Cheng, L., Yang, T., Kuang, Y., Kong, B., Yu, S., Shu, H., Zhou, H., and Gu, J. (2014). MicroRNA-23a promotes neuroblastoma cell metastasis by targeting CDH1. Oncology Letters 7, 839-845.

Cheng, L.Y., Bailey, A.P., Leevers, S.J., Ragan, T.J., Driscoll, P.C., and Gould, A.P. (2011). Anaplastic lymphoma kinase spares organ growth during nutrient restriction in Drosophila. Cell 146, 435-447.

Cheng, M., and Ott, G.R. (2010). Anaplastic lymphoma kinase as a therapeutic target in anaplastic large cell lymphoma, non-small cell lung cancer and neuroblastoma. Anti- cancer agents in medicinal chemistry 10, 236-249.

Chesler, L., Schlieve, C., Goldenberg, D.D., Kenney, A., Kim, G., McMillan, A., Matthay, K.K., Rowitch, D., and Weiss, W.A. (2006). Inhibition of phosphatidylinositol 3-kinase destabilizes Mycn protein and blocks malignant progression in neuroblastoma. Cancer Res 66, 8139-8146.

Ching, Y.P., Leong, V.Y., Lee, M.F., Xu, H.T., Jin, D.Y., and Ng, I.O. (2007). P21- activated protein kinase is overexpressed in hepatocellular carcinoma and enhances cancer metastasis involving c-Jun NH2-terminal kinase activation and paxillin phosphorylation. Cancer Res 67, 3601-3608.

Cho, H.Y., Gao, H., Srinivasan, A., Hong, J., Bencherif, S.A., Siegwart, D.J., Paik, H.J., Hollinger, J.O., and Matyjaszewski, K. (2010). Rapid cellular internalization of multifunctional star polymers prepared by atom transfer radical polymerization. Biomacromolecules 11, 2199-2203.

Cho, H.Y., Srinivasan, A., Hong, J., Hsu, E., Liu, S., Shrivats, A., Kwak, D., Bohaty, A.K., Paik, H.J., Hollinger, J.O., et al. (2011). Synthesis of biocompatible PEG-Based star polymers with cationic and degradable core for siRNA delivery. Biomacromolecules 12, 3478-3486.

Chou, T.C. (2010). Drug combination studies and their synergy quantification using the Chou-Talalay method. Cancer Res 70, 440-446.

References 255 Clemens, M.J. (1997). PKR--a protein kinase regulated by double-stranded RNA. The international journal of biochemistry & cell biology 29, 945-949.

Cleveland, J.L., Huleihel, M., Bressler, P., Siebenlist, U., Akiyama, L., Eisenman, R.N., and Rapp, U.R. (1988). Negative regulation of c-myc transcription involves myc family proteins. Oncogene research 3, 357-375.

Cohn, S.L., Pearson, A.D., London, W.B., Monclair, T., Ambros, P.F., Brodeur, G.M., Faldum, A., Hero, B., Iehara, T., Machin, D., et al. (2009). The International Neuroblastoma Risk Group (INRG) classification system: an INRG Task Force report. Journal of clinical oncology : official journal of the American Society of Clinical Oncology 27, 289-297.

Cole, M.D. (1986). The myc oncogene: its role in transformation and differentiation. Annual review of genetics 20, 361-384.

Cory, S., Huang, D.C., and Adams, J.M. (2003). The Bcl-2 family: roles in cell survival and oncogenesis. Oncogene 22, 8590-8607.

Cowling, V.H., Chandriani, S., Whitfield, M.L., and Cole, M.D. (2006). A conserved Myc protein domain, MBIV, regulates DNA binding, apoptosis, transformation, and G2 arrest. Molecular and cellular biology 26, 4226-4239.

Cowling, V.H., and Cole, M.D. (2006). Mechanism of transcriptional activation by the Myc oncoproteins. Semin Cancer Biol 16, 242-252.

Croce, C.M. (2008). Oncogenes and Cancer. New England Journal of Medicine 358, 502-511.

Dang, C.V. (2012). MYC on the path to cancer. Cell 149, 22-35.

Dang, C.V., Le, A., and Gao, P. (2009). MYC-induced cancer cell energy metabolism and therapeutic opportunities. Clinical cancer research : an official journal of the American Association for Cancer Research 15, 6479-6483.

Dang, C.V., Resar, L.M., Emison, E., Kim, S., Li, Q., Prescott, J.E., Wonsey, D., and Zeller, K. (1999). Function of the c-Myc oncogenic transcription factor. Exp Cell Res 253, 63-77.

Dawson, M.A., Prinjha, R.K., Dittmann, A., Giotopoulos, G., Bantscheff, M., Chan, W.I., Robson, S.C., Chung, C.W., Hopf, C., Savitski, M.M., et al. (2011). Inhibition of BET recruitment to chromatin as an effective treatment for MLL-fusion leukaemia. Nature 478, 529-533.

De Bernardi, B., Gerrard, M., Boni, L., Rubie, H., Canete, A., Di Cataldo, A., Castel, V., Forjaz de Lacerda, A., Ladenstein, R., Ruud, E., et al. (2009). Excellent outcome with reduced treatment for infants with disseminated neuroblastoma without MYCN

References 256 gene amplification. Journal of clinical oncology : official journal of the American Society of Clinical Oncology 27, 1034-1040.

De Bernardi, B., Nicolas, B., Boni, L., Indolfi, P., Carli, M., Cordero Di Montezemolo, L., Donfrancesco, A., Pession, A., Provenzi, M., di Cataldo, A., et al. (2003). Disseminated neuroblastoma in children older than one year at diagnosis: comparable results with three consecutive high-dose protocols adopted by the Italian Co-Operative Group for Neuroblastoma. Journal of clinical oncology : official journal of the American Society of Clinical Oncology 21, 1592-1601. de Pontual, L., Kettaneh, D., Gordon, C.T., Oufadem, M., Boddaert, N., Lees, M., Balu, L., Lachassinne, E., Petros, A., Mollet, J., et al. (2011). Germline gain-of-function mutations of ALK disrupt central nervous system development. Human mutation 32, 272-276.

Degoutin, J., Brunet-de Carvalho, N., Cifuentes-Diaz, C., and Vigny, M. (2009). ALK (Anaplastic Lymphoma Kinase) expression in DRG neurons and its involvement in neuron-Schwann cells interaction. The European journal of neuroscience 29, 275-286.

Delmore, J.E., Issa, G.C., Lemieux, M.E., Rahl, P.B., Shi, J., Jacobs, H.M., Kastritis, E., Gilpatrick, T., Paranal, R.M., Qi, J., et al. (2011). BET bromodomain inhibition as a therapeutic strategy to target c-Myc. Cell 146, 904-917.

Dey, A., Chitsaz, F., Abbasi, A., Misteli, T., and Ozato, K. (2003). The double bromodomain protein Brd4 binds to acetylated chromatin during interphase and mitosis. Proc Natl Acad Sci U S A 100, 8758-8763.

Di Paolo, D., Brignole, C., Pastorino, F., Carosio, R., Zorzoli, A., Rossi, M., Loi, M., Pagnan, G., Emionite, L., Cilli, M., et al. (2011). Neuroblastoma-targeted nanoparticles entrapping siRNA specifically knockdown ALK. Molecular therapy : the journal of the American Society of Gene Therapy 19, 1131-1140.

DuBois, S.G., Kalika, Y., Lukens, J.N., Brodeur, G.M., Seeger, R.C., Atkinson, J.B., Haase, G.M., Black, C.T., Perez, C., Shimada, H., et al. (1999). Metastatic sites in stage IV and IVS neuroblastoma correlate with age, tumor biology, and survival. Journal of pediatric hematology/oncology 21, 181-189.

Eberhardy, S.R., and Farnham, P.J. (2002). Myc recruits P-TEFb to mediate the final step in the transcriptional activation of the cad promoter. The Journal of biological chemistry 277, 40156-40162. el Alaoui, S., Lawry, J., and Griffin, M. (1997). The cell cycle and induction of apoptosis in a hamster fibrosarcoma cell line treated with anti-cancer drugs: its importance to solid tumour chemotherapy. Journal of neuro-oncology 31, 195-207.

Evans, A.E., D'Angio, G.J., Propert, K., Anderson, J., and Hann, H.W. (1987). Prognostic factor in neuroblastoma. Cancer 59, 1853-1859.

References 257 Evans, L., Chen, L., Milazzo, G., Gherardi, S., Perini, G., Willmore, E., Newell, D.R., and Tweddle, D.A. (2015). SKP2 is a direct transcriptional target of MYCN and a potential therapeutic target in neuroblastoma. Cancer Letters 363, 37-45.

Fakih, M.G. (2015). Metastatic Colorectal Cancer: Current State and Future Directions. Journal of Clinical Oncology 33, 1809-+.

Farrell, A.S., and Sears, R.C. (2014). MYC Degradation. Cold Spring Harbor Perspectives in Medicine 4.

Felsher, D.W. (2010). MYC Inactivation Elicits Oncogene Addiction through Both Tumor Cell-Intrinsic and Host-Dependent Mechanisms. Genes & cancer 1, 597-604.

Felsher, D.W., and Bishop, J.M. (1999). Reversible tumorigenesis by MYC in hematopoietic lineages. Mol Cell 4, 199-207.

Filippakopoulos, P., Qi, J., Picaud, S., Shen, Y., Smith, W.B., Fedorov, O., Morse, E.M., Keates, T., Hickman, T.T., Felletar, I., et al. (2010). Selective inhibition of BET bromodomains. Nature 468, 1067-1073.

Flinn, E.M., Busch, C.M., and Wright, A.P. (1998). myc boxes, which are conserved in myc family proteins, are signals for protein degradation via the proteasome. Molecular and cellular biology 18, 5961-5969.

Foley, N.H., Bray, I.M., Tivnan, A., Bryan, K., Murphy, D.M., Buckley, P.G., Ryan, J., O'Meara, A., O'Sullivan, M., and Stallings, R.L. (2010). MicroRNA-184 inhibits neuroblastoma cell survival through targeting the serine/threonine kinase AKT2. Mol Cancer 9, 83.

Fong, C.T., Dracopoli, N.C., White, P.S., Merrill, P.T., Griffith, R.C., Housman, D.E., and Brodeur, G.M. (1989). Loss of heterozygosity for the short arm of chromosome 1 in human neuroblastomas: correlation with N-myc amplification. Proc Natl Acad Sci U S A 86, 3753-3757.

Fraley, S.I., Feng, Y., Giri, A., Longmore, G.D., and Wirtz, D. (2012). Dimensional and temporal controls of three-dimensional cell migration by zyxin and binding partners. Nature communications 3, 719.

Frank, S.R., Parisi, T., Taubert, S., Fernandez, P., Fuchs, M., Chan, H.M., Livingston, D.M., and Amati, B. (2003). MYC recruits the TIP60 histone acetyltransferase complex to chromatin. EMBO reports 4, 575-580.

Frank, S.R., Schroeder, M., Fernandez, P., Taubert, S., and Amati, B. (2001). Binding of c-Myc to chromatin mediates mitogen-induced acetylation of histone H4 and gene activation. Genes & development 15, 2069-2082.

Frenzel, A., Loven, J., and Henriksson, M.A. (2010). Targeting MYC-Regulated miRNAs to Combat Cancer. Genes & cancer 1, 660-667.

References 258 Friboulet, L., Li, N., Katayama, R., Lee, C.C., Gainor, J.F., Crystal, A.S., Michellys, P.Y., Awad, M.M., Yanagitani, N., Kim, S., et al. (2014). The ALK inhibitor ceritinib overcomes crizotinib resistance in non-small cell lung cancer. Cancer Discov 4, 662- 673.

Fulda, S. (2009). The PI3K/Akt/mTOR pathway as therapeutic target in neuroblastoma. Current cancer drug targets 9, 729-737.

Fulda, S., Lutz, W., Schwab, M., and Debatin, K.M. (2000). MycN sensitizes neuroblastoma cells for drug-triggered apoptosis. Med Pediatr Oncol 35, 582-584.

Funa, K., Steinholtz, L., Nou, E., and Bergh, J. (1987). Increased expression of N-myc in human small cell lung cancer biopsies predicts lack of response to chemotherapy and poor prognosis. American journal of clinical pathology 88, 216-220.

Galkin, A.V., Melnick, J.S., Kim, S., Hood, T.L., Li, N., Li, L., Xia, G., Steensma, R., Chopiuk, G., Jiang, J., et al. (2007). Identification of NVP-TAE684, a potent, selective, and efficacious inhibitor of NPM-ALK. Proc Natl Acad Sci U S A 104, 270-275.

