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Molecular characterization of pediatric spindle cell tumors Knezevich, Stevan Robert 2000

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MOLECULAR CHARACTERIZATION OF PEDIATRIC SPINDLE CELL TUMORS by STEVAN ROBERT KNEZEVICH B.Sc. The University of British Columbia, 1995 A THESIS SUBMITTED I N P A R T I A L F U L F I L L M E N T OF T H E R E Q U I R E M E N T S FOR T H E D E G R E E OF D O C T O R OF P H I L O S O P H Y i n T H E F A C U L T Y OF G R A D U A T E STUDIES (Department of Pathology and Laboratory Medicine) We accept this thesis as conforming to the required standard T H E UNIVERSITY OF BRITISH C O L U M B I A 2000 © S T E V A N ROBERT K N E Z E V I C H , 2000 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of Pfrrmi-OQY to*l> C/j&aRAWZY' M£PTCZW£-The University of British Columbia Vancouver, Canada Date fl-pr-s ' / 5~/ OQ DE-6 (2/88) ABSTRACT Congenital fibrosarcoma (CFS) is a cellular, mitotically active neoplasm of soft tissues. It affects infants less than two years of age, has a low metastatic rate and a relatively high propensity for local recurrence. One of the predominant clinical issues surrounding CFS is its distinction from other histologically identical and virtually indistinguishable pediatric spindle cell tumors including adult-type fibrosarcoma (ATFS) and infantile fibromatosis (IFB). ATFS is a malignant lesion that is treated more aggressively than CFS, while IFB is a benign lesion which is treated less aggressively. Reliable distinction between these entities is therefore clinically very important. We therefore wanted to identify a diagnostic tool to distinguish CFS from other fibroblastic tumors such as ATFS and IFB. Cytogenetic analysis of CFS cases has shown a nonrandom gain in chromosomes 8, 11, 17, and 20 with trisomy for chromosome 11 being present in most cases. Cytogeneticists at the Department of Pathology of B.C.C.H. recently identified recurrent cytogenetic alterations involving chromosome 12pl3 and 15q25 in three CFS cases, which were not present in ATFS, IFB, and aggressive fibromatosis. Cloning of the chromosomal breakpoints revealed a novel fusion between the ETS transcription factor member, ETV6, and the gene encoding the neurotrophin-3 cell surface receptor, NTRK3. This fusion results in the juxtaposition of the H L H dimerization domain of ETV6 to the protein tyrosine kinase (PTK) domain of NTRK3. We hypothesized that this molecule acts as an aberrant PTK signaling molecule i n which the H L H domain mediates ligand independent dimerization resulting i n I l l constitutive P T K activation. The fusion protein exists as a 70-80 kDa doublet and was found to undergo homodimerization as wel l as heterodimerization w i t h ETV6. Furthermore, we were able to show that the E T V 6 - N T R K 3 protein acts as a P T K that was capable of interacting wi th PLOyl , but not wi th other known N T R K 3 interactors including SHC, SH2Bp\ GRB2 and PI3K. Moreover, E T V 6 - N T R K 3 was shown to localize mainly in the cytoplasm. Our data support the notion that CFS is a biologically distinct entity, and ETV6-NTRK3 detection provides a diagnostic screening tool potentially useful in the clinical evaluation of children wi th spindle cell tumors. i v TABLE OF CONTENTS Page ABSTRACT ii TABLE OF CONTENTS i v LIST OF TABLES viii LIST OF FIGURES ix LIST OF ABBREVIATIONS xi ACKNOWLEDGEMENTS xiv Chapter I INTRODUCTION 1 1.1 Synopsis and Rationale for the Thesis 1 1.2 Pediatric Spindle Ce l l Sarcomas 2 1.2.1 Pathologic Workup, Radiology, and Cl inical Features 3 1.2.2 Congenital Fibrosarcoma 5 1.3 General Aspects of Normal Growth Regulation 7 1.3.1 Signal Transduction Involved in Ce l l Proliferation 8 The P D G F receptor and Ras pathway 9 The phosphatidylinositol-3 kinase and protein kinase B pathway 12 1.3.2 Signal Transduction Involved in L imi t ing Growth 16 1.3.3 Cel l Cycle 20 1.4 Mechanisms of Oncogenesis 24 1.4.1 Oncogenes 25 Proviral Insertion 25 Gene Amplif icat ion 26 Point Mutations 28 Chromosomal Rearrangements 28 1.4.2 Tumor Suppressor Genes 29 R B I 29 p53 31 1.5 Genetic Aspects of Pediatric Solid Tumors 32 1.5.1 Aneup lo idy 34 1.5.2 Tumor Specific Translocations 36 1.5.3 Tumor Specific Translocations Result In Functional Gene Fusions In Solid Tumors 41 V Ewing's Sarcoma Family Of Peripheral Primitive Neuroecto-Dermal Tumors 42 Myxoid Liposarcoma 45 Alveolar Rhabdomyosarcoma 46 Synovial Sarcoma 47 1.6 Aims and Objectives 49 Chapter II MATERIALS A N D METHODS 51 2.1 Clinical Features of Index Case 51 2.1.1 Pathology of Index Case 51 2.1.2 Cytogenetic Analysis of Index Case 52 2.2 Clinical Samples, Tissue Culture Techniques and Cytogenetic Analysis 52 2.3 Y A C and Cosmid Probes 59 2.4 D N A and R N A Isolation 59 2.5 Southern and Northern Blot Analysis 61 2.6 Fluorescence In Situ Hybridization (FISH) Studies 62 2.7 3' and 5' Rapid Amplification of c D N A Ends (RACE) 63 2.8 RT-PCR Analysis of Tumor Samples 64 2.9 Preparation of Protein Lysates for Immunoprecipitation and Immunoblotting 65 2.10 Immunoprecipitation 67 2.11 Immunoblotting 67 2.12 Generation of GST-ETV6-NTRK3 Fusion Proteins 68 2.13 In Vitro Protein Association Studies 70 2.14 Subcellular Localization by Immunofluorescence 71 Chapter III A NOVEL t(12; 15)(pl3; q25) IN CONGENITAL FIBROSARCOMA 73 3.1 Introduction 73 3.2 Results 74 3.2.1 Cytogenetic Analysis 74 3.2.2 Fish Analysis Identifies a Common Derivative Chromosome 74 3.2.3 Identification of the Breakpoint Region by Y A C Mapping 76 3.2.4 Micromapping the Breakpoint with Cosmid Probes 81 3.3 Discussion 83 v i Chapter IV CLONING A N D CHARACTERIZATION OF THE t(12;15) IN CFS 91 4.1 Introduction 91 4.2 Results 92 4.2.1 Cloning the t(12;15) Breakpoint in CFS 92 4.2.2 Reciprocal Fusions were Not Detected 94 4.2.3 RT-PCR Analysis of CFS and Other 94 Morphologically Similar Tumors 4.2.4 Northern A n d Southern Blot Analysis 95 4.3 Discussion 95 Chapter V ETV6-NTRK3 GENE FUSIONS A N D TRISOMY 11 ESTABLISH A HISTOGENETIC LINK BETWEEN MESOBLASTIC NEPHROMA A N D CONGENITAL FIBROSARCOMA 104 5.1 Introduction 104 5.2 Results 106 5.2.1 Clinical History and Cytogenetics 106 5.2.2 RT-PCR Analysis of C M N Cases 108 5.2.3 Northern Blot Analysis 110 5.2.4 FISH Analysis 110 5.3 Discussion 110 Chapter VI MOLECULAR STUDIES OF THE ETV6-NTRK3 FUSION PROTEIN 115 6.1 Introduction 115 6.2 Results 116 6.2.1 Expression and Phosphorylation Status of ETV6-N T R K 3 and ETV6-NTRK3 Mutant Proteins in NIH3T3 Cells 116 6.2.2 E T V 6 - N T R K 3 Homodimerizes and Heterodimerizes wi th ETV6 119 6.2.3 Downstream Interactors Affected by the ETV6-N T R K 3 Molecule 121 6.2.4 Subcellular Localization 123 6.3 Discussion 123 Chapter VII SUMMARY A N D CONCLUSIONS 132 7.1 Identification of a Recurring t(12;15) in Congenital Fibrosarcoma 133 v i i 7.2 The ETV6-NTRK3 Gene Fusion Characterizes Congenital Fibrosarcoma 134 7.3 Trisomy 11 and the ETV6-NTRK3 Gene Fusion L ink Congenital Fibrosarcoma to Congenital Mesoblastic N e p h r o m a 134 7.4 Molecular Studies of the E T V 6 - N T R K 3 Fusion Protein 135 7.5 General Comments 138 R E F E R E N C E S 141 v i i i LIST OF TABLES Page Table 1. Various Classes of Oncogenes and their Mode of Action Within Tumors 30 Table 2. Tumor Suppressors and the Tumors Affected by their Loss 33 Table 3. Summary of the Various