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The role of the insulin-like growth factor signaling axis in ETV6-NTRK3- mediated anchorage-independent… Martin, Matthew Joseph 2006

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T H E R O L E O F T H E INSULIN-LIKE G R O W T H F A C T O R S IGNALING AXIS IN ETV6-NTRK3-M ED I A T E D A N C H O R A G E - I N D E P E N D E N T G R O W T H A N D T R A N S F O R M A T I O N by MATTHEW JOSEPH MARTIN B.Sc. (Hon.), University of Guelph, 1999 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES (Experimental Medicine) THE UNIVERSITY OF BRITISH COLUMBIA October 2006 © Matthew Joseph Martin, 2006 ABSTRACT The insulin-like growth factor signaling axis is an important regulator of normal cell growth and proliferation, and is frequently dysregulated in cancer. Most dominantly acting oncoproteins tested to date require the type-1 insulin-like growth factor receptor (IGF-IR) for cellular transformation. ETV6-NTRK3 (EN) - the product of a t(12;15)(p13;q25) translocation that occurs in human pediatric spindle cell sarcomas and secretory breast carcinoma - is one such protein, as it fails to transform IGF-IR-null fibroblasts. EN binds and tyrosine phosphorylates the insulin-receptor substrate (IRS)-1, a major substrate of IGF-IR, which links EN to activation of the PI3K/Akt survival pathway and the Ras/Erk proliferative cascade. Here we show that EN specifically interacts with the phosphotyrosine-binding (PTB) domain of IRS-1, and in the absence of IRS-1 can bind its closely related homolog IRS-2. Disruption of these EN«IRS interactions through overexpression of the IRS-1 PTB domain, or depleting the cell of IRS-1 and IRS-2 together, inhibit EN-induced anchorage-independent growth. Further, we find that IGF-IR, through its ability to bind IRS molecules, serves to localize EN to the plasma membrane, which leads to acivation of the PI3K/Akt pathway. Non-transformed IGF-IR-null fibroblasts fail to activate this pathway in response to EN expression when plated under anchorage-indpendent conditions, and undergo detachment-induced cell death. Chemical inhibition of PI3K, or its downstream effector mTOR, significantly impairs EN-mediated transformation. Finally, I demonstrate that EN expression induces the ligand-independent tyrosine phosphorylation of IGF-IR. Both IGF-IR IRS-binding and kinase activity are required for this phenomenon, and IGF-IR mutants lacking either function do not display EN-mediated PI3K/Akt activation or subsequent oncogenesis. These observations point to EN as a regulator of a novel multicomponent membrane-localized signaling complex which potently stimulates the PI3K/Akt survival cascade, and they suggest that blocking the formation or activity of this complex would be a promising way to target EN-expressing tumors in vivo. 11 TABLE OF CONTENTS ABSTRACT ii TABLE OF CONTENTS iii LIST OF FIGURES viii LIST OF ABBREVIATIONS xi ACKNOWLDEGEMENTS ..xiv CHAPTER I: INTRODUCTION 1 1.1 SYNOPSIS AND RATIONALE FOR THE THESIS ...1 1.2 INSULIN-LIKE GROWTH FACTOR SIGNALING IN NORMAL AND ONCOGENIC GROWTH 3 1.2.1 Insulin-Like Growth Factors 3 1.2.2 IGF Binding Proteins 5 1.2.3 Insulin-Like Growth Factor Receptors... 7 1.2.4 IRS Proteins ...10 1.3 REGULATION OF CELL GROWTH, PROLIFERATION, AND SURVIVAL 14 1.3.1 The Extracellular Regulated Kinase Pathway 14 1.3.2 PI3K/Akt Signaling Pathway 17 1.3.2.1 PI3K Structure and Regulation 18 1.3.2.2 Regulation of Akt Activation 19 1.3.2.3 Akt Downstream Targets 22 1.3.2.4 Negative Regulation of PI3K/Akt Signaling 26 1.3.3 Regulation of the Cell Cycle .26 1.3.3.1 Cyclins and Cyclin-Dependent Kinases 27 1.3.3.2 Cyclin Dependent Kinase Inhibitors 29 1.3.4 Regulation of Cell Death 30 1.3.4.1 Apoptotic Pathways 31 1.3.4.2 Role of PI3K/Akt Signaling in Prevention of Apoptosis 32 1.3.4.3 Anoikis 33 1.3.4.4 Other Forms of Cell Death 34 1.4 THREE-DIMENSIONAL MODELS OF TUMOR CELL GROWTH 35 1.4.1 Multicellular spheroids 36 1.4.2 Other 3D Culture Models 37 iii 1.5 THE ETV6-NTRK3 CHIMERIC TYROSINE KINASE 38 1.5.1 ETV6 40 1.5.2 NTRK3 41 1.5.3 ETV6-NTRK3 Fusion Expression in Human Tumors .43 1.5.3.1 Congenital Fibrosarcoma 43 1.5.3.2 Congenital Mesoblastic Nephroma ..44 1.5.3.3 Acute Myelogenous Leukemia .45 1.5.3.4 Secretory Breast Carcinoma 45 1.5.4 ETV6-NTRK3 Signal Transduction 46 1.5.4.1 Activation of Growth Factor-Regulated Pathways 46 1.5.4.2 Polymer Formation 46 1.5.4.3 TGF-p Signaling 47 1.5.4.4 Role of the IGF-IR Pathway 48 1.6 AIMS AND HYPOTHESES 50 CHAPTER II: MATERIALS AND METHODS 51 2.1 CELL LINES 51 2.2 CLONING OF DNA CONSTRUCTS 52 2.2.1 MSCV Plasmids 52 2.2.2 ETV6-NTRK3 Constructs 52 2.2.3 IRS-1 Constructs 53 2.2.4 IGF-IR Constructs 54 2.2.5 Akt-myr 54 2.3 CELL TRANSFECTION 55 2.3.1 Transient Transfections 55 2.3.2 Transduction of Genes Using MSCV Retroviral Vectors 55 2.3.3 siRNA-Mediated Gene Knockdown 55 2.4 ASSESSMENT OF ANCHORAGE-INDEPENDENT GROWTH 56 2.4.1 Soft Agar Colony Formation Assay 56 2.4.2 Growth of Fibroblasts as Multicellular Spheroids 57 2.4.3 In vivo Tumor Growth in Nude Mice. .57 2.4.4 Kinase Inhibitor Studies 58 2.5 PROTEIN ANALYSIS ...58 2.5.1 Western Blotting 58 iv 2.5.2 Immunoprecipitation 59 2.5.3 Subcellular Fractionation 59 2.5.4 Antibodies 60 2.5.5 IGF ELISAs 61 2.6 IMMUNOFLUORESCENCE MICROSCOPY 61 2.7 CELL PROLIFERATION ASSAYS 62 2.7.1 BrdU Proliferation Assay 62 2.7.2 FACS Analysis 63 2.8 MEASUREMENT OF CELL DEATH 63 2.8.1 Trypan Blue Exclusion Assay .63 2.8.2 Caspase-3 Activity Assay 63 CHAPTER III: ETV6-NTRK3 BINDS THE PTB DOMAIN OF IRS-1/IRS-2: AN ESSENTIAL STEP IN ETV6-NTRK3-MEDIATED TRANSFORMATION .65 3.1 INTRODUCTION.... ..65 3^ 2 RESULTS 68 3.2.1 The PTB Domain of IRS-1 Mediates its Association with EN 68 3.2.2 Co-Expression of IRS-1 PTB Domain Fragment Inhibits EN-Mediated Transformation 71 3.2.3 IRS-1 Overexpression Potentiates EN-Mediated Transformation 73 3.2.4 IRS-1 Overexpression Fails to Restore Transformation to R-EN Fibroblasts 75 3.2.5 EN Shows Transforming Ability in IRS-1-Null Mouse Embryo Fibroblasts .....75 3.2.6 EN Binds and Tyrosine Phosphorylates IRS-2 :...77 3.2.7 Concurrent siRNA-Mediated Knockdown of IRS-1/IRS-2 Expression Inhibits Transformation of EN-Expressing Fibroblasts 81 3.2.8 EN Transformation of IRS-11-2 Deficient 32D Hematopoietic Cells Requires Re-Expression of IRS-1 88 3.3 DISCUSSION 89 CHAPTER IV: THE INSULIN-LIKE GROWTH FACTOR I RECEPTOR IS REQUIRED FOR AKT ACTIVATION AND SUPPRESSION OF ANOIKIS IN CELLS TRANSFORMED BY THE ETV6-NTRK3 CHIMERIC TYROSINE KINASE 96 4.1 INTRODUCTION 96 v 4.2 RESULTS 99 4.2.1 Anchorage-Independent Growth of ETV6-NTRK3-Expressing Cells Requires IGF-IR 99 4.2.2 ETV6-NTRK3 Expression in Cells Lacking IGF-IR are Defective in Akt Activation 100 4.2.3 Expression of Activated Akt Restores Anchorage-Independent Growth and Transformation Activity to ETV6-NTRK3-Expressing IGF-IR-Null Fibroblasts. 105 4.2.4 ETV6-NTRK3 and IRS-1 Membrane Localization are Increased by IGF-IR Expression 108 4.2.5 Membrane-Targeted EN Confers Anchorage-Independent growth and Transforms IGF-IR-Null Fibroblasts 112 4.2.6 IGF-IR Suppresses Anoikis in Anchorage-Independent EN-Transformed Fibroblasts 118 4.2.7 PI3K Inhibitors Block EN-lnduced Anchorage-Independent Growth 125 4.3 DISCUSSION 128 CHAPTER V: ROLE OF THE mTOR SIGNALING PATHWAY IN ETV6-NTRK3 TRANSFORMATION 134 5.1 INTRODUCTION 134 5.2 RESULTS 135 5.2.1 mTOR Signaling is Elevated in ETV6-NTRK3-Expressing Fibroblasts 135 5.2.2 Effect of Rapamycin Treatment on EN-lnduced Signaling 136 5.2.3 Differential Effects of Rapamycin on EN-lnduced Anchorage-Independent Growth .....141 5.2.4 Co-Inhibition of PI3K/Akt and mTOR Synergistically Impair Growth of ETV6-NTRK3-Expressing Multicellular Spheroids 143 5.3 DISCUSSION 147 CHAPTER VI: INTERACTION OF THE EN/IRS-1 TRANSFORMING COMPLEX WITH IGF-IR LEADS TO LIGAND-INDEPENDENT IGF-IR PHOSPHORYLATION, AKT ACTIVATION, AND TRANSFORMATION 151 6.1 INTRODUCTION 151 6.2 RESULTS 153 6.2.1 EN Expression Leads to Constitutive IGF-IR Tyrosine Phosphorylation 153 6.2.2 EN Expression does not Lead to Elevated Production of IGF Ligands 156 6.2.3 IGF-IR Mutants Lacking IRS-1 Binding Ability or Kinase Activity Fail to Support ETV6-NTRK3 Transformation 158 6.2.4 Membrane-Targeted IGF-IR Intracellular Domain is Tyrosine Phosphorylated when Co-Expressed with EN 160 6.2.5 Expression of Membrane-Targeted IGF-IR Intracellular Domain Supports Akt Activation and Transformation of EN-Expressing Fibroblasts ..162 6.3 DISCUSSION 165 CHAPTER VII: SUMMARY AND FUTURE DIRECTIONS 169 7.1 GENERAL SUMMARY 169 7.2 ROLE OF IRS MOLECULES IN TRANSFORMATION INDUCED BY ONCOGENIC KINASES 170 7.3 ETV6-NTRK3 MEMBRANE LOCALIZATION 175 7.4 IGF-IR LIGAND-INDEPENDENT ACTIVATION BY EN ...180 REFERENCES 183 APPENDIX: Animal Care Certificate 224 v i i LIST OF FIGURES FIGURE # PAGE Figure 1.1 The type-1 Insulin-Like Growth Factor Receptor (IGF-IR)... 8 Figure 1.2 Insulin-Receptor Substrate (IRS)-1 12 Figure 1.3 The ETV6-NTRK3 (EN) chimeric oncoprotein 39 Figure 3.1 ETV6-NTRK3 fusion binds to the phophotyrosine domain of IRS-1 69-70 Figure 3.2 Coexpression of IRS-1C (PTB/PH domains) disrupts ENIRS-1 complexes and inhibits transformation 72 Figure 3.3 Overexpression of IRS-1 potentiates EN-mediated transformation 74 Figure 3.4 Overexpression of IRS-1 does not rescue transformation of R-EN fibroblasts 76 Figure 3.5 EN transformation in IRS-1-null mouse embryo fibroblasts 78 Figure 3.6 EN binds and tyrosine phosphorylates IRS-2 80 Figure 3.7 siRNA-mediated knockdown of IRS-1 and IRS-2 protein levels 82 Figure 3.8 IRS-1/2 knockdown inhibits anchorage-independent growth of R+ (HA)EN fibroblasts 84-5 Figure 3.9 IRS-1/IRS-2 co-knockdown downregulates Akt activation in R+ (HA)EN fibroblasts 87 •\ Figure 3.10 Full transformation of IRS-1/2 deficient hematopoietic cells by EN requires re-expression of IRS-1 90-1 Figure 4.1 ETV6-NTRK3-induced growth of multicellular spheroids requires IGF-IR expression 101 Figure 4.2 Akt activation in EN-expressing cells requires IGF-IR 103 Figure 4.3 Restoration of Akt activation in R-EN cells rescues anchorage-independent growth 106-7 Figure 4.4 Membrane localization of ETV6-NTRK3 in the presence of IGF-IR or following N-terminal myristoylation of ETV6-NTRK3 109 Figure 4.5 IGF-IR expression or myristoylation of EN leads to membrane localization of EN and tyrosine-phosphorylated IRS-1 111 Figure 4.6 ENmyr restores anchorage-independent growth to IGF-IR-null cells 114 Figure 4.7 ENmyr restores Akt activation to IGF-IR-null cells 116 Figure 4.8 Restoration of Akt signaling in R-EN cells rescues in vivo Transformatio 117 Figure 4.9 R-EN fibroblasts proliferate in anchorage-independent cultures 120-1 Figure 4.10 R-EN fibroblasts undergo apoptotic death in anchorage-independent cultures 123-4 Figure 4.11 Inhibition of PI3K-Akt signaling blocks anchorage-independent growth induced by EN 126-7 Figure 5.1 ETV6-NTRK3 expression leads to constitutive activation of the mTOR signaling pathway 7 137 Figure 5.2 Rapamycin inhibits mTOR but fails to upregulate Akt in R+EN Cells 139-40 Figure 5.3 Rapamycin inhibits R+EN soft agar colony formation but not spheroid growth 142 Figure 5.4 Inhibition of Akt signaling sensitizes EN-expressing spheroids to rapamycin 144-6 Figure 6.1 IGF-IR(Y950F) and IGF-IR(K1003A) mutants fail to restore Akt activation and anchorage-independent growth to R-EN Fibroblast 155 Figure 6.2 Wildtype IGF-IR is constitutively tyrosine phosphorylated upon EN expression 157 Figure 6.3 Constitutive tyrosine phosphorylation of IGF-IR is not due to EN-induced upregulation of IGF levels 159 Figure 6.4 Membrane-targeted IGF-IR intracellular domain supports EN-induced Akt activation and transformation 161 Figure 6.5 IGF-IR(ICmyr) rescues Akt activation and transformation in R-EN fibroblasts 164 Figure 7.1 Cooperation between the IGF signaling axis and the ETV6-NTRK3 chimeric tyrosine kinase in activating transformation-associated pathways 171 -2 Figure 7.2 ENmyr but not EN localizes to detergent insoluble membrane fractions 177 Figure 7.3 Differential transforming potential of activated Ha-Ras and K-Ras in IGF-IR-null fibroblasts 179 x LIST OF ABBREVIATIONS 4E-BP1 4E binding protein-1 Akt AKR mouse thymoma AML acute myeloid leukemia AP-1 activator protein-1 ATFS adult type fibrosarcoma ATM gene mutated in ataxia telangiectasia ATP adenosine triphosphate BAD bcl-2 antagonist of cell death bp base pair Bcl-2 b-cell CLL/lymphoma 2 BCR breakpoint cluster region BSA bovine serum albumin Cbl Casitas B-lineage lymphoma CDK cyclin-dependent serine/threonine kinase cDNA complimentary deoxyribonucleic acid CHAPS 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate CFS congenital fibrosarcoma CIP calf intestinal phosphatase CMN congenital mesoblastic nephroma CREB cyclic AMP response element binding protein CS calf serum DAP I diamidino-2-phenylindole dihydrochloride hydrate DMEM Dulbecco's Modified Eagle Medium DMSO dimethylsulfoxide DNA deoxyribonucleic acid DNA-PK DNA-dependent protein kinase DOK1/2 docking protein-112 DTT dithiothreitol ECM extracellular matrix EDTA ethylene-diamine-tetraacetic acid EGF epidermal growth factor EGTA ethylene glycol-bis(2-aminoethylether)-N,N,N',N'-tetraacetic acid elF4E eukaryotic initiation factor 4E EN ETV6-NTRK3 ErbB2 v-erb-b2 erythroblastic leukemia viral oncogene Erk extracellular signal regulated kinase ETS E-26 transforming specific ETV6 ets variant gene 6 EWS Ewing's sarcoma FACS fluorescence activated cell sorting FAK focal adhesion kinase FBS fetal bovine serum FKBP FK506 binding protein x i FKHR forkhead in rhabdomyosarcoma FN friend leukemia virus integration GAB1 Grb2-associated binder-1 GDP guanosine diphosphate GEF guanine nucleotide exchange factor GH growth hormone Grb2 growth factor receptor-bound protein 2 GSK3 glycogen synthase kinase-3 GTP guanosine triphosphate HA hemagglutinin HEK293T human embryonic kidney cells transformed by the large T antigen HEPES N-(2-hyroxyethel)piperazine-N'-(2-ethanesulfonic acid) HLH helix-loop-helix IDC infiltrating ductal carcinoma IGFBP insulin-like growth factor binding protein IGF-IR insulin-like growth factor-l receptor IGF insulin-like growth factor IL interleukin ILK integrin linked kinase IR insulin receptor IRS insulin receptor substrate kb kilo-base kDa kilo-daltons LCK lymphocyte specific kinase MAPK mitogen-activated protein kinase MEK map kinase/erk-activating kinase MEFs mouse embryo fibroblasts MMP matrix metalloproteinase MMTV mouse mammary tumor virus mRNA messenger ribonucleic acid MSCV murine stem cell virus mTOR mammalian target of rapapmycin MYR myristoylated N F - K B nuclear factor-kappa B NGF nerve growth factor nM nanomolar nt nucleotide NT-3 neurotrophin-3 NTRK3 neurotrophic tyrosine kinase receptor type 3 -P phosphorylated PARP poly(ADP-ribose) polymerase PBS phosphate buffered saline PCR polymerase chain reaction PDGF platelet derived growth factor PDK phosphoinositide dependent protein kinase PH pleckstrin homology x i i PI3K phosphoinositol-3' kinase PIKK phosphoinositide 3-kinase related kinase PKB protein kinase B PKC protein kinase C PLC phospholipase C PM plasma membrane PMSF phenylmethylsulfonyl fluoride PP2A protein phosphatase 2A PTB phosphotyrosine binding Ptdlns phosphatidyl inositol PTEN phosphatase and tensin homolog deleted on chromosome ten PTK protein tyrosine kinase RB retinoblastoma Rheb Ras homolog enriched in brain RNA ribonucleic acid RSK ribosomal S6 kinase RTK receptor tyrosine kinase S6K1 S6 kinase-1 SAIN She and IRS-1 NPXY-binding SAPK stress activated protein kinase SAM sterile alpha motif SBC secretory breast carcinoma SDS sodium dodecyl sulfate SDS-PAGE SDS polyacrylamide gel electrophoresis siRNA small interfering ribonucleic acid SH2 src-homology-2 SH3 src-homology-3 She src homology and collagen SHIP Src homology 2-containing-inositol 5'-phosphatase SOS son of sevenless Src Schmidt-Ruppin A-2 viral oncogene homolog SV40 simian vacuolating virus 40 TBS tris-buffered saline TEL translocation, ets, leukemia TGF transforming growth factor TK tyrosine kinase TOP terminal oligopyrimidine tract TSC tuberous sclerosis complex UTR untranslated region ACKNOWLEDGEMENTS To begin, I must thank my supervisor Dr. Poul Sorensen. Throughout the PhD process he has provided an environment where I can take my ideas and run with them, all the while providing insight and encouragement. The essence of his techincal advice - "just get it in there" was simple yet strangely effective. Next I'd like to recognize the support of my thesis committee: Drs. Michael Cox, Sandra Dunn and Catherine Pallen. They were there to kick my butt to get me through some dark early days, and were very helpful in guiding me along the way to becoming an independent scientist. The members of the Sorensen lab, past and present, must be thanked for their support, advice, patience, and forgiveness (for contaminating the lab after that nasty TEMED spill). I would especially like to thank the other EN experts, Drs! Cristina Tognon and Chris Lannon, for many insightful scientific (and non-scientific) discussions, technical help, and laughs. I would also like to thank Joan Mathers, Mary Bowden, Hon Leong and Dr. Thomas Podor for technical assitance with experiments critical to the completion of this thesis. I acknowledge both the Canadian Institutes of Health Research and the University of British Columbia for their financial support. My family have always been my biggest fans, and their love arid support has followed me throughout this long odyssey. Finally, to Jill: these last few years have been so joyful and inspirational. Not to mention my Westerns have never looked better. Thank you for everything. NB: Throughout the thesis I have used "I" to indicate experiments that I myself have carried out directly, as well as the resulting conclusions derived from these studies. It should be noted that as science is a collaborative endeavour, the material and intellectual assistance of others forms a vital component of all data contained herein. xiv CHAPTER I INTRODUCTION 1.1 SYNOPSIS AND RATIONALE FOR THESIS Unlike most adult tumours, pediatric sarcomas commonly exhibit a simple set of genetic changes that give rise to cancer. This is most evident in tumours that arise as a result of chromosomal translocations, whereby the occurrence of one genetic event -the expression of a dominantly acting fusion oncogene - is sufficient to induce carcinogenesis. This is the case for the soft tissue sarcoma of early childhood known as congenital fibrosarcoma, where a t(12;15)(p13;q25) chromosomal translocation gives rise to the expression of the ETV6-NTRK3 (EN) fusion protein, which leads to uncontrolled cell growth and clinical disease. Despite the genetic simplicity of pediatric malignancies expressing fusion oncogenes, it is known that tumour cells rely heavily on growth factor signaling cascades - which under normal conditions regulate cell proliferation, survival, and other cellular events - to carry out their oncogenic program. Rather than developing strategies to directly inhibit fusion proteins themselves, which often vary in their specific structure, one can look to common pathways that underlie the transformation induced by chimeric oncoproteins for anti-cancer therapies. One such pathway is the insulin-like growth factor signaling axis. It has been shown that most dominantly acting oncogenes require the expression of the insulin-like growth factor receptor (IGF-IR) to transform cells, and targeting IGF-IR activity preferentially 1 induces cell death in tumour cells as compared to normal cells. Thus, insulin-like growth factor signaling represents a promising target for intervention against pediatric sarcomas. Previous work in our laboratory found that EN requires IGF-IR expression for cellular transformation, and that EN can physically bind and tyrosine phosphorylate the major IGF-IR substrate, insulin receptor substrate (IRS)-1. In this thesis, I have expanded upon these studies by clarifying the roles of each of these molecules in the anchorage-independent growth induced by EN expression. First, I have shown that IRS-1 binding to EN is an essential step in EN carcinogenesis, and that in the absence of IRS-1 expression, IRS-2 can substitute to support EN transformation. Next, I have established that the IGF-IR is required to localize EN and IRS-1 to the plasma membrane, where this complex activates the PI3-kinase/Akt signaling pathway to prevent cell death under anchorage-independent conditions. Further, I show hyperactivation of the mTOR cascade in EN-expressing cells, while EN-induced anchorage-independent growth is significantly impaired upon mTOR inhibition through rapamycin treatment. Finally, I have found that EN expression induces ligand-independent phosphorylation of wildtype IGF-IR, while specific IGF-IR mutants that are not similarly phosphorylated are unable to support EN transformation. In the following sections, a general overview of growth factor signaling will be given, with emphasis on pathways activated by the insulin-like growth factor receptor. These pathways will be discussed both in the context of normal cell biology and their participation in dysregulation of cell growth during cancer development. A particular focus will be given to signaling in cell proliferation, cell cycle progression, and survival. 2 Finally, current knowledge of cellular events induced by ETV6-NTRK3 expression will be presented. 1.2 INSULIN-LIKE GROWTH FACTOR SIGNALING IN NORMAL AND ONCOGENIC GROWTH The insulin-like growth factor (IGF) signaling axis, consisting of IGF ligands, binding proteins, receptors and members of the insulin receptor substrate (IRS) family of adaptor proteins, is a system that plays a key role in the development and maintenance of normal mammalian tissues. IGF signals can induce proliferation, differentiation, and potent anti-apoptotic signals. A large amount of evidence has accumulated indicating that the IGF cascade is central to the development of many types of cancer. In this section, the individual components of this signaling pathway will be described in terms of their role in normal cells, as well as how IGF signaling can be dysregulated in cancer. 1.2.1 Insulin-Like Growth Factors The insulin-like growth factors I and II (IGF-I/-II) (also known as somatomedins) were first isolated more than 50 years ago as growth hormone (GH)-induced serum factors which lead to the sulfation of chondroitin, a component of cartilage (1). These single chain amino acid peptides consist of four separate domains. IGF-I shares significant homology to IGF-I I and - to a lesser extent - insulin, especially in the A and B domains of the peptide (2). However, differences in peptide sequence result in alterations in the affinity each ligand has with various cell receptors and binding proteins. 3 Based on the original discovery of IGFs as factors mediating the sulfation of cartilage, the so-called "somatomedin hypothesis" postulated that local induction of IGF expression by GH leads to bone growth, and thus overall growth of the organism. Much work since then has established that IGF ligands play an essential role in growth during development. This was most definitively shown in IGF-I (3), and I G F - I I (4) knockout mice, which exhibit defects in fetal (IGF-I and I G F - I I) and post-natal (IGF-I) growth. IGF ligands are primarily produced in the liver (5). Direct evidence for this comes from a liver-specific IGF-I knockout mouse, which found a > 75% decrease in IGF-I circulating in the blood (6, 7). However, these mice did not show any defect in post-natal growth, indicating other sources of IGF-I are important after development has occurred (7). Subsequent work has established that insulin-like ligands have a multitude of effects at the cellular level. IGFs are effective at stimulating DNA synthesis and cell proliferation. A wide spectrum of cell types are able to respond to the mitogenic effects of IGFs, including cells of epithelial, mesenchymal and endothelial origin (8). Perhaps the most important role for IGFs is in the prevention of apoptotic cell death. IGF-I and I G F - I I are potent inducers of Akt activation, which has well-established anti-apoptotic functions (see below) (9). IGF-I expression can also modulate the expression of apoptotic genes. For example, IGF-I down-regulates the pro-death Fas receptor (10), while it increases the expression of the anti-apoptotic Bcl-XL protein (11, 12). Finally, emerging evidence points to a role for IGFs in cell motility (13). Normal osteoblasts migrate towards both IGF-I and IGF-II, which is important in bone remodeling after injury (14). Chemotaxis towards IGF-I by breast cancer cells (15), and towards IGF-II by 4 rhabdomyosarcoma cells (16), indicates these ligands may contribute to the motility and invasion of tumour cells during metastasis. A link between the GH/IGF axis and cancer was first established when it was observed that women with breast cancer who underwent removal of the pituitary gland -the site of GH production - showed improved prognosis (17). Due to their growth promoting, anti-apoptotic and pro-migration effects, IGFs have been the subject of intense study in the cancer field. Expression of IGF ligands is now a well-established marker for cancer incidence and progression. Elevated levels of circulating IGF-I are a relevant risk factor for epithelial cancers such as prostate, colorectal, lung and breast (18). This implies that these tumours are responding to IGF-I produced at distant sites. On the other hand, IGF-I I overexpression by tumour cells themselves, which activates IGF signaling in an autocrine manner, is a commonly seen occurrence (19). Often, this increase in IGF-II results from a loss of genomic imprinting in the tumour cell, whereby the IGF-II maternal allele, which is normally transcriptionally inactive, becomes actively transcribed for reasons that are not entirely clear (19). This phenomenon has been documented in several tumour types including rhabdomyosarcoma (20), adrenocortical tumours, (21) and those of liver tissue (22). 1.2.2 IGF B i n d i n g P r o t e i n s In addition to controlling levels of IGF-I/-II ligand, cells and tissues can modulate the IGF response via IGF binding proteins (IGFBPs) (23). There have been six such proteins characterized to date, each of which is a cysteine-rich secreted protein that binds to IGF ligands with high affinity in the extracellular matrix. IGFBPs 1 through 5 5 show increased binding affinity towards IGF-I vs. IGF-II, while IGFBP6 has ~100x greater affinity towards IGF-II compared to IGF-I (24). In vivo, most IGF-I/-II molecules exist in complex with IGFBP3 and the acid labile subunit (ALS) glycoprotein (25). The remainder is bound to other IGFBPs, with only a small proportion - 1% - present as a single molecule. Transcription of IGFBP mRNA can be controlled by multiple factors. \ Examples include GH treatment, which leads to upregulation of IGFBP-5 levels (26), and the onset of senescence, which induces transcription of IGFBP-3 (27). IGFBP levels can also be regulated through degradation by extracellular proteases, including the matrix metalloproteinases (MMP)-7 and -9 (28, 29). IGFBPs generally act to inhibit the IGF signal by sequestering ligand molecules away from IGF-IR. For example, IGFBP3 was shown to inhibit the proliferation of myeloid cells in response to IGF-I, an effect not seen when an IGF-I molecule that is unable to bind IGFBP3 was added to cultured cells (30). However, in some cases IGFBPs can act as a 'reservoir' for IGF molecules to increase the half-life of the ligand, while slowly releasing it to the IGF-IR (31). Though this dampens the initial IGF signal, the net effect is increased signaling through IGF-IR by preventing receptor degradation in response to high IGF exposure. IGFBPs can also affect signaling in an IGF-independent manner, by binding a variety of molecules both at the cell surface and within the nucleus (32). Although it has been postulated that IGFBPs play an important role in cancer, the available data do not point to a general mechanism. Low serum levels of IGFBP-3 have been associated with poor prognosis in a wide range of tumour types, including those of breast, prostate, and colon (33). This is corroborated by in vitro experiments, where 6 overexpression of IGFBP3 inhibits the growth and tumourigenicity of non-small cell lung cancer cells (34). On the other hand, IGFBP2 expression is increased in several cancers, such as glioblastoma (35) and prostate cancer (36). 1.2.3. Insulin-Like Growth Factor Receptors The type 1 insulin-like growth factor receptor (IGF-IR) is a tyrosine kinase receptor that shares 70% homology with the insulin receptor (IR) (37). Rather than playing a role in glucose metabolism, however, IGF-IR exerts its effects over cell proliferation, survival, and transformation. It is present at the plasma membrane in a tetrameric complex of two a- (extracellular) and two (3- (transmembrane + intracellular) subunits (see Fig. 1.1). It is initially synthesized as a 200 kDa immature receptor containing both a- and p-sequences, and is subsequently proteolytically cleaved into the separate subunits (37). The tetrameric complex is held together by disulphide bonds between individual subunits. In cells that express both IGF-IR and IR, hybrid receptor complexes consisting of one IGF-IR and one IR" hemireceptor have been documented (38). These receptors have a much greater binding affinity for IGF-I, and thus are thought to be mediators of IGF, rather than insulin, signaling (39). Binding of IGF-I or IGF-II to the extracellular domain of IGF-IR induces a conformational change in the transmembrane protein that activates its tyrosine kinase domain (40). A key part of this process is trans-phosphorylation, where one p-subunit kinase phosphorylates the other. Several tyrosines on the molecule become phosphorylated upon receptor activation, forming sites for intracellular proteins to bind 7 IGF binding Y950 K1003A Y1131 Y1135 Y1136 Y1250 Y1251 Y1316 P a a juxtamembrane domain Kinase domain C-terminus P IRS-1/-2, She binding ATP binding Kinase activation loop Unique Y residues (not present in insulin receptor) PI3K binding Figure 1.1: The Type-1 Insulin-Like Growth Factor-I Receptor (IGF-IR): Schematic diagram of IGF-IR, showing the heterotetrameric structure of two extracellular c t -subunits and two transmembrane p-subunits - held together by disulphide bonds. Tyrosine (Y) and lysine (K) residues important in IGF-IR function and signaling are highlighted on the left side of the receptor complex, with the corresponding function designated at the same position on the right side. 8 via phosphotyrosine binding (PTB) or Src homology-2 (SH2) domain interactions. One notable site is tyrosine 950, which can bind IRS-1, IRS-2, and She when phosphorylated (41) . The IRS proteins are themselves phosphorylated on multiple tyrosines by IGF-IR (42) , which leads to an even greater number of PTB/SH2 binding sites, and allows the formation of a multi-component signaling complex. IGF ligands acting through IGF-IR have been shown to induce cell division in multiple cell lines, (43, 44). This phenomenon is underscored by the ability of a-IGF-IR antibodies to block the mitogenic signal induced by IGF-I in MCF-7 breast cancer cells (45). IGF-IR also plays a vital role in cell survival. Both antisense oligonucleotides (46) and a dominant-negative IGF-IR (47) are able to induce massive levels of apoptosis in cells grown in anchorage-independent conditions. IGF-I ligand is able to protect fibroblasts from c-Myc-induced apoptosis (48), evidence that this anti-apoptotic effect of IGF-IR is especially relevant in transformation by dominantly acting oncogenes. The importance of IGF-IR in the oncogenic process has been highlighted by the failure of many viral and cellular oncogenes to transform mouse embryo fibroblasts (MEFs) derived from IGF-IR-null transgenic mice (so-called R- cells). These include SV40 Large T antigen (49), c-src (50), and the fusion oncogenes EWS-FLI1 (51) and PAX3-FKHR (52). However, clinical samples of tumour tissue have failed to display significant incidences of IGF-IR mutation (53). Further, there is conflicting data with respect to the relevance of IGF-IR overexpression in tumourigenesis. For example, upregulation of IGF-IR expression levels has been documented in a screen of prostate cancer samples (54), while another group found a downregulation of IGF-IR in tumour vs. normal prostate (55). Therefore, it is thought that normal physiological levels of IGF-9 IR are required for supporting oncogenesis in vivo, but IGF-IR does not represent a transforming signal on its own. iGF-ll receptor In addition to the type-1 IGF receptor, there exists a specific receptor for IGF-II (IGF-IIR). However, unlike IGF-IR, IGF-llR is a monomeric receptor that possesses no intrinsic kinase activity. Instead, it consists of an extensive extracellular region - with separate binding domains for both IGF-II and proteins bearing a mannose-6-phosphate post-translational modification - a transmembrane domain, and a relatively short cytoplasmic tail (56). This latter domain mediates the interaction with the serine proteinase granzyme B found on the surface of cytotoxic that leads to the induction of apoptosis on target cells bearing this receptor (57). It is thought to largely function as a negative regulator for IGF-II activity, whereby free ligand is internalized and degraded upon receptor binding (58). IGF-IIR loss-of-function mutations have been documented in hepatocellular carcinoma (59) and breast carcinoma (60) further implicating the IGF signaling axis in tumours of liver and breast tissue. 