Gannon, P.O., Koumakpayi, I.H., Le Page, C., Karakiewicz, P.I., Mes-Masson, A.M., and Saad, F. (2008). Ebp1 expression in benign and malignant prostate. Cancer Cell Int 8.

Garaventa, A., Luksch, R., Biasotti, S., Severi, G., Pizzitola, M.R., Viscardi, E., Prete, A., Mastrangelo, S., Podda, M., Haupt, R., et al. (2003). A phase II study of topotecan with vincristine and doxorubicin in children with recurrent/refractory neuroblastoma. Cancer 98, 2488-2494.

Gasparian, A.V., Burkhart, C.A., Purmal, A.A., Brodsky, L., Pal, M., Saranadasa, M., Bosykh, D.A., Commane, M., Guryanova, O.A., Pal, S., et al. (2011). Curaxins: anticancer compounds that simultaneously suppress NF-kappaB and activate p53 by targeting FACT. Science translational medicine 3, 95ra74.

Geatti, O., Shapiro, B., Sisson, J.C., Hutchinson, R.J., Mallette, S., Eyre, P., and Beierwaltes, W.H. (1985). Iodine-131 metaiodobenzylguanidine scintigraphy for the location of neuroblastoma: preliminary experience in ten cases. Journal of nuclear medicine : official publication, Society of Nuclear Medicine 26, 736-742.

George, R.E., Sanda, T., Hanna, M., Frohling, S., Luther, W., 2nd, Zhang, J., Ahn, Y., Zhou, W., London, W.B., McGrady, P., et al. (2008). Activating mutations in ALK provide a therapeutic target in neuroblastoma. Nature 455, 975-978.

Germain, M., Affar, E.B., D'Amours, D., Dixit, V.M., Salvesen, G.S., and Poirier, G.G. (1999). Cleavage of automodified poly(ADP-ribose) polymerase during apoptosis. Evidence for involvement of caspase-7. The Journal of biological chemistry 274, 28379-28384.

References 259 Gerner, E.W., and Meyskens, F.L., Jr. (2004). Polyamines and cancer: old molecules, new understanding. Nature reviews Cancer 4, 781-792.

Gilbert, F., Feder, M., Balaban, G., Brangman, D., Lurie, D.K., Podolsky, R., Rinaldt, V., Vinikoor, N., and Weisband, J. (1984). Human neuroblastomas and abnormalities of chromosomes 1 and 17. Cancer Res 44, 5444-5449.

Giuriato, S., Ryeom, S., Fan, A.C., Bachireddy, P., Lynch, R.C., Rioth, M.J., van Riggelen, J., Kopelman, A.M., Passegue, E., Tang, F., et al. (2006). Sustained regression of tumors upon MYC inactivation requires p53 or thrombospondin-1 to reverse the angiogenic switch. Proc Natl Acad Sci U S A 103, 16266-16271.

Glover, D.M., Leibowitz, M.H., McLean, D.A., and Parry, H. (1995). Mutations in aurora prevent centrosome separation leading to the formation of monopolar spindles. Cell 81, 95-105.

Goc, A., Al-Azayzih, A., Abdalla, M., Al-Husein, B., Kavuri, S., Lee, J., Moses, K., and Somanath, P.R. (2013). P21 activated kinase-1 (Pak1) promotes prostate tumor growth and microinvasion via inhibition of transforming growth factor beta expression and enhanced matrix metalloproteinase 9 secretion. The Journal of biological chemistry 288, 3025-3035.

Goldman, R., Levy, R.B., Peles, E., and Yarden, Y. (1990). Heterodimerization of the erbB-1 and erbB-2 receptors in human breast carcinoma cells: a mechanism for receptor transregulation. Biochemistry 29, 11024-11028.

Goldsby, R.E., and Matthay, K.K. (2004). Neuroblastoma: evolving therapies for a disease with many faces. Paediatric drugs 6, 107-122.

Gossen, M., Freundlieb, S., Bender, G., Muller, G., Hillen, W., and Bujard, H. (1995). Transcriptional activation by tetracyclines in mammalian cells. Science 268, 1766-1769.

Greengard, E.G., and Park, J.R. (1993). ALK-Related Neuroblastic Tumor Susceptibility. In GeneReviews(R), R.A. Pagon, M.P. Adam, H.H. Ardinger, S.E. Wallace, A. Amemiya, L.J.H. Bean, T.D. Bird, C.T. Fong, H.C. Mefford, R.J.H. Smith, et al., eds. (Seattle (WA)).

Gregory, M.A., and Hann, S.R. (2000). c-Myc proteolysis by the ubiquitin-proteasome pathway: stabilization of c-Myc in Burkitt's lymphoma cells. Molecular and cellular biology 20, 2423-2435.

Grignani, F., Lombardi, L., Inghirami, G., Sternas, L., Cechova, K., and Dalla-Favera, R. (1990). Negative autoregulation of c-myc gene expression is inactivated in transformed cells. The EMBO journal 9, 3913-3922.

Gullick, W.J. (1991). Prevalence of aberrant expression of the epidermal growth factor receptor in human cancers. British medical bulletin 47, 87-98.

References 260 Gupta, S., Seth, A., and Davis, R.J. (1993). Transactivation of gene expression by Myc is inhibited by mutation at the phosphorylation sites Thr-58 and Ser-62. Proc Natl Acad Sci U S A 90, 3216-3220.

Gustafson, W.C., Meyerowitz, J.G., Nekritz, E.A., Chen, J., Benes, C., Charron, E., Simonds, E.F., Seeger, R., Matthay, K.K., Hertz, N.T., et al. (2014). Drugging MYCN through an allosteric transition in Aurora kinase A. Cancer Cell 26, 414-427.

Gustafson, W.C., and Weiss, W.A. (2010). Myc proteins as therapeutic targets. Oncogene 29, 1249-1259.

Hande, K.R. (1998). Etoposide: four decades of development of a topoisomerase II inhibitor. Eur J Cancer 34, 1514-1521.

Harmon, B.V., Takano, Y.S., Winterford, C.M., and Potten, C.S. (1992). Cell death induced by vincristine in the intestinal crypts of mice and in a human Burkitt's lymphoma cell line. Cell proliferation 25, 523-536.

Hartmann, O., Valteau-Couanet, D., Vassal, G., Lapierre, V., Brugieres, L., Delgado, R., Couanet, D., Lumbroso, J., and Benhamou, E. (1999). Prognostic factors in metastatic neuroblastoma in patients over 1 year of age treated with high-dose chemotherapy and stem cell transplantation: a multivariate analysis in 218 patients treated in a single institution. Bone marrow transplantation 23, 789-795.

Heery, D.M., Kalkhoven, E., Hoare, S., and Parker, M.G. (1997). A signature motif in transcriptional co-activators mediates binding to nuclear receptors. Nature 387, 733- 736.

Henriksson, M., Bakardjiev, A., Klein, G., and Luscher, B. (1993). Phosphorylation sites mapping in the N-terminal domain of c-myc modulate its transforming potential. Oncogene 8, 3199-3209.

Henriksson, M., and Luscher, B. (1996). Proteins of the Myc network: essential regulators of cell growth and differentiation. Adv Cancer Res 68, 109-182.

Hershko, A., and Ciechanover, A. (1998). The ubiquitin system. Annual review of biochemistry 67, 425-479.

Hidalgo, M., Amant, F., Biankin, A.V., Budinska, E., Byrne, A.T., Caldas, C., Clarke, R.B., de Jong, S., Jonkers, J., Maelandsmo, G.M., et al. (2014). Patient-derived xenograft models: an emerging platform for translational cancer research. Cancer Discov 4, 998-1013.

Hoelder, S., Clarke, P.A., and Workman, P. (2012). Discovery of small molecule cancer drugs: successes, challenges and opportunities. Molecular oncology 6, 155-176.

Hogarty, M.D., Norris, M.D., Davis, K., Liu, X., Evageliou, N.F., Hayes, C.S., Pawel, B., Guo, R., Zhao, H., Sekyere, E., et al. (2008). ODC1 is a critical determinant of

References 261 MYCN oncogenesis and a therapeutic target in neuroblastoma. Cancer Res 68, 9735- 9745.

Hu, Y.F., Luscher, B., Admon, A., Mermod, N., and Tjian, R. (1990). Transcription factor AP-4 contains multiple dimerization domains that regulate dimer specificity. Genes & development 4, 1741-1752.

Huang, M., and Weiss, W.A. (2013). Neuroblastoma and MYCN. Cold Spring Harb Perspect Med 3, a014415.

Hunger, S.P., Lu, X., Devidas, M., Camitta, B.M., Gaynon, P.S., Winick, N.J., Reaman, G.H., and Carroll, W.L. (2012). Improved survival for children and adolescents with acute lymphoblastic leukemia between 1990 and 2005: a report from the children's oncology group. Journal of clinical oncology : official journal of the American Society of Clinical Oncology 30, 1663-1669.

Ikegaki, N., Bukovsky, J., and Kennett, R.H. (1986). Identification and characterization of the NMYC gene product in human neuroblastoma cells by monoclonal antibodies with defined specificities. Proc Natl Acad Sci U S A 83, 5929-5933.

Ikegaki, N., Gotoh, T., Kung, B., Riceberg, J.S., Kim, D.Y., Zhao, H., Rappaport, E.F., Hicks, S.L., Seeger, R.C., and Tang, X.X. (2007). De novo identification of MIZ-1 (ZBTB17) encoding a MYC-interacting zinc-finger protein as a new favorable neuroblastoma gene. Clinical cancer research : an official journal of the American Association for Cancer Research 13, 6001-6009.

Ingvarsson, S., Asker, C., Axelson, H., Klein, G., and Sumegi, J. (1988). Structure and expression of B-myc, a new member of the myc gene family. Molecular and cellular biology 8, 3168-3174.

Iraci, N., Diolaiti, D., Papa, A., Porro, A., Valli, E., Gherardi, S., Herold, S., Eilers, M., Bernardoni, R., Della Valle, G., et al. (2011). A SP1/MIZ1/MYCN repression complex recruits HDAC1 at the TRKA and p75NTR promoters and affects neuroblastoma malignancy by inhibiting the cell response to NGF. Cancer Res 71, 404-412.

Iragavarapu, C., Mustafa, M., Akinleye, A., Furqan, M., Mittal, V., Cang, S., and Liu, D. (2015). Novel ALK inhibitors in clinical use and development. Journal of hematology & oncology 8, 17.

Jackstadt, R., Roh, S., Neumann, J., Jung, P., Hoffmann, R., Horst, D., Berens, C., Bornkamm, G.W., Kirchner, T., Menssen, A., et al. (2013). AP4 is a mediator of epithelial-mesenchymal transition and metastasis in colorectal cancer. Journal of Experimental Medicine 210, 1331-1350.

Jacobs, J.F., van Bokhoven, H., van Leeuwen, F.N., Hulsbergen-van de Kaa, C.A., de Vries, I.J., Adema, G.J., Hoogerbrugge, P.M., and de Brouwer, A.P. (2009). Regulation of MYCN expression in human neuroblastoma cells. Bmc Cancer 9, 239.

References 262 Jain, M., Arvanitis, C., Chu, K., Dewey, W., Leonhardt, E., Trinh, M., Sundberg, C.D., Bishop, J.M., and Felsher, D.W. (2002). Sustained loss of a neoplastic phenotype by brief inactivation of MYC. Science 297, 102-104.

Jang, M.K., Mochizuki, K., Zhou, M., Jeong, H.S., Brady, J.N., and Ozato, K. (2005). The bromodomain protein Brd4 is a positive regulatory component of P-TEFb and stimulates RNA polymerase II-dependent transcription. Mol Cell 19, 523-534.

Janoueix-Lerosey, I., Lequin, D., Brugieres, L., Ribeiro, A., de Pontual, L., Combaret, V., Raynal, V., Puisieux, A., Schleiermacher, G., Pierron, G., et al. (2008). Somatic and germline activating mutations of the ALK kinase receptor in neuroblastoma. Nature 455, 967-970.

Jiang, M., Stanke, J., and Lahti, J.M. (2011). The connections between neural crest development and neuroblastoma. Current topics in developmental biology 94, 77-127.

Johnson, S.M., Grosshans, H., Shingara, J., Byrom, M., Jarvis, R., Cheng, A., Labourier, E., Reinert, K.L., Brown, D., and Slack, F.J. (2005). RAS is regulated by the let-7 microRNA family. Cell 120, 635-647.