Recurring Chromosomal Abnormalities Found in Pediatric Soft Tissue Tumors 35 Table 4. Summary of Cytogenetic Analysis of Initial B C C H CFS Cases 58 Table 5. Summary of the Various Constructs Used to Transfect NIH3T3 Cells 66 Table 6. Summary of Various Antibodies Used for Immunoblotting, their Source and Required Concentrations 69 Table 7. Summary of ETV6 Rearrangements in Human Neoplasia 82 Table 8. Clinical Characteristics and Molecular Genetic Findings in C M N Cases 107 Table 9. Summary of ETV6-NTRK3 (TEL-TRKC) Analysis 136 ix LIST OF FIGURES Page(s) Figure 1. Ras Signaling in the Eukaryotic Cell 13-14 Figure 2. Partial Schematic of Signaling Mechanisms Involved in Growth Control 18-19 Figure 3. The Cell Cycle 21 Figure 4. The Possible Outcomes of Chromosomal Translocations 38-40 Figure 5. Schematic Representation of EWS-ETS Fusions 43 Figure 6. Histologic Analysis of CFS and IFB 53-54 Figure 7. G-Banded Karyotype of Index Case 55-56 Figure 8. Mapping of Chromosomal 12pl3 and 15q25-26 Breakpoints in CFS 75 Figure 9. Dual-Coloured FISH of CFS 77-78 Figure 10. FISH Analysis for CFS Breakpoints 79-80 Figure 11. Schematic Representation of the cDNA for ETV6 as well as Some of the More Common Rearrangements Involving the ETV6 Gene 87-88 Figure 12. ETV6-NTRK3 Gene Fusions in CFS 93 Figure 13. Northern Analysis of CFS Cases 96 Figure 14. Southern Analysis of CFS Cases 97 Figure 15. Schematic Representation of the Predicted ETV6-NTRK3 Protein 102 Figure 16. ETV6-NTRK3 Detection in CMN 109 Figure 17. Northern Analysis of CMN Cases 111 Figure 18. FISH Analysis for Trisomy 11 112 Figure 19. Western Blot Analysis of NIH3T3 Cells Expressing ETV6-NTRK3 and Various Mutants Using CC-NTRK3 (C-14) 118 X Figure 20. Immunoprecipitation and Western Blot Analysis Demonstrates E T V 6 - N T R K 3 Tyrosine Phosphorylation 120 Figure 21. Immunoblot Analysis Demonstrating H L H - D o m a i n Dependant Homo- and Heterodimerization of E T V 6 - N T R K 3 122 Figure 22. ETV6-NTRK3 Interacts with P L C y but not wi th S H C , GRB2, or PI-3K p85 Subunit 124 Figure 23. Analysis of P L C y Binding Mutants for Abi l i ty to Associate wi th P L C y l 125 Figure 24. Confocal Microscopy of ETV6-NTRK3 and A H L H Expressing NIH3T3 Cells 126 xi LIST O F A B B R E V I A T I O N S A adenine O P / K I P A E L acute eosinophilic C M L leukemia A F B aggressive fibromatosis C M M L A J adherens junction A L D activation loop dead C M N A L L acute lymphoid leukemia CS A M L acute myeloid leukemia D A G A P C adenomatosis polyposis D A P I col i A R M S alveolar rhabdomyosarcoma D B D A T F S adult-type fibrosarcoma DDIT3 A T P adenosine triphosphate B A D Bcl-2 antagonist of cell Del death Der B C C H British Columbia DFSP Children's Hospital B C R B cell antigen receptor D A G B - N H L B-cell non-Hodgkin's A H L H l y m p h o m a d m i n bp base pair D M E M B S A bovine serum albumin C cytosine D N A C C H T N Cooperative H u m a n D N A - P K Tissue Network C C S K clear cell sarcoma of the Drk kidney C D K cyclin dependent ECD serine/threonine kinases C D K I cyclin dependent E D T A serine/threonine kinases inhibi tor EGF c D N A complimentary E G F R deoxyribonucleic acid CFS congenital fibrosarcoma E R K C G H comparative genomic hybridization E R M S C H L A Children's Hospital Los Angeles ES C i Cur ie ETS CID chemically-induced dimerizat ion E T V 6 CDK-interacting proteins chronic myeloid leukemia chronic myelomonocytic leukemia congenital mesoblastic nephroma calf serum diacylglycerol diamidino-2-phenyl indole dihydrochloride hydrate D N A binding domain D N A damage-inducible transcript 3 deletion derivative dermatofibrosarcoma protuberans diacylglycerol deleted H L H domain double minute Dulbecco's Modif ied Eagle M e d i u m deoxyribonucleic acid DNA-dependent protein kinase Drosophila receptor kinase extracellular l igand binding domain ethylene-diamine-tetraacetic acid epidermal growth factor epidermal growth factor receptor extracellular signal regulated kinase embryonal rhabdomyosarcoma Ewings sarcoma E-26 transforming specific Ets variant gene 6 FCS fetal calf serum F G F fibroblast growth factor F G F R fibroblast growth factor receptor F I S H fluorescence in situ hybridization F K H R L 1 forkhead in rhabdomyosarcoma-like 1 G guanine GADD153 growth arrest and D N A damage-inducible gene 153 G6PD glucose-6-phosphate dehydrogenase G A P GTPase activating protein G S K 3 glycogen synthase kinase-GST *J glutathione S-transferase GTPase guanosine triphosphatase H & E hematoxylin and eosin H L H helix-loop-helix H P V human papil lomavirus H S R heterogeneously staining region H U V E human umbil ical vein endothelial cells IFB infantile fibromatosis IGF2 insulin growth factor 2 InsP 3 inositol (1, 4, 5)-triphosphate IPs inositol triphosphate ITF inducible transcription factor J A K Janus family of tyrosine kinases kb kilo-base K D kinase dead kDa kilo-Daltons K R A B Kruppel-associated box L O H loss of heterozygosity LPS liposarcoma M A P K mitogen-activated protein kinase x n M D S myelodysplastic syndrome M E K M A P k inase /ERK-activating kinase M F B infantile myofibromatosis m R N A messenger ribonucleic acid M S C V murine stem cell virus N F neurofibromatosis N F T P neurofilament triplet protein n M nanomolar N S E neuron-specific enolase nt nucleotide N T - 3 neurotrophin-3 N T R K 3 neurotrophic tyrosine kinase receptor type 3 N W T S G Nat ional W i l m s ' Tumor Study Group PBS phosphate buffered saline P C N A proliferating cell nuclear antigen P C R polymerase-chain reaction PDGF platelet derived growth factor P D G F R platelet derived growth factor receptor PI-3K phosphoinositol-3' kinase P K C protein kinase C PLCy phospholipase-Cy P M S F Phenylmethylsulfonyl Fluor ide p P N E T Peripheral primit ive neuroectodermal tumors PTB phosphotyrosine binding doma in Ptdlns phosphatidylinositol PtdInsP 2 phosphatidylinositol diphosphate PTP protein tyrosine phosphatases Xlll PSF penici l l in streptomycin fungazone P T K protein tyrosine kinase R A C E rapid amplification of c D N A ends R A E B refractory anemia wi th excess blasts (with basophilia) R B retinoblastoma R M S rhabdomyosarcoma R N A ribonucleic acid R N P ribonucleoprotein R T K receptor tyrosine kinase SDS sodium dodecyl sulfate SH2 Src homology 2 SH3 Src homology 3 S N T sucl-associated neurotrophin factor target Sos Son of Sevenless T thymine T A D transactivation domain TBS tris-buffered saline Tcf4 T-cell transcription factor-4 T C R T cell antigen receptor TEL translocation, Ets, l eukemia TF transcription factor TLS translocated in liposarcoma T N F S F 6 tumor necrosis factor ligand superfamily member 6 T R K C tropomyosin receptor kinase C T S G tumor-suppressor gene U T R untranslated region V E G F vascular endothelic growth factor W T W i l m s ' tumor Y A C yeast artificial chromosome Z N F zinc finger x i v A C K N O W L E D G E M E N T S First and foremost, I would like to thank Dr. Poul H B Sorensen for his expertise, patience, leadership, relaxing personality and for having been an integral part in my maturation over the four years I spent under his supervis ion. Moreover, this thesis and the work represented wi th in wou ld never have been completed without his help and instruction. From the Sorensen laboratory, I w i s h to thank Jerian L i m for her immense technical knowledge and close friendship. In addition, I wish to thank Beth Lawlor, Jessica Palmer, Wen Tao, and Daniel W a i for their friendship and never ending support. In addition, I wish to thank the members of my supervisory committee, Dr. Doug Horsman, Dr. Kei th Humphr ies , and Dr. Janet Chantler, for their interest and continued enthusiasm. Finally, I thank my parents who have given me the opportunity and continuous support i n following my dreams. 