"Medicine, Faculty of"@en . "Pathology and Laboratory Medicine, Department of"@en . "DSpace"@en . "UBCV"@en . "Lawlor, Elizabeth Rachel"@en . "2009-09-23T20:00:16Z"@en . "2002"@en . "Doctor of Philosophy - PhD"@en . "University of British Columbia"@en . "The Ewing family of peripheral primitive neuroectodermal tumours (ET or pPNET)\r\ncomprises a genetically related group of bone and soft tissue tumours that primarily affects\r\nchildren and adolescents. All members of this tumour family are characterized by tumour\r\nspecific translocations involving the EWS gene from chromosome llq23 and a second gene\r\ncontributed by a variety of chromosomal partners. In all cases, the partner gene is a member\r\nof the ETS family of transcription factors and the resultant EWS-ETS gene fusions encode\r\nnovel chimeric proteins comprised of the EWS-amino-terminal domain and an ETS-DNA\r\nbinding domain. These fusion proteins are oncogenic and the accumulated evidence suggests\r\nthat they are the primary pathologic lesions contributing to the development of ET. Despite\r\nits genetic characterization, little is known about the mechanisms of EWS-ETS mediated\r\noncogenesis specifically, or in fact, the biologic features that contribute to the malignant\r\nnature of ET cells. We have endeavoured to study proliferative signaling in ET cells in an\r\neffort to better characterize the biology of these aggressive and often fatal tumours. Using\r\ndifferential-display PCR, our laboratory had previously identified differential expression of\r\nthe human gastrin-releasing peptide (GRP) gene among EWS-ETS expressing tumour cell lines. Further studies confirmed that 100% of ET cell lines and approximately 50% of primary ET samples express the GRP gene and the gene encoding its receptor. Protein and functional studies demonstrated that the mature GRP peptide is secreted by ET cells and that GRP acts as an autocrine growth factor in ET both in vitro and in vivo. The proliferative pathways initiated in response to GRP stimulation are still not completely understood but are known to be both cell-type and situation specific. Our attempts at characterizing GRP-mediated proliferative signaling in ET suggest that the effects of this peptide may not be primarily mitogenic and we hypothesize that involvement of cytoskeletal proteins and differentiation pathways may be more important to the autocrine growth-factor capability of GRP in ET cells. Furthermore, studies of ET cell proliferation grown as multi-cellular spheroids indicate that in vivo growth and proliferation may be better represented by this anchorage-independent cell culture model. In comparing adherent and anchorage-independent\r\nproliferation, we also observed that fundamental differences exist in the regulation of cyclin DI between these two models. Moreover, the PI3K\u00E2\u0080\u0094AKT pathway was shown to be of key importance to the regulation of both cyclin DI expression and\r\nproliferation in ET cells. Our results suggest that an autocrine growth factor pathway\r\nmediated by GRP exists in ET and that proliferative signaling in these tumours is critically dependent on cell-cell and/or cell-matrix adhesion and the PI3K\u00E2\u0080\u0094AKT pathway."@en . "https://circle.library.ubc.ca/rest/handle/2429/13114?expand=metadata"@en . "18736963 bytes"@en . "application/pdf"@en . "STUDIES OF SIGNALING PATHWAYS THAT REGULATE EWING TUMOUR CELL GROWTH IN VITRO. by ELIZABETH R A C H E L L A W L O R B.Sc. McMaster University, 1986 M . D . McMaster University, 1989 A THESIS SUBMITTED IN PARTIAL F U L F I L L M E N T OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE F A C U L T Y OF G R A D U A T E STUDIES (Department of Pathology and Laboratory Medicine) We accept this thesis as conformiru^jDjhej^q^ired standard THE UNIVERSITY OF BRITISH C O L U M B I A 2001 \u00C2\u00A9 E L I Z A B E T H R A C H E L L A W L O R , 2001 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of p A - T i i ^ u r - c ^ j L*S> rUHMCiMcr The University of British Columbia Vancouver, Canada Date IT 2 O 0 j DE-6 (2/88) 11 ABSTRACT The Ewing family of peripheral primitive neuroectodermal tumours (ET or pPNET) comprises a genetically related group of bone and soft tissue tumours that primarily affects children and adolescents. A l l members of this tumour family are characterized by tumour specific translocations involving the EWS gene from chromosome l l q 2 3 and a second gene contributed by a variety of chromosomal partners. In all cases, the partner gene is a member of the E T S family of transcription factors and the resultant EWS-ETS gene fusions encode novel chimeric proteins comprised of the EWS-amino-terminal domain and an E T S - D N A binding domain. These fusion proteins are oncogenic and the accumulated evidence suggests that they are the primary pathologic lesions contributing to the development of ET. Despite its genetic characterization, little is known about the mechanisms of E W S - E T S mediated oncogenesis specifically, or in fact, the biologic features that contribute to the malignant nature of E T cells. We have endeavoured to study proliferative signaling in E T cells in an effort to better characterize the biology of these aggressive and often fatal tumours. Using differential-display P C R , our laboratory had previously identified differential expression of the human gastrin-releasing peptide (GRP) gene among EWS-ETS expressing tumour cell lines. Further studies confirmed that 100% of E T cell lines and approximately 50% of primary E T samples express the GRP gene and the gene encoding its receptor. Protein and functional studies demonstrated that the mature G R P peptide is secreted by E T cells and that G R P acts as an autocrine growth factor in E T both in vitro and in vivo. The proliferative pathways initiated in response to G R P stimulation are still not completely understood but are known to be both cell-type and situation specific. Our attempts at characterizing G R P -mediated proliferative signaling in E T suggest that the effects of this peptide may not be i i i primarily mitogenic and we hypothesize that involvement of cytoskeletal proteins and differentiation pathways may be more important to the autocrine growth-factor capability of G R P in E T cells. Furthermore, studies of E T cell proliferation grown as multi-cellular spheroids indicate that in vivo growth and proliferation may be better represented by this anchorage-independent cel l culture model. In comparing adherent and anchorage-independent proliferation, we also observed that fundamental differences exist in the regulation of cyclin D I between these two models. Moreover, the P I 3 K \u00E2\u0080\u0094 A K T pathway was shown to be of key importance to the regulation of both cyc l in D I expression and proliferation in E T cells. Our results suggest that an autocrine growth factor pathway mediated by G R P exists in E T and that proliferative signaling in these tumours is critically dependent on cell-cell and/or cell-matrix adhesion and the P I 3 K \u00E2\u0080\u0094 A K T pathway. IV TABLE OF CONTENTS ABSTRACT TABLE OF CONTENTS iv LIST OF TABLES ix LIST OF FIGURES x LIST OF ABBREVIATIONS x i i ACKNOWLEDGEMENTS xiv CHAPTER I: INTRODUCTION 1 1.1 Synopsis and rationale for thesis 1 7.3 The Ewing Family of Peripheral Primitive 2 Neuroectodermal Tumours 1.2.1 Clinical & Pathologic Features 2 1.2.2 Genetics of the Ewing Tumour Family 4 1.2.2.1 EWS-ETS Gene Fusions 5 1.2.2.2 E W S - E T S Chimeric Oncoproteins 6 1.2.2.3 Mechanisms of E W S - E T S Mediated 6 Oncogenesis 1.2.2.4 Clinical Importance of EWS-ETS Gene 10 Fusion Type 1.2.2.5 Other Genetic Alterations in E T 10 1.2.3 The Role of the IGFI-Receptor and other 11 Growth Factro Receptor Pathways in E T 1.3 Regulation of Normal Cel l Growth & Proliferation 12 1.3.1 Growth Factor-Mediated Cel l Signaling 12 1.3.1.1 Growth Factor Receptors 12 1.3.1.2 The R A S \u00E2\u0080\u0094 R A F 1 \u00E2\u0080\u0094 M E K \u00E2\u0080\u0094 E R K 17 Mitogen Activated Protein Kinase Pathway 1.3.1.3 The P I 3 K \u00E2\u0080\u0094 A K T Pathway 21 1.3.1.4 Cross-talk between Growth Factor- 22 Mediated Signaling Pathways 1.3.2 The Cel l Cycle 26 1.3.2.1 Cyclins and Cyclin-Dependent Kinases 27 1.3.2.2 Inhibitors of Cel l Cycle Progression 30 1.3.2.3 Regulation of Cycl in D 33 1.3.3 Differentiation 35 1.3.4 Apoptosis 35 1.3.5 Anchorage Dependent Growth 37 1.4 Mechanisms of Oncogenesis 39 1.4.1 Oncogenes 39 1.4.2 Chromosomal Translocations & Gene 42 Rearrangements 1.4.3 Tumour Suppressor Genes 45 1.4.3.1 p53 45 1.4.3.2 Rb 49 1.4.3.3 A R F 49 1.4.4 Autocrine Growth Factors 50 1.5 Aims & Objectives 51 C H A P T E R II: M A T E R I A L S & M E T H O D S 52 2.1 Cel l Culture & Clinical Samples 52 2.1.1 Cel l lines and tissue culture 52 2.1.2 Anchorage-independent multicellular spheroid 52 cultures 2.1.3 Primary tumour specimens 55 2.2 Analysis of Gene Expression 56 2.2.1 Isolation of R N A 56 2.2.2 Reverse Transcriptase-PCR 56 2.2.3 Southern & Northern Analysis 57 2.2.4 Differential Display-PCR 58 2.3 Analysis of Protein Expression & Cel l Structure 59 2.3.1 Radioimmunoassay 59 2.3.2 Immunohistochemistry & Electron Microscopy 59 2.3.3 Protein Lysate Preparation 60 2.3.4 Immunoprecipitation 61 2.3.5 Immunoblotting 62 2.4 Functional Studies & Proliferation Assays 63 2.4.1 EWS-FLI1 & GflP-promoter Reporter Gene 63 Assays 2.4.2 Cel l Growth in vitro Following Treatment with 63 G R P - R Antagonists & Agonists 2.4.3 Cel l Growth in vivo Following Treatment with 65 G R P - R Antagonists 2.4.4 Signal Transduction in Ewing cells following 66 Treatment with G R P - R Antagonists & Agonists 2.4.5 B r d U Proliferation Assays 66 2.4.6 Cel l cycle analysis by F A C S 67 2.4.7 Kinase inhibitor studies 67 2.4.8 Myris t i la ted-AKT & Dominant-negative I L K 68 Transfections 2.5 Clinical Correlates Study 68 VI CHAPTER III: HUMAN GASTRIN-RELEASING PEPTIDE IS 70 DIFFERENTIALLY EXPRESSED BY THE EWING FAMILY OF TUMOURS 3.1 Introduction 70 3.2 Results 71 3.2.1 Differential expression of GRP in E T cell lines 71 3.2.2 Expression of GRP-R in E T cell lines 75 3.2.3 Expression of GRP and GRP-R in Primary 75 Ewing Tumours 3.2.4 Expression of Bioactive G R P peptide in ET 78 3.2.5 GRP is not a direct target of E W S - E T S chimeric proteins 3.2.6 Clinical Correlates Study 81 3.3 Discussion 83 CHAPTER IV: GASTRIN-RELEASING PEPTIDE FUNCTIONS AS AN AUTOCRINE GROWTH FACTOR IN THE EWING FAMILY OF TUMOURS 4.1 4.2 Introduction Results 4.2.1 in vitro Ce l l Proliferation Studies 4.2.2 Collaborative studies on GRP-mediated Autocrine growth in vitro & in vivo 4.2.3 Blockade of G R P signaling in E T does not appear to affect E R K or A K T activation 4.2.4 Cyc l in D protein Levels are unaffected by blockade of G R P Signaling in E T cells 4.2.5 The effects of G R P - R blockade on the adhesion related kinases F A K & P Y K 2 4.3 Discussion 88 88 89 89 91 91 94 97 97 CHAPTER V: PROLIFERATION IN EWING TUMOUR CELLS GROWN AS ANCHORAGE-INDEPENDENT SPHEROIDS 5.1 Introduction 5.2 Results 5.2.1 Ewing tumour cells spontaneously form spheroids when grown in suspension 5.2.2 Adherent monolayer E T cells have significantly higher rates of proliferation than either spheroid or primary tumour cells 5.2.3 Cyc l in D protein expression in E T cells grown in suspension requires cell-cell adhesion and is serum dependent 103 103 104 104 106 108 vii 5.2.4 Differences in cyclin D I expression between 112 spheroids and monolayers are post-transcriptional and are associated with differences in subcellular localization 5.3 Discussion 118 C H A P T E R V I : T H E R O L E S O F T H E R A S \u00E2\u0080\u0094 R A F \u00E2\u0080\u0094 M E K \u00E2\u0080\u0094 E R K A N D 122 P I 3 K \u00E2\u0080\u0094 A K T P A T H W A Y S IN E W I N G T U M O U R C E L L P R O L I F E R A T I O N 6.1 Introduction 122 6.2 Results 123 6.2.1 The E R K 1 / 2 M A P K and P I 3 K \u00E2\u0080\u0094 A K T pathways 123 are upregulated in E T cells in suspension 6.2.2 M E K inhibition does not appreciably affect 124 cyclin D I protein expression or cellular proliferation in E T cells 6.2.3 PI3K inhibition blocks cyclin D I protein 127 expression in E T cells and significantly reduces proliferation 6.2.4 Down-regulation of cyclin D I expression in 130 PI3K inhibitor treated E T cells is post-transcriptional but does not correlate with GSK3(3 phosphorylation 6.2.5 Primary Ewing tumours demonstrate patterns of 133 cyclin D I expression and E R K 1 / 2 and A K T activation that resemble those of E T spheroids 6.2.6 Expression of constitutively active A K T by E T 136 cells leads to spontaneous formation of anchorage independent spheroids 6.2.7 Preliminary studies evaluating the role of 139 integrin-linked kinase ( ILK) in A K T phosphorylation in anchorage-independent E T cells. 6.3 Discussion 141 C H A P T E R VII: S U M M A R Y & F U T U R E D I R E C T I O N S 149 7.1 General Summary 149 7.2 G R P Autocrine Growth Signaling in E T 150 7.3 The Role of Adhesion Molecules in E T Proliferative 153 Signaling 7.4 Cycl in D I Regulation and Subcellular 155 Compartmentalization 7.5 Final Comments 158 R E F E R E N C E S 160 LIST OF TABLES TABLE # TITLE OF TABLE PAGE TABLE 1. Selected Oncogenes in Human Cancer 40 TABLE 2. Summary of recurrent chromosomal translocations found in 43 pediatric soft-tissue tumours TABLE 3. Selected human tumour suppressor genes 46 TABLE 4. Ewing tumour family cell lines 53 TABLE 5. Non-Ewing tumour cell lines 54 TABLE 6. Immunoreactive G R P peptide expression demonstrated by 79 radioimmunoassay TABLE 7. GRP/GRP-R Status of Primary Tumour Cohort and its Relationship 82 to Event-Free Survival TABLE 8. Cel l cycle analysis of culture TC32 cells shows decreased cycling 111 and increased serum sensitivity of spheroids X LIST OF FIGURES FIGURE # TITLE OF FIGURE PAGE FIGURE 1. Chromosomal translocations in Ewing tumours. 7-8 FIGURE 2. Growth signaling mediated by receptor protein tyrosine 15-16 kinases FIGURE 3. Proliferative signaling via G-protein coupled receptors 18-19 FIGURE 4. P I 3 K \u00E2\u0080\u0094 A K T survival signaling 23-24 FIGURE 5. The cell cycle 28 FIGURE 6. Regulation of cell cycle progression 31-32 FIGURE 7. Chromosomal translocation can result in a potentially 44 oncogenic gene rearrangement FIGURE 8. Mult iple pathways mediate effects of p53 activation 48 FIGURE 9. D D - P C R analysis of S R C T cell lines 72 FIGURE 10. Screening of S R C T cell lines for GRP expression by Northern 74 analysis FIGURE 11. R T - P C R Analysis of S R C T Cel l Lines 76 FIGURE 12. R T - P C R Analysis of Primary S R C T Samples 77 FIGURE 13. Immunohistochemistry of primary S R C T s with a G R P - 80 specific antibody FIGURE 14. In vitro response of S R C T cell lines to G R P - R antagonist & 90 agonist FIGURE 15. A ) , in vitro proliferation rate of E T cell line TC71 in response 92 to increasing concentrations of G R P - R antagonists RC-3095 & RC-3940II B) . in vivo growth of human E T xenografts is slowed in 93 response to treatment with G R P - R antagonist RC-3095 FIGURE 16. Manipulation of the G R P - R pathway does not affect levels of 95 E R K 1 / 2 phosphorylation in TC32 cells XI FIGURE 17. Cyc l in D levels remain unchanged in E T cells despite G R P stimulation or G R P - R pathway inhibition FIGURE 18. G R P - R antagonist treatment alters phosphorylation of Pyk2-related proteins FIGURE 19. E T cells form multi-cellular spheroids when grown in suspension FIGURE 20. E T spheroids are morphologically similar to primary Ewing tumours 96 98 105 107 FIGURE 21. The proliferative index of E T spheroids is similar to primary 109-10 Ewing tumours FIGURE 22. Cyc l in D I protein expresssion is dependent on cell-cell 113-114 adhesion and serum stimulation in suspension cultures of E T cells FIGURE 23. FIGURE 24. FIGURE 25. FIGURE 26. FIGURE 27. FIGURE 28. FIGURE 29. FIGURE 30. FIGURE 31. FIGURE 32. Variations in cyclin D I are post-transcriptional 116 Subcellular distribution of cyclin D1 differs between 119 monolayer and spheroid E T cells. Differential activation of the E R K 1 and E R K 2 M A P kinases 125-126 and of A K T in E T suspension cultures M E K inhibition has little effect on E T cell proliferation 128-129 PI3K inhibition blocks cyclin D I protein expression in E T 131-132 cells and significantly reduces cell proliferation Down-regulation of cyclin D I in inhibitor treated cells is post- 134-135 transcriptional Primary Ewing tumours express activated E R K 1 / 2 & A K T 137 and variable levels of cyclin D I M y r - A K T induces spontaneous spheroid formation 138 Effect of PI3K inhibition on expression of cyclin D I in M y r - 140 A K T transfected TC32 cells Effect of dominant-negative I L K expression on A K T 142 phosphorylation in cultured E T cells xii LIST OF ABBREVIATIONS A adenine E R K extra-cellular signal A L L acute lymphoblastic regulated kinase leukemia E R M S embryonal A M P adenosine mono-phosphate rhabdomyosarcoma A P A F 1 apoptotic protease E T Ewing tumour activating factor 1 ETS E-26 transforming specific A R F alternate reading frame E W S Ewing sarcoma A R M S alveolar F A C S fluorescent analysis cell rhabdomyosarcoma sorting A T P adenosine tri-phosphate F A K focal adhesion kinase B A D Bcl -2 antagonist of cell F B S fetal bovine serum death F G F fibroblast growth factor B C R breakpoint cluster region FKFfR forkhead in bp base pair rhabdomyosarcoma B R D U bromo-deoxyuridine F L U Friend leukemia virus C cytosine integration site 1) C A K C D K activating kinase F R N K FAK-rela ted non kinase C D N A complementary D N A G guanine C D K cyclin dependent kinase G A P GTPase activating protein C K I cyclin dependent kinase G D P guanosine diphosphate inhibitor G P C R G-protein coupled receptor CIP /KIP CDK-interacting G R P gastrin releasing peptide proteins/kinase inhibitory G R P - R G R P receptor proteins G S K 3 glycogen synthase kinase-3 C N S central nervous system G T P guanosine tri-phosphate C T P cytosine tri-phosphate GTPase guanosine tri-phosphatase D A G di-acyl glycerol H & E hematoxylin and eosin D B D D N A binding domain IGFI/II insulin-like growth factor D D - P C R differential display P C R type I and II der derivative IGFI-R IGFI receptor D F S P dermatofibrosarcoma I L K integrin linked kinase protruberans I N K 4 inhibitor of C D K - 4 D M S O dimethyl sulphoxide IRS insulin receptor substrate D N A deoxyribonucleic acid kDa kilodalton D S R C T desmoplastic small round M A P K mitogen activated protein cell tumour kinase E C M extra-cellular matrix M E K M A P K - E R K \u00E2\u0080\u0094 a c t i v a t i n g E D T A ethylene-diamine- kinase tetraacetic acid M E M malignant EFS event-free survival ectomesenchymoma E G F epidermal growth factor M L monolayer E G F R EGF-receptor m R N A messenger R N A E R G ETS-related gene xiii M T T methylthiazol tetrazolium P T K protein tyrosine kinase bromide P Y K 2 protein-tyrosine kinase 2 M Y R myristilated R A F T K related adhesion focal N B neuroblastoma tyrosine kinase N O S not otherwise specified R B retinoblastoma N T D N-terminal domain R B D RNA-bind ing domain O C T optimal cutting temperature R G D arginine-glycine-aspartic O N B olfactory neuroblastoma acid OS overall survival R I A radioimmunoassay P A G E poly-acrylamide gel R M S rhabdomyosarcoma electrophoresis R N A ribonucleic acid P B S phosphate buffered saline R P T K receptor protein tyrosine P C N A proliferating cell nuclear kinase antigen R T - P C R reverse-transcriptase P C R P C R polymerase chain reaction S C L C small cell lung cancer P D K 3-phosphoinosifi de- SDS sodium dodecyl sulphate dependent kinase 1 S E R serine P D G F platelet derived growth SH2/3 Src-homology 2 and 3 factor SOS son of sevenless P D G F - R P D G F receptor S R C T small round cell tumour P D K phosphotidylinositol 3 -OH T thymine kinase T A D transactivation domain P K B protein kinase B T B S tris-buffered saline P K C protein kinase C T G F p transforming growth factor P L C phospholipase C P P M S F phenylmethylsulfonyl T G F p T l - R T G F P type II receptor fluoride T H R threonine P N peripheral T L S translocated in liposarcoma neuroepithelioma T N F tumour necrosis factor p P N E T peripheral primitive T R A I L TNF-related apoptosis neuroectodermal tumour inducing ligand P R N K PYK2-related non-kinase T S G tumour suppressor gene P S B phosphorylation V E G F vascular endothelial growth solubilization buffer factor P T B phospho-tyrosine binding xiv ACKNOWLEDGEMENTS T may not have gone where I intended to go, but I have ended up where I intended to be.\" ~ Douglas Adams ~ This thesis would not have been completed, nor in fact undertaken at all , i f it were not for the continued and unwavering support of many. First and foremost, I must thank my supervisor and mentor Dr. Poul Ff.B. Sorensen. When I initially approached him with the idea of spending a \"short time\" in his lab to learn more about childhood cancer genetics, neither of us had any concept of the commitment he would be making. A t that time, I had no visions of pursuing either a PhD or a career as a clinician scientist. Now, largely because of his training and guidance, my career focus has shifted and I am embarking upon a completely new and challenging course. I am indebted to Dr. Sorensen for his patience, his wisdom and his friendship. Secondly, I owe tremendous thanks to all members of the Sorensen lab family, past and present. Because of their support, humour and camaraderie I was able to endure even the most frustrating months of slow progress and no results. In particular, I must recognize and thank Jerian L i m for her incredible patience at the very beginning when I was truly a neophyte in the lab. Much of my success in the lab can be attributed to her initial instruction. To my family and friends who have always believed in me and encouraged me to follow my dreams, thank you for your love and your unwavering support. Finally, and most importantly, I must thank the people who inspire me on a daily basis. To my patients, the children who endure so much, thank you for all that you continue to teach me about courage, dignity and grace. I am honoured to know you and promise to do my very best to lessen your suffering. 1 CHAPTER I INTRODUCTION 1.1 SYNOPSIS AND RATIONALE FOR THESIS Each year, approximately 1200 children in Canada are diagnosed with a malignancy but, because of the tremendous advances in diagnosis and treatment over the past 25 years, greater than 70% of afflicted children w i l l be long term survivors (1). Unfortunately, however, there are still a number of childhood cancers for which cures elude us and the majority of children diagnosed with these tumours succumb to their disease despite state-of-the-art treatment. Thus, there is still a real need for the development of novel treatment strategies in childhood cancer. A m o n g the pediatric malignancies with continued high mortality rates are brain tumours, relapsed leukemias and the metastatic solid tumours. Advances in treatment for these aggressive tumours depend on the development of an improved understanding of their biology. In this thesis, my focus is on growth regulation in the Ewing family of tumours (ET), the second most common family of pediatric bone tumours and soft tissue sarcomas (2). Despite increasingly intensive therapeutic protocols, the mortality rate for metastatic and relapsed E T is extremely high. Innovative approaches to E T therapy need to be developed that target the cancer cells specifically by exploiting the biologic features that differentiate them from normal cells. In this thesis, I have studied the growth of E T cells in vitro in an effort to better define the signal transduction pathways that are important to its growth in vivo. I have shown that E T differentially expresses human gastrin releasing peptide (GRP) 2 and that this hormone acts as an autocrine growth factor in vitro and in a xenograft mouse model. Observations on the role of GRP in ET cell growth and its potential implications for novel treatment strategies will be discussed in Chapters III and IV. I have also demonstrated that ET cell growth in vitro is radically different between cells grown as anchorage independent spheroids and those grown as traditional adherent monolayers. The potential mechanisms behind these differences were investigated with respect to critical proliferative pathways and these results and their relevance to the in vivo setting will be discussed in Chapters V and VI. In the following sections, our current understanding of ET cell biology will be discussed with reference to the currently published literature. This will be followed by a general review of the normal mechanisms of cell growth regulation, in particular signal transduction and the cell cycle. Finally, a brief discussion of the general mechanisms of oncogenesis will complete this introductory chapter. For more in depth discussions of normal and dysregulated growth, the reader is referred to the list of references at the end of this thesis. 1.2 THE EWING TUMOUR FAMILY OF PERIPHERAL PRIMITIVE NEUROECTODERMAL TUMOURS 1.2.1 C L I N I C A L & P A T H O L O G I C F E A T U R E S The Ewing family of tumours (ET), also referred to as peripheral primitive neuroectodermal tumours (pPNETs), comprises a group of genetically related aggressive bone and soft tissue malignancies including Ewing sarcoma, peripheral neuroepithelioma, and Askin's tumour (3). Although ET predominantly affects children and adolescents, it does occur in older individuals and its incidence in adults is likely underestimated (4). The incidence of E T in Caucasians under the age of 14 is approximately 2.4 per mil l ion, with males and females being equally represented (2). Interestingly, E T almost never occurs in individuals of African or Asian origin for reasons that are not yet understood. This may be explained by subtle inter-racial differences in the sequence of the EWS gene, which is always rearranged in E T (see below)(5). Tumours can develop in almost any bone or soft tissue and presenting symptoms usually include pain and swelling. A t least a quarter of patients have evidence of gross metastatic disease at diagnosis and, given the 10% cure rate attained with local therapy alone, most have micrometastases (3). The most commonly involved primary sites are the pelvis and long bones and metastases are found most frequently in the lungs and bone marrow. Therapy for both localized and metastatic disease is multi-modal, requiring aggressive local control with surgery and/or radiotherapy in addition to multi-agent cytotoxic drug therapy. In cases of metastatic or recurrent disease, therapy has been intensified to the degree that, in some cases, myeloablative doses of chemotherapy are administered followed by autologous or allogeneic bone marrow transplant. Unfortunately, despite such aggressive approaches, the mortality rate of metastatic and relapsed E T is still greater than 75% and over a third of non-metastatic patients w i l l also succumb to their disease (6). In addition, treatment related morbidity is not insignificant and long-term complications of therapy continue to plague survivors for many years following the cure of their cancer. Histologically, E T is characterized by the appearance of small round blue cells, a feature it shares with other pediatric malignancies inc lud ing neuroblastoma, rhabdomyosarcoma and lymphoma. Morphologically, these tumour cells are characterized by a very primitive appearance without any specific defining features (7). Thus, successful differentiation of E T from other histologically similar tumours is extremely important and 4 often very difficult. Typically, the histology of E T is that of monomorphous sheets of small round cells with hyperchromatic nuclei, inconspicuous nucleoli, and scant cytoplasm (3). The histogenetic origins of E T remain unclear. Immunohistochemistry and ultrastructural analysis confirm the very primitive nature of E T cells and support a neuroectodermal derivation (3). Also , E T cell lines w i l l display neural differentiation after treatment with nerve growth factors (2, 8). However, E T can also demonstrate epithelial and mesenchymal characteristics and can arise in organs not directly related to the neural crest (3). Hence, although neuroectodermal origin is currently deemed most l ikely, the histogenetic orgins of E T are still not known with certainty. A n important immunohistochemical feature of E T that aids in its diagnosis and may be important to understanding its biology is its positive staining with the 013 antibody. This antibody recognizes the M I C 2 ( C D 9 9 M I C 2 ) antigen, which is a membrane glycoprotein involved in cell adhesion that may be important in the control of cell cycle progression and cell morphology (3, 6). Detection of membrane localized M I C 2 in a tumour specimen is a sensitive diagnostic marker for E T but, given its very low degree of specificity, it cannot be used exclusively to differentiate E T from other histologically similar tumours. Therefore, until the advent of genetic diagnosis, E T was largely a diagnosis of exclusion. 