Jung, P., and Hermeking, H. (2009). The c-MYC-AP4-p21 cascade. Cell Cycle 8, 982- 989.

Jung, P., Menssen, A., Mayr, D., and Hermeking, H. (2008). AP4 encodes a c-MYC- inducible repressor of p21. Proc Natl Acad Sci U S A 105, 15046-15051.

Kaneko, Y., Kanda, N., Maseki, N., Sakurai, M., Tsuchida, Y., Takeda, T., and Okabe, I. (1987). Different karyotypic patterns in early and advanced stage neuroblastomas. Cancer Res 47, 311-318.

Kang, J.H., Rychahou, P.G., Ishola, T.A., Qiao, J., Evers, B.M., and Chung, D.H. (2006). MYCN silencing induces differentiation and apoptosis in human neuroblastoma cells. Biochem Biophys Res Commun 351, 192-197.

Kapeli, K., and Hurlin, P.J. (2011). Differential regulation of N-Myc and c-Myc synthesis, degradation, and transcriptional activity by the Ras/mitogen-activated protein kinase pathway. The Journal of biological chemistry 286, 38498-38508.

Karin, M., and Delhase, M. (2000). The I kappa B kinase (IKK) and NF-kappa B: key elements of proinflammatory signalling. Seminars in immunology 12, 85-98.

Kato, G.J., Barrett, J., Villa-Garcia, M., and Dang, C.V. (1990). An amino-terminal c- myc domain required for neoplastic transformation activates transcription. Molecular and cellular biology 10, 5914-5920.

Khanna, C., Jaboin, J.J., Drakos, E., Tsokos, M., and Thiele, C.J. (2002). Biologically relevant orthotopic neuroblastoma xenograft models: primary adrenal tumor growth and spontaneous distant metastasis. In Vivo 16, 77-85.

References 263 Killion, J.J., Radinsky, R., and Fidler, I.J. (1998). Orthotopic models are necessary to predict therapy of transplantable tumors in mice. Cancer metastasis reviews 17, 279- 284.

Kim, C.K., Lee, S.B., Nguyen, T.L., Lee, K.H., Um, S.H., Kim, J., and Ahn, J.Y. (2012). Long isoform of ErbB3 binding protein, p48, mediates protein kinase B/Akt- dependent HDM2 stabilization and nuclear localization. Exp Cell Res 318, 136-143.

Kim, C.K., Nguyen, T.L.X., Joo, K.M., Nam, D.H., Park, J., Lee, K.H., Cho, S.W., and Ahn, J.Y. (2010). Negative Regulation of p53 by the Long Isoform of ErbB3 Binding Protein Ebp1 in Brain Tumors. Cancer Research 70, 9730-9741.

Kim, H.H., Sierke, S.L., and Koland, J.G. (1994). Epidermal growth factor-dependent association of phosphatidylinositol 3-kinase with the erbB3 gene product. The Journal of biological chemistry 269, 24747-24755.

Kim, S.Y., Herbst, A., Tworkowski, K.A., Salghetti, S.E., and Tansey, W.P. (2003). Skp2 regulates Myc protein stability and activity. Molecular Cell 11, 1177-1188.

Knoepfler, P.S., Cheng, P.F., and Eisenman, R.N. (2002). N-myc is essential during neurogenesis for the rapid expansion of progenitor cell populations and the inhibition of neuronal differentiation. Genes & development 16, 2699-2712.

Knudson, A.G., Jr., and Amromin, G.D. (1966). Neuroblastoma and ganglioneuroma in a child with multiple neurofibromatosis. Implications for the mutational origin of neuroblastoma. Cancer 19, 1032-1037.

Ko, H.R., Nguyen, T.L., Kim, C.K., Park, Y., Lee, K.H., and Ahn, J.Y. (2015). P42 Ebp1 functions as a tumor suppressor in non-small cell lung cancer. BMB reports 48, 159-165.

Koehler, A.N., Shamji, A.F., and Schreiber, S.L. (2003). Discovery of an inhibitor of a transcription factor using small molecule microarrays and diversity-oriented synthesis. Journal of the American Chemical Society 125, 8420-8421.

Kohl, N.E., Kanda, N., Schreck, R.R., Bruns, G., Latt, S.A., Gilbert, F., and Alt, F.W. (1983). Transposition and Amplification of Oncogene-Related Sequences in Human Neuroblastomas. Cell 35, 359-367.

Kohl, N.E., Legouy, E., DePinho, R.A., Nisen, P.D., Smith, R.K., Gee, C.E., and Alt, F.W. (1986). Human N-myc is closely related in organization and nucleotide sequence to c-myc. Nature 319, 73-77.

Kohler, J.A., Rubie, H., Castel, V., Beiske, K., Holmes, K., Gambini, C., Casale, F., Munzer, C., Erminio, G., Parodi, S., et al. (2013). Treatment of children over the age of one year with unresectable localised neuroblastoma without MYCN amplification: results of the SIOPEN study. Eur J Cancer 49, 3671-3679.

References 264 Kowalinski, E., Bange, G., Bradatsch, B., Hurt, E., Wild, K., and Sinning, I. (2007). The crystal structure of Ebp1 reveals a methionine aminopeptidase fold as binding platform for multiple interactions. FEBS letters 581, 4450-4454.

Kraus, M.H., Issing, W., Miki, T., Popescu, N.C., and Aaronson, S.A. (1989). Isolation and characterization of ERBB3, a third member of the ERBB/epidermal growth factor receptor family: evidence for overexpression in a subset of human mammary tumors. Proc Natl Acad Sci U S A 86, 9193-9197.

Kumar, R., Gururaj, A.E., and Barnes, C.J. (2006). p21-activated kinases in cancer. Nature reviews Cancer 6, 459-471.

Kumimoto, H., Hamajima, N., Nishimoto, Y., Matsuo, K., Shinoda, M., Hatooka, S., and Ishizaki, K. (2002). L-myc genotype is associated with different susceptibility to lung cancer in smokers. Japanese journal of cancer research : Gann 93, 1-5.

Kushner, B.H., and Cheung, N.K. (1989). GM-CSF enhances 3F8 monoclonal antibody-dependent cellular cytotoxicity against human melanoma and neuroblastoma. Blood 73, 1936-1941.

Kushner, B.H., Kramer, K., Modak, S., Qin, L.X., and Cheung, N.K. (2010). Differential impact of high-dose cyclophosphamide, topotecan, and vincristine in clinical subsets of patients with chemoresistant neuroblastoma. Cancer 116, 3054-3060.

Kushner, B.H., LaQuaglia, M.P., Bonilla, M.A., Lindsley, K., Rosenfield, N., Yeh, S., Eddy, J., Gerald, W.L., Heller, G., and Cheung, N.K. (1994). Highly effective induction therapy for stage 4 neuroblastoma in children over 1 year of age. Journal of clinical oncology : official journal of the American Society of Clinical Oncology 12, 2607- 2613.

Kwak, E.L., Bang, Y.J., Camidge, D.R., Shaw, A.T., Solomon, B., Maki, R.G., Ou, S.H., Dezube, B.J., Janne, P.A., Costa, D.B., et al. (2010). Anaplastic lymphoma kinase inhibition in non-small-cell lung cancer. The New England journal of medicine 363, 1693-1703.

Ladenstein, R., Poetschger, U., Luksch, R., and al:, e. (2011). Busulphan-melphalan is the superior myeloablative Therapy (mat) for high risk neuroblastoma: Results From the hr-nbl1/siopen trial. J Clin Oncol 29:5s (suppl; abstr 2).

Ladenstein, R., Valteau-Couanet, D., Brock, P., Yaniv, I., Castel, V., Laureys, G., Malis, J., Papadakis, V., Lacerda, A., Ruud, E., et al. (2010). Randomized Trial of prophylactic granulocyte colony-stimulating factor during rapid COJEC induction in pediatric patients with high-risk neuroblastoma: the European HR-NBL1/SIOPEN study. Journal of clinical oncology : official journal of the American Society of Clinical Oncology 28, 3516-3524.

Lagos-Quintana, M., Rauhut, R., Lendeckel, W., and Tuschl, T. (2001). Identification of novel genes coding for small expressed RNAs. Science 294, 853-858.

References 265 Lai, A., Kennedy, B.K., Barbie, D.A., Bertos, N.R., Yang, X.J., Theberge, M.C., Tsai, S.C., Seto, E., Zhang, Y., Kuzmichev, A., et al. (2001). RBP1 recruits the mSIN3- histone deacetylase complex to the pocket of retinoblastoma tumor suppressor family proteins found in limited discrete regions of the nucleus at growth arrest. Molecular and cellular biology 21, 2918-2932.

Lamartine, J., Seri, M., Cinti, R., Heitzmann, F., Creaven, M., Radomski, N., Jost, E., Lenoir, G.M., Romeo, G., and Sylla, B.S. (1997). Molecular cloning and mapping of a human cDNA (PA2G4) that encodes a protein highly homologous to the mouse cell cycle protein p38-2G4. Cytogenet Cell Genet 78, 31-35.

Larsson, L.G., and Henriksson, M.A. (2010). The Yin and Yang functions of the Myc oncoprotein in cancer development and as targets for therapy. Exp Cell Res 316, 1429- 1437.

Lavorgna, G., Dahary, D., Lehner, B., Sorek, R., Sanderson, C.M., and Casari, G. (2004). In search of antisense. Trends in biochemical sciences 29, 88-94.

Le Dourin, N.M., and Kalcheim, C. (1999). The Neural Crest 2nd ed, 2nd ed. edn (Cambridge University Press.).

Lee, W.H., Murphree, A.L., and Benedict, W.F. (1984). Expression and amplification of the N-myc gene in primary retinoblastoma. Nature 309, 458-460.

Legouy, E., DePinho, R., Zimmerman, K., Collum, R., Yancopoulos, G., Mitsock, L., Kriz, R., and Alt, F.W. (1987). Structure and expression of the murine L-myc gene. The EMBO journal 6, 3359-3366.

Lemoine, N.R., Barnes, D.M., Hollywood, D.P., Hughes, C.M., Smith, P., Dublin, E., Prigent, S.A., Gullick, W.J., and Hurst, H.C. (1992). Expression of the ERBB3 gene product in breast cancer. Br J Cancer 66, 1116-1121.

Lessor, T.J., and Hamburger, A.W. (2001). Regulation of the ErbB3 binding protein Ebp1 by protein kinase C. Mol Cell Endocrinol 175, 185-191.

Lessor, T.J., Yoo, J.Y., Xia, X.M., Woodford, N., and Hamburger, A.W. (2000). Ectopic expression of the ErbB-3 binding protein Ebp1 inhibits growth and induces differentiation of human breast cancer cell lines. J Cell Physiol 183, 321-329.

Leung, C.H., Chan, D.S., Ma, V.P., and Ma, D.L. (2013). DNA-binding small molecules as inhibitors of transcription factors. Medicinal research reviews 33, 823-846.

Li, F., Zhao, C., and Wang, L. (2014). Molecular-targeted agents combination therapy for cancer: developments and potentials. International journal of cancer Journal international du cancer 134, 1257-1269.

Li, Z.W., Chu, W., Hu, Y., Delhase, M., Deerinck, T., Ellisman, M., Johnson, R., and Karin, M. (1999). The IKKbeta subunit of IkappaB kinase (IKK) is essential for nuclear

References 266 factor kappaB activation and prevention of apoptosis. The Journal of experimental medicine 189, 1839-1845.

Lin, C.Y., Loven, J., Rahl, P.B., Paranal, R.M., Burge, C.B., Bradner, J.E., Lee, T.I., and Young, R.A. (2012). Transcriptional amplification in tumor cells with elevated c- Myc. Cell 151, 56-67.

Liu, P.Y., Erriquez, D., Marshall, G.M., Tee, A.E., Polly, P., Wong, M., Liu, B., Bell, J.L., Zhang, X.D., Milazzo, G., et al. (2014). Effects of a novel long noncoding RNA, lncUSMycN, on N-Myc expression and neuroblastoma progression. Journal of the National Cancer Institute 106.

Liu, P.Y., Xu, N., Malyukova, A., Scarlett, C.J., Sun, Y.T., Zhang, X.D., Ling, D., Su, S.P., Nelson, C., Chang, D.K., et al. (2013). The histone deacetylase SIRT2 stabilizes Myc oncoproteins. Cell death and differentiation 20, 503-514.