1 CHAPTER I INTRODUCTION 1.1 SYNOPSIS A N D R A T I O N A L E FOR T H E THESIS The studies to be described in this thesis were initially performed on a series of congenital fibrosarcoma cases, which belong to the family of spindle cell lesions of childhood. This family also includes infantile fibromatosis, aggressive fibromatosis and adult-type fibrosarcoma, all of which appear histologically s imi lar under the microscope. Pediatric spindle cell lesions of early childhood pose significant diagnostic challenges for the pathologist, as they are difficult to differentiate from one another due to their similar morphologic appearance. Differentiating these tumors is of great importance clinically as they show different clinical behaviors and require distinct treatment protocols. We were therefore interested i n finding a specific recurring genetic anomaly which wou ld provide the pathologist wi th a molecular tool for accurately diagnosing these tumors. W e studied and identified a recurrent t(12;15)(pl3;q25) i n congenital fibrosarcoma, which lead to the identification of a novel gene fusion between ETV6 (also k n o w n as TEL) and NTRK3 (also known as TRKC) from chromosomes 12 and 15, respectively. Further studies showed that the fusion gene encoded a chimeric tyrosine kinase protein which we hypothesized functioned by dysregulating n o r m a l signaling pathways within the malignant cell. Given these findings, the remainder of this chapter w i l l deal wi th pediatric spindle cell tumors (with an emphasis o n congenital fibrosarcoma and other morphologically similar lesions), general aspects 2 of cancer biology and genetics, normal and abnormal growth (signal transduction and cell cycle), pediatric solid tumors, as wel l as signal transduction as it relates to tyrosine kinase receptors. 1.2 P E D I A T R I C SPINDLE C E L L S A R C O M A S H u m a n malignant tumors can be categorized into sarcomas, carcinomas and hematopoietic (including lymphoid) malignancies. Sarcomas are tumors w h i c h have arisen from mesenchymal tissue while carcinomas derive from epithelial tissue. Hematopoietic malignancies arise from blood forming and lymphoid cells which originate from mesoderm. During embryogenesis there are three primary germ layers; endoderm, mesoderm and ectoderm. The ectoderm gives rise to the epithelium, the entire nervous system, the lens and retina of the eye as wel l as a broad range of structures in the head and pharynx. The mesoderm gives rise to mesenchyme which is responsible for forming the skeletal muscles, vertebrae and skull , connective tissues and blood vessels of the body wal l , the skeletal elements of the body wal l , girdles and limbs, the smooth muscles and connective tissue of the digestive tract, the heart, the blood vessels of the viscera and blood. The endoderm gives rise to the epithelium of the digestive tract and to the epitheliod components of all organs that arise as evaginations from the embryonic foregut, midgut, or hindgut. Sarcomas are malignant mesenchymally-derived tumors which exhibit local recurrence and metastatic behavior, and have a high proliferative rate. Sarcomas are categorized on the basis of cell of origin. For example, there are rhabdomyosarcomas (derived from skeletal muscle precursor cells), 3 leiomyosarcomas (smooth muscle cells), chondrosarcomas (cartilage cells), liposarcomas (fat cells), hemangiosarcomas (blood vessels), fibrosarcomas (fibroblasts), osteosarcomas (bone cells), synovial sarcomas (synovial cells), and sarcomas of unknown origin. Several of the sarcomas mentioned above can also have spindle cell morphology. The word "spindle" refers to the shape of the cells, being long and drawn out in the shape of a spindle. Some examples of spindle cell tumors include fibrosarcoma, leiomyosarcoma, synovial sarcoma and some forms of rhabdomyosarcoma. One of the predominant issues in tumor pathology is the difficulty in differentiating malignant sarcomas from each other as well as from benign lesions such as fibromatoses (benign lesions involving fibroblast cells). These benign lesions can be virtually indistinguishable from their malignant counterparts by morphological criteria, but have no metastatic behavior, have a low recurrence rate, and are less aggressive than sarcomas. A variety of approaches commonly used by the pathologist in diagnosing tumors are discussed below. 1.2.1 Pathologic Workup of Pediatric Sarcomas Histology is the primary method for evaluating tumors and is based on the study of the morphology of the various tumors. Briefly, the tumor specimen is fixed in formalin and embedded in paraffin. A 5pm section of the tumor is then placed onto a microscope slide and stained with hematoxylin and eosin. This is known as an H&E section and the characteristic staining patterns it produces is the primary diagnostic modality used in evaluating tumors. 4 Immunohistochemistry helps determine the cell of origin. A thin section of the tumor is placed onto a slide and is then incubated wi th antibodies against specific antigens such as neuron-specific enolase (NSE), Leu7, neurofilament triplet protein (NFTP), desmin, muscle-specific actin, vimentin, S100, keratin, leukocyte common antigen (CD45), and the surface antigen MIC2. For example, a neural tumor wou ld be positive for NSE , Leu7, and N F T P , while myogenic tumors w o u l d be positive for desmin, muscle-specific actin, and M Y O D (a transcription factor that appears early i n myogenesis and activates later gene expression). Therefore, the combination of positive and negative staining for the above antigens helps the pathologist form an accurate diagnosis. Another analytical technique often used is electron microscopy. The electron microscope is used to evaluate ultrastructural features. Neuroblastomas, for example, contain dense core granules, rhabdomyosarcomas contain myofilaments, and acute megakaryocytic leukemia contains platelet granules each of which can be identified by electron microscopy. Cytogenetics is a technique which involves the analysis of chromosomes from short term cultures of tumors. Recurrent chromosomal abnormalities can be used as a diagnostic tool, especially i n pediatric tumors where there are many examples of recurring chromosomal deletions, translocations and whole chromosome gains and losses (discussed in further detail below). The results from histologic, immunohistochemistry, electron microscopic and cytogenetic analysis provide the pathologist wi th useful diagnostic information. This information needs to be correlated wi th the clinical history and 5 radiological features of the tumor. In summary, histology and other pathologic modalities, coupled wi th the clinical history (including radiological results) is the init ial route taken i n the accurate diagnosis of many soft tissue pediatric tumors. Often however, it is extremely difficult if not impossible to distinguish different sarcomas from each other or sarcomas from benign lesions as they may be v i r tua l ly identical morphologically. This has lead to the relatively new field of molecular pathology, i n which the tumor is further analyzed by cytogenetic and molecular techniques to make an accurate diagnosis (discussed i n further detail below). 