1.2.2 GENETICS OF T H E E W I N G T U M O U R F A M I L Y In the mid-1980's , it was recognized that E w i n g sarcoma and peripheral neuroepithelioma both demonstrated recurrent and identical chromosomal translocations (9, 10). Identification of the t ( l l ;22)(q24;ql2) translocation in these tumours led to the realization that they were genetically related and provided pathologists with a diagnostic tool 5 that was specific for the E T family. It is now accepted that approximately 85% of E T cases exhibit a t ( l I ;22)(q24;ql2) translocation while the remainder demonstrate alternate translocations involving 22ql2 and another chromosomal partner, most commonly 21q22 (reviewed in (11)). Molecular cloning studies have since revealed that gene disruption and rearrangement occur as a result of ET-specific translocations, and identification of the affected genes has led to a dramatic increase in our understanding of the origins and biology of this tumour family. 1.2.2.1 EWS-ETS Gene Fusions Molecular cloning experiments of ET-specif ic translocations have successfully identified that two disparate genes are disrupted and then joined together as a result of the chromosomal translocations. In all cases, the EWS gene (for E w i n g sarcoma) on chromosome 22q l2 is disrupted and, in most cases, it is fused to the FLU gene on chromosome l l q 2 4 (12, 13). In the majority of the remainder of cases, EWS is fused to the ERG gene from chromosome 21q22 (14). Rare gene fusions resulting from t(2;22), t(7;22) and t(17;22) translocations have also been described in which EWS is fused to the genes FEV, ETV1, and E1AF, respectively (15-17). A l l EWS partners identified thus far in E T -specific translocations are members of the ETS family of transcription factors. Additionally, all EWS-ETS fusions occur in-frame such that the resulting fused sequences create completely novel genes that encode chimeric proteins containing functional domains contributed by each of the involved partner genes. 6 1.2.2.2 EWS-ETS Chimeric Oncoproteins E W S is a ubiquitously expressed RNA-binding protein of unknown function that may be involved in m R N A transcription (3). It is closely related to the TLS/FUS gene, which is involved in myxoid liposarcoma-specific gene translocations, and contains an amino-terminal transactivation domain (NTD) and a carboxy-terminal R N A binding domain. E W S -E T S chimeric products retain the N T D of E W S and replace its carboxyterminus with domains from the E T S partner. The E T S family of transcription factors is a large family of D N A - b i n d i n g proteins that are implicated in the control of cellular proliferation, development and tumorigenesis (18). They are characterized by a highly conserved 85-amino acid domain termed the erythroblastosis virus-transforming sequence (ETS) domain that mediates specific D N A binding to purine-rich sequences with a GGA(ATT) core element (3). In E T translocations, the E W S - N T D is fused to the E T S domain of the gene partner, creating a gene with D N A binding properties of an ETS gene under the control of EWS regulatory elements (see Figure 1)(11). E W S - E T S proteins are oncogenic and transformation requires both the N T D - E W S and a functional E T S D N A binding domain (reviewed in (11)). Although the precise mechanisms of E W S - E T S mediated oncogenesis are as yet unknown, many studies have sought to determine the nature of E W S - E T S induced transformation. 1.2.2.3 Mechanisms of EWS-ETS Mediated Oncogenesis Several lines of evidence support the notion that E W S - E T S oncoproteins function as aberrant transcription factors. First, E W S - F L I 1 localizes to the nucleus (19). Second, E W S -FLI1 and E W S - E R G bind in vitro ETS target sequences with similar specificities and FIGURE 1. Chromosomal Translocations in Ewing Tumours. Chromosomal breakage of chromosome 22ql2 results in disruption of the EWS gene at one of several potential breakpoints between exons 7 & 10. This results in splitting of the N-terminal transactivation domain (NTD) from the R N A binding (RBD) domain and the N T D then fuses to the D N A binding domain (DBD) of an E T S partner gene that has been similarly disrupted by chromosomal breakage. Approximately 85% of EWS-ETS fusions involve fusion of N T D - \u00C2\u00A3 W S with F L U . The resultant fusion gene encodes a novel chimeric protein that is oncogenic and acts as an aberrant transcription factor. Target genes of E W S - E T S chimeras are still being identified. Several are listed in the figure and discussed further in the text. 9 affinities as the native FLI1 and E R G molecules, respectively (20, 21). Third, E W S - F L I 1 is a more potent transactivator than native F L U (22-24). The genes specifically regulated by E W S - E T S chimeric transcription factors remain largely unknown; however, several potential targets have been identified and their activation may not be solely dependent on the ETS domain but may also be affected by protein-protein interactions that are unique to the chimeric molecule. Thus, the spectrum of fusion gene targets may be different from native E T S targets (25). Among putative E W S - E T S target genes identified to date are: MFNG, a member of the Fringe gene family of signaling molecules; the metalloproteinase stromelysin; the SH2-domain containing molecule EAT-2; the cyclin-specific ubiquitin-conjugating enzyme E2-C; c-myc; and serum response element-containing genes such as c-fos or egrl (see (3) for review). Recent evidence also indicates that the gene for the TGF(3-type II receptor is transcriptionally repressed by the E W S - E T S oncoproteins (26). Knowledge of genes transcriptionally activated or repressed by chimeric E W S - E T S oncoproteins may lead to a better understanding of the mechanisms underlying malignant transformation in E T and many investigators continue to endeavour to identify E W S - E T S transcriptional targets. Further evidence supporting the oncogenic role of EWS-ETS gene fusions comes from studies of E T cell lines. Transfection of antisense molecules into E T cell lines results in diminished E W S - F L I 1 protein expression and growth inhibition, both in vitro and in tumour xenografts (27). In addition, transfection of E T cells with dominant negative inhibitors to E W S - F L I 1 also results in diminished growth (28). Both of these experimental observations suggest that antagonism of the E W S - E T S proteins in vivo may provide a novel therapeutic strategy for E T . 10 1.2.2.4 Clinical Importance of EWS-ETS Gene Fusion Type Analysis of large numbers of cases has documented that virtually all E T carry some form of EWS-ETS gene fusion, and this marker is now considered pathognomonic of the E T family (29). Moreover, screening of a variety of tumour subtypes for EWS-ETS fusion transcripts has led to the inclusion of at least some forms of malignant ectomesenchymoma ( M E M ) and olfactory neuroblastoma (ONB) in the E T family based on the expression of similar gene fusions (30, 31). Molecular analysis of the breakpoints within EWS and its partners has also revealed genetic heterogeneity such that as many as 18 different combinatorial fusion products are possible (3, 32). The most common fusion transcript links exon 7 of EWS with exon 6 of FLU, yielding the so-called type I fusion (32). Identification of the fusion type is potentially important to the understanding of the biology of these tumours given that patients with the type I fusion have a better prognosis than patients harbouring the less common EWS-FLI1 variants (33). Thus far, the clinical features of patients with EWS-FL11 and EWS-ERG fusions appear to be similar (34). 1.2.2.5 Other Genetic Alterations in ET While EWS-ETS gene rearrangements are uniformly present and likely represent the primary genetic events in E T , other recurrent genetic lesions have also been described that may contribute to E T progression (reviewed in (3)). Frequent gains of chromosomes 8 and 12 have been observed by conventional and molecular cytogenetic techniques as well as losses of l p and an unbalanced derl6t(l ;16). Clinico-pathologic cohort studies of primary tumour samples have also identified changes in tumour-associated genes. Homozygous deletion of p l 6 I N K 4 a was detected in 18% of cases and p53 mutations in 11% and both lesions 11 conferred a poor outcome (35, 36). Clearly, secondary genetic events are also important to the biology and clinical characteristics of E T and warrant further investigation. 1.2.3 T H E R O L E O F T H E I G F 1 - R E C E P T O R A N D O T H E R G R O W T H F A C T O R R E C E P T O R P A T H W A Y S IN ET Most of the current knowledge surrounding the pathogenesis of E T involves the study of its characteristic genetic lesions. Recently however, more efforts have been placed on defining the roles of specific growth factor-mediated pathways in regulating the growth of these tumours. A general review of normal growth factor-mediated proliferative and survival pathways can be found in section 1.3. The evidence supporting their dysregulation in E T is presented here. The insulin-like growth factor I receptor (IGF1-R) pathway was the first pathway that was identified to be of critical importance to E T pathogenesis. In 1997, Toretsky et al. reported that E W S - F L I 1 induced transformation of fibroblasts requires expression of the IGFl-receptor (37). Exactly how E W S - F L I 1 and IGF1-R interact to promote oncogenesis is not known and is the subject of intense ongoing study. Disruption of the IGF1-R pathway by receptor-targeted antibodies or blockade of the downstream signaling molecule phosphotidylinosistol-3-kinase (PI3K; see section 1.3.1.3) results in reduced proliferation and increased apoptosis of E T cells (38, 39). Thus, whatever the mechanism, the IGF1-R pathway is a key regulator of the pathogenesis and continued proliferation of E T cells. Other growth-factor pathways recently implicated in E T cell growth include the basic fibroblast growth factor (bFGF) pathway (40), stem cell factor and its receptor c-kit (41, 42), 12 and phospholipase C-(3 pathways (43). The precise roles of these pathways in regulating E T cell growth and proliferation remain to be determined, as do their relationships to the E W S -E T S oncoproteins. In this thesis, we explore the role of another growth factor, gastrin-releasing peptide, in mediating E T growth and endeavour to elucidate the growth signaling pathways that are critical for E T cell proliferation. 1.3 REGULATION OF NORMAL CELL GROWTH & PROLIFERATION 1.3.1 G R O W T H F A C T O R - M E D I A T E D C E L L S I G N A L I N G The processes of cell growth, differentiation and death are tightly regulated in normal cells by complex biochemical events that govern cellular fate. Upon receipt of a signal from the extra-cellular environment or from within itself, a cell w i l l respond in the manner that is appropriate to the signal. In other words, a normal cel l can recognize and respond accordingly to stimuli that signal it to divide, differentiate or die. Disruption of any part of this intricate homeostatic mechanism can lead to the aberrations of growth and differentiation that are the hallmarks of cancer. In this section, the cellular processes that control these key events wi l l be reviewed. 1.3.1.1 Growth Factor Receptors Signal transduction is responsible for modulating or altering cellular behaviour and its precise coordination and integration is necessary for normal growth and differentiation in embryonic and adult life (44). Signal transduction cascades commence with the binding of extra-cellular growth factors to trans-membrane receptors that, once activated, bind 13 cytoplasmic proteins. Among these proteins are the cytoplasmic kinases whose phosphorylation at tyrosine or serine/threonine residues leads to conformational change and activation of kinase domains. This initiates a complex sequential cascade of phosphorylation and dephosphorylation that results in propagation of the signal to the nucleus (see (45) for review). Growth factor receptors include those with intrinsic tyrosine kinase activity, such as the receptor protein tyrosine kinase ( R P T K ) family, those which are indirectly coupled to tyrosine kinases, such as integrins, and those which are coupled to guanine nucleotide binding proteins, the G-protein coupled receptors (GPCR) . In very broad terms, activation of R P T K s usually results in cellular proliferation while G P C R signaling is more often linked to tissue-specific, fully differentiated cell functions (46). However, as w i l l be discussed in more detail later, it is becoming increasingly apparent that extensive cross-talk exists between proliferative and differentiation pathways. Ligands for the R P T K S are diverse and can be small monomelic polypeptides such as epidermal growth factor (EGF) and fibroblast growth factor (FGF) , or dimeric polypeptides such as platelet-derived growth factor (PDGF) . There are currently 58 known R P T K s , distributed into 20 subfamilies. A l l are transmembrane proteins with protruding growth factor binding amino termini that are several hundred amino acids in length (reviewed in (44)). Examples of R P T K s are the insulin-like growth factor type 1 receptor (IGF1-R), the E G F receptor and the P D G F receptor. The cytoplasmic portion of the R P T K s contains a catalytic tyrosine kinase domain that is typically about 260 amino acids long and is conserved with up to 90% sequence identity from one R P T K to another (45). Signaling by R P T K s requires ligand-induced receptor ol igomerizat ion, which induces tyrosine autophosphorylation. That is, ligand binding brings together two or more receptors and their 14 catalytic subunits trans-phosphorylate one another. Once dimerized, R P T K s are catalytically active and the phosphorylated residues act as docking sites for cytoplasmic signaling proteins that recognize specific phospho-tyrosine residues. Thus, active cell signaling molecules complex at the cell surface with growth factor receptors, and with each other, through the recognition of phospho-tyrosines by specific binding domains. These domains, are evolutionarily conserved and include the Src homology-2 (SH2) and other protein tyrosine binding (PTB) domains (44). SH2 and P T B domains lie outside of the catalytically active kinase domains of signaling molecules and bind to phosphorylated tyrosines that are adjacent to specific flanking amino acids (45). Binding of S H 2 / P T B proteins to their recognized phospho-tyrosine targets facilitates auto-phosphorylation, resulting in alteration of enzymatic activities and propagation of the signal. In addition to catalytically active molecules such as protein tyrosine kinases (e.g. phosphotidylinositol-3 kinase; P I3K) , phospholipases (e.g. phospholipase C ; P L C ) , and protein tyrosine phosphatases (e.g. Src homology 2-protein tyrosine phosphatase 1; SHP1), other non- catalytically active SH2/PTB proteins exist. Such SH2-adaptor molecules include Grb2, insulin-receptor substrate 1 (IRS-1) and IRS-2, all of which act as links between signaling molecules within and among different signaling pathways. In summary, R P T K s igna l ing involves l igand- induced tyrosine phosphorylation/activation of the receptor followed by sequential phosphorylation/activation of kinases, lipases and phosphatases, either directly or through adaptor molecules, such that the signal is propagated from the cell membrane to the nucleus. Proliferative signaling via the E G F R is shown schematically in Figure 2. G-protein coupled receptor (GPCR) signaling is less well characterized than that of the R P T K s . G P C R s are serpentine, seven-membrane spanning receptors that associate 15 FIGURE 2. Growth signaling mediated by receptor protein tyrosine kinases (RPTKs). In this figure, proliferative signaling induced by binding of the extracellular growth factor epidermal growth factor (EGF) to its transmembranous receptor E G F - R is shown schematically. Ligation of the receptor by E G F leads to receptor dimerization, intrinsic kinase activation, and auto-phosphorylation of cytoplasmic tyrosine residues. These residues then act as docking sites for cytoplasmic proteins with SH2 or other protein tyrosine binding (PTB) domains such as Grb2. Binding and activation of cytoplasmic kinases initiates a cascade of phosphorylation and dephosphorylation of signaling proteins, resulting in transmission of the signal to the nucleus, where gene transcription occurs. In this figure, the most common proliferative cascade, the R A S \u00E2\u0080\u0094 R A F \u00E2\u0080\u0094 M E K \u00E2\u0080\u0094 E R K mitogen activated protein kinase ( M A P K ) pathway is shown. 17 cytoplasmically with GTP-binding proteins (G-proteins). G proteins are small heterotrimeric molecules with a , (3, and y sub-units that are bound to G D P in their inactive state. Activation of a G P C R by its ligand causes the G-protein to exchange G D P for G T P and dissociate into a G a - G T P bound subunit and a G(3y heterodimer. Both components subsequently activate downstream effectors, most importantly phospholipase C and c y c l i c - A M P , that in turn acitvate protein kinase C and protein kinase A , respectively, initiating proliferation and differentiation in calcium- and cAMP-dependent fashions (47, 48). Futhermore, G proteins can directly regulate ion channel or kinase function (46). In addition to being linked to cell growth and differentiation, G P C R mediated signaling is also involved in the regulation of neurotransmission, endocrine and exocrine gland function, chemotaxis and embryogenesis (46). Examples of G P C R s include the receptors for thrombin, bombesin, bradykinin, substance P, acetylcholine and serotonin (46). G P C R mediated mitogenic signaling via the bombesin receptor is shown in Figure 3. Human gastrin releasing peptide is the mammalian equivalent of bombesin and its signaling wi l l be discussed in more detail in Chapters III and I V in relation to its role as a growth factor in E T . 1.3.1.2 The RAS\u00E2\u0080\u0094RAF1\u00E2\u0080\u0094MEK\u00E2\u0080\u0094ERK Mitogen Activated Protein Kinase Pathway Despite numerous divergent initiating signals, proliferative signaling via R P T K s and G P C R s most often converges on a common pathway that culminates in nuclear propagation of the signal. This pathway is the R A S \u00E2\u0080\u0094 R A F \u00E2\u0080\u0094 M E K \u00E2\u0080\u0094 E R K mitogen activated protein kinase ( M A P K ) pathway. R A S is a 21 kDa molecular weight protein that cycles between active GTP-bound and inactive GDP-bound configurations (49). Inactivation of the R A S 18 FIGURE 3. Proliferative signaling via G-protein coupled receptors (GPCRs). G P C R s are linked to cellular proliferation via complex inter-connected pathways that are cell-type and situation specific. In this diagram, several well-characterized (solid arrows) and putative (dashed arrows) pathways l inking bombesin/gastrin-releasing peptide (GRP) and its receptor to G l - S phase transition are shown. Proliferative signaling largely converges on the E R K M A P K pathway following activation of such upstream molecules as phospholipase C (PLC) , protein kinase C ( P K C ) and S R C . G R P signaling also links the adhesion related proteins Pyk2 and p l 2 5 F A K to proliferation, possibly via activation of cytoskeletal proteins such as Rho and R O K (50) 20 protein occurs via its intrinsic GTPase activity, which is facilitated by the family of GTPase-activating proteins (GAPs) . R A S activation occurs at the cell membrane, downstream of growth factor receptor activation and is accomplished by guanine nucleotide exchange factors such as SOS (sons of sevenless) that exchange free G T P for RAS-bound G D P (49). In the case of R P T K signaling, receptor activation leads to direct activation of R A S via the SH2-adaptor molecule G R B 2 , which links the activated receptor to SOS, thereby facilitating activation of R A S . Activated R A S leads to the activation of several downstream effector molecules, which in turn mediate the multitude of biologic effects that are governed by the R A S molecule (51). Among R A S effectors is R A F 1 , the next critical molecule in the R A S proliferative pathway. R A F 1 is a serine/threonine kinase that becomes phosphorylated upon binding to activated R A S . R A F 1 phosphorylates and activates M E K 1 and M E K 2 ( M A P K -E R K kinases), dual-specificity kinases that in turn activate E R K 1 and E R K 2 (extracellular stimulus regulated kinases) by phosphorylating threonine and tyrosine residues (49). The E R K s are members of the M A P K family of signaling molecules and their activation leads to cell d ivis ion. Act ivat ion of the other M A P K s , S A P K / J N K (stress-activated protein kinase/jun amino-terminal kinase) and p 3 8 H O G , occurs via structurally related molecules in parallel signaling pathways but leads to a different outcome, growth arrest (49). Once activated, the E R K molecules translocate to the nucleus where they regulate gene transcription by phosphorylating transcription factors such as E L K 1 , ETS1 and c-myc (52). Activation of these transcription factors leads to D N A replication and cell division. Figure 2 shows E G F receptor-induced R A S pathway activation. In addition, cytoplasmic targets of E R K exist, including the E R K regulators SOS, R A F 1 and M E K , such that the potential for positive feedback loops exists within the R A S \u00E2\u0080\u0094 R A F 1 \u00E2\u0080\u0094 M E K \u00E2\u0080\u0094 E R K pathway (52). 21 1.3.1.3 The P I 3 K \u00E2\u0080\u0094 A K T Pathway In recent years it has become apparent that cellular proliferation is critically linked to cellular survival. That is, growth factors often initiate both proliferative and survival signals concomitantly. L ike proliferative signals, survival signals generally originate from ligation of transmembrane receptors and converge on a single pathway, the phosphotidylinositol 3-O H kinase ( P I 3 K ) \u00E2\u0080\u0094 A K T pathway (53). One form of PI3K, which is activated by R P T K s , exists as a heterodimer of an 85 kDa regulatory subunit and a 110 k D a catalytic subunit. Activation of the catalytic subunit occurs when the p85 regulatory subunit binds to tyrosine phosphorylated growth factor receptors via two SH2 domains (49). Additionally, P I3K can be activated indirectly via intermediate activation of R A S ; thus, like R A F , PI3K is a R A S effector and links proliferative and survival pathways (54). Once the P I 3 K p i 10 catalytic subunit is activated, it catalyzes the transfer of phosphate from A T P to membrane-localized phospholipids termed phosphoinositides. The principally generated 3'-phosphorylated phosphoinositides are phosphoinositol 3,4 bisphosphate (PT3,4P) and phosphoinositol 3,4,5 trisphospate (PI3,4,5P) which function as signaling intermediates regulating downstream signal transduction cascades (53). The 3'-phosphorylated phosphoinositides bind to the serine/threonine kinase A K T (also termed P K B ) at its pleckstrin homology domain resulting in a conformational change and translocation of A K T to the plasma membrane where it becomes phosphorylated. Fu l l activation of A K T is dependent on phosphorylation of two key residues, serine-473 and threonine-308. Phosphorylation of A K T at these residues is accomplished by the direct binding of phosphoinositides and by the combined activities of three kinases that function independently and in concert. These three kinases are termed 3-phosphoinositide-dependent protein kinase 1 (PDK1) , P R K 2 , and integrin-linked kinase 22 ( ILK) (53). Activation of A K T is generally accomplished via activation of P I3K as just described. However, A K T can also be activated by non-PI3K dependent means such as agonists of the protein kinase A pathway and the calcium/calmodulin dependent kinase kinase ( C a M K K ) , which lead to phosphorylation of A K T (55, 56). O f particular relevance to pediatric solid tumours in general and E T specifically, the P I 3 K \u00E2\u0080\u0094 A K T survival pathway is initiated upon ligand binding of the IGFl-receptor (see Figure 4). Various in vitro studies using dominant negative and constitutively active A K T alleles have demonstrated that growth factor-mediated survival is dependent on A K T activation and that A K T activation is sufficient for survival even i f upstream kinases such as P I 3 K are blocked (reviewed in (53)). This critical role for A K T in cell survival has now been explained by the observation that A K T effectors include molecules which are key to the regulation of programmed cell death or apoptosis. Among the reported direct targets of A K T are the cell death mediators B A D , caspase 9, the Forkhead family of transcription factors, and the N F - K B regulator I K K (53). Whether or not all of these are true A K T targets in human cells is a subject of ongoing debate; in particular, the phosphorylation of B A D by A K T has been disputed (57). Identification of definitive A K T effectors, therefore, remains an area of intense study. 1.3.1.4 Cross-talk between Growth Factor-Mediated Signaling Pathways It is becoming increasingly apparent that the R A S \u00E2\u0080\u0094 R A F 1 \u00E2\u0080\u0094 M E K \u00E2\u0080\u0094 E R K and the P I 3 K \u00E2\u0080\u0094 A K T pathways are integrally linked (reviewed in (58)). Several recent studies have demonstrated that A K T phosphorylates and negatively regulates R A F 1 (59, 60) and, as discussed above, PI3K has been shown to be an effector of R A S (54). Also , Treinies et al. 23 F I G U R E 4. P I 3 K \u00E2\u0080\u0094 A K T su rv iva l s ignal ing . In this figure, activation of the P I 3 K \u00E2\u0080\u0094 A K T survival pathway is shown as occurs upon activation of the insulin-like growth factor I receptor (IGFI-R). Activation results in tyrosine phosphorylation of key cytoplasmic domains, which then activate P I 3 K (see text). Through the generation of intermediary 3'-phosphoinositides, P I 3 K activates A K T / P K B . A K T effectors are numerous and regulate apoptosis. Thus, the net effect of A K T activation is inhibition of apoptosis. A K T is also a negative regulator of R A F and therefore links the P I 3 K \u00E2\u0080\u0094 A K T pathway to the R A S proliferative pathway. A s shown in the left side of the diagram, IGFI -R is a PTK-receptor that also activates the R A S \u00E2\u0080\u0094 R A F \u00E2\u0080\u0094 M E K \u00E2\u0080\u0094 E R K M A P K pathway in a manner similar to the E G F - R shown in Figure 1. 