Liu, T., Tee, A.E.L., Porro, A., Smith, S.A., Dwarte, T., Liu, P.Y., Iraci, N., Sekyere, E., Haber, M., Norris, M.D., et al. (2007). Activation of tissue transglutaminase transcription by histone deacetylase inhibition as a therapeutic approach for Myc oncogenesis. Proc Natl Acad Sci U S A 104, 18682-18687.

Liu, Z.X., Ahn, J.Y., Liu, X., and Ye, K.Q. (2006). Ebp1 isoforms distinctively regulate cell survival and differentiation. Proc Natl Acad Sci U S A 103, 10917-10922.

Liu, Z.X., Oh, S.M., Okada, M., Liu, X., Cheng, D.M., Peng, J.M., Brat, D.J., Sun, S.Y., Zhou, W., Gu, W., et al. (2009). Human BRE1 Is an E3 Ubiquitin Ligase for Ebp1 Tumor Suppressor. Molecular Biology of the Cell 20, 757-768.

Loi, M., Di Paolo, D., Soster, M., Brignole, C., Bartolini, A., Emionite, L., Sun, J., Becherini, P., Curnis, F., Petretto, A., et al. (2013). Novel phage display-derived neuroblastoma-targeting peptides potentiate the effect of drug nanocarriers in preclinical settings. Journal of controlled release : official journal of the Controlled Release Society 170, 233-241.

Loike, J.D., and Horwitz, S.B. (1976). Effects of podophyllotoxin and VP-16-213 on microtubule assembly in vitro and nucleoside transport in HeLa cells. Biochemistry 15, 5435-5443.

London, W.B., Castel, V., Monclair, T., Ambros, P.F., Pearson, A.D., Cohn, S.L., Berthold, F., Nakagawara, A., Ladenstein, R.L., Iehara, T., et al. (2011). Clinical and biologic features predictive of survival after relapse of neuroblastoma: a report from the International Neuroblastoma Risk Group project. Journal of clinical oncology : official journal of the American Society of Clinical Oncology 29, 3286-3292.

London, W.B., Castleberry, R.P., Matthay, K.K., Look, A.T., Seeger, R.C., Shimada, H., Thorner, P., Brodeur, G., Maris, J.M., Reynolds, C.P., et al. (2005). Evidence for an age cutoff greater than 365 days for neuroblastoma risk group stratification in the Children's Oncology Group. Journal of clinical oncology : official journal of the American Society of Clinical Oncology 23, 6459-6465.

References 267 London, W.B., Frantz, C.N., Campbell, L.A., Seeger, R.C., Brumback, B.A., Cohn, S.L., Matthay, K.K., Castleberry, R.P., and Diller, L. (2010). Phase II randomized comparison of topotecan plus cyclophosphamide versus topotecan alone in children with recurrent or refractory neuroblastoma: a Children's Oncology Group study. Journal of clinical oncology : official journal of the American Society of Clinical Oncology 28, 3808-3815.

Look, A.T., Hayes, F.A., Nitschke, R., McWilliams, N.B., and Green, A.A. (1984). Cellular DNA content as a predictor of response to chemotherapy in infants with unresectable neuroblastoma. The New England journal of medicine 311, 231-235.

Luscher, B., and Larsson, L.G. (1999). The basic region/helix-loop-helix/leucine zipper domain of Myc proto-oncoproteins: function and regulation. Oncogene 18, 2955-2966.

Ma, L., Young, J., Prabhala, H., Pan, E., Mestdagh, P., Muth, D., Teruya-Feldstein, J., Reinhardt, F., Onder, T.T., Valastyan, S., et al. (2010). miR-9, a MYC/MYCN- activated microRNA, regulates E-cadherin and cancer metastasis. Nature cell biology 12, 247-256.

Malynn, B.A., de Alboran, I.M., O'Hagan, R.C., Bronson, R., Davidson, L., DePinho, R.A., and Alt, F.W. (2000). N-myc can functionally replace c-myc in murine development, cellular growth, and differentiation. Genes & development 14, 1390- 1399.

Maris, J.M. (2005). The biologic basis for neuroblastoma heterogeneity and risk stratification. Current opinion in pediatrics 17, 7-13.

Maris, J.M. (2010). Recent advances in neuroblastoma. The New England journal of medicine 362, 2202-2211.

Maris, J.M., Chatten, J., Meadows, A.T., Biegel, J.A., and Brodeur, G.M. (1997). Familial neuroblastoma: a three-generation pedigree and a further association with Hirschsprung disease. Med Pediatr Oncol 28, 1-5.

Maris, J.M., Hogarty, M.D., Bagatell, R., and Cohn, S.L. (2007). Neuroblastoma. Lancet 369, 2106-2120.

Maris, J.M., Morton, C.L., Gorlick, R., Kolb, E.A., Lock, R., Carol, H., Keir, S.T., Reynolds, C.P., Kang, M.H., Wu, J., et al. (2010). Initial testing of the aurora kinase A inhibitor MLN8237 by the Pediatric Preclinical Testing Program (PPTP). Pediatric blood & cancer 55, 26-34.

Maris, J.M., Weiss, M.J., Mosse, Y., Hii, G., Guo, C., White, P.S., Hogarty, M.D., Mirensky, T., Brodeur, G.M., Rebbeck, T.R., et al. (2002). Evidence for a hereditary neuroblastoma predisposition locus at chromosome 16p12-13. Cancer Res 62, 6651- 6658.

References 268 Marshall, G.M., Carter, D.R., Cheung, B.B., Liu, T., Mateos, M.K., Meyerowitz, J.G., and Weiss, W.A. (2014). The prenatal origins of cancer. Nature reviews Cancer 14, 277-289.

Marshall, G.M., Liu, P.Y., Gherardi, S., Scarlett, C.J., Bedalov, A., Xu, N., Iraci, N., Valli, E., Ling, D., Thomas, W., et al. (2011). SIRT1 promotes N-Myc oncogenesis through a positive feedback loop involving the effects of MKP3 and ERK on N-Myc protein stability. PLoS genetics 7, e1002135.

Martinez, M.E., O'Brien, T.G., Fultz, K.E., Babbar, N., Yerushalmi, H., Qu, N., Guo, Y., Boorman, D., Einspahr, J., Alberts, D.S., et al. (2003). Pronounced reduction in adenoma recurrence associated with aspirin use and a polymorphism in the ornithine decarboxylase gene. Proc Natl Acad Sci U S A 100, 7859-7864.

Marumoto, T., Honda, S., Hara, T., Nitta, M., Hirota, T., Kohmura, E., and Saya, H. (2003). Aurora-A kinase maintains the fidelity of early and late mitotic events in HeLa cells. The Journal of biological chemistry 278, 51786-51795.

Masdehors, P., Glaisner, S., Maciorowski, Z., Magdelenat, H., and Delic, J. (2000). Ubiquitin-dependent protein processing controls radiation-induced apoptosis through the N-end rule pathway. Exp Cell Res 257, 48-57.

Matthay, K.K., and Reynolds, C.P. (2000). Is there a role for retinoids to treat minimal residual disease in neuroblastoma? Br J Cancer 83, 1121-1123.

Matthay, K.K., Reynolds, C.P., Seeger, R.C., Shimada, H., Adkins, E.S., Haas-Kogan, D., Gerbing, R.B., London, W.B., and Villablanca, J.G. (2009). Long-Term Results for Children With High-Risk Neuroblastoma Treated on a Randomized Trial of Myeloablative Therapy Followed by 13-cis-Retinoic Acid: A Children's Oncology Group Study. Journal of Clinical Oncology 27, 1007-1013.

Matthay, K.K., Villablanca, J.G., Seeger, R.C., Stram, D.O., Harris, R.E., Ramsay, N.K., Swift, P., Shimada, H., Black, C.T., Brodeur, G.M., et al. (1999a). Treatment of high-risk neuroblastoma with intensive chemotherapy, radiotherapy, autologous bone marrow transplantation, and 13-cis-retinoic acid. Children's Cancer Group. The New England journal of medicine 341, 1165-1173.

Matthay, K.K., Villablanca, J.G., Seeger, R.C., Stram, D.O., Harris, R.E., Ramsay, N.K., Swift, P., Shimada, H., Black, C.T., Brodeur, G.M., et al. (1999b). Treatment of high-risk neuroblastoma with intensive chemotherapy, radiotherapy, autologous bone marrow transplantation, and 13-cis-retinoic acid. New England Journal of Medicine 341, 1165-1173.

McEwan, I.J., Dahlman-Wright, K., Ford, J., and Wright, A.P. (1996). Functional interaction of the c-Myc transactivation domain with the TATA binding protein: evidence for an induced fit model of transactivation domain folding. Biochemistry 35, 9584-9593.

References 269 McMahon, S.B., Wood, M.A., and Cole, M.D. (2000). The essential cofactor TRRAP recruits the histone acetyltransferase hGCN5 to c-Myc. Molecular and cellular biology 20, 556-562.

Mei, H., Lin, Z.-Y., and Tong, Q.-S. (2014a). The roles of microRNAs in neuroblastoma. World Journal of Pediatrics 10, 10-16.

Mei, Y., Zhang, P., Zuo, H., Clark, D., Xia, R., Li, J., Liu, Z., and Mao, L. (2014b). Ebp1 activates podoplanin expression and contributes to oral tumorigenesis. Oncogene 33, 3839-3850.

Mercer, T.R., Dinger, M.E., and Mattick, J.S. (2009). Long non-coding RNAs: insights into functions. Nature reviews Genetics 10, 155-159.

Mertz, J.A., Conery, A.R., Bryant, B.M., Sandy, P., Balasubramanian, S., Mele, D.A., Bergeron, L., and Sims, R.J., 3rd (2011). Targeting MYC dependence in cancer by inhibiting BET bromodomains. Proc Natl Acad Sci U S A 108, 16669-16674.

Mestdagh, P., Fredlund, E., Pattyn, F., Rihani, A., Van Maerken, T., Vermeulen, J., Kumps, C., Menten, B., De Preter, K., Schramm, A., et al. (2010a). An integrative genomics screen uncovers ncRNA T-UCR functions in neuroblastoma tumours. Oncogene 29, 3583-3592.

Mestdagh, P., Fredlund, E., Pattyn, F., Schulte, J.H., Muth, D., Vermeulen, J., Kumps, C., Schlierf, S., De Preter, K., Van Roy, N., et al. (2010b). MYCN/c-MYC-induced microRNAs repress coding gene networks associated with poor outcome in MYCN/c- MYC-activated tumors. Oncogene 29, 1394-1404.

Meyer, N., and Penn, L.Z. (2008). Reflecting on 25 years with MYC. Nature reviews Cancer 8, 976-990.

Minard, V., Hartmann, O., Peyroulet, M.C., Michon, J., Coze, C., Defachelle, A.S., Lejars, O., Perel, Y., Bergeron, C., Boutard, P., et al. (2000). Adverse outcome of infants with metastatic neuroblastoma, MYCN amplification and/or bone lesions: results of the French society of pediatric oncology. Br J Cancer 83, 973-979.

Mizukami, Y., Nonomura, A., Takizawa, T., Noguchi, M., Michigishi, T., Nakamura, S., and Ishizaki, T. (1995). N-myc protein expression in human breast carcinoma: prognostic implications. Anticancer research 15, 2899-2905.

Mo, H., and Henriksson, M. (2006). Identification of small molecules that induce apoptosis in a Myc-dependent manner and inhibit Myc-driven transformation. Proc Natl Acad Sci U S A 103, 6344-6349.

Monclair, T., Brodeur, G.M., Ambros, P.F., Brisse, H.J., Cecchetto, G., Holmes, K., Kaneko, M., London, W.B., Matthay, K.K., Nuchtern, J.G., et al. (2009). The International Neuroblastoma Risk Group (INRG) Staging System: An INRG Task Force Report. Journal of Clinical Oncology 27, 298-303.

References 270 Monie, T.P., Perrin, A.J., Birtley, J.R., Sweeney, T.R., Karakasiliotis, I., Chaudhry, Y., Roberts, L.O., Matthews, S., Goodfellow, I.G., and Curry, S. (2007). Structural insights into the transcriptional and translational roles of Ebp1. Embo J 26, 3936-3944.

Moore, H.C., Wood, K.M., Jackson, M.S., Lastowska, M.A., Hall, D., Imrie, H., Redfern, C.P., Lovat, P.E., Ponthan, F., O'Toole, K., et al. (2008). Histological profile of tumours from MYCN transgenic mice. Journal of clinical pathology 61, 1098-1103.