1.2.4. IRS Proteins Over 20 years ago, it was observed that insulin treatment resulted in the rapid tyrosine phosphorylation of a 185 kDa protein (61). Since this phosphorylation closely followed the dose response profile of insulin receptor (IR) activation, it was concluded that this protein was the major endogenous substrate of insulin kinase activity, and thus was dubbed insulin receptor substrate (IRS)-1. Soon after, it was found that IGF-IR activation also stimulated the tyrosine phosphorylation of IRS-1 (62). Following the 10 discovery of IRS-1, additional members of the human IRS family of proteins were identified; namely, IRS-2 and IRS-4. There is also an IRS-3 protein which is expressed in mouse - but not human - cells, the function of which is thought to be restricted to insulin signaling in adipose tissue (63). Not only do IRS-2-4 share the ability to be phosphorylated by IR and IGF-IR, but they share significant homology with IRS-1 as well. Other IR and IGF-IR substrates outside the IRS family have been identified, including She (64), GAB1 (65, 66), DOK1/2 (67) and Cbl (68). A commonality among the major IR substrates is that they are adaptor proteins. IRS-1 and IRS-2 possess no catalytic activity, but instead organize a localized signaling complex in response to IGF or insulin stimulation. Three important functional domains lie at the N-terminus of IRS-1/-2 (Fig. 1.2); the pleckstrin homology (PH) domain, which facilitates interactions with the plasma membrane (see below) (63); the phosphotyrosine binding (PTB) domain, which binds molecules that possess phosphorylated tyrosine within the context of an NPXY motif (63); and a Shc-and-IRS-1 NPXY binding (SAIN) domain, which is similar in function to the PTB domain. The minimal region required for IRS-1 to bind IGF-IR includes the complete SAIN domain, and a portion of the PTB domain (41). Within the remainder of the molecule exist multiple tyrosine, serine, and threonine residues which, when phosphorylated, regulate many aspects of insulin or IGF signaling. Phosphorylated tyrosines on IRS-1/-2 serve as docking sites for SH2-containing proteins, thereby localizing signaling molecules to the plasma membrane. For example, the p85 regulatory subunit of PI3-kinase can bind via its SH2 domain to multiple phosphorylated tyrosines within IRS-1 (69), while the Grb2 protein binds at Y895 (70). Differences among SH2 domains result in preferences for particular motifs that surround 11 PIP3 IGF-IR/IR i / \ PH | PTB / p 8 \ J3HP-2 Y608 Y939 Y1172 Y1222 t She/IRS-1 S307 S636/9 j IRS-1 downregulation Y895 t Grb2 Figure 1.2: Insulin Receptor Substrate (IRS)-1. Schematic diagram of IRS-1, showing the major functional domains: pleckstrin homology (PH), phosphotyrosine binding (PTB), and She and IRS-1 NPXY binding (SAIN). Arrows indicate the potential molecular interactions of these domains, and those of important residues within the IRS-1 C-terminal portion. 12 the phosphorylated tyrosine, allowing multiple SH2-containing proteins to bind distinct residues simultaneously (71). Serine and threonine phosphorylation are thought to be negative regulatory events towards IGF/insulin signaling, by promoting dissociation of IRS-1 from the activated receptor. PKC^ (72), Akt (73, 74), tumour necrosis factor (TNF)-a (75) and S6-kinase-1 (S6K1) (76) are all capable of mediating this type of IRS-1 phosphorylation. Although evidence for IRS-1/-2 mutations in human cancer is scarce, a growing body of work points to an important role ,for these proteins in oncogenesis. Overexpression of IRS-1 in NIH 3T3 fibroblasts leads to potent activation of the Erk pathway and in vitro transformation (77). Conversely, a dominant-negative IRS-1, with all tyrosine residues mutated to phenylalanine, was able to block anchorage-independent growth of breast cancer cells (78). Murine hematopoietic 32D cells, which do not express IRS-1, cannot be transformed by IGF-IR overexpression, and instead undergo terminal differentiation (79). However, forced expression of IRS-1 pushes these cells out of the differentiation program, and they become tumourigenic. In vivo, overexpression of IRS molecules is a commonly seen phenomenon in tumour tissue. This seems to be especially relevant to tumours of the liver, which have been shown to overexpress both IRS-1 (80) and IRS-2 (81). IRS-2 overexpression has also been documented in metastatic breast cancer (82, 83) and pancreatic tumours (84). Recently, a study was undertaken to examine levels of tyrosine phosphorylated IRS-1 in a panel of human tumour samples (78). Strikingly, it was found that 27 of 34 samples showed constitutive IRS-1 tyrosine phosphorylation, implicating activation of this signaling pathway as an event in a wide range of human tumours. 13 1.3 REGULATION OF CELL GROWTH, PROLIFERATION, AND SURVIVAL Normal cells tightly regulate the processes of cell growth, differentiation, and survival via a network of intimately linked biochemical events. Signals from the extracellular environment or from within the cell itself, determine its phenotypic fate. Extracellular growth factors can significantly influence these processes by binding to specific receptors on the cell surface. A subset of receptors known as receptor tyrosine kinases (RTKs), of which IGF-IR and NTRK3 are important examples, are particularly suited to propagating proliferative, anti-apoptotic, and differentiation signals. Disruption of these tightly controlled events can lead to aberrations in proliferation and survival, which are the hallmarks of cellular transformation. This section will describe the major growth factor controlled signaling pathways, and their role in various models of oncogenesis. 1.3.1 The Extracellular Regulated Kinase Pathway The most extensively studied response to growth factor stimulation is that of cell proliferation. Central to this process is the Ras/Raf/MEK/Erk pathway (hereafter called Ras/Erk). This cascade is often referred to as the mitogen activated protein kinase (MAPK) pathway, although Ras/Erk activation represents just one arm of MAPK signaling. Structurally related MAPK pathways that are activated in response to various cellular stresses, and generally lead to growth arrest, include the stress-activated protein kinase (SAPK) and p38 cascades (85). Ras/Erk signaling can be activated by a 14 multitude of growth factors, cytokines, hormones, and other stimuli to transmit signals from outside the cell to the nucleus (85). The canonical Erk pathway begins with activation of the Ras small GTPase by receptor tyrosine kinases (RTKs) after ligand binding and auto-phosphorylation. Ras is a 21 kDa protein which is targeted to the plasma membrane by a lipid modification known as farnesylation (86). When Ras is bound to GTP it is in an active conformation. The slow GTPase activity intrinsic to the molecule converts GTP to GDP, and Ras becomes inactivated. Proteins known as GTPase activating proteins (GAPs) facilitate this enzymatic event, and promote inactivation of Ras. Conversely, guanine nucleotide exchange factors (GEFs) activate Ras by promoting the exchange of Ras-bound GDP for free GTP (86). Upon RTK activation, phosphorylated tyrosine residues within the receptor serve as docking sites for Src-homology-2 (SH2) domain-containing adaptor proteins such as She and Grb2. These molecules are able to bind Son of Sevenless (SOS), a Ras GEF, thereby localizing SOS to the plasma membrane where it can activate Ras. Ras has multiple effector molecules that are recruited after activation. Among these is the Raf family of serine/threonine kinases, of which there are three members (Raf-1, A-Raf and B-Raf) (85). Raf isoforms can phosphorylate the MEK1/2 dual specificity kinases, which in turn activate extracellular regulated kinases Erkl and Erk2 through phosphorylation on both threonine and tyrosine residues (85). The multistep activation of Erk1/2 serves to amplify this growth factor-induced signal, whereby activation of -5% of Ras molecules in a cell leads to full Erk stimulation (87). Phosphorylated Erk1/2 translocates to the nucleus where it participates in the activation 15 of multiple transcription factors. These include N F - K B , Ets-1, AP-1, and c-Myc (88). Erks can also activate the ribosomal S6 kinase (RSK), which in turn activates CREB-mediated transcription (89). The net effect of Erk stimulation is the transcription of early response genes, which include many genes that promote cell cycle progression (88). Chief among these are cyclins and the cyclin-dependent kinases (cdks), which are discussed in more detail below. In addition to stimulating proliferation, Ras/Erk signaling can induce other cellular responses, such as differentiation and prevention of apoptosis (88). The cellular outcome in response to activation of this pathway is now known to depend both on the magnitude and the timing of activation. It had been noted in early studies of Erk signaling that growth factors induce biphasic Erk phosphorylation; an immediate response 5-10 minutes after stimulation with ligand, followed by a late sustained phase that persists for as long as 4 hours after growth factor treatment (90, 91). In PC12 rat nerve progenitor cells, nerve growth factor (NGF) induces sustained Erk activation and leads to differentiation, while the Erk response to epidermal growth factor (EGF) is only transient and results in cell proliferation (92). While the importance of Ras/Erk cascade in the prevention of apoptosis is somewhat controversial, there is some evidence that the magnitude of Erk activation can affect this process. Detachment-induced cell death of primary mouse fibroblasts is accompanied by a moderate elevation in Erk phosphorylation (93), whereas strong Erk activation via overexpression of an activated Raf-1 construct can prevent apoptosis in CCL39 Chinese hamster lung fibroblasts (94). Since Ras/Erk signaling provides such a strong proliferative signal, dysregulation of the pathway at various steps is a frequent event in human cancer. Ras genes were 1 6 among the first oncogenes discovered, in the form of the Harvey and Kirsten sarcoma viruses (Ha-Ras and K-Ras, respectively). Cellular homologs for Ha-Ras and K-Ras were cloned soon after, and activating genetic mutations were implicated in the oncogenesis of bladder and lung tumours (95-97). It is now known that 20-30% of human malignancies possess Ras mutations, with the incidence being especially high in pancreatic (-90%), colon (-50%) and thyroid (-50%) tumours (98). While it was initially thought that Raf was only important in oncogenesis in that it carried out the actions of mutant Ras proteins, mutations in B-Raf have been observed in several tumour types, most notably malignant melanoma (99). Although constitutive activation of MEK1/2 is able to transform cells in vitro (100), there have been no activating mutations of MEK1/2 detected in human cancer (101). Nevertheless, the fact that Erk is hyperactivated in -30% of cancer (102) and that Erkl and Erk2 are the only known substrates of MEK1/2 (103), make MEK1/2 an attractive therapeutic target. 1.3.2 P13K / Akt Signaling Pathway A second major signaling pathway which is activated by growth factors is the phosphotidylinositide 3'-kinase (PI3K)/Akt cascade. PI3K/Akt signaling plays a vital regulatory role in multiple cellular events, including proliferation, protein translation, cell size determination, and prevention of cell death (discussed in detail below). Multiple growth factors possess the ability to activate this pathway, including G-protein coupled receptors and those with intrinsic tyrosine kinase activity. The insulin-like growth factor receptor shows a particularly potent ability to activate PI3K in response to ligand stimulation (104). 17 PI3K/Akt signaling has emerged as a crucial component in the initiation and progression of cancer (reviewed in (105)). Akt was initially discovered as the oncogenic component of a transforming retrovirus, and the human homologue was detected as an amplified gene in human gastric tumours (106). It was subsequently found that an activated form of the catalytic subunit of PI3K (p110a) is encoded by the avian sarcoma virus (107). Recent work has established that activating mutations in p110a are a frequent event in human tumours (107), making this pathway a potential target for intervention against cancer. 1.3.2.1 PI3K Structure and Regulation PI3K is a member of the phosphoinositide 3-kinase related kinases (PIKKs), a group of kinases that includes mTOR, the gene mutated in ataxia telangiectasia (ATM), and DNA-dependent protein kinase (DNA-PK) (108). However, unlike these kinases, PI3K substrates are lipids rather than proteins. Specifically, PI3K phosphorylates the 3' position of the inositol ring in phosphatidylinositol (Ptdlns) compounds - lipid molecules that reside within the inner leaflet of the plasma membrane. These consist of Ptdlns, Ptdlns 4-P and Ptdlns (4,5)-P2, giving rise to Ptdlns 3-P, Ptdlns (3,4)-P2 and Ptdlns (3,4,5)-P3, respectively. Class I PI3Ks, the most widely expressed group of PI3K proteins, have equal affinity for each substrate (109). The PI3K enzyme consists of two subunits that are expressed from separate genes, both of which have multiple isoforms (109). The p110 catalytic subunit binds to the p85 regulatory subunit, an adaptor protein which contains an N-terminal SH3 domain and two C-terminal SH2 domains (110). The binding of p110 to p85 is mediated 18 through the latter's intra-SH2 (IS) domain (111), and this interaction is required for p110 catalytic activity (112). Regulation of PI3K catalysis is determined through access to PI substrates and is therefore dependent on localization to the plasma membrane. The p85 regulatory subunit is able to achieve PM localization by binding to phosphorylated tyrosines that result from growth factor activation through either of its two SH2 domains. p85 binding can be direct, as in the case of activated PDGF (110) or EGF receptors (113) , or indirect, through binding to activated IRS-1 in response to insulin (69) or IGF (114) signaling. Thus, PI3K activity is able to couple to growth factor signaling, and mediates an important facet of the downstream response. The discovery that PI3K is a Ras effector (115), adds another layer of complexity to the growth factor regulation of this pathway. GTP-bound Ras can directly interact with p110, and Ras overexpression leads to a dramatic increase in PI3K lipid products. These multiple levels of PI3K regulation serve to subtly and distinctly modulate the response to a specific growth factor in a given cell type. Finally, in addition to receptor tyrosine kinases, ligand binding to G-protein coupled receptors is capable of inducing PI3K enzymatic activity (116). Free G-protein (3y subunits activate a unique group of PI3K isoforms known as class 1b PI3K, to stimulate this cascade (117). 1.3.2.2 Regulation of Akt Activation As a consequence of the production of 3' phosphorylated phosphatidylinositol compounds, multiple proteins are activated. This is achieved by the specific binding of proteins containing the conserved pleckstrin homology (PH) domain to Ptdlns 3-P, Ptdlns (3,4)-P2 and Ptdlns (3,4,5)-P3 molecules. PH domains have been shown to 19 mediate interactions with the plasma membrane for many proteins. The PH domain of IRS-1 helps stabilize its interaction with the insulin and insulin-like growth factor receptors (118), and the resulting membrane localization positively influences PI3K signaling by its interaction with p85 (119). PH domain/plasma membrane interactions are also key to the PI3K mediated activation of the ACG family of protein kinases. These include serum- and glucocorticoid-regulated kinases (SGKs 1-3), p70 S6 kinase-1 (S6K1), and atypical protein kinase C isoforms (120). However, the most well characterized ACG kinase is Akt (also known as Protein Kinase B (PKB)), which is central to cell physiology due to its wide range of substrates. Each ACG kinase contains two highly conserved Ser/Thr residues, which become phosphorylated upon activation: one in the kinase activation loop and another in the C-terminal hydrophobic domain (120). The 3'-phosphoinositide-dependent protein kinase-1 (PDK1), a PH domain-containing, constitutively active kinase is the major protein that phosphorylates the activation loop residue. Since aberrant Akt activation is thought to play a key role in cancer progression, its regulation and downstream signaling have been the subject of much study. There are three closely related isoforms of Akt (Akt1-3), which have similar (but not completely redundant) functions. Each possesses an N-terminal PH domain which binds to both Ptdlns (3,4)-P2 and Ptdlns (3,4,5)-P3 with equal affinity (121). Once PI3K activity is stimulated, Akt translocates to the membrane in a PH dependent manner (c!22). As described above, Akt possesses two important regulatory sites that become phosphorylated upon its activation. In the case of Akt1, Thr308 is found within the activation loop and is the target of phosphorylation by PDK1 (123). This phosphorylation 20 event requires the PH domain of Akt to bind Ptdlns (3,4)-P2 or Ptdlns (3,4,5)-P3 (122, 124). 7 The identity of the Akt hydrophobic motif kinase, which phosphorylates Ser 473 and is often referred to as "PDK2", has been more elusive and is the subject of much debate. Akt autophosphorylation at Ser 473 was an early mechanism proposed, as one group found the kinase activity of Akt was required for phosphorylation at this site (125). However, subsequent studies showed that drugs which inhibit Akt kinase activity do not affect Ser 473 phosphorylation (126), and that isolation of subcellular fractions possessing PDK2 activity were distinct from Akt itself (127, 128). PDK1 was shown to acquire Ser 473 kinase activity in vitro when interacting with a subdomain of the protein kinase C-related kinase-2 (PRK2) termed PDK1-interacting fragment (PIF) (129). The relevance of this finding was questioned by the observation that Ser 473 phosphorylation is not impaired in PDK1 -/- ES cells (130). The integrin-linked kinase (ILK), a PI3K regulated kinase that suppresses detachment-induced cell death, was shown by one group to be an Akt Ser473 kinase (131), and a conditional knockout of this protein caused hypophosphorylation of Ser 473 (132). However, this finding has been disputed by another group, who observed no effect on Akt phosphorylation in ILK knockout fibroblasts (133) or chondrocytes (134). A recent study provides evidence that ILK phosphorylation of Akt may be more relevant in transformed, as opposed to normal, cells (135). Finally, a strong case for PDK2 activity has been made for members of the PIKK family, including DNA-PK (136), ATM (137), and mTOR (138). In the case of mTOR, Akt Ser 473 kinase activity specifically resides within the pool of mTOR molecules that are in a rapamycin-insensitive complex with the protein rictor. RNA 21 interference knockdown of both rictor and mTOR inhibited Ser 473 phosphorylation (138), making the evidence for this mechanism of Ser 473 phosphorylation particularly compelling. 1.3.2.3 Akt Downstream Targets Once activated, Akt has a myriad of potential substrates whose phosphorylation can have diverse effects on cellular phenotypes. Unlike Ras/Erk signaling, where the cascade consists of a series of activating phosphorylation events, phosphorylation by Akt generally inhibits the function of the target protein. In terms of cell proliferation, these Akt substrate proteins are most often inhibitory themselves, therefore activated Akt has the net effect of positively regulating both normal and cancer cell growth. Below is a brief description of selected examples of Akt substrates. GSK-3 One Akt-regulated protein that is involved in multiple cellular processes, including metabolism and proliferation, is glycogen synthase kinase-3 (GSK-3). There are two distinct GSK-3 isoforms, a and p\ which are expressed from separate genes (139). Unlike most protein kinases, GSK-3 has a high basal activity, and is inactivated rather than activated by phosphorylation. This is achieved by phosphorylation of an N-terminal serine (Ser 21 for GSK-3a and Ser 9 for GSK-3P) that acts as a "pseudosubstrate" for the, enzyme's kinase domain, leading to intramolecular folding to an inactive conformation. Akt is a major kinase for both GSK-3 isoforms (140), although S6K1 (141), RSK (142) and protein kinase A (143) can each inactivate GSK-3 by Ser 21/Ser 9 phosphorylation. The soluble factor Wnt also leads to GSK-3 phosphorylation and 22 inactivation, thus preventing phosphorylation and subsequent degradation of p-catenin (144). This leads to accumulation of p-catenin in the nucleus, where it binds to the T-cell factor (TCF) and lymphoid-enhancing factor (LEF) transcription factors, promoting transcription of specific genes (145). There are many other GSK-3 substrates, the majority of which have their activity inhibited by phosphorylation, and are thus released from inhibition when Akt phosphorylates GSK-3. One such substrate is IRS-1. Phosphorylation of IRS-1 serine residues by GSK-3 attenuates insulin signaling by decreasing the amount of tyrosine phosphorylated IRS-1 (146). GSK-3 phosphorylation of cyclin D1 leads to its export from the nucleus, and targets it for proteasomal degradation (147). Therefore Akt inactivation of GSK-3 can promote cell proliferation by both supporting growth factor responses and stabilizing molecules that push the cell through the cell cycle. i TSC1/2 Both PI3K and mTOR are key growth-factor responsive regulators of cell proliferation and growth. mTOR was first isolated in a screen for proteins that are inhibited by the immunosuppressive drug rapamycin. Rapamycin forms an inhibitory complex with FK506 binding protein-12 (FKBP12) that binds to the C-terminus of mTOR (148, 149). mTOR is a serine/threonine kinase which itself has multiple downstream targets, including S6K1 (150) and 4E-BP1 (151). Cell growth through mTOR activation is thought to primarily stem from its ability to promote protein translation. The phosphorylation of 4E-BP1 induces its release from the ribosomal protein elF4E, which is then able to bind to the 5' cap structure of mRNA and allow translation to proceed (152, 153). Activated S6K1 has the ability to specifically promote the translation of 23 mRNAs with a 5' terminal oligopyrimidine. tract (TOP) (154), which exclusively encode for proteins in the translational machinery. The idea that PI3K/Akt and mTOR are part of a linear signaling pathway is r supported by several lines of evidence. PI3K inhibitors LY294002 and wortmannin inhibit the activity of mTOR (155), while expression of a dominant negative p85 was shown to block insulin-mediated activation of S6K1 (156). Conversely, phosphatase and tensin homologue deleted on chromosome ten (PTEN)-null cells, which have constitutively high levels of Akt activation (see below), have similarly high levels of 4E-BP1 and S6K1 phosphorylation (157, 158). However, the fact that mTOR itself is a poor Akt substrate meant there was additional complexity to the PI3K/mTOR story. The direct link between the PI3K/Akt and mTOR pathways has come from recent experiments investigating the regulation of the tuberous sclerosis complex (TSC) proteins. TSC1 (also known as hamartin) and TSC2 (tuberin) have each been found to be mutated in the genetic disorder tuberous sclerosis complex, which is characterized by the formation of tumour-like growths known as hamartomas in multiple tissues (159). TSC1 and TSC2 physically interact via their N-termini to form a large protein complex (160). An early observation that TSC1/2 mutations in Drosophila had similar phenotypic consequences as PTEN mutations (161, 162) suggested TSC proteins were functionally related to PI3K/Akt. Upon searching for Akt conserved phosphorylation motifs, it was found that human TSC2 contained up to 3 such sites (163, 164). Moreover, mutation of these serine or threonine residues to alanine, thus preventing Akt regulation, caused a loss of mTOR activation in response to growth factors (163). Further work confirmed the negative regulation of mTOR by the TSC proteins, where overexpression of TSC1/2 24 lead to decreased S6K1 and 4E-BP1 phosphorylation (165), while TSC2-null cells exhibited hyperphosphorylation of these same proteins (166). Although the mechanism remains unclear, Akt phosphorylation acts to inhibit the GTPase Activating Protein activity of TSC2 towards the small G-protein Rheb (Ras homolog enriched in brain) (167), which favours its active, GTP-bound state. Overexpressed Rheb leads to growth factor-independent activation of mTOR (163), though the mechanism of this activation has yet to be elucidated. It should be noted that a recent report has described an Erk-dependent phosphorylation of TSC2 via the ribosomal S6 kinase-1 (RSK1), which can also activate mTOR signaling (168). This provides even greater evidence that these two pathways work in concert to promote growth signaling. Raf As mentioned above, the ability of activated Ras to stimulate both the PI3K/Akt and Ras/Erk cascades supports the idea that there is significant crosstalk between these pathways. Further confirmation that this is an important phenomenon comes from the ability of Akt to regulate Raf-1 activity. In both HEK293 and MCF-7 cell lines, Zimmerman and Moelling found that Akt bound to Raf-1, phosphorylating it on Ser 259 to inhibit its kinase activity, and thus its ability to propagate a signal through to Erk (169). This was suggested as a mechanism to explain the observed phenomenon that IGF-I stimulation of MCF-7 cells induces sustained PI3K/Akt activation but only a transient Raf/Erk signal (170). Additional work established that this event may be cell-type or cell-stage dependent, as inhibitory Raf-1 phosphorylation by Akt was observed in differentiated myotubes but not myoblast progenitors (171). Negative regulation of B-Raf via Akt phosphorylation has also been observed (172). 25 1.3.2.4 Negative Regulation of PI3K/Akt Signaling Turning off the PI3K/Akt signal induced by growth factors is important in regulating growth homeostasis within the cell. PTEN is a dual specificity phosphatase that has the ability to reverse the reaction catalyzed by PI3K to remove the 3' phosphate from Ptdlns molecules. The activity of PTEN is unique in its ability to reverse this reaction, and thus has emerged as an important player in cancer biology. Overexpression of PTEN results in a reduction of Akt activity, and leads to growth inhibition of transformed cells (173), (174). Transgenic mice with only one copy of the PTEN gene display a high incidence of cancer (175). These tumours are of wide ranging types (lymph node, endometrium, breast etc.) (176), but share the characteristic of loss of PTEN heterozygosity, and hyperactive Akt signaling (175). In fact, PTEN is one of the most commonly mutated tumour suppressor genes in human cancer (177). Perhaps nothing supports this fact more than the frequent occurrence of tumours in patients with Cowden's syndrome, which is characterized by an inherited mutation in ' one copy of the PTEN gene (178). Sre homology 2-containing-inositol 5'-phosphatase (SHIP), and its more widely expressed homologue SHIP2, also dephosphorylate the inositol ring of Ptdlns, in this case at the 5' position. However, they do not play a similar negative regulatory role in PI3K/Akt signaling (174). 1.3.3 Regulation of the Cell Cycle Normal tissues must maintain a balance between cell proliferation, growth arrest, differentiation, and cell death. To prevent uncontrolled division, cells possess a multi-1 \ 2 6 layered system of checkpoints, which require specific signals for the cycle to proceed (reviewed in (179)). After cells have divided, they undertake a growth phase (termed G1), where gene expression, protein synthesis, and cellular metabolism take place. When a signal to divide is received, cells enter what is referred to as S-phase, where DNA replication is undertaken. After a second growth phase (G2), physical division of the cell into two daughter cells, a process known as mitosis (M), takes place. Cancer can be thought of as an upsetting of this balance towards unregulated cell proliferation. Accordingly, molecules that are involved in regulating the transition from one cell cycle stage to another have been directly implicated in oncogenesis. Thus, a brief discussion of the major players in cell cycle control is in order. 1.3.3.1 Cyclins and Cyclin-Dependent Kinases Passage through key cell cycle checkpoints is largely promoted by two sets of proteins - the cyclins and cyclin-dependent kinases (CDKs) (179). As their name suggests, levels of cyclin proteins oscillate at regular intervals, showing greatest abundance at particular stages of the cell cycle. The amount of cyclin protein present in the cell can be regulated at many levels, including transcription, translation, and protein stability. On their own, CDKs are catalytically inactive, but become functional kinases upon interaction with cyclin molecules. Therefore CDK enzyme activity also acts in a cyclical fashion. The transition from G1 to S phase is largely controlled by cyclin D- and cyclin E-dependent kinases (179). The D-type cyclins, of which there are three members (cyclin D1, D2 and D3), activate both the cdk4 and cdk6 enzymes, forming up to 6 potential 27 holoenzyme complexes. cdk4 and cdk6 are relatively stable proteins, whereas cyclin D undergoes rapid turnover. This means that control over G1/S transition is dependent on steady-state levels of cyclins, as opposed to CDK levels. The kinase activity of cyclin D«cdk4/6 complexes is almost exclusively directed against the retinoblastoma (Rb) protein (180, 181). Hypophosphorylated Rb represses the transcription of genes that promote DNA synthesis, through its interactions with the E2F transcription factor (182). Rb phosphorylation by cyclin D*cdk4/6 causes the release of E2F, allowing it to promote transcription of its target genes. As cyclin E is one of these E2F-controlled genes (183), cyclin D»cdk4/6 activity has the effect of promoting cyclin E«cdk2 activation. When present at high levels, cyclin D»cdk4/6 complexes can further enhance cyclin E-cdk2 activity by binding and "titrating-out" cdk inhibitors (CDKIs), pushing cells through the G1/S restriction point (184). Due to their important position at the restriction point of the cell cycle, and the high level of regulation attached to their protein levels, cyclins have been the subject of much study in the context of oncogenesis. Cyclin E overexpression has been implicated in a variety of cancers, including breast carcinoma, lymphoma, and non-small cell lung cancer (185). Cyclin D1 and the cdks it regulates have emerged as particularly relevant proteins in the development of breast cancer. Gene amplification of cyclin D1 is seen in 15-20% of human breast carcinomas (186, 187), while a majority of breast tumour tissue samples show elevated cyclin D1 protein levels (188, 189). Mice with expression of cyclin D1 under the control of the mouse mammary tumour virus (MMTV) promoter develop mammary adenocarcinomas (190). Conversely, mammary tumours that develop in MMTV-Ras or MMTV-ErbB-2 mice are not seen when these mice are 2 8 crossed to a cyclin D1 -/- genetic background (191, 192). A recent study showed that this requirement for cyclin D1 in mammary tumourigenesis was specifically due to its activation of cdks (193). Mice where the normal cyclin D1 allele was replaced with a mutant which fails to activate cdk4/6, yet is still able to promote cyclinE»cdk2 activation, are resistant to ErbB-2 mediated tumours. Cyclin D-regulated kinases were further implicated in carcinogenesis by work which demonstrated that cdk4 is required for initiation and maintenance of ErbB-2- and neu-induced mammary tumours (194, 195). In each of these studies, inactivation of cdk4 had little effect on normal mammary development, indicating this kinase may be of particular interest as a target for breast cancer that is likely to have minimal side effects. 1.3.3.2 Cyclin Dependent Kinase Inhibitors An additional level of control upon the cell cycle is exerted by several classes of cdk inhibitor proteins. The two major classes of such proteins are the Cip/Kip family, which includes p21 C i p 1, p27 K i p 1, and p57 K i p 2, and the INK4A family, whose members include p16 I N K 4 a , p15 I N K 4 b , p18 I N K 4 C , and p19 I N K 4 D (196). These proteins directly bind to cdk molecules, and generally inhibit their function. For example, p16 I N K 4 a directly binds cdk4 and cdk6, inhibiting the function of cyclin D«cdk4/6 complexes (196). Similarly, both p21 C I P 1 and p27 K i p 1 bind to cdk2, which inhibits the activation of cyclins A and E, leading to G1 cell cycle arrest (197). However, several groups have found that Cip/Kip members can actually facilitate cell cycle progression by promoting the formation cyclin D»cdk4 complexes (198, 199). 29 Connections between PI3K/Akt signaling and regulation of the cell cycle have recently been established by the discovery that Akt can phosphorylate p27 k i p 1 (200-202). An Akt consensus sequence lies within the nuclear localization sequence of p27 k i p 1, and Akt phosphorylation inhibits its movement into the nucleus. The accumulation of p27 k i p 1 in the cytoplasm means that it cannot bind to nuclear cdk2, and has the net effect of overcoming G1 arrest. The implication that this promotion of cell cycle progression is relevant to carcinogenesis is underscored by the fact that cytoplasmic p27 k i p 1 is a feature of primary breast tumour tissue (200-202) and correlates with poor prognosis (200). Likewise, it was recently demonstrated that Akt can phosphorylate p21 C I P 1 in a manner that increases its stability (203). This phenomenon leads to the binding and activation of the cyclin D»CDK4 complex, pushing cells through the G1-S checkpoint. 1.3.4 Regulation of Cell Death Multicellular organisms have evolved a process of programmed cell death, known as apoptosis, whereby unwanted cells undergo a specific set of controlled events that eliminate them from the body. Cells undergoing apoptosis exhibit a characteristic set of phenotypic changes, including chromatin condensation, DNA fragmentation, and plasma membrane blebbing (204). This process can be triggered by DNA damage, loss of attachment to the normal substratum or alterations in cell cycle. The normal balance between cell proliferation and cell death is nearly always lost upon the transition to an oncogenic state, as cells inappropriately survive conditions that would usually trigger 30 apoptosis. This phenomenon has important implications for anti-tumour chemotherapy, which is often reliant on induction of apoptosis for its killing of cancer cells (205). 