25 demonstrated that while an activated form of M E K can induce cell division in quiescent fibroblasts, P I 3 K signals are required for this effect (61). Thus, two seemingly parallel signaling pathways have cross-talk capability by which they communicate with and impact on one another. Such cross-talk ensures a balance between cell proliferation and cell death, thus maintaining cellular homeostasis. For example, it is well established that constitutive expression of R A S can induce apoptosis or cell cycle arrest through mechanisms involving p l 9 A R F , p21 C I P 1 , and p53 (reviewed in (62)). Parallel activation of the P I 3 K - A K T pathway wi l l prevent R A S induced cell cycle arrest or apoptosis and allow proliferation to occur (58, 61, 63). Moreover, Reusch et al. have recently demonstrated that the effect of cross-talk between A K T and R A F is dependent on the origins of the initiating signal at the cell membrane (64). They observed that while thrombin and P D G F activated both the R A S \u00E2\u0080\u0094 R A F \u00E2\u0080\u0094 M E K \u00E2\u0080\u0094 E R K and the P I 3 K \u00E2\u0080\u0094 A K T pathways in vascular smooth muscle cells, thrombin stimulation led to cellular differentiation rather than proliferation while P D G F acted as a potent mitogen, having no effect of the cell phenotype. These divergent results were shown to be a consequence of differential phosphorylation of R A F by A K T in response to the two different growth factors. Thus, cross-talk between the R A S \u00E2\u0080\u0094 R A F \u00E2\u0080\u0094 M E K \u00E2\u0080\u0094 E R K and P I 3 K \u00E2\u0080\u0094 A K T pathways is important for the regulation of cell proliferation, death and differentiation and is mediated in large part by growth factor-induced signaling cascades. Disruption of these cascades and their mechanisms of cross-talk leads to dysregulated cell growth and is a common feature of many malignancies. Their respective importance in the regulation of E T cell growth was studied for this thesis and wi l l be discussed in Chapters V and V I . 26 1.3.2 T H E C E L L C Y C L E Homeostasis of normal tissues is maintained by an intricately regulated balance between cell proliferation, growth arrest and differentiation, and cell death. In cancer this balance is lost and cellular proliferation continues unchecked. In the previous sections, the major pathways governing proliferation and survival of cells were discussed. These pathways ultimately converge upon the cell cycle, which controls D N A synthesis and division of a cell . Understanding the cell cycle and its regulation is critical to understanding key elements of oncogenesis. In 1951, Howard and Pelc first described that D N A synthesis in a dividing cell occurs during a discrete temporal phase, rather than continuously as had been previously held (65). Subsequently, further experiments revealed that a cell divides into two daughter cells by passing through 4 distinct phases. Among these phases are S phase, wherein D N A synthesis occurs and the parental cell chromosomal complement is duplicated, and M phase, for mitosis, where replicated D N A is divided and two genetically identical daughter cells are formed. The other 2 phases of the cell cycle, G l and G2 , are gaps between mitosis and S phase and S phase and mitosis, respectively. In G l , the cell responds to mitogenic signaling and prepares itself for the initiation of D N A synthesis. In G2 , D N A synthesis is terminated and cell growth continues with accumulation of proteins and organelles to be divided between the two daughter cells during mitosis. The duration of each of these phases may vary among cells of a population (49). Non-proliferating cells do not perpetually move through successive cell cycles. Rather, these cells exist in a quiescent state between M and S phases in a phase termed GO. Most cells in normal adult tissues exist in GO. Cells in GO and G l are receptive to proliferative signals and, as such, quiescent cells that have maintained the 27 ability to proliferate wi l l leave GO and enter G l in response to an appropriate signal (see (49) for review). A schematic diagram of the cell cycle is shown in Figure 5. Within the cell cycle, passage from one phase to another is strictly regulated by a complex interplay between enablers and inhibitors. The most well-studied checkpoint is that regulating passage between G l and S phases, termed the restriction point or \" R point\". In cultured cells, once a cell passes the R point, it is committed to enter S phase. In vivo, however, cells may arrest at different points within G l in response to different inhibitory signals; thus, in reality, there may be several R points in different cell types that restrict cell cycle progression (49). When cells are stimulated by growth factors to enter G l from GO, they generally require continuous mitogenic stimulation to reach the R point, after which mitogens can be withdrawn and cells wi l l continue through the cell cycle in their absence (66). In the following sections, the major regulators of cel l cycle progression wi l l be discussed. 1.3.2.1 Cyclins and Cyclin-Dependent Kinases Passage of a cell from one phase to the next within the cell cycle is largely regulated by the interactions of cyclins and cyclin-dependent kinases (CDKs) . C D K s are inactive until they bind with their respective cyclin to form a holoenzyme complex that is catalytically active (66). C y c l i n levels oscillate during the cel l cycle due to variations in gene transcription, protein translation and protein degradation, ensuring that levels peak at the time of maximal kinase activation (49). Mammalian cyclin family members include cyclins A to H , which all share a conserved sequence of about 100 amino acids. In mammalian cells, the C D K family includes 7 members that are conserved in size between 32-40 kDa, and share F I G U R E 5. The Cell Cycle. Most quiescent adult cells exist in GO. Mitogenic stimulation can cause them to enter the cell cycle where they passage through an intricately regulated sequence of events culminating in mitosis or cell division. Once the R-(restriction) point has been passed, the cell is committed to enter S-phase and cell cycle transition ceases to be mitogen-dependent. 29 approximately 40% sequence homology (49). C D K s are expressed at constant levels throughout the cell cycle and, once bound to cyclins, are active serine/threonine kinases. Ful l activation of the c y c l i n - C D K complex depends both on phosphorylation of a conserved threonine in the catalytic cleft by CDK-activating kinase, or C A K , and on dephosphorylation of inhibitory threonine sites by phosphatases of the cdc25 family (49). Once fully activated, the c y c l i n - C D K holoenzyme complexes phosphorylate their effectors, facilitating cell cycle progression. Passage through G l into S phase through the R point is mediated by cyclin D - and cyclin E-dependent kinases (66). Cycl in D family members (DI , D 2 and D3) bind to C D K 4 and C D K 6 to yield at least 6 possible holoenyzmes that are expressed in tissue specific patterns (66). Activated cyclin D - C D K 4 / 6 complexes phosphorylate, and thus inactivate, the retinoblastoma protein, Rb. Ffypophosphorylated Rb represses the transcription of genes whose products are required for D N A synthesis, largely by binding transcription factors such as the E2Fs (67). C y c l i n D - C D K 4 / 6 mediated hyperphosphorylation of Rb disrupts the interaction between E2Fs and Rb, E2Fs are released and transcriptional activation ensues (66). Completion of Rb phosphorylation is accomplished by the cyclin E - C D K 2 complex which is activated in response to E 2 F mediated induction of the cycl in E gene (66). Thus, phosphorylation of Rb by cyclin D - C D K 4 / 6 is a pivotal event in R checkpoint passage. Once a cell has passed through the R point, cyclin A - C D K 2 activation occurs and is essential for the initiation of and progression through S phase (49). During S phase, levels of the B-cycl ins rise and entry into and out of mitosis is controlled by cycl in B - C D K 1 complexes ( C D K 1 is also known as cdc2) (49). A t the completion of mitosis, Rb is dephosphorylated and cyclin B is degraded. Degradation of the cyclin proteins, especially 30 cyclin D , is essential for controlled cell cycling and wil l be discussed in greater detail in a subsequent section. A summary of the c y c l i n - C D K complexes and their respective inhibitors (see below) is shown on a diagram of the cell cycle in Figure 6. 1.3.2.2 Inhibitors of Cell Cycle Progression The cell cycle is tightly regulated by effectors, such as the cyclins and C D K s , and by inhibitors. C e l l cycle inhibitors largely inhibit the activity of the C D K s and can be segregated into two categories: the CIP /KIP family of inhibitors, which interact with cyclin-C D K complexes in all phases of the cell cycle, and the more specific I N K 4 family that specifically inhibits cyclin D-dependent kinases (see Figure 6) (66). The C I P / K I P family of polypeptide inhibitors includes p 2 1 C I P 1 , p27 K I P 1 and p57 K 1 P 2 (66). A l l members of this family bind efficiently to c y c l i n - C D K holoenzyme complexes containing any of the C D K s from 1-6, and inhibition is accomplished by induction of conformational changes within the C D K molecules themselves (49). Addit ional ly , all C I P / K I P molecules have been found to directly inhibit C D K 2 in a dose dependent fashion such that high inhibitor levels are required to effect growth suppression (49, 68). Quiescent cells generally express high levels of p27 K I P 1 that diminish as the cell enters cycle and accumulates cyclin D - C D K 4 / 6 (66). Rather than exerting an inhibitory effect similar to that on C D K 2 , K I P proteins bound to C D K 4 / 6 complexes are merely sequestered and the kinase remains catalytically active (66). In cycling cells, therefore, cyclin D - C D K 4 / 6 complexes act as effectors of cell cycle progression in a second manner: they sequester virtually all of the p27 K I P I molecules thereby diminishing their availability to negatively regulate cyclin E -C D K 2 (reviewed in (69)). Once cyclin E - C D K 2 is activated, it phosphorylates p27 K I P 1 , 31 FIGURE 6. Regulation of cell cycle progression. Progression of a cell through the cell cycle is dependent on regulated expression and activation of cyclin-cyclin dependent kinase ( C D K ) complexes and their inhibitors. Two classes of C D K inhibitors exist. The I N K 4 family specifically inhibits Gl-phase cycl in D - C D K 4 / 6 complexes while the C I P / K I P family act throughout the cell cycle. Successful transition from one phase to the next requires appropriately timed degradation of cyclins that were active in the previous phase, (adapted from Slingerland, 1998 (49)). 33 targeting it for ubiquitin-mediated proteosomal degradation; thus, cyclin E - C D K 2 targets its own inhibitor for degradation ensuring completion of Rb phosphorylation and passage beyond the R point (70). The I N K 4 proteins specifically inhibit the cyclin D-dependent kinases, C D K 4 and C D K 6 . There are four known members of the family to date: p l 6 I N K 4 a , p l 5 I N K 4 b , p l 8 I N K 4 c , and p l 9 1 N K 4 d . I N K 4 proteins inhibit the cell cycle by sequestering C D K 4 and C D K 6 into binary C D K - I N K 4 complexes (66). Sequestration of C D K 4 and C D K 6 has two inhibitory effects. First, it prevents interaction between C D K s and cyclin D , thus preventing kinase activation. Second, disruption of cyclin D - C D K complexes effectively releases bound CIP /KIP proteins, making them available to inhibit cyclin E - C D K 2 complexes as discussed above. Inhibition of cell cycle progression is also accomplished by growth inhibitory signaling. Growth-inhibitory cytokines, such as the interferons and TGF[3, mediate G l arrest by diverse mechanisms including induction of C D K inhibitor m R N A and protein, suppression of Rb phosphorylation, and downregulation of cycl in D I and c -Myc protein levels (49, 71). 1.3.2.3 Regulation of Cyclin D A s discussed above, phosphorylation of the Rb protein by cycl in D - C D K 4 / 6 is a pivotal event in R checkpoint passage. Therefore, it is not surprising that cyclin D levels are tightly regulated by both transcriptional and post-transcriptional mechanisms in normal, proliferating cells. Induction of cycl in D I m R N A in early G l requires both mitogen-induced, sustained activation of the E R K s and co-stimulation of integrins via attachment to the extracellular matrix (reviewed in (62)). Either loss of adhesion or loss of mitogenic 34 stimulation leads to abrogation of E R K activation and diminished cycl in D I m R N A transcription. Recent evidence indicates that R A S \u00E2\u0080\u0094 R A F \u00E2\u0080\u0094 M E K \u00E2\u0080\u0094 E R K activation must be accompanied by an intact P I3K signal for induction of cyclin D I m R N A synthesis (61, 63). In addition to its role in the regulation of cyclin D gene transcription, the P I 3 K \u00E2\u0080\u0094 A K T pathway is also integrally l inked to the post-transcriptional regulation of cycl in D I expression (72, 73). Muise-Ffelmericks et al. reported that serum induction of cyclin D I protein expression results from enhanced translation of its m R N A via a P I 3 K \u00E2\u0080\u0094 A K T -dependent pathway (72) and Diehl et al. first l inked the AKT-effector GSK3-(3 to post-translational cycl in D regulation (73). Thus, both the R A S \u00E2\u0080\u0094 R A F \u00E2\u0080\u0094 M E K \u00E2\u0080\u0094 E R K and P I 3 K \u00E2\u0080\u0094 A K T pathways are key players in cyclin D regulation. Ubiquitin-mediated proteosomal degradation is another means by which cyclin D protein levels within a cell are controlled. Proteosomes are cytoplasmic protease complexes that rapidly degrade ubiquitinated-proteins into short peptides (74). Brief ly, a series of enzymatic reactions involving ubiquitin-associated enzymes ligate ubiquitin molecules to targeted proteins in multimeric chains. The 26S proteosome then recognizes and degrades these poly-ubiquitinated substrates. Cyc l in D is targeted for ubiquitination, and therefore degradation, when it has been phosphorylated at threonine residue 286 (Thr-286) located near the carboxyl terminus (75). Phosphorylation of Thr-286 is catalyzed by GSK3(3 (73). Moreover, GSK3(3 -mediated phosphorylation of cycl in D I redirects the protein from the nucleus to the cytoplasm where proteosomal degradation can occur (73). This export of cyclin D I out of the nucleus is actively mediated by the nuclear exportin C R M 1 and appears to be essential for regulated cell division (76). 35 1.3.3 D I F F E R E N T I A T I O N In its most basic form, the process of cellular differentiation is essential for normal embyrogenesis and tissue development. It is the ability of pluripotent stem cells to give rise to differentiated progeny that enables the development of multi-cellular organisms comprised of cells and organs with different phenotypes and functions. A s discussed above, cellular differentiation pathways are often coupled to proliferative pathways and it is the balance between these that allows for normal growth. In the past ten years, the genetics of normal development and cancer have converged as aberrant regulation of normal embryonic developmental pathways have been found in many cancer cells (77). In general, the proliferative potential of a cell is inversely correlated to its degree of differentiation and, therefore, most cancerous cells exist in undifferentiated forms with variable degrees of differentiation potential (78). While a detailed discussion of normal and aberrant cellular differentiation is outside the scope of this thesis, it is worth noting that many pediatric solid tumours are embryonal and, therefore, altered differentiation pathways are likely involved in oncogenesis. The most compelling evidence for this comes from studies of Wilms tumour and neuroblastoma which demonstrate distinct biologic similarities between normal embryonic cells and the corresponding tumour cells (79, 80). The potential role of autocrine G R P production in altering cellular differentiation pathways wi l l be discussed in Chapter I V in relation to the Ewing family of tumours. 1.3.4 A P O P T O S I S Apoptosis is the active mechanism of programmed cell death (reviewed in (49)). Unl ike cell necrosis, which is a passive response to injury in which cells swell and lyse, 36 apoptosis is the activation of a genetic program in which cells lose their viability, fragment and are ingested before losing membrane integrity. Apoptosis can be initiatied by intrinsic signals in response to injury or stress, such as occurs upon withdrawal of growth factors or nutrient supply and following exposures to ultra-violet radiation, ionizing radiation or toxins (49). Extrinsic signals can also initiate apoptotic pathways through ligation of trans-membrane death receptors such as FAS (CD95), tumour-necrosis factor (TNF) and TRAIL (71). Ultimately, intrinsic and extrinsic pathways converge upon the common final degradation phase in which the cell dies and is endocytosed. An extensive discussion of apoptosis is not relevant to this thesis; however, the key molecules and events involved in intrinsic death signaling will be briefly reviewed. Initiation of apoptosis in response to injury or stress is dependent on a functional p53 gene. p53 functions as a tumour suppressor causing either G l - S phase arrest or induction of apoptosis (81). Once initiated, apoptosis is controlled by the molecular regulatory families of caspases and Bcl-2 which are directly and indirectly controlled by p53 and other triggers of apoptosis (49). Caspases are cysteine proteases which cleave their target molecules at aspartic acid residues (81). More than ten different caspases have been identified in humans and many are active in apoptosis. Although ubiquitously expressed, the caspases required for apoptosis vary depending on cell type. They become activated upon cleavage from precursor molecules and substrates include other caspases, D N A polymerases, cytoskeletal proteins and other proteins critical for cell survival. Activation of caspases therefore proceeds through an amplifying proteolytic cascade ultimately resulting in cell death. The Bcl-2 family includes the pro-apoptotic proteins B A X , Bcl-Xs and B A D and the inhibitory proteins Bcl-2 and Bc l -X L . Interaction and binding between different members of the Bcl-2 37 family is a critical mechanism by which the ratio between effectors and inhibitors is determined. At a molecular level, members of the Bc l -2 family have a hydrophobic tail that allows them to bind the outside surface of their target organelles: the mitochondria, endoplasmic reticulum and the nucleus. For example, B A X enacts its pro-apoptotic function by binding to the mitochondria, interfering with its function and causing release of cytochrome c (81). Once cytochrome c has been released from the mitochondria, it orchestrates assembly of the apoptosome complex containing A P A F 1 and caspase 9 which then culminates in activation of caspase 3 and cell death (71). Thus, intrinsic cell death pathways are critically linked to functional Bcl -2 proteins, caspases and mitochondria. 1.3.5 ANCHORAGE DEPENDENT GROWTH Thus far, the key mechanisms involved in cell signaling and cell division have been discussed with little reference to the role of the extra-cellular environment in the regulation of these processes. In truth however, cellular proliferation is critically dependent on cellular adhesion to an extra-cellular matrix ( E C M ) . This reliance on adhesion is termed anchorage-dependence and it is a key feature that differentiates normal cells from transformed or cancerous cells, which demonstrate anchorage-independent growth (82). The E C M of mammalian cells varies in structure and composition between cell type, stage of development and location (83). The E C M of epithelial cells is highly structured in the form of a basement membrane that attaches cells to each other and to the membrane. On the other hand, mesenchymal cells do not attach to each other and the E C M surrounds the cells in a less structured manner. Components of the E C M of mesenchymal cells include the interstitial collagens, type I to III, elastin, fibronectin, proteoglycans and vitronectin (83). Interaction 38 between cells and the E C M is essential for survival and proliferation, can play a role in cellular differentiation, and is mediated by structural and function domains within E C M proteins. These domains include the arginine-glycine-aspartic acid (RGD) domains, which interact with and attach to cells via integrins, and non-RGD domains that recognize other components within the E C M itself. These interactions result in the formation of a highly organized three-dimensional matrix in which E C M proteins associate with each other and with cells (83). Integrins are cell adhesion molecules that function as trans-membrane receptors, linking R G D proteins of the E C M with the initiation of signal transduction cascades (83). Disruption of R G D protein-integrin interactions leads to the induction of apoptosis in epithelial and endothelial cells (84) and cell cycle arrest in fibroblasts (85). Integrins are obligate heterodimers comprised of trans-membranous a and 6 subunits that interact with the E C M , and short cytoplasmic domains that interact directly with cytoskeletal proteins and protein tyrosine kinases (83). Once activated, integrins can activate the proliferative R A S \u00E2\u0080\u0094 R A F \u00E2\u0080\u0094 M E K \u00E2\u0080\u0094 E R K pathway via interaction with the protein tyrosine kinase She (86) and the P I 3 K \u00E2\u0080\u0094 A K T via the serine/threonine kinase I L K (integrin-linked kinase) (87). Addit ional ly , a key function of integrin-mediated signaling is to regulate cytoskeletal reorganization and cell shape via activation of focal adhesion kinase ( F A K ) and other protein kinases that are components of focal adhesion plaques, which act as the cytoplasmic points of contact between integrins and the E C M (83). Thus, interactions between cells and E C M proteins regulate growth, survival, differentiation, cell shape and motility. Since these regulatory pathways are often disrupted in cancer and transformed cells are able to circumvent anchorage-dependence, it follows that 39 growth signaling must be different in anchorage-independent conditions. In this thesis, we test this hypothesis with respect to E T cell growth and present our results in Chapters V and V I . 1.4 MECHANISMS OF ONCOGENESIS 1.4.1 Oncogenes Oncogenes, first identified in cancer-causing viruses, are now well established as major contributors to the development of cancer in humans. Oncogenes may be viral in origin or may be derived from normal cellular genes referred to as proto-oncogenes. To date, about 100 potential oncogenes of cellular or viral origin have been recognized (88). Proto-oncogenes are highly conserved in evolution and their products are important in the regulation of cell growth and differentiation in organisms ranging from primitive eukaryotes to humans (89). The expression of cellular proto-oncogenes is tightly regulated in normal cells, but i f converted to oncogenes, they can induce tumour formation. Examples of cellular proto-oncogenes include many growth factors, growth factor receptors, cytoplasmic protein kinases, G-proteins, cyclins and transcription factors (see Table 1). Conversion of proto-oncogenes into oncogenes can occur by several mechanisms including proviral insertion, gene amplification, point mutation, and chromosomal rearrangement (90). Chromosomal rearrangement wi l l be discussed separately in a subsequent section. Activation of oncogenes by proviral insertion is complex and involves recombination between viral and cellular genomes following infection and integration of the virus into the cell . In this manner, viral sequence is integrated adjacent to the cellular proto-oncogene resulting in alterations that convert the normal gene to its oncogenic counterpart (90). The 40 TABLE 1. Selected Oncogenes in Human Cancer (adapted from Park, 1998 and Kopnin, 2000 (88, 89)) Oncogene Function of protein Lesion Tumours KS3 F G F family member D N A transfection Kaposi 's sarcoma HST F G F family member D N A transfection Stomach carcinoma E G F R Receptor tyrosine kinase Gene amplification Squamous cell carcinoma PDGFR Receptor tyrosine kinase Gene rearrangement Leukemia ( C M L / A M L ) TRK Receptor tyrosine kinase Gene rearrangement Colon cancer NEU Receptor tyrosine kinase Gene amplification Breast cancer Point mutation Neuroblastoma RET Receptor tyrosine kinase Point mutation Thyroid cancer S R C Tyrosine kinase Point mutation Colon carcinoma BCR-ABL Tyrosine kinase Gene rearrangement Leukemia ( C M L / A L L ) c-mos Serine/threonine Sarcoma kinase c-raf Serine/threonine Sarcoma kinase Pim-1 Serine/threonine Proviral insertion T-cell lymphoma kinase ETV6-NTRK3* Tyrosine kinase Gene rearrangement Congenital fibrosarcoma H - R A S G Protein Point mutation Colon, lung carcinoma K-RAS G Protein Point mutation A M L , thyroid cancer N - R A S G Protein Point mutation Melanoma D b l G E F Gene rearrangement Lymphoma Ost G E F osteosarcoma crk SH2/SH3 adaptor N-myc Transcription factor Gene amplification Neuroblastoma c-myc Transcription factor Gene amplification Many neoplasms Gene rearrangement Bukitt 's lymphoma c-fos Transcription factor Osteosarcoma c-jun Transcription factor Sarcoma Cyclin Dl/pradl Cell cycle regulator Gene amplification Breast cancer *see Knezevich, et al. 1998 (91) 41 first described example of proviral insertion leading to oncogene activation was the 50- to 100- fold elevation of c-myc transcription observed in bursal lymphomas induced by avian leukosis virus (92). In this situation, viral sequence insertion leads to enhanced and unregulated expression of the c-myc proto-oncogene, thus converting it to an oncogene. A second mechanism of oncogene activation observed both in transformed cells in vitro and in tumours in vivo is gene amplification. Amplified copies of proto-oncogenes can be seen microscopically as homogeneously staining chromosomal regions and double-minute chromosomes (89). The proto-oncogene may be amplified at its native chromosomal location or may be found at another locus as a result of chromosomal rearrangement (see below). While the precise mechanisms of gene amplification are not yet completely understood, it appears to result from several rounds of unscheduled D N A synthesis during a single cell cycle and amplicons usually include several genes (90). Amplified proto-oncogenes are found in human tumours, often in tumour-specific patterns, and the presence of multiple copies of proto-oncogenes in tumour cells is associated with a poor prognosis (89). Examples include amplification of n-myc in neuroblastoma and HER2/neu (c-erbB2) in breast cancer. Point mutations in proto-oncogenes can also result in oncogene activation, such as occurs with the ras mutations that are characteristic of epithelial malignancies. Such single-base mutations alter the amino acid sequences of the RAS proteins, causing diminished intrinsic GTP-ase activity and constitutive activation of RAS and its downstream proliferative pathways (89). 42 1.4.2 Chromosomal Translocations and Gene Rearrangements Two patterns of chromosome translocation have been observed in human cancers. The first pattern is observed in many solid tumours and can result in gains and losses of large portions of chromosomal material. These complex translocations appear to be random and are not tumour-specific (93). The second pattern of chromosomal translocation (referred to as the simple type) is characterized by distinctive rearrangements of chromosomal segments in disease-specific manners. These tumour specific translocations are often felt to be primary oncogenic lesions and they are particularly common in sarcomas and some leukemias (see Table 2). Common sites of chromosome translocations in malignant cells have breakpoints that are frequently near proto-oncogenes, suggesting that chromosomal breaks may lead to activation of oncogenes (94). Alternatively, chromosomal translocation may result in deletion of a tumour suppressor gene or fusion of two unrelated genes in frame creating a novel fusion gene with oncogenic potential (90). This latter phenomenon was first described with the cloning of the bcr-abl fusion oncogene from the Philadelphia chromosome, t(9;22) translocation, in chronic myelogenous leukemia (95). In this situation, fusion of the bcr gene from chromosome 22 with the abl gene from chromosome 9 leads to translation of a novel fusion protein with constitutive tyrosine kinase activity that is oncogenic. As discussed in detail earlier, the oncogenic EWS-ETS gene fusions of E T are created as a result of such tumour specific chromosomal translocations. The potential effects of chromosomal translocation are shown schematically in Figure 7. 