Morris, S.W., Kirstein, M.N., Valentine, M.B., Dittmer, K., Shapiro, D.N., Look, A.T., and Saltman, D.L. (1995). Fusion of a kinase gene, ALK, to a nucleolar protein gene, NPM, in non-Hodgkin's lymphoma. Science 267, 316-317.

Mosquera, J.M., Beltran, H., Park, K., MacDonald, T.Y., Robinson, B.D., Tagawa, S.T., Perner, S., Bismar, T.A., Erbersdobler, A., Dhir, R., et al. (2013). Concurrent AURKA and MYCN gene amplifications are harbingers of lethal treatment-related neuroendocrine prostate cancer. Neoplasia 15, 1-10.

Mosse, Y.P., Laudenslager, M., Khazi, D., Carlisle, A.J., Winter, C.L., Rappaport, E., and Maris, J.M. (2004). Germline PHOX2B mutation in hereditary neuroblastoma. American journal of human genetics 75, 727-730.

Mosse, Y.P., Laudenslager, M., Longo, L., Cole, K.A., Wood, A., Attiyeh, E.F., Laquaglia, M.J., Sennett, R., Lynch, J.E., Perri, P., et al. (2008a). Identification of ALK as a major familial neuroblastoma predisposition gene. Nature 455, 930-935.

Mosse, Y.P., Laudenslager, M., Longo, L., Cole, K.A., Wood, A., Attiyeh, E.F., Laquaglia, M.J., Sennett, R., Lynch, J.E., Perri, P., et al. (2008b). Identification of ALK as a major familial neuroblastoma predisposition gene. Nature 455, 930-U922.

Mosse, Y.P., Lim, M.S., Voss, S.D., Wilner, K., Ruffner, K., Laliberte, J., Rolland, D., Balis, F.M., Maris, J.M., Weigel, B.J., et al. (2013). Safety and activity of crizotinib for paediatric patients with refractory solid tumours or anaplastic large-cell lymphoma: a Children's Oncology Group phase 1 consortium study. The Lancet Oncology 14, 472- 480.

Mosse, Y.P., Wood, A., and Maris, J.M. (2009). Inhibition of ALK signaling for cancer therapy. Clinical cancer research : an official journal of the American Association for Cancer Research 15, 5609-5614.

Muller, J., and Eilers, M. (2008). Ubiquitination of Myc: proteasomal degradation and beyond. Ernst Schering Foundation symposium proceedings, 99-113.

Murphy, D.M., Buckley, P.G., Bryan, K., Das, S., Alcock, L., Foley, N.H., Prenter, S., Bray, I., Watters, K.M., Higgins, D., et al. (2009). Global MYCN transcription factor binding analysis in neuroblastoma reveals association with distinct E-box motifs and regions of DNA hypermethylation. PloS one 4, e8154.

References 271 Nakagawa, M., Takizawa, N., Narita, M., Ichisaka, T., and Yamanaka, S. (2010). Promotion of direct reprogramming by transformation-deficient Myc. Proc Natl Acad Sci U S A 107, 14152-14157.

Nau, M.M., Brooks, B.J., Battey, J., Sausville, E., Gazdar, A.F., Kirsch, I.R., McBride, O.W., Bertness, V., Hollis, G.F., and Minna, J.D. (1985). L-myc, a new myc-related gene amplified and expressed in human small cell lung cancer. Nature 318, 69-73.

Nau, M.M., Brooks, B.J., Jr., Carney, D.N., Gazdar, A.F., Battey, J.F., Sausville, E.A., and Minna, J.D. (1986). Human small-cell lung cancers show amplification and expression of the N-myc gene. Proc Natl Acad Sci U S A 83, 1092-1096.

Negroni, A., Scarpa, S., Romeo, A., Ferrari, S., Modesti, A., and Raschella, G. (1991). DECREASE OF PROLIFERATION RATE AND INDUCTION OF DIFFERENTIATION BY A MYCN ANTISENSE DNA OLIGOMER IN A HUMAN NEUROBLASTOMA CELL-LINE. Cell Growth & Differentiation 2, 511-518.

Nesbit, C.E., Tersak, J.M., and Prochownik, E.V. (1999). MYC oncogenes and human neoplastic disease. Oncogene 18, 3004-3016.

Neznanov, N., Gorbachev, A.V., Neznanova, L., Komarov, A.P., Gurova, K.V., Gasparian, A.V., Banerjee, A.K., Almasan, A., Fairchild, R.L., and Gudkov, A.V. (2009). Anti-malaria drug blocks proteotoxic stress response: anti-cancer implications. Cell Cycle 8, 3960-3970.

Nicholson, D.W., Ali, A., Thornberry, N.A., Vaillancourt, J.P., Ding, C.K., Gallant, M., Gareau, Y., Griffin, P.R., Labelle, M., Lazebnik, Y.A., et al. (1995). Identification and inhibition of the ICE/CED-3 protease necessary for mammalian apoptosis. Nature 376, 37-43.

Nisen, P.D., Zimmerman, K.A., Cotter, S.V., Gilbert, F., and Alt, F.W. (1986). Enhanced expression of the N-myc gene in Wilms' tumors. Cancer Res 46, 6217-6222.

O'Donnell, K.A., Wentzel, E.A., Zeller, K.I., Dang, C.V., and Mendell, J.T. (2005). c- Myc-regulated microRNAs modulate E2F1 expression. Nature 435, 839-843.

Oberthuer, A., Theissen, J., Westermann, F., Hero, B., and Fischer, M. (2009). Molecular characterization and classification of neuroblastoma. Future Oncol 5, 625- 639.

Okada, M., Jang, S.W., and Ye, K.Q. (2007). Ebp1 association with nucleophosmin/B23 is essential for regulating cell proliferation and suppressing apoptosis. J Biol Chem 282, 36744-36754.

Olayioye, M.A., Neve, R.M., Lane, H.A., and Hynes, N.E. (2000). The ErbB signaling network: receptor heterodimerization in development and cancer. The EMBO journal 19, 3159-3167.

References 272 Orphanides, G., Wu, W.H., Lane, W.S., Hampsey, M., and Reinberg, D. (1999). The chromatin-specific transcription elongation factor FACT comprises human SPT16 and SSRP1 proteins. Nature 400, 284-288.

Ott, C.J., Kopp, N., Bird, L., Paranal, R.M., Qi, J., Bowman, T., Rodig, S.J., Kung, A.L., Bradner, J.E., and Weinstock, D.M. (2012). BET bromodomain inhibition targets both c-Myc and IL7R in high-risk acute lymphoblastic leukemia. Blood 120, 2843- 2852.

Otto, T., Horn, S., Brockmann, M., Eilers, U., Schuttrumpf, L., Popov, N., Kenney, A.M., Schulte, J.H., Beijersbergen, R., Christiansen, H., et al. (2009). Stabilization of N-Myc is a critical function of Aurora A in human neuroblastoma. Cancer Cell 15, 67- 78.

Ou, K., Kesuma, D., Ganesan, K., Yu, K., Soon, S.Y., Lee, S.Y., Goh, X.P., Hooi, M., Chen, W., Jikuya, H., et al. (2006). Quantitative profiling of drug-associated proteomic alterations by combined 2-nitrobenzenesulfenyl chloride (NBS) isotope labeling and 2DE/MS identification. Journal of proteome research 5, 2194-2206.

Palmer, R.H., Vernersson, E., Grabbe, C., and Hallberg, B. (2009). Anaplastic lymphoma kinase: signalling in development and disease. The Biochemical journal 420, 345-361.

Papathanasiou, N.D., Gaze, M.N., Sullivan, K., Aldridge, M., Waddington, W., Almuhaideb, A., and Bomanji, J.B. (2011). 18F-FDG PET/CT and 123I- metaiodobenzylguanidine imaging in high-risk neuroblastoma: diagnostic comparison and survival analysis. Journal of nuclear medicine : official publication, Society of Nuclear Medicine 52, 519-525.

Park, J.R., Scott, J.R., Stewart, C.F., London, W.B., Naranjo, A., Santana, V.M., Shaw, P.J., Cohn, S.L., and Matthay, K.K. (2011). Pilot Induction Regimen Incorporating Pharmacokinetically Guided Topotecan for Treatment of Newly Diagnosed High-Risk Neuroblastoma: A Children's Oncology Group Study. Journal of Clinical Oncology 29, 4351-4357.

Parodi, M.T., Varesio, L., and Tonini, G.P. (1989). Morphological change and cellular differentiation induced by cisplatin in human neuroblastoma cell lines. Cancer chemotherapy and pharmacology 25, 114-116.

Pearson, A.D., Pinkerton, C.R., Lewis, I.J., Imeson, J., Ellershaw, C., and Machin, D. (2008). High-dose rapid and standard induction chemotherapy for patients aged over 1 year with stage 4 neuroblastoma: a randomised trial. The Lancet Oncology 9, 247-256.

Peer, D., and Lieberman, J. (2011). Special delivery: targeted therapy with small RNAs. Gene therapy 18, 1127-1133.

Pegg, A.E. (1988). Polyamine metabolism and its importance in neoplastic growth and a target for chemotherapy. Cancer Res 48, 759-774.

References 273 Pelengaris, S., Abouna, S., Cheung, L., Ifandi, V., Zervou, S., and Khan, M. (2004). Brief inactivation of c-Myc is not sufficient for sustained regression of c-Myc-induced tumours of pancreatic islets and skin epidermis. BMC biology 2, 26.

Pelengaris, S., and Khan, M. (2003). The many faces of c-MYC. Archives of biochemistry and biophysics 416, 129-136.

Pelengaris, S., Littlewood, T., Khan, M., Elia, G., and Evan, G. (1999). Reversible activation of c-Myc in skin: induction of a complex neoplastic phenotype by a single oncogenic lesion. Mol Cell 3, 565-577.

Penn, L.J., Brooks, M.W., Laufer, E.M., and Land, H. (1990a). Negative autoregulation of c-myc transcription. The EMBO journal 9, 1113-1121.

Penn, L.J., Brooks, M.W., Laufer, E.M., Littlewood, T.D., Morgenstern, J.P., Evan, G.I., Lee, W.M., and Land, H. (1990b). Domains of human c-myc protein required for autosuppression and cooperation with ras oncogenes are overlapping. Molecular and cellular biology 10, 4961-4966.

Perri, P., Longo, L., Cusano, R., McConville, C.M., Rees, S.A., Devoto, M., Conte, M., Ferrara, G.B., Seri, M., Romeo, G., et al. (2002). Weak linkage at 4p16 to predisposition for human neuroblastoma. Oncogene 21, 8356-8360.

Petrocca, F., and Lieberman, J. (2011). Promise and challenge of RNA interference- based therapy for cancer. Journal of clinical oncology : official journal of the American Society of Clinical Oncology 29, 747-754.

Peukert, K., Staller, P., Schneider, A., Carmichael, G., Hanel, F., and Eilers, M. (1997). An alternative pathway for gene regulation by Myc. The EMBO journal 16, 5672-5686.

Pilipenko, E.V., Pestova, T.V., Kolupaeva, V.G., Khitrina, E.V., Poperechnaya, A.N., Agol, V.I., and Hellen, C.U. (2000). A cell cycle-dependent protein serves as a template-specific translation initiation factor. Genes & development 14, 2028-2045.

Pinto, N.R., Applebaum, M.A., Volchenboum, S.L., Matthay, K.K., London, W.B., Ambros, P.F., Nakagawara, A., Berthold, F., Schleiermacher, G., Park, J.R., et al. (2015). Advances in Risk Classification and Treatment Strategies for Neuroblastoma. Journal of clinical oncology : official journal of the American Society of Clinical Oncology 33, 3008-3017.

Plevin, M.J., Mills, M.M., and Ikura, M. (2005). The LxxLL motif: a multifunctional binding sequence in transcriptional regulation. Trends in biochemical sciences 30, 66- 69.

Plowman, G.D., Whitney, G.S., Neubauer, M.G., Green, J.M., McDonald, V.L., Todaro, G.J., and Shoyab, M. (1990). Molecular cloning and expression of an additional epidermal growth factor receptor-related gene. Proc Natl Acad Sci U S A 87, 4905- 4909.

References 274 Pugh, T.J., Morozova, O., Attiyeh, E.F., Asgharzadeh, S., Wei, J.S., Auclair, D., Carter, S.L., Cibulskis, K., Hanna, M., Kiezun, A., et al. (2013). The genetic landscape of high- risk neuroblastoma. Nature genetics 45, 279-284.