1.3.4.1 Apoptotic Pathways Two major signaling pathways that elicit an apoptotic response exist within cells (206). Both of these pathways converge on the caspase-3 protein, which when activated initiates the phenotypic changes associated with apoptotic cell death. Caspase-3 is a protease, and cleaves numerous cellular proteins, which include poly(ADP-ribose) polymerase (PARP), the retinoblastoma protein (RB), and other caspases (207). The intrinsic pathway refers to signaling events that originate at the mitochondrion. The key event after a pro-death signal has been received is the release of cytochrome c from the mitochondrion intermembrane space. Then, along with apoptotic protease-activating factor-1 (APAF1) and dATP, cytochrome c activates caspase-9, which in turn activates caspase-3. The release of cytochrome c is primarily controlled by members of the BCL2 protein family. This group of molecules is divided into pro-apoptotic members (e.g. Bad, Bax, Bid), which promote cytochrome c release, and anti-apoptotic members (BCL2 and BCL-XL), which inhibit this event. The second major pathway for induction of apoptosis is referred to as the extrinsic pathway, as it is initiated by the binding of an extracellular signal peptide to a membrane receptor. The classic example of a ligand that activates the extrinsic pathway is FAS ligand (FASL), a member of the tumour necrosis factor family of proteins. Binding of FASL to the FAS receptor triggers a series of events, which are dependent on interactions between modular protein motifs, which include the death domain (DD) and the death effector 31 domain (DED). Specifically, activation of FAS enables the death domain (DD) on its cytoplasmic face to bind a similar DD in the FAS-associated death domain (FADD) adaptor protein. This allows the DED of FADD to recruit caspase-8 via its DED, leading to caspase-8 activation, and subsequent activation of caspase-3 (206). 1.3.4.2 Role of PI3K/Akt Signaling in Prevention of Apoptosis The physiological response over which PI3K/Akt signaling exerts its most dramatic effects is programmed cell death. It was observed several years ago that inhibition of PI3K through LY294002 or wortmannin treatment leads to a potent induction of apoptosis (208, 209). Conversely, expression of activated PI3K or Akt constructs were found to prevent cell death under wide-ranging conditions (210-212). Clues to possible mechanisms for Akt's ability to block apoptosis arose when yet more Akt substrates were discovered. The pro-apoptotic Bcl-2 family member BAD becomes phosphorylated Akt at Ser 136 (213, 214). This causes BAD to dissociate from the anti-apoptotic protein Bcl-XL in favour of binding 14-3-3 adaptor proteins, releasing Bcl-XL from inhibition and preventing cell death (215). 14-3-3 interactions are also promoted by Akt phosphorylation of the FOXO family of transcription factors (216). This has the effect of preventing nuclear localization of FOXO proteins, so that they cannot induce expression of their target genes (217). These targets include the pro-apoptotic proteins FASL (217) and Bim (218). Finally, caspase-9 is a substrate of Akt (219). Phosphorylated caspase-9 is unable to propagate the apoptotic cascade, and can block apoptosis even if cytochrome c has been released from mitochondria. 32 1.3.4.3 Anoikis Anoikis, the Greek word for "homelessness", refers to the programmed cell death triggered by loss of cell anchorage. This phenomenon was first observed in human endothelial cells, which underwent apoptosis upon withdrawal from the extracellular matrix (ECM) (220). Subsequent studies have shown that anoikis occurs in most cell types of the endothelial, epithelial and mesenchymal cell lineages (221). It is hypothesized that avoidance of anoikis is an essential adaptation for cancer cells if they are to detach from their primary site and metastasize. Integrin/ECM interactions are a key component of the survival signal provided by anchorage to a substrate (222). Integrins consist of a heterodimer composed of one a-chain and one p-chain. There are multiple isoforms of oA} dimers, each of which has a particular binding affinity for various ECM constituents. The most well characterized of these is the a 5Pi interaction with fibronectin, which has been shown to prevent apoptosis (223). On the intracellular side of integrin molecules, large protein complexes known as focal adhesions are formed (224). Several non-receptor kinases are important components of this complex - focal adhesion kinase (FAK), Src-family kinases, and ILK. FAK becomes phosphorylated upon integrin engagement (225), and can associate via its tyrosine 397 autophosphorylation site with the SH2 domains of Src (226) and PI3K p85 (227). This latter interaction links integrin engagement with activation of Akt (228, 229), which promotes cell survival as discussed above. ILK is also activated by integrin-fibronectin interactions (131), and its overexpression leads to anchorage-independent survival (230). The potential for ILK to phosphorylate Akt at serine 473 and thus activate it is the suggested mechanism for this observation. Recently, a genomic screen for suppressors 33 of anoikis was undertaken, with TrkB emerging as a kinase capable of inducing survival under anchorage-independent conditions (231). Activation of PI3K/Akt signaling was an essential downstream event in TrkB's prevention of detachment-induced death, i implicating Akt signaling as a key mediator of anoikis resistance. 1.3.4.4 Other Forms of Cell Death While apoptosis was first being described, it was observed that a distinct form of cell death known as necrosis could take place (204). Phenotypically, necrosis is quite different from apoptosis; cells swell and lyse as opposed to the shrinking seen in apoptotic cells, while necrotic cells do not exhibit nuclear condensation or DNA fragmentation. Necrosis was thought to be a passive process that resulted from pathological events Asuch as radiation exposure, infection, or inflammation (232). However, there is some recent evidence that necrosis is relevant to normal physiological processes, especially in cells of the digestive tract (232). While signaling pathways regulating necrotic death are poorly understood, it is possible that concentrations of reactive oxygen species (ROS) and calcium ions play a role (232). Since transformed cells that exhibit defects in the ability to undergo apoptosis usually retain the potential for necrotic cell death, it has been suggested that agents which promote necrosis may be useful for treating cancer (233) Autophagy is a process whereby cells achieve degradation of intracellular protein complexes and organelles in response to nutrient deprivation or other types of stress (234). Material to be broken down is engulfed in a bilayer membrane referred to as an autophagosome, which eventually fuses with lysosomal vesicles where degradation 34 takes place (234). Whether autophagy is a bona fide form of cell death or an "emergency" mechanism for cells to survive unfavourable conditions has not been firmly established. From a signaling perspective, it is becoming clear that the PI3K/Akt/mTOR pathway plays a role in preventing autophagy (234). As mTOR acts as a sensor for cellular nutrient levels, it is logical that it plays an important role in autophagy, which is a response to nutrient deprivation. 1.4 THREE-DIMENSIONAL MODELS OF TUMOUR CELL GROWTH Research into the signals regulating the growth of normal cells, and the mechanisms by which these signals become dysregulated in tumour cells has been the subject of intense study over the past two decades. Initial signal transduction studies were limited to immortalized cell lines grown on plastic tissue culture plates, resulting in adherent monolayer cultures. While this cell culture model is still used at the present time, and has been incredibly informative, it is not without its drawbacks. Most notably, it is thought that monolayer culture poorly replicates the dynamic three-dimensional (3D) structure of tumours. Signaling pathways in particular can be dramatically affected by the growth conditions to which cells are subjected. Potential targets for therapeutic intervention may have much different behaviors or even relevance depending on the culture model used. Therefore, much study has gone into developing culture models that better reflect the growth of tumour cells in their natural microenvironment. A common characteristic of these models is that they exploit the ability of transformed cells to grow under anchorage-independent conditions. Examples include the 35 multicellular spheroid model, the soft agar colony forming assay, and mouse tumour xenografts. i 1.4.1 Multicellular Spheroids An important step in the transformation of a cell to the oncogenic state is acquiring the ability to grow under anchorage-independent conditions. While normal cells undergo detachment-induced apoptosis, transformed cells possess the ability to survive and aggregate into three-dimensional structures. Anchorage-independent spheroid cultures create conditions whereby the adhesion affinity for a cell to the substrate is less than that between two cells (235). This can be achieved by covering the plastic surface of a culture dish, to which cells have a high binding affinity, with a material of low affinity. Such materials include poly-hema and solidified agar. Several aspects of the multicellular spheroid model more closely re-create the tumour microenvironment. Firstly, cells grown in these conditions can mimic the network of extracellular matrix proteins and undergo complex cell-cell and cell-matrix interactions, which are largely absent in monolayer culture (236). For example, Ewing's sarcoma cell lines secrete a network of fibronectin when grown as multicellular spheroids, a process which is hypothesized to be key in promoting their continued growth under anchorage-independent conditions (B. McLean and P. Sorensen, unpublished observations). Secondly, spheroids more closely resemble cells from human tumours in their rate of replication. Lawlor et al. found this to be the case for Ewing's sarcoma cells grown in 3D culture (237). This observation was thought to be a consequence of cyclin D levels which were much more similar when comparing 36 spheroids to tumour than monolayer to tumour. Finally, another advantage of this model is that it allows the co-culture of tumour cells with normal cells such as stromal fibroblasts or endothelial cells. Again, this serves to better recapitulate the conditions within which tumour cells find themselves. 1.4.2 Other 3D Culture Models Soft Agar Colony Forming Assay Growth of cells in soft agar has long been used as a standard assay for cellular transformation (238). Single cells are suspended in a solution of liquid agar and growth media, which then solidifies to form a matrix. As most normal cells require engagement with a substratum to survive and proliferate, they will die or undergo growth arrest under these non-adherent conditions. Transformed cells, on the other hand, are able to survive these conditions, to form macroscopic colonies of multiple cells. As soft agar colony formation requires a single cell to possess the potential to not only survive and proliferate, but presumably to degrade the surrounding agar matrix, it can be considered a more stringent measurement of anchorage-independent growth potential than the spheroid model. However, one major limitation to the soft agar system is the difficulty in conducting signaling studies, as obtaining cell lysates for Western blotting is impractical. In vivo Models Ideally, any model of 3D anchorage-independent growth would mimic the conditions encountered by a tumour cell as closely as possible. Mouse models, therefore, have been extensively used not only to determine the tumourigenicity of a given cell line, but to assess the efficacy of anti-cancer drugs (239). In the mouse 37 tumour xenograft model, cells are injected subcutaneously into immunocompromised mice (so as not to elicit an immune response against the cells under study). This strategy has been used to confirm the oncogenic nature of a multitude of activated signaling molecules (240-242). Evaluation of potential therapeutic agents has met with mixed results, with certain tumour types being more predictive than others (239). Another major drawback is that the subcutaneous compartment is an artificial tumour environment, lacking the vasculature and stromal cells normally encountered by transformed cells. This problem can be at least partially overcome by the development of transgenic mice. Engineering animals that overexpress oncogenes, or carry inactivating mutations in tumour suppressor genes, has led to a greater understanding of the initiation and development of cancer. That being said, significant differences in the genetic requirements for cancer initiation exist between mice and humans (243), meaning that no model will perfectly replicate the anchorage-independent growth of in vivo human tumours. 1.5 THE ETV6-NTRK3 CHIMERIC TYROSINE KINASE Soft-tissue tumours of childhood, which include Ewing's Sarcoma and alveolar rhabdomyosarcoma, are often characterized by disease-specific chromosomal translocations. The ETV6-NTRK3 gene fusion that is the subject of this thesis was first isolated in cases of congenital fibrosarcoma, which exhibit a recurring t(12; 15) translocation. The resulting chimeric protein fuses the sterile alpha motif (SAM) domain of the ETV6 transcriptional repressor to the tyrosine kinase domain of the NTRK3 receptor tyrosine kinase (Fig. 1.3). This results in a constitutively active tyrosine kinase, 38 ETV6 A93 ' v*112 S A M DNA binding > NTRK3 She/Gab 1 ATP activation I I loop Y516 K555 ligand binding TM PTK £LC Y Y803 1 - r > i ETV6-NTRK3 A93 V112 S A M ATP activation • loop K380 r-^ |>LCY Y615 Y628 P T K Figure 1.3: The ETV6-NTRK3 (EN) chimeric oncoprotein. EN consists of the sterile alpha motif (SAM) domain of the ETV6 transcriptional repressor fused to the protein tyrosine kinase (PTK) domain of the NTRK3 neurotrophin receptor. Note that EN does not contain NTRK3 tyrosine 516 (Y516), the site of She and Gab1 binding. 3 9 which has potent transforming activity in fibroblasts (242), breast epithelial cells (244), and cells of hematopoietic lineage (245). It is hoped that the study of EN-activated signal transduction pathways, which underlie the oncogenic process, can lead to the development of potential therapies against EN-expressing tumours, as well as provide a greater general understanding of cellular transformation. 1.5.1 ETV6 The ETS variant gene 6 (ETV6; also known as TEL) is a member of the E26 transformation-specific (ETS) family of transcription factors. It contains two. distinct functional domains; the N-terminal sterile alpha motif (SAM) domain (also known as the pointed or helix-loop-helix domain), which achieves dimerization and oligomerization by its possession of two binding interfaces (246); and the C-terminal DNA binding domain which binds the consensus sequence (GGAA/T) (247). The general activity of ETV6 is that of a transcriptional repressor - complexing with the co-repressor molecules histone deacetylase-3, N-CoR (248), mSin3a, and SMRT (249). Sumoylation - a small protein modification similar to ubiquitination (though less likely to lead to degradation) - can also modulate ETV6 transcriptional activity (250, 251). ETV6 is expressed widely in both embryonic and adult tissues (252). The embryonic lethality exhibited by ETV6 -/- mice at day 10.5 to 11.5 is thought to arise due to a defect in yolk sac angiogenesis (253). A role for ETV6 in early hematopoiesis has also been observed (254). ETV6-mediated transcriptional repression appears to have a growth suppressive effect as its overexpression in Ras-transformed fibroblasts results in inhibited growth and impaired soft agar colony formation (255). Moreover, loss of 40 heterozygosity at the ETV6 locus has been implicated in tumours of the blood and ovary (256-258). Therefore ETV6 is thought to act as a tumour suppressor. ETV6 is the frequent target of chromosomal translocations that give rise to human tumours, particularly to leukemias. Oncogenic ETV6 fusion proteins contain the N-terminal SAM domain partnered with a protein containing a DNA binding domain (AML1 (259)), or more likely a tyrosine kinase domain (ABL (260), ARG (261), JAK2 (262), and PDGFRp (263)). For this latter group, the polymerization of ETV6 is sufficient to result in a constitutively active PTK, leading to transformation. It is not clear whether inactivation of wildtype ETV6 by binding to fusion protein molecules also plays a role in oncogenesis mediated by ETV6 fusion proteins, although it has been found that ETV6-AML1 fusions are frequently accompanied by the deletion of the normal ETV6 allele on chromosome 12 (264). 1.5.2 NTRK3 Neuronal differentiation and survival is greatly influenced by a class of peptide ligands known as neurotrophins. These molecules bind with high affinity to receptors of the neurotrophic tyrosine receptor kinase (NTRK) family, of which there are three members (NTRK1, NTRK2 and NTRK3, formerly known as TrkA, TrkB and TrkC, respectively) (265). Neurotrophins can also bind to the low-affinity p75 receptor, a member of the tumour necrosis factor (TNF) death receptor superfamily (266). Each NTRK has a preferred ligand partner, with nerve growth factor (NGF) binding NTRK1, brain-derived neurotrophic factor (BDNF) binding NTRK2 and neurotrophin-3 (NT-3) binding NTRK3 (265). However, at higher concentrations NT-3 can bind and activate 41 both NTRK1 and NTRK2 (267). Similar to most RTKs, ligand binding to NTRK molecules induces dimerization and activation of tyrosine kinase activity. Interestingly, both NTRK2 and NTRK3 have alternatively spliced forms of unknown function that include an insert within the kinase domain, suppressing its enzymatic activity (268). Subsequent effects on proliferation, differentiation, and survival are mediated by stimulation of downstream signaling pathways by activated NTRK molecules. In the case of NTRK3, autophosphorylation of tyrosine 516 serves as a docking site for the SH2 domains of both She (leading to Ras/Erk activation) and Gab1 (activating the PI3K/Akt cascade through Gab1*p85 interactions) (269). Phosphorylation of tyrosine 820 allows for the binding of phospholipase-Cy (PLCyl), leading to its activation. Active PLCyl will eventually activate protein kinase C isoforms, and this may play a role in neurotrophin-mediated survival and differentiation (270, 271). Other SH2-domain-containing proteins that have been shown to bind activated NTRK3, and may affect the signaling response to neurotrophins are SH2BP and rAPS (272). As normal NTRK expression is limited almost exclusively to neuronal cells, it has been hypothesized that aberrant NTRK expression in other tissues, free from the regulatory controls present in nerve cells, can lead to oncogenesis. Several lines of evidence support this idea. NTRK1 was initially isolated as a sequence that undergoes genetic rearrangement in colon carcinoma biopsies (273). Eventually it was determined that this sequence consisted of a fusion of tropomyosin (TPM3) and the tyrosine kinase domain of NTRK1. TPM3-NTRK1 (also known as Trk-T1) fusions have since been detected in papillary thyroid carcinomas (274). There is evidence that NTRK1 supports the progression of prostate (275) and breast (276) tumours, as well as those of the 42 nervous system (277, 278). Finally/activating NTRK1 mutations have been described in AML (279). As mentioned above, a genome-wide study to find genes that are required for anoikis-resistance and metastasis identified NTRK2 as a major mediator of these processes (231). In neuroblastoma, NTRK2 has also been implicated in prevention of apoptosis during tumour progression (280), and NTRK2 can co-operate with c-Met to promote invasion (281). NTRK3 expression was detected in a majority (86%) of soft tissue sarcomas tested in a recent report (282), while NTRK3 mutations have been detected in colon carcinomas (283). However, NTRK3 expression is an indicator of positive prognostic outcome in neuroblastomas and medulloblastomas, possibly relating to its ability to induce differentiation in neuronal cells. Nevertheless, there is accumulating evidence for NTRK family members playing a role in cancer progression. Thus, by determining the signaling pathways that support ETV6-NTRK3-mediated transformation, potential avenues for therapeutic intervention against a wide range of tumours may be uncovered. 1.5.3 ETV6-NTRK3 Fusion Expression in Human Tumours 1.5.3.1 Congenital Fibrosarcoma Congenital fibrosarcoma (CFS) is a soft-tissue spindle-cell tumour which.occurs exclusively in patients less than two years of age. Despite its high rate of recurrence, CFS has an excellent prognosis and low metastatic rate (284). Histologically, CFS is indistinguishable from adult-type fibrosarcoma (ATFS), which has a poorer prognosis and requires a more aggressive treatment regime (284). Furthermore, CFS may resemble other, benign lesions of fibroblasts such as infantile fibromatosis and 43 myofibromatosis (285). Therefore, molecular markers were sought to distinguish CFS from these other pathological entities so as to ensure that a proper course of treatment would be available for these young patients. Knezevich et al., discovered a recurrent t(12; 15)(p13;q25) rearrangement in CFS tumour samples, and upon cloning of the breakpoint established the occurrence of ETV6-NTRK3 fusion transcripts (286). EN transcripts were absent from ATFS and the benign fibromatoses. Thus, EN expression is unique to CFS among fibrous lesions and is now a useful diagnostic tool for the disease. ' q 1.5.3.2 Congenital Mesoblastic Nephroma Congenital mesoblastic nephroma (CMN) is an infantile spindle cell tumour of the kidney that is relatively benign in its progression (287). CMN is subdivided into "classical" and "cellular" forms based on the degree of cellularity and mitotic activity. As cellular CMN shares clinical features with CFS, such as low metastatic potential and good prognosis, several groups examined cases of cellular CMN and detected EN expression (288-290), while expression of EN transcripts was absent in the classical subtype of the disease (288). Additionally, both cellular CMN and CFS were shown to have a recurrent trisomy of chromosome 11 (284, 288, 291). While the significance of this is unknown, it has been postulated that this trisomy may contribute to the pathology of the disease by providing an extra copy of the insulin-like growth factor-ll (IGF-II) gene. The relevance of this will be discussed below. 44 1.5.3.3 Acute Myelogenous Leukemia It had been widely accepted that fusion oncogenes are restricted in their expression to a specific cell lineage (292). To explain the strong association between acquired chromosomal translocations and specific tumour type, it had been proposed that only certain cell lineages would allow expression of a given fusion transcript, or that expression of this transcript would only have oncogenic outcome in a particular cell type (293). However, evidence that the ability of ETV6-NTRK3 to induce transformation was free from the constraints of lineage specificity came from a reported EN fusion in a single case of adult acute myelogenous leukemia (294). Unlike EN transcripts isolated from CFS and CMN samples, which contained the first five exons of ETV6, this case showed a form of EN containing only ETV6 exons 1-4. When this EN variant was expressed in the Ba/F3 murine hematopoietic cell line, it was able to induce a rapid myeloproliferative disorder when transplanted into the bone marrow of lethally irradiated mice (245). 1.5.3.4 Secretory Breast Carcinoma Further evidence that EN has transforming activity in multiple cell lineages, comes from a study detecting EN transcripts in a majority (12/13) of secretory breast carcinomas (SBCs) (244), a tumour of epithelial origin. These tumours showed a (12; 15) translocation with an identical breakpoint to that seen in CFS cases, although unlike CFS, no trisomy of chromosome 11 was detected in SBC. Moreover, overexpression of EN in mouse mammary epithelial cell lines Eph4 and Scg6 led to a transformed phenotype (244). 45 1.5.4 ETV6-NTRK3 Signal Transduction 1.5.4.1 Activation of Growth Factor-Regulated Pathways Soon after EN transcripts were isolated from the original CFS cases, the gene was cloned and stably introduced into mouse NIH 3T3 fibroblasts, so that signaling properties of this tyrosine kinase could be easily studied (242). It was found that EN could potently transform these cells, both in vitro and in vivo, while mutational analysis established that both the ETV6 SAM domain and an active kinase were required for transformation. Phospholipase-C (PLC)-y could be co-immunoprecipitated with EN. However mutation of tyrosine 516 - the site of PLC-y binding - to phenylalanine, had no effect on the transformed phenotype. Not surprisingly, neither SHC, Grb2 nor p85 could be shown to associate with EN, as the residues from wildtype NTRK3 that are involved in the binding of these molecules are not present in the EN fusion molecule (242). Despite this fact, subsequent work determined that EN expression could robustly activate both the PI3K/Akt and Ras/Erk cascades in the absence of serum, leading to an upregulation in cyclin D1 levels (295). Abrogation of either of these pathways dramatically inhibited transformation, indicating these signaling cascades work in concert to initiate EN-mediated oncogenesis. 1.5.4.2 Polymer Formation Greater insight into the mechanism of EN transformation was gained through studies that examined the binding capabilities of the EN SAM domain (296). Recent work has determined that wildtype ETV6 is able to form a helical polymer through 46 homotypic interactions of its SAM domain. This domain contains two interfaces of binding whose structure must be maintained for polymer formation (297). To assess whether EN can also undergo polymer formation, and whether this process was required for transformation, wildtype EN, and EN constructs with various mutations in the ETV6 SAM domain were expressed in NIH 3T3 fibroblasts (296). Wildtype EN did indeed form large polymer structures, which could be detected by electron microscopy. Not only did mutations in either of the SAM binding interface regions block polymer formation, they blocked transformation as well. Since these molecules are able to dimerize, but not polymerize, it indicated that polymerization is required for EN transformation. To confirm this, the ETV6 domain of EN was replaced with the FKBP inducible dimerizer. Upon induction of dimerization, this mutant EN molecule had constitutive kinase activity, but did not transform cells. Finally, an isolated EN SAM domain acted in a dominant negative manner to block oncogenesis, implying a potential avenue of therapeutic intervention for EN-based tumours, as well as the wide variety of malignancies which express an ETV6-containing fusion protein. 1.5.4.3 TGF-p Signaling Increasing complexity in the signaling pathways affected by ETV6-NTRK3 expression was uncovered in a recent study examining its effects on the transforming growth factor (TGF)-p pathway (298). TGF-p ligand binds to the type II TGF-p receptor (TpRII), which undergoes an activating conformational change allowing it to phosphorylate the type I TGF-p receptor (TpRI) on serine residues (299). Activated TpRI then phosphorylates the intracellular proteins Smad2 and Smad3, which can then 4 7 couple with Smad4 to induce transcriptional upregulation of TGF-p-controlled genes (299). Contrary to its name, the TGF-p pathway has a general inhibitory role on early carcinogenesis, and both mutations and transcriptional repression of the type II TGF-p (TpRII) receptor have been catalogued in tumour tissue (299). Initially, it was observed that a reporter construct that measured TGF-p-induced transcription was inhibited by EN expression (298). Further work showed that this was caused by EN binding directly to TpRII, blocking its interaction with TpRI and preventing Smad2/3 phosphorylation (298). Somewhat paradoxically, TGF-p ligand mRNA and protein was found to overexpressed in EN-transformed cells (C. Tognon and P. Sorensen, unpublished observations). However, as EN is able to down-regulate the ability of the transformed cell to respond to TGF-p signals, it is hypothesized that the negative regulatory effects of secreted TGF-p are limited to cells surrounding the actual tumour. This may serve to modulate the response of the immune system and allow EN-expressing cells to evade the negative impact this would have on tumour growth. 1.5.4.4 Role of the IGF-IR Pathway Besides the recurring t(12; 15) translocation seen in CFS samples, a consistent feature of cells from these tumours is a trisomy of chromosome 11. A prominent cancer-associated gene on this chromosome is IGF-II, and a previous report indicated IGF-II transcripts were elevated in cases of the histogenetically related CMN (300). Therefore CFS samples were examined for IGF-II transcript levels (301). Both CFS and CMN showed elevated IGF-II mRNA, while this was not the case for infantile fibromatosis or the classical form of mesoblastic nephroma. It was thus postulated that the IGF 48 signaling pathway played an important role in EN-mediated transformation. Further evidence for the importance of IGF signaling came from experiments where EN was expressed in mouse embryo fibroblasts (MEFs) derived from animals with a targeted deletion of the IGF-I receptor (IGF-IR) gene (301). Other groups had shown that these cells were refractory to transformation by a wide-ranging set of oncogenes (302), and this was found to be the case for EN as well (301). Although it had previously been established that EN activated both PI3K/Akt and Ras/Erk signaling (295), the mechanism for this had remained unclear. As mentioned, EN is unable to directly bind SHC, Grb2 or p85 due to the position of the fusion point (242). It was therefore hypothesized that another protein was able to bind EN to link it to these signaling pathways. A clue as to the identity of this protein came from phosphotyrosine Western blots performed on lysates from EN expressing cells. A phosphorylated band of ~160-180 kDa was consistently seen in these samples which was not present in control cells. As this was the approximate size of the insulin receptor substrate (IRS)-1, and this protein is the major adaptor protein in JGF-IR signaling, these blots were re-probed with a-IRS-1 antibodies. Not only did this confirm the identity of the tyrosine-phosphorylated band as IRS-1, but further co-immunoprecipitation experiments showed a physical interaction between EN and IRS-1. Interestingly, although IGF-IR-null cells that expressed EN were not transformed, EN was still able to bind and phosphorylate IRS-1 in this context. Thus, the precise role of IGF-IR in EN transformation has yet to be determined. 49 1.6 AIMS AND HYPOTHESES Despite preliminary evidence that the insulin-like growth factor / insulin receptor substrate signaling axis contributes to the cellular transformation induced by expression of ETV6-NTRK3, the exact role of several components of this pathway have yet to be fully defined. Therefore, the studies presented in this thesis were undertaken with the following aims: 1) To determine the domain(s) of IRS-1 that are required for physical binding to EN, use this information to inhibit this interaction, and determine if EN»IRS interactions are a required step for EN-induced transformation. 2) To elucidate the role of IGF-IR expression in EN-mediated transformation 3) To determine the role of the mTOR pathway in anchorage-independent growth induced by EN. 4) To examine the potential for EN to activate IGF-IR signaling in a ligand-independent manner. 50 CHAPTER II MATERIALS AND METHODS 2.1 CELL LINES The human embryonic kidney cell line HEK293T was obtained from the American Type Culture Collection (ATCC) (cat. # CRL-1573) and grown in Dulbecco's modified Eagle's medium (DMEM) containing 9% fetal bovine serum (FBS) (Invitrogen). NIH 3T3 fibroblast cells were obtained from ATCC (cat. # CRL-1658) and maintained in DMEM containing 9% calf serum (CS) (Invitrogen). R- IGF-IR knockout mouse embryonic fibroblasts were a kind gift of Dr. Renato Baserga (Kimmel Cancer Center, Philadelphia, PA, USA). They were engineered to overexpress IGF-IR by retroviral transfection with IGF-IR cDNA in the MSCV hygromycin vector (see below) and designated R+ cells. R-and derivative cells were grown in 9% FBS and DMEM. IRS-1 knockout mouse embryo fibroblasts (S-) were a gift of Dr. Stuart Orkin (Dana Farber Cancer Institute, Boston, MA, USA). Cells engineered to overexpress IRS-1 by retroviral transfection with IRS-1 cDNA in the MSCV neomycin vector were designated S+ cells. S- and derivative cells were grown in 9% FBS and DMEM. The BOSC23 packaging cell line was obtained from Dr. R. Kay (Terry Fox Laboratory, Vancouver, BC, Canada) and grown in 9% FBS and DMEM. 32D mouse myeloid progenitor cells were a kind gift of Dr. Rob Kay. They were grown in RPMI medium containing 9% fetal bovine serum (FBS) (Invitrogen). Hepa 1-6 mouse hepatoma cells were obtained from ATCC (cat. # CRL-1830), and maintained in DMEM containing 9% FBS. All culture media was supplemented with 51 antibiotic/antimycotic solution (Gibco). Starvation of cells was accomplished by replacing the high-serum (9% FBS) media with low-serum (0.25% FBS) media for 16 -24 h, after washing once in phosphate-buffered saline (PBS). 2.2 CLONING OF DNA CONSTRUCTS Restriction digest analysis and DNA sequencing confirmed the accuracy of cloning of all plasmid DNA constructs. 2.2.1 MSCV Plasmids The murine stem cell virus (MSCV) vector (Clontech), was derived from the murine embryonic stem cell virus and the LN retroviral vectors (303, 304). After transfection, MSCV vectors express a transcript containing the extended viral packaging signal, an antibiotic resistance gene (either puromycin (puro), hygromycin (hygro), or neomycin (neo)), and a gene of interest inserted into the multiple cloning site. The vectors achieve stable high-level gene expression through a specifically designed 5' long terminal repeat. 2.2.2 ETV6-NTRK3 Constructs cDNA encoding EN was C-terminally tagged with V5-His or hemagglutinin (HA) using pcDNA3.1/V5-His-TOPO (Invitrogen) and pcDNA3.1(HA) (a gift of Dr. Valentina Evdokimova, BC Cancer Research Centre, Vancouver BC), respectively. Full length EN in MSCVpuro was cloned as described in (242). The cDNA encoding N-myristoylated ETV6-NTRK3 was produced via PCR amplification of EN residues 43-633 using a 5' 52 primer containing a Hpa\ restriction site and the N-myristoylation sequence from the Lck tyrosine kinase with a 3' primer containing an EcoR\ restriction site. 5' Hemagglutinin (HA) tagged versions of wildtype and N-myristoylated EN cDNA were produced by PCR amplification of EN residues 43-633 using 5' primers containing a H p a l restriction site and two tandem HA DNA sequences (for HA EN-wildtype) or a H p a l restriction site, the Lck N-myristoylation sequence and two tandem HA DNA sequences (for HA EN-myristoylated) with a 3' primer containing an EcoRI restriction site. Polymerase chain reaction was performed using high fidelity Pfu DNA polymerase (Stratagene). A l l EN PCR products were digested with Hpa\/EcoR\ and cloned into MSCVpuro. - ' 2.2.2 IRS-1 Constructs HA-tagged portions of IRS-1 were created by PCR and cloned into the pcDNA3.1 plasmid as follows: IRS-1 B, PH domain alone (open reading frame nucleotides 16-351); IRS-1 C, PH/PTB domains (nucleotides 16-779); IRS-1 D, PH/PTB/SAIN domains (nucleotides 16-1391); IRS-1 E, PTB domain alone (nucleotides 436-779). The MSCVneo/IRS-1 and MSCVhygro/IRS-1 retroviral expression vectors were created by excising the mouse IRS-1 cDNA from pBluescript SKII/IRS-1 plasmid (Dr. R. Baserga) using EcoRV and cloning into the H p a l site of MSCVneo or MSCVhygro. MSCVneo/HA-IRS-1C was created by excising HA-IRS-1C from pcDNA3.1 with Hind\\\ and Not\, treating with DNA polymerase Klenow fragment to give blunt ends, and cloning into the Hpa\ site of MSCVneo. HA-tagged IRS-1 was created by PCR amplification of full-length IRS-1 (5'-primer, 5'-TCCACAAGCTTTTATGGCGAGCCCTCC-3'; 3'-primer, 5'-53 CCCCCCATGCGGCCGCAATTGACGGTCCTCTGGTTG-3') and ligation into pMH (Roche) with Hind\\\ and Not\ restriction sites. 