43 TABLE 2 . Summary of recurrent chromosomal translocations found in pediatric soft tissue tumours, (adapted from de Alava, 1998)(3) Tumour type Translocation Gene Fusion Incidence (%) E T t(ll ;22)(q24;ql2) EWS-FLI1 85 E T t(21;22)(q22;ql2) EWS-ERG 10 E T t(7;22)(q22;ql2) EWS-ETV1 Rare E T t(17;22)(ql2;ql2) EWS-E1AF Rare E T t(2;22)(q33;ql2) EWS-FEV Rare D S R C T t ( l l ;22)(q l3 ;q l2) EWS-WT1 95 M y x o i d liposarcoma t(12;16)(ql3;pll) TLS-CHOP 95 M y x o i d liposarcoma t(12;22)(ql3;ql2) EWS-CHOP 5 Extraskeletal myxoid t(9;22)(q22;ql2) EWS-CHN 75 chondrosarcoma Malignant melanoma of soft t(12;22)(ql3;ql2) EWS-ATF1 N K parts Synovial sarcoma t (X;18) (p l l .23 ;q l l ) SYT-SSX1 65 Synovial sarcoma t (X;18) (p l l .21 ;q l l ) SYT-SSX2 35 Alveolar rhabdomyosarcoma t(2;13)(q35;ql4) PAX3-FKHR 75 Alveolar rhabdomyosarcoma t(l;13)(p36;ql4) PAX7-FKHR 10 D F S P t(17;22)(q22;ql3) COL1A1-PDGFB N K Congenital fibrosarcoma & t(12;15)(pl3;q25) ETV6-NTRK3 N K mesoblastic nephroma Abbreviations: D F S P , dermatofibrosarcoma protuberans; D S R C T , desmoplastic small round-cell tumour; N K , not known 44 5' genel 3' gene2 *'5'gene2 x 3 ' g e n e l Possible results of chromosomal translocation: Breaks in heterochromatin Deletion of gene 1 or gene 2 No observed effect Potential deletion of tumour suppressor gene Stop codon created in gene 1 or gene 2 Truncated protein Non-in frame fusion between genes 1&2 In-frame fusion between genes 1&2 Non-functional protein Novel , functional fusion protein (e.g. E W S - F L I 1 ) F I G U R E 7 . Chromosomal translocation can result in a potentially oncogenic gene rearrangement. 45 1.4.3 Tumour Suppressor Genes Tumour suppressor genes (TSGs) are cellular genes whose inactivation increases the probability of tumour formation. Moreover, reinstitution of their function may suppress the growth of already established tumour cells (88). Vira l oncogenes and genetic alterations can, therefore, effect tumour formation by either activating cellular proto-oncogenes as described above, or by inactivating TSGs . Currently, twenty tumour suppressors have been recognized and several are listed in Table 3. Three key TSGs involved in the evolution of most human tumours are p53, Rb, and ARF. Unl ike the dominant oncogenes, T S G s are recessive genes and, as such, inheritance of an abnormal or deleted T S G locus predisposes an individual to the development of tumours. This phenomenon was first described by Knudson in relation to the Rb gene as the \"two-hit\" hypothesis (96). As with oncogenes, it is now apparent that the overwhelming majority of T S G s are components of the pathways that regulate cellular proliferation, differentiation, apoptosis, cell cycle, and genomic integrity (88). 1.4.3.1 p53 p53 is a frequent target of mutations in many human and rodent malignancies such that loss of p53 protein function is observed in over half of all human cancers. p53 encodes a nuclear D N A - b i n d i n g phosphoprotein that normally exists as a tetramer, which binds to specific D N A sequences (97). p53 can be activated by a variety of genotoxic stimuli that cause increased transcription, increased protein half-life, and/or increased translational initiation of its m R N A . Through a multitude of biologic pathways, activated p53 then exerts it net effects of cell cycle arrest, apoptosis, maintenance of genomic integrity, and D N A 46 T A B L E 3. Selected Human Tumour Suppressor Genes (adapted from Kopnin, 2000) (88) Gene Funct ion of Protein Tumours p53 Transcription factor Cel l cycle regulation Regulation of apoptosis Control of genomic integrity Li-Fraumeni syndrome Most forms of sporadic malignancy in adults Rb Cel l cycle regulation ( G l - S phase transition) Hereditary retinoblasoma Many sporadic tumours INK4a/ARF Cell cycle regulation (CDK4/6 inhibitor)/p53 activation Hereditary melanomas Many sporadic tumours NF1 ( neurofibromin) N F 2 (merlin) G A P protein (RAS inactivation) Cytoskeleton-membrane link Neurofibromas Schwannomas Meningiomas BRCA1 BRCA2 D N A repair p53 activation D N A repair Transcription factor Hereditary breast and ovarian cancer Various forms of sporadic tumours Hereditary breast and ovarian cancer Various forms of sporadic tumours WT1 Transcription factor p53 regulation Hereditary Wi lms ' tumour APC fi-catenin regulation Hereditary adenomatosis polyposis coli Sporadic colon cancer VHL Suppresses expression of VEGF(angiogenic factor) Von Hippel-Lindau syndrome Clear-cell carcinoma 47 repair (see (97) and (98) for reviews). As shown in Figure 8, the pathways regulated by p53 are numerous and complex and a detailed review is beyond the scope of this thesis. In the following paragraphs, the role of p53 in the induction of cell cycle arrest and apoptosis wi l l be briefly discussed. The effects of p53 on proliferation are mainly effected through actions on the G l phase of cell cycle progression, although it is now apparent that p53 also functions at the G2-M checkpoint (97). In response to oncogenic or hyperproliferative stimuli (e.g. M y c , Ras, E l A ) , D N A damage, toxin exposure, or withdrawal of growth factors, p53 is activated. Activated p53 causes G l arrest by inducing expression of the cell cycle inhibitor p21 C I P 1 . The mechanism behind G 2 - M growth arrest is unknown, but it is thought that p53 may directly decrease cyclin B l levels and indirectly inactivate C D K 1 (cdc2) (97). Induction of apoptosis in response to p53 can also occur following exposure to the aforementioned s t imuli . The mechanisms of p53-induced apoptosis include both transcriptional and non-transcriptional means. The pro-apoptotic proteins B A X and IGF-binding protein 3 are transcriptional targets of p53, as is the death-receptor ligand F A S (97). Addit ionally, p53 is a transcriptional repressor of the anti-apoptotic Bcl-2 gene. Non-transcriptional effects of p53 largely involve protein-protein interactions between p53 and factors involved in D N A repair (97). Whether a cell undergoes p53 induced cell cycle arrest or apoptosis is cell type and situation dependent. In addition, the presence of functional p21 C I P 1 and cross-talk with Rb are critical determinants in p53's role (97). From a clinical perspective, germ-line mutations of p53 exist among patients with the Li-Fraumeni syndrome (99). These patients have inherited only one functional copy of p53 and are therefore predisposed to the development of malignancies. 49 1.4.3.2 R b Rb is a nuclear protein with a molecular weight of lOOkDa that is expressed by all cells (99). The importance of Rb as a tumour suppressor was first noted in hereditary retinoblastoma where germ-line deletions of chromosome 13ql4 containing the Rb locus were first described (100). The function of the retinoblastoma protein (Rb) in cell cycle regulation has been previously discussed and its role as a suppressor of proliferation is well characterized. In addition to its role in G l - S phase transition, Rb also acts as an anti-apoptotic factor (97). As such, Rb interacts with p53 pathways in regulating cell cycle arrest and apoptosis and evidence suggests that malignant transformation of a normal mammalian cell requires inactivation of both Rb and p53 function (99). 1.4.3.3 A R F In 1995, the tumour suppressor gene pl4ARt was first described (101). Interestingly, this gene is encoded by the same locus as the INK4a locus but ARF is inititiated from a different first exon and is transcribed in an alternate reading frame. Thus, the two proteins are distinctly different from one another even though they are encoded by the same genetic locus. L ike p l 6 I N K 4 a , A R F is capable of inducing cell cycle arrest and more recent studies have identified it to be a tumour suppressor with the critical role of l inking the p53 and Rb pathways (66). A R F acts upstream of p53 in its induction of cell cycle arrest and also acts to stabilize p53 by antagonizing the p53-negative regulator M D M 2 (66). A R F expression is activated by abnormal mitogenic signals that overstimulate the cyclin D - C D K \u00E2\u0080\u0094 R b \u00E2\u0080\u0094 E 2 F circuit, thus leading to activation of p53 and cell cycle arrest or apoptosis. In this manner, A R F is responsible for tumour surveillance and functions as a tumour suppressor. The 50 accumulated data suggest that disruption of A R F , M D M 2 , and p53, like mutations in the p l 6 I N K 4 c t - c y c l i n D - C D K \u00E2\u0080\u0094 R b pathway, seem to be part of the evolution of all cancer cells, irrespective of patient age or tumour type (66). 1.4.4 Autocrine Growth Factors Tumour cells are dependent on extra-cellular growth factors for proliferation and survival. In a well-vascularized tumour critical nutrients and growth factors are readily available to individual cells from endocrine sources. However, even in the absence of external growth factor supply tumour cells can continue to proliferate and survive. The mechanisms that allow such growth include those mentioned above: e.g. activation of oncogenes or inactivation of tumour suppressor genes leading to the dysregulation of normal proliferative, survival and apoptotic pathways. Additionally, tumour cells can circumvent the need for an endocrine supply of growth factors by secreting their own autocrine growth factors. The genes that encode these autocrine peptides can, therefore, be classified as oncogenes and examples of autocrine growth factors in human malignancy include P D G F in gliomas, V E G F and F G F in Kaposi ' s sarcoma, IGFII in rhabdomyosarcoma and IGFI in Ewing tumours (89, 102, 103). Autocrine production of growth factors by tumour cells leads to constitutive activation of growth factor receptors and dysregulation of signal transduction pathways. We have found that G R P acts as an autocrine growth factor in E T and discuss these findings in Chapters III and IV. 51 1.5 AIMS & OBJECTIVES Despite the extensive knowledge that has been gained about the genetics of E T , relatively little is known about the signal transduction pathways that govern its proliferation. Insights into the biologic pathways that mediate the tremendous proliferative and survival potential of E T cells w i l l be important to the development of novel treatment strategies that can target these tumours in vivo. Therefore, the studies presented in this thesis were undertaken with the following aims: 1) To characterize the expression of human gastrin releasing peptide in E T cells in vitro and in vivo and explore its role as a potential diagnostic and/or prognostic variable in E T patients. 2) To determine the role of G R P as an autocrine growth factor in E T and characterize the signaling pathways through which it acts. 3) To study E T cell proliferation and signal transduction pathways in an anchorage independent culture environment in an effort to better represent the in vivo setting. 4) To study the roles of the R A S \u00E2\u0080\u0094 R A F \u00E2\u0080\u0094 M E K \u00E2\u0080\u0094 E R K and P I 3 K \u00E2\u0080\u0094 A K T pathways in the regulation of E T cell growth and proliferation. 52 C H A P T E R II MATERIALS & METHODS 2.1 C E L L C U L T U R E A N D C L I N I C A L S A M P L E S 2.1.1 Cell lines and Tissue Culture A l l Ewing and other small round cell tumour cell lines were originally obtained from Children's and Women's Health Centre of Bri t ish Columbia, from the American Type Culture Collection, or from Dr. T i m Triche at Children's Hospital Los Angeles. Ewing tumour cell lines used in this study are listed in Table 4 along with their respective EWS-ETS gene fusion type. Table 5 lists the non-Ewing tumour cell lines that were used as controls as detailed in later sections. Cells were grown under standard conditions at 37\u00C2\u00B0C in 5% C O , in R P M I medium supplemented with 15% fetal bovine serum, 2 m M glutamine and antibiotic-antimycotic ( G I B C O - B R L , Life Technologies, Burlington). For analysis of gene and protein expression in low-serum and serum-free conditions, media containing 2.5% and 0.25% F B S respectively, was used. Cells were grown as adherent monolayers except where specifically indicated and were collected for analysis by trypsinization (0.25% Trypsin, G I B C O B R L ) following washes with phosphate buffered saline (PBS) while in logarithmic growth. A l l cell lines were tested by R T - P C R (see below) for expression of EWS-ETS fusion genes. 2.1.2 Anchorage-independent Multi-cellular Spheroid Cultures For anchorage independent suspension cultures, ambient environmental conditions and media were identical to adherent monolayer cultures. To establish suspension cultures, TABLE 4 . Ewing Tumour Family Cell Lines Cell line Diagnosis Gene Fusion Reference TC-71 ES EWS-FLI1 (10) TC-32 P N EWS-FLI1 (10) A4573 E S EWS-FLI1 (104) B C - E S 1 A T EWS-FLI1 unpublished results 6647 E S EWS-FLI1 (104) TC-174 M E M EWS-FLI1 (31) TC-253 M E M EWS-FLI1 (31) TC-547 M E M EWS-FLI1 (31) J F E N O N B EWS-FLI1 (30) 633 P N EWS-ERG (14) 466 ES EWS-ERG (14) 5838 E S EWS-ERG (105) T A B L E 5. Non-Ewing T u m o u r C e l l Lines Ce l l line Diagnosis Reference IMR-32 N B (106) San2 N B (106) CT-10 E R M S (107) R D E R M S (108) Birch E R M S (31) R h l 8 A R M S (109) TTC-487 A R M S (31) A204 Sarcoma N O S (108) Jurkat A L L (108) H345 S C L C (108) H E L 92.1.7 erythroleukemia (108) 55 confluent monolayers were trypsinized, resuspended as single cells, and replated on tissue culture dishes that had been coated with 1.4% agar. Suspended cells were collected at intervals up to 24 hours post-suspension for time course studies. After 48 hours in suspension, stable spheroids were evident and were separated from single and dead cells by low speed (500 rpm) centrifugation for 3 minutes and replated in new media on fresh agar-coated plates. Starvation of cells was accomplished by replacing the high-serum (15% FBS) media with serum-free (0.25% F B S ) media for 20-24 hours prior to experimentation. Suspension cells were harvested by low-speed centrifugation, washed in P B S , then subsequently analyzed for protein and R N A expression as discussed in later sections. 2.1.3 P r i m a r y tumour specimens Except where indicated below for the clinical correlates study, primary small round cell tumour samples were all obtained from the tumour bank at Children's & Women's Health Centre of Bri t ish Columbia. A l l samples were obtained prior to therapy and diagnoses were confirmed by standard histopathologic and molecular pathologic techniques. A l l cases of primary Ewing tumours were confirmed to express EWS-FLI1 gene fusions by reverse-transcriptase P C R (see below). Fresh tissue samples flash-frozen in liquid nitrogen and stored at - 7 0 \u00C2\u00B0 C were used for R N A and protein studies. Formalin-fixed paraffin-embedded blocks of tissue were used for immunohistochemical analysis (see below). For gene cloning and sequencing of gastrin releasing peptide (GRP) , frozen fetal lung tissue resected at Children 's & Women's Health Centre of Bri t ish Columbia was used and bronchial carcinoid tumour tissue obtained from St. Paul 's Hospital, Vancouver, British Columbia was used as a positive control for G R P expression studies. 56 2.2 A N A L Y S I S OF G E N E EXPRESSION 2.2.1 Isolation of RNA Total R N A was extracted from cell lines and primary tissues using the acid-guanidinium-phenol/chloroform method of Chomczynski and Sacchi (110). Briefly, cell pellets or approximately ten 5p,m-thick sections of primary tumour were resuspended and homogenized in 1 ml of Trizol (Gibco-BRL, Life Technologies) and R N A then isolated from the aqueous phase. Purity and integrity of R N A was assessed by 1% agarose gel electrophoresis and spectrophotometric analysis of the O D A 2 6 0 / A 2 8 0 ratio. 2.2.2 Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) Five micrograms of total R N A from cell lines and primary tumours was reverse transcribed to complementary-DNA ( c D N A ) using random hexamer nucleotide primers under previously described reaction conditions (111). One microlitre of the generated c D N A was then amplified using the polymerase chain reaction ( P C R ) for analysis of gene expression as published previously (111). To amplify EWS-ETS fusion transcripts, primers capable of amplifying EWS-FLI1 and EWS-ERG fusions were utilized. These primers included the FLU and ERG consensus primer 11.3: 5 ' - A C T C C C C G T T G G T C C C C T C C - 3 ' , and the \u00C2\u00A3WS-specif ic primers 22.3: 5 ' - T C C T A C A G C C A A G C T C C A A G T C - 3 ' (111) and E S B P - 1 : 5' - C G A C T A G T T A T G A T C A G A G C A G T - 3 ' (14). R T - P C R amplification of the human gastrin-releasing peptide ( G R P ) gene and its receptor ( G R P - R ) was also accomplished using previously described conditions (112). GRP primers included GRP-1 ( 5 ' - T G C A A G G A A T T T G C T G G G T C T C - 3 ' ) corresponding to GenBank positions 295-316, and GRP-2 ( 5 ' - T G T G A A T G G T A A C A G C T C G G G G - 3 ' ) corresponding to positions 758-779 57 (112). Bronchial carcinoid tumours and the small cell lung cancer cell line H345 were used as pos i t i ve con t ro l s . P r imers for GRP-R i n c l u d e d G R P - R 1 : 5 ' -C T C C C C G T G A A C G A T G A C T G G - 3 ' corresponding to GenBank position 65-85, and G R P -R2: 5 ' - A T C T T C A T C A G G G C A T G G G A G - 3 ' corresponding to position 468-488 (112). One microlitre of each c D N A product was also amplified with primers for the ubiquitously expressed antigen M I C 2 to ascertain the integrity of the c D N A (113). A l l amplified P C R products were visualized on a 2% agarose gel stained with ethidium bromide. The identity of the amplified fragments was confirmed by size and by blotting the P C R products onto nylon filters and hybridizing them with gene specific probes (see below). 2.2.3 Southern & Nor thern Analysis To confirm the identity of P C R products, amplified c D N A was transferred from 2% agarose gels to Nytran filters (Schleicher and Schwell Inc., N H ) by standard protocols, and the filters were then probed with radioactively labeled probes. For EWS-ETS gene fusions, blots were probed with a [y- 3 2 P]dCTP (Amersham)-end-labeled E W S oligonucleotide probe ( 5 ' - A G C C A A C A G A G C A G C A G C T A C G G G C A G C A G A - 3 ' ) . GRP and GRP-R were identified by probing with [a- 3 2 P]dCTP (Amersham)-labeled c D N A probes (114) (gift of Dr. J. Battey, NTH). Northern blotting was performed using standard methods (115). Briefly, 10 to 25 ug of denatured total R N A (amount consistent within each experiment) was separated by agarose gel electrophoresis, blotted onto nylon membranes, and prepared for hybridization with lOOng of [a - 3 2 P]dCTP (Amersham)-labeled c D N A probe. Probes utilized included the aforementioned G R P and G R P - R probes, c D N A fragments recovered from differential 58 display-PCR gels (see below), a cycl in D I c D N A probe (provided by Dr . T i m Triche, C H L A ) and an E W S c D N A probe (provided by Dr. Chris Denny, U C L A ) . 2.2.4 Differential D i s p l a y - P C R ( D D - P C R ) D D - P C R was performed by M s . Jerian L i m using the RNAimage Kit\u00E2\u0084\u00A2 (GenHunter) following the manufacturer's instructions. Briefly, DNase l treated total R N A was subjected to reverse transcription using 3 separate one-base-anchored oligo-dT primers ( d A - d T n , dC-d T u , or d G - d T u ) , to allow for initial subdivision of the m R N A populations. Complementary D N A was then amplified using the same oligo-dT primer used in first strand synthesis in combination with one of a series of 8 arbitrary 13mer 5' primers as described in the GenHunter K i t using a Perkin-Elmer 9600 thermocycler. Ampl i f i ed products from the different cell lines were run side-by-side on a 6% polyacrylamide sequencing gel (Bio-Rad) and D N A from differentially expressed bands was recovered, reamplified, and gel purified using the Qiex II gel extraction kit (Qiagen). Differentially expressed bands from D D - P C R were cloned and sequenced using the pCRII plasmid vector and T A cloning kits (Invitrogen, U S A ) using standard methods. The same methods were used for cloning and sequencing of bands following R T - P C R of cell lines, primary tumours, bronchial carcinoids, and fetal lung tissue using primers for GRP (see above). A l l sequences were submitted to GenBank for analysis. Confirmation of differential gene expression was accomplished by probing Northern blots with cloned D D - P C R c D N A fragments and with the G R P c D N A probe. 59 2.3 A N A L Y S I S OF P R O T E I N E X P R E S S I O N & C E L L S T R U C T U R E 2.3.1 Radioimmunoassay (RIA) Radioimmunoassay was performed on cell culture media and cell lysates for quantitation of G R P protein expression. H-345 was used as a positive control and test samples were obtained from 3 small round cell tumour cell lines. Ce l l culture media was collected 48hrs after a media change from cells in logarithmic growth phase (lOml/flask). Confluent cells were collected from T-75 flasks, lysed in single detergent 1% NP-40 (Sigma, U S A ) lysis buffer, and frozen at -70\u00C2\u00B0C. Protease inhibitors aprotinin ( l^g /ml ) , leupeptin (l j ig/ml) and phenylmethylsulfonyl fluoride ( P M S F ) (100/zg/ml) were added to collected culture media and cell lysates. A commercially available kit was obtained for R I A (Phoenix Pharmaceuticals, C A ) and the assay carried out as per the manufacturer's instructions. Lyophi l ized culture media and cell lysate samples were resuspended in R I A buffer and assayed in duplicate for quantitation of G R P expression. Aliquots of lyophilized lysis buffer and R P M I with F B S were used to assess background G R P expression of the cell lysate and culture media preparations, respectively. For all samples, net G R P expression was calculated by subtracting background expression from the total quantity of measured G R P and results were expressed as picograms of G R P per 10 6cells. 2.3.2 Immunohistochemistry & Electron Microscopy Histologic and immunohistochemical analysis was done on 4pm sections of formalin-fixed paraffin-embedded tissue/cell blocks. Morphology of cells was assessed by light microscopy following staining with hematoxylin and eosin. Immunostaining with all primary antibodies was done following established protocols using a standard three-step 60 streptavidin-biotin peroxidase method, with 3-amino-9-ethylcarbazole as the chromogen. Antibodies used to detect G R P protein expression by primary E T cells were the anti-GRP antibody LR-148 , and rabbit anti-human G R P antiserum (Dako) at dilutions of 1:1000 and 1:250 respectively. LR-148 was raised in rabbits against synthetic porcine GRP( l -27) and has been previously described (116). Both antibodies detect the secreted bioactive form of human G R P . Negative controls for immunohistochemistry experiments included both non-Ewing tumour samples and E T tissue sections that were processed without primary antibody to rule out non-specific peroxidase activity. For analysis of cell proliferation, similar immunohistochemical analysis was performed on primary Ewing tumour tissue and Ewing tumour cel l blocks using antibodies against proliferating cel l nuclear antigen ( B D Transduction Labs, Mississauga) and bromo-deoxyuridine (Sigma) (see below for more details). Electron microscopy of primary tumor specimens and T C 3 2 cells was done using standard procedures after cells were fixed in 3% glutaraldehyde. 2.3.3 Protein Lysate Preparation. Ewing tumour cell lines growing as monolayers or in suspension were rinsed once in PBS and then lysed in the appropriate lysis buffer for analysis of protein expression. For most studies of whole cell lysates, phosphorylation solubilization buffer (PSB: 5 0 m M Hepes, lOOmM NaF, l O m M N a 4 P 2 O v , 2 m M N a 3 V 0 4 , 2 m M E D T A , 2 m M N a M o 0 4 \u00C2\u00AB 2 H 2 0 , 1% Tnton-XlOO) containing protease inhibitors (leupeptin 10u,g/mL, aprotinin 10u,g/mL, and P M S F 250uM) and 0.01% H 2 0 2 w a s used. Primary tumor and xenograft lysates were obtained by cutt ing sections of frozen tissue directly into 500ul P S B and homogenization/solubilization was accomplished by high-speed vortexing and passage of 61 tissues through a 21-gauge needle. A l l lysates were incubated in 1.5ml Eppendorf tubes on a nutator at 4\u00C2\u00B0C for a minimum of 30 minutes and insoluble fractions removed by centrifugation at 12000 R P M for 5 minutes. For preparation of cytoplasmic and nuclear lysate fractions, cells were lysed initially in 150u,l low salt buffer (20mM Hepes, 5 m M KC1, 5 m M M g C l 2 , 0.5% Triton-XlOO) with protease inhibitors (leupeptin 10ixg/mL, aprotinin 10Lig/mL, and P M S F 250uM). Fol lowing low salt lysis, the cytoplasmic fraction (the supernatant) was separated by centrifugation of the sample at 4000 R P M for 4 minutes. The nuclear pellets were then washed once in low salt buffer and resuspended in high salt buffer (low salt buffer with 250mM N a C l added) with added protease inhibitors for lysis of the nuclear membranes. Following incubation on ice for 15 minutes, nucleoplasm was separated from insoluble membrane fractions by centrifugation at 4000 R P M for 4 minutes. For more stringent lysis of cells for whole cell lysate preparations, P S B was replaced by modified R I P A buffer (20mM Tris p H 7.4, 120mM N a C l , 1% Tri ton-XlOO, 0.5%Na deoxycholate, 0.1% S D S , 10% glycerol, 5 m M E D T A , 5 0 m M NaF, 0 . 5 m M N a 3 V 0 4 ) containing protease inhibitors (leupeptin lOug/mL, aprotinin 10pg/mL, and P M S F 250uM) and 0.01% H 2 0 2 . Lysis of cells in R I P A buffer followed by passage of lysates through a 21-gauge needle ensured lysis of the nuclear membranes and inclusion of nuclear and other less soluble proteins in whole-cell supernatants. Fol lowing solubilization in appropriate buffer, protein concentrations were determined and standardized using the D C Bio -Rad Protein assay kit (Bio-Rad Laboratories, C A ) . 62 2.3.4 Immunoprecipitation Between 500 and 1000u,g of total protein lysates was incubated in 1.5ml Eppendorf tubes on a nutator at 4\u00C2\u00B0C for 3 hours with 20ul of Protein-A conjugated sepharose beads (Pharmacia) and 2-5 u.g of primary antibody to the protein of interest. Primary antibodies used for immunoprecipitation included Pyk2 ( B D Transduction Labs), Src (Santa Cruz Biotechnology), F l i l (Santa Cruz Biotechnology), and the IGF-1 receptor beta-subunit (Santa Cruz Biotechnology). Fol lowing incubation, tubes were centrifuged at 2000 rpm for 5 minutes and the supernatants discarded. Pellets were washed three times in wash buffer (PSB containing 0.1% instead of 1% Tri ton-X 100) and prepared for immunoblotting as detailed below. 2.3.5 Immunoblotting Immunoprecipitated samples or 30u,g of total protein lysates were boiled in Laemmli SDS-sample buffer (115), and resolved by S D S - P A G E . Briefly, boiled samples were loaded onto a Protean I l /x i cell electrophoresis system (Bio-Rad) and electrophoresed on 8-15% polyacrylamide gels at 4-15mA according to standard methods (117). Fo l lowing electrophoretic transfer to Immobilon-P P V D F membranes (Mil l ipore , M A ) , western blot analysis was performed using the indicated antibodies at dilutions according to the manufacturer's instructions. P h o s p h o - E R K l / 2 , to ta l -ERK, phospho-AKT, to t a l -AKT, phospho-GSK3p\ total F A K , and total P Y K 2 antibodies were obtained from New England Biolabs ( N E B / C e l l Signaling, Mississauga, Ontario). Antibodies to cyclin D1/D2, p27 K I P 1 and p21 C I P 1 were obtained from Upstate Biotechnology (Lake Placid, N Y ) . The F l i l and IGFl-receptor beta-subunit antibodies were obtained from Santa Cruz Biotechnology. 