Pui, C.H., Pei, D., Campana, D., Bowman, W.P., Sandlund, J.T., Kaste, S.C., Ribeiro, R.C., Rubnitz, J.E., Coustan-Smith, E., Jeha, S., et al. (2011). Improved prognosis for older adolescents with acute lymphoblastic leukemia. Journal of clinical oncology : official journal of the American Society of Clinical Oncology 29, 386-391.

Puissant, A., Frumm, S.M., Alexe, G., Bassil, C.F., Qi, J., Chanthery, Y.H., Nekritz, E.A., Zeid, R., Gustafson, W.C., Greninger, P., et al. (2013). Targeting MYCN in neuroblastoma by BET bromodomain inhibition. Cancer Discov 3, 308-323.

Pulverer, B.J., Fisher, C., Vousden, K., Littlewood, T., Evan, G., and Woodgett, J.R. (1994). Site-specific modulation of c-Myc cotransformation by residues phosphorylated in vivo. Oncogene 9, 59-70.

Pylayeva-Gupta, Y., Grabocka, E., and Bar-Sagi, D. (2011). RAS oncogenes: weaving a tumorigenic web. Nature reviews Cancer 11, 761-774.

Raabe, E.H., Laudenslager, M., Winter, C., Wasserman, N., Cole, K., LaQuaglia, M., Maris, D.J., Mosse, Y.P., and Maris, J.M. (2008). Prevalence and functional consequence of PHOX2B mutations in neuroblastoma. Oncogene 27, 469-476.

Rahman, S., Sowa, M.E., Ottinger, M., Smith, J.A., Shi, Y., Harper, J.W., and Howley, P.M. (2011). The Brd4 extraterminal domain confers transcription activation independent of pTEFb by recruiting multiple proteins, including NSD3. Molecular and cellular biology 31, 2641-2652.

Raica, M., Cimpean, A.M., and Ribatti, D. (2008). The role of podoplanin in tumor progression and metastasis. Anticancer research 28, 2997-3006.

Rana, T.M. (2007). Illuminating the silence: understanding the structure and function of small RNAs. Nature reviews Molecular cell biology 8, 23-36.

Rathert, P., Roth, M., Neumann, T., Muerdter, F., Roe, J.S., Muhar, M., Deswal, S., Cerny-Reiterer, S., Peter, B., Jude, J., et al. (2015). Transcriptional plasticity promotes primary and acquired resistance to BET inhibition. Nature 525, 543-547.

Reiff, T., Huber, L., Kramer, M., Delattre, O., Janoueix-Lerosey, I., and Rohrer, H. (2011). Midkine and Alk signaling in sympathetic neuron proliferation and neuroblastoma predisposition. Development 138, 4699-4708.

Reynolds, C.P., Kane, D.J., Einhorn, P.A., Matthay, K.K., Crouse, V.L., Wilbur, J.R., Shurin, S.B., and Seeger, R.C. (1991). Response of neuroblastoma to retinoic acid in vitro and in vivo. Progress in clinical and biological research 366, 203-211.

References 275 Reynolds, C.P., Wang, Y., Melton, L.J., Einhorn, P.A., Slamon, D.J., and Maurer, B.J. (2000). Retinoic-acid-resistant neuroblastoma cell lines show altered MYC regulation and high sensitivity to fenretinide. Medical and Pediatric Oncology 35, 597-602.

Roe, J.S., Mercan, F., Rivera, K., Pappin, D.J., and Vakoc, C.R. (2015). BET Bromodomain Inhibition Suppresses the Function of Hematopoietic Transcription Factors in Acute Myeloid Leukemia. Mol Cell 58, 1028-1039.

Rous, P. (1911). A Sarcoma of the Fowl Transmissible by an Agent Separable from the Tumor Cells. The Journal of experimental medicine 13, 397-411.

Roussel, M., Saule, S., Lagrou, C., Rommens, C., Beug, H., Graf, T., and Stehelin, D. (1979). Three new types of viral oncogene of cellular origin specific for haematopoietic cell transformation. Nature 281, 452-455.

Rubie, H., De Bernardi, B., Gerrard, M., Canete, A., Ladenstein, R., Couturier, J., Ambros, P., Munzer, C., Pearson, A.D., Garaventa, A., et al. (2011). Excellent outcome with reduced treatment in infants with nonmetastatic and unresectable neuroblastoma without MYCN amplification: results of the prospective INES 99.1. Journal of clinical oncology : official journal of the American Society of Clinical Oncology 29, 449-455.

Sahai, E., and Marshall, C.J. (2003). Differing modes of tumour cell invasion have distinct requirements for Rho/ROCK signalling and extracellular proteolysis. Nature cell biology 5, 711-719.

Salvesen, G.S., and Dixit, V.M. (1997). Caspases: intracellular signaling by proteolysis. Cell 91, 443-446.

Santegoets, S., Schreurs, M.W.J., Reurs, A.W., Lindenberg, J.J., Kueter, E.W.M., van den Eertwegh, A.J.M., Hooijberg, E., Brandwijk, R.J., Hufton, S.E., Hoogenboom, H.R., et al. (2007). Identification and characterization of ErbB-3-binding protein-1 as a target for immunotherapy. Journal of Immunology 179, 2005-2012.

Saulnier Sholler, G.L., Gerner, E.W., Bergendahl, G., MacArthur, R.B., VanderWerff, A., Ashikaga, T., Bond, J.P., Ferguson, W., Roberts, W., Wada, R.K., et al. (2015). A Phase I Trial of DFMO Targeting Polyamine Addiction in Patients with Relapsed/Refractory Neuroblastoma. PloS one 10, e0127246.

Scherl, A., Coute, Y., Deon, C., Calle, A., Kindbeiter, K., Sanchez, J.C., Greco, A., Hochstrasser, D., and Diaz, J.J. (2002). Functional proteomic analysis of human nucleolus. Mol Biol Cell 13, 4100-4109.

Schleiermacher, G., Javanmardi, N., Bernard, V., Leroy, Q., Cappo, J., Rio Frio, T., Pierron, G., Lapouble, E., Combaret, V., Speleman, F., et al. (2014). Emergence of new ALK mutations at relapse of neuroblastoma. Journal of clinical oncology : official journal of the American Society of Clinical Oncology 32, 2727-2734.

References 276 Schleiermacher, G., Michon, J., Huon, I., d'Enghien, C.D., Klijanienko, J., Brisse, H., Ribeiro, A., Mosseri, V., Rubie, H., Munzer, C., et al. (2007). Chromosomal CGH identifies patients with a higher risk of relapse in neuroblastoma without MYCN amplification. Br J Cancer 97, 238-246.

Schleiermacher, G., Raynal, V., Janoueix-Lerosey, I., Combaret, V., Aurias, A., and Delattre, O. (2004). Variety and complexity of chromosome 17 translocations in neuroblastoma. Genes, chromosomes & cancer 39, 143-150.

Schneider, A., Peukert, K., Eilers, M., and Hanel, F. (1997). Association of Myc with the zinc-finger protein Miz-1 defines a novel pathway for gene regulation by Myc. Current topics in microbiology and immunology 224, 137-146.

Schramm, A., Koester, J., Assenov, Y., Althoff, K., Peifer, M., Mahlow, E., Odersky, A., Beisser, D., Ernst, C., Henssen, A.G., et al. (2015). Mutational dynamics between primary and relapse neuroblastomas. Nature genetics 47, 872-+.

Schulte, J.H., Horn, S., Otto, T., Samans, B., Heukamp, L.C., Eilers, U.C., Krause, M., Astrahantseff, K., Klein-Hitpass, L., Buettner, R., et al. (2008). MYCN regulates oncogenic MicroRNAs in neuroblastoma. International journal of cancer Journal international du cancer 122, 699-704.

Schwab, M., Alitalo, K., Klempnauer, K.H., Varmus, H.E., Bishop, J.M., Gilbert, F., Brodeur, G., Goldstein, M., and Trent, J. (1983). Amplified DNA with limited homology to myc cellular oncogene is shared by human neuroblastoma cell lines and a neuroblastoma tumour. Nature 305, 245-248.

Schwab, M., Varmus, H.E., and Bishop, J.M. (1985). Human N-myc gene contributes to neoplastic transformation of mammalian cells in culture. Nature 316, 160-162.

Seeger, R.C., Brodeur, G.M., Sather, H., Dalton, A., Siegel, S.E., Wong, K.Y., and Hammond, D. (1985). ASSOCIATION OF MULTIPLE COPIES OF THE N-MYC ONCOGENE WITH RAPID PROGRESSION OF NEUROBLASTOMAS. New England Journal of Medicine 313, 1111-1116.

Segerstrom, L., Baryawno, N., Sveinbjornsson, B., Wickstrom, M., Elfman, L., Kogner, P., and Johnsen, J.I. (2011). Effects of small molecule inhibitors of PI3K/Akt/mTOR signaling on neuroblastoma growth in vitro and in vivo. International journal of cancer Journal international du cancer 129, 2958-2965.

Seoane, J., Pouponnot, C., Staller, P., Schader, M., Eilers, M., and Massague, J. (2001). TGFbeta influences Myc, Miz-1 and Smad to control the CDK inhibitor p15INK4b. Nature cell biology 3, 400-408.

Shang, X., Burlingame, S.M., Okcu, M.F., Ge, N., Russell, H.V., Egler, R.A., David, R.D., Vasudevan, S.A., Yang, J., and Nuchtern, J.G. (2009). Aurora A is a negative prognostic factor and a new therapeutic target in human neuroblastoma. Mol Cancer Ther 8, 2461-2469.

References 277 Sharma, M., and Sun, Z. (2001). 5'TG3' interacting factor interacts with Sin3A and represses AR-mediated transcription. Mol Endocrinol 15, 1918-1928.

Sheiness, D., and Bishop, J.M. (1979). DNA and RNA from uninfected vertebrate cells contain nucleotide sequences related to the putative transforming gene of avian myelocytomatosis virus. Journal of virology 31, 514-521.

Sheiness, D., Fanshier, L., and Bishop, J.M. (1978). Identification of nucleotide sequences which may encode the oncogenic capacity of avian retrovirus MC29. Journal of virology 28, 600-610.

Shen, H., Sun, T., and Ferrari, M. (2012). Nanovector delivery of siRNA for cancer therapy. Cancer gene therapy 19, 367-373.

Shen, W., Tremblay, M.S., Deshmukh, V.A., Wang, W., Filippi, C.M., Harb, G., Zhang, Y.Q., Kamireddy, A., Baaten, J.E., Jin, Q., et al. (2013). Small-molecule inducer of beta cell proliferation identified by high-throughput screening. Journal of the American Chemical Society 135, 1669-1672.

Shenkman, M., Tolchinsky, S., and Lederkremer, G.Z. (2007). ER stress induces alternative nonproteasomal degradation of ER proteins but not of cytosolic ones. Cell stress & chaperones 12, 373-383.

Shimada, H., Ambros, I.M., Dehner, L.P., Hata, J., Joshi, V.V., Roald, B., Stram, D.O., Gerbing, R.B., Lukens, J.N., Matthay, K.K., et al. (1999). The International Neuroblastoma Pathology Classification (the Shimada system). Cancer 86, 364-372.

Shimada, H., Chatten, J., Newton, W.A., Jr., Sachs, N., Hamoudi, A.B., Chiba, T., Marsden, H.B., and Misugi, K. (1984). Histopathologic prognostic factors in neuroblastic tumors: definition of subtypes of ganglioneuroblastoma and an age-linked classification of neuroblastomas. Journal of the National Cancer Institute 73, 405-416.

Shimada, H., Umehara, S., Monobe, Y., Hachitanda, Y., Nakagawa, A., Goto, S., Gerbing, R.B., Stram, D.O., Lukens, J.N., and Matthay, K.K. (2001). International neuroblastoma pathology classification for prognostic evaluation of patients with peripheral neuroblastic tumors: a report from the Children's Cancer Group. Cancer 92, 2451-2461.

Shojaei-Brosseau, T., Chompret, A., Abel, A., de Vathaire, F., Raquin, M.A., Brugieres, L., Feunteun, J., Hartmann, O., and Bonaiti-Pellie, C. (2004). Genetic epidemiology of neuroblastoma: a study of 426 cases at the Institut Gustave-Roussy in France. Pediatric blood & cancer 42, 99-105.

Shou, Y., Martelli, M.L., Gabrea, A., Qi, Y., Brents, L.A., Roschke, A., Dewald, G., Kirsch, I.R., Bergsagel, P.L., and Kuehl, W.M. (2000). Diverse karyotypic abnormalities of the c-myc locus associated with c-myc dysregulation and tumor progression in multiple myeloma. Proc Natl Acad Sci U S A 97, 228-233.