2.2.4 IGF-IR Constructs MSCVhygro/IGF-IR and MSCVneo/IGF-IR(Y950F) plasmids were a gift of Dr. R Baserga. IGF-IR(K1003A) was excised from pBPV(KA) (Dr. R Baserga) using Sad and Not\, treated with mung bean nuclease to give blunt DNA ends, and cloned into the Hpal site of MSCVneo. cDNAs encoding N-myristoylated IGF-IR intracellular (IC) domain (wildtype, Y950F, and K1003A) were produced via PCR amplification of IGF-IR residues 2920-4104 using a 5' primer containing a EcoRI restriction site and the N-myristoylation sequence from the Lck tyrosine kinase with a 3' primer containing an HA tag followed by a Hpal restriction site. IGF-IR(IC) was similarly generated using a 5' primer that omitted the N-myristoylation sequence. Note that the numbering system for IGF-IR amino acids refers to the mature peptide, where amino acid 1 corresponds to amino acid 31 of the peptide synthesized from IGF-IR cDNA. 2.2.5 Akt-myr Akt cDNA with a 5' myristoylation sequence and a 3' Myc-tag was excised from pUSE(Akt-myr) (a gift of Dr. Sandra Dunn, Child & Family Research Institute, Vancouver BC) using Xbal, treated with mung bean nuclease, and cloned into the Hpa\ site of MSCV neo. 54 2.3 CELL TRANSFECTION 2.3.1 Transient Transfections Different combinations of HA- and V5-tagged constructs were transiently co-transfected in HEK293T cells using FuGENE 6 transfection reagent (Roche Applied Science). Lysates were collected 36h after transfection (as described below), and immunoprecipitations (a-HA, a-V5) were immediately performed, followed by V5 or HA Western blotting. 2.3.2 Transduction of Genes Using MSCV Retroviral Vectors Infections of fibroblasts and 32D cells were carried out using the MSCV retroviral expression system. The BOSC23 packaging cell line was transfected with target plasmids using the calcium phosphate method as described previously (294). Cells co-expressing two different constructs were made by transfecting and selecting for the control (MSCV) or ETV6-NTRK3 constructs first then co-expressing and selecting for the second construct (e.g. wild-type and mutant IGF-IR). Expression of all proteins was confirmed by Western blotting. 2.3.3 siRNA-Mediated Gene Knockdown siRNA constructs were obtained to mouse IRS-T; 507 (targeting nt 194-219), and mouse IRS-2; 824 (nt 1810-1834); 825(nt 1419-1443); 826 (nt 3525-3549) (Invitrogen). A non-specific medium GC content siRNA was used as control. R+(HA)EN fibroblasts were grown to 25% confluency in 35mm dishes. 100 pmol of each siRNA, in duplicate, was introduced into cells using the Lipofectamine 2000 transfection reagent (Invitrogen). 5 5 When IRS-1 and IRS-2 siRNA were transfected in tandem, 50 pmol of each RNA was used for a total of 100 pmol. siRNA/lipofectamine duplexes were removed after an 8-hour incubation by replacement with fresh media. Efficacy of knockdown was determined by Western blotting against IRS-1 or IRS-2 (measured 3, 5 and 7 days post-transfection). For growth of these cells in spheroid or soft agar culture (see below), monolayer cells were trypsinized 48h post-transfection, and plated under anchorage-independent conditions. 2.4 ASSESSMENT OF ANCHORAGE-INDEPENDENT GROWTH 2.4.1 Soft Agar Colony Formation Assay Fibroblast cells were seeded in triplicate at a concentration of 8 x 103 cells/35-mm dish. Bottom layers were made up of 0.4% agar in DMEM with 9% CS or FBS. Cells were resuspended in a top layer of 0.2% agar in DMEM with 9% CS/FBS. Cells were fed every other day by placing two drops of medium on the top layer. After 10-14 days at 37 °C the number of single cells and colonies/high power field were counted. Results were formulated as a percentage of macroscopic (> 0.1 mm) colonies formed/total number of cells plated. Cell lines were examined at 5 sites per well for a total of 15 fields in a minimum of three separate soft agar experiments. Statistical significance of differences in the respective groups was evaluated using the student's t-test; p'values < 0.05 were considered to be of statistical significance. 56 2.4.2 Growth of Fibroblasts as Multicellular Spheroids Confluent monolayers were trypsinized, resuspended as single cells, and replated at a concentration of 1.0 x 105 cells/ml on tissue culture dishes that had been coated with sterile 1.4% agar for anchorage independent suspension cultures. Cells were isolated by centrifugation (1000 r.p.m.) every 48 hours and replated in new media on fresh agar-coated plates. 2.4.3 In vivo Tumor Growth in Nude Mice Pathogen-free male athymic nude mice, 6-8 weeks old were obtained from Harlan. One million R- and derivative cells were injected subcutaneously at three sites/animal (five animals/group), for a total of 15 monitored sites for each cell line. Animals were housed in laminar flow racks arid microisolator cages under specific pathogen-free conditions and received autoclaved food and water. Nude mice were evaluated for tumor growth periodically until 20 days after injection, a time point at which tumor growth in the R+EN group necessitated the termination of the experiment. Tumor volume was estimated using the following equation: tumor length x (tumor width)2 x 0.5236. The statistical significance of differences in tumor size in the respective groups was evaluated using the Mann-Whitney ranks test; p values < 0.05 were considered to be of statistical significance. These studies were carried out with ethical approval from the University of British Columbia Animal Care Committee (for certificate, see Appendix). 57 2.4.4 Kinase Inhibitor Studies The following kinase, inhibitors were used: LY294002 (5-25 u,M), PI3-kinase inhibitor; U0126 (25 uM), MEK1/2 inhibitor; rapamycin (20-50 nM), mTOR inhibitor. Inhibitors or vehicle control (DMSO) were added directly to the media of cells grown as anchorage-independent spheroids. For soft agar experiments involving LY294002 or rapamycin, the inhibitors or vehicle control were included at the indicated concentration in both layers as well as in the feeding media. 2.5 PROTEIN ANALYSIS 2.5.1 Western Blotting Monolayer cells grown to ~ 90% confluence in 10 cm dishes were rinsed once with PBS and lysed with either 400-1000 pi of phosphorylation solubilization buffer (PSB; 50 mM HEPES, 100 mM NaF, 10 mM Na4P 20 7, 2 mM Na3V0 4, 2 mM EDTA, 2 mM NaMo0 4 and 0.5% NP40) or Radio Irnmunoprecipitation Assay (RIPA) buffer (50 mM Tris HCI, 150 mM NaCl, 0.1% SDS, 0.25% sodium deoxycholate and 0.5% NP-40) containing protease and phosphatase inhibitors (10 ug/ml Leupeptin, 10 ug/ml Aprotinin, and 250 pM PMSF). Anchorage-independent cultures were spun down at 1000 rpm, washed once with PBS and lysed in 250 pi of the same buffer with 15 passages through a 22-gauge syringe to break apart spheroid structures. All cells were solubilized for 30 min at 4°C on a shaking platform. Lysates were cleared by centrifugation at 12,000 x g for 10 min at 4°C. Protein quantification of the lysates was performed using a detergent-compatible protein assay kit from Bio-Rad. Total cell lysate (10-30 pg) was mixed with Laemmli buffer and electrophoresed on 7.5-12% SDS-58 polyacrylamide gels according to standard methods. Electrophoresed proteins were transferred to Nitrocellulose membranes (Bio-Rad) before immunoblot analysis with the indicated antibodies. 2.5.2 Immunoprecipitation Cell lysates to be subjected to immunoprecipitation (IP) were collected as described above. 0.5 - 1.5 mg of protein was then incubated in 1.5 ml Eppendorf tubes with 30 u.l of either Protein A- or Protein G-conjugated sepharose beads (mouse monoclonal antibodies: Protein G; rabbit polyclonal antibodies: Protein A - see below) and 2 u,g of antibody to the protein of interest. For each experiment total volumes were equilibrated to ensure equal concentrations of lysate, beads, and antibody. After incubation overnight at 4 °C on a rotating platform, tubes were centrifuged at 2000 rpm for 5 minutes and the supernatants discarded. Pellets were washed two times in wash buffer (PSB containing 0.1% NP-40) and prepared for Western blotting as described above. 2.5.3 Subcellular Fractionation R-EN(HA), R+EN(HA), R-ENmyr(HA) and R+ENmyr(HA) monolayers at -90% confluence in 15 cm dishes were starved overnight (16 h) in 0.25% serum media, washed with PBS and then resuspended in. 1.5 ml of homogenization buffer (20 mM HEPES, 50 mM KCI, 2 mM MgCI2, 1 mM dithiothreitol, 0.25% NP-40 + protease and phosphatase inhibitors). Cells were incubated in homogenization buffer on ice for 10 min. and then lysed with 10 passages through a 22-gauge needle. Samples were 5 9 centrifuged at 800 x g for 10 min. at 4 °C to remove nuclei and cell debris. Supernatants were collected and centrifuged at 15,000 x g for 15 min. at 4 °C. The resulting supernatant was removed and used as the cytoplasmic fraction. The heavy membrane pellet was resuspended in homogenization buffer and designated the membrane fraction. 30 pg total protein from each fraction was loaded onto SDS-PAGE gels, and immunoblotted with a-HA antibodies. Purity of the cytoplasmic and membrane fractions was validated with a-Grb2 and oc-caveolin-1 antibodies, respectively. 2.5.4 Antibodies The antibodies used were as follows: HA mouse monoclonal (IB 1:1000; IP 5 pi of 1/50 dilution; BAbCO #MMS-101P) - V5 mouse monoclonal (IB 1:1000; IP 2 ug/ml; Invitrogen #R960-25) - IRS-1 rabbit polyclonal (IB 1:1000; IP 2 pg/ml; Upstate Biotechnology #06-248) - IRS-2 rabbit polyclonal (IB 1:1000; IP 2 ug/ml; Upstate Biotechnology #06-506) - NTRK rabbit polyclonal (IB 1:1000; IP 1 pg/ml; Santa Cruz #sc-139) - 4G10 Anti-phosphotyrosine (IB 1:3000; IP 2 ug/ml Upstate Biotechnology) -phospho-Akt (Ser 473) rabbit polyclonal (IB 1:1000; Cell Signaling #9271) - total Akt rabbit polyclonal (IB 1MO0O; Cell Signaling #9272) - phospho-MEK1/2 (Ser217/221) rabbit polyclonal (IB 1:1000; Cell Signaling #9121) - total MEK1/2 rabbit polyclonal (IB 1:1000; Cell Signaling #9126) - Cyclin D1 mouse monoclonal (IB 1:2000; Cell Signaling #2926) - actin goat polyclonal (IB 1:5000; Santa Cruz #sc-1616) - phospho-GSK-3p (Ser 9) rabbit polyclonal (IB 1:1000; Cell Signaling #9336) - Grb2 mouse monoclonal (IB 1:5000; BD Transduction Labs #610111) - caveolin-1 mouse monoclonal (IB 1:1000; BD Transduction Labs #610406) - PARP (IB 1:1000; Cell Signaling #9542) -60 total 4E-BP1 (IB 1:1000; Cell Signaling #9452) - phospho-S6 (Ser240/244) rabbit polyclonal (IB 1:5000; Cell Signaling #2215) - IGF-IR p-subunit rabbit polyclonal (IB 1:1000; IP 2 ug/ml Santa Cruz #sc-713). 2.5.5 IGF ELISAs Quantification of levels of IGF-I and IGF-II in media collected from R+ derived cell lines grown in DMEM + 0.25% FBS was achieved using the Enzyme Linked Immunosorbent Assay (ELISA) method (R&D Systems). Briefly, 50 u.l of two-fold diluted conditioned media, media alone, or IGF standard, was added to 96-well plates coated with antibodies towards either mouse IGF-I or IGF-II and incubated for 2h at room temperature. After five washes, an enzyme-linked polyclonal antibody specific for mouse IGF-I or IGF-II was added to the wells, and plates were incubated for 2h at room temperature. Plates were again washed five times and then incubated with 100 u.l of substrate solution for 30 min. at room temperature. Finally, 100 uJ of stop solution was added to each well and optical density at 450 nm was determined using a Versa Max plate reader (Molecular Devices). Concentration of IGF ligand in the collected media was calculated from the standard curve generated in parallel. Average concentrations represent data collected from three separate experiments. 2.6 IMMUNOFLUORESCENCE MICROSCOPY Fibroblast cells exponentially growing on coverslips were rinsed with PBS and fixed with cold methanol at 20°C for 10 min. To detect HA-tagged EN ((HA)EN) or the N-myristoylated HA-tagged EN construct ((HA)ENmyr), coverslips were incubated 61 \ overnight with mouse anti-HA antibody (1:1,000 dilution) followed by the secondary antibody Oregon green 514-conjugated goat anti-mouse antibody (Molecular Probes). Slides were counterstained with DAPI (4 ,6 -diamidino-2-phenylindole) and analyzed using a Zeiss Axioplan epifluorescent microscope equipped with a COHU charge coupled-device camera. For confocal images, fibroblast cells grown on coverslips;were starved overnight (16 h) in 0.25% serum medium, rinsed with PBS, and fixed with 4% paraformaldehyde for 10 min followed by permeabilization with 0.01% Triton X-100 for 10 min. Coverslips were incubated for 1.5 h with mouse anti-HA antibody (1:200 dilution) followed by incubation with Oregon green 514-conjugated goat anti-mouse antibody. Slides were counterstained with DAPI, and images were obtained using a Leica DM IRE2 inverted confocal microscope. For each immunofluorescence experiment, control staining in the absence of primary antibodies or using pre-immune antibodies showed no signals. 2.7 CELL PROLIFERATION ASSAYS 2.7.1 BrdU Proliferation Assay R-EN and R+EN spheroids were grown in media containing 9% FBS for the times indicated. Fresh media containing 9% FBS and 100 u.M bromo-deoxyuridine (BrdU; Sigma) was then added for a further 24h prior to harvesting. 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. All conditions were repeated in triplicate and the percentage of BrdU positive cells was calculated by counting cells in five representative high-power, fields 6 2 (hpf) for each condition (approx. 200400 cells/hpf). 2.7.2 FACS Analysis Monolayer and spheroid cells were resuspended in a 1:1 solution of PBS and Accumax cell dissociation reagent (Innovative Cell Technologies), and incubated for 15 min. at 37 °C with periodic vortexing to break apart cell clumps. Cells were then passed through a 70 pM nylon strainer, fixed in 70% ethanol, treated with 100 g/ml RNase, stained with 50 g/ml propidium iodide, and live cells analysed for DNA content using the FACSCalibur Flow cytometry system with CellQuest and Modfit LT analytic software (Becton Dickinson, San Jose CA). 2.8 MEASUREMENT OF CELL DEATH 2.8.1 Trypan Blue Exclusion Assay Cell viability of 32D mouse hematopoietic cells was measured by their ability to exclude trypan blue. 5.0 x 104 cells per 3.5 mm dish were plated at time 0 and collected at the indicated times. Cells were then spun down and resuspended in a 1:1 solution of DMEM and trypan blue (final trypan blue concentration = 0.2%). Numbers of stained and unstained cells were counted using a hemocytometer. The calculated percentage of unstained cells represents the percentage of viable (surviving) cells. 2.8.2 Caspase-3 Activity Assay The activity of caspase-3 was determined by using the fluorogenic caspase-3 substrate (Z-Asp-Glu-Val-Asp)2-Rhodamine 110-bisamide (Calbiochem). Fibroblast cells 63 were plated as monolayer or spheroids in 10 cm dishes and grown for the times and conditions indicated. Cells were collected by centrifugation at 1000 rpm, washed once in PBS, and lysed in Caspase-3 lysis buffer (10 mM HEPES pH 7.4, 50 mM NaCl, 2mM MgCI2, 5 mM EGTA, 0.2% CHAPS and protease inhibitors as described above). 100 ng of protein lysate was combined with DTT-containing 2x Reaction buffer and 1 mM caspase-3 substrate and incubated at 37 °C for 1 h. The fluorescence intensity was measured at an excitation wavelength of 485 nm and an emission wavelength of 530 nm with a Wallac 1420 multilabel counter (Perkin Elmer). The relative amount of caspase-3 activity was expressed as the fluorescence ratio between the various treatments and untreated R+EN fibroblast monolayer grown to 80% confluence. 3 64 CHAPTER III ETV6-NTRK3 BINDS THE PTB DOMAIN OF IRS-1/IRS-2: AN ESSENTIAL STEP IN ETV6-NTRK3-MEDIATED TRANSFORMATION 3.1 INTRODUCTION Receptor tyrosine kinases are a highly regulated family of proteins in normal cells, but may undergo mutations or structural alterations to become oncoproteins in human malignancies. One such mechanism is chromosomal rearrangement, which can lead to the formation of a gene fusion and is known to involve receptor tyrosine kinase-encoding genes (305). A oncogenic fusion protein is created in which the tyrosine kinase domain is fused to a dimerization domain contributed by the fusion partner (305). Resulting chimeric tyrosine kinases are thought to constitutively activate downstream signaling cascades of the wild-type receptor tyrosine kinases, potentially leading to oncogenesis. However, the mechanisms by which many of these oncogenic tyrosine kinases link to effector pathways have not been fully characterized. A version of this chapter has been published: Lannon, CL, Martin MJ, Tognon CE, Jin W, Kim SJ, Sorensen PH. J. Biol. Chem 2004. Feb 20;279(8):6225-34. Cristina Tognon performed the experiments in Fig. 3.1B and 3.1C. 65 The t(12;15)(p13; q25) translocation associated with * congenital fibrosarcoma (286), cellular mesoblastic nephroma (288, 289), adult acute myeloid leukemia (294) and human secretory breast carcinoma (244), generates a gene fusion encoding the sterile alpha motif dimerization domain of the ETV6 transcription factor linked to the protein-tyrosine kinase domain of the neurotrophin-3 receptor NTRK3 (286). ETV6-NTRK3 (EN) expression leads to elevation of cyclin D1 mRNA and protein levels, which correlates with increased cell cycle progression (295). Additionally, previous work in the Sorensen laboratory demonstrated that EN expression in NIH 3T3 fibroblasts leads to constitutive activation of two of the major effector pathways of wild-type NTRK3 - the Ras/Erk mitogenic pathway and the phosphatidylinositol 3-kinase (PI3K) pathway leading to activation of the Akt survival factor (295). Phenotypic transformation and soft agar colony formation by EN-expressing cells are blocked by inhibition of either MEK1/2 or PI3K. However, interactions between EN and adaptor molecules known to link NTRK3 to Ras/Erk and PI3K/Akt pathways such as She, Grb2, SH2B, or the p85 subunit of PI3K (242), as well as ABL, Sre, and SHIP2 (CL Lannon, MJ Martin, CE Tognon, and PH Sorensen, unpublished data), were not detected. Wild-type NTRK proteins utilize juxtamembrane tyrosine residues (e.g. NTRK3 Tyr 516) to interact with several of these adaptors including She (306), Grb2 (307), and p85 (307, 308), but this residue is not present in the EN oncoprotein because of the position of the fusion point (242). Previous studies in the Sorensen laboratory found that EN fails to transform mouse embryo fibroblasts derived from mice with a targeted disruption of the insulin-like growth factor-l receptor (IGF-IR) gene, but that reintroduction of IGF-IR into these cells 66 restores EN transformation activity (301). This led to the examination of the relationship between IGF-IR signaling and EN transformation in more detail. A direct physical interaction between EN and the major IGF-IR substrate, insulin receptor substrate-1 (IRS-1) was observed. It was further found that IRS-1 is constitutively tyrosine-phosphorylated in EN-transformed cells and that EN«IRS-1 complexes bind both Grb2 and the PI3K p85 regulatory subunit. This strongly suggests that IRS-1 is functioning as the adaptor molecule linking EN to Ras/Erk and PI3K/Akt signaling (301). However, the mechanism by which IRS-1 interacts with EN remains unknown. IRS-1 along with IRS-2-4 comprise a family of tyrosine-phosphorylated scaffold proteins that are substrates for IGF-IR and the insulin receptor (63). Although IRS proteins lack enzymatic activity, they play key adaptor roles in linking the IGF-IR and insulin receptor to downstream pathways. IRS-1 and IRS-2 can each promote cell growth through activation of the PI3K/Akt and Ras/Erk pathways (309), whereas IRS-3 and IRS-4 appear to play a negative role in IRS-1/-2 signaling (310). Three different domains in IRS-1/-2 have been identified as potentially contributing to IGF-IR and insulin receptor binding: the pleckstrin homology (PH) domain, the phosphotyrosine binding (PTB) domain, and the She and IRS-1 NPXY binding (SAIN) domain (311). PH domains bind phospholipids, thereby mediating the interaction of signaling proteins such as IRS-1 with the plasma membrane (312). PTB domains of adaptor proteins bind to phosphorylated tyrosines within NPXY motifs in interacting proteins such as cell surface receptors (313), thus promoting receptor-adaptor interactions. The SAIN domain of IRS-1/-2 is postulated play a similar role in binding phosphorylated tyrosines, as it too has affinity for NPXY motifs (311, 314). In this chapter I now show that EN interacts 67 specifically with ,the PTB domain of IRS-1 and that this interaction is essential for EN transformation activity. Moreover, the transformation activity of EN can be inhibited by a dominant-negative IRS-1 construct, whereas IRS-1 overexpression in EN-transformed cells enhances the tumourigenic activity of this oncoprotein. In IRS-1 null cells, IRS-2 can substitute for IRS-1 to promote EN transformation. However, knocking down expression of both IRS-1 and IRS-2 together blocks EN-mediated anchorage-independent growth. These data highlight the essential role for EN«IRS interactions in the transformation of fibroblasts. 3.2 RESULTS 3.2.1 The PTB Domain of IRS-1 Mediates its Association with EN. Others in our laboratory previously demonstrated that IRS-1 is constitutively tyrosine phosphorylated in EN-transformed cells, that EN associates with IRS-1 in vitro, and that IRS-1 functions as the adaptor protein linking EN to the Ras/Erk and PI3K/Akt cascades which are essential for EN transformation (301). To further characterize the EN*IRS-1 interaction, I created a series of HA-tagged IRS-1 constructs expressing specific portions of the protein (Fig. 3.1 A). These constructs were transiently transfected into HEK293T cells along with V5-tagged EN. Expression of each construct was confirmed by a-HA irnmunoprecipitation followed by a-HA immunoblotting (Fig. 3.1 B). (V5)EN expression was confirmed by a-V5 irnmunoprecipitation followed by a-V5 immunoblotting (Fig. 3.1C). In order to determine the region of IRS-1 responsible for binding to EN, lysates 68 Figure 3.1: ETV6-NTRK3 fusion binds to the phosphotyrosine domain of IRS-1. (A) Schematic diagram describing the five HA-tagged IRS-1 constructs. (B) Immunoblots of HA immunoprecipitations from HEK293T cells co-transfected with V5-tagged EN and either 1) HA alone 2) HA-tagged EN or 3) one of the five HA-tagged IRS-1 constructs. Blots were probed with anti-HA and anti-V5 to detect immunoprecipitated HA-tagged IRS-1 constructs and co-immunoprecipitated V5-tagged EN protein, respectively. V5-tagged EN was used as a positive control. (C) Immunoblots of V5 immunoprecipitations from HEK293T cells co-transfected with V5-tagged EN and 1) HA alone, 2) HA-tagged EN, or 3) one of the five HA-tagged IRS-1 constructs. Blots were probed with anti-HA and anti-V5 antibodies to detect co-immunoprecipitated HA-tagged IRS-1 constructs and immunoprecipitated V5-EN, respectively. 69 oz, prj T J < X m < C D O o C Q O C D T J u J I I 331" COCO mco JJ JJ CO GO O 6 > > ( H A ) E N GO H A I R S 1 FL (HA) ( H A ) I R S 1 B ( H A ) I R S 1 C ( H A ) I R S 1 D ( H A ) I R S 1 E + rn z i < X > 55 CO m JJ CO X > 55 GO o „ JJ X GO > ~=: -n 33 JZ oo x w > in > CO > o rjj T J cn cn O O 1 .1 < — Oi X" GO m O X > 55 GO O TT I t t X > 55 GO ( H A ) E N H A ( H A ) I R S 1 B ( H A ) I R S 1 C ( H A ) I R S 1 D ( H A ) I R S 1 E J < > were immunoprecipitated with either a-V5 or a-HA antibodies, followed by immunoblotting with a-HA or a-V5 antibodies. HA-tagged EN, IRS-1 full-length (FL), (HA)IRS1C, (HA)IRS-1D and (HA)IRS-1E were all able to pull down (V5)EN (Fig. 3.1 B). , V5-tagged EN was able to immunoprecipitate HA-tagged EN as well as all IRS-1 constructs containing the phosphotyrosine binding (PTB) domain ((HA)IRS-1C, (HA)IRS-1D, and (HA)IRS-1E (Fig. 3.1C)). Only the fragment consisting of the IRS-1 PH domain alone was incapable of associating with (V5)EN, indicating that the PTB domain of IRS-1 is required for its interaction with EN (Fig. 3.1C). 3.2.2 Co-expression of the IRS-1 PTB Domain Fragment Inhibits EN-Mediated Transformation. Since EN was shown to specifically interact with the PTB domain of IRS-1, it was postulated that overexpression of an IRS-1 fragment containing this domain ((HA)IRS-1C) should be able to block the EN»IRS-1 interaction, and thus block EN transformation in a dominant-negative fashion. Retroviral gene transfer was used to co-express EN along with either (HA)IRS-1C or empty vector in NIH 3T3 fibroblasts. Levels of EN expression were confirmed by immunoprecipitation with a-NTRK3 antibodies (Fig. 3.2A), and expression of (HA)IRS-1C was confirmed by a-HA Western blotting. Lysates from the same cells were then subjected to immunoprecipitation with an a-IRS-1 antibody that recognizes the C-terminus (but does not bind IRS-1 C). Significantly less IRS-1 associated with EN in cells co-expressing EN and (HA)IRS-1C compared to cells expressing EN alone, despite higher levels of EN in the co-expressing cells (Fig. 3.2A). These cells were then assessed for their ability to form colonies in soft agar. Cells co-expressing both EN and (HA)IRS-1C showed a significant 7 1 MSCV EN (HA)IRS-1C IP: NTRK3 IB: NTRK3 IP: IRS-1 IB: NTRK3 IB: HA IB: Actin B I m I ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ 0 ^ ^ ^ ^ ^ ^ ^^^^^^P ^H^^P ^^^^^ 0.5 -T 3 * I I 1 0.4 -C L _ a) 0.3 -O 1 • • •§ 0.1 -o * 0 • 1 1 / Figure 3.2: Co expression of IRS-1C (PTB/PH domains) disrupts ENIRS-1 complexes and inhibits transformation. A) Immunoprecipitation analysis of NIH 3T3 fibroblasts expressing EN and/or IRS-1 C constructs. Anti-IRS-1 antibodies pull down less EN protein in cells expressing (HA)IRS-1C compared with those expressing EN alone. Anti-NTRK3 immunoprecipitation demonstrates levels of EN expression. B) Anchorage-independent growth was assessed by the ability to form macroscopic colonies in soft agar, as described in Materials and Methods. Each cell line was assessed in triplicate in 35-mm wells, and the experiment was repeated five times. Cells expressing both (HA)IRS-1C and EN show a significantly lower rate of colony formation than those expressing EN alone (* p < 0.0001), as assessed by the Student's t test. 72 reduction in colony formation compared with cells expressing EN alone (p<0.0001; Fig. 3.2B). Together, these data indicate that (HA)IRS-1C can function as a dominant-negative regulator of EN-mediated transformation by reducing the interaction with endogenous IRS-1. 3.2.3 IRS-1 Overexpression Potentiates EN-Mediated Transformation. The above findings underscore the importance of IRS-1 in EN-mediated transformation. I next wished to assess the influence of IRS-1 expression levels on EN transformation activity. To study this, IRS-1 was retrovirally overexpressed in EN-transformed fibroblasts. A different murine fibroblast cell line was used for these studies, namely murine R+ cells. These cells are derived from IGF-IR null R- cells (3, 315) that have been engineered to re-express IGF-IR (301). R+ cells were chosen as it had previously been shown that while they are transformed by EN, transformation activity is lower than in NIH 3T3 cells (301). IRS-1 infected R+ and R+EN cells were shown by a-IRS-1 Western blot analysis to have significantly higher (at least 5-fold) levels of IRS-1 compared to levels of endogenous IRS-1 in R+EN cells alone (see Fig. 3.3A). Soft agar colony assays were used to assess transformation activity. R+ and R+/IRS-1 cells showed little or no colony formation as expected (Fig. 3.3B). Consistent with previous results (300), R+EN cells f^ormed macroscopic colonies at a rate of ~20%. However, in the presence of IRS-1 overexpression, EN-mediated anchorage-independent growth was greatly increased, with up to 80% of plated cells forming colonies (p<0.0001; Fig. 3.3B). Furthermore, the rate of colony formation was greatly accelerated, and colonies were larger in R+ cells expressing both EN and IRS-1 constructs compared to EN alone (Fig. 3.3C). These 73 Figure 3.3 Overexpression of IRS-1 potentiates EN transformation. A) The R+ fibroblast cell line was retrovirally transfected with expression constructs of EN and/or full-length IRS-1 (IRS-1 o/e). R+EN cells transfected with IRS-1 resulted in at least a 10-fold increase in protein levels compared to R+EN cells alone B) Soft agar colony formation: histogram comparing R+ control (MSCV) cells with those overexpressing EN, IRS-1, or both EN and IRS-1. Results are expressed as the ratio between colonies and number of cells plated. Differences were found to be statistically significant using the Student's t test (* p < 0.0001; n=5). C) R+EN/IRS-1 cells rapidly form large colonies in soft agar. Colonies are visible at 24 h and are significantly larger than those of R+EN at 72h. ' 7 4 findings not only confirm the crucial role of IRS-1 in EN transformation, but also suggest that actual IRS-1 protein levels might influence EN transformation activity. 3.2.4 IRS-1 Overexpression Fails to Restore Transformation to R-EN Fibroblasts. The observation that overexpression of IRS-1 could dramatically enhance the transforming potential of EN led me to ask whether similar overexpression could rescue transformation in the R-EN cell line. When IRS-1 is expressed significantly above physiological levels, NIH 3T3 fibroblasts show phenotypic transformation (77), while IRS-1 overexpression is able to restore transformation to R- cells that co-express the SV40 Large T antigen (316). Thus, R- and R-EN cells were engineered to express excess IRS-1 protein to an approximately 5-fold level (see Fig. 3.4A), and assessed for their growth potential in soft agar. As seen in Fig. 3.4B, whereas R+EN fibroblasts form significant numbers of colonies in soft agar, IRS-1 overexpression fails to induce even a marginal increase in anchorage-independent growth in the R-EN cell line. Thus, the loss of IGF-IR expression compromises the ability of EN to transform fibroblasts in a manner that cannot be overcome by increasing IRS-1 levels. 3.2.5 EN shows transforming ability in IRS-1-null mouse embryo fibroblasts. To further confirm the requirement for IRS-1 in EN-mediated transformation, I obtained mouse embryo fibroblasts derived from an IRS-1 knockout mouse (hereafter referred to as S- cells; a kind gift of Dr. Stuart Orkin, Boston MA), and assessed the potential for EN to induce transformation. S- and R+ fibroblasts were retrovirally transfected with HA-75 A IGF-IR EN IRS-1 o/e IRS-1 Actin + + B lated 0.25 i Q_ __ 0.2-13 O 0.15 ies / 0.1 -c o 0.05-o O 0 -+ + Figure 3.4: Overexpression of IRS-1 does not rescue transformation of R-EN fibroblasts. A) The R- fibroblast cell line was retrovirally transfected with expression constructs of EN and/or full-length IRS-1 (IRS-1 o/e). R-EN cells transfected with IRS-1 resulted in at least a 10-fold increase in protein levels compared to R-EN cells alone B) Soft agar colony formation: histogram comparing R+EN cells to R- cells overexpressing EN, IRS-1, or both EN and IRS-1. Results are expressed as the ratio between colonies and number of cells plated (n=5). 76 tagged EN (see Fig. 3.5A). Additionally, S- cells were transfected with an IRS-1 construct to control for any peculiarities of the parental cell line (here referred to as S+) and transfected with either empty vector (S+) or (HA)EN (S+ (HA)EN). S+ and S+ (HA)EN cells expressed IRS-1 at approximately equal levels to R+ (HA)EN (see Fig. 3.5A). Interestingly, S- derived cell lines showed a significant increase in the levels of IRS-2 protein expression compared to R+ (HA)EN. Next, these cell lines were plated in soft agar to assess their ability to grow in an anchorage-independent manner (Fig. 3.5B). As expected, S- MSCV and S+ cells did not show anchorage-independent growth, while S+ (HA)EN and R+ (HA)EN cells formed macroscopic colonies at rates previously seen with EN expression. However, contrary to expectations, S- (HA)EN fibroblasts showed no deficiency in their ability to grow in soft agar, despite confirmation of a lack of IRS-1 expression (Fig. 3.5A). Therefore, IRS-1 is not an absolute requirement for EN transformation, indicating S- cells possess an alternative mechanism for linking EN to activation of downstream signaling pathways and subsequent oncogenesis. 