63 Biosource International ( U S A ) was the source of the phospho-PYK2 antibody. A n t i -phosphotyrosine (RC20) and Grb2 primary antibodies and secondary anti-mouse and anti-rabbit antibodies conjugated to horseradish peroxidase were obtained from B D Transduction Labs (Mississauga, Ontario) and blotted proteins were visualized using enhanced chemi-luminescence (Amersham Pharmacia, Quebec). 2.4 F U N C T I O N A L STUDIES & P R O L I F E R A T I O N ASSAYS 2.4.1 EWS-FLI1 & GflP-promoter Reporter Gene Assays The cell lines Birch, S A N 2 , A4573 and TC-32 cells were cultured as described above and luciferase reporter gene assays done by Dr . Wen Tao. N I H 3 T 3 and NIH3T3 cells expressing EWS-FLI1 (19) (kindly provided by Dr. C . Denny, U C L A ) were maintained in Dulbecco's Modif ied Eagle Medium with high glucose ( D - M E M ) containing 5% F B S and antibiotics. The cells were transfected with constructs using Lipofectamine reagent ( G I B C O / B R L ) using established methods. Plasmids used included S7 and S6, containing 0.5-kb and 1.0-kb fragments of human G R P promoter-luciferase constructs respectively (118) (gift of Dr . E . Spindel, Oregon Regional Primate Research Center, Oregon). In addition, EWS/FLI1 expressing plasmids and the empty retroviral vector p S R a M S V were obtained from Dr . C . Denny (19). Cells were harvested 48 hours after the start of transfection, lysed by freeze-thawing and resuspended in 70 ul of extraction buffer ( lOOmM K 2 H P 0 4 , pH7.8, I m M D T T ) . The extracts were assayed for total protein using a Bio-Rad Protein Assay K i t (Bio-Rad Laboratories). The (3-gal activity was assayed colormetrically and expressed as A 4 2 0 / ( m g protein x minute) (115). The luciferase activity was measured as 64 described (119). The luciferase assays were corrected for transfection efficiency using the (3-gal assay. 2.4.2 Cell Growth in vitro Following Treatment with GRP-R Antagonist and Agonist. Studies on the effects of G R P - R antagonist treatment of E T cell lines were done both at Children's & Women's Hospital, Vancouver and at and the Gerhard-Domagk-Institut fiir Pathologie, Munster, Germany in collaboration with Dr . Christopher Poremba. In Vancouver, the E T cell line 6647 and R M S cell line R D were treated with the G R P - R antagonist DC-28-33B (H-tyr-Gln-Trp-Ala-Val-Gly-His-Leu-OH3) and agonist DC-28-45B (H-phe-Gln-Trp-Ala-Val -Gly-His -Leu-Met-NH2) (peptides kindly provided by Dr . David Coy, Tulane University School of Medicine, New Orleans). Ce l l growth in low or optimal serum conditions was then measured directly by cell counting or indirectly by measurment of 3 H-thymidine uptake'as previously described (120). Briefly, cells were plated in 24-well plates, treated after 48 hours with 0, 10 or lOOnM concentrations of either peptide, and then counted after 24 hours either manually with a hemacytometer or with a scintillation counter after a four hour incubation with 3H-thymidine. Data were collected in triplicate for each experimental condition and the results averaged. Antagonist experiments carried out in Munster used the E T cel l lines R D - E S and T C 7 1 . Equal numbers of cells (2x l0 4 ) were plated in 96-well plates in lOOu.1 R P M I containing 10% fetal calf serum. Cells were allowed to grow for 24 hours and then either RC-3095 or RC-3940-II was added to final concentrations of 1, 5, or l O n M for incubation times of 24, 48 or 72 hours. RC-3095 and RC-3940-II are both synthetic G R P - R antagonists 65 manufactured by A S T A Medica, Frankfurt, Germany with the following chemical structures: R C - 3 0 9 5 ( [ D - T p i 6 , L e u 1 3 \ ) / ( C H 2 N H ) L e u l 4 ] B n ( 6 - 1 4 ) ) and R C - 3 9 4 0 - I I ( [ H c a 6 , Leu' 3 \ j / (CH 2 NH)Tac 1 4 ]Bn(6-14)). Fol lowing incubation with antagonist, cells were counted manually or indirectly by spectrophotometric measurement of formazan using the M T T assay (Boehringer Mannheim). This assay is dependent on the ability of viable cells to reduce M T T (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide) thus producing a blue crystallized fomazan product which can be measured spectrophotometrically and interpreted using an E L I S A reader (121). Data were collected at least 4 times for each experimental condition and the results averaged. 2.4.3 Cell Growth in vivo Following Treatment with GRP-R Antagonists. A l l in vivo experiments were completed in Minister, Germany by Dr. Christopher Poremba. Athymic nude mice ( N M R I mice) were housed in sterile cages under laminar flow hoods in a temperature controlled room with a 12 hour light/12 hour dark schedule. A l l studies were carried out with full institutional approval in accordance with guidelines for the care and use of experimental animals at the Gerhard-Domagk-Institut fur Pathologie. Ewing tumour xenografts were initiated in two nude mice by subcutaneous injection of 5 mil l ion C A D O - E S cells (a local ly derived human E T cel l line). After 6 weeks of tumour development, animals were sacrificed, tumours dissected and minced aseptically. Three-mm 3 pieces of minced tumour were then transplanted subcutaneously by trocar needle into the right or left flanks of 10 mice. Two weeks following tumour transplantation, at a tumour volume of approximately 50mm 3 , the mice were divided into 2 experimental groups of 5 66 animals each. Group 1 animals received vehicle control injections of 0.9% saline/0.1% D M S O while Group 2 animals received injections of RC-3095 at a dose of 10u,g/animal. Injections were given subcutaneously once daily. Tumour volumes were measured every other day with microcalipers using the calculation formula of volume = length x width x height x 0.5236. After 33 days of treatment, mice were sacrificed, tumours dissected, cleaned in P B S and weighed. 2.4.4 Signal Transduction in Ewing Cells Following Treatment with GRP/Bombesin, Bombesin Receptor Antagonist & GRP Antisense. Ewing tumour cell lines grown as adherent monolayers or anchorage independent spheroids were starved overnight in serum-free media and then stimulated with 100-500nM bombesin or human G R P (both from Sigma Canada) for variable lengths of time as detailed in figure legends. For antagonist experiments, cells were incubated with 500-1000nM bombesin receptor antagonist (Sigma, Canada) for 24-72 hours in serum-free (0.25%) or low- serum (2.5%) media prior to lysis. GRP-antisense and control oligonucleotides were purchased from Biognostik, Germany. Monolayer and spheroid cells were incubated in low-serum media with 2 u M concentrations of oligonucleotide for 24-72 hours prior to lysis. Incorporation of oligonucleotides into Ewing cells was confirmed prior to antisense experiments by demonstrating uptake of a fluorescein-labeled randomized-sequence phosphorothioate oligo using fluorescent microscopy. A l l agonist, antagonist and antisense treated cells were then lysed in P S B lysis buffer and lysates analyzed for protein expression by immunoprecipitation and/or immunoblotting as detailed above section 2.3. 67 2.4.5 BrdU Proliferation Assays. Stable T C 3 2 spheroids and logarithmic growth phase monolayers were starved in serum-free media for 24 hours to synchronize cells as much as possible. Media was then changed to fresh media containing 15% F B S and 100p.M bromo-deoxyuridine (BrdU; Sigma) and cells allowed to grow for a subsequent 20 hours. Fo l lowing the 20 hour incubation, cells were pelleted, formalin fixed and embedded in paraffin as per standard protocols. Immunostaining with an anti-BrdU antibody (Sigma) was performed following the manufacturer's instructions. A l l conditions were repeated in triplicate and the percentage of B r d U positive cells was calculated by counting cells in 5 representative high-power fields for each condition (approx. 200-400 cells/hpf). 2.4.6 Cell Cycle Analysis by Fluorescent Activated Cell Sorting (FACS). T C 3 2 monolayer and spheroid cells were resuspended in P B S and vortexed to break apart cell clumps. Cells were then fixed in 70% ethanol, treated with 100u,g/ml RNase, stained with 50u.g/ml propidium iodide, and live cells analyzed for D N A content using the F A C S C a l i b u r F low cytometry system with CellQuest and Modfi t LT analytic software (Becton Dickinson, San Jose). 2.4.7 Kinase Inhibitor Studies. Starved Ewing tumour cells were treated with either the M E K 1 inhibitor U0126 (Calbiochem, San Diego) at 2 0 u M for 30 minutes, the P I 3 K inhibitor L Y 2 9 4 0 0 2 (Calbiochem) or wortmannin (Sigma) at 5 0 u M or 200 n M , respectively, for 3 hours, or an equivalent volume dimethylsulfoxide ( D M S O ) vehicle control. Fo l lowing inhibitor 68 treatment cells were harvested without serum stimulation or were stimulated with serum for 30 minutes or 3 hours prior to lysis. Total protein lysates and immunoblotting were carried out as above. To assess the effects of M E K and P I 3 K inhibition on cell proliferation, E T cells were cultured in 20u,M U0126 or 5 0 u M LY294002 , respectively in the presence of B r d U and serum for 20 hours and then cells collected for B r d U immunohistochemistry as described above. 2.4.8 Myristilated-AKT and Dominant-Negative ILK Transfections. T C 3 2 cells were transfected with a Myc-tagged myrist i lated-AKT c D N A construct (Upstate Biotechnology, N Y ) , a dominant-negative integrin-linked kinase ( D N - I L K ) c D N A construct (gift of Dr . S. Dedhar), or empty vector controls using lipofectamine ( G I B C O B R L ) and standard protocols. Two days following transfection, cells were transferred to media containing 250u,g/ml G418 for selection of successfully transfected cells. (Geneticin; Sigma). Cells were maintained in selection media until harvested for analysis of protein expression. M y r - A K T and control transfected cells were treated with 5 0 u M LY294002 or an equivalent volume of D M S O vehicle control for 3 hours fol lowing 21 hours serum-starvation. Cells were then harvested immediately or fol lowing 30 minutes or 3 hours serum-stimulation. Harvested cells were washed in P B S then lysed in P S B lysis buffer and processed for immunoblotting as detailed above. 2.5 C L I N I C A L C O R R E L A T E S STUDY Primary tumour specimens from 63 patients diagnosed with Ewing tumours at Children's and Women's Health Centre, Vancouver, B C and the Gerhard-Domagk-Institut 69 fur Pathologic Munster, Germany were analyzed by RT-PCR for expression of GRP and GRP-R genes. A l l tumours expressed EWS-ETS gene fusions. Clinical and follow-up information was available for 48 patients and their charts were reviewed for clinical (age, sex, stage & site of disease), pathologic (histology, fusion type) and outcome (relapse, status at last follow-up) data. Statistical analysis incorporated use of Kaplan-Meier estimates for event-free (EFS) and overall survival (OS) using the SPSS Version 7.0 statistical package. Events were defined as recurrence, progression of disease and death. Death resulting from therapy complications was not counted as an event but censored for survival and EFS analysis. GRP/GRP-R positive patients were compared to non-expressers using the log-rank test. Fisher's exact test or the X 2 test were used where appropriate to examine a potential association between dichotomous study variables of interest. P values of <0.05 were considered to be of statistical significance. 70 C H A P T E R III HUMAN GASTRIN-RELEASING PEPTIDE IS DIFFERENTIALLY EXPRESSED BY THE EWING FAMILY OF TUMOURS 3.1 I N T R O D U C T I O N E W S - F L I 1 and E W S - E R G proteins have been demonstrated to be oncogenic (19), and E W S - E T S oncoproteins likely function as aberrant transcription factors (11, 19, 24). Although the genes specifically regulated by E W S - E T S fusions remain largely unknown, recent studies have identified several potential targets of E W S - F L I 1 , as discussed in Chapter 1 (reviewed in (3) and (6)). Knowledge of genes transcriptionally activated or repressed by chimeric E W S - E T S oncoproteins may lead to a better understanding of the mechanisms underlying malignant transformation in E T . A s a strategy to identify genes differentially expressed in tumours with EWS-ETS gene fusions, we used the technique of differential d isplay-PCR ( D D - P C R ) (122). The utility of D D - P C R in comparing gene expression patterns in human tumours is well documented (123, 124). One advantage of this technique is that it allows for the simultaneous comparison of multiple tissue samples in the same experiment. We therefore used D D - P C R to compare gene expression patterns in E T cell lines expressing various EWS-ETS gene fusions with those of other pediatric small round cell tumour (SRCT) cell lines including rhabdomyosarcoma (RMS) and neuroblastoma (NB) . These studies demonstrated the differential expression of a gene with virtually complete sequence homology to human gastrin releasing peptide (GRP) in tumour cells positive for EWS-ETS gene fusions. GRP 71 expression was present in E T cell lines and primary tumours, but was not detectable in other pediatric S R C T s tested. In this study, the expression of GRP and GRP receptor (GRP-R) in E T cell lines and primary tumours was characterized. Fol lowing expression profiling, a clinical correlates study was done to determine i f expression of either the GRP or GRP-R genes con-elated with clinical or pathologic features of primary E T in a multi-center cohort of patients. 3.2 R E S U L T S 3.2.1 Differential Expression of GRP in ET Cell Lines To detect genes differentially expressed in ETs , we initially compared D D - P C R generated gene expression profiles of the three E T cell lines, TC-32 , TC-174, and TTC-547, to those of four R M S cell lines, R h l 8 , TTC-487, Birch, and CT10. Numerous differentially expressed P C R bands specific to either E T or R M S cell lines were observed using various combinations of 3'-oligo-dT primers and 5' arbitrary primers. Of these, we focussed only on those bands showing obvious differences between the two tumour subgroups. A representative example is shown in Figure 9 A , in which a band, designated p5.1, was identified only in E T lines after P C R with primers H - T , , G and H - A P 4 . Differentially expressed fragments were next excised from gels and P C R amplified, and then used to probe Northern blots of the above E T and R M S cell lines (data not shown). Only those fragments for which Northern analysis confirmed differential expression were further characterized. These were cloned and sequenced, and sequences were compared to Genbank databases. Three ET-specific sequences generated using Ff-T, ,G or H - T H C primers in combination with arbitrary primer H - A P 4 , including clone p5.1, were found to have virtually complete 1 2 3 4 5 6 7 B. p5.1 AACGTGAAGGAAGGAACCCCCAGCTGAACCAGCAATGATAATGATGGCCT 397 hGRP AACGTGAAGGAAGGAACCCCCAGCTGAACCAGCAATGATAATGATGGCCT 514 p5.1 C T C T C A A A A G A G G A A A A C A A A A C C C C T A A G A G A C T G C G T T C T G C A A G C A T 347 hGRP C T C T C A A A A G A G A A A A A C A A A A C C C C T A A G A G A C T G A G T T C T G C A A G C A T 564 p5.1 CAGTTCTACGGATCATCAACAAGATTTTCCTTGTGCAAAATATTTGACTA 297 hGRP CAGTTCTACGGATCATCAACAAGATTT-CCTTGTGCAAAATATTTGACTA 613 p5.1 TTCTGTATCTTTCATCCTTGACTAAATTCGTGATTTTCAAGCAGCATCTT 247 hGRP TTCTGTATCTTTCATCCTTGACTAAATTCGTGATTrTCAAGCAGCATCTT 663 p5.1 CTGGTTTAAACTTGTTTGCTGTGAACAATTGTCGAAAAGAGTCTTCCAAT 197 hGRP CTGGTTTAAACTTGTTTGCTGTGAACAATTGTCGAAAAGAGTCTTCCAAT 713 p5.1 TAATGCTTTTTTATATCTAGGCTACCTGTTGGTTAGATTCAAGGCCCCGA 147 hGRP TAATGCTTTTTTATATCTAGGCTACCTGTTGGTTAGATTCAAGGCCCCGA 763 p5.1 GCTGTTACCATTCACAATAAAAGCTTAAACACAT 113 hGRP GCTGTTACCATTCACAATAAAAGCTTAAACACAT 797 F I G U R E 9. D D - P C R analysis of S R C T cell lines. (A) . Differential expression of P C R fragment p5.1 (arrow) in cell lines expressing EWS-ETS gene fusions. L a n e l , TC-32 (ET); Lane 2, R h l 8 ( A R M S ) ; Lane 3, TC-174 ( M E M ) ; Lane 4, TTC-547 ( M E M ) ; Lane 5, TTC-487 ( A R M S ) ; Lane 6, Birch ( E R M S ) ; Lane 7, CT-10 ( E R M S ) . (B). c D N A sequence shows homology to human GRP. D D - P C R product, fragment p5.1, was cloned into vector pCRII and sequenced, revealing virtually complete sequence identity to the 3'-terminus of the human preproG-RP gene (nucleotides 465-797). 73 sequence homology to the 3'-terminus of the human preproG-KP gene. As shown in Figure 9B, the sequence of clone p5.1 is almost identical to nucleotides 465-797 of preproGPP, representing the terminal 332 nucleotides of this gene (125). Cloning and sequencing of GRP R T - P C R products from H345, a small cell lung carcinoma cell line, the E T cell line 6647, 2 primary ETs, a bronchial carcinoid and a human fetal lung tissue sample all revealed the identical substitution of cytosine for adenine at position 551 compared to published GRP sequences (data not shown). Additionally, a more 5'-alteration was discovered at position 365 where thymine is replaced by cytosine in 4 of the sequenced samples. Therefore, our studies may have detected previously undetected species of GRP or errors in previously submitted G R P sequence data. The potential significance of these sequence changes is discussed below. To confirm differential expression of GRP in E T cell lines, both clone p5.1 and full length GRP c D N A were used to probe Northern blots of multiple S R C T cell lines. R N A s from 2 bronchial carcinoid tumours known to express GRP were used as positive controls. In total, all 6 cell lines with EWS-ETS gene fusions were positive for GRP, including 5 EWS-FLI1 and one EWS-ERG expressing cell lines (Figure 10). None of the nine other S R C T s tested showed detectable GRP expression. A second Northern analysis of other E T cell lines also demonstrated positive but variable degrees of GRP expression (data not shown): the EWS-FLI1 expressing cell lines I A R C - E W 2 , F P B H , R M 8 2 , S K - E S - 1 , and STA-ET8 .2 were all positive; EWS-ERG expressers G R - O H - 1 and K N - O H - 1 were weakly and strongly positive, respectively. Furthermore, S T A - E T - 1 0 , a cell line containing the newly described EWS-ETS fusion, EWS-FEV (15), was also weakly positive for GRP (data not shown). A B C E T R M S Other ^ 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 18S-p5.1 B-actin F I G U R E 10. Screening of S R C T cell lines for GRP expression by Northern analysis. Using full length preproGPP c D N A as a probe, EWS-ETS expressing cell lines were shown to differentially express GRP, while other S R C T lines were negative. Equivalent R N A loading was confirmed with a P-actin probe and two human bronchial carcinoid tumours, known to express GRP, were used as positive controls. Lanes 1 & 2 : bronchial carcinoids. Lanes 3-8: E T cell lines TC-32 , T C - 7 1 , TC-174, TTC-547, J F E N and 466, respectively. Lanes 9 -13: R M S cell lines R h l 8 , TTC-487, Bi rch , CT-10 and R D , respectively. Lanes 14 & 15: N B cell lines San-2 and I M R - 3 2 . Lane 16: undifferentiated sarcoma cell line, A204. 75 further Ewing cell line, I A R C - E W 7 , in which a fusion transcript could not be identified was negative for G R P (cell lines reviewed in (126)). Using R T - P C R , we next screened 19 S R C T cell lines for GRP expression. A l l 11 E T samples tested were positive for GRP including EWS-FLI1 and EWS-ERG expressing cell lines. These results were confirmed by Southern analysis (Figure 11 A ) . None of the R M S or N B cel l lines tested demonstrated GRP expression. Two leukemia cel l lines known to express wild-type FLU were also screened to test whether the presence of the F L U transcription factor correlates with GRP expression, but both were found to be negative. These results indicate that GRP is specifically expressed in E T among S R C T cell lines. 3.2.2 Expression of GRP-R in ET Cell Lines R T - P C R and Southern analysis demonstrated transcripts for GRP-R in 6 (54.5%) of the 11 p P N E T cell lines tested. Additionally, 1 of 2 N B and 1 of 4 R M S cell lines also expressed GRP-R (Figure 11B). 3.2.3 Expression of GRP and GRP-R in Primary Ewing Tumours To rule out that the above in vitro results represented artifacts of tissue culture, we tested whether GRP and GRP-R expression could be demonstrated in primary E T tissue. Twenty-six primary tumours were screened by R T - P C R for both GRP and GRP-R expression, including 16 ETs and 10 other SRCTs . Of the 16 ETs , 7 (43.7%) were positive for GRP, while none of the other 10 SRCTs including 7 R M S , 2 N B , and 1 intra-abdominal desmoplastic small round cell tumour expressed the gene (Figure 12A). GRP-R expression was found in 4 of 16 ETs (25%), all of which also expressed GRP, in both N B cases tested and in 4 of 7 R M S (Figure 12B). A. GRP Expression in Cell Lines SC 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 -4\u00E2\u0080\u0094485 bp i. GRP-R Expression in Cell Lines SC 1 2 3 4 5 6 7 8 9 10 11 1213 14 15 16 17 <-390 bp F I G U R E 11. R T - P C R Analysis of S R C T C e l l Lines. C e l l lines were tested by R T - P C R for GRP and GRP-R expression. The S C L C cell line H345 was used as a positive control in both assays. The identity of the amplified bands was confirmed with Southern analysis using c D N A probes for GRP and GRP-R, respectively. (A.) Southern blot showing uniform expression of GRP by E T cell lines. Lane S C : H345; Lanes 1-9: EWS-FLU expressing E T cell lines TC-71 , TC-32 , A4573, B C - E S 1 , 6647, TC-174, TC-253, TC-547 and J F E N . Lanes 10 & 11: EWS-ERG expressing E T cell lines 633 and 466. Lanes 12 & 13: R M S cell lines CT-10 and R h l 8 . Lanes 14 &15: N B cell lines I M R - 3 2 and San2. Lanes 16 & 17: Jurkat and H E L , leukemia cell lines expressing wild-type FLIL (B.) Southern blot showing expression of GRP-R by 8 of 19 S R C T cell lines tested, including 6 ETs , 1 N B and 1 R M S . Lane assignments 1-6 as in A . Lane 7: R h l 8 , Lane 8: San2, Lane 9: CT-10, Lane 10: IMR-32 . A . GRP Expression in Primary Tumours SC 1 2 3 4 5 6 7 8 9 10 11 12 <-485bp B. GRP-R Expression in Primary Tumours SC 1 2 3 4 5 6 7 8 9 10 <-390bp F I G U R E 12. RT-PCR Analysis of Primary SRCT Samples. (A.) A representative Southern blot of 12 primary tumours, including all 7 ET tumours shown to be positive for GRP. No other primary SRCTs tested expressed GRP. Lane SC: H345; Lanes 1-7: ETs; Lanes 8 & 9: RMS; Lanes 10 & 11: NB; Lane 12: intra-abdominal desmoplastic small round cell tumour. (B.) Ten of 26 primary tumours were positive for GRP-R, including 4 ETs, 4 RMS and 2 NB. A representative Southern blot of 10 primary tumours is shown in this figure. Lane SC: H345; Lanes 1-4: ETs; Lanes 5 & 6: RMS; Lanes 7 & 8: NB; Lane 9: RMS; Lane 10: intra-abdominal desmoplastic small round cell tumour. 78 3.2.4 Expression of Immunoreactive G R P Peptide in E T . Using a commercially available G R P radioimmunoassay (RIA) kit we were able to demonstrate the presence of the immunoreactive G R P peptide in culture media and cell lysates from the E T cell line 6647 while little G R P peptide was detected in media or lysates collected from the N B and R M S cell lines S A N 2 and R D , respectively (Table 6). The levels detected in 6647 cells were similar to those observed in the positive control cell line H345. For all R I A results, the quantity of G R P expressed was indicative of biologically active peptide produced by the cells themselves, as background G R P activity in the lysis buffers and culture media was first subtracted. Immunohistochemistry of S R C T s using 2 different antibodies directed against bioactive G R P was also used in an attempt to show peptide expression, but the results were inconclusive. Twenty E T samples from 18 patients were tested. There was positive staining for G R P peptide in 7 (35%) of 20 ETs, but expression was highly variable ranging from diffusely positive intracellular staining (Figure 13A) to staining of the extra-cellular matrix, to positivity in only rare cells (data not shown). More consistent, however, was the complete absence of staining in other S R C T s including N B and R M S using either antibody (Figure 13B). Control slides using secondary antibody alone were uniformly negative in both E T and other tumour tissue sections. 79 T A B L E 6. Immunoreactive GRP peptide expression demonstrated by radioimmunoassay. C E L L L I N E C E L L L Y S A T E S (net pg G R P / 1 0 6 cells) C U L T U R E M E D I A (net pg G R P / 1 0 6 cells) H345 42.55\u00C2\u00B15.30 100.00\u00C2\u00B19.76 6647 27.90\u00C2\u00B14.10 71.60\u00C2\u00B110.04 San2 5.20\u00C2\u00B11.41 0 R D 10.55\u00C2\u00B11.06 4.45\u00C2\u00B16.29 Ce l l lines were grown to confluence and their respective cell lysate and culture media preparations tested for expression of G R P using a radioimmunoassay. Quantitation of G R P was standardized per 10 6 cells after first subtracting background G R P activity from the total. F I G U R E 13. Immunohistochemistry of primary S R C T s with a GRP-specific antibody (A). Positive staining in a case of E T . (B). Negative staining in a case of R M S . (x400) 81 3.2.5 GRP is not a Direct Target of EWS-ETS Chimeric Proteins E W S - E T S oncoproteins appear to act as aberrant transcription factors in E T cells (reviewed in (11)). It is therefore conceivable that E W S - E T S proteins may bind directly to and activate the GRP promoter. However, luciferase reporter assays using luciferase constructs containing G R P promoter inserts revealed no differences between E T , R M S , and N B cell lines despite similar transfection efficiencies (data not shown). Second, the same reporter constructs expressed in murine NIH3T3 fibroblasts stably expressing EWS-FLI1 or parental NIH3T3 cells alone (19, 20) failed to detect differences. Third, reporter constructs were transiently co-transfected along with an EWS-FLI1 expression construct (19, 20) or vector alone into parental NIH3T3 cells, but no difference in luciferase activity was observed (data not shown). Finally, Northern blots of EWS-FLI1 positive cell lines transfected with EWS-FLI1 antisense R N A (28) did not appear to affect GRP expression (data not shown). We therefore conclude that it is unlikely that E W S - E T S chimeric proteins directly activate the GRP promoter. 3.2.6 Clinical Correlates Study Sixty-three cases of primary E T were identified, of which 48 had sufficient clinical and follow-up data to be included in the study. Patients ranged in age from newborn to 60 years with a median age at presentation of 14 years. Twenty-five patients were male, 23 female and 16 of the 48 presented with metastatic disease. Site of primary disease was almost equally split between central (N=26) and peripheral (N=20) skeleton while 2 patients had primary soft-tissue Ewings. At the time of this study, 32 patients were still alive (median follow-up 42 months). Review of histopathology revealed 31 patients with typical Ewing sarcoma, 6 with atypical Ewing sarcoma and the remainder with peripheral primitive 82 neuroectodermal tumours. Molecular analysis confirmed the presence of an EWS-ETS gene fusion in all cases; 26 were EWS-FLI1 1/6 (type I), 15 were EWS-FLI1 7/5 (type II), and 7 showed other rearrangements. R N A from all 63 cases was analyzed by R T - P C R for expression of GRP and GRP-R and results showed almost equivalent numbers of expressers and non-expressers as shown in Table 7. Data were then analyzed for any significant differences in cl inical or pathologic features between GRP/GRP-R positive and negative cases. A summary of event-free survival data is presented in Table 7. Data analysis showed no statistically significant difference between positive and negative cases for any of the fol lowing variables: age at diagnosis, sex, site of primary tumour, stage of disease, histopathology, gene fusion type, event-free survival, and overall survival. TABLE 7. GRP/GRP-R STATUS OF PRIMARY TUMOUR COHORT AND ITS RELATIONSHIP TO EVENT-FREE SURVIVAL T O T A L G R P + G R P - G R P - R + G R P - R Total number of cases studied 63 34 (54%) 29 (46%) 33 (52%) 30 (48%) Number of patients with follow-up data 48 24 (50%) 24 (50%) 20 (43%) 27 (57%) Al ive at last follow-up: Range 11-115 mo. (median 42 mo.) 32 16 (50%) 16 (50%) 13 (41%) 19 (59%) Dead 16 8 (50%) 8 (50%) 7 (47%) 8 (53%) GRP+ G R P -5 Y E A R E V E N T - F R E E S U R V I V A L (P>0.05) 33% 24% 83 3.3 DISCUSSION Using D D - P C R , we have shown that ETs differentially express a gene with virtually complete sequence homology to human GRP. Expression of GRP, although variable, was found in 100% of a series of E T cell lines, all of which are known to be positive for EWS-ETS gene fusions. In contrast, GRP expression was not detectable in other S R C T tumour cell lines screened, including N B , embryonal and alveolar R M S and one E T lacking demonstrable EWS-ETS gene fusion. These results were confirmed by Northern blot analysis of a series of S R C T cell lines using a GRP c D N A probe. Moreover, using R I A we were able to demonstrate immunoreactive G R P peptide expression from a GRP expressing E T cell line. Little to no G R P peptide was detectable in 2 non-ET S R C T cell lines tested in the same series of R I A experiments. Gastrin releasing peptide is a member of the bombesin family of peptides and plays an important role in the control of gut motility and gastroenteropancreatic secretion, in thermoregulation and other CN-mediated regulatory processes (reviewed in (127)). It also functions as a growth factor in developing gastrointestinal and lung tissues and is expressed at high levels in fetal lung (127). In mature lung, pulmonary neuroendocrine cells express G R P , as do many of the small cell lung cancers ( S C L C ) which arise from these cells (128). G R P has been shown to function as an autocrine growth factor in S C L C (129) and other tumours that produce the peptide, such as pancreatic (130), colon (131) and breast cancers (132). Inhibition of the growth of these and other tumours has been demonstrated using GRP-receptor antagonists in vitro and in animal models (120, 133-135). One of these antagonists is currently undergoing clinical trials in S C L C studies (136). 