References 278 Siddik, Z.H. (2003). Cisplatin: mode of cytotoxic action and molecular basis of resistance. Oncogene 22, 7265-7279.

Sidell, N. (1982). Retinoic acid-induced growth inhibition and morphologic differentiation of human neuroblastoma cells in vitro. Journal of the National Cancer Institute 68, 589-596.

Sidell, N., Altman, A., Haussler, M.R., and Seeger, R.C. (1983). EFFECTS OF RETINOIC ACID (RA) ON THE GROWTH AND PHENOTYPIC-EXPRESSION OF SEVERAL HUMAN NEURO-BLASTOMA CELL-LINES. Experimental Cell Research 148, 21-30.

Simbulan-Rosenthal, C.M., Rosenthal, D.S., Iyer, S., Boulares, H., and Smulson, M.E. (1999). Involvement of PARP and poly(ADP-ribosyl)ation in the early stages of apoptosis and DNA replication. Molecular and cellular biochemistry 193, 137-148.

Sjostrom, S.K., Finn, G., Hahn, W.C., Rowitch, D.H., and Kenney, A.M. (2005). The Cdk1 complex plays a prime role in regulating N-myc phosphorylation and turnover in neural precursors. Developmental cell 9, 327-338.

Skoulidis, F., Byers, L.A., Diao, L., Papadimitrakopoulou, V.A., Tong, P., Izzo, J., Behrens, C., Kadara, H., Parra, E.R., Canales, J.R., et al. (2015). Co-occurring Genomic Alterations Define Major Subsets of KRAS-Mutant Lung Adenocarcinoma with Distinct Biology, Immune Profiles, and Therapeutic Vulnerabilities. Cancer Discovery 5, 860-877.

Slack, A., Chen, Z., Tonelli, R., Pule, M., Hunt, L., Pession, A., and Shohet, J.M. (2005). The p53 regulatory gene MDM2 is a direct transcriptional target of MYCN in neuroblastoma. Proc Natl Acad Sci U S A 102, 731-736.

Smalle, J., and Vierstra, R.D. (2004). The ubiquitin 26S proteasome proteolytic pathway. Annual review of plant biology 55, 555-590.

Smith, K.P., Byron, M., O'Connell, B.C., Tam, R., Schorl, C., Guney, I., Hall, L.L., Agrawal, P., Sedivy, J.M., and Lawrence, J.B. (2004). c-Myc localization within the nucleus: Evidence for association with the PML nuclear body. Journal of cellular biochemistry 93, 1282-1296.

Smith, M.A., Seibel, N.L., Altekruse, S.F., Ries, L.A., Melbert, D.L., O'Leary, M., Smith, F.O., and Reaman, G.H. (2010). Outcomes for children and adolescents with cancer: challenges for the twenty-first century. Journal of clinical oncology : official journal of the American Society of Clinical Oncology 28, 2625-2634.

Smulson, M.E., Kang, V.H., Ntambi, J.M., Rosenthal, D.S., Ding, R., and Simbulan, C.M. (1995). Requirement for the expression of poly(ADP-ribose) polymerase during the early stages of differentiation of 3T3-L1 preadipocytes, as studied by antisense RNA induction. The Journal of biological chemistry 270, 119-127.

References 279 Sordella, R., Bell, D.W., Haber, D.A., and Settleman, J. (2004). Gefitinib-sensitizing EGFR mutations in lung cancer activate anti-apoptotic pathways. Science 305, 1163- 1167.

Soucek, L., and Evan, G.I. (2010). The ups and downs of Myc biology. Current opinion in genetics & development 20, 91-95.

Soucek, L., Helmer-Citterich, M., Sacco, A., Jucker, R., Cesareni, G., and Nasi, S. (1998). Design and properties of a Myc derivative that efficiently homodimerizes. Oncogene 17, 2463-2472.

Soucek, L., Whitfield, J., Martins, C.P., Finch, A.J., Murphy, D.J., Sodir, N.M., Karnezis, A.N., Swigart, L.B., Nasi, S., and Evan, G.I. (2008). Modelling Myc inhibition as a cancer therapy. Nature 455, 679-683.

Spencer, C.A., and Groudine, M. (1991). Control of c-myc regulation in normal and neoplastic cells. Adv Cancer Res 56, 1-48.

Squatrito, M., Mancino, M., Donzelli, M., Areces, L.B., and Draetta, G.F. (2004). EBP1 is a nucleolar growth-regulating protein that is part of pre-ribosomal ribonucleoprotein complexes. Oncogene 23, 4454-4465.

Squatrito, M., Mancino, M., Sala, L., and Draetta, G.F. (2006). Ebp1 is a dsRNA- binding protein associated with ribosomes that modulates eIF2 alpha phosphorylation. Biochem Biophys Res Commun 344, 859-868.

Srivastava, S.P., Kumar, K.U., and Kaufman, R.J. (1998). Phosphorylation of eukaryotic translation initiation factor 2 mediates apoptosis in response to activation of the double-stranded RNA-dependent protein kinase. The Journal of biological chemistry 273, 2416-2423.

Staller, P., Peukert, K., Kiermaier, A., Seoane, J., Lukas, J., Karsunky, H., Moroy, T., Bartek, J., Massague, J., Hanel, F., et al. (2001). Repression of p15INK4b expression by Myc through association with Miz-1. Nature cell biology 3, 392-399.

Stanton, B.R., Perkins, A.S., Tessarollo, L., Sassoon, D.A., and Parada, L.F. (1992). Loss of N-myc function results in embryonic lethality and failure of the epithelial component of the embryo to develop. Genes & development 6, 2235-2247.

Strasser, A., Harris, A.W., Bath, M.L., and Cory, S. (1990). Novel primitive lymphoid tumours induced in transgenic mice by cooperation between myc and bcl-2. Nature 348, 331-333.

Strother, D.R., London, W.B., Schmidt, M.L., Brodeur, G.M., Shimada, H., Thorner, P., Collins, M.H., Tagge, E., Adkins, S., Reynolds, C.P., et al. (2012). Outcome after surgery alone or with restricted use of chemotherapy for patients with low-risk neuroblastoma: results of Children's Oncology Group study P9641. Journal of clinical

References 280 oncology : official journal of the American Society of Clinical Oncology 30, 1842- 1848.

Sugiyama, A., Kume, A., Nemoto, K., Lee, S.Y., Asami, Y., Nemoto, F., Nishimura, S., and Kuchino, Y. (1989). Isolation and characterization of s-myc, a member of the rat myc gene family. Proc Natl Acad Sci U S A 86, 9144-9148.

Sun, J., Luo, Y.X., Tian, Z., Gu, L., Xia, S.C., and Yu, Y.C. (2012). Expression of ERBB3 binding protein 1 (EBP1) in salivary adenoid cystic carcinoma and its clinicopathological relevance. Bmc Cancer 12.

Swartling, F.J., Grimmer, M.R., Hackett, C.S., Northcott, P.A., Fan, Q.W., Goldenberg, D.D., Lau, J., Masic, S., Nguyen, K., Yakovenko, S., et al. (2010). Pleiotropic role for MYCN in medulloblastoma. Genes & development 24, 1059-1072.

Swartling, F.J., Savov, V., Persson, A.I., Chen, J., Hackett, C.S., Northcott, P.A., Grimmer, M.R., Lau, J., Chesler, L., Perry, A., et al. (2012). Distinct neural stem cell populations give rise to disparate brain tumors in response to N-MYC. Cancer Cell 21, 601-613.

Tao, W., and Levine, A.J. (1999). Nucleocytoplasmic shuttling of oncoprotein Hdm2 is required for Hdm2-mediated degradation of p53. Proc Natl Acad Sci U S A 96, 3077- 3080.

Thiele, C.J., Reynolds, C.P., and Israel, M.A. (1985). Decreased expression of N-myc precedes retinoic acid-induced morphological differentiation of human neuroblastoma. Nature 313, 404-406.

Touriol, C., Greenland, C., Lamant, L., Pulford, K., Bernard, F., Rousset, T., Mason, D.Y., and Delsol, G. (2000). Further demonstration of the diversity of chromosomal changes involving 2p23 in ALK-positive lymphoma: 2 cases expressing ALK kinase fused to CLTCL (clathrin chain polypeptide-like). Blood 95, 3204-3207.

Trochet, D., Bourdeaut, F., Janoueix-Lerosey, I., Deville, A., de Pontual, L., Schleiermacher, G., Coze, C., Philip, N., Frebourg, T., Munnich, A., et al. (2004). Germline mutations of the paired-like homeobox 2B (PHOX2B) gene in neuroblastoma. American journal of human genetics 74, 761-764.

Trochet, D., Mathieu, Y., Pontual, L., Savarirayan, R., Munnich, A., Brunet, J.F., Lyonnet, S., Goridis, C., and Amiel, J. (2009). In Vitro studies of non poly alanine PHOX2B mutations argue against a loss-of-function mechanism for congenital central hypoventilation. Human mutation 30, E421-431.

Ullrich, A., Coussens, L., Hayflick, J.S., Dull, T.J., Gray, A., Tam, A.W., Lee, J., Yarden, Y., Libermann, T.A., Schlessinger, J., et al. (1984). Human epidermal growth factor receptor cDNA sequence and aberrant expression of the amplified gene in A431 epidermoid carcinoma cells. Nature 309, 418-425.

References 281 Vandesompele, J., Baudis, M., De Preter, K., Van Roy, N., Ambros, P., Bown, N., Brinkschmidt, C., Christiansen, H., Combaret, V., Lastowska, M., et al. (2005). Unequivocal delineation of clinicogenetic subgroups and development of a new model for improved outcome prediction in neuroblastoma. Journal of clinical oncology : official journal of the American Society of Clinical Oncology 23, 2280-2299.

Varmus, H.E. (1984). The molecular genetics of cellular oncogenes. Annual review of genetics 18, 553-612.

Veal, G.J., Nguyen, L., Paci, A., Riggi, M., Amiel, M., Valteau-Couanet, D., Brock, P., Ladenstein, R., and Vassal, G. (2012). Busulfan pharmacokinetics following intravenous and oral dosing regimens in children receiving high-dose myeloablative chemotherapy for high-risk neuroblastoma as part of the HR-NBL-1/SIOPEN trial. Eur J Cancer 48, 3063-3072.

Vinci, M., Gowan, S., Boxall, F., Patterson, L., Zimmermann, M., Court, W., Lomas, C., Mendiola, M., Hardisson, D., and Eccles, S.A. (2012). Advances in establishment and analysis of three-dimensional tumor spheroid-based functional assays for target validation and drug evaluation. BMC biology 10, 29.

Vita, M., and Henriksson, M. (2006). The Myc oncoprotein as a therapeutic target for human cancer. Semin Cancer Biol 16, 318-330. von der Lehr, N., Johansson, S., Wu, S.Q., Bahram, F., Castell, A., Cetinkaya, C., Hydbring, P., Weidung, I., Nakayama, K., Nakayama, K.I., et al. (2003). The F-Box protein Skp2 participates in c-Myc protelosornal degradation and acts as a cofactor for c-Myc-regulated transcription. Molecular Cell 11, 1189-1200.

Wada, R.K., Seeger, R.C., Reynolds, C.P., Alloggiamento, T., Yamashiro, J.M., Ruland, C., Black, A.C., and Rosenblatt, J.D. (1992). Cell type-specific expression and negative regulation by retinoic acid of the human N-myc promoter in neuroblastoma cells. Oncogene 7, 711-717.

Wakamatsu, Y., Watanabe, Y., Nakamura, H., and Kondoh, H. (1997). Regulation of the neural crest cell fate by N-myc: promotion of ventral migration and neuronal differentiation. Development 124, 1953-1962.

Wang, W., Walker, J.R., Wang, X., Tremblay, M.S., Lee, J.W., Wu, X., and Schultz, P.G. (2009). Identification of small-molecule inducers of pancreatic beta-cell expansion. Proc Natl Acad Sci U S A 106, 1427-1432.

Wanzel, M., Herold, S., and Eilers, M. (2003). Transcriptional repression by Myc. Trends in cell biology 13, 146-150.

Watt, R., Nishikura, K., Sorrentino, J., ar-Rushdi, A., Croce, C.M., and Rovera, G. (1983). The structure and nucleotide sequence of the 5' end of the human c-myc oncogene. Proc Natl Acad Sci U S A 80, 6307-6311.

References 282 Watters, K.M., Bryan, K., Foley, N.H., Meehan, M., and Stallings, R.L. (2013). Expressional alterations in functional ultra-conserved non-coding rnas in response to all-trans retinoic acid-induced differentiation in neuroblastoma cells. Bmc Cancer 13.