3.2.6 EN Binds and Tyrosine Phosphorylates IRS-2. Functional redundancy is a phenomenon often seen among signaling molecules, where the absence of a particular protein can be compensated for by related homologues. Since I found that EN could transform in the absence of IRS-1 expression, I postulated that a redundant mechanism for linking EN to downstream pathway activation must exist in S- cells. An obvious candidate for such a protein is IRS-2, which is expressed at relatively high levels in this 77 IRS-1 (HA)EN IRS-1 IRS-2 HA Actin + + + + Figure 3.5: EN transformation in IRS-1-null mouse embryo fibroblasts. A) IRS-1 -/-mouse embryo fibroblasts (S- cells, where "S" refers to substrate) were retrovirally transfected with HA-tagged EN ((HA)EN). S- cells transfected with IRS-1 are designated S+. Levels of reconstituted IRS-1 expression in S+ cells are similar to R+ (HA)EN fibroblasts, as assessed by Western blot. B) Cells were plated in soft agar as described above. S- (HA)EN cells formed a significantly greater percentage of colonies compared with S- or S+ (n=3; * represents P < 0.0001 as assessed by the Student's t test). No statistically significant differences were detected in the rate of colony formation when comparing S- (HA)EN to S+ (HA)EN or R+ (HA)EN. 78 cell line (Fig. 3.5A), and shares 36% homology with IRS-1. More significantly, the IRS-2 PTB domain is 78% identical and 93% similar to that of IRS-1. Both IRS-1 and IRS-2 have been shown to bind the Trk-T1 fusion oncogene (317), making an EN«IRS-2 interaction a likely possibility, especially in the absence of IRS-1 expression. To determine whether EN expression led to tyrosine phosphorylation of IRS-2 in the absence of IGF-1 stimulation, S- MSCV, S- (HA)EN and R+ (HA)EN cells were starved for 16h in 0.25% serum, after which time cell lysates were immunoprecipitated with a-IRS-2 antibodies and immunoblotted for phosphotyrosine. As seen in Fig. 3.6A, expression of (HA)EN led to constitutive tyrosine phosphorylation of IRS-2 in both S-and R+ cells - a phenomenon not seen in S- MSCV cells. The levels of IRS-2 tyrosine phosphorylation due to (HA)EN expression in serum-starved R+ cells was similar in magnitude to that seen in all cell lines after treatment with IGF-I for 10 minutes (Fig. 3.6A, compare lane 3 to lanes 4, 5, and 6). A lower level of constitutive IRS-2 tyrosine phosphorylation was seen in S- (HA)EN cells, which correlates with decreased (HA)EN expression levels seen after repeated passaging of this cell line. To investigate the possibility that EN and IRS-2 were physically linked in a protein complex, a co-immunoprecipitation experiment was undertaken (Fig. 3.6B). Anti-NTRK3 antibodies, but not non-specific rabbit antibodies (anti-Ephrin A1), were able to pull down IRS-2 protein in both S- (HA)EN and R+ (HA)EN cells. Interestingly, significantly more IRS-2 was detected in anti-NTRK3 immunoprecipitates in the S-(HA)EN vs. R+ (HA)EN cells. This is likely due to the available IRS binding sites on EN 79 Starved 16h Starved; 5' IGF-{ V IRS-2 IP; P-Tyr IB IRS-2 HA Actin NTRK3 IP IRS-2 Figure 3.6: EN binds and tyrosine phosphorylates IRS-2. A) IRS-2 is constitutively tyrosine phosphorylated in both S- and R+ cells expressing (HA)EN. S-, S-(HA)EN and R+(HA)EN cells were grown in 10 cm dishes to 90% confluence and then serum-deprived for 16 h in 0.25% serum. Cells were then stimulated with 50 ng/ml IGF-I in 9% FBS/DMEM as indicated for 10 min. and harvested in cell lysis buffer. Lysates were either subjected to a-IRS-2 irnmunoprecipitation followed by Western blotting against phospho-tyrosine (P-Tyr), or loaded directly onto SDS-PAGE and blotted against HA and IRS-2. P-Tyr antibodies demonstrated differential tyrosine phosphorylation of a 185 kDa band when comparing unstimulated S- to S-(HA)EN and R+ (HA)EN. Levels of actin were determined as a loading control B) Irnmunoprecipitation analysis: S-, S-(HA)EN and R+(HA)EN cells were serum-deprived as in (A). Equal protein amounts (750 pg) from cell lysates were subjected to irnmunoprecipitation using either a-IRS-2 or a-NTRK3 antibodies, followed by Western blotting with a-IRS-2 antibodies. Irnmunoprecipitation with a non-specific rabbit antibody (*; a-Ephrin A1) was used as a negative control for IRS-2 irnmunoprecipitation. 80 being occupied by IRS-1 in R+ (HA)EN cells. Therefore I conclude that EN can bind and tyrosine phosphorylate IRS-2, an event that may explain the ability of EN to transform IRS-1-null fibroblasts. 3.2.7 Concurrent siRNA-Mediated Knockdown of IRS-1/IRS-2 Expression Inhibits Transformation of EN-Expressing Fibroblasts. The experiments detailed above provide evidence that EN binding to IRS molecules is a crucial step in EN mediated oncogenesis. To confirm this requirement for IRS-1/-2 I obtained small interfering RNA (siRNA) oligonucleotides (oligos) to knock down protein levels of both IRS-1 and IRS-2, either alone or in combination, and determined the effect this had on EN-induced anchorage-independent growth. To assess the efficacy of these siRNA constructs at knocking down IRS levels compared to a non-specific siRNA control, R+ (HA)EN cells were transfected with one oligo directed against IRS-1 mRNA (designated 507), and three oligos directed against IRS-2 (824, 825 and 826). Cells were trypsinized and re-plated 2 and 6 days post-transfection (to prevent overgrowth in cell culture dishes), and harvested at either 3, 5 or 7 days. As seen in Fig. 3.7A, the IRS-1 (507) oligonucleotide provided consistent knockdown (>90%) of IRS-1 protein levels when transfected into R+ (HA)EN cells, measured 3 days post-transfection. Three separate IRS-2 siRNA constructs (824, 825 and 826) were able to reduce IRS-2 levels greater than 90% 3 days after transfection into R+ (HA)EN cells. After 5 days, IRS-1 (507) maintained significant knockdown (-75%), as did IRS-2(825) and IRS-2(826), whereas IRS-2(824) was less effective, showing about 50% knockdown (Fig. 3.7B). However, by 7 days post-transfection there was neither IRS-1 knockdown with IRS-1 (507) nor IRS-2 81 P ^ 4 ? IRS-1 IRS-2 Actin B IRS-1 IRS-2 Actin ^ <^ & ^ <^ <$> <$> IRS-1 IRS-2 Actin Figure 3.7: siRNA-mediated knockdown of IRS-1 and IRS-2 protein levels. R+ (HA)EN cells were transfected with non-specific (N.S.) siRNA, or siRNA specific for either IRS-1 or IRS-2 as described in the Materials and Methods. One IRS-1 siRNA construct (507) and three IRS-2 siRNA constructs (824, 825 and 826) were assayed. Cells were split into larger dishes 48h post-transfection to prevent overgrowth. To assess the degree and longevity of IRS-1 and IRS-2 knockdown, cells were lysed 3, 5 and 7 days after initial transfection ((A), (B) and (C) respectively) and immunoblotted for IRS-1 and IRS-2. Actin was used as a loading control. 82 knockdown with IRS-2(824), while IRS-2(825) and IRS-2(826) showed a modest ability to decrease IRS-2 levels (Fig. 3.7C). It is likely that two rounds of passaging these cells significantly diluted the oligos available to reduce IRS levels. In all cases, the IRS siRNA molecules showed specificity, with IRS-1 (507) having no effect on IRS-2 levels and the IRS-2 oligos having no effect on IRS-1 levels. R+ (HA)EN cells were next transfected with IRS-1 (507) and IRS-2(826) siRNA constructs, either alone or in combination. Cells were then plated under anchorage-independent conditions 48h after siRNA transfection, to assess the effects of IRS knockdown on three-dimensional growth. When plated as spheroids, cells transfected with either non-specific oligonucleotides or IRS-1 siRNA, were not prevented from forming multicellular spheroids (Fig. 3.8A and B). IRS-2 siRNA had a small inhibitory effect on R+ (HA)EN spheroid formation, slightly delaying aggregate formation (Fig. 3.8A), and limiting the average size of the spheroid colonies (Fig. 3.8B). Interestingly, when IRS-1 and IRS-2 siRNA constructs were combined, they were able to greatly inhibit spheroid formation, and by 72h of culture, these cells appeared to be undergoing significant cell death (Fig. 3.8B). To confirm this effect on anchorage-independent growth, R+ (HA)EN cells transfected with either non-specific siRNA, IRS-1 (507) alone, IRS-2 siRNA alone, or IRS-1 (507) in combination with each of the IRS-2 siRNA constructs, were plated in soft agar. As seen in Fig. 3.8C, R+ (HA)EN cells transfected with either a non-specific oligo, IRS-1 (507) alone, or any of the IRS-2 oligos alone, resulted in robust growth in soft agar, with a rate of colony formation of approximately 83 N.S. IRS-1 (507) IRS-2(826) N.S. IRS-1 (507) IRS-2(826) Figure 3.8: IRS-1/2 knockdown inhibits anchorage-independent growth of R+ (HA)EN fibroblasts. R+ (HA)EN fibroblasts were transfected with non-specific siRNA, IRS-1 siRNA (507), IRS-2 siRNA (826), or siRNA against IRS-1 and IRS-2 in combination (507/826). 48h post-transfection, cells were trypsinized and transferred to dishes coated with 1.4% agar to prevent adhesion and undergo anchorage-independent spheroid formation (150,000 cells per 35 mm dish). Photos (100X magnification) were taken after 24h (3 days post-transfection; (A)) or 72h (5 days post-transfection; (B)) of spheroid culture. 8 4 c Figure 3.8 (ctd.): IRS-1/2 knockdown inhibits anchorage-independent growth of R+ (HA)EN fibroblasts. (C) R+ (HA)EN fibroblasts were transfected with non-specific siRNA, IRS-1 siRNA (507), IRS-2 siRNA (824, 825 or 826), or siRNA against IRS-1 and IRS-2 in combination (507/824, 507/ 825 or 507/826). 48h post-transfection, cells were trypsinized and cultured in soft agar as described in the Materials and Methods. R+ (HA)EN cells transfected with non-specific siRNA or with IRS-1 or IRS-2 siRNA alone formed a greater percentage of colonies compared with R+ (HA)EN cells transfected with IRS-1 and IRS-2 siRNA in combination (n=2; * represents P < 0.001 as assessed by the Student's f test). 85 30%. However, when IRS-1 (507) was co-transfected with each of the IRS-2 oligos, soft agar colony formation was significantly inhibited. The effect was most striking with the combination of IRS-1 (507) and IRS-2(824) - reducing the rate of colony formation from 30.8% (non-specific control siRNA) to 10.4% (n=2). It can therefore be concluded that significantly reducing the level of both IRS-1. and IRS-2 protein greatly impairs EN-mediated anchorage independent growth. However if either IRS-1 or IRS-2 is available, this is sufficient to support EN transformation. EN expression has been shown to constitutively activate both the PI3K/Akt and Ras/Erk pathways, leading to an up-regulation of cyclin D1 expression (295). Therefore, the R+ (HA)EN cells transfected with IRS-1/-2 siRNA from the previous soft agar experiment were plated as spheroids in parallel, and examined for the level of activation of these signaling pathways. Figure 3.9 represents the results of a typical experiment with respect to the response of signaling pathways to knockdown of IRS-1 and IRS-2, either alone or in combination. Singular transfection with either IRS-1 (507), IRS-2(825) or IRS-2(826) had no effect on levels of phospho-Akt, phospho-MEK or cyclin D1 compared to those seen in cells transfected with non-specific oligos, even though significant knockdown of their target proteins was achieved. Interestingly, R+ (HA)EN cells in which targeted knockdown of both IRS-1 and IRS-2 protein levels was achieved (namely 507/824 and 507/826), levels of phospho-Akt were significantly reduced compared to cells transfected with non-specific oligos, or those with targeted knockdown of IRS-1 or IRS-2 alone. It was noted that cells treated with the 507/825 combination (which have near complete knockdown of IRS-1 but retain relatively high 86 scrambled IRS-1 (507) IRS-2 (824) IRS-2 (825) IRS-2 (826) IRS-1 IRS-2 Ser-473-P Akt Mek-1/2-P Mek-1/2 Cyclin D1 + + ft-1 ** Actin Figure 3.9: IRS-1/IRS-2 co-knockdown downregulates Akt activation in R+ (HA)EN fibroblasts. R+ (HA)EN fibroblasts were transfected with non-specific siRNA, IRS-1 siRNA (507), IRS-2 siRNA (824, 825 or 826), or siRNA against IRS-1 and IRS-2 in combination (507/824, 507/ 825 or 507/826). 48h post-transfection, cells were trypsinized and cultured as spheroids as described above. 24h after initiation of spheroid culture (3 days post-transfection) cells were lysed and subjected to immunoblotting using the antibodies indicated. Activated Akt (Ser-473-P) and MEK1/2 (MEK1/2-P) were assessed using antibodies to phosphorylated proteins. Actin was used as a loading control. 87 IRS-2 levels in this particular experiment) did not show inhibition of Akt signaling. Further, transfection of R+(HA)EN cells with IRS-2 (824) alone caused some reduction in the level of phospho-Akt, indicating the possibility that off-target effects are mediated by this siRNA molecule. The cells with impaired Akt phosphorylation showed a moderate decrease in levels of phospho-MEK1/2 in this experiment, which was likely due to decreased total MEK1/2 levels, while cyclin D1 expression was largely unaffected by downregulation of IRS proteins. Thus, in the context of signal transduction, reducing both IRS-1 and IRS-2 protein levels is primarily reflected in reduced PI3K/Akt activation in EN-expressing cells, and correlates with reduced transformation potential. 3.2.8EN Transformation of IRS-1/-2 Deficient 32D Hematopoietic Cells Requires Re-Expression of IRS-1. Aside from the pediatric malignancy known as congenital fibrosarcoma, EN expression has been reported in a case of acute myeloid leukemia (294). To determine the requirement for IRS protein expression in EN-mediated transformation of hematopoietic cells, I examined the potential for EN to transform 32D murine myeloid cells, as they do not express IRS-1 or IRS-2 (318). This cell line is IL-3 dependent, undergoing rapid apoptosis upon withdrawal of IL-3 from the culture medium. Both constitutively active Ha-Ras (319) and the SV40 Large T antigen (320) require co-expression of IRS-1 to confer IL-3 independent growth and oncogenic potential in vivo, although transformation is not induced by IRS-1 expression alone (321). 32D cells were thus transfected with vector control, EN, IRS-1 or EN and IRS-1 in combination manner. These five cell lines were plated in either IL-3-deficient or IL-3-88 containing medium and assessed for their ability to grow (cell number) and survive (trypan blue exclusion assay) under each growth condition. All cell lines examined showed proliferation'and survival in IL-3 containing media (Fig. 3.10A and 3.10C). As expected, 32D cells expressing IL-3 were able to robustly grow in IL-3 deficient media, and maintained survival over a period of 120h (see Fig. 3.1 OB and D). Conversely, 32D (MSCV) or 32D (IRS-1) could not proliferate and showed significant cell death within 24h when plated in IL-3-free conditions. 32D (EN) cells showed a modest ability to proliferate in IL-3 deficient conditions. IL-3 independent survival of 32D (EN) cells was extended as compared to 32D (MSCV) or 32D (IRS-1), though significant cell death was observed by 120h in culture. However, concurrent expression of EN and IRS-1 induced IL-3-independent growth and survival to levels similar to 32D (IL-3) cells. Therefore it can be concluded that EN is able to partially transform 32D myeloid cells, but requires co-expression of IRS-1 to fully induce an oncogenic phenotype. 3.3 DISCUSSION The t(12;15)-associated ETV6-NTRK3 oncoprotein is similar to other chimeric protein-tyrosine kinases such as BCR-ABL (322), ETV6-PDGFR (263), ETV6-JAK2(262), and NPM-ALK (323) in that it functions as a constitutively active tyrosine kinase. Activation of the EN protein-tyrosine kinase is linked to induction of downstream signaling pathways of wild-type NTRK3 including Ras/Erk and PI3K/Akt, leading to elevated cyclin D1 expression and aberrant cell cycle progression (295). Rather than utilizing known NTRK3 adaptors to link to these pathways, EN does so through an interaction with the IGF-IR substrate IRS-1, and EN transformation is associated with 89 A 120 100 o 80 - 6 0 w g 40 * 20 0 B o 5, v> "S3 o 140 120 100 80 60 40 20 0 > c r ^ ( — 1 =8-—^—-%— £ 1 6 1 A 1 0 24 48 72 Time (h) 96 120 0 24 48 72 Time (h) 96 120 — A — MSCV — • — IL-3 • EN • IRS-1 —x— EN/IRS-1 Figure 3.10: Full transformation of IRS-1/2 deficient hematopoietic cells by EN requires re-expression of IRS-1. 32D hematopoeitic cells were retrovirally transfected with either empty vector, EN, IRS-1 or EN and IRS-1 in combination, and assessed for their ability to grow in the presence or absence of IL-3. A separate 32D line was transfected with IL-3 as a positive control. Cells were grown for 24 -120 h in RPMI supplemented with 9% FBS. At the indicated times, cells were spun down, resuspended in a 1:1 mixture of PBS and trypan blue reagent and counted. Clear cells that excluded trypan blue were counted as viable, while blue cells were considered dead. Cell survival was calculated as the ratio of live cells to total cells. The plots represent data from three separate experiments. Error bars represent +/- one standard deviation. (A) Cell growth (-) IL-3; (B) Cell Growth (+) IL-3. 9 0 c Figure 3.10 (ctd.): Full transformation of IRS-1/2 deficient hematopoietic cells by EN requires re-expression of IRS-1. (C) Cell survival (-) IL-3; (D) Cell survival (+) IL-3, as measured by the trypan blue exclusion assay (see Materials and Methods). 91 constitutive IRS-1 tyrosine phosphorylation (301). Here I demonstrate that the EN»IRS-1 interaction occurs via the PTB domain of IRS-1 binding to EN. Disrupting the EN»IRS-1 interaction was effective in inhibiting anchorage-independent growth, while IRS-1 overexpression enhanced EN transformation. In IRS-1 deficient cells, IRS-2 is able to substitute as a binding partner for EN, and supports tumorigenesis. Both fibroblasts and myeloid cells with reduced or absent levels of both IRS-1 and IRS-2 are significantly impaired in their transformation potential. Therefore, EN binding to IRS-1/-2 is an essential step in transformation. Tyrosine-phosphorylated IRS-1/-2 proteins are known to bind efficiently to a number of SH2 domain-containing proteins involved in activation of downstream signaling pathways, including PI3K p85, Grb2, SHP-2, Nek, and Crk (for review, see (324)). There is increasing interest in the potential role of IRS molecules in oncogenesis. Overexpression of IRS-1 in NIH 3T3 fibroblasts leads to increased activation of Erk and cell transformation (77, 325). Although the LNCaP prostate cancer cell line does not express IRS-1 or IRS-2, introduction of either protein in combination with IGF-IR converts these cells to a more aggressive phenotype (326). A recent study examining endogenous IRS-1 shows that it is constitutively tyrosine-phosphorylated in a wide range of human tumour samples, suggesting that IRS-1 activation may be a common phenomenon in tumours (78). IRS-2 phosphorylation and subsequent activation of PI3K/Akt signaling has been implicated in transformation by the Ret proto-oncogene (327). Moreover, a relationship between IRS-1/-2 activation and fusion oncoproteins has already been established. IRS adaptor binding and tyrosine phosphorylation has been established for BCR-ABL (IRS-1; (328)), Trk-T1 (IRS-1 and 92 IRS-2; (317)) and Ret-PTC3 (IRS-2; (329)). Therefore IRS-1/-2 activation may be a more general mechanism for transformation mediated by fusion oncogenes than is currently recognized. I found that a 5-fold elevation in IRS-1 levels leads to a dramatic increase in the transformation potential of the EN oncoprotein. I hypothesize that this leads to an increased number of IRS-1 molecules being phosphorylated by EN, thus providing an increased number of docking sites for activation of downstream signaling cascades. Concurrent work in our laboratory established that the C-terminus of EN is involved in IRS-1 binding. Specifically, the last 19 amino acids of EN are essential for this interaction. A mutant EN protein lacking the distal C-terminal 19 amino acids (EN A614), though still tyrosine-phosphorylated when expressed in NIH 3T3 cells, does not bind IRS-1 and is non-transforming. In contrast to EN-expressing cells that are characterized by strong constitutive IRS-1 tyrosine phosphorylation even when serum-starved, those expressing EN A614 show a complete block in total and EN-associated IRS-1 tyrosine phosphorylation under these conditions. Moreover, MEK1/2 and Akt activation as well as cyclin D1 elevation, hallmarks of EN transformation (242, 295), were reduced markedly in EN A614 expressing cells after serum starvation compared with cells expressing EN. This correlated directly with the ability of IRS-1 to recruit Grb2 and the p85 subunit of PI3K, which link EN to the Ras/Erk and PI3K/Akt pathway, respectively (295, 301). These observations provide further evidence that the interaction of the C terminus of EN with IRS-1 (and likely IRS-2) is essential for activation of signaling pathways underlying EN-mediated oncogenesis. 93 If, as my studies suggest, IRS-1/-2 plays a pivotal role in EN transformation (and potentially that of activated NTRK3 in other tumours), then blocking the EN«IRS-1/-2 interactions offers an interesting avenue for potential cancer therapeutics. A dominant-negative IRS-1 construct partially blocked EN transformation by preventing association of EN and IRS-1. As the PTB domains of IRS-1 and IRS-2 are highly homologous, it is likely that EN«IRS-2 interactions would be inhibited by expression of this construct as well. This would prevent the compensatory effect of IRS-2 that is observed in IRS-1-deficient cells. In IRS-1/-2 deficient 32D myeloid cells, EN induced only a moderately transformed phenotype. When co-expressed with IRS-1, EN expression resulted in a greatly increased rate of proliferation and delayed the onset of apoptosis in response to IL-3 withdrawal. These data suggest that stoichiometric relationships between EN and IRS-11-2 are important in EN transformation. This is further supported by the fact that siRNA-mediated knockdown of both IRS-1 and IRS-2 in tandem significantly inhibited EN-induced anchorage-independent growth compared to knockdown of either IRS isoform alone. This double knockdown was accompanied by a significant reduction in PI3K/Akt activation, a pathway that is normally required for EN transformation (294). The effect on signaling was specific to Akt, as there was little to no effect on levels of phospho-MEK1/2, and IRS-1/-2 co-knockdown did not affect the amount of cyclin D1 in R+ (HA)EN cells. A recent report observed that siRNA-mediated knockdown of IRS-1 levels induced apoptosis and enhanced the cytotoxic effects of tamoxifen in MCF-7 breast cancer cells (330). As IRS-2 activation is minimal in these cells (331), it can be concluded that blocking IRS function in transformed cells is an effective mechanism for inhibiting their growth. Further, overexpression of the dual specificity phosphatase 94 PTEN reduces the overall level of IRS-1 phosphorylation and induces growth arrest in MCF-7 cells (332). Expression of an N-terminal portion of IRS-1 blocks the tumourigenic phenotype of human hepatocellular carcinoma (333). A recent report shows that overexpression of the IRS-1 PH/PTB domains blocks binding of IRS-1 to the human JC virus T-antigen and prevents subsequent cellular transformation by this protein (334). Therefore agents blocking the interaction between the C terminus of EN and IRS adaptor proteins, or those which lead to a down-regulation of IRS-11-2 levels may provide a novel approach for treatment of EN-expressing tumours. 95 CHAPTER IV THE INSULIN-LIKE GROWTH FACTOR I RECEPTOR IS REQUIRED FOR AKT ACTIVATION AND SUPPRESSION OF ANOIKIS IN CELLS TRANSFORMED BY THE ETV6-NTRK3 ; CHIMERIC TYROSINE KINASE 4.1 INTRODUCTION The IGF-I receptor (IGF-IR) is a membrane protein tyrosine kinase (PTK) which binds the insulin-like growth factors, IGF-I and IGF-II (reviewed in (335)). Ligand binding autoactivates the PTK domain and links the receptor to downstream signaling pathways including the Ras/Erk (336) and PI3K/Akt pathways (337, 338). IGF-IR has a multitude of physiological functions in normal cells including mitogenesis, suppression of apoptosis, cell adhesion, and regulation of cell size and lifespan (339, 340). In addition, abundant literature points to a critical role for the IGF-IR axis in cellular transformation A version of this chapter has been published. Moi Cell Biol. 2006 Mar;26(5): 1754-69. Martin MJ, Melnyk N, Pollard M, Bowden M, Leong M, Podor TJ, Gleave M, Sorensen PH. Moi Cell Biol. 2006 Mar;26(5):1754-69. 96 (341, 342). Antibodies to IGF-IR (343) or IGFs (344), receptor or IGF antisense (345, 346), or dominant negative IGF-IR mutants (344), all reverse the transformed phenotype or inhibit tumourigenesis. IGF-IR over-expression transforms mouse fibroblasts (347), and IGF-IR gene amplification has been reported in human malignancies including breast cancer (348-350). IGF-II over-expression is well documented in both pediatric and adult tumours (reviewed in (351)). Recently, selective inhibition of the IGF-IR PTK was shown to block tumour growth in vivo in animal studies (352). Perhaps most compelling evidence for a role of IGF-IR in oncogenesis is the observation that many dominantly-acting oncoproteins such as activated Ras, c-Src, SV40 large T antigen, and the fusion oncogenes EWS-Fli1 and PAX3-FKHR, fail to transform cells lacking IGF-IR (reviewed in (353)). These studies analyzed transforming properties of the respective oncoproteins in R- cells, which are IGF-IR -I- mouse embryo fibroblasts (MEFs) derived from mice with a targeted disruption of the IGF-IR locus (3, 315). Transformation can be restored by re-introduction of IGF-IR (301, 354) but not by over-expression of the major IGF-IR adaptor, insulin receptor substrate-1 (IRS-1) (353). These observations have led to speculation that IGF-IR activation provides a second, or complementary, signal that is essential for oncogenesis by dominantly-acting transforming proteins (341, 342). Many hypotheses have been put forth to explain how IGF-IR might contribute to oncoprotein function, including enhancement of mitogenesis or suppression of apoptosis (reviewed in (302)). However, other studies indicate that the mitogenic and anti-apoptotic functions of IGF-IR are distinct from its transformation activity (355, 356). 97 Indeed, no theory fully explains the phenomenon, and the mechanism by which loss of IGF-IR signaling blocks transformation remains contentious (reviewed in (342)). Prevailing evidence suggests that resistance to apoptosis is at least partially involved, as inhibition of this pathway leads to massive apoptosis of tumour cells but much less so of normal cells (341). The IGF-IR axis may be particularly important for survival of tumour cells under anchorage independent conditions; i.e. for suppression of anoikis (reviewed in (342)). IGF-IR signaling protects suspended cells from anoikis (79, 357), and inhibition of the IGF-IR axis blocks anchorage independent soft agar colony formation of tumour cells (358). Moreover, while antisense inhibition of IGF-IR had little effect on survival of anchorage dependent monolayer cultures of melanoma cells, it profoundly blocked their ability to form soft agar colonies and in vivo tumourigenicity (46). This is consistent with previous reports indicating that the apoptotic effect of IGF-IR blockade is more pronounced in metastatic cells (359, 360), as it is hypothesized that ability to survive under anchorage independent conditions is a requirement of metastatic cells. To specifically address how IGF-IR contributes to transformation by dominantly acting oncoproteins, I assessed its ability to complement the transforming properties of the ETV6-NTRK3 (EN) chimeric tyrosine kinase. Ras-ERK and PI3K-Akt cascades, and both are required for EN transformation (295). Previous studies in the Sorensen lab showed that EN fails to transform R- cells, and that transformation is restored in R- cells engineered to re-express IGF-IR (R+ cells) (301). However, EN can still bind and tyrosine phosphorylate IRS-1 in the absence of IGF-IR expression, which suggests IGF-IR has an anti-tumour function not related to IRS-1 phosphorylation. It is here 98 hypothesized that IGF-IR, through its ability to interact with IRS-1, serves to dock EN/IRS-1 complexes at the plasma membrane where they can activate downstream signaling pathway activation - most importantly PI3K/Akt and Ras/Erk. Therefore I have now utilized EN expressing R- and R+ in a 3-dimensional spheroid culture model, which is thought to more closely replicate signaling taking place within tumour tissue, as a model system in which to study the mechanism by which IGF-IR complements EN in transformation. I have found that while the absence of IGF-IR does not affect Ras/Erk induction of cyclin D1 and cell cycle progression, this receptor appears to be essential for anchorage independent growth and Akt activation in EN transformed cells. Moreover, targeting the EN molecule to the plasma membrane through N-myristoylation increases membrane-associated IRS-1, activates Akt signaling, and induces transformation in the absence of IGF-IR, suggesting a role for this receptor in EN membrane targeting. 4.2 RESULTS 4.2.1 Anchorage-Independent Growth of ETV6-NTRK3-Expresslng Cells Requires IGF-IR. Several studies have postulated a role for IGF-IR in supporting anchorage independent growth of transformed cells (reviewed in (341)). Previous studies in our laboratory reported that when cultured under anchorage dependent monolayer conditions, EN-expressing R- fibroblasts (R-EN cells) have similar rates of growth and cell viability as EN-expressing R- cells engineered to re-express IGF-IR (R+EN cells (301)). However, R-EN cells are defective in the ability to form colonies in soft agar compared to R+EN cells (301). Since soft agar colony formation, widely used as a 99 criterion for cell transformation, is a measure of a cell's capacity to grow under anchorage independent conditions (361), I hypothesized that IGF-IR may complement EN by conferring non-adherent growth ability to EN transformed cells. To model anchorage independent growth, the culturing of Ewing tumour cell lines as multicellular spheroids was previously undertaken (237). This approach utilizes tissue culture plates overlayed with a non-adherent 1.4% agar coating to prevent attachment to the plastic plate surfaces (362). Whereas most tumour cell lines form matrix-deficient multicellular spheroids through cell-cell adhesion under these conditions, non-malignant cells generally fail to do so and undergo massive cell death in culture (362). Therefore cell lines were cultured on agar-coated plates for 24-96 h as described (236). As shown in Fig. 4.1A and 4.1B, R- fibroblasts undergo rapid celldeath as single cells while R+ form only tiny clusters that undergo morphological changes suggestive of cell death after several days in culture. Although R-EN fibroblasts initially formed larger spheroid structures over the first 24-48 hours, I observed that after 96 hours R-EN cultures consisted of poorly formed aggregates of dead or dying cells (Fig. 4.1B). In contrast, R+EN cells readily form multicellular spheroids within 6-24 h that are stable indefinitely in culture. Therefore the ability of fibroblasts to grow as anchorage independent multicellular spheroids requires the presence of both EN and IGF-IR. 4.2.2ETV6-NTRK3 Expressing Cells Lacking IGF-IR are Defective in Akt Activation. It has been previously shown that EN strongly activates the Ras/Erk pathway leading to cyclin D1 up-regulation and aberrant cell cycle progression, as well as the PI3K-Akt cell survival cascade (295). Since both cascades are required for EN 100 B Figure 4.1: ETV6-NTRK3-induced growth of multicellular spheroids requires IGF-IR expression. Adherent monolayer EN-expressing and vector control fibroblasts either lacking (R-) or re-expressing (R+) IGF-IR were grown to confluence, trypsinized, resuspended and then replated on agar-coated dishes at a density of 1.0 x 105 cells/ml. Photos of anchorage-independent cultures (100x magnification) were taken at 24 (A) or 96 (B) hours. EN-expression led to formation of multicellular spheroid structures by 24 h regardless of IGF-IR expression, while vector control cells showed only a weak ability to form colonies. At 96 hours R+EN fibroblasts had formed large viable spheroid structures, while R-EN cultures showed poorly formed aggregates of dead or dying cells. 101 transformation (295), I asked whether IGF-IR might contribute to one or both pathways in EN transformed cells. Earlier studies of anchorage dependent monolayer cultures failed to detect differences in activation of these pathways in R-EN versus R+EN cells under serum-free conditions (301). However, a deficiency in the ability of R-EN cells to fully activate Akt after serum stimulation was observed, while MEK activation was less obviously affected (301). Given the requirements for IGF-IR in anchorage independent growth as shown above, I next wished to determine whether these pathways were differentially activated in cells grown as spheroid cultures. R+, R-EN, and R+EN cell lines (R- cells alone are not viable under these conditions) were cultured on agar-coated plates for 48h in low (0.25%) and high (9%) serum as above. Cell lysates were then immunoblotted for phosphorylated MEK1/2, cyclin D1, and phosphorylated Akt (Ser-473). As shown in Fig. 4.2A, R+ spheroid cultures grown in low serum show minimal activation of MEK1/2 compared to R-EN and R+EN cell lines. All cell lines responded to serum stimulation by enhanced MEK1/2 activation, indicating that the Ras/Erk pathway is intact in these cells (Fig. 4.2B). R-EN and R+EN spheroids also both show serum independent cyclin D1 up-regulation, while in R+ spheroid cultures cyclin D1 expression remains low. Therefore MEK1/2 activation and cyclin D1 induction both appear to be serum and IGF-IR independent in EN-expressing spheroids. This is consistent with our findings in monolayer cultures of EN transformed fibroblasts, which show serum independent MEK1/2 and cyclin D1 induction (300, 350). However, while R+ and R+EN spheroid cultures both showed Akt activation (as detected by Ser-473 phosphorylation) that could be enhanced by serum stimulation, Akt phosphorylation was severely impaired in R-EN spheroids in both low and high serum. To confirm that defects in 102 A B IGF-1 R + - + IGF-1 R + - + EN - + + EN + + IGF-1 R + - + EN + + Ser 473-P Akt PTEN Actin Figure 4.2: Akt activation in EN-expressing cells requires IGF-IR. R+, R-EN and R+EN fibroblasts were cultured in anchorage-independent conditions in either 0.25% (A) or 9% (B) serum for 48 h, lysed and subjected to immunoblotting. Activated Akt (Ser-473-P), GSK-3(3 (GSK-3p-P), and MEK1/2 (MEK1/2-P) were assessed using antibodies to phosphorylated proteins. Detection of actin was used as a loading control. (C) Cells cultured similarly to (B) were lysed and assayed for PTEN protein expression. 