84 Sequencing of GRP c D N A amplified from tumour cell lines, primary tumours and human fetal lung tissue revealed consistent base changes at base pairs 365 and 551 as compared to the originally published c D N A sequence (125). The switch from thymine to cytosine at position 365 likely represents a polymorphism as it produces no change in the amino acid sequence of the peptide product. Likewise, the replacement of the adenine residue at position 551 occurs in the 3'-untranslated region of preproGRP. Whether this replacement affects the stability of the m R N A or the translation of the prepro-, pro-, or mature G R P molecules wi l l require further investigation. In contrast to GRP, which was expressed by 100% of E T cell lines and no other S R C T cell lines, expression of GRP-R was variable and equivocal in both cell line and primary tumour analysis. While there appeared to be a trend towards differential expression of the receptor among E T as compared with other S R C T , this was not definitive nor easily reproducible. G R P and other bombesin-like peptides exert their function by binding to high affinity receptors on the target cell 's surface. At least four different receptors have been described and because of the high homology among the bombesin-like peptides, there is potential for interaction between different ligands and receptors (137). We tested cell lines and primary tumours for the presence of the GRP-preferring subtype of receptor alone (112). It is possible that other members of the GRP-receptor family are important in E T and this could explain the lack of detection of GRP-R among all GRP expressing tumours. Also , there is often low level expression of growth factor receptor m R N A s in cells even when protein expression may be high. In fact, screening for GRP-R m R N A expression is reported to be problematic even in S C L C cells (112). Therefore, R T - P C R may not be the best method by which to study receptor expression and quantitation may be better established using 1 2 5 I -85 GRP binding assays. Detection of GRP-receptor mRNA in non-ET tumours lacking concomitant GRP expression may simply indicate that other related peptides are important in the growth of these SRCTs and that these growth factors may interact with the bombesin family of receptors. Expression of GRP mRNA has been described previously in S K - N - M C I X C , an undifferentiated peripheral neuroepithelioma cell line (138). These cells co-expressed several neuropeptide genes, suggestive of a pluripotent neuronal cell. Additionally Sawin et al. have described GRP production from several primary childhood retroperitoneal tumours, including an ET (139) and have demonstrated its role as an autocrine growth factor in a neuroblastoma cell line, SK-N-SH (140). These data, together with our own, support the proposed neural origin of ET and suggest that they might represent another tumour group whose growth is influenced by GRP. We found variable immunoreactivity for GRP in primary ET tumours. The prepro-GRP gene demonstrates a 3 exon structure and encodes the 3 known forms of the prepro-GRP transcript by alternative R N A processing (114). Common to all 3 transcripts is the mature 27-amino acid biologically active peptide which has carboxyl-terminal domain homology with amphibian bombesin and is responsible for the biological activities of GRP and its related peptides (reviewed in (127)). Once cleaved from the biologically inactive pro-GRP hormone, GRP is detectable using LR-148 and the commercially available D A K O antibody to GRP. Previous investigators have shown that immunostaining for the proGRP peptides appears to be the most sensitive way of detecting GRP gene expression in routinely processed tumour tissues (141). This may be because intracellular GRP is rapidly secreted from cells once it is cleaved from the pro-hormone or, alternatively, mature GRP may be 86 much more unstable than proGRP. Either way, it is not surprising that many of our primary tumour specimens showed variable GRP immunoreactivity and it is possible that antibodies to proGRP may show more consistent staining of ET cells. Histopathologically, the members of the ET family express varying degrees of neural differentiation. This may also contribute to the variability of GRP expression among ETs given that identification of GRP mRNA in lung tumours and its translation to the mature peptide appears to depend on the degree of neuroendocrine differentiation (127). Studies with lung tumours have demonstrated that GRP and its receptors can be upregulated in non-SCLC cells if they are grown in serum-free and growth-factor free conditions (112). Cell culture of ET may facilitate expression of neuroendocrine elements leading to increased levels of GRP expression in cell lines as compared to primary tumours. Transfection studies and reporter assays failed to demonstrate direct targeting of the GRP promoter by EWS-FLI1 oncoproteins. Also, Northern analysis of ET cell lines transfected with EWS-FLI1 anti-sense mRNA did not reveal a direct relationship between EWS-FLI1 expression and the level of GRP expression. These data suggest that while ET differentially expresses GRP, this expression does not appear to be directly activated by EWS-ETS chimeric transcription factors. GRP expression in these tumours may instead reflect the differentiation capacity of the cell of origin of ET, and provides further evidence for neuroectodermal histogenesis of these tumours. Whether expression of the GRP gene, the peptide and/or its receptor is somehow indirectly influenced by EWS-ETS fusion products remains to be determined. While GRP was uniformly expressed by ET cell lines, only 43.7% of the initial group of 16 primary tumours tested were positive for gene expression. It is well known that 87 aggressive primary tumours can be established in cell culture with greater ease than less aggressive tumours. This led us to hypothesize that G R P expression may be correlated with the degree of tumour aggressivity among ETs and, thus, tumour cells from established cell culture would be more likely to express G R P than a cohort of primary tumour samples. In order to study this hypothesis, R T - P C R analysis of a further 47 tumours (for a total of 63) was undertaken along with an analysis of available clinical and pathologic data. R T - P C R results confirmed expression rates of approximately 50% for both GRP and GRP-R. However, we were unable to discern any significant differences between GRP/GRP-R expressers in any of the studied variables. Our evidence suggests that although the expression of G R P may not influence clinical outcome, the peptide appears to be involved in the modulation of E T cell growth. Thus, we next attempted to define this role by investigating the growth pathways that are activated by G R P in E T cells. Defining G R P signaling in E T is of importance for several reasons. First, if G R P acts as a growth factor in vivo, G R P - R antagonists may prove to be useful therapeutic agents for patients with E T . Second, G R P expression may prove to be useful as a marker for microscopic metastases, minimal residual disease or disease recurrence, given the demonstration of immunoreactive G R P in the plasma of patients with S C L C (142). In the following Chapter our studies investigating the role of G R P as an autocrine growth factor in E T wi l l be presented. 88 C H A P T E R IV GASTRIN-RELEASING PEPTIDE FUNCTIONS AS AN AUTOCRINE GROWTH FACTOR IN THE EWING FAMILY OF TUMOURS 4.1 I N T R O D U C T I O N We have found that the Ewing family of tumours expresses G R P in 100% of cultured cell lines and 50% of primary tumours. Human G R P is a member of the bombesin family of proteins and is normally expressed in the human brain and neuroendocrine cells of lung, gut, and pancreas. It also functions as a growth factor in developing gastrointestinal and lung tissues and is expressed at high levels in fetal lung (127). O f importance to our studies, G R P has been shown to function as an autocrine growth factor in several malignancies including small cell lung cancer (129) pancreatic cancer (130), colon cancer (131) and breast cancer (132). Inhibition of the growth of these and other tumours has been demonstrated using GRP-receptor antagonists in vitro and in animal models (120, 133-135). The receptor for G R P is a member of the seven-membrane spanning family of G -protein coupled receptors whose general structure and function was discussed in Chapter I (Section 1.3.1.1 and Figure 3). Normal proliferative signaling via the GRP/bombesin receptor is still under study, but the literature to date has shown downstream effects to be both cell type and situation dependent (reviewed in (50)). In general, however, the mitogenic response of cells to G R P usually results from activation of the R A S \u00E2\u0080\u0094 R A F \u00E2\u0080\u0094 M E K \u00E2\u0080\u0094 E R K M A P K pathway and initiation of G l - S phase transition (50). 89 In this chapter, our results supporting the hypothesis that G R P functions as an autocrine growth factor in E T wi l l be presented. In addition, preliminary data suggesting that G R P induced mitogenesis in E T may not directly invo lve activation of the R A S \u00E2\u0080\u0094 R A F \u00E2\u0080\u0094 M E K \u00E2\u0080\u0094 E R K pathway wi l l be discussed. 4.2 R E S U L T S 4.2.1 in vitro Ce l l Proliferat ion Studies Initial in vitro experiments assessing the effect of G R P - R antagonists and agonists on E T cell growth were carried out by myself and Dr. Chris Poremba. Treatment of the E T cell line 6647 with the G R P - R antagonist DC-28-33B for 24 hours in low-serum media resulted in a slowing of cell growth as measured by 3F\u00C2\u00A3-thymidine uptake. Conversely, similar treatment of these cells with the G R P - R agonist, D C - 2 8 - 4 5 B , stimulated cell growth compared to the R M S cell line R D (see Figure 14). Likewise, exposure of 6647 to the receptor antagonist for 24 hours resulted in a 50% decrease in cel l growth compared to control cells as demonstrated by direct cell counting while agonist stimulated growth by 140% (data not shown). These early results supported the potential role of G R P as an autocrine growth factor in E T and further collaborative studies were continued by Dr . Poremba in Munster, Germany to elucidate the autocrine nature of G R P growth stimulation in E T in vitro and in vivo. 90 F I G U R E 14. In Vitro Response of S R C T Ce l l Lines to G R P - R Antagonist & Agonist. Treatment of p P N E T cel l line 6647 with G R P - R antagonist, DC-28-33B resulted in a net decrease in cell growth compared to control levels as measured by 3F\u00C2\u00A3-thymidine uptake. Treatment with G R P - R agonist, DC-28-45B, resulted in stimulation of cell growth of 6647 while neither peptide had any appreciable effect on the growth of the R M S cel l line R D . Data points: mean of triplicate experiments. Bars: standard deviation. 91 4.2.2 Collaborative Studies on GRP Mediated Autocrine Growth in vitro & in vivo In an effort to corroborate our preliminary results of the effect of G R P - R blockade on E T cell growth, Dr. Chris Poremba completed the following in vitro and in vivo studies in Muenster, Germany. First, he treated the E T cell lines R D - E S & TC71 with the synthetic G R P - R antagonists RC-3095 and RC-3940-II. Increasing concentrations of RC-3095 and RC-3940-II were observed to significantly slow the growth of E T cells as compared to untreated controls (Figure 15A). N o effect of these antagonists could be observed in an identically treated rhabdomyosarcoma cel l line (data not shown). Second, RC-3095 treatment of nude mice transplanted with human E T xenografts resulted in suppressed tumour growth. Growth inhibition was observed in treated mice as compared to controls within a week. After 33 days, the final tumour volume in RC-3095 treated mice was 924 \u00C2\u00B1 245 m m 3 versus a final tumour volume of 2292 + 261 m m 3 in control treated mice. The tumour growth curves for the first 28 days of control and G R P - R antagonist treated mice are shown in Figure 15B. 4.2.3 Blockade of GRP Signaling in ET does not appear to affect ERK or AKT Activation Having established both in vitro and in vivo evidence for the role of G R P as an autocrine growth factor in E T , we next sought to determine which signaling pathways mediated this effect. In view of the previously published literature, the effects of G R P - R stimulation and inhibition on the E R K M A P K s were first assessed. Stimulation of cells with serum for 30 minutes following a 24 hour period of serum starvation led to dramatic c \u00E2\u0080\u00A2 03 1\" '\u00E2\u0080\u0094 \u00E2\u0080\u00A2: \u00E2\u0080\u00A2\u00E2\u0080\u00A2\u00E2\u0080\u00A2-\"\u00E2\u0080\u00A2>i \u00E2\u0080\u00A2; | | | | | \u00E2\u0080\u00A2 \u00C2\u00AB'\u00E2\u0080\u00A2 IP?** \u00E2\u0080\u00A2 =) O CO IF ... 3 f, \u00E2\u0080\u00A2 \u00E2\u0080\u00A24* O CO 106 Moreover, ultrastructural analysis of TC32 spheroids showed numerous well-developed tight junctions between neighbouring spheroid cells that were indistinguishable from those of primary E T , in contrast to the poorly-formed junctions observed in monolayer cultures (see Figure 20B). The survival of E T cells in suspension was highly dependent on the formation of multi-cellular aggregates. If less than 0 .5x l0 6 cells were initially plated in a 10 cm agar-coated dish, the vast majority of cells remained as single cells which were dead after 6-12 hrs in suspension, as measured by trypan blue exclusion (data not shown). Spheroids readily formed when cells were plated at higher densities (at least l - 2 x l 0 6 cells/plate), with almost 100% of clumped cells remaining viable after 24 hrs while single cells were dead. 5.2.2 Adherent monolayer ET cells have significantly higher rates of proliferation than either spheroid or primary tumor cells. Several studies have reported proliferative rates ranging from 7-14% for primary E T as measured by immunohistochemistry or flow cytometric assessment of cells in G 2 / M or S-phase (155-159). Visual comparisons of E T cell growth in vitro suggested that monolayer cells proliferated much more quickly than spheroids in all three cell lines tested. Therefore, we used three different methods to determine the proliferative state of T C 3 2 cells grown in monolayer or spheroid cultures. First, using an antibody to proliferating cell nuclear antigen ( P C N A ) , which is a well-established marker of cells in S phase or mitosis (160), we performed immunohistochemical analysis of fixed monolayer and spheroid T C 3 2 cells in addition to primary E T biopsy specimens. TC32 monolayer cells showed P C N A staining in a large majority of cells. In contrast, T C 3 2 spheroids and the primary E T sections showed very similar staining patterns, with fewer than 10% of cells being P C N A positive (Figure 107 o \u00E2\u0080\u0094 u & on SH a >^ o c o 1 i ' t %-7 55 J = w .9 2 5 C3 108 21A). Second, cell cycle was assessed using F A C S analysis of D N A content in propidium iodide stained cells. Consistent with the P C N A results, we observed much higher proliferative rates in monolayer compared to spheroid cells and a marked decrease in the proliferative index of spheroids in response to serum starvation. After overnight serum-starvation, F A C S analysis of D N A content revealed 46% of monolayer cells to be proliferating as compared to only 9% of spheroid cells. Twelve hours of serum stimulation increased these proliferative rates to 64% and 16%, respectively (Table 8). Finally, cells were assessed for uptake of bromo-deoxyuridine (BrdU) as a measure of proliferation. As shown in figure 21B & C, the proliferative index of monolayer cells over a 20 hr period was significantly greater than spheroid cells and the latter were more responsive to serum stimulation. In keeping with F A C S and P C N A data, 60% of serum-stimulated monolayers were proliferating compared to only 9% of serum-stimulated spheroid cells. Therefore, the proliferative index of E T spheroid cells is significantly lower than monolayer cells and is much more in keeping with the proliferative rates of primary Ewing tumors in vivo. 5.2.3 Cyclin DI protein expression in ET cells grown in suspension requires cell-cell adhesion and is serum-dependent. We next wished to assess whether the observed differences in proliferation were associated with differences in cyclin D I expression in E T monolayer and spheroid cells. Constitutive up-regulation of cyclin D I has been described in numerous tumor types and is implicated in oncogenesis (reviewed in (66)). Monolayer T C 3 2 cells demonstrated high 109 FIGURE 21. The proliferative index of ET spheroids is similar to primary Ewing tumors. (A) Immunohistochemical analysis of a primary Ewing tumor and T C 3 2 cells using an antibody to proliferating cel l nuclear antigen ( P C N A ) demonstrating rare positive cells in primary tumor and spheroid sections (arrows), compared to frequent positive cells in monolayers. (B) T C 3 2 cells growing in monolayer and spheroid cultures were incubated with B r d U for 20 hours. Cells were then pelleted, fixed, paraffin-blocked and processed for immunohistochemical staining with an anti-BrdU antibody. Significantly greater B r d U uptake (brown-stained nuclei) was observed in monolayer cells whether or not serum was present. (C) Histograms of B r d U uptake in TC32 monolayer and spheroid cells. The percentage of positive cells is an average of 5 high power fields (total of 1000-2000 cells) \u00C2\u00B1 standard deviation. 110 Starved Stimulated 'um 'um CD CO ocl \u00E2\u0080\u00A2 \u00E2\u0080\u00A2 CN + O CN T\u00E2\u0080\u0094( +! oo C N CO r> OO m o s-CU a C/3 o Q CD o C O CO o CM - i \u00E2\u0080\u0094 S | | B 3 SAIJISOd fll> ' 3 JO % o CU \u00E2\u0080\u00A2 a CO oh * \u00E2\u0080\u00A2 * \u00E2\u0080\u00A2 \u00C2\u00AB\u00E2\u0080\u00A2 *i M i Ill Table 8: Cell Cycle Analysis of Cultured TC32 Cells Shows Decreased Cycling and Increased Serum Sensitivity of Spheroids Monolaver % cvcling cells CG2/M + S) Spheroid % cvcline cells ( G 2 / M + S) Serum starved 46 9 + 3 Hours serum 41 10 + 6 Hours serum 35 13 + 12 Hours serum 64 16 112 levels of cyclin DI expression that were independent of serum stimulation (see left panel, Figure22A). However, we observed immediate and virtually complete loss of cyclin DI protein expression when cells were placed in suspension, either under high-serum or serum-free conditions (left and middle panels, Figure 22A). Cyclin DI expression slowly increased over time coincident with cell clumping but only when cells were suspended in serum-containing media (middle panels, Figure 22A). In contrast, cyclin DI remained undetectable even in the presence of serum when suspended cells were prevented from clumping by continuous rocking of the cultures (see right panel, Figure 22A). We also tested whether cyclin DI expression remained serum-dependent once stable spheroids had formed. TC32 spheroids were grown as above for 48 hrs in the presence of serum, and then subjected to overnight serum starvation. This resulted in a reduction of cyclin DI protein which could be recovered by addition of serum to the medium (see right panel, Figure 22B). These results indicate that, in contrast to monolayer TC32 cells which are characterized by constitutive serum-independent cyclin DI expression, anchorage-independent TC32 cells appear to require both serum stimulation and cell-cell adhesion for cyclin DI protein expression. Similar to TC32, monolayer cultures of the ET cell lines A4573 and 5838 also expressed high, serum-independent cyclin DI expression while spheroid cultures of these cell lines had lower levels of expression which were serum-responsive (see Figure 22C). 5.2.4 Differences i n cycl in D I expression between spheroids and monolayers are post- t ranscr ipt ional and are associated wi th differences in subcel lular localization. Cyclin DI protein levels are tightly regulated by both transcriptional and post-113 FIGURE 22. Cyclin DI protein expression is dependent on cell-cell adhesion and serum stimulation in suspension cultures of ET cells. (A) Monolayer ( M L ) TC32 cells were starved for 24 hours and then replated on agar-coated dishes at a density of 3-5 x l 0 6 cells/ lOcm dish, in either serum-free (left panel), 15% serum-containing medium (right panel), or in 15% serum with continuous rocking of the plates to prevent spheroid formation (right panel). Suspension cells were then collected at the indicated time-points and analyzed for expression of cyclin D I by Western analysis. Total A K T levels were used to demonstrate equal loading. (B) T C 3 2 cells were grown in 15% serum on regular plates as monolayers or on agar-coated dishes for 48 hours to form stable spheroids. Cells were then starved for 24 hrs in 0.25% serum, followed by treatment with (+) or without (-) 15% serum for the indicated times. Cyc l i n D I levels were assessed by Western analysis as above. Total A K T levels were used to demonstrate equal loading. (C) 5838 and A4573 monolayer ( M L ) and stable spheroid cells grown in 15% serum-containing medium were starved for 24 hrs in 0.25% serum and then treated with (+) and without (-) serum for 3 hrs, prior to assessment of cyclin D I levels as above. Grb2 levels were used to demonstrate equal loading. 115 transcriptional mechanisms (66, 72, 161). In order to assess whether the observed down-regulation of cycl in D I protein in E T spheroids might be occurring through a post-transcriptional process, we performed Northern-blot analysis of T C 3 2 cell cultures using a cyclin D I specific c D N A probe. We observed that cyclin D I transcript levels were similar in monolayer and spheroid cells (see Figure 23A). Moreover, neither serum starvation nor starvation followed by serum stimulation had an appreciable effect on cyclin D I m R N A levels in either culture condition. These data suggest that regulation of cyclin D I protein expression in E T cells may be largely post-transcriptional, at least in vitro. Glycogen synthase kinase 3(3 (GSK3P) phosphorylates cyclin D I at residue threonine 286, thereby targeting it for proteosomal degradation (73). This GSK3(3 activity is inhibited by AKT-induced phosphorylation at G S K 3 P serine residue 9 (162). We therefore compared the phosphorylation status of GSK3p(Ser9) in TC32 monolayers and spheroids. As shown in Figure 23B, no appreciable differences were observed and therefore, the reduced cyclin D I levels observed in E T spheroids could not be explained on the basis of diminished GSK3P(Ser9) phosphorylation in these cells. Cyc l i n D I regulation is also dependent on shuttling of the protein between the cytoplasm and nucleus during different phases of the cell cycle (73, 76). Protein lysates were therefore prepared from the nuclear and cytoplasmic fractions of E T monolayer and spheroid cells to assess potential differences in subcellular cyclin D I localization. As shown in Figure 24A, while cyclin D I appeared to be entirely cytoplasmic in monolayer cells, in spheroid cells cycl in D I was almost equally distributed between the nucleus and cytoplasm. Importantly, these results also demonstrated that total cycl in D I levels (nuclear plus cytoplasmic fractions) were actually equivalent between monolayer and spheroid cells. This 116 Q CO LU TJ 0 a s: a. t/J si CO + co| + 1 a >. re o c T3 0 x : a CO > ro \u00C2\u00BB O c/5 PQ 117 finding was in direct contradiction to our earlier findings (Figure 22) and was initially difficult to explain. However, upon review of our protein lysis protocols, it became apparent that the standard protocol for whole cell lysis using phosphorylation solubilization buffer (PSB; see Materials and Methods for complete composition) may have led to a loss of cyclin D I in the discarded insoluble pellets. This may have occurred for two reasons. First, the single detergent P S B may have been insufficiently stringent to lyse both cytoplasmic and nuclear membranes in the same lysis procedure and nucleoplasm^ proteins would be under-represented. Second, the final spin that is used to separate soluble proteins from insoluble fractions in whole cell lysates is done at maximal velocity or 12000 rpm, while nuclear-cytoplasmic preparations call for lower speeds of only 4000 rpm. Thus, i f cyclin D I was present in insoluble cellular compartments, it would be pelleted and discarded during high-speed centrifugation in whole cell preparations. Thus, in the preparation of whole cell lysates with P S B , it is possible that nuclear membranes were not effectively lysed and proteins, such as cyclin D I , that were localized to the nucleus were being precipitated in the discarded insoluble pellets. This led us to interpret that total levels of cyclin D I were less in spheroids when, in fact, they were just differently distributed within the cells. To test this hypothesis, whole cell lysates from serum starved and serum stimulated E T cells were prepared using a modified R I P A buffer (see Materials and Methods), which is a more stringent 3-detergent buffer that w i l l solubilize subcellular fractions that may have been insoluble in P S B . These lysates expressed equal levels of total cyclin D I , confirming our hypothesis and explaining our seemingly contradictory results (Figure 24B). We also found that the differences in cyclin D I subcellular localization corresponded to differences in the subcellular localization of P-GSK3(3 (Figure 24A). Therefore, although total levels of P-118 GSK3f3 remained unchanged between culture conditions, its subcellular distribution differed. We therefore conclude that the differences in cyclin D I regulation observed in anchorage independent spheroids appear to be related to differences in its subcellular localization, likely mediated by GSK3p\ 5.3 DISCUSSION Cel l cycle progression of non-transformed cells is dependent on the coordinated control of cyclin\u00E2\u0080\u0094cyclin-dependent kinase complexes as induced by growth factors as well as adherence to an extracellular matrix (82). In contrast, transformed cells are capable of anchorage-independent proliferation (153). We therefore reasoned that conventional in vitro growth of E T cells as adherent monolayers might be expected to activate a number of signaling pathways, secondary to phenomena