1.2.2 Congenital Fibrosarcoma Congenital (infantile) fibrosarcoma (CFS) arises from fibroblasts and is a mitotically active spindle cell lesion affecting soft tissue. CFS acquired its name because of its histologic similarity to adult fibrosarcoma [1]. Fibrosarcomas tend to be categorized into two distinct age groups (the upper and lower limits of which are poorly defined): those occurring before the age of 2 years (most under one year of age), known as CFS, and those occurring in patients aged 10 years or older, known as adult-type fibrosarcoma (ATFS). Correlating wi th these age groups are distinct differences i n clinical behavior. Whi le CFS has a metastatic and recurrence rate of 10% and up to 40%, respectively [2-4], it is unique among human sarcomas for its excellent prognosis wi th an 80-90% overall survival rate [5, 6]. O n the other hand, A T F S is an aggressive lesion wi th a poor prognosis similar to that of adult fibrosarcoma [7]. 6 Under the microscope, CFS is morphologically similar to other fibroblastic tumors such as aggressive fibromatosis (AFB), A T F S and infantile fibromatosis (IFB) and has, historically, been difficult to diagnose [8-10]. This is evidenced by CFS having been misdiagnosed in the past for a lymphatic malformation [11, 12], hydrops fetalis [13], or congenital hemangioma [11,14]. CFS was also postulated as being histogenetically related to infantile myofibromatosis (MFB) and congenital hemangiopericytoma [1,15,16]. CFS is predominantly found i n the extremities (71%) and is primari ly treated by surgical excision [17], often wi th adjuvant preoperative and postoperative chemotherapy as wel l as combination chemotherapy [8, 18-22]. The propensity for CFS tumors to metastasize seems to depend on the primary tumor site: 7-8% of primary CFS tumors located in the extremities metastasize while 26% of those located wi th in the abdomen, pelvic and chest area metastasize [9, 23]. The benign counterpart to fibrosarcoma is infantile fibromatosis (IFB). IFB is a lesion that arises from fibroblasts and/or myofibroblasts and exhibits lower cellularity and mitotic activity than fibrosarcomas [1]. Infantile fibromatosis (IFB) primari ly occurs i n children aged 2 years or younger and, i n addition to the above characteristics, contains more collagenous matrix than CFS. A variant of IFB known as aggressive fibromatosis (AFB) is clinically more aggressive than IFB and is inclined to increased local invasion and recurrence [1]. Cytogenetic analyses of CFS cases to date have shown non-random whole chromosome gains for chromosomes 2, 8, 11, 17 and 20 [6, 24-26]. Other tumors which share some of these specific chromosomal abnormalities include: +20 and +8 7 i n desmoid tumors [27-29], +8 i n Dupuytren's contracture, Peyronie's disease and hematologic malignancies [30-33], +20, +17, +11, and +8 i n congenital mesoblastic nephroma ( C M N ) [34, 35], and +11 i n acute myeloid leukemia ( A M L ) [36]. Currently, there have been no reports of any recurring chromosomal abnormalities for IFB and ATFS. As w i l l be discussed in later chapters, we have now identified a recurrent t(12;15)(pl3;q25) translocation in CFS. 1.3 GENERAL ASPECTS OF NORMAL GROWTH REGULATION Understanding the mechanisms of oncogenesis (see section 1.4 below), requires the familiarization wi th the way an extracellular "message" is transferred from the cell membrane into the nucleus. Once i n the nucleus the initial message results i n the activation or suppression of transcription of certain genes required either for growth and progression through the cell cycle or differentiation. A cell maintains a normal rate of growth by a variety of important biological mechanisms. The following discussion w i l l briefly cover three mechanisms pertaining to no rma l growth regulation: 1. Signal Transduction involved in Cel l Proliferation: the concept of how a cell receives an extracellular "message" and the intracellular ( including nuclear) consequences. 2. Signal Transduction Involved i n Limi t ing Cel l Growth: the concept of how a cell "knows" when to stop growing i n the context of its environment . 8 3. Ce l l Cycle: the nuclear machinery responsible for determining w h e n it is appropriate for a cell to proliferate and divide. 1.3.1 Signal Transduction Involved in Cell Proliferation In general, signal transduction refers to the specific molecular interactions and subsequent modifications in response to an external st imulus, such as g rowth factors and cytokines. Messages from extracellular growth regulatory molecules are transferred into the cell via receptors that b ind these molecules. There are several different classes of receptors inc luding growth factor receptors (eg., fibroblast g rowth factor receptor), steroid receptors (eg., glucocorticoid receptor), neurotransmitter receptors (eg., acetylcholine receptor), seven transmembrane-spanning or serpentine receptors (eg., gastrin releasing peptide receptor), B-cell receptors (eg., B cell ant igen receptor (BCR)), and T-cell receptors (eg., TCR). Since our studies resulted in the discovery of a chimeric fusion protein containing a port ion of a tyrosine kinase receptor (a member of the growth factor receptor family), the remainder of this discussion wi l l focus on signal transduction as it pertains to the protein tyrosine kinase family of growth factor receptors. M a n y of the signals responsible for inducing prol iferation or di f ferent iat ion act through growth factors b ind ing to cell surface receptor tyrosine kinases (RTKs). The process of transducing a signal from RTKs on the cell surface to the nucleus can be d iv ided into 4 steps (other receptors, such as the steroid receptors, contain a slightly different series of events): 9 1. l igand binding 2. receptor dimerization and P T K activation 3. phosphorylation of cytoplasmic proteins 4. phosphorylation of nuclear proteins (i.e. transcription factors responsible for the activation or inhibition of genes involved i n growth control) These steps w i l l now be discussed below i n the context of the P D G F receptor and the wel l established R A S pathway [37-39]. The P D G F receptor and R A S pathway The way many extracellular hormone or growth factors deliver their messages to the intracellular environment of a cell is by binding to a specific protein tyrosine kinase (PTK) receptor. Each P T K molecule has an extracellular l igand binding domain, a transmembrane domain, and an intracellular tyrosine kinase domain [37]. The ligand (e.g. EGF, FGF or PDGF), w i l l b ind to the appropriate extracellular portion of the receptor, thus beginning the process of signal transduction. A n example of this is the platelet derived growth factor (PDGF) binding to PDGFR. U p o n ligand binding, the PDGF receptor undergoes homodimerizat ion [37] bringing the kinase domains i n close proximity. This interaction results i n the auto- or cross-phosphorylation of