103 phosphorylation of Akt at serine 473 represented impaired Akt kinase activity, cell lysates from R+, R-EN and R+EN cells grown as spheroids in low serum were findings in monolayer cultures of EN transformed fibroblasts, which show serum independent MEK1/2 and cyclin D1 induction (300, 350). However, while R+ and R+EN spheroid cultures both showed Akt activation (as detected by Ser-473 phosphorylation) that could be enhanced by serum stimulation, Akt phosphorylation was severely impaired in R-EN spheroids in both low and high serum. To confirm that defects in phosphorylation of Akt at serine 473 represented impaired Akt kinase activity, cell lysates from R+, R-EN and R+EN cells grown as spheroids in low serum were examined for their level of Ser-9 phosphorylation of glycogen synthase kinase-3(3 (GSK3(3), a known Akt substrate (363). GSK-3P showed significantly lower levels of phosphorylation in R-EN spheroids when compared to either R+ or R-EN in the absence of serum, indicating a functional loss of Akt activity in this cell line. From these observations I conclude that the transformation-deficient R-EN cell line is impaired in activation of the PI3K-Akt pathway under anchorage-independent conditions. However, Ras/Erk pathway activation and cyclin D1 up-regulation appear to be intact in these cells, indicating that induction of this cascade is dependent on EN expression but is independent of IGF-IR in non-adherent cultures. One possible explanation for impaired Akt signaling in R-EN fibroblasts, even in the presence of serum, is upregulation of negative regulators of PI3K/Akt signaling. The dual specificity phosphatase PTEN reverses the reaction catalyzed by PI3K and potently inhibits Akt activation when expressed at high levels (365). Therefore, one explanation for impaired Akt activation in R-EN fibroblasts is upregulation of PTEN expression. Levels of PTEN protein expression were examined in R+, R-EN and R+EN 104 spheroids grown for 24 hours in 9% serum-containing media (Fig. 4.2C). As seen before, levels of Akt phosphorylated at serine 473 were significantly lower in R-EN fibroblasts, indicating an impairment of the PI3K/Akt signaling. However, no significant differences could be detected in the levels of PTEN protein among the three cell lines examined. Thus, overexpression of PTEN can be ruled out as a cause for impaired Akt activation in R-EN fibroblasts. 4.2.3 Expression of Activated Akt Restores Anchorage-Independent Growth and Transformation Activity to ETV6-NTRK3-Expressing IGF-IR-Null Fibroblasts. To determine whether the critical deficiency preventing R-EN cells from anchorage independent growth and cellular transformation is their inability to activate the PI3K-Akt pathway, a constitutively active myristoylated Akt construct (122) was expressed in these cells. Stable expression of Akt-myr in R- and R-EN cell lines was documented in Western blots as a slight band shift upwards in mobility, due to the presence of a Myc epitope tag on the exogenous protein (Fig. 4.3A). Despite the fact that total Akt immunoblotting indicated only a small increase in overall Akt expression due to the presence of Akt-myr, I saw a significant increase in Akt phosphorylation at Ser-473 (almost exclusively attributed to the exogenous band), indicating restoration of PI3K-Akt signaling in R-EN/Akt-myr cells. Increased phosphorylation of the downstream Akt substrate GSK3P correlated with Akt-myr expression, indicating functional activation of this pathway. In contrast, expression of Akt-myr had no effect on the Ras/Erk pathway, as MEK activation remained low in the R-/Akt-myr cells and comparable for 105 Figure 4.3: Restoration of Akt activation in R-EN cells rescues anchorage-independent growth. A) Myristoylated Akt (Akt-myr) was expressed in both R- and R-EN cells (R-/Akt-myr and R-EN/Akt-myr, respectively). Spheroid cultures were grown in 0.25% serum, lysed and immunoblotted. Expression of the Akt-myr protein could be seen as an upward band shift in both P-Akt and total Akt immunoblots (arrows). B) Spheroid growth of Akt-myr expressing R- and R-EN cells at both 24 and 96 hours. Expression of Akt-myr rescued long-term growth and survival of R-EN anchorage-independent cultures, but did not induce spheroid formation in R- cells. 106 c Figure 4.3 (ctd.): Restoration of Akt activation in R-EN cells rescues anchorage-independent growth and in vivo transformation. C) Cells were plated in soft agar as described above. R-EN/Akt-myr cells formed a significantly greater percentage of colonies compared with R-EN or R-/Akt-myr cells (n = 5; * represents P < 0.0001 as assessed by the Student's t test). D) Average diameter of R-EN/Akt-myr colonies is significantly smaller than R+EN colonies (n=75; f represents P < 0.005 by the Mann-Whitney ranks test). 107 R-EN/Akt-myr, R-EN, and R+EN cells (see Fig. 4.3A; compare lanes 2,3 and 5). R-EN/Akt-myr cells readily formed stable spheroids under anchorage independent conditions (Fig. 4.3B), and soft agar colony formation was restored to levels comparable to R+EN cells (see Fig. 4.3C). However, expression of Akt-myr in the absence of EN was insufficient to support spheroid (Fig. 4.3B) or soft agar colony formation (Fig. 4.3C) of R- fibroblasts. A small decrease in average soft agar colony size in R-EN/Akt-myr versus R+EN cells (4.3D) was noted, possibly indicating that the myristoylated Akt construct is expressed at sub-optimal levels or that additional functions of the EN complex are important for this process. However, these data provide strong evidence that the activation of PI3K-Akt signaling is critical for EN induced anchorage independent growth. 4.2.4 ETV6-NTRK3 and IRS-1 Membrane Localization are Increased by IGF-IR Expression. Since IGF-IR is a plasma membrane receptor, one possibility suggested by the above findings is that IGF-IR functions to localize EN/IRS-1 complexes to the plasma membrane at sites of activation of the PI3K/Akt cascade. I therefore tested the effects of expressing a membrane-targeted EN construct in IGF-IR-null cells. An N-myristoylated, HA-tagged EN construct ((HA)ENmyr) was generated (Fig. 4.4A) and expressed in both R- and R+ fibroblasts and its cellular localization was assessed as compared to HA-tagged EN ((HA)EN) alone. (HA)EN proteins showed a diffuse cytosolic distribution in R+ cells (Fig. 4.4C) as previously shown for EN-transformed NIH 3T3 cells (296), while a punctate perinuclear and cytosolic pattern was observed in R-cells (see Fig. 4.4B). (HA)ENmyr showed a prominent plasma membrane 108 Figure 4.4: Membrane localization of ETV6-NTRK3 in the presence of IGF-IR or following N-terminal myristoylation of ETV6-NTRK3. (A) Schematic of HA-tagged N-terminal myristoylated (N-myr) ETV6-NTRK3 (ENmyr) possessing the Lck myristoylation sequence and two tandem HA epitope tags at the N terminus of the EN molecule. (B to D) Immunofluorescence detection of EN molecules. Stable expression of HA-tagged EN or HA-tagged ENmyr in R- or R+ mouse embryo fibroblasts is depicted. (B) R-(HA)EN, (C) R+(HA)EN, and (D) R-(HA)ENmyr cells were grown on coverslips in 9% serum, fixed, and subjected to immunofluorescence using a-HA antibodies (green labeling). Cell nuclei were stained with DAPI (blue staining). (E to G) Confocal microscopic images of similarly stained R-(HA)EN (E), R+(HA)EN (F), and R-(HA)ENmyr (G) fibroblasts grown in low (0.25%) serum conditions. White arrow represents 100 pm. 109 distribution in R- cells (Fig. 4.4D). Using confocal microscopy to more precisely examine cell membranes, localization of (HA)EN to. the plasma membrane was readily observed in cells expressing IGF-IR (Fig. 4.4F) while (HA)EN localized exclusively within the cytoplasm in R-EN cells (Fig. 4.4E). Of particular note, (HA)EN localized to small membrane extensions in R+ cells, which was not evident in R- cells expressing this construct. Extensive plasma membrane localization of (HA)ENmyr was confirmed by confocal microscopy in the absence of IGF-IR expression (Fig. 4.4G). Interestingly, (HA)EN association with the membrane was observed in both the presence and absence of serum, suggesting that its localization may be serum independent. These findings were confirmed by subcellular fractionation studies of the (HA)EN and (HA)ENmyr molecules. R- and R+ cells expressing these constructs were starved overnight in 0.25% serum, lysed, and separated into cytoplasmic and membrane-associated fractions using differential centrifugation (see Materials and Methods). Total protein aliquots from each fraction were resolved by SDS-PAGE and immunoblotted with a-HA antibodies to determine the relative abundance of EN molecules in each fraction (Fig. 4.5A). R- cells showed virtually no membrane-associated (HA)EN, while a small but significant proportion of (HA)EN could be detected in R+ cell plasma membrane fractions (Fig. 4.5A lane 6). (HA)ENmyr demonstrated strong membrane localization regardless of IGF-IR expression, with a 2-3 fold increase compared to cytoplasmic fractions in both R- and R+ cells. Interestingly, a slight band shift occurs in potentially indicating multiple tyrosine phosphorylation events for this pool of EN molecules. a-Grb2 and a-caveolin-1 immunoblotting confirmed the identity of the 110 R+ M R-i H A ) E N C M R+ R- R+ (HA)EN (HA)ENmyr (HA)ENmvr C M 1 l~c rvP ic MI HA IRS-1 Grb2 Caveolin-1 B IGF-1 R EN ENmyr + - + + + P-Tyr IRS-1 P-Tyr NTRK3 h - 185 KDa 185 KDa 70 KDa 70 KDa Figure 4.5: IGF-IR expression or myristoylation of EN leads to membrane localization of EN and tyrosine-phosphorylated IRS-1. (A) EN and IRS-1 subcellular localization. Monolayer R- or R+ cells expressing (HA)EN or (HA)ENmyr were starved for 16 h in medium containing 0.25% serum, washed, and harvested. Cell lysates were separated into cytoplasmic (C) and membrane (M) fractions by differential centrifugation, followed by immunoblotting with antibodies directed against HA, IRS-1, Grb2 (as a cytosolic marker), and caveolin-1 (as a membrane marker). (B) Aliquots of 500 u.g protein isolated from membrane fractions of cell lines treated as in (A) were subjected to a-IRS-1 immunoprecipitation, followed by immunoblotting with a -phosphotyrosine (P-Tyr), a-IRS-1, or a-NTRK3 antibodies as indicated. Approximate sizes of protein bands (in kiloDaltons) are shown to the right of each panel. I l l cytoplasmic and membrane fractions, respectively. I next determined whether IRS-1 was also differentially localized in R-EN versus R+EN cells. Using a-IRS-1 antibodies, abundant cytosolic IRS-1 was (365). However, compared to R-EN cells, the proportion of IRS-1 associated with membrane fractions was dramatically elevated in R+EN cells and in either R- or R+ cells expressing ENmyr, (Fig. 4.5B compare lane 4 to lanes 6, 8 and 10). To confirm this I performed immunoprecipitation from membrane fractions from cells starved overnight using antibodies to IRS-1 followed by immunoblotting using D-phosphotyrosine antibodies. This showed prominent tyrosine phosphorylation of an ~185 kDa band only in membranes from R+EN, R-ENmyr, and R+ENmyr cells, which detected in all cell lines tested (see Fig. 4.5B) as expected based on previous studies was confirmed to be IRS-1 by reprobing with a-IRS-1 antibodies (see Fig. 4.5B). Moreover, tyrosine phosphorylated EN could readily be pulled down in association with IRS-1 from these fractions but not from those of R-EN or R+ cells (see Fig. 4.5B). This indicates that at least a portion of membrane-localized EN and IRS-1 molecules are associated with each other in R+EN, R-ENmyr, and R+ENmyr cells. From these studies I can conclude that 1) EN localization to membranes is dependent on IGF-IR expression or N-myristoylation, with the latter possibly being a more efficient mechanism; and 2) that IRS-1 membrane localization is enhanced in R+ cells by the presence of EN, or in R-and R+ cells by the expression of ENmyr. 4.2.5 Membrane-Targeted EN Confers Anchorage-Independent Growth and Transforms IGF-IR-Null Fibroblasts. If localization of EN and IRS-1 to the plasma 112 membrane requires IGF-IR and is important for transformation, then expression of membrane-targeted EN should circumvent the requirement for IGF-IR in transformation-i related functions of EN. Therefore the effects of myristoylated EN on the ability of R-and R+ cells to grow under anchorage independent conditions were assessed. ENmyr is expressed at similar levels and is tyrosine phosphorylated to the same degree as EN in corresponding cells (Fig. 4.6A). As shown in Fig. 4.6B, either R- or R+ cells engineered to express ENmyr readily formed multicellular spheroids identical to those of R+EN cells within 48 hours which are stable indefinitely in anchorage independent cultures. To confirm this we next performed soft agar colony assays as shown in Fig.4.6C. Similar to previous observations (301), R-MSCV (containing vector alone), R+, and R-EN cell lines showed minimal rates of macroscopic colony formation (averages of 1.8%, 6.9% and 3.7%, respectively), while R+EN fibroblasts demonstrated potent colony forming ability, with an average of 33.0% of cells forming macroscopic colonies. Interestingly, myristoylated EN was able to induce anchorage-independent growth (43.1% and 41.5% macroscopic colony formation for R-ENmyr and R+ENmyr cells, respectively). Thus, targeting EN to the plasma membrane is sufficient to induce transformation irrespective of IGF-IR expression levels. Fig.4.6C. Similar to previous observations (301), R-MSCV (containing vector alone), R+, and R-EN cell lines showed minimal rates of macroscopic colony formation (averages of 1.8%, 6.9% and 3.7%, respectively), while R+EN fibroblasts demonstrated potent colony forming ability, with an average of 33.0% of cells forming macroscopic colonies. Interestingly, myristoylated EN was able to induce anchorage-independent growth (43.1% and 41.5% macroscopic 113 A IGF-1R EN ENmyr P-Tyr NTRK3 B + + + + m mm* 1 m c TJ 0.6 ro 0.5 0.4 ai O 0.3 co ' c 0.2 o o O 0.1 0 R-ENmyr R+ENmyr <5> Figure 4.6: ENmyr restores anchorage-independent growth to IGF-IR-null cells. (A) 0C-NTRK3 irnmunoprecipitation on whole-cell lysates of EN- and ENmyr-expressing cells shows the level of oncogene expression. Blots were immunoblotted for a-phosphotyrosine, stripped, and reprobed with a-NTRK3. (B) R-ENmyr and R+ENmyr readily formed viable multicellular spheroids after 96 h in anchorage independent culture (100X magnification). (C) Soft agar colony-forming assays of EN- and ENmyr-expressing cells. Cells were plated in soft agar as described in the legend of Fig. 3C. R-EN cells form a significantly lower percentage of colonies than R+EN, R-ENmyr, and R+ENmyr cells. * represents a P value of <0.0001, as assessed by the Student's t test. 114 colony formation for R-ENmyr and R+ENmyr cells, respectively). Thus, targeting EN to the plasma membrane is sufficient to induce transformation irrespective of IGF-IR expression levels. I next tested signaling properties of ENmyr expressing cells. As shown in Fig. 4.7A and 4.7B, lanes 4 and 5, R-ENmyr and R+ENmyr spheroids showed similar constitutive activation of MEK1/2 and cyclin D1 induction in low and high serum as in R+EN cells. Of particular note, ENmyr was able to restore Akt activation in R- cells to levels comparable to those observed in R+EN cells under either low or high serum conditions. Constitutive phosphorylation of GSK3P serine 9 in low serum conditions indicates that this increase in Akt serine 473 manifests itself as increased Akt kinase activity. These studies indicate that membrane targeting of EN induces cellular Akt activation, anchorage independent growth, and soft agar colony formation of cells lacking IGF-IR. This supports our hypothesis that an important function of IGF-IR in EN oncogenesis is to enhance EN membrane localization, and that this likely involves the IRS-1 protein. Finally, I sought to determine if the observed effects of IGF-IR- independent Akt activation on anchorage independent growth correlate with in vivo transformation activity in EN expressing cells. I thus tested the ability of the R-EN/Akt-myr and R-ENmyr cell lines - both of which activate Akt signaling despite the absence of IGF-IR expression - to form tumours in nude mice, as compared to R+, R-/Akt-myr, R-EN and R+EN fibroblasts (Fig. 4.8). R+EN cells readily formed large palpable tumours after subcutaneous injection at rates comparable to those previously reported for EN-expressing NIH 3T3 cells (52, 295) (see Fig. 3D). In contrast, R-EN cells along with R-115 B IGF-1R EN ENmyr Ser 473-P Akt GSK-33-P MEK1/2-P Cyclin D1 Actin IGF-1 R EN ENmvr Ser 473-P Akt MEK1/2-P Cvclin D1 Actin + + + + Figure 4.7. ENmyr restores Akt activation to IGF-IR-null cells. Spheroid cultures of R- or R+ cells expressing EN, ENmyr, or vector alone were grown in 0.25% serum (A) or 9% serum (B) for 48 h, lysed, and subjected to immunoblotting as described in the legend of Fig. 4.2. Total levels of cyclin D1 protein were detected using a specific antibody, while activated Akt (Ser-473-P), GSK-3P (GSK-3p-P), and MEK1/2 (MEK1/2-P) were assessed using antibodies to phosphorylated proteins. Detection of actin was used as a loading control.. 116 2500 2000 1500 1000 500 0 4» R-EN x R+EN A - R-ENmyr --©•-- R-/Akt-myr -^R-EN/Akt-myr - i — i — i — i — i — m — t i — i v i ^ i 0 2 4 6 8 10 12 14 16 18 20 22 24 Days Post-Injection Figure 4.8 Restoration of Akt signaling in R-EN cells rescues in vivo transformation. Tumourigenesis in nude mice injected with R- and R+ derived fibroblast cell lines. R+, R-EN, R+EN, R-ENmyr, R-/Akt-myr, and R-EN/Akt-myr cells were injected subcutaneously into five mice per cell line and three sites per mouse. The y axis represents the average size of tumours from all sites for all mice in each given group, as measured every 2 to 3 days. Akt activation, either through EN membrane-targeting or expression of Akt-myr, rescued tumour formation in R-EN cells. Statistical analysis of tumour volume at day 20 was performed using the Mann-Whitney rank test, with the P value comparing R-EN and R-ENmyr cells < 0.0001. 117 and R+ control cells failed to form tumours even after four weeks post-injection. However, restoration of Akt activation through expression of Akt-myr in R-EN cells or ENmyr in R- cells restored tumourigenic activity; R-EN/Akt-myr tumours grew to similar sizes as R+EN tumours (average volume > 2000 mm3), although there was a lag of 2-4 days in attaining comparable tumour volumes in the R-EN/Akt-myr mice. These data confirm that the ability of activated Akt to restore anchorage independent growth to R-EN cells correlates with tumourigenesis and therefore with transformation potential. 4.2.6 IGF-IR Suppresses Anoikis in Anchorage-Independent EN-Transformed Fibroblasts. The above data indicate that IGF-IR contributes to maximal Akt activation in EN transformed cells. I next wished to determine whether this predominantly influences cell proliferation or survival, as Akt has been linked to both functions (367). R-EN and R+EN cells have similar growth and survival rates in monolayer cultures (300). To characterize these processes under anchorage-independent conditions, spheroid cultures of the various cell lines were assessed for indices of cell proliferation and apoptosis. First, R-EN and R+EN spheroids were cultured for either 48 or 96 hours, dissociated into single cells, fixed, stained with propidium iodide (PI) as an indicator of DNA content, and subjected to FACS analysis (Fig. 4.9A). Upon plotting a curve of DNA content, the typical result is two major peaks - the area under the first peak represents non-dividing cells in the G1 phase of the cell cycle; the second peak represents cells which have replicated their DNA content and are in G2 phase in preparation for mitosis; finally, the area between the two peaks represents cells actively replicating their DNA in S phase. At 48h, R-EN spheroids were found to have 60% of cells in the non-dividing 118 G1 phase and 31% of cells in either G2 or S phase, as compared to 48%:39% (G1:G2/S) for R+EN. While this indicated that R+EN spheroids might have a slight proliferative advantage over R-EN, it was postulated that this difference was not sufficient to explain the profound difference in transformation potential when comparing these two cell lines. This is in keeping with the comparable levels of MEK1/2 activation and cyclin D1 observed in R-EN and R+EN spheroid cultures (Fig. 4.2), as Ras/Erk induction of cyclin D1 is thought to underlie aberrant cell cycle progression in EN transformed cells (295). After 96 hours in spheroid culture I observed a significant proportion of sub G1 DNA content in R-EN cells (13%), a phenomenon often associated with late-stage apoptosis (368). To confirm these data, cells were assessed for uptake of bromo-deoxyuridine (BrdU) as a measure of proliferation. As shown in Figure 4.9B and 4.9C, the proliferative index of R-EN spheroids was identical to that of R+EN spheroids after 24 hours in culture, with each cell line showing ~41% BrdU positive nuclei. Interestingly, proliferating cells were distributed evenly throughout the spheroid structures for both cell lines at this time point (Fig. 4.9B). At the 48 and 96h time points, R+EN spheroids had a slightly increased proliferative index (48h: 27.6% for R-EN vs. 34.1% for R+EN (p = 0.055); 96h: 24.0% for R-EN vs. 29.4% for R+EN (p = 0.03), Fig 4.9C). However, R-EN spheroids at 96h (Fig. 4.9B) showed obvious structural disintegration, likely indicating that cell death was contributing to the observed differences in proliferation. 119 A 800 i =2 600 0 O 400 200 0 60% R-EN 48h 16% 0 200 400 600 800 FL2-H R-EN 96h 0 200 400 600 800 FL2-H 0 200 400 600 800 FL2-H R+EN 96h 0 2 0 0 4 0 0 b"00 8 0 0 FL2-H Figure 4.9: R-EN fibroblasts proliferate in anchorage-independent cultures. (A) R-EN and R+EN spheroids were grown for 48 and 96 h as indicated. Multicellular spheroids were separated into single cells, immediately fixed, stained with propidium iodide, and subjected to FACS analysis. Numbers represent the approximate percentages of cells in G1/S/G2, respectively. FL2-H, intensity of fluorescence channel 2. 120 B R-EN R+EN *V 24h > H * v ' . ' * - t ^ * * If » \ 24h R-EN R+EN 96h 96h - ^ |V ' **** • R-EN • R+EN 24h 48h 96h Figure 4.9: R-EN fibroblasts proliferate in anchorage-independent cultures (B) R-EN and R+EN spheroids were grown for the times indicated in the presence of BrdU, harvested, fixed, and embedded in paraffin. Sections from the spheroids were cut, and the samples were immunoassayed with a-BrdU antibodies. Pictures were taken from a typical field at 100X magnification. Dark staining indicates BrdU positivity. (C) BrdU-positive nuclei were counted and are represented as a percentage of total cells (n=15). Error bars represent 1 standard deviation. 121 Apoptotic rates in spheroids were then compared, as prominent morphologic features of cell death were observed in R-EN cells after extended days in anchorage-independent cultures (see Figs. 4.1B and 4.9B). Cell lysates were collected from R-EN, R+EN, R-ENmyr and R-EN/Akt-myr spheroid cultures in a time course from 24-96 hours post-plating, and caspase-3 activation was measured by fluorometry. As shown in Fig. 4.1 OA, R-EN spheroid cells showed significant increases in caspase-3 activation by 24h relative to R+EN monolayers which was maintained until 72h. By 96h post-plating, caspase-3 activity levels returned to baseline, presumably because most cells were non-viable or in late stages of apoptosis. In contrast, R+EN and R-ENmyr spheroids showed no detectable increases in caspase-3 activation (Fig. 4.10A), even after 96 hours in culture. Similarly, no enhanced caspase-3 activation was observed in R-EN/Akt-myr cells (Fig. 4.10A), as these cells remain viable in long-term spheroid cultures. To further document increased apoptosis in R-EN vs. R+EN spheroids I performed a - P A R P immunoblots on cell lysates from R-EN, R+EN, R-ENmyr or R-EN/Akt-myr fibroblasts grown for 24 or 48 hours in anchorage-independent culture (Fig. 4.1 OB). Lysates from R+EN monolayers treated with 10 u.g/ml doxorubicin were included as a positive control for PARP cleavage. R-EN cells showed significant levels of cleaved PARP at both 24 and 48 hours of anchorage-independent culture, whereby -50% of the total cellular amount was of the cleaved form, indicating that significant induction of caspase-3 mediated apoptosis was occurring. In contrast, cleaved PARP were undetectable or present at very low levels relative to the uncleaved form in R+EN, R-ENmyr and R-EN/Akt-myr cells at both time points tested. These findings strongly 122 A 3.5 3 -I 2.5 2 1.5 1 0.5 0 0 .-. i - • - R-EN • R+EN R-ENmyr ^^R-EN Akt + 24 48 72 Time Post-Plating (h) 1 96 Figure 4.10: R-EN fibroblasts undergo apoptotic death in anchorage-independent cultures. (A) Caspase-3 activation in spheroid cultures of R- and R+ fibroblasts. R-EN, R+EN, R-ENmyr, and R-EN/Akt-myr cells were plated in 9% serum under anchorage-independent conditions for 0 to 96 h as indicated, lysed, and assayed for caspase-3 activity by fluorometry as described in Materials and Methods. The relative level of caspase-3 activation was normalized to a value of 1.0 for R+EN fibroblasts growing in subconfluent monolayer cultures. Error bars represent 1 standard deviation. 123 B 24h spheroid culture 48h spheroid culture PARP Actin Figure 4.10 (ctd.): R-EN fibroblasts undergo apoptotic death in anchorage-independent cultures. (B) R-EN, R+EN, R-ENmyr, and R-EN/Akt-myr fibroblasts were grown in spheroid cultures for 24 or 48 h as indicated, lysed, and subjected to oc-PARP and a-actin immunoblotting. The 116-kDa and 89-kDa bands represent uncleaved (uc) and cleaved (cl) PARP, respectively, as indicated. R+EN monolayers treated with 10 u.g/ml doxorubicin (R+EN ML + dox) are included as a positive control for PARP cleavage. Detection of actin was used as a loading control. 124 indicate that the IGF-IR axis is not essential for cell cycle progression in EN transformed cells, but instead supports cell survival through enhanced Akt activation. Since this is only observed in cells placed under anchorage independent conditions, I conclude that IGF-IR signaling functions to suppress anoikis in EN transformed cells. 4.2.7 PI3K Inhibitors Block EN-lnduced Anchorage Independent Growth. If PI3K-Akt signaling is crucial for suppression of anoikis in EN transformed cells, then blocking this pathway should inhibit EN-induced anchorage independent growth. Previous studies have shown that the PI3K inhibitor LY294002 blocks soft agar colony formation of EN transformed NIH 3T3 fibroblasts (294). LY294002 also impairs soft agar colony formation of R+EN, R-ENmyr, and R-EN/Akt-myr cells, which it does in a dose-dependent manner to concentrations as low as 2.5 wVI (Fig. 4.11B). As expected, EN myristoylation does not confer resistance to LY294002. Soft agar colony formation of the R-EN/Akt-myr cell line was also inhibited by LY294002, since activation of the Akt-myr construct is dependent on PI3K to activate PDK1 and PDK2 functions (121). Inhibition of colony formation by LY294002 correlates with dose-dependent loss of Akt phosphorylation, as shown for R+EN cells in Fig. 4.11A. Moreover, LY294002 inhibits spheroid growth of EN transformed fibroblasts (see Fig 4.11C for R+EN cells), while the U0126 MEK1 inhibitor, which effectively blocked Erk1/2 phosphorylation (Fig.4.11A), had no demonstrable effect on spheroid formation or morphology (Fig. 4.11C). R+EN fibroblasts were next plated in either monolayer or non-adherent cultures and treated cells with varying doses of LY294002 for 24 hours and assessed caspase-3 activation as a measure of apoptotic cell death. Interestingly, it was found that while LY294002 125 R-EN R+EN LY294002 (uM) U0126 Ser 473-P Akt Erk1/2-P Actin B 0 0 2.5 10 25 0 + — - mWm ^ ^ ^ ^ ^ ^^^^^k MHjjj^^tt mtm} ^^ B^r * W ^^^(P mm — mm — — -a 0.5 CD I 0.4 o3 0.3 o 102 I 0.1 o * 0 X Li • DMSO • 2.5 U M L\ • 10 u M LY ^25 U M LY h i Figure 4.11: Inhibition of PI3K-Akt signaling blocks anchorage-independent growth induced by EN. (A) R+EN fibroblasts were grown under anchorage-independent culture conditions in medium containing 9% serum and were treated with the indicated doses of LY294002, DMSO control, or 25 pM U0126 for 4 h. Similarly cultured R-EN cells were treated with vehicle control only. Cells were then lysed and immunoblotted using antibodies to the indicated proteins. (B) R+, R-EN, R+EN, R-ENmyr, and R-EN/Akt-myr fibroblasts were grown in soft agar as described in the legend of Fig. 3B except that indicated doses of LY294002 or vehicle control were included. Cultures were fed every 2 days with 2 drops of medium containing the appropriate concentration of LY294002 (LY) or vehicle control. R+EN, R-ENmyr, and R-EN/Akt-myr all showed significant differences when DMSO treatment was compared to 10 uM LY294002 treatment (P < 0.0001 by Student's t test). 126 DMSO LY294002 U0126 I 1 r >, 7 5 e o u < 5 I c/> 4 co | 3 (0 ° 2 > _5 1 o n cn 0 • Monolayer • Spheroid 11111 DMSO dox 2.5n M 10 nM 25 „ M LY LY LY 25n M U0126 Figure 4.11 (ctd.): Inhibition of PI3K-Akt signaling blocks anchorage-independent growth induced by EN. (C) Photomicrographs (100X magnification) of the effects of LY294002 on R+EN spheroid formation after 24 and 72 hours. Cells were plated under anchorage-independent conditions and treated with 25 j^ M LY294002 or 25 u.M U0126 at the time of plating. (D) LY294002 induces apoptosis in R+EN spheroid cultures but not in monolayers. R+EN cells were plated in either anchorage-independent cultures or subconfluent monolayers and treated with vehicle control (DMSO), 10 p.g/ml doxorubicin (dox), or the indicated doses of LY294002 for 24 h. Cells were then collected, lysed, and assayed for caspase-3 activity as described in the legend of Fig. 4.10. Caspase-3 activation is plotted relative to untreated R+EN monolayer cells. Error bars represent 1 standard deviation (n=3). 127 treatment specifically induces apoptosis in anchorage independent cultures, monolayer cells remain unaffected (Fig. 4.11D). For example, treatment of R+EN spheroids with 10 \M LY294002, which effectively inhibits Akt Ser-473 phosphorylation (Fig. 4.11A) induces similar levels of caspase-3 activation as observed in R-EN spheroid cells grown for 24h in spheroid culture with no treatment. Although U0126 induced low-level caspase-3 activation in R+EN spheroids, a significant proportion of cells survived to form stable spheroid cultures (see Fig. 4.11C). Therefore, while it cannot be ruled out that the Ras/Erk pathway contributes to suppression of anoikis in EN transformed fibroblasts, I conclude that inhibition of Akt signaling through PI3K blockade can significantly impair anchorage independent growth of these cells. 4.3 DISCUSSION A large body of literature points to an essential role for the IGF-I receptor in transformation by dominantly acting oncogenes. Besides ETV6-NTRK3, other childhood tumour fusion oncoproteins such as the Ewing tumour EWS-FLI1 molecule and PAX3-FKHR of alveolar rhabdomyosarcoma also require an intact IGF-IR axis for oncogenesis (51, 52, 301). In this chapter I have shown that IGF-IR is necessary for suppression of anoikis in anchorage independent EN transformed murine fibroblasts. IGF-IR null R-EN cells fail to form colonies in soft agar and do not survive in anchorage independent spheroid cultures. This correlates with the inability of these cells to form tumours when injected into nude mice, compared to R+EN cells that are highly tumourigenic. It was found that expression of EN in R- cells is specifically associated with a defect in Akt activation, while induction of the Ras/Erk pathway leading to cyclin D1 up-regulation and 128 cell cycle progression are not impaired. Soft agar colony formation, spheroid formation, and tumourigenesis are all restored by ectopic expression of an activated form of Akt in R-EN cells, but not in R- cells expressing activated Akt alone. These data imply that the major component of IGF-IR's contribution to EN transformation is activation of Akt. The inability of EN to transform IGF-IR null cells could be circumvented by membrane targeting of the chimeric oncoprotein through N-terminal myristoylation. This suggests that IGF-IR functions to localize EN to the plasma membrane for optimal activation of downstream signaling pathways. As R+ENmyr cells had no increase in rate of transfomation as compared to R-ENmyr, I conclude that the critical signal provided by IGF-IR in wildtype EN transformation is merely facilitating membrane localization, rather than activation of a particular IGF-IR substrate. Membrane localization of EN correlated with a dramatic increase in the amount of tyrosine phosphorylated IRS-1 at the plasma membrane, and EN and IRS-1 were associated with each other in this compartment. The ability of IGF-IR to directly bind IRS-1 is presumably responsible for this phenomenon. Interestingly, insulin receptor is abundantly expressed in R- cells (48), but it cannot similarly localize EN/IRS-1 complexes to the plasma membrane to support transformation. Expression of the ENrriyr molecule in IGF-IR null cells is sufficient for stimulating the full spectrum of EN downstream signals including IRS-1 membrane targeting and Akt activation, suggesting that localization of EN along with IRS-1 to membranes may be particularly important for induction of the PI3K-Akt cascade. In order to survive under anchorage independent conditions and ultimately metastasize, cancer cells must be capable of suppressing detachment-induced apoptosis, or anoikis. Many studies point to a critical role for the pro-survival activity of 129 Akt in suppressing anoikis (369). Akt substrates such as Bad (213, 214) and caspase-9 (219) are direct apoptotic effectors which are inactivated by phosphorylation, while Akt phosphorylation of the FOXO subfamily of Forkhead transcription factors prevents their nuclear localization and subsequent up-regulation of pro-apoptotic genes (217, 370). Akt activation through expression of oncogenic Ras (229), or ectopic expression of constitutively activate Akt (371), is sufficient to prevent anoikis. Others have reported that inhibitors of PI3K-Akt signaling, but not of the Ras/Erk pathway, inhibit anchorage independent growth of fibroblasts induced by the v-Ros and EGFR-Ros oncogenes (372). Interestingly, a genome-wide screen for suppressors of anoikis identified the TrkB (NTRK2) neurotrophin receptor as a key molecule in this process (231). Ectopic expression of TrkB in rat intestinal epithelial cells led to up-regulation of phosphorylated Akt and prevention of caspase-3 mediated apoptosis, and these cells could be maintained under anchorage-independent conditions. Whether this involves IGF-IR was not evaluated. The NTRK3 kinase domain shares 79% identity with that of NTRK2, and therefore EN may promote anchorage independent survival of fibroblasts in a related fashion. In the current study, growth of R-EN cells in nonadherent cultures resulted in marked caspase-3 activation and cell death that was reversed by ectopic expression of myristoylated Akt. I also found that treatment of R+EN cell spheroids with LY294002 resulted in a significant induction of caspase-3 activity which was not observed in monolayer cells treated in parallel (see Fig. 4.11B). Taken together, these observations establish an essential role for Akt activation in maintaining the survival of EN-expressing fibroblasts under anchorage independent conditions, and implicate IGF-IR in this process. 