such as cell-plastic adhesion and cel l -spreading, that are not actually representative of in vivo tumour growth. Several studies have indicated that anchorage-independent survival and proliferation of tumour cells are dependent on cell-cell adhesion which is mimicked when tumour cells form multi-cellular spheroids (reviewed in (154)and (163, 164)). We therefore cultured E T cell lines on agar-coated plates and found that they spontaneously form multi-cellular spheroids. Stable spheroids demonstrated numerous similarities to primary E T including small round cell morphology, a marked increase in well-developed ce l l -ce l l junctions evident ultra-structurally, and a proliferative index of approximately 10%. Several studies have reported similar proliferative rates of 7-14% in primary E T (155-159). In contrast, proliferative rates were significantly higher in monolayer cells and these cells showed only rare poorly-formed 119 120 junctions. Our data support the concept that the in vitro growth of Ewing tumour cells as spheroids may be more representative of primary E T than conventional monolayer cultures. The observed differences in cellular proliferation between monolayer and spheroid cells were accompanied by differences in the regulation of cyclin D I . These differences were apparent at the protein level but not at the level of m R N A indicating that the variability was largely mediated in a post-transcriptional fashion. Moreover, variations in cyclin D I protein expression between serum starved and serum stimulated spheroid cells were also due to post-transcriptional changes. Post-transcriptional regulation of cycl in D I is largely mediated by altered rates of m R N A translation (72), and by subcellular trafficking and proteosomal degradation (73, 75, 76). We have shown that subcellular localization of cyclin D I varies between adherent monolayer cells and anchorage-independent spheroids, and also between serum-starved and serum-stimulated spheroid cells. In non-proliferating normal cells in GO, cycl in D I subcellular localization is heterogeneous and is maintained at very low levels by reduced transcriptional activity and GSK3(3-mediated phosphorylation and proteosomal degradation. Upon receipt of a mitogenic signal, transcription is upregulated and cycl in D I protein is stabilized by the inhibition of GSK3(3 in an AKT-dependent manner (162, 165). Stabilized, unphosphorylated cyclin D I then localizes to the nucleus during late G l where, in conjunction with C D K 4 and C D K 6 , it phosphorylates R B allowing for R-checkpoint passage and G l - S phase transition. Once the cel l is in S phase, cyc l in D is again susceptible to G S K 3 ( 3 - m e d i a t e d phosphorylation, nuclear export and ubiquitin-targeted proteosomal degradation in the cytoplasm. Nuclear export of cyclin D I from the nucleus is essential for completion of regulated cell division (76). Thus, cycl in D I is predominantly cytoplasmic in cells in S-121 phase and nuclear in cells in G l . In keeping with these observations, we have shown that rapidly proliferating monolayer E T cells express cycl in D I almost exclusively in the cytoplasm. In comparison, the more quiescent spheroid cells demonstrate a heterogeneous pattern of subcellular cyclin D I expression that is sensitive to mitogen stimulation. Whether there are any differences in the efficiency of proteosomal degradation of cyclin D I between E T cells in different culture milieus remains to be determined. However, there is at least one prior report of increased cycl in D I stability as a mechanism of increased cycl in D I expression in cultured sarcoma cells (166). This may prove to be another factor contributing to the constitutively high cyclin D I levels and high proliferative rates observed in monolayer E T cells. In summary, we hypothesize that the biology of E T spheroid cells closely resembles the biology of E T cells in vivo and therefore, spheroids provide a more relevant model for the study of tumour behaviour. We have shown that multi-cellular spheroids proliferate at a much slower rate than adherent monolayer E T cells and this difference is accompanied by differences in the post-transcriptional regulation of c y c l i n D I . Bo th the R A S \u00E2\u0080\u0094 R A F 1 \u00E2\u0080\u0094 M E K \u00E2\u0080\u0094 E R K and P I 3 K \u00E2\u0080\u0094 A K T signaling pathways are integrally linked to cyclin D I regulation and proliferation. Therefore, we next evaluated whether differences in either or both of these pathways could be responsible for the proliferative differences between E T monolayer and spheroid cells. The results of these studies wi l l be presented in the next chapter. 122 CHAPTER VI THE ROLES OF THE RAS\u00E2\u0080\u0094RAF\u00E2\u0080\u0094MEK\u00E2\u0080\u0094ERK AND PI3K\u00E2\u0080\u0094AKT PATHWAYS IN EWING TUMOUR CELL PROLIFERATION 6.1 INTRODUCTION The signal transduction cascades governing the proliferation of E T cells remain to be fully elucidated. In addition to our studies on gastrin releasing peptide (GRP) (167), other growth-factors recently implicated in E T cell growth include IGF1 (37, 102), b F G F (40), and stem cell factor and its receptor c-kit (41, 42). The exact mechanisms by which these growth factors affect proliferation and survival in E T are unclear and remain an area of intense study among E T cancer biologists. Possible downstream effectors include phospholipase C-P2 and -(33 (43), P I3K (39) and mediators of T G F P signaling (26). In normal cells, proliferative and survival pathways most often converge on the R A S \u00E2\u0080\u0094 R A F 1 \u00E2\u0080\u0094 M E K \u00E2\u0080\u0094 M A P K and/or the P I 3 K \u00E2\u0080\u0094 A K T pathways. Si lvany et al. have recently reported that activation of the R A S \u00E2\u0080\u0094 R A F 1 \u00E2\u0080\u0094 M E K \u00E2\u0080\u0094 E R K pathway is critical for E T cell transformation and proliferation (168) and survival of E T cells is impaired in the face of P I 3 K blockade (39). It is not yet known whether there is functional redundancy or cross-talk between or among any of these pathways, although this is possibly the case. In the preceding chapter we identified that variable cyc l in D I regulation and proliferation rates exist between E T cell spheroids and adherent monolayers. These differences are secondary to differences in post-transcriptional control of cyclin D I resulting in differences in subcellular localization and cell cycle passage. We therefore investigated 123 whether differences in R A S \u00E2\u0080\u0094 R A F 1 \u00E2\u0080\u0094 M E K \u00E2\u0080\u0094 E R K and/or P I 3 K \u00E2\u0080\u0094 A K T pathways could explain our findings. Our results indicate that significant differences do exist between the two culture models. Studies with inhibitors of M E K and PI3K suggest that the P I 3 K \u00E2\u0080\u0094 A K T pathway may be more critical than E R K activation to cyc l in D I expression and cell proliferation in E T cells, particularly in an anchorage-independent setting. Furthermore, patterns of cyclin D I expression and kinase activation in a cohort of primary E T samples support the use of spheroids as an in vitro representation of in vivo growth. We also show that transfection of a constitutively active A K T construct into adherent E T cells leads to the spontaneous formation of spheroids, providing further evidence that the A K T pathway may be a key regulator of anchorage-independent growth in these tumours. Finally, we evaluate the roles of known upstream activators of A K T in an effort to better understand which growth-receptor pathways mediate A K T phosphorylation in anchorage-independent E T cells. 6.2 RESULTS 6.2.1 The ERK1/2 MAPK and PI3K\u00E2\u0080\u0094AKT pathways are upregulated in ET cells in suspension. Constitutive activation of the M A P kinases E R K 1 and E R K 2 and the survival factor A K T have been implicated in the uncontrolled growth of numerous malignancies including E T (39, 168, 169). As shown in Figure 25a (left panel), starved T C 3 2 monolayer cells responded to serum stimulation by markedly increasing levels of E R K 1 and E R K 2 phosphorylation. However, when these cells were placed in suspension as described in Chapter V , E R K 1 and E R K 2 became activated in a serum-independent fashion (see right 124 panel of Figure 25A). This constitutive activation of E R K 1 / 2 was sustained, being observed in stable spheroids that had been in suspension for 48 hrs or more (Figure 25A). Screening for phosphorylation of A K T at serine residue 473 was used to assess activation of the P I 3 K \u00E2\u0080\u0094 A K T pathway in T C 3 2 cells. A K T phosphorylation was readily induced by serum in monolayer cells, but suspension of these cells resulted in A K T phosphorylation even in the absence of serum (Figure 25B) . However , A K T phosphorylation in T C 3 2 spheroids was only transiently serum-independent, in contrast to the sustained E R K 1 / 2 activation described above. B y 48 hrs, corresponding to the formation of stable spheroids, the basal level of A K T phosphorylation had decreased to that of monolayer cells and was again serum-inducible (Figure 25B). A n identical pattern of phosphorylation of the other major A K T phosphorylation site, threonine 308, was observed (data not shown). A s shown in Figure 25C (top panel), 5838 and A4573 spheroids showed similar increases in E R K activation relative to monolayer cultures. A s with TC32 , A K T activation was serum-independent when cells were placed in suspension (data not shown), but then became serum-responsive after stable spheroids had formed (middle panel, Figure 25C). Interestingly, basal levels of phosphorylated A K T were higher in A4573 spheroid cells compared with the other E T cell lines even in the absence of serum (Figure 25C). 6.2.2 M E K inhibition does not appreciably affect cyclin DI protein expression or cellular proliferation in ET cells. Activation of E R K 1 / 2 and A K T have both been linked to induction of cyclin D I (reviewed in (62)). In T C 3 2 monolayers, activation of E R K 1 / 2 did not correlate with changes in cyclin D I expression as the latter was already elevated whether or not serum 125 FIGURE 25. Differential activation of the ERK1 and ERK2 MAP kinases and of AKT in ET suspension cultures. Confluent E T cells were trypsinized and replated on regular or agar-coated dishes, allowed to grow for 48 hours, and then starved for 24 hrs in 0.25% serum prior to stimulation with (+) or without (-) 15% serum for 30 minutes. For those cells that were in suspension for only 1 hr, monolayer cells were starved for 24 hrs prior to being replated on agar-coated dishes for 1 hr with or without serum. Cells were then lysed for Western blot analysis using phospho-specific antibodies. (A) Western blot analysis of T C 3 2 whole cell lysates using an antibody that recognizes phosphorylated E R K 1 / 2 (P-ERK1/2) . Total E R K / 2 levels demonstrate equal loading. (B) Western analysis of TC32 whole cell lysates for expression of A K T phosphorylated at serine 473 ( P - A K T ) . Total A K T levels were used to demonstrate equal loading. (C) Western analysis of 5838 and A4573 E T monolayer ( M L ) and spheroid cells treated identically as T C 3 2 cells for the 48 hr time-point, using the same a - P - E R K l / 2 and a-P-A K T antibodies. Total Grb2 levels demonstrate equal loading. 126 A . Monolayer Serum: - + Suspension lhr 48hr + - + P-ERK 1/2 ,&mmtA, mmm: s$ i P - E R K 1/2 Cycl in D I Total A K T B. D 70 m 60 \u00E2\u0080\u00A22 50 \u00E2\u0080\u00A21 40 o Q. 30 ! 20 as # 0 58.9+7.3 Monolayer \u00E2\u0080\u00A2 No Inhibitor \u00E2\u0080\u00A2 +U0126 9.0+4.2 g.5\u00C2\u00B12.1 _ T - r Spheroid 130 stimulation of cells and similar findings were observed with another P I3K inhibitor, wortmannin (data not shown). LY294002 treatment of A4573 E T cells resulted in a similar reduction of cyclin D I protein levels, even though A K T phosphorylation was not completely blocked by this agent (Figure 27B). Moreover, incubation of either monolayer or spheroid TC32 cells with LY294002 resulted in a marked reduction in cell proliferation as measured by B r d U uptake (Figure 27C). Therefore, PI3K activation appears to be essential for cyclin D I protein expression and cellular proliferation in both monolayer and spheroid cultures of E T cells. Interestingly, we found that PI3K inhibition reproducibly led to serum-independent increases in E R K 1 / 2 phosphorylation in T C 3 2 monolayer cells. In fact, levels of phosphorylated E R K 1 / 2 after LY294002 treatment were similar to those observed in untreated T C 3 2 spheroids (compare P - E R K 1/2 panels in Figure 27A) . This suggests potential cross-talk between P I 3 K \u00E2\u0080\u0094 A K T and R A S \u00E2\u0080\u0094 E R K 1 / 2 pathways in E T cells, which we are currently evaluating in more detail. Despite its dramatic effects on cyclin D I expression, LY294002 did not appear to affect E R K 1 / 2 activation in T C 3 2 spheroids (see Figure 27A), further underscoring the possibility that the P I 3 K \u00E2\u0080\u0094 A K T pathway may be more critical than E R K 1 / 2 activation in regulating cycl in D I levels in E T cells grown in anchorage-independent conditions. 6.2.4 Down-regulation of cyclin DI expression in PI3K inhibitor treated E T cells is post-transcriptional but does not correlate with GSK(3 phosphorylation. Using Northern blot analysis, we examined whether inhibition of E R K 1 / 2 or PI3K activation resulted in altered cyclin D I transcript levels. In keeping with our earlier results, 131 FIGURE 27. PI3K inhibition blocks cyclin DI protein expression in ET cells and significantly reduces cell proliferation. (A) . T C 3 2 monolayers and stable spheroids were starved and then treated with a PI3K inhibitor (LY294002; + L Y ) or vehicle control. Prior to lysis, cells were serum-stimulated as described in Figure 26 then analyzed for expression of P - A K T , P - E R K 1 / 2 and cyclin D I . (B) . Protein expression and kinase activation in A4573 monolayer and spheroid cells treated with LY294002 as in (A). (C). T C 3 2 cells were grown for 20 hours in B r d U containing media with (+LY) or without LY294002 and then analyzed by immunohistochemistry for uptake of B r d U as a measure of proliferation. The percentage of positive cells is an average of 5 high power fields (total of 1000-2000 cells) \u00C2\u00B1 standard deviation. 132 Monolayer Spheroid Serum: control +30' +3h + L Y +30' +3h control + L Y - +30' +3h - +30' +3h \u00E2\u0080\u00A2 M S 8 8 8 MSB \u00E2\u0080\u00A2 \" \" I R l P - A K T Cyc l in D1/D2 P - E R K 1 / 2 Total A K T B . Monolayer control + L Y Serum: - +3h . - +3h Spheroid control +3h + L Y +3h \u00C2\u00A5 P - A K T Cyc l in D1/D2 C. 70 I 60 50 '140 o 5 30 a 20 f 10 0 58.9+7.3 19.3+2.0 \u00E2\u0080\u00A2 Control B U + L Y 9.0+4.2 1.6+1.2 Monolayer Spheroid 133 (see Chapter V ) , cyclin D I regulation in E T cells appeared to be largely post-transcriptional, even in the presence of M E K or P I3K inhibitors. A s shown in Figure 28, neither M E K (Figure 28A) nor P I 3 K inhibition (Figure 28B) significantly altered levels of cycl in D I m R N A . Given the marked effect of P I 3 K inhibition on cyc l in D I protein and on proliferation, the PI3K-mediated regulation of cyclin D I must mainly occur at the protein level. Interestingly, when we treated either monolayer or spheroid T C 3 2 cells with PI3K inhibitors at levels which blocked A K T phosphorylation and cyclin D I protein expression, there were still appreciable levels of phosphorylated GSK3(3 that could be further induced with serum (Figure 28C). This suggests that in E T cells there may be a P I 3 K \u00E2\u0080\u0094 A K T independent pathway contributing to G S K 3 P phosphorylation. 6.2.5 Primary Ewing tumors demonstrate patterns of cyclin DI expression and ERK1/2 and AKT activation that resemble those of ET spheroids. Given the differences in cycl in D I expression and activation of key signaling pathways that we observed between E T cell monolayers and spheroids, we sought to determine which culture condition more closely mimicked E T growth in vivo. Seven fresh-frozen primary E T biopsy samples were obtained and total protein lysates were analyzed as described for monolayer and spheroid cultures. A l l samples were obtained prior to initiation of therapy and were positive for EWS-FLll gene fusions. Six cases expressed the most common EWS exon 1IFLI1 6 gene fusion while a single case (case 6) expressed an exon 10/5 fusion (reviewed in (32)) (data not shown). A s shown in Figure 29, high levels of phosphorylated E R K 1 / 2 were present in all of the primary tumor samples. Furthermore, A K T phosphorylation could be detected in 6 of the 7 primary tumors. Cyc l in D I protein expression appeared to correlate with levels of A K T phosphorylation in these tumors, as low 134 F I G U R E 28. Downregulation of cyclin DI in inhibitor treated cells is post-transcriptional. ( A ) C y c l i n D I transcript levels in T C 3 2 cells treated with M E K inhibitor U0126 as described in Figure 26. Northern analysis shows equal expression of cyclin D I m R N A between serum-starved and serum-stimulated monolayer and spheroid cells. A n E W S c D N A probe was used to compare total R N A levels. (B) Northern analysis shows that monolayer and spheroid cells treated with the P I 3 K inhibitor LY294002 (as described in Figure 27) express equal levels of cycl in D I transcript. Comparison of (A) and (B) with cyclin D I transcript levels in control cells reveals no significant differences (see Figure 23A). (C) Western blot of T C 3 2 monolayer and spheroid cells treated with or without the P I3K inhibitor LY294002 (as described in Figure 27A). Despite dramatic effects on cyclin D I protein expression P-GSK3(3(Ser9) levels are similar in all experimental conditions. 135 c N i \u00E2\u0080\u0094 i O + V, \u00E2\u0080\u00A2p CN * u + >^ o c o PQ -a o \u00E2\u0080\u00A2a CO c CO U W CO Q c U CO m 3 CO CO 136 or undetectable levels of cyclin D I were observed in several tumors even in the presence of high E R K 1 / 2 phosphorylation (see lanes 1, 3, and 7 of Figure 29). Although the sample size is small, these results provide evidence that cyclin D I expression of in vivo E T cells may be more closely linked to A K T rather than E R K activation, as was found for E T cell lines. Moreover, the pattern of E R K 1 / 2 and A K T phosphorylation as well as cyclin D I expression of the primary tumors was more in keeping with E T spheroids than monolayers, indicating that primary E T growth signaling in vivo may be better represented in vitro by anchorage-independent spheroid culture systems. 6.2.6 Expression of constitutively active AKT by ET cells leads to spontaneous formation of anchorage independent spheroids. In order to determine i f the observed effects of PI3K inhibition were dependent on inhibi t ion of A K T phosphorylation, we transiently transfected T C 3 2 cells with a myrist i lated-AKT ( M y r - A K T ) c D N A construct. When expressed, this Myc-tagged A K T protein localizes to the cell membrane and is constitutively activated independent of PI3K (170). We observed that transfected monolayer T C 3 2 cells spontaneously grew as 3-dimensional foci and then anchorage-independent spheroids after 10 days in selection media (Figure 30A). Growth as spheroids continued despite culturing cells on standard plastic culture dishes and the proliferative rate was noted to be slower than cells transfected with control vector alone (visual observations). Baseline levels of cyclin D I protein were high in both cell populations and slightly more serum inducible in M y r - A K T cells than in control transfectants (Figure 30B). Subcellular localization of cycl in D I in these cells was not assessed and it remains to be determined if differences exist. T C 3 2 P r i m a r y E w i n g Tumors ML SP 1 2 3 4 5 6 7 F I G U R E 29. Primary Ewing tumors express activated E R K 1 / 2 & A K T and variable levels of cyclin D I . Total protein lysates were obtained from 7 primary E T samples as detailed in Materials and Methods. Western blots for expression of P - E R K 1 / 2 , P - A K T , G S K 3 P phosphorylated at serine 9 (P-GSK3P), and cyclin D I were performed as for E T cell lines, and show patterns similar to E T spheroid cultures. TC32 cell lysates from monolayer ( M L ) and spheroid (SP) cells stimulated for 3 hrs with serum are used for comparison. 138 S2 < l I < 0,1 c o U fa 6 u 139 Treatment of transfected cells with LY294002 was carried out exactly as described for non-transfected cells. As shown in Figure 31, while PI3K inhibition effectively blocked cyclin D I expression in control transfectants, TC32 cells expressing M y r - A K T continued to express cyclin D I , albeit at diminished levels. This suggests that PI3K-mediated regulation of cyclin D I is, at least in part, dependent on A K T activation. 6.2.7 Preliminary studies evaluating the role of integrin-linked kinase (ILK) in AKT phosphorylation in anchorage-independent ET cells. A s discussed in Chapter I, full activation of A K T requires phosphorylation of Ser 473 and this may be facilitated by the serine-threonine kinase I L K (87, 171). Furthermore, activation of I L K can induce anchorage independent growth (171). Having observed that anchorage-independent growth of E T cells was correlated with increased phosphorylation of A K T , we hypothesized that the cell-cell adhesions responsible for proliferation in spheroids may act through integrin-mediated activation of I L K . Therefore, we expressed a dominant-negative I L K c D N A construct in T C 3 2 E T cells and observed its effect on A K T (Ser 473) phosphorylation in both monolayer and spheroid cell cultures. This D N - I L K c D N A has been previously demonstrated to be effective in diminishing native I L K activity and A K T phosphorylation in other cell types (87). As shown in Figure 32, T C 3 2 cells expressed high levels of D N - I L K (lanes 1-3 and 7-9) following transient transfection using lipofectamine. However, despite readily detectable D N - I L K expression, no differences in A K T activation were observed between D N - I L K and control transfectants. Specifically, 140 CN o o \"^ M P ^ + ?^ s s 4\u00E2\u0080\u0094\u00C2\u00BB c o U C N O o C N 3 + W C o U P c \"o >> U CN o H < cn + o m + + O CO + O r o + C O + O r o + E a CO I I I bo o < S \"S 11 3 ^ in C J T 3 P o o B C N >> U O \" c o oo oo o r-a 1 o < It jo x) 1c c c \u00C2\u00AB w 1 en S o O T j c IE 00 ^ C3 o .3 T3 CO l-i C o O o 1*8 c .2 E \" 3 S .\u00C2\u00A7 \u00C2\u00A3 \u00E2\u0080\u00A24\u00E2\u0080\u0094< 5 cn CU w CO ,23 * 8 o > Lu C N ^ c n e\u00C2\u00A3 o g \u00C2\u00A3 S3 s fa hi c c n p i CO O o 0\ 3 3 _1 cn o oo p-, s s iZ 00 c n c 0 H U - J \"2 B c ed o 2 5 . 2 CO c3 c cd 00 ed c .S o 15 c- ^ w w 4\u00E2\u0080\u0094> M P \"E, 6 o o o s e T3 \u00E2\u0080\u00941 M to Q i) s 3 E ^oo E SH CU CO O \"cd cd \u00C2\u00AB >,p c .S \u00C2\u00B0 o .s ^ 141 TC32 cells grown in suspension culture as spheroids demonstrated the same levels of serum-independent A K T activity regardless of expression of D N - I L K (Figure 32, lanes 7-12). 6.3 DISCUSSION The R A S \u00E2\u0080\u0094 - E R K 1/2 and P I 3 K \u00E2\u0080\u0094 A K T pathways both become activated in numerous cell types in response to diverse extra-cellular stimuli, and aberrant activation of these signaling cascades is also associated with cellular transformation (88). A well-established link exists between R A S activation and induction of cyclin D I expression in different cell types (172-175). This is thought to occur predominantly via transcriptional regulation, with activation of the M A P kinase pathway leading to induction of the cycl in D I promoter (reviewed in (62)). Paradoxically, however, constitutive activation of E R K 1 / 2 did not correlate with increased cycl in D I expression in E T spheroids. Rather, cyclin D I protein expression in spheroids appeared to be dependent on cel l -ce l l adhesion and serum stimulation in a largely E R K 1 / 2 M A P kinase independent manner. Blockade of M E K 1 had no effect on monolayer cycl in D I levels and only minimal effects on spheroid cyclin D I expression. M E K 1 inhibition did not alter levels of cyclin D I m R N A in either culture setting and only slightly decreased proliferation rates. These results indicate not only that the R A S \u00E2\u0080\u0094 R A F 1 \u00E2\u0080\u0094 M E K \u00E2\u0080\u0094 E R K 1 / 2 cascade is regulated in a different manner in monolayer and spheroid E T cells, but also that early cell cycle progression of E T cells may not be critically dependent on E R K activation. This was corroborated by examination of a set of primary Ewing tumors, which demonstrated high levels of E R K 1 / 2 activation that also did not correlate with levels of cyclin D I expression. It is conceivable that adhesion of E T cells to 142 53 31 o CU l l OO CU \"o c o o 2 O c + o U -oo W + P P o <2 o > c o U C/3 + d o p oo, H \"3 \u00E2\u0080\u00A2J o H W) C3 *3 CO I i n > P i Z Q c l CO CN 143 plastic induces cyclin D I expression, via alternate pathways to those mediated by cell-cell adhesion in E T spheroids or primary tumors. One possibility is that cell shape changes induced by adherence of E T cells to plastic facilitate constitutive cyclin D I expression and cellular proliferation by activating proteins that link the actin cytoskeleton to the cell cycle. Potential candidates include the GTPases Rho or Rac (51, 176) and the adhesion-related cytoplasmic protein tyrosine kinase pl25 1 \" A K (177). The relative activation of these proteins in E T monolayers versus spheroids and their relevance to primary tumors remains to be determined. Our results are in contrast to those of Silvany et al, who found that E R K 1 / 2 activation was constitutive in cells transformed by E W S - F L I 1 , and that M E K 1 inhibition blocked soft agar colony formation of these cells (168). However, these studies were performed using murine NIH3T3 cells expressing E W S - F L I 1 , as opposed to the human E T cell lines used in our studies. Therefore the observed variation might be cell type specific, and the requirement for E R K 1 / 2 activation in NIH3T3 transformation may involve pathways that are inherently different from those in E T cells. P I3K inhibition of monolayer or spheroid E T cells resulted in virtually complete loss of cyclin D I protein expression and a marked reduction in proliferative rates. Consistent with these findings, A K T phosphorylation was readily detectable in 6 of 7 primary E T samples\" and appeared to correlate with cyclin D I expression. Our data are therefore consistent with a role for the P I 3 K \u00E2\u0080\u0094 A K T pathway in cyclin D I regulation and cell cycle progression of E T cells in vitro and in vivo. Muise-Helmericks et al. have reported that cyclin D I expression is controlled via a P I 3 K \u00E2\u0080\u0094 A K T dependent pathway in breast and colon carcinoma cell lines (72). They found that translation of cyclin D I m R N A was enhanced in these cells by a P I 3 K \u00E2\u0080\u0094 A K T dependent pathway. Cyc l i n D I down-regulation in both 144 monolayer and spheroid E T cells in response to P I3K inhibition was post-transcriptional as m R N A levels were not altered by LY294002. It is known that GSK3(3 inhibits cyclin D I expression by a mechanism involving reduced gene transcription and more directly by phosphorylation of cyclin D I threonine 286 leading to enhanced protein degradation (73, 161). Furthermore, these activities are known to be inhibited by A K T phosphorylation of GSK3(3 at serine residue 9 (162, 165). We found no differences in the phosphorylation status of G S K 3 P at this residue in E T spheroids versus monolayers, even under conditions in which cyclin D I levels were dramatically different. While this would suggest that the regulation of cycl in D I expression in E T is independent of GSK3(3, it is also possible that other phosphorylation sites on GSK3(3 regulate its kinase activity and substrate specificity in E T cells. Alternatively, differences in subcellular localization of G S K 3 P