specific tyrosine moieties wi th in the intracellular domain of the receptor. The structure responsible for this is known as the activation loop and consists of tyrosine residues wi th in the kinase domain . Phosphorylated tyrosine residues outside the tyrosine kinase domain act as anchors for downstream molecular interactions (see below) [40, 41]. In addition to these 10 tyrosine residues are tyrosines which do not get phosphorylated and act as structural amino acids. Other types of receptors which undergo ligand induced d imer iza t ion (and even oligomerization) include B-cell receptors, T-cell receptors, hormone and cytokine receptors [42]. Once the ligand interaction has taken place, the P D G F / P D G F R complex acts as a tyrosine kinase and phosphorylates interacting proteins (including other tyrosine kinases) which can then interact wi th other molecules and phosphorylate them [37]. The P D G F receptor contains several different phosphorylated tyrosines responsible for its interaction wi th associating molecules such as Src [43], phosphatidylinositol-3 kinase (PI-3K) [44], G A P [45], Syp [46], S H C [47], growth factor receptor-bound protein 2 (GRB2) [48, 49] and PLC-y l [44]. These downstream interacting molecules possess an SH2 domain (for Src homology 2) which allows for the specific interaction w i t h phosphorylated tyrosines [50]. The specificity for a particular phosphorylated tyrosine is determined by the 3 dimensional shape of the SH2 domain i n the downstream molecule and the 3 dimensional context of the phosphorylated tyrosine moiety on the receptor. Another domain which has recently been implicated i n phosphorylated tyrosine binding is known as the phosphotyrosine binding domain (PTB) [51, 52]. GRB2 is the 217 amino acid homologue of drk i n Drosophila and Sem-5 i n Cuenorhubditis [53, 54]. It contains an SH2 d o m a i n responsible for interacting wi th one of the phosphorylated tyrosines on the intracellular portion of the activated EGF receptor and a SH3 domain w h i c h interacts wi th proline rich sequences on downstream molecules such as Sos [55]. GRB2 interaction wi th Sos (the 1596-residue product of the Son of Sevenless gene, 11 so named because Sos interacts w i th the Sevenless gene product, a P T K receptor that regulates the development of the R7 photoreceptor cell i n the Drosophila compound eye [42]) results i n the relocalization of Sos to the plasma membrane where it can convert inactive R A S (RAS-GDP) into active R A S (RAS-GTP) [56, 57]. One of the pathways affected by R A S is the mitogen activated protein kinases ( M A P K ) pathway which is a series of cytosolic serine/threonine kinases [58]. One of the molecules which leads to the activation of the M A P K pathway is R A F 1 , which is a serine/threonine protein kinase (also known as M A P kinase kinase kinase ( M A P K K K ) ) [59]. Phosphorylation of multiple serine and threonine residues activates R A F 1 allowing it to interact wi th and phosphorylate M E K ( M A P kinase/ERK-activating Kinase or M A P kinase kinase ( M A P K K ) ) [39, 60]. Activated M E K then interacts wi th and phosphorylates a family of proteins, known as the M A P kinases or ERKs (extracellular-regulated kinases) which require the phosphorylation of both serine/threonine and tyrosine residues in order for proper activation. Before proliferation or differentiation can occur, certain genes involved i n growth regulation need to be turned on or off. One mechanism involves the phosphorylation of particular transcription factors found in the nucleus by activated cytoplasmic kinases that have migrated into the nucleus. In the R A S pathway, activated (phosphorylated) E R K migrates from the cytoplasm into the nucleus and phosphorylates various transcription factors, such as J u n / A P l , Fos, Myc , and Sap l which subsequently turn their respective target genes on or off [60-62]. These target genes are most likely responsible for controlling the cell cycle and 12 proliferation associated signaling pathways, but more research is needed to elucidate the exact targets and functions of these transcription factors (see Figure 1). In addition to controlling the R A F kinase cascade, R A S has been shown to either directly or indirectly interact wi th a number of other molecules including PI-3K [63], Bcl-2 [64-66], protein kinase CC, (PKCCJ [67, 68], Rin , the guanine nucleotide exchange factors for Ral , AF6 and p l 2 0 G A P which can result i n a variety of cellular responses including cell survival , mitogenesis, and differentiation [69]. Since the purpose of this introduction is to familiarize the reader wi th the important mechanisms involved in growth regulation, a brief discussion of the PI-3K pathway w i l l now be included as it is important i n activating cell survival pathways. The phosphatidylinositol-3 kinase and protein kinase B pathway PI-3K is a heterodimeric enzyme composed of a p85 and a p l l O subunit (other isoforms exist for both of these molecules) [70]. R A S can protect a cell f rom detachment-induced programmed cell death (apoptosis) and other forms of apoptosis through the activation of the PI-3K pathway [57]. R A S activation of PI-3K is mediated through the p l l O subunit of PI-3K [63]. The PI-3K pathway is implicated i n apoptosis, cell motility, and vesicle trafficking and secretion [71]. Its role i n apoptosis w i l l be discussed further. Activated PI-3K (either through the interaction wi th an activated tyrosine kinase receptor such as PDGF receptor or through RAS) leads to the phosphorylation of phosphatidylinositol (Ptdlns) [71]. In addition, activated PI-3K can phosphorylate Ptdlns 4-P and Ptdlns 4, 5-P2 [71]. Yao and Cooper found that the 1 3 F I G U R E 1. R A S signaling in the eukaryotic cell. The init ial event, w h i c h ultimately results i n the activation of nuclear transcription factors, is l igand induced dimerization of the cell surface receptor. This results i n the autophosphorylation of specific tyrosine residues of which only a few are i n v o l v e d i n attracting cytosolic molecules to the receptor. A n adaptor molecule, G R B 2 , attaches itself to one of these phosphotyrosines and is responsible for recruiting Sos (son of sevenless) to the plasma membrane, which facilitates the dissociation of G D P from R A S , thus activating RAS. This process is reversed by another molecule known as GTPase activating protein (GAP). R A S activation can result i n the activation of mitogen activated protein kinase pathways like R A F , M E K (mitogen activated, E R K activating protein), and E R K (extracellular signal-regulated protein kinase). Phosphorylated E R K can phosphorylate and thus activate nuclear transcription factors such as Elk, Myc and c-Jun, which results i n physiological changes i n the cell (ie. Cel l cycle activation). 