130 Impaired Akt activation in R-EN spheroids persisted even in the presence of serum. This is surprising since these cells presumably possess other growth factor receptors that would be expected to activate the PI3K-Akt pathway in the absence of IGF-IR. One possible explanation for this observation is overexpression of PTEN, which has been linked to growth suppression and enhanced apoptosis in several tumour cell lines (373-375). However, Western blot analysis showed PTEN levels remained consistent between R-EN and R+EN spheroids. An alternative mechanism for constitutive Akt downregulation in R-EN cells is suggested by a recent study indicating that monomeric p85 forms cytosolic sequestration complexes with IRS-1 that act to dampen IRS-1/PI3K p110 signaling (376). Previous work in the Sorensen laboratory observed comparable or even increased binding of the PI3K p85 regulatory subunit to phospho-IRS-1 in serum-stimulated R-EN versus R+EN cells (301). In the absence of IGF-IR to localize EN and IRS-1 to the membrane, EN/IRS-1/p85 complexes might remain predominantly cytosolic to function in a dominant negative fashion to sequester IRS-1 and the PI3K p110 catalytic subunit away from the plasma membrane at sites of phosphoinositol-3,4,5-trisphosphate (PIP3) and phosphoinositol-3,4-bisphosphate (PIP2) generation (377). This would impact Akt activation, which requires recruitment to the plasma membrane through association of the Akt pleckstrin homology (PH) domain with PIP3 and PIP2. It is tempting to speculate that an active IGF-IR axis (or membrane-targeted EN) may modulate IRS-1 in such a way as to shift its equilibrium to the plasma membrane at sites of p110 function and Akt activation. Previous reports of a membrane-targeted IRS-1 molecule showed increased PI3K-Akt signaling, emphasizing the importance of proper IRS-1 localization for the activation of this pathway (366). 131 The fact that Ras/Erk pathway signaling remains intact in R-EN cells implies that activation of this cascade is independent of EN/IRS-1 complex localization to the plasma membrane. Whereas PI3K/Akt signaling takes places almost exclusively at the plasma membrane, Ras/Erk signaling can take place on Golgi and endoplasmic reticulum membranes (378). An HA-tagged EN construct showed a perinuclear expression pattern when expressed in R- cells (Fig. 4.4B), indicative of localization to internal membrane compartments. This allows for the possibility that Ras/Erk signaling is activated exclusively on endomembranes in R-EN cells. Increases in the output of Ras/Erk signaling in R-EN cells may also be due to their chronic inability to activate Akt. Akt can directly phosphorylate Raf on Serine 259 (169) in an inhibitory fashion, thus any residual amount of Ras signal in R-EN cells will be amplified when Raf is unencumbered by this phosphorylation event. In summary, I demonstrate here that a kinase active IGF-IR molecule that retains IRS-1 binding complements EN oncogenesis by contributing optimal activation of the PI3K-Akt cascade, a pathway that is essential for transformation by the EN oncoprotein. In contrast, Ras/Erk activation, cyclin D1 induction, and cell cycle progression appear to be IGF-IR independent in EN transformed cells. This data suggests that localization of EN and IRS-1 to the plasma membrane is key for Akt activation during EN transformation, and that such localization is enhanced by IGF-IR. Whether IGF-IR-mediated IRS-1 membrane localization is also essential for transformation by other fusion oncoproteins remains to be determined. The presence of IGF-IR confers anchorage independent growth potential to EN expressing cells through suppression of anoikis, likely by facilitating maximal PI3K-Akt activation. Based on my results, inhibition 132 of the IGF-IR/PI3K/Akt pathway may represent a clinically relevant strategy for tumours that express EN. 133 CHAPTER V ROLE OF THE mTOR SIGNALING PATHWAY IN ETV6-NTRK3 TRANSFORMATION 5.1 INTRODUCTION The abnormal cell growth observed in cancer is not only described in terms of increased proliferation, but also by an increase in cell size. An important component in the determination of cell size is translational control. Optimally growing cells, in the presence of the appropriate nutrients and growth factors, demand a high rate of protein synthesis to accomplish all the tasks occurring in the cell. The mTOR signaling pathway has evolved as a major regulator of translation and cell growth (379). Specifically, mTOR acts to control ribosomal proteins involved in the translation of a subset of mRNAs which possess a cap structure at their 5' untranslated region (UTR). Recent work has established that aberrant activity of this pathway leads to increased translation and subsequent cellular transformation. The specific mTOR inhibitor rapamycin is an inhibitor of tumour cell growth (380), and has shown promise in clinical trials as an anti-cancer agent (381). A major regulator of the mTOR signaling pathway is Akt, though its ability to inhibit the TSC1/2 tumour suppressor complex (as described in Chapter I). ETV6-NTRK3 potently activates Akt when expressed in fibroblasts, leading to the constitutive phosphorylation of the Akt substrate GSK3p\ Therefore, it was hypothesized EN-134 expressing cells would show constitutive activation of mTOR signaling under serum-starved conditions. Non-transformed R-EN cells show a particular defect in the activity state of Akt, thus I postulated that these would show similarly low levels of mTOR activity, and this would contribute to the non-oncogenic phenotype. Strong activation of mTOR effectors was observed in both the presence and absence of serum. In low serum conditions, I found that EN-induced mTOR activity was dependent on the presence IGF-IR. However, this effect was lost in 9% serum, as R-EN cells showed abundant S6 and 4E-BP1 phosphorylation. Inhibition of mTOR activity through rapamycin treatment effectively blocked growth of EN-expressing fibroblasts in soft agar. In contrast, inhibition of mTOR signaling failed to attenuate spheroid colony formation when levels of activated Akt were high in the cell, but did when PI3K/Akt signaling was also blocked. This illustrates a fundamental difference in signaling requirements for these two models of anchorage-independent growth. 5.2 RESULTS 5.2.1 mTOR Signaling is Elevated in ETV6-NTRK3-Expressing Fibroblasts. mTOR is a downstream effector of Akt, and plays an important role in protein translation, cell growth and proliferation (379). mTOR activity is potently inhibited by the bacterial macrolide rapamycin, and much current study is devoted to analysis of its use as a potential anti-cancer drug. As transformation-defective R-EN fibroblasts show a constitutive impairment in Akt signaling, components of the mTOR pathway were examined to assess whether this downstream cascade is important in EN-mediated oncogenesis. Thus R+, R-EN, and R+EN fibroblasts were grown in anchorage-135 independent culture, both in low (0.25%) and high (9%) serum, lysed, and subjected to Western blotting. The activity of mTOR was assessed by determining the levels of phosphorylated 4E-BP1, a direct mTOR target, and phosphorylated ribosomal S6 protein (S6-P), which is phosphorylated by S6K1, a second major mTOR substrate (379). In the case of 4E-BP1, the phosphorylated protein is represented by the slower-migrating upper band in an immunoblot, using antibodies directed against total 4E-BP1 protein. Interestingly, under low serum conditions, both phospho-4E-BP1 and phospho-S6 are low or absent in both R+ and R-EN spheroids, whereas R+EN cells show constitutive phosphorylation of these proteins (Fig. 5.1 A). When these same cells are plated in serum-containing media, the R+ fibroblasts maintain low levels of phospho-4E-BP1 and S6 (Fig. 5.1 B). However, R-EN spheroids show significant activation of the mTOR pathway in high serum conditions, as shown by the abundant amount of phospho-4E-BP1 and phospho-S6. As expected, levels of both phosphoproteins remain high in R+EN spheroids grown in high serum. Therefore it can be concluded that EN induces constitutive activation of mTOR signaling, and this phenomenon is IGF-IR-dependent in low, but not high, serum conditions. 5.2.2 Effect of Rapamycin Treatment on EN-lnduced Signaling. Rapamycin, through interactions with its intracellular receptor FKBP12, binds to mTOR and potently inhibits its kinase activity (378). This is reflected in decreased levels of phosphorylated S6 and 136 0.5% Serum 9% Serum Figure 5.1: ETV6-NTRK3 expression leads to constitutive activation of the mTOR signaling pathway. R+, R-EN and R+EN fibroblasts were cultured under anchorage-independent conditions in either 0.5% (left) or 9% (right) serum for 48h, lysed and subjected to immunoblotting. Levels of phosphorylated S6 (S6-P) were assessed using a pnospho-specific antibody. Hyper-phosphorylated 4E-BP-1 is detected with a total 4E-BP1 antibody as the slow-migrating (upper) band. Detection of total Akt was used as a loading control. 137 4E-BP1 ribosomal proteins. When examining the effects of 4h rapamycin treatment on R+EN spheroids it was noted that either 20 nM or 50 nM rapamycin blocked phosphorylation of these proteins to a similar extent in both low and high serum (Fig. 5.2 A and B). The upstream position of Akt in the mTOR cascade was confirmed by the observation that LY294002 had similar, though less potent, effects on both S6 and 4E-BP1 phosphorylation. LY294002-mediated reduction in activated Akt and S6-P levels was maintained after 24h in 9% serum, though its effect on the phosphorylation state of 4E-BP1 was greatly diminished (Fig. 5.2C). However, 24h rapamycin treatment caused a dramatic increase in the ratio of non-phosphorylated to phosphorylated 4E-BP1 (Fig. 5.2C compare lower band lane 3 vs. lanes 5 & 6), while continuing to block phosphorylation of S6. Therefore rapamycin treatment is able to effectively inhibit mTOR signaling when incubated with R+EN cells grown in spheroid culture. Recent work with rapamycin treatment of transformed cells has uncovered a negative feedback loop where mTOR-induced S6K1 activation leads to the suppression of PI3K/Akt signaling via degradation of IRS-1 (381). Since I have demonstrated in Chapter IV that EN-transformed fibroblasts are particularly dependent on IRS-1 mediated PI3K/Akt activation, we sought to determine whether rapamycin treatment of R+EN spheroids would lead to increases in IRS-1 protein levels and upregulate Akt signaling to an even greater extent. At the 4-hour time point, there was no significant difference in IRS-1 levels when comparing untreated to rapamycin or LY294002-treated R+EN cells. Akt phosphorylation remained similarly unchanged in rapamycin treated spheroids, whereas LY294002 was able to block Akt phosphorylation as expected. 24h 138 + + + + + + + + + 20 50 B + + + + + + + + + 1 IGF-IR EN LY RAPA(nM) IRS-1 P-S6 4EBP-1 P-Ser 473 Akt Actin IGF-IR EN LY RAPA(nM) IRS-1 P-S6 4EBP-1 P-Ser 473 Akt Actin Figure 5.2: Rapamycin inhibits mTOR but fails to upregulate Akt in R+EN cells. R+EN fibroblasts were grown under anchorage-independent culture conditions in medium containing 9% serum and were treated with either DMSO control, 10 pi of LY294002 (LY), or the indicated doses of rapamycin (RAPA). Similarly cultured R+ and R-EN cells were treated with DMSO only. Cells were then lysed and immunoblotted using antibodies to the indicated proteins. A) 4 h culture; 0.25% serum. B) 4h culture; 9% serum. 20 50 139 IGF-IR EN LY RAPA(nM) IRS-1 P-S6 4EBP-1 S473-P Akt Mek1/2-P Cyclin D1 Actin + + + + + + + + + 20 50 Figure 5.2 (ctd.): Rapamycin inhibits mTOR but fails to upregulate Akt in R+EN cells. R+EN fibroblasts were grown under anchorage-independent culture conditions in medium containing 9% serum and were treated with either DMSO control, 10 uJ of LY294002, or the indicated doses of rapamycin. Similarly cultured R+ and R-EN cells were treated with DMSO only. Cells were then lysed and immunoblotted using antibodies to the indicated proteins. C) 24 h culture; 0.25% serum. 140 treatment with either rapamycin or LY294002 caused phosphorylation as expected. 24h treatment with either rapamycin or LY294002 caused a modest increase in IRS-1 levels (Fig. 5.2C), however this rapamycin-induced elevation was not reflected by increased phospho-Akt activation. In fact, levels of Ser 473 phosphorylation were seen to decrease with 24h rapamycin treatment, though not to the low level seen in untreated R-EN spheroids. This indicates that an mTOR regulated negative feedback mechanism on Akt activation does not exist in EN-transformed fibroblasts, and that rapamycin may actually inhibit Akt phosphorylation after 24h treatment. This effect was specific to Akt, as rapamycin induced no variation in levels of phosphorylated MEK1/2 or cyclin D1. 5.2.3 Differential Effects of Rapamycin on EN-induced Anchorage-Independent Growth. As mTOR signaling is hyperactivated in EN-transformed cells, I postulated that this might contribute to EN oncogenesis and thus make rapamycin a potential therapeutic agent. Upon examination of R+EN fibroblasts grown in anchorage-independent conditions and treated with the above inhibitors, it was determined that unlike LY294002, rapamycin is unable to block formation of multicellular spheroids under any of the conditions tested (Fig. 5.3A). However, when these same cells were plated in soft agar in the presence of rapamycin, mTOR inhibition was able to significantly reduce both the number (Fig. 5.3B) and size (compare figures 5.3C and 5.3D) of colonies formed. This result is remarkably similar to that of U0126 treatment, which is unable to block spheroid formation (Fig. 4.11C), but completely abolishes soft agar colony formation (295). The magnitude of Akt activation maintained in rapamycin 141 DMSO LY294002 Rapamycin 0.25% 9% B r </ </ </ # # ^ # # ^ # # Figure 5.3: Rapamycin inhibits R+EN soft agar colony formation but not spheroid growth. A) R+EN fibroblasts were grown under anchorage-independent culture conditions in medium containing 0.25% or 9% serum and treated with either DMSO control, 10 pi of LY294002, or 50 nM rapamycin. Photos of anchorage-independent cultures (100x magnification) were taken 48h post-plating. B) (B) R+EN fibroblasts were grown in soft agar which included DMSO, 10 pM LY294002 or the indicated doses of rapamycin. Cultures were fed every 2 days with 2 drops of medium containing the appropriate concentration of drug or vehicle control. Similarly cultured R+ and R-EN cells were grown in the presence of DMSO. Colonies formed by DMSO-treated R+EN cells (C) were significantly larger than those formed by R+EN cells treated with rapamycin (D) (200x magnification). 142 treated R+EN cells (Fig. 5.2) is likely sufficient for cell survival in the spheroid assay, whereas other mTOR-regulated processes necessary for 3D growth, such as proliferation or protein translation may be more relevant in soft agar growth. Thus, these two models of anchorage-independent growth do not completely correlate with one another, especially in the context of drug treatment. 5.2.4 Co-Inhibition of PI3K/Akt and mTOR Synergistically Impair Growth of ETV6-NTRK3-Expressing Multicellular Spheroids. To further delineate the relative contributions of Akt and mTOR to EN-induced anchorage-independent growth, R-EN cells, which show a constitutive impairment in Akt activation, were treated with rapamycin, and compared to vehicle treated control cells. If the rapamycin insensitivity seen in R+EN spheroids was due to high levels of PI3K/Akt signaling, this would suggest that their receptor-null counterparts would be highly sensitive to inhibition of mTOR signaling. Figure 5.4A shows that this is indeed the case. Whereas R-EN spheroids treated with vehicle control are relatively stable 48h after plating, rapamycin treated R-EN spheroids appear as small loose clumps of dead or dying cells. As this phenotype does not occur in DMSO treated EN spheroids until ~96-120h post-plating, rapamycin can be said to accelerate the cell death induced by anchorage-independent culture conditions in R-EN cells. To confirm the rapamycin sensitivity of EN-expressing fibroblasts that possess low PI3K/Akt activity, R+EN fibroblasts were treated with the PI3K inhibitor LY294002, either alone or in combination with rapamycin. Unlike a dose of 25 u.M, which effectively blocks spheroid formation of R+EN cells by 24h (Fig. 4.11C), 143 Figure 5.4: Inhibition of PI3K sensitizes EN-expressing fibroblasts to the rapamycin inhibition. A) R-EN fibroblasts were grown under anchorage-independent culture conditions in medium containing 9% serum and treated with either DMSO control or 20 nM rapamycin. Photos of anchorage-independent cultures (100x magnification) were taken 48h and 96h post-plating. B) R+EN fibroblasts were grown under anchorage-independent culture conditions in medium containing 9% serum and treated with either DMSO control, 5 uM LY294002 (LY), 20 nM rapamycin (RAPA) or LY and RAPA together. Photos of anchorage-independent cultures (100x magnification) were taken 48h post-plating. 144 IGF-IR EN LY RAPA IRS-1 S6-P 4E-BP1 Ser 473-P Akt IGF-IR EN LY RAPA IRS-1 S6-P 4E-BP1 Ser 473-P Akt + + + + + + + + + + + + + + + + + + + + + + Figure 5.4 (ctd.): Inhibition of PI3K sensitizes EN-expressing fibroblasts to the rapamycin inhibition. EN-expressing fibroblasts were grown under anchorage-independent culture conditions in medium containing 9% serum and were treated with either DMSO control (R-EN and R+EN), 5 uM of LY (R+EN), 20 nM RAPA (R-EN and R+EN) or LY and RAPA together (R+EN). Cells were then lysed and immunoblotted using antibodies to the indicated proteins. C) 6h treatment D) 12h treatment. 145 IGF-IR EN LY RAPA IRS-1 S6-P 4E-BP1 Ser 473-P + + + + + + + + + + + + + + Akt Figure 5.4 (ctd.): Inhibition of PI3K sensitizes EN-expressing fibroblasts to the rapamycin inhibition. EN-expressing fibroblasts were grown under anchorage-independent culture conditions in medium containing 9% serum and were treated with either DMSO control (R-EN and R+EN), 5 \iM of LY (R+EN), 20 nM RAPA (R-EN and R+EN) or LY and RAPA together (R+EN). Cells were then lysed and immunoblotted using antibodies to the indicated proteins. E) 24h treatment. 146 5 pM LY294002 alone did not prevent anchorage-independent growth after 48h of treatment, as compared to DMSO control (Fig. 5.4B). As seen previously, rapamycin alone was unable to inhibit R+EN spheroid formation. However, when both the PI3K/Akt and mTOR pathways were concurrently inhibited, R+EN anchorage-independent growth was severely compromised. Thus, sensitivity to rapamycin was confirmed in EN expressing cells that have low Akt activity, either through a lack of IGF-IR expression or inhibition via LY294002 treatment. As expected, 20 nM rapamycin treatment of R-EN fibroblasts led to the potent inhibition of S6 and 4E-BP1 phosphorylation. In the case of 4E-BP1, this inhibition was progressive; at 6h an approximately equal ratio of the phosphorylated (upper) and non-phosphorylated (lower) forms existed, whereas by 24h of treatment this had shifted to an almost exclusively hypophosphorylated population of 4E-BP1. A similar pattern emerged for R+EN cells, where inhibition of both PI3K and mTOR was required for full inhibition of 4E-BP1 phosphorylation, at least at the 6h and 12h time points. 24h of treatment with LY294002 alone, however, was sufficient to ensure that the majority of 4E-BP1 was in the hypophosphorylated state. Such early inhibition of 4E-BP1 phosphorylation correlated well with apparent cell death in spheroid cultures, although whether this is a direct causative factor remains to be determined. 5.3 DISCUSSION Much recent work has been carried out to target mTOR signaling in oncogenesis. The findings that the high levels of mTOR activation in EN transformed cells lead to rapamycin sensitivity provides interesting insight into the relative contribution of various 147 signaling pathways in' EN oncogenesis. I observed that under serum-free conditions R+EN cells exhibited constitutive phosphorylation of mTOR downstream targets 4E-BP1 and S6. Non-transformed R-EN cells did not show activation of this pathway in the absence of serum, but IGF-IR was not required for mTOR activation in the presence of serum. R-EN cells have been shown to have very high levels of Ras/Erk pathway activation when grown in 9% serum (Fig. 4.2B). Activated Erk has been shown to stimulate p90 ribosomal S6 kinase (RSK) activity (168), which has now been linked to mTOR activation, as RSK was recently demonstrated to be a kinase which phosphorylates the TSC1/2 complex (168). This is one potential mechanism to explain the persistence of mTOR activity in R-EN cells with low levels of phospho-Akt. Rapamycin effectively inhibited the growth of EN-expressing fibroblasts in soft agar. Surprisingly, rapamycin had no phenotypic effect on R+EN spheroid culture, even with up to 7 days treatment. It is therefore likely that spheroid growth in particular is highly dependent on PI3K/Akt activation, and failure to adequately propagate the Akt signal leads to anoikis. A dependence on Akt for spheroid growth was observed in the Ewing's Sarcoma cell line TC32 (237). Since rapamycin significantly inhibits soft agar colony formation, clearly there are other mTOR-dependent aspects of growth in soft agar. mTOR inhibition results in decreased rate of translation, which manifests as a decrease in cell size. As can be seen in Figs. 5.3C and 5.3D, the R+EN colonies which are able to form in the presence of rapamycin are significantly smaller than their untreated counterparts, which may reflect decreased size of individual cells within the colony. This result is similar to treatment with the MEK1/2 inhibitor U0126 (see Fig 148 4.11C and (295)), where inhibition of MEK1/2 has little effect on spheroid cultures but effectively blocks soft agar colony formation through its effects on cell proliferation. Cells with lowered Akt signaling - either through lack of IGF-IR or PI3K inhibition via LY294002 - show significantly attenuated spheroid growth. In both cases it appeared that rapamycin treatment of Akt-deficient cells accelerated cell death in spheroid culture compared to Akt-deficient cells alone. Since the spheroid assay is essentially an anoikis assay, this provides evidence that mTOR activation can play a role in EN-induced survival, though it is only relevant if Akt is in an inactive state. A role of mTOR in preventing apoptosis has been demonstrated in other cell types, including cells derived from the childhood tumour rhabdomyosarcoma (382). Concurrent inhibition of both the mTOR and PI3K/Akt signaling pathways has been the subject of recent study. O'Reilly et al. (383) found that rapamycin resistant tumour cells could be made rapamycin sensitive with co-inhibition of Akt signaling through the NVP-AEW541 IGF-IR inhibitor. This report showed that rapamycin treatment leads to an up-regulation of both IRS-1 and P-Akt levels. The mTOR/S6K1 pathway has been shown to phosphorylate IRS-1 on serine and threonine residues (384). Serine/threonine phosphorylation of IRS-1 promotes its proteasomal degradation (385), which dramatically compromises the activation of PI3K/Akt. In R+EN cells, it was found that inhibition of mTOR by rapamycin does not lead to an increase in P-Akt levels. In fact, after 24h in culture, rapamycin appeared to cause decrease in P-Akt. This corroborates a recent study, where prolonged rapamycin treatment was inhibitory towards Akt signaling (386). The authors show that after 24h with the drug, mTOR is much less likely to be complexed with rictor than in untreated cells. They suggest this results from mTOR being partially 1 4 9 sequestered in complex with Raptor, FKBP12, and rapamycin. This has the effect of reducing the amount of mTOR molecules available to bind rictor and phosphorylate Akt on Ser 473. Furthermore, they found that cells fall into 3 groups with respect to the effect of rapamycin on Akt activity; cells in which activity increases, cells which show no effect and cells where Akt is inhibited. It would appear that EN transformed fibroblasts fall into the latter category. It is unlikely that the S6K1/IRS-1 negative feedback loop is relevant in R+EN cells, as they don't show an increase in Akt phosphorylation upon rapamycin treatment. The constitutive tyrosine phosphorylation of IRS-1 by EN would act to inhibit serine/threonine phosphorylation (387) and stabilize IRS-1 protein levels. However, rapamycin treatment does not completely abolish Akt activation; meaning agents which inhibit of PI3K/Akt signaling are likely to enhance the anti-tumour effects of rapamycin treatment in EN transformed cells. 150 C H A P T E R V I INTERACTION OF THE EN/IRS-1 TRANSFORMING COMPLEX WITH IGF-IR LEADS TO LIGAND-INDEPENDENT IGF-IR PHOSPHORYLATION, AKT ACTIVATION, AND TRANSFORMATION 6.1 INTRODUCTION Growth homeostasis in normal cells is maintained by the transient nature of growth factor-induced signals. Negative regulatory mechanisms that are simultaneously triggered upon ligand binding are essential to balance the proliferative signals propagated by growth factor receptors. Through a variety of mechanisms, receptor tyrosine kinases (RTKs) can be activated in a ligand-independent manner. This phenomenon is often devoid of the accompanying negative signal. Therefore ligand-independent RTK activation represents a potentially powerful mechanism for inducing oncogenic transformation. A portion of this chapter has been published. Moi Cell Biol. 2006 Mar;26(5): 1754-69. Martin MJ, Melnyk N, Pollard M, Bowden M, Leong H, Podor TJ, Gleave M, Sorensen PH. Moi Cell Biol. 2006 Mar;26(5):1754-69. 151 Numerous examples of RTK activation in the absence of ligand can be found in the cancer literature. One mechanism is through mutations that result in a constitutively active RTK. For example, several epidermal growth factor receptor (EGFR) mutations have been documented which result in a constitutively active tyrosine kinase domain (reviewed in (388)). Their importance in oncogenic transformation is underscored by the fact that glioblastoma multiforme patients who display one such EGFR mutation show very poor prognosis (389). Other RTKs displaying oncogenic mutations that lead to ligand-independent kinase activity include c-kit (390) and PDGFRa (391). Furthermore, altered cell-cell interactions in transformed cells can also lead to ligand-independent RTK activation. Adhesion can activate the Met receptor in the absence of its ligand hepatocyte growth factor (HGF) in mouse melanoma cells, but not in normal endothelial cells (392). E-cadherin mediated cell adhesion is able to activate EGFR in a ligand-independent manner in both ovarian (393) and squamous cell carcinomas (394). In this case, it is thought that E-cadherin can directly bind to EGFR leading to receptor aggregation and activation of PI3K/Akt and Ras/Erk proliferative signaling. As described in Chapter I, oncogenic fusion proteins often result from chromosomal translocations that juxtapose a tyrosine kinase alongside a dimerization domain, leading to.a constitutively active kinase and aberrant activation of growth-promoting pathways. In the case of ETV6-NTRK3, the chromosomal translocation effectively results in the expression of a ligand-independent NTRK3 receptor, leading to constitutive activation of PI3K/Akt and Ras/Erk signaling. Since I have shown that another RTK - the IGF-IR - plays an essential supporting role in EN transformation, I asked whether EN could activate IGF-IR in the absence of IGF-I or IGF-II. A previous 152 report showed that the Src intracellular tyrosine kinase was capable of ligand-independent activation of IGF-IR by direct phosphorylation of its kinase activation loop, and that this event was key to Src-mediated oncogenic transformation (395). Evidence from Chapter IV strongly indicates that IGF-IR is able to localize EN to the plasma membrane, through each protein's ability to interact with IRS molecules. I hypothesize that ligand-independent activation of IGF-IR would occur via direct tyrosine phosphorylation by the EN«IRS transforming complex, and thus require the IRS»IGF-IR interaction to take place. To this end, I here show that expression of EN results in constitutive tyrosine phosphorylation of wildtype IGF-IR. Further, IRS-binding and kinase dead mutants do not exhibit this phenomenon, which is reflected by their inability to rescue PI3K/Akt activation and transformation in R-EN fibroblasts. Finally, a membrane-targeted IGF-IR intracellular domain, which lacks the extracellular domain and is therefore unable to bind IGF ligand, was expressed in R-EN cells. This modified IGF-IR undergoes phosphorylation upon EN expression, and can support EN transformation on its own, strengthening the case for EN ligand-independent activation of IGF-IR as an essential step in EN transformation. 6.2 RESULTS 6.2.1 EN Expression Leads to Constitutive IGF-IR Tyrosine Phosphorylation. Data from Chapter IV provides strong evidence that IGF-IR localizes ETV6-NTRK3 to the plasma membrane, where it activates PI3K/Akt signaling to support transformation of fibroblasts. I hypothesized that this co-localization of EN and IGF-IR would result in the ability of the former to tyrosine phosphorylate IGF-IR in the absence of ligand 153 stimulation, and that this would be a necessary step in EN-mediated induction of anchorage-independent growth. I further postulated that IRS-1 acts to bridge IGF-IR and EN, and thus an IGF-IR mutant unable to bind IRS-1 (Y950F) would not exhibit ligand-independent tyrosine phosphorylation upon EN expression. Thus, R+, R-EN, R+EN, R- (Y950F) EN and R-EN cells expressing a kinase-dead IGF-IR (K1003A) were starved for 16h in 0.25% serum, after which time cell were lysed in Radio Immunoprecipitation Assay (RIPA) buffer, immunoprecipitated with anti-IGF-IR a-subunit antibodies, and immunoblotted for phosphotyrosine. Results were compared to similarly starved cells that had been stimulated with 50 ng/ml IGF-I for 10 minutes. As seen in Fig. 6:1, expression of EN in R+ cells led to constitutive tyrosine phosphorylation of IGF-IR (lane 3) - an event not seen in R+ cells transfected with vector alone. Interestingly, IGF-IR (Y950F) did not show ligand-independent phosphorylation, despite its ability to be strongly phosphorylated after IGF-I stimulation (lane 9). No tyrosine phosphorylation of IGF-IR (K1003A) could be detected when co-expressed with EN, implying the, kinase activity of IGF-IR is required for the ligand-independent phosphorylation that accompanies EN expression. I therefore conclude that EN expression induces constitutive tyrosine phosphorylation of IGF-IR in the absence of serum, and is a process that requires IGF-IR's IRS-binding and kinase activities. 154 r IGF-IR + Y950F -K1003 EN Starved^-24h IGF-IR IP P-Tyr IB Total Cell Lysate IGF-IR IB Actin IB Starved; 10 min. IGF-I + + igG Figure 6.1: Wildtype IGF-IR displays ligand-independent tyrosine phosphorylation upon EN expression. R-EN fibroblasts were engineered to express wild-type IGF-IR (IGF-IR +), IGF-IR with a tyrosine-to-phenylalanine mutation at residue 950 (Y950F +), or IGF-IR with a lysine-to-alanine mutation at residue 1003 (K1003A +) as indicated. These cells along with R+ and R-EN cells were starved overnight (16h) in 0.25% media and either lysed immediately in Radio Irnmunoprecipitation Assay (RIPA) buffer, or stimulated with 50 ng/ml of IGF-I for 10 min. prior to lysis. Lysates were next subjected to irnmunoprecipitation (IP) with a-IGF-IR antibodies (ct-subunit), followed by a-phosphotyrosine Western blotting. Western blots for total IGF-IR (p-subunit antibody) were performed on total cell lysates, while actin was used as a loading control. (*) 90 kDa IGF-IR p-subunit, (**) 200 kDa IGF-IR precursor, IgG: heavy-chain immunoglobulin. 155 6.2.2 EN Expression does not Lead to Elevated Production of IGF Ligands. One possible explanation for the constitutive tyrosine phosphorylation seen in R+EN cells is that they preferentially upregulate expression of either IGF-I or IGF-II to activate IGF-IR in an autocrine manner. To determine if this was indeed the case, conditioned media from R+, R-EN, R+EN, R-(Y950F) EN and R-(K1003A) EN cells starved for 16h in 0.25% serum-containing media was collected and subjected to IGF-I and IGF-II ELISA (see Materials and Methods). Conditioned media from Hepa 1-6 cells, a transformed cell line derived from mouse hepatocytes, was also collected as a positive control for IGF ligand production (396). As seen in Fig. 6.2, Hepa 1-6 cells produced an average of 531 pg/ml of IGF-I. However, levels of IGF-I in culture media collected from R+ derived cell lines were barely detectable, regardless of EN or IGF-IR expression. When levels of IGF-II were examined by ELISA, it was found that media collected from R+ cells contained an average of 568 pg/ml of the ligand. Upon EN expression, this level rose to 858 pg/ml. However, conditioned media from R-(Y950F) EN cells showed an even greater concentration of IGF-II, with an average of 1218 pg/ml. These cells do not display constitutive IGF-IR phosphorylation, despite their ability to respond to IGF treatment (Fig. 6.1), implying that this level of IGF-II in the media is insufficient for detectable tyrosine phosphorylation of IGF-IR under serum-free conditions. Therefore the hypothesis that autocrine production of IGF ligands explains the ability of R+EN to undergo constitutive tyrosine phosphorylation can be discounted, and allows for the possibility that EN is an IGF-IR kinase. 156 1500 n £ 1000 Ui o 5 0 0 0 JL • IGF-I • IGF-II 3> Figure 6.2: Constitutive tyrosine phosphorylation of IGF-IR is not due to EN-induced upregulation of IGF levels. R+, R-EN, R+EN, R-(Y950F)EN and R-(K1003A)EN were grown to 70% confluence in 1.0 ml of 0.25% serum-containing media per 35 mm dish. Samples of conditioned media were collected and subjected to IGF-I and IGF-II ELISA as described in the Materials and Methods. Media was also collected from Hepa 1-6 mouse hepatoma cells as a positive control for IGF production. Concentrations of IGF ligands found in the collected media were calculated as a measure of fluorescent intensity based on a comparison to a standard curve of known IGF-I and IGF-II concentrations. Data was collected from three separate experiments. Error bars represent 1 standard deviation. 