rather than phosphorylation status may be important in regulating cyclin D I protein expression in E T spheroid cells (see Figure 24). Interestingly, we observed high level phosphorylation of GSK3P(Ser 9) even when A K T activation was blocked by P I 3 K inhibitors. This indicates that there may be an additional level of complexity involving P I 3 K \u00E2\u0080\u0094 A K T independent phosphorylation of GSK3p(Ser9) in E T cells. P I3K blockade in monolayer cells was also associated with constitutive activation of E R K 1 / 2 , suggesting that inhibition of PI3K not only blocks cyclin D I expression but at the same time activates the R A S \u00E2\u0080\u0094 R A F 1 \u00E2\u0080\u0094 M E K \u00E2\u0080\u0094 E R K 1 / 2 pathway in these cells. It is becoming increasingly apparent that the P I 3 K \u00E2\u0080\u0094 A K T and the R A S \u00E2\u0080\u0094 R A F 1 \u00E2\u0080\u0094 M E K \u00E2\u0080\u0094 E R K pathways are integrally linked (reviewed in (58)). In fact, several recent studies have demonstrated negative regulation of R A F 1 through phosphorylation by A K T (59, 60). Also, Treinies et al. demonstrated that while an activated form of M E K can induce cyclin D I 145 expression and cell cycle entry in NIH3T3 cells, P I3K signals are required for this effect (61). It is well established that constitutive expression of R A S can induce apoptosis or cell cycle arrest through mechanisms involving p l 9 A R F , p21 C I P 1 , and p53 (reviewed in (62)). A n emerging model is that unless the P I 3 K - A K T pathway is activated in parallel, continuously high R A S activity wi l l lead to cell cycle arrest or apoptosis (58, 61, 63). It is therefore possible that the observed effect of PI3K on cyclin D I regulation in E T cells is secondary to suppression of R A F 1 to a level where E R K 1 / 2 activation induces cell cycle progression and not cell cycle arrest (or apoptosis). In this scenario, PI3K inhibition of E T cells would lead to higher levels of E R K 1 / 2 activation (as observed), which would in turn drive the cells into arrest rather than cell cycle progression. It is therefore apparent from our results that regulation of proliferation of E T cells in vitro involves cross-talk between the R A S \u00E2\u0080\u0094 R A F 1 \u00E2\u0080\u0094 M E K \u00E2\u0080\u0094 E R K and P I 3 K \u00E2\u0080\u0094 A K T pathways. The precise mechanisms of this cross-talk are still being investigated; however, our studies thus far suggest that PI3K plays a key role in E T cell proliferation, as has already been described in other non-malignant cell model systems (61). We have also sought to determine whether the role of PI3K in the regulation of cyclin D I and proliferation in E T may be dependent on as yet undetermined effectors other than A K T . This possibility was raised following several observations that suggested cyclin D I might be expressed in a PI3K-dependent but AKT-independent fashion in E T cells. First, we found that while serum starvation of T C 3 2 monolayers or stable spheroids resulted in markedly reduced A K T phosphorylation this was not accompanied by a significant reduction in cyclin D I expression (see Figure 27A). Second, although suspension of E T cells resulted in immediate activation of A K T , this was accompanied by a complete loss of cyclin D I 146 expression (see Figures 22 and 25). Third, treatment of A4573 spheroids with LY294002 resulted in blockade of cyc l in D I despite having only a moderate effect on A K T phosphorylation (see Figure 27B). In an effort to test our hypothesis of PI3K-dependent, AKT-independent cycl in D I regulation, we overexpressed a constitutively active A K T c D N A in an E T cell line. Interestingly, expression of M y r - A K T led to spontaneous, preferential growth of T C 3 2 E T cells as multi-cellular spheroids rather than adherent monolayers. This suggests that A K T activation is integrally involved in anchorage-independent growth in E T cells and further corroborates our earlier observations. Cycl in D I was eliminated entirely in PI3K-inhibi tor treated control transfectants but was only moderately reduced in M y r - A K T expressing cells. This could support the existence of a secondary PI3K-dependent pathway which acts in parallel with A K T to maintain cyclin D I levels. Alternatively, it is possible that reduction of cyclin D I protein levels in the PI3K-inhibitor treated M y r - A K T cells occurred simply because of the inhibitor's effects on native A K T activation rather than on an alternative pathway. Either way, we have still not ruled out the possibility of a parallel AKT-independent pathway. More studies are needed to rigorously test this possibility and to determine what targets of P I3K other than A K T might be involved in cyclin D I regulation in E T cells. Ce l l survival studies confirmed that E T cells could not survive indefinitely as single cells in suspension i f they were prevented from clumping. One well-established function of A K T is to act as a survival factor (178). This role of A K T in cell survival has already been reported in E T (39), likely functioning through the insulin-like growth factor 1 receptor (37). We found that activation of A K T in monolayer TC32 cells was serum-dependent, but that transfer of cells to suspension cultures led to immediate serum-independent A K T activation. 147 This was transient with A K T activation becoming serum-dependent once stable spheroids had formed. It is therefore possible that this transient activation of A K T allows cells to survive until they can clump and form cell-cell junctions that are critical for continued survival and cell cycle progression (163, 164). Wong et al. have shown that transformed cells which overexpress the anti-apoptotic protein Bcl -2 have a greater propensity for the formation of lung metastases, presumably because they can evade apoptosis long enough to allow successful implantation (179). Activation of the A K T survival pathway may be an important mechanism facilitating metastasis of E T cells in vivo by allowing potentially metastatic anchorage-independent cells to survive until they can adhere to the extra-cellular matrix or to other circulating tumor cells. The upstream molecules and pathways involved in the activation of A K T in anchorage-independent E T cells remains unclear. The i n t e g r i n \u00E2\u0080\u0094 I L K \u00E2\u0080\u0094 P I 3 K \u00E2\u0080\u0094 A K T pathway is an obvious candidate given the role of integrins in cell-cell adhesion and the association of I L K activation with stimulation of anchorage independent growth (83, 87, 171). However, we found that expression of a dominant-negative I L K construct did not lead to reduced levels of A K T activation in spheroid E T cells. These preliminary results suggest that the increase in A K T activation observed in anchorage-independent E T cells may not be secondary to I L K activity or, therefore, to integrin signaling. However, since we did not measure I L K activity in our experiments, we cannot be certain that D N - I L K transfection efficiently downregulated kinase activity. Thus, given the importance of cell-cell adhesion to anchorage-independent E T cell proliferation, further studies of I L K , integrins and integrin-mediated signaling are warranted before they are conclusively eliminated as upstream activators of A K T in E T spheroid cells. 148 In conclusion, our results indicate that proliferation of E T cells may be better studied in spheroid cultures where a number of physical and biochemical characteristics of the cells more closely resemble those of primary tumor cells. Compared to conventional monolayer cultures, key differences in the regulation of cycl in D I expression and early cell cycle progression exist in this anchorage-independent model and at least some of the differences likely occur as a direct consequence of cell-cell adhesion and the observed variations in key signal transduction pathways. These cell-cell interactions involve as yet undetermined adhesion molecules that may initiate signal transduction cascades such as the P I 3 K \u00E2\u0080\u0094 A K T pathway to ensure survival and proliferation of the tumor cells in their anchorage-independent milieu. It is also probable that E T cells grown in suspension have an increased dependency on autocrine growth pathways such as those mediated by IGF1 and G R P given their increased sensitivity to mitogen withdrawal. Such key differences in anchorage independent growth signaling pathways wi l l need to be considered as novel pathway-targeted biologic therapies for Ewing tumors are developed. 149 CHAPTER VII SUMMARY & FUTURE DIRECTIONS 7.1 General Summary The Ewing family of tumours presents a challenge to clinicians and cancer biologists alike as E T continues to be a difficult tumour to cure despite modern multi-modal approaches to therapy. Cytotoxic therapies have been maximized to the degree that therapy associated morbidity and mortality rates prohibit further increases in drug or radiation doses. Novel therapeutic approaches are necessary and this requires that we first develop a better understanding of E T cell biology in order to develop targeted therapies. While the genetics of E T have been largely characterized and the identification of EWS-ETS gene fusions has improved diagnostic capabilities, very little is yet known about the function of these chimeric oncogenes, which likely represent the primary genetic events in E T pathogenesis. Moreover, we have yet to understand the mechanisms behind the dysregulated growth of E T cells that afford these tumours their characteristic local invasiveness and high metastatic potential. In the past decade there has been an explosion of knowledge in the field of normal cell biology and in particular, in our understanding of the regulatory processes governing cell proliferation. For this thesis I have used this general knowledge of proliferative signaling pathways and cell cycle regulation in my studies of E T in an attempt to understand how they may be dysregulated in E T cells. Specifically, I have characterized the expression of the growth factor G R P by E T cells and have identified it to be an autocrine growth factor in these tumours. More generally, I have studied the proliferation of E T cells in two different 150 ce l l culture models and have attempted to define the contributions of the R A S \u00E2\u0080\u0094 R A F \u00E2\u0080\u0094 M E K \u00E2\u0080\u0094 E R K M A P K and P I 3 K \u00E2\u0080\u0094 A K T pathways to the control of E T cell proliferation in vitro. In so doing, I have identified that growth regulation is different between cells grown as traditional adherent monolayers and those grown as anchorage-independent multi-cellular spheroids. I have also determined, based on morphologic, ultra-structural and functional data, that the proliferation of E T cells in vivo may be better represented in vitro by anchorage-independent spheroids. Based on these results, I believe that further studies of growth regulatory pathways in anchorage-independent E T cells are warranted. In particular, I hypothesize that identification of adhesion molecules involved in cell-cell and c e l l - E C M adhesion and characterization of their respective signaling pathways w i l l afford key insights into the regulation of E T cell growth in vivo. In the following sections, potential avenues of future investigation wi l l be discussed with reference to the major observations presented in this thesis. 7.2 G R P Autocrine Growth Signaling in ET Our studies of E T confirmed expression of GRP and its receptor in 100% of cell lines and approximately 50% of primary tumour samples. Although a clinico-pathologic correlates study did not identify a relationship between GRP/GRP-R expression and prognosis, in vitro and in vivo experimental evidence suggested that the peptide functions as an autocrine growth factor in E T . These observations are interesting for several reasons. First, G R P is normally secreted by embryonic and adult cells of neuroendocrine lineage and it has been previously demonstrated to act as an autocrine growth factor in malignancies of neuroendocrine origin such as S C L C , breast cancer and prostate cancer (see (127) for 151 review). This provides further evidence for the putative neural origins of E T . Second, investigators studying S C L C have recently published that detection of GRP by R T - P C R in peripheral blood, sputum and cerebrospinal fluid can be used as a sensitive marker of microscopic residual or metastatic disease (180 ; 181). Thus, although G R P may not be a prognostic marker in E T , there is potential for its use in tumour diagnosis and detection, particularly when gross disease is not apparent. Finally, recognition of G R P - R detection in E T may provide therapeutic opportunities. Direct blockade of the G R P - R has resulted in reduced growth of several different tumour types, including E T (120, 133-135, 167). It is conceivable that patients with bulk disease may not respond dramatically to G R P - R blockade given the relatively small contribution of G R P signaling to overall tumour growth. However, the use of G R P - R antagonists in E T patients with microscopic or minimal residual disease may delay tumour recurrence and should be evaluated in clinical trials similar to those being conducted in prostate cancer (182). Moreover, cytotoxic bombesin-analogues have been developed that specifically target cells expressing G R P - R (136, 183). Such agents may prove to be useful in the development of targeted therapies that are specifically cytotoxic to E T cells. O f course, the development of optimal targeted therapies that exploit GRP-mediated growth wi l l require a more complete understanding of GRP-signaling pathways in E T cells. Our evidence supports the role of G R P as a growth factor but does not directly implicate the R A S \u00E2\u0080\u0094 R A F \u00E2\u0080\u0094 M E K \u00E2\u0080\u0094 E R K pathway, which has been most often linked to GPCR-induced mitogenesis. Rather, our results and those of others suggest that the adhesion-related proteins Pyk2 and/or p l 2 5 F A K may be key regulators in GRP-induced proliferation (50, 184). The potential importance of adhesion-mediated signaling pathways in the mitogenic and 152 morpnogenic functions of GRP has already been proposed for other cancer cell systems (148, 185). Additionally, studies of the related neuromedin-B receptor have demonstrated that integrity of the actin cytoskeleton is necessary for neuromedin-B stimulated phosphorylation of p l 2 5 F A K and that the Ras-related small GTP-binding protein Rho is at least partially involved (186). Preliminary studies by our collaborator Dr. C. Poremba have shown that GRP-positive primary ET tumours express higher levels of Rho than do GRP-negative tumours and that elevated Rho is associated with decreased levels of expression of the cell cycle inhibitor p21 C I P 1 (unpublished observations). Thus, we hypothesize that GRP signaling in ET likely facilitates cell cycle passage via Rho\u00E2\u0080\u0094pl25 F A K\u00E2\u0080\u0094p21 C I P 1 dependent mechanisms and that future studies should focus on evaluation of this pathway. Furthermore, the effect of GRP on this pathway may be critically linked to the actin cytoskeleton and, therefore, to cell-cell and/or cel l -ECM adhesion. Given the significant changes in morphology and cell-cell adhesion that we have demonstrated in ET cells grown as adherent monolayers, I believe GRP signaling in ET cells would be better studied in multi-cellular spheroids where cell-cell and cel l-ECM adhesion may be more representative of primary in vivo tumours. Other potential mechanisms of GRP-mediated growth in ET are also worthy of consideration. First, the hypothesis tested in this thesis presumes that GRP exacts its effects by acting directly on the ET cells themselves in a pro-proliferative fashion. However, it is possible that GRP provides a survival signal to ET causing a reduction in the rate of apoptosis rather than a true increase in proliferation. While there is no evidence to support the role of GRP as an anti-apoptotic factor in other normal or malignant tissues, this potential mechanism of action should be definitively studied in ET before it is ruled out completely. Moreover, it is possible that the pro-proliferative effects of GRP are mediated by interactions 153 with the E C M or with other surrounding cells. These effects would not be apparent in the current adherent in vitro model and future studies could evaluate the role of G R P on the expression of growth inhibitory molecules, such as TGF(3 and the interferons, by neighbouring E T cells, lymphocytes or other non-tumour cells. It is also not clear from our studies whether G R P is constitutively expressed by E T cells or whether its expression is in some way regulated, e.g. in a cell cycle or differentiation-state-dependent fashion. If G R P expression and secretion are regulated in E T cells in vivo, it is plausible that random, non-regulated blockade and stimulation of the GRP-receptor in these in vitro studies may not have an appreciable effect on cellular biochemistry. Final ly , the potential relationship between G R P and E W S - E T S proteins requires further study. The ability of EWS-FLI1 to transform fibroblasts depends on the presence of the IGF-1 receptor and in its absence, apoptosis is induced (37). It remains to be determined whether G R P also in some way contributes to the permissive cellular envrionment facilitating E W S - E T S induced malignant transformation. Studies evaluating the transformation efficiency of EWS-ETS genes in cells expressing and not expressing G R P and its receptor would help to clarify this relationship. 7.3 The Role of Adhesion Molecules in E T Proliferative Signaling The results of our studies on GRP-mediated signaling and the conclusions that cell shape, cellular adhesion and the actin cytoskeleton might be crit ical regulators of this pathway led us to hypothesize that the model we were using may not have been representative of the in vivo situation. Therefore, we developed an anchorage independent multi-cellular spheroid model in which to study E T cell growth. Previous studies of other cancer cell spheroids have demonstrated that they express cell surface adhesion molecules 154 and E C M components of an intermediate level between monolayer cultures and in vivo tumours (154, 164, 187, 188). Our studies of E T cell morphology, ultra-structure and proliferation confirmed that spheroid cells were more similar to primary tumour cells than were monolayer cells. Adhesion of tumour cells to one another or to components of the E C M results in the activation of adhesion molecules and their down-stream signaling cascades. The identity of these molecules and their respective signaling pathways remains to be determined in E T . However, we believe that there must be differences between multi-cellular spheroids and adherent monolayers cells and that these differences most likely result in differential activation of the P I 3 K \u00E2\u0080\u0094 A K T pathway. Ongoing studies in our laboratory are currently testing this hypothesis. To date, there has been little investigation into adhesion molecules and their signaling pathways in E T . There is no published evidence to support expression of either e-cadherin or n-cadherin in these tumours and studies of N - C A M expression have been contradictory (189, 190). Reports of integrin expression by E T cells have also been conflicting (190, 191); however, the expression of high levels of I L K by primary E T tumours (192) suggests that integrin signaling may be important in this tumour family. Moreover, the direct link between integrins, I L K and the P I 3 K \u00E2\u0080\u0094 A K T pathway (87) provides further evidence to support the study of integrins in E T adhesion-related signaling. Previous studies have demonstrated that E T cells synthesize both laminin and fibronectin, components of the E C M (193). Preliminary analysis of gene expression profiles of E T monolayer and spheroid cells using microarray analysis has revealed increased expression of the fibronectin gene in spheroid cells (unpublished observations from our laboratory). Ongoing studies have confirmed increased expression of fibronectin in 155 spheroids by both Western blot and fluorescent immunocytochemistry. One aspect of integrin signaling that is worthy of further investigation, therefore, is the association between fibronectin\u00E2\u0080\u0094integrin binding and protection from apoptosis or anoikis (194, 195). Anoikis is the apoptotic cell death that is induced upon loss of adhesion to an extracellular matrix (196). A s transformed cells, E T cells have established mechanisms by which to avoid anoikis. It is possible that integrin molecules on the surface of E T cells bind to autonomously produced fibronectin, thus protecting the cells from anoikis. This effect would be critical to the survival of E T cells in suspension and would therefore be expected to be more apparent in spheroids. Integrin-mediated protection from anoikis can be controlled mechanistically in different ways, depending on the cel l and integrin type, including upregulation of Bc l -2 , suppression of p53 induced apoptosis and activation of She (see (195) for review). Further studies of integrin expression are warranted in the study of E T cell biology in general and in the study of anchorage-independent E T growth signaling specifically. Understanding the mechanisms of survival of anchorage-independent E T cells could greatly impact on the development of novel targeted therapeutic agents for the treatment of metastatic disease. 7.4 EWS -FLI1 , C y c l i n D I Regula t ion and Subce l lu l a r Compartmentalization We have found that subcellular localization of cycl in D I and P-GSK3(3 varies between monolayer and spheroid E T cells, consistent with the observed variations in cell cycle status. These differences only became apparent when we specifically looked at cytoplasmic and nucleoplasm^ cellular fractions by Western blot analysis. The functional 156 status of cyclins and other cell cycle regulators is known to be influenced by their subcellular location (197), and this must be considered when studies of cell cycle regulation are interpreted. From our results we can conclude that evaluation of the function of cell cycle regulatory proteins in E T requires an assessment not only of their phosphorylation status but also of their localization within the cell . Moreover, proteins that associate themselves with insoluble membrane fractions or that localize to the nucleus require cell lysis with an appropriately stringent lysis buffer if they are to be detected by standard immunoblotting techniques. The subcellular localization of E W S - F L I 1 and its role in cyclin D I regulation in E T cells also requires further investigation. Treatment of E T cells with EWS-FLI1 antisense results in G l arrest and reduced proliferation (120, 133-135) and, it has been recently reported that cyclin D I may in fact be a downstream target of E W S - F L I 1 (198). How E W S -FLI1 affects cyclin D I levels is not yet known but may involve either transcriptional and/or post-transcriptional mechanisms. In the past decade, much effort has been placed into the identification of transcriptional targets of E W S - F L I 1 . This approach has not met with great success for, although putative target genes have been identified, the mechanism(s) of action of E W S - E T S fusions remain unknown. Silvany et al. have observed that E R K 1 / 2 activation is dependent on the ability of E W S - F L I 1 to translocate to the nucleus and to bind D N A (168). Shuttling of the E W S - E T S oncoproteins between cytoplasm and nucleus may, therefore, be a critical parameter in the regulation of E T cell proliferation. To date, in vitro studies of E W S - F L I 1 localization and target gene identification have been done exclusively in cell lines grown as adherent monolayers. In this thesis, I have presented data demonstrating that the subcellular localization of proteins varies in E T cells between 157 adherent and anchorage independent culture conditions. I have also shown that the regulation of cyclin D I is similarly variable. Therefore, it is possible that the subcellular localization of E W S - E T S fusion proteins varies between models and this variability may contribute to the differential regulation of cyclin D I expression. It is known that multiple domains of the EWS-FLI1 fusion gene are required for transformation and that protein-protein interactions involving the N T D - E W S segment are key to the function of the oncoprotein (reviewed in (11)). E W S and the related protein T L S (also involved in oncogenic gene fusions (199, 200)), are both R N A - b i n d i n g proteins that appear to be involved in the splicing and stabilization of m R N A (201). Disruption of these genes by chromosomal translocation may alter m R N A processing and, therefore, affect protein expression (202). Further investigation into the potential role of E W S - E T S fusions in protein modification needs to be pursued. In particular, whether E W S - E T S proteins contribute to the post-transcriptional regulation of cyclin D I expression should be among the first questions addressed. This w i l l require studying E T cells in a system in which the fusion proteins readily shuttle to the cytoplasm. Based on the data presented in this thesis, I believe that multi-cellular spheroids wi l l provide such an in vitro model. It w i l l also be interesting to assess whether Pyk2 affects E W S - E T S subcellular localization. It is known that nuclear localization of wild-type E W S is influenced in neural cells by the phosphorylation status of Pyk2 (147). We have shown that Pyk2 is expressed by E T cells and that its pattern of activation may be affected by G R P - R signaling. Further studies measuring Pyk2 kinase activity in E T cells are warranted as it is possible that activation of Pyk2 may affect the subcellular localization of E W S - E T S oncoproteins and facilitate oncogenesis and maintenance of the malignant phenotype. Once again, it w i l l be 158 important to study this relationship in multicellular spheroids where Pyk2 activation and EWS-ETS location may be more representative of in vivo tumours. Finally, it should be determined whether regulation of cyclin DI in ET is integrally linked to the IGFI-R autocrine growth pathway. PI3K is activated downstream of the IGF1-R and an intact IGFI-R is necessary for EWS-FLI1 induced cellular transformation (37, 203). This thesis has identified a critical role for the P I 3 K \u00E2\u0080\u0094 A K T pathway in the post-transcriptional regulation of cyclin DI in ET, particularly in anchorage-independent cells. Thus, the relationship between EWS-ETS fusions, the IGFI-R and their potential interaction in regulating cyclin DI protein expression and ET cell proliferation are areas of study being further pursued in our laboratory. 7.5 Final Comments For this thesis I have studied growth signaling pathways in ET cells in vitro and have gained significant insights into potential mechanisms of proliferative control in these cells. I have determined that the neuropeptide GRP functions as an autocrine growth factor in these tumours, that proliferation differs markedly between anchorage-independent and adherent monolayer cultures, and that the P I 3 K \u00E2\u0080\u0094 A K T pathway is a key regulator of cyclin DI expression and cell cycle progression in ET cells. 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"@en . "Thesis/Dissertation"@en . "2002-05"@en . "10.14288/1.0090557"@en . "eng"@en . "Pathology"@en . "Vancouver : University of British Columbia Library"@en . "University of British Columbia"@en . "For non-commercial purposes only, such as research, private study and education. Additional conditions apply, see Terms of Use https://open.library.ubc.ca/terms_of_use."@en . "Graduate"@en . "Studies of signaling pathways that regulate Ewing tumour cell growth in vitro"@en . "Text"@en . "http://hdl.handle.net/2429/13114"@en .