14 15 PI-3K inhibitor, wortmannin, caused apoptosis in PC12 cells suggesting that the PI-3K pathway is involved in cell survival [72]. One of the downstream interactors for PI-3K responsible for cell survival is the protein kinase A K T (also known as "protein kinase B " (PKB)) [73]. A K T is homologous to the protein kinase A and C families and to the retroviral oncogene v-akt and encodes a serine/threonine protein kinase that is ubiquitously expressed [74]. A K T differs from P K A and P K C i n that the amino terminal portion contains an A K T homology domain part of which is related to the Pleckstrin homology domain. This domain is found i n a number of signaling molecules including the phospholipase family of tyrosine kinases (see chapter 6) and are thought to mediate protein-lipid and/or protein-protein interactions [75, 76]. Serine and threonine phosphorylation of A K T by PI-3K activated PDK1 activates A K T . This leads to the phosphorylation by A K T of the Bcl-2 antagonist of cell death (BAD). B A D may also be phosphorylated by an as of yet unknown kinase which does not involve PI-3K or A K T [77]. In its unphosphorylated form, B A D can interact wi th the Bel family members Bcl-XL and Bcl-2 inducing apoptosis. There has been a report on non -BAD mediated apoptosis, however further elucidation of the pathways involve are required [78]. Once phosphorylated by A K T however , B A D associates wi th another protein called 14-3-3 (a group of scaffolding proteins that bind to phosphorylated serine residues and are thought to be implicated i n cell cycle control and various signal transduction pathways) and is no longer able to interact wi th Bcl-XL or Bcl-2 and apoptosis is abrogated [79, 80]. Expression of B A D 16 is not ubiquitous however, suggesting that other cell survival proteins must exist for A K T [81]. Recently, Brunet et al. showed that A K T can directly phosphorylate and inactivate the forkhead in rhabdomyosarcoma-like 1 transcription factor (FKHRL1) [82]. F K H R L 1 is a member of the forkhead family of transcription factors and is thought to transcribe genes responsible for cell death. Phosphorylation causes F K H R L 1 to associate wi th 14-3-3 proteins [83, 84]. This association causes F K H R L 1 to remain i n the cytoplasm thus inhibiting its transcriptional activity. In the absence of survival factors, the F K H R L 1 transcription factor becomes dephosphorylated, translocates to the nucleus and transactivates target genes critical for cell death, such as the tumor necrosis factor ligand superfamily member 6 (TNFSF6) gene [85]. 1.3.2 Signal Transduction Involved i n L imi t ing Growth Cells need to know when to stop growing. It is thought that when a n o r m a l cell is i n contact wi th a neighboring cell, certain proteins on the surface of the cell recognize similar proteins on the neighboring cell, sending signals to the nucleus to stop proliferation. Adherens junctions (AJ or zonula adherens) mediate adhesion between cells, communicate a signal that neighboring cells are present (contact inhibition), and anchor the actin cytoskeleton. Beta-catenin (p-catenin) is an A J protein which is critical for the establishment and maintenance of epithelial layers, such as those l ining organ surfaces [86]. AJs are therefore responsible for regulating normal cell growth and behavior. 17 The A J is a multiprotein complex assembled around calcium-regulated cell adhesion molecules called cadherins [86]. Cadherins are membrane spanning proteins that mediate interactions wi th neighboring cells also containing cadherins. The intracellular domain of the cadherin molecule transmits the adhesion signal resulting i n the anchoring of the A J to the actin cytoskeleton. The cytoplasmic proteins responsible for transmitting the signal include the a-, [}-, and y-catenins [87]. Korinek et al. and M o r i n et al. showed that the adenomatosis polyposis col i (APC) gene (mutated in adenomatosis polyposis of the colon), is a negative regulator of beta-catenin signaling [88, 89]. The A P C protein normally binds to (3-catenin, which interacts wi th the Tcf and Lef transcription factors. Studies by Korinek et al. showed nuclei of A P C - / - colon carcinoma cells contained a stable (3-catenin/Tcf4 (T-cell transcription factor-4) complex that was constitutively active. Reintroduction of A P C removed beta-catenin from Tcf4 and ablated transcriptional activation. They concluded that A P C loss of function leads to constitutively activated Tcf4 and may be an important step towards early transformation of colonic epithelium. Some colorectal tumors have been found to contain intact A P C genes, while the (3-catenin gene contains an activating mutant. These studies suggest that regulation of |3-catenin is critical for normal growth, that the A P C gene acts as a growth inhibitor (tumor suppressor gene; see below), and that mutations in either A P C or (3-catenin can lead to tumorigenesis (see Fig. 2). 18 FIGURE 2. Partial schematic of signaling mechanisms involved in growth control. In order for a cell to become tumorigenic and/or malignant, the cell must acquire immortality, increase its growth rate, and even develop motility in order to become metastatic. A number of these requirements are met by the constitutive activation of RAS. RAS can become constitutively activated by a point mutation or by another aberrantly regulated molecule which could lead to increased proliferation through the activation of certain M A P K pathways. RAS can also activate N F K B (responsible for the expression of anti-apoptotic proteins) and PI-3K, which can activate A K T which stimulates B A D and ultimately results in the activation of anti-apoptotic pathways. Activated A K T can phosphorylate and block the activity of another molecule, namely GSK3 (glycogen synthase kinase-3) to block either gene transcription mediated through p-catenin or motility regulated by A P C (adenomatous polyposis of the colon). Some of these pathways are duplicated by the cell surface receptor itself. For example, when a ligand binds to the extracellular ligand binding domain (ECD), the protein tyrosine kinase domain (PTK) can activate PI3-K or RAS. Moreover, the activated cell surface receptor can activate a-catenin, which is involved in cytoskeletal modifications. 1 9 20 1.3.3 C e l l Cyc le Since many of the pathways discussed above converge on the cell cycle, regulation of the cell cycle will now be summarized. The process of cell division and differentiation is essentially determined by the impact of external stimuli (e.g., growth factors, lack of nutrients, stress, DNA damage) on the cell cycle machinery within the cell [90]. The cell cycle machinery is composed of cyclins, cyclin dependent serine/threonine kinases (CDKs) and their regulatory kinases and phosphatases. Briefly, the cell cycle consists of four phases known as Gv S, G 2 , and M. Gj (GAP1) refers to the first growth phase of the cell cycle and represents the time frame during which the various growth factors can act upon the cell. The synthesis of DNA occurs during S phase, where the normal diploid content, 2n, becomes tetraploid, 4n [90]. G 2 (GAP2) follows S phase and represents the termination of DNA synthesis and the continuation of cell growth (organelles and proteins). Mitosis (M phase) is the point when the cell divides and distributes the newly synthesized DNA into two identical daughter cells. Another phase exists in which the cell is static. There is neither growth nor differentiation and this is known as G 0 . When nutrients are in short supply or the cells are touching one another (contact inhibition), the cell enters G 0. Surgical removal of tissue, on the other hand, will cause the surrounding cells to re-enter the cell cycle and begin the regeneration process until growth is arrested due to contact inhibition (see Fig. 3). The cell cycle is tightly controlled by cyclins (regulatory subunits) [91], cyclin dependent kinases (CDKs) (enzymatic subunits) [91, 92], and cyclin-dependent kinase inhibitors (CDKIs) [93,94]. Briefly, the level of expression of cyclins and their rapid 21 Cyclin D Cyclin E • • • • • • Cyclin A ^ - ^ • * * Cycl in B \ ^ A""N \ N \ s • \ N \ v m * x \ \ x \ x Gl s G2 M Figure 3. The cell cycle. The chart (top panel) displays the variation of expression of some of the cyclins which are important in regulating the transition from one phase to another. There are 4 phases in the cell cycle (bottom) including G l , S, G2 (which comprise interphase) and M (mitosis). The amount of time spent in each phase and the amount of D N A present within a phase is also shown. Quiescent cells are in a phase known as GO and can re-enter the cell cycle with certain stimuli such as growth factors. 