157 6.2.3 IGF-IR Mutants Lacking IRS-1 Binding Ability or Kinase Activity Fail to Support ETV6-NTRK3 Transformation. The above experiments point to a mechanism whereby EN expression leads to ligand-independent activation of IGF-IR. Expression of wildtype IGF-IR restores Akt phosphorylation and transformation to R-EN fibroblasts (Figs. 4.1 and 4.3), through its ability to localize EN«IRS complexes to the plasma membrane. I hypothesized that these events are dependent on the constitutive activation of IGF-IR by EN, and that IGF-IR IRS-binding and kinase-activity mutants that do not undergo EN-induced tyrosine phosphorylation would be unable to restore survival signaling and resulting anchorage-independent growth to R-EN cells. The potential for IGF-IR(Y950F) or IGF-IR(K1003A) to support EN-mediated Akt activation and transformation in R- cells was therefore tested. Cells were plated in spheroid cultures for 48 hours in 0.25% serum and then lysed to determine relative activation of the Ras/Erk and PI3K/Akt pathways (Fig. 6.3A). MEK1/2 activation in cells co-expressing EN plus either IGF-IR (Y950F) or IGF-IR (K1003A) was similar to that in R-EN and R+EN cells, and enhanced compared to lines lacking EN. As before, Akt phosphorylation was impaired in R-EN cells compared to R+EN cells, and this was not rescued by expression of IGF-IR(Y950F) or IGF-IR(K1003A). The ability of the IGF-IR mutants to restore transformation activity to R-EN fibroblasts was next assessed by soft agar colony assays. Figure 6.3B demonstrates that, unlike wildtype IGF-IR, the Y950F and K1003A mutants were unable to support macroscopic colony formation of R-EN cells. These findings indicate that the kinase and IRS binding activities of IGF-IR are required for EN-associated Akt activation and transformation. 158 A I G F - I R Y 9 5 0 F K 1 0 0 3 A E N IGF-1R3 Ser 473-P Akt GSK-33-P MEK1/2-P Actin B S 0.5 ro K 0.4 w « 0.3 O w 0.2 CD o 0.1 o 2 o + + c ^ Figure 6.3: IGF-IR(Y950F) and IGF-IR(K1003A) mutants fail to restore Akt activation and anchorage-independent growth to R-EN fibroblasts. (A) R + , R - E N , R + E N , R - ( Y 9 5 0 F ) E N and R - ( K 1 0 0 3 A ) E N fibroblasts were grown as spheroids in 0 . 2 5 % serum for 2 4 h, lysed, and subjected to immunoblotting using antibodies to the indicated proteins. Detection of actin was used as a loading control. (B) Soft agar colony-forming assays were performed for each of these cell lines. (*) represents a P value < 0 . 0001 using the Student's t test to compare R + E N to R - ( Y 9 5 0 F ) E N and R - ( K 1 0 0 3 A ) E N . 159 6.2.4 Membrane-Targeted IGF-IR Intracellular Domain is Tyrosine Phosphorylated when Co-Expressed with EN. To confirm that EN-induced phosphorylation of the IGF-IR is truly ligand-independent, a membrane-targeted construct was created, consisting of the IGF-IR intracellular domain alone (ICmyr) (Fig. 6.4A). This molecule is unable to bind ligand, and would therefore be dependent on cytoplasmic kinases for its tyrosine phosphorylation. I also synthesized similar constructs that lacked either IRS-binding (ICmyr(YF)) or kinase (ICmyr(KA)) activity. Finally, to verify that this phosphorylation event takes place at the plasma membrane, an IGF-IR intracellular domain construct that does not localize to the plasma membrane (IC) was synthesized. ICmyr was then expressed in IGF-IR-null R- cells grown in 0.25% serum, and its ability to be tyrosine phosphorylated in the presence or absence of EN was assessed. Retroviral expression of ICmyr(YF), ICmyr(KA) and IC was also achieved in R-EN cells and they were grown under similar conditions. Cells were then lysed in RIPA buffer, immunoprecipitated with anti-phosphotyrosine antibodies and immunoblotted for the IGF-IR p-subunit (Fig. 6.4B). R+ cells stimulated for 10 min. with 50 ng/ml IGF-I and R+EN cells grown in low-serum media were included as a positive control for detection of phosphorylated IGF-IR. As expected, lysates from these cells showed abundant IGF- IR when immunoprecipitated with a-phosphotyrosine antibodies (90 kDa band). R+ cells grown in serum free conditions did show a low background level of tyrosine phosphorylation, which may be explained by the increased efficiency of anti-phosphotyrosine compared to anti-IGF-IRa immunoprecipitation (see Fig. 6.1 lane 3 vs. Fig. 6.4B lane 4), and the low but detectable level of IGF-II in media collected from 160 ATG I Y950 K1003 Y1136 Y1250 5' — N-myr IGF-IR intracellular domain 3' B 4r A A. n < 0 Total Cel l Lysate JS 4r P-Tyr IP IGF-IRfl IB Hi 9 90 k D a 4 5 k D a Total Cel l Lysate KaF-IRp IB Act in < — 90 k D a + — 4 5 k D a Figure 6.4: Membrane-targeted IGF-IR intracellular domain is tyrosine phosphorylated when co-expressed with EN. A) Schematic diagram of N-myristoylated IGF-IR intracellular (IC) domain (IC-myr). Note important tyrosine (Y) and lysine (K) residues. B) ICmyr was expressed in both R- and R-EN cells, while IRS-binding mutant (IC-myr(YF)), kinase dead mutant (ICmyr(KA)) and non-myristoylated (IC) constructs were expressed in R-EN cells alone. Cells were lysed in RIPA buffer and lysates were subjected to immunoprecipitation with a-phospho-tyrosine antibodies, followed by a-IGF-IRp Western blotting. Total lysates were blotted with a-IGF-IRp antibodies (top panel, right three lanes and middle panel), while actin was used as a loading control. Arrow indicates position of IC constructs. 161 R+ cells (Fig. 6.2). IC constructs appeared as two separate bands, possibly owing to the multiple in-frame ATG codons at the 5' end of the construct. Only the full-length upper band undergoes N-myristoylation and subsequent membrane localization. Interestingly, a-phosphotyrosine antibodies were able to pull down significant amounts of IGF-IR (ICmyr) (45 kDa band) when EN is also expressed. Significantly, ICmyr was not similarly pulled down in the absence of EN expression (Fig. 6.4B, lane 5), indicating that EN leads to the ligand-independent tyrosine phosphorylation of the IGF-IR IC domain. Further, it was found that this phosphorylation was dependent on the IRS-binding and kinase functions of IGF-IR, as ICmyr(YF) and ICmyr(KA) were not immunoprecipitated with anti-phosphotyrosine antibodies (Fig. 6.4B). I also conclude that this event occurs much more efficiently when localized to the plasma membrane, as only low amounts IGF-IR (IC) are pulled down in R-EN cells (Fig. 6.4B). 6.2.5 Expression of Membrane-Targeted IGF-IR Intracellular Domain Supports Akt Activation and Transformation of EN-Expressing Fibroblasts. I hypothesized that ligand-independent activation of IGF-IR by EN is necessary for the latter's ability to activate PI3K/Akt signaling and subsequently transform fibroblasts. Therefore it would follow that a membrane-targeted IGF-IR intracellular domain, which has been shown to be tyrosine phosphorylated in EN expressing cells, could rescue the defect in Akt activation and anchorage-independent growth seen in R-EN cells. To this end, R-EN (ICmyr) fibroblasts were grown as spheroids in low-serum media, and the level of Akt pathway activation as compared to R- (ICmyr) cells, which do not express EN, as well as R+EN fibroblasts that constitutively activate PI3K/Akt signaling, was assessed. I also 162 sought to determine whether the lack of EN-induced phosphorylation of ICmyr constructs bearing a mutation in either the IRS-binding or kinase domains correlated with an inability to restore Akt activation to R-EN cells. As seen in Fig 6.5A, expression of ICmyr in R- cells fails to upregulate phosphorylation of Akt, or its downstream target GSK-3(3. However, when expressed in R-EN cells, ICmyr induces phosphorylation of Akt to a magnitude similar to what is seen with expression of full-length IGF-IR (compare lanes 3 and 5). Furthermore, GSK-3P is robustly phosphorylated in R-EN (ICmyr), indicating that the observed Akt phosphorylation translates into functional activation of this kinase. As expected, ICmyr(YF) and ICmyr(KA) do not support full Akt activation in R-EN cells, as this process requires the IRS-binding and kinase activities of IGF-IR. Non-myristoylated IGF-IR (IC) was similarly unable to restore Akt pathway activity, further supporting the hypothesis that IGF-IR promotes localization of EN and PI3K/Akt activation at the plasma membrane. To assess the ability of ICmyr to restore anchorage-independent growth to R-EN fibroblasts, R-EN (ICmyr) cells were plated in soft agar along with the other cell lines analyzed for Akt signaling above (Fig. 6.5B). Whereas ICmyr was unable to induce significant soft agar colony formation when expressed in R- cells, R-EN (ICmyr) fibroblasts formed colonies at a rate that approached that seen for R+EN cells (26.4% vs. 34.4%; n=4). In concordance with their inability to show ligand-independent tyrosine phosphorylation or support Akt activation in concert with EN expression, neither ICmyr(YF), ICmyr(KA), nor IC alone were able to restore anchorage-independent 163 IGF-IR IC Figure 6.5: IGF-IR(ICmyr) rescues Akt activation and transformation in R-EN fibroblasts. A) R+, R-EN, R+EN, R-IC myr, R-EN ICmyr, R-EN ICmyr(YF), R-EN ICmyr(KA) and R-EN IC cells were grown under anchorage-independent conditions for 24h in DMEM containing 0.25% serum. Cells were lysed and subjected to Western blotting as described in Fig. 4.2. B) These same 8 cell lines were plated in soft agar as described above and assessed for colony formation after 10 days in culture (n = 3; * represents P < 0.0001 by the Student's f test). 164 growth when expressed in R-EN cells. It can thus be concluded that ligand-independent phosphorylation of IGF-IR by EN, requiring both the IRS-binding potential and kinase activity of IGF-IR, serves to promote PI3K/Akt activation, leading to cellular transformation. 6.4 DISCUSSION Ligand-independent activation of receptor tyrosine kinases is a commonly observed phenomenon in tumour cells, and leads to aberrant activation of proliferative and survival pathways, which are central to the pathogenesis of cancer. Here I provide evidence that the ETV6-NTRK3 fusion oncogene (a ligand-independent RTK in its own right) is able to induce constitutive tyrosine phosphorylation of IGF-IR, and this event does not require binding of IGF ligands to their receptor. This EN-induced phosphorylation of IGF-IR correlates with potent activation of the PI3K/Akt signaling cascade and promotion of anchorage-independent growth. As hypothesized, the IRS-1/-2 binding site (Y950) on IGF-IR is required for its ligand-independent tyrosine phosphorylation in EN-expressing cells, as IGF-IR(Y950F) mutants fail to display this phosphorylation. Since both EN (through binding the IRS PTB domain (Fig. 3.1)) and IGF-IR (through binding the IRS SAIN domain (41)) are both able to bind IRS-1/-2, our model dictates that IRS adaptors can act as a bridge to facilitate the phosphorylation event. Moreover, unlike wildtype IGF-IR, this mutant does not rescue PI3K/Akt activation and transformation in R-EN cells. Previous studies have shown that an intact tyrosine residue at position 950 is required for the mitogenic and transforming properties of IGF-IR (397). The data presented here support this notion, 165 and lend further support to the hypothesis that the primary function of IGF-IR in EN transformation is to localize EN»IRS complexes to the plasma membrane, as described in Chapter IV. It is tempting to speculate that the observed ligand-independent phosphorylation of IGF-IR stabilizes this membrane complex, which serves as a 'signaling node' to activate pro-proliferative and anti-apoptotic pathways. It was perhaps somewhat unexpected that the kinase activity of IGF-IR itself is required for its EN-induced tyrosine phosphorylation under serum-free conditions. If IGF-IR is truly an EN substrate, one might hypothesize that it would remain so even if non-tyrosine residues underwent mutation. As this is not the case, the proposed model of the interplay between IGF-IR and EN»IRS must necessarily be more dynamic. The observed results are reminiscent of a recent study showing ligand-independent propagation of EGFR activation in response to phosphatase inhibition, an event that is not seen with kinase-dead EGFR expression (398). EGFR polymerizes in a manner not seen with IGF-IR, however EN does have this capability (296). If a low frequency EN-induced activating phosphorylation of the IGF-IR can take place at its kinase loop, then subsequent IGF-IR-induced phosphorylation of Y950 would stabilize EN«IRS complexes at the plasma membrane. This might be initiated by EN, through it's interaction with IRS, binding to non-phosphorylated IGF-IR; there is some evidence that IRS-1 can bind the Y950 equivalent of insulin receptor (Y960) when non-phosphorylated, albeit at greatly reduced affinity (399). Polymerized EN could then activate IGF-IR molecules in the local vicinity, to amplify the ligand-independent phosphorylation of IGF-IR. Perhaps this at least partially explains why EN SAM domain mutants which can dimerize, but not polymerize, fail to induce transformation (296). It will be intriguing to assess whether 166 ligand-independent phosphorylation of IGF-IR is achieved upon expression of these mutant EN molecules. Alternatively, it is possible that the K1003A mutation alters the conformation IGF-IR to the point where it cannot interact with the EN»IRS complex. One potential way to address this question would be the creation of an IGF-IR (Y950D; K1003A) double mutant that lacks IGF-IR kinase activity but carries an aspartic acid residue, which mimics phosphorylated tyrosine, at the IRS binding site. If this mutant is able to restore transformation to R-EN cells, it would imply that the sole function of IGF-IR kinase activity is to phosphorylate the Y950 site to solidify the formation of the tri-molecular complex at the plasma membrane. A construct consisting of the IGF-IR intracellular domain alone, targeted to the plasma membrane with a myristoylation sequence (ICmyr), exhibited EN-induced tyrosine phosphorylation, and supported PI3K/Akt pathway activation and transformation when expressed in R-EN cells. As this molecule does not undergo tyrosine phosphorylation in R- cells with no EN expression, it is unlikely that clustering at the membrane causes an auto-phosphorylation, but rather indicates that EN mediates its activation. This construct is somewhat similar to the EGFR vlll mutant, which is expressed in glioblastomas, as well as lung and ovarian tumours (388). EGFR vlll lacks a significant portion of the extracellular domain, and thus does not bind ligand. Despite this fact, it is constitutively active (400), and its expression leads to potent activation of both the Ras/Erk (401, 402) and PI3K/Akt (403) cascades. Interestingly, patients who display this mutation respond favourably to treatment with the EGFR inhibitor gefitinib (Iressa) (404). 1 6 7 Much effort is currently being invested in targeting the IGF signaling axis, and IGF-IR in particular, for potential anti-oncogenic therapies (reviewed in (405)). While there have been examples of tyrosine kinase inhibitors that have demonstrated success in the clinic (e.g. imatinib (406) and gefitinib (407)), many others have failed to live up to initial promise. It is thought that IGF-IR may be a particularly difficult kinase to specifically target, due to its high degree of homology to the insulin receptor, and the resulting potential for disrupting normal glucose metabolism. Therefore an alternative strategy of inhibiting IGF ligand interaction and activation of IGF-IR through humanized interfering IGF-IR antibodies has been undertaken, and many such antibodies are in pre-clinical development (405). If our hypothesis that EN activates IGF-IR in a ligand-independent manner is correct, one would predict that IGF-IR antibody therapy would have little effect on the growth of EN-expressing tumours. As the list of oncogenic kinases that require IGF-IR for their transformation grows, so do the potential mechanisms for ligand-independent activation of this RTK. This phenomenon will need to be taken into account when designing anti-cancer therapies that target the insulin-like growth factor signaling axis. 168 C H A P T E R V I I SUMMARY AND FUTURE DIRECTIONS 7.1 GENERAL SUMMARY The ETV6-NTRK3 fusion tyrosine kinase is a potent oncogene that has transforming potential in multiple cell lineages. Previous work in our laboratory indicated that the IGF-IR axis plays a critical role in supporting EN tumorigenesis (300). The studies presented here expand upon this notion. Specifically, I have shown that the EN interaction with the PTB domain of IRS adaptor proteins is essential for its ability to transform either fibroblasts or hematopoietic cells. Reducing IRS-1/-2 expression through siRNA mediated gene knockdown, or disrupting EN«IRS interactions through overexpression of ah IRS-1 PTB domain, is sufficient to inhibit transformation. Further work showed that the insulin-like growth factor receptor, through its ability to interact with IRS molecules, localizes EN to the plasma membrane, which facilitates the activation of the PI3K/Akt survival pathway. Inhibition of PI3K, or its downstream effector mTOR, blocks soft agar colony formation of EN-expressing cells, highlighting the importance of this signaling cascade in the pathogenesis of EN-induced tumours. Finally the experiments detailed in this thesis provide evidence that EN expression causes ligand-independent tyrosine phosphorylation of the IGF-IR, which likely serves to stabilize the EN«IRS complex at the plasma membrane to trigger activation of signaling pathways associated with transformation. The data presented in this thesis has considerably updated the model for EN-induced signal transduction, and highlights 169 the essential role for the IGF signaling axis in EN-mediated oncogenesis. A schematic summary of these findings is represented in Figure 7.1. In the following sections, major observations of this thesis will be discussed in the context of potential avenues for further experimentation. 7.2 ROLE OF IRS MOLECULES IN TRANSFORMATION INDUCED BY ONCOGENIC KINASES Work in both fibroblasts and hematopoietic cells that have reduced or absent levels of IRS-1 and IRS-2 has demonstrated their importance in EN-mediated anchorage independent growth. Further evidence that IRS adaptor function is key for EN's ability to activate pro-proliferative and survival pathways comes from recent experiments with the Eph4 breast epithelial cell line transformed by EN. These studies have shown that IRS-1/-2 siRNA can disrupt the three-dimensional morphology of EN-expressing Eph4 multicellular spheroids grown in Matrigel (a growth medium consisting of reconstituted basement membrane components), and attenuate the activation of signaling pathways associated with EN expression (C. Tognon, unpublished observations). Thus, the importance of IRS adaptors to the pathogenesis of tumours exhibiting this t(12;15) translocation applies to each of the cell lineages in which it is found. It had been earlier hypothesized that chimeric oncoproteins resulting from chromosomal translocations were restricted to a specific cell lineage (292). One potential reason given for this idea was that only certain cell types possessed the 170 Figure 7.1: Cooperation between the IGF signaling axis and the ETV6-NTRK3 chimeric tyrosine kinase in activating transformation-associated pathways. Note that EN induced activation of PI3K/Akt signaling is dependent on IGF-IR-mediated localization of EN/IRS complexes to the plasma membrane. Conversely, Ras/Erk activation and cyclin D1/2 upregulation are IGF-IR- and therefore plasma membrane-independent. Our model indicates that EN-induced tyrosine phosphorylation of IGF-IR leads to its ligand-independent activation - an event which stabilizes the EN/IRS transforming complex at the plasma membrane to allow for oncogenic signaling. 1 7 1 \y IGF i/n i IGF-1R Q Q IGF-1R Protein Translation T Proliferation suitable signaling machinery with which the fusion protein could co-operate to promote transformation (293). The fact that IRS-1 and IRS-2 have a wide spectrum of expression is a possible explanation as to why EN oncogenesis is not restricted to a particular cell type. Besides EN, several other fusion oncoproteins have a similar structure that pairs a dimerization domain with a protein tyrosine kinase. Of these, Trk-T1 (317), Bcr-Abl (328), and NPM-ALK (408) have each been shown to interact with IRS proteins. PI3K/Akt pathway activation is an important signal resulting from expression of these chimeric kinases (409, 410). This raises the intriguing possibility that IGF-IR localizes them, through their binding to IRS-1/-2, to the plasma membrane to activate PI3K/Akt in a similar fashion. I would hypothesize that R- cells would be resistant to transformation by Trk-T1, Brc-Abl or NPM-ALK, and cells expressing these fusion proteins would be susceptible to agents which block IGF-IR kinase activity or reduce expression of IRS molecules. In the case of Bcr-Abl, whose expression is the causative factor in chronic myelogenous leukemia (CML), there already exists a potent kinase inhibitor (imatinib) that has shown clinical effectiveness for patients with this disease (406). However, the appearance of Bcr-Abl mutations that confer imatinib resistance have led to the search for new drugs to combat CML (411). If the IGF axis plays as an important of a role in Bcr-Abl signal transduction as has been shown for EN, then drugs which target this pathway would be an effective complementary treatment for CML, especially for those cases which show imatinib resistance. EN expression leads to constitutive activation of the PI3K/Akt and Ras/Erk pathways (295). Though EN cannot bind to the upstream components of these 173 pathways directly, EN-associated IRS-1 has been shown to bind both p85 (to activate PI3K) and Grb2 (to activate Ras) (301). However IRS-1 can potentially bind a number of other signaling molecules whose downstream effectors have been directly implicated in oncogenic transfomation. These include Nek (71), Crk (412) and SHP2 (413). Nek is an adaptor protein that is able to bind and p21 activated kinase (PAK1) (414), which can bring PAK1 into proximity with Rho GTPases (415). PAK1 is the major Rho family effector, and among other functions can promote the cytoskeletal reorganization, which is essential for invasion (415). Crk is also an adaptor that plays a role in cytoskeletal rearrangement, and its overexpression has been documented to promote transformation in vitro (416) and tumour progression in vivo (417). Tyrosine phosphorylation of IRS-1 is maintained in R-EN cells, despite the fact that they are not transformed (301). Data from Chapter IV points to a lack of membrane localization for tyrosine phosphorylated IRS-1 in R-EN cells as the cause for this phenotype. However, this may not fully explain the transformation defect. While it is clear that R-EN-derived IRS-1 is tyrosine phosphorylated and associated with the p85 subunit of PI3K, it is not known whether any of the 18 potential tyrosine residues are differentially phosphorylated compared to the IRS-1 found in R+EN cells. Mass-spectometry on tryptic digests of IRS-1 or IRS-2 proteins isolated from R-EN and R+EN cells could determine if specific tyrosines are hypophosphorylated in R-EN cells. Further experiments could reveal whether this lack of phosphorylation translated into a lack of binding affinity for corresponding proteins. If this were the case, defects in the pathways which these proteins activate would be hypothesized to play a role in the non-malignant phenotype displayed by R-EN cells. 174 The experiments detailed in Fig. 4.6B demonstrated a correlation between EN transformation and the presence of tyrosine phosphorylated IRS-1 at the plasma membrane. In our membrane fractionation experiments, however, cell nuclei were removed in the initial centrifugation step. Recent work has shown that IRS-1 localization to the nucleus is associated with oncogenic transformation (320, 334). Moreover, IRS-1 mediates the IGF-I stimulated nuclear translocation of p-catenin, and leads to the upregulation of specific genes, including cyclin D1 (418). However, this process is unlikely to be relevant in EN-mediated transformation, as there is no upregulation in p-catenin-controlled genes upon EN expression (Cristina Tognon, unpublished observations). Nevertheless, the potential exists for preferential accumulation of tyrosine phosphorylated IRS-1 in the nucleus of R+EN cells, compared to the nuclei of R+ or R-EN cells. Since PI3K/Akt can also function in the nucleus (419), and this pathway is upregulated in EN-expressing cells, it would be interesting to determine if nuclear signaling plays any role in EN-mediated transformation. 7.3 E T V 6 - N T R K 3 MEMBRANE LOCALIZATION Once thought to be a uniform lipid bilayer, the plasma membrane is now regarded as a dynamic structure that consists of several microdomains that vary in their lipid and protein composition. One such microdomain is the "lipid raft", which is a detergent insoluble structure enriched for cholesterol and glycosphingolipids (420). It is thought that lipid rafts display an increased concentration of signaling proteins, allowing for clustering of functionally related proteins and efficient propagation of signals from outside the cell to the nucleus. It should be noted, however, that the relevance or even 175 the existence of lipid rafts in vivo has been disputed (420). IGF-IR has been detected in a subset of lipid rafts known as caveolae (421, 422), which are present as small membrane invaginations and characterized by the presence of caveolin-1 (423). IGF-IR activation leads to the tyrosine phosphorylation of caveolin-1 (424), and this event facilitates the localization of IRS-1 to caveolae (425). Myristoylated proteins such as c-src also localize to caveolae, and caveolin-1 has been shown to be a c-src substrate (426). Evidence that IGF-IR localizes EN via IRS-1/-2 interactions to the plasma membrane may indicate the formation of a signaling complex at this specific membrane microdomain. However, immunoprecipitating EN from detergent insoluble fractions (fraction 3.) in which caveolin-1 and c-src are found was unsuccessful (Fig. 7.2). Instead, the majority of EN was pulled out in cytoplasmic fractions (fractions 6-8). This may have been due to cell lysis conditions causing disruption of EN membrane interactions, as only low amounts of (HA)EN were seen in membrane fractions as measured by direct Western blot (see Fig. 4.5A). N-myristoylated EN, on the other hand, clearly co-fractionated with caveolin-1 and c-src. Strong localization to these caveolae microdomains induced by the myristoyl lipid modification may explain the potent ability of ENmyr to transform fibroblasts, even in the absence of IGF-IR expression. Whether EN association with lipid rafts/caveolae is necessary for transformation could be determined by experiments employing cholesterol-depleting compounds. They are able to disrupt formation of these membrane microdomains, and would block EN-induced oncogenesis if such structures are indeed essential for the downstream signaling initiated by EN expression. 176 Sucrose gradient fraction 1 2 3 4 5 6 7 8 caveolin-1 w\ c-src Sucrose gradient fraction 1 2 3 4 5 6 7 8 NIH 3T3 - MSCV NIH3T3-EN NIH 3T3-ENmyr NTRK3 IP y NTRK3IB J Figure 7.2: ENmyr but not EN localizes to detergent insoluble membrane fractions. NIH 3T3 fibroblasts were stably transfected with empty vector (MSCV), EN or ENmyr, grown in DMEM + 9% serum and then lysed 1% Triton X-100-containing buffer. Lysates were subjected to sucrose density gradient centrifugation as described in (426). Fraction 3 represents the detergent-insoluble precipitate. Fractions were removed, beginning at the top of the tube with fraction 1, and were either immunoprecipitated with a-NTRK3 antibodies followed by NTRK3 immunoblotting, or probed directly for caveolin-1 and c-src. 177 N-myristoylated EN was able to induce PI3K/Akt activation and transformation to R- cells. This is one of the rare cases in which a dominantly acting oncoprotein is able to overcome the requirement for the IGF-IR in oncogenesis. A similar phenomenon has been noted with regards to the Src tyrosine kinase. Expression of an activated form of c-src (Y527F) was unable to transform R- cells, but v-src (the sequence originally derived from a transforming virus of chickens) is able to induce transformation in the absence of IGF-IR (428). Like EN and ENmyr, non-transforming c-src (Y527F) and transforming v-src each lead to constitutive IRS-1 tyrosine phosphorylation when expressed in R- cells. However, phosphorylation of FAK is significantly increased in R-cells expressing v-src compared to those expressing c-src(Y527F). As FAK can potentially act upstream of PI3K activation (227), one might postulate that activation of PI3K/Akt signaling by oncoproteins in the absence of IGF-IR is sufficient for transformation, v-src's increased potential to activate FAK may reflect a greater efficiency in associating with focal adhesions, and underscore the importance of subcellular localization in the propagation of specific intracellular signals. I have observed that the activated form of K-Ras is also able to transform R- cells (Fig. 7.3). In contrast, Ha-Ras transformation shows an absolute requirement for IGF-IR expression, as has been previously found (354). Differences in lipid modifications amongst individual Ras isoforms leads to differences in localization to membrane microdomains (429). For example, Ha-Ras appears to have a much higher affinity for lipid rafts than K-Ras (430). If the IGF-IR is required for localizing Ha-Ras to caveolae/lipid rafts, and for subsequent activation of PI3K/Akt signaling, this may explain why R- cells are resistant to Ha-Ras-induced anchorage-independent growth. It 178 Figure 7.3: Differential transforming potential of activated Ha-Ras and K-Ras in IGF-IR-null fibroblasts. R- or R+ cells were stably transfected with either EN, activated Ha-Ras or activated K-Ras, and plated in soft agar as described in the Materials and Methods. (* represents a P < 0.0001 as measured by the student's t-test; n=3). 179 would be tempting to speculate that the particular lipid domain in which K-Ras is found allows for IGF-IR-independent activation of PI3K. However, data showing Ha-Ras as a more potent activator of PI3K signaling compared to K-Ras (which has a stronger affinity for Raf-1) (431), casts some doubt as to the validity of this hypothesis. 7.4 IGF-IR LIGAND-INDEPENDENT ACTIVATION Data from chapter VI of this thesis show that EN expression leads to constitutive IGF-IR tyrosine phosphorylation, and is dependent on the IRS-1 binding and kinase activities of the receptor. I hypothesize that this phosphorylation event, likely through targeting of the activation-loop tyrosines of IGF-IR, would serve to turn on IGF-IR kinase activity. Future work would endeavour to assess whether this is the case by performing kinase assays on IGF-IR immunoprecipitated from EN-expressing and control cells. To determine the specific tyrosines in IGF-IR that undergo phosphorylation, phospho-peptide mapping could be performed. If it were indeed to be found that EN phosphorylation leads to ligand independent IGF-IR kinase activity via phosphorylation of its activation loop, this would parallel the similar activation of IGF-IR by Src (395). Interestingly, there is some evidence that Src may play a role in EN transformation, as Src appears to bind EN, and the Src inhibitor SU6656 can impair anchorage-independent growth of EN expressing NIH 3T3 cells (Wook Jin and Seong-Jin Kim, unpublished observations). If this same inhibitor blocked EN-induced phosphorylation of IGF-IR, it would point to a role for EN as an organizing protein for a large signaling complex featuring IRS molecules, PI3-kinase, IGF-IR, Src, and EN itself. 180 Inhibition of the IGF signaling axis has been actively pursued as a mechanism for blocking tumour cell growth. As mentioned above, strategies to directly inhibit the IGF-IR fall into two groups: humanized antibodies that block IGF ligand/receptor interaction (and may also lead to receptor internalization), and chemical kinase inhibitors. The IGF-IR inhibitor NVP-AEW541 has been shown to potently inhibit IGF-IR and shows in vivo anti-tumour activity (352). Ewing's tumour, osteosarcoma and rhabdomyosarcoma cell lines, each of which is derived from a pediatric neoplasm, were shown to be susceptible to NVP-AEW541-induced growth arrest in a mouse xenograft model (432). It should be noted, however, that this drug has a similar IC 5 0 value with respect to the insulin receptor, and thus may interfere with glucose metabolism if used in the clinic. However, the mice used in the original in vivo study showed normal levels of plasma glucose and insulin after NVP-AEW541 treatment (352). Moreover, in vitro experiments with NVP-AEW541 have been very informative regarding the growth inhibitory effects of blocking IGF-IR signaling, and the interplay between activated IGF-IR and mTOR signaling (383). NVP-AEW541 and rapamycin act synergistically to block proliferation of transformed breast cells. There is evidence that both IGF-IR and mTOR kinase activities are required for EN transformation, and that inhibition of IGF-IR-dependent PI3K activation sensitizes EN spheroids to the effects of rapamycin (Fig. 5.4). Therefore the Sorensen laboratory has undertaken a collaboration* to determine if NVP-AEW541 * collaboration with the Genomics Institute of the Novartis Research Foundation; La Jolla, CA. 181 can block EN-induced tumour formation in vivo, and if so, we will assess the co-operative effects of treating with rapamycin simultaneously. 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Scotlandi, K., Manara, M. C, Nicoletti, G., Lollini, P. L, Lukas, S., Benini, S., Croci, S., Perdichizzi, S., Zambelli, D., Serra, M., Garcia-Echeverria, C , Hofmann, F., and Picci, P. Antitumor activity of the insulin-like growth factor-l receptor kinase inhibitor NVP-AEW541 in musculoskeletal tumors. Cancer Res, 65:3868-3876,2005. 223 \ APPENDIX U B C THE UNIVERSITY OF BRITISH COLUMBIA ANIMAL CARE CERTIFICATE Application Number: A03-0190 Investigator or Course Director: Poul H . B . Sorensen Department: Pathology & Laboratory Medicine Animals: Mice N u / N u C D - 1 , N u / N u or SCID 85 Start Date: October 1,2004 Date™™1 February 22,2006 Funding Sources: Grant Agency: Canadian Institutes of Health Research Grant Title: Role of the IGF's and Type 1 IGF receptor in pediatric solid tumours Unfunded X T / A . . . . N /A title: The Animal Care Committee has examined and approved the use of animals for the above experimental project. This certificate is valid for one year from the above start or approval date (whichever is later) provided there is no change in the experimental procedures. Annual review is required by the CCAC and some granting agencies. A copy of this certificate must be displayed in your animal facility. 224 

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