Weese-Mayer, D.E., Berry-Kravis, E.M., Zhou, L., Maher, B.S., Silvestri, J.M., Curran, M.E., and Marazita, M.L. (2003). Idiopathic congenital central hypoventilation syndrome: analysis of genes pertinent to early autonomic nervous system embryologic development and identification of mutations in PHOX2b. American journal of medical genetics Part A 123A, 267-278.

Weiss, W.A., Aldape, K., Mohapatra, G., Feuerstein, B.G., and Bishop, J.M. (1997). Targeted expression of MYCN causes neuroblastoma in transgenic mice. The EMBO journal 16, 2985-2995.

Weiss, W.A., Godfrey, T., Francisco, C., and Bishop, J.M. (2000). Genome-wide screen for allelic imbalance in a mouse model for neuroblastoma. Cancer Res 60, 2483-2487.

Welcker, M., Orian, A., Jin, J., Grim, J.E., Harper, J.W., Eisenman, R.N., and Clurman, B.E. (2004). The Fbw7 tumor suppressor regulates glycogen synthase kinase 3 phosphorylation-dependent c-Myc protein degradation. Proc Natl Acad Sci U S A 101, 9085-9090.

Wenzel, A., Cziepluch, C., Hamann, U., Schurmann, J., and Schwab, M. (1991). The N- Myc oncoprotein is associated in vivo with the phosphoprotein Max(p20/22) in human neuroblastoma cells. The EMBO journal 10, 3703-3712.

Westermark, U.K., Wilhelm, M., Frenzel, A., and Henriksson, M.A. (2011). The MYCN oncogene and differentiation in neuroblastoma. Semin Cancer Biol 21, 256-266.

Wirtz, D., Konstantopoulos, K., and Searson, P.C. (2011). The physics of cancer: the role of physical interactions and mechanical forces in metastasis. Nature reviews Cancer 11, 512-522.

Xia, X.M., Cheng, A.W., Lessor, T., Zhang, Y.X., and Hamburger, A.W. (2001). Ebp1, an ErbB-3 binding protein, interacts with Rb and affects Rb transcriptional regulation. J Cell Physiol 187, 209-217.

Xu, H., Cheung, I.Y., Guo, H.F., and Cheung, N.K. (2009). MicroRNA miR-29 modulates expression of immunoinhibitory molecule B7-H3: potential implications for immune based therapy of human solid tumors. Cancer Res 69, 6275-6281.

Yaari, S., Jacob-Hirsch, J., Amariglio, N., Haklai, R., Rechavi, G., and Kloog, Y. (2005). Disruption of cooperation between Ras and MycN in human neuroblastoma cells promotes growth arrest. Clinical cancer research : an official journal of the American Association for Cancer Research 11, 4321-4330.

Yamamoto, K., Hanada, R., Kikuchi, A., Ichikawa, M., Aihara, T., Oguma, E., Moritani, T., Shimanuki, Y., Tanimura, M., and Hayashi, Y. (1998). Spontaneous

References 283 regression of localized neuroblastoma detected by mass screening. Journal of clinical oncology : official journal of the American Society of Clinical Oncology 16, 1265- 1269.

Yancopoulos, G.D., Nisen, P.D., Tesfaye, A., Kohl, N.E., Goldfarb, M.P., and Alt, F.W. (1985). N-myc can cooperate with ras to transform normal cells in culture. Proc Natl Acad Sci U S A 82, 5455-5459.

Yang, Y., Zhao, S., and Song, J.G. (2004). Caspase-dependent apoptosis and - independent poly(ADP-ribose) polymerase cleavage induced by transforming growth factor beta 1. International Journal of Biochemistry & Cell Biology 36, 223-234.

Yang, Z., Yik, J.H., Chen, R., He, N., Jang, M.K., Ozato, K., and Zhou, Q. (2005). Recruitment of P-TEFb for stimulation of transcriptional elongation by the bromodomain protein Brd4. Mol Cell 19, 535-545.

Yaylim-Eraltan, I., Bozkurt, N., Ergen, A., Zeybek, U., Ozturk, O., Arikan, S., Erbil, Y., Uslu, I., Camlica, H., and Isbir, T. (2008). L-myc gene polymorphism and risk of thyroid cancer. Experimental oncology 30, 117-120.

Yeap, B.B., Wilce, J.A., and Leedman, P.J. (2004). The androgen receptor mRNA. BioEssays : news and reviews in molecular, cellular and developmental biology 26, 672-682.

Yin, X., Giap, C., Lazo, J.S., and Prochownik, E.V. (2003). Low molecular weight inhibitors of Myc-Max interaction and function. Oncogene 22, 6151-6159.

Yip, K.W., and Reed, J.C. (2008). Bcl-2 family proteins and cancer. Oncogene 27, 6398-6406.

Yoo, J.Y., Wang, X.W., Rishi, A.K., Lessor, T., Xia, X.M., Gustafson, T.A., and Hamburger, A.W. (2000). Interaction of the PA2G4 (EBP1) protein with ErbB-3 and regulation of this binding by heregulin. Br J Cancer 82, 683-690.

Young, J.L., Jr., Ries, L.G., Silverberg, E., Horm, J.W., and Miller, R.W. (1986). Cancer incidence, survival, and mortality for children younger than age 15 years. Cancer 58, 598-602.

Yu, A.L., Gilman, A.L., Ozkaynak, M.F., London, W.B., Kreissman, S.G., Chen, H.X., Smith, M., Anderson, B., Villablanca, J.G., Matthay, K.K., et al. (2010). Anti-GD2 antibody with GM-CSF, interleukin-2, and isotretinoin for neuroblastoma. The New England journal of medicine 363, 1324-1334.

Yu, J.Y., DeRuiter, S.L., and Turner, D.L. (2002). RNA interference by expression of short-interfering RNAs and hairpin RNAs in mammalian cells. Proc Natl Acad Sci U S A 99, 6047-6052.

References 284 Yu, M., Ohira, M., Li, Y., Niizuma, H., Oo, M.L., Zhu, Y., Ozaki, T., Isogai, E., Nakamura, Y., Koda, T., et al. (2009a). High expression of ncRAN, a novel non-coding RNA mapped to chromosome 17q25.1, is associated with poor prognosis in neuroblastoma. Int J Oncol 34, 931-938.

Yu, W., Lee, H.K., Hariharan, S., Bu, W., and Ahmed, S. (2009b). Quantitative neurite outgrowth measurement based on image segmentation with topological dependence. Cytometry Part A : the journal of the International Society for Analytical Cytology 75, 289-297.

Yu, Y., Chen, W., Zhang, Y., Hamburger, A.W., Pan, H., and Zhang, Z. (2007). Suppression of salivary adenoid cystic carcinoma growth and metastasis by ErbB3 binding protein Ebp1 gene transfer. International journal of cancer Journal international du cancer 120, 1909-1913.

Yuan, P., Temam, S., El-Naggar, A., Zhou, X., Liu, D.D., Lee, J.J., and Mao, L. (2006). Overexpression of podoplanin in oral cancer and its association with poor clinical outcome. Cancer 107, 563-569.

Zaman, M.H., Trapani, L.M., Sieminski, A.L., Mackellar, D., Gong, H., Kamm, R.D., Wells, A., Lauffenburger, D.A., and Matsudaira, P. (2006). Migration of tumor cells in 3D matrices is governed by matrix stiffness along with cell-matrix adhesion and proteolysis. Proc Natl Acad Sci U S A 103, 10889-10894.

Zariwala, M., Liu, J., and Xiong, Y. (1998). Cyclin E2, a novel human G1 cyclin and activating partner of CDK2 and CDK3, is induced by viral oncoproteins. Oncogene 17, 2787-2798.

Zelinski, T., Verville, G., White, L., Hamerton, J.L., McAlpine, P.J., and Lewis, M. (1988). Confirmation of the assignment of MYCL to chromosome 1 in humans and its position relative to RH, UMPK, and PGM1. Genomics 2, 154-156.

Zeng, S.X., Li, Y., Jin, Y., Zhang, Q., Keller, D.M., McQuaw, C.M., Barklis, E., Stone, S., Hoatlin, M., Zhao, Y., et al. (2010). Structure-specific recognition protein 1 facilitates microtubule growth and bundling required for mitosis. Molecular and cellular biology 30, 935-947.

Zhang, S., Zhang, B., Xu, X., Wang, L., Feng, X., Wang, Q., Huang, H., Wu, J., Li, P., and Wang, J. (2014). HIV-1 viral protein R downregulates Ebp1 and stabilizes p53 in glioblastoma U87MG cells. Clinical & translational oncology : official publication of the Federation of Spanish Oncology Societies and of the National Cancer Institute of Mexico 16, 293-300.

Zhang, Y., Akinmade, D., and Hamburger, A.W. (2008a). Inhibition of heregulin mediated MCF-7 breast cancer cell growth by the ErbB3 binding protein EBP1. Cancer Lett 265, 298-306.

References 285 Zhang, Y., Linn, D., Liu, Z., Melamed, J., Tavora, F., Young, C.Y., Burger, A.M., and Hamburger, A.W. (2008b). EBP1, an ErbB3-binding protein, is decreased in prostate cancer and implicated in hormone resistance. Mol Cancer Ther 7, 3176-3186.

Zhang, Y., Wang, X.W., Jelovac, D., Nakanishi, T., Yu, M.H., Akinmade, D., Goloubeva, O., Ross, D.D., Brodie, A., and Hamburger, A.W. (2005a). The ErbB3- binding protein Ebp1 suppresses androgen receptor-mediated gene transcription and tumorigenesis of prostate cancer cells. Proc Natl Acad Sci U S A 102, 9890-9895.

Zhang, Y.X., Akinmade, D., and Hamburger, A.W. (2005b). The ErbB3 binding protein Ebp1 interacts with Sin3A to repress E2F1 and AR-mediated transcription. Nucleic Acids Res 33, 6024-6033.

Zhang, Y.X., Fondell, J.D., Wang, Q.B., Xia, X.M., Cheng, A.W., Lu, M.L., and Hamburger, A.W. (2002). Repression of androgen receptor mediated transcription by the ErbB-3 binding protein, Ebp1. Oncogene 21, 5609-5618.

Zhang, Y.X., and Hamburger, A.W. (2004). Heregulin regulates the ability of the ErbB3-binding protein Ebp1 to bind E2F promoter elements and repress E2F-mediated transcription. J Biol Chem 279, 26126-26133.

Zhang, Y.X., Lu, Y., Zhou, H., Lee, M., Liu, Z.Q., Hassel, B.A., and Hamburger, A.W. (2008c). Alterations in cell growth and signaling in ErbB3 binding protein-1 (Ebp1) deficient mice. Bmc Cell Biology 9.

Zhang, Y.X., Woodford, N., Xia, X.M., and Hamburger, A.W. (2003). Repression of E2F1-mediated transcription by the ErbB3 binding protein Ebp1 involves histone deacetylases. Nucleic Acids Res 31, 2168-2177.

Zhou, H., Mazan-Mamczarz, K., Martindale, J.L., Barker, A., Liu, Z.Q., Gorospe, M., Leedman, P.J., Gartenhaus, R.B., Hamburger, A.W., and Zhang, Y.X. (2010a). Post- transcriptional regulation of androgen receptor mRNA by an ErbB3 binding protein 1 in prostate cancer. Nucleic Acids Res 38, 3619-3631.

Zhou, X., Chen, W.T., Zhang, Y.X., Sun, J., Wang, Q., and Yu, Y.C. (2010b). Potential therapeutic strategy for oral squamous cell carcinoma by ErbB3-binding protein 1 gene transfer. J Cancer Res Clin Oncol 136, 891-896.

Zimmerman, K.A., Yancopoulos, G.D., Collum, R.G., Smith, R.K., Kohl, N.E., Denis, K.A., Nau, M.M., Witte, O.N., Toran-Allerand, D., Gee, C.E., et al. (1986). Differential expression of myc family genes during murine development. Nature 319, 780-783.

Zuber, J., Tchernitsa, O.I., Hinzmann, B., Schmitz, A.C., Grips, M., Hellriegel, M., Sers, C., Rosenthal, A., and Schafer, R. (2000). A genome-wide survey of RAS transformation targets. Nature genetics 24, 144-152.

References 286 APPENDIX A: Log rank test

Appendix A 287

Appendix A 288

Appendix A 289

Appendix A 290

Appendix A 291

Appendix A 292

Appendix A 293