22 degradation, determines when a transition from one phase to another w i l l occur. In addit ion, the cyclins can interact wi th C D K proteins to form complexes whose phosphorylat ion status determines if it is active or not, thus augmenting the contro l of the cell cycle [90]. Phosphorylat ion and dephosphorylat ion play a major role in the control of the cell cycle. The Gj to S transition is controlled by the retinoblastoma gene product, RB [95]. W h e n RB is hypophosphorylated, it can interact w i th E2F, a transcription factor wh ich can activate transcription of a number of genes [96-98]. W h e n the RB protein is hyperphosphorylated by the c y c l i n - D / C D K 4/6 complex, it no longer has the ability to interact wi th E2F. Hyperphosphory lat ion of RB needs to take place in order for the transition f rom G 5 to S to occur [99-102]. This is commonly referred to as the G1 to S checkpoint. Once the signal wh ich initiated the phosphory lat ion is terminated, the phosphate groups need to be removed in order to terminate downs t ream activations [103-107]. This is carried out by proteins k n o w n as phosphatases such as CDC25. A n addit ional level of control is achieved by directly inhib i t ing C D K molecules. C D K inhibitors, such as p ^ 4 8 , pltf1™*, p l 8 I N K 4 C , p ^ 4 0 , p 2 1 C I P 1 ' W A F 1 - S D n -CAI™ p 2 7 K I P 1 / a n d p57 r a p 2 are responsible for directly coupl ing w i th C D K molecu les and inhibit ing their regulative role in the cell cycle [93]. The INK4 (cyclin-dependent kinase inhibitor) proteins are responsible for inhibit ing C y c l i n D / C D K 4 / 6 complexes, whi le the CIP/KIP (CDK-interacting proteins) mo lecu les inhibit Cyc l i n A , B, and E / C D K 2 complexes [93]. 23 Another important gene which is involved in the regulation of the cell cycle is p53. Like RB, p53 is also a tumor suppressor gene whose activity increases w h e n there is damage to D N A [108-111]. p53 is a nuclear phosphoprotein wi th two D N A binding domains [112], two SV40 large T-antigen binding sites [113, 114], a nuclear localizing signal [115], an oligomerization domain [116, 117], and several phosphorylation sites [118]. It is thought to act as a transcription factor for other growth regulatory genes either activating or inhibiting their transcription [109, 119]. In addition to acting as a transcription factor, p53 can interact wi th proteins such as p21 (CIP1) [120-122]. CIP1 inactivates G 2 c y c l i n / C D K complexes and i n addition, binds to the D N A polymerase cofactor, proliferating cell nuclear antigen ( P C N A ) , thus preventing D N A replication, but allowing for D N A repair [123]. Th is event effectively blocks the G^ to S transition and again acts as a checkpoint w i t h i n the cell cycle. Bunz et al. demonstrated that upon D N A damage, cells enter a sustained arrest in the G 2 phase only when p53 was present i n the cell and capable of transcriptionally activating the cyclin-dependent kinase inhibitor CD?1 [124]. After disruption of either the p53 or the CIP1 gene, gamma-radiated cells progressed into mitosis, but failed to undergo cytokinesis. The cells therefore exhibited a G 2 (tetraploid) D N A content. In addition, Shieh et al. showed that D N A damage leads to the phosphorylation of p53 and that this event reduces the ability of p53 to interact wi th M D M 2 . M D M 2 is a negative regulator of p53 that normally binds to p53 inhibit ing its function [125]. Furthermore, they demonstrated that the phosphorylation of p53 by purified DNA-dependent protein kinase ( D N A - P K ) impairs the ability of M D M 2 to inhibit p53-dependenf transactivation. 24 Finally, p53 has been implicated in apoptosis [126-128]. One group, Polyak et al, examined i n detail the transcripts induced by p53 expression before the onset of apoptosis [129]. Of the 7,202 transcripts identified, only 14 (0.19%) were found to be markedly increased in p53-expressing cells compared wi th controls. Str ikingly, many of these genes were predicted to encode proteins that could generate or respond to oxidative stress. p53 levels are normally very low, but have been s h o w n to rapidly increase after D N A damage or viral infection. It is known that p53 induces B A X transcription, a member of the Bcl-2 gene family [130]. The exact mechanism of p53 associated apoptosis, however, is not known and further research is needed to elucidate this pathway. 1.4 MECHANISMS OF ONCOGENESIS The activation or inactivation of specific molecules or cellular pathways, such as those discussed above, can result i n the transformation of normal cells into oncogenic ones. For example, inactivating mutations of p53 results i n the tolerance of D N A damage throughout the cell cycle leading to increased genetic instability and possibly an oncogenic advantage. Tumor cell metastasis is thought to be a result of a disrupted (3-catenin pathway where contact inhibi t ion is no longer present thus allowing the cell to continue growing in the presence of neighboring cells and invade other tissues in the body [131]. Vi ra l oncoproteins (such as the SV40, adenovirus and papillomavirus) have the capability to bind RB and p53 and thus disrupt their ability to inhibit growth [132-134]. It is important to note that cancer is not due to a single aberration, but the disruption of multiple pathways. A n increase 25 i n the expression of an oncogene or the loss of tumor suppressor factor expression alone, for example, is insufficient to produce a malignant tumor. Genes which are responsible for enhancing the proliferative rate of a cell are known as oncogenes while growth inhibit ing genes are known as tumor suppressor genes (discussed i n further detail below). 1.4.1 Oncogenes Oncogenes are mutated forms of their normal cellular counterparts, proto-oncogenes. Briefly, these proto-oncogenes can be converted to an oncogene by a chromosomal rearrangement, proviral insertion, gene amplification or a point mutation (discussed in further detail below). Proto-oncogenes have been found at virtually every level of the proliferation associated signal transduction pathway and include: growth factors, growth factor receptors, guanine nucleotide like proteins, guanine nucleotide exchange factors (GNEFs), cytoplasmic serine/threonine or tyrosine protein kinases, or nuclear proteins (transcription factors). Oncogenes were first discovered as the transforming elements in the R N A viruses responsible for causing sarcomas i n fowl [135]. The following discussion w i l l detail some of the mechanisms by which a proto-oncogene is converted into an oncogene. Proviral Insertion When a retrovirus inserts itself adjacent to a proto-oncogene, it places the expression of the proto-oncogene under the control of the enhancer elements of the retrovirus. This type of conversion (proto-oncogene to oncogene) was first identified wi th avian leukosis virus-induced bursal lymphomas where the level of 26 transcription of c-myc was 50 to 100 fold higher than normal due to p rov i ra l insertion upstream of the c-myc proto-oncogene locus [136]. Gene Amplif icat ion Genomic amplification has been seen i n many solid tumors and represents another mechanism by which an oncogene may be overexpressed [137]. The actual mechanism by which this amplification occurs is not clear but the end result may be seen cytogenetically as structures referred to as double minutes (dmins) and homogenously staining regions (HSRs) [138, 139]. dmins are sma l l extrachromosomal, circular structures that occur i n pairs and contain specific chromosomal regions, but lack telomeres [140]. These regions (amplicons) contain a number of genes (typically proto-oncogenes) and the number of dmins per cell determines the level of amplification of the genes involved. Tumorigenesis usually results when one or more of the genes amplified are responsible for cell proliferation [141,142]. HSRs are dmins which have integrated into the genome. They appear as homogenously staining regions (hence the name) wi th in a chromosome. The mechanism(s) by which a cell acquires dmins and HSRs is not fully understood. The fact that both forms of gene amplification may be seen i n a particular cancer is interesting. For example, one cell (or subline) may have an H S R while another cell (or subline) wi th in the same tumor may have a dmin. It has been postulated that the dmins represent the unstable form of HSRs [143]. These dmins however, w i l l integrate and form HSRs i n culture [144]. One of the most extensively studied tumors where gene amplification plays an important role i n tumorigenesis is neuroblastoma. The NMYC oncogene is 27 located on chromosome 2p24.1 and is amplified 25- to 700-fold i n h u m a n neuroblastomas by means of dmins and HSRs [145-149]. The high levels of N M Y C protein production is thought to deregulate transcription and lead to a proliferative advantage for the cell. NMYC amplification i n neuroblastoma is associated w i t h advanced stage disease and a poor prognosis [146,150,151]. A recent review has summarized the various reports of recurrent D N A sequence copy number amplifications i n human neoplasms detected by comparative genomic hybridization (CGH) [152]. C G H is a technique similar to F I S H which allows for the detection of deletions, duplications and amplifications. Amplicons have been identified for almost every chromosome. One of these regions, namely 12ql3-q21, encompasses the GU, CHOP, SAS, WNT1, WNTlOb, CDK2, MDM2 and CDK4 genes, and has been shown to be amplified i n a variety of tumors. Portions of this region have also been shown to be amplified i n a number of tumors including osteosarcoma (12ql3-ql4, specifically invo lv ing CDK4, MDM2 and SAS), chondrosarcoma (12cen-ql5 and 12q24.1), liposarcoma (12ql4-q21 specifically invo lv ing the CDK4 and MDM2 genes), synovial sarcoma (12ql5), embryonal and alveolar rhabdomyosarcoma (12ql3-ql5), breast carcinoma (12ql5), hereditary ovarian cancer (12ql3-q21), colon carcinoma (12ql3), bladder carcinoma (12ql3-ql5), diffuse large cell lymphoma (12ql3-ql4), follicular lymphoma (12ql3-ql4, specifically involv ing the GLI gene), neuroglial tumors (12ql3-ql5), non-smal l cell lung cancer (12ql4-q21), and squamous cell carcinomas of the head and neck (12ql3-ql4) [153-176]. 28 Point Mutations Another mechanism by which a proto-oncogene can be converted to an oncogene is by single base pair substitutions (mutations), which can have drastic effects on the translated protein. These mutations can arise due to replication errors, or from direct D N A damage such as ultraviolet radiation [42]. The RAS gene family (HRAS, KRAS, and NRAS) have all been found to contain point mutations i n various malignancies [177-179]. These point mutations result i n the constitutive activation of the R A S molecule by ablating its need for a guanine nucleotide exchange factor thus mimicking a constitutively activated growth factor receptor, such as E G F R or P D G F R [177-182]. Sekiya et al. found a point mutation in the second exon of the HRAS1 gene substituting an adenine residue to a thymine residue i n a melanoma [183]. Activat ion of the NRAS gene by point muta t ion occurs i n about 15% of all human melanomas [184]. In these cases, mutated NRAS was found to contribute to tumor growth by enhancing cellular proliferation and by blocking apoptosis. Other changes observed include: g ly l2val (bladder carcinoma), glyl2asp (mammary carcinosarcoma), gln611eu (lung carcinoma), and gln61 to arg (renal pelvic carcinoma) for the HRAS gene, and gln61 to arg (lung carcinoma) for the NRAS gene [185]. Chromosomal Rearrangements Finally, proto-oncogene activation can arise when a chromosomal rearrangement, such as a translocation, places a proto-oncogene downstream of a promoter of an IgG locus. This results i n the constitutive expression of an otherwise tightly regulated gene. A n example of this is seen wi th the M Y C gene and 29 its translocation to an IgG locus i n Burkitt's lymphoma due to a t(8;14) [186]. Alternatively a translocation can produce a fusion gene where part of gene A is fused to part of gene B (this topic w i l l be discussed in further detail below). Table 1 summarizes the more wel l known oncogenes. 1.4.2 Tumor Suppressor Genes Generally, tumor suppressor genes are responsible for inhibit ing cellular growth. Mul t ip le molecular approaches that have typically been used to identify tumor suppressor genes, including: cytogenetic analysis to determine the extent of chromosomal loss (deletions) or rearrangements; linkage analysis to determine which region-specific markers are l inked to the disease (which helps lead to the identification of the gene); loss of heterozygosity (LOH) analysis, which detects the loss of an allele or other molecular marker. Oncogenic transformation of a cell can also occur if both copies of a tumor suppressor gene are inactivated. Inactivation of tumor suppressor genes can occur via a variety of mechanisms including loss of the gene (deletion) or mutation (point, missense or nonsense) and must include both copies. Some wel l characterized examples of tumor suppressor genes include APC (discussed above), RBI, and p53. A brief discussion on their inactivation w i l l now be discussed using RBI and p53 as examples. RBI The retinoblastoma gene, RBI (see cell cycle above), is a tumor suppressor since it is responsible for controlling the transition from G l to S i n the cell cycle. Retinoblastoma occurs when both copies of the RBI gene have been inactivated 30 TABLE 1. Various classes of oncogenes and their mode of action wi th in tumors. From Vogelstein, 1998 [90]. Oncogene Mechanism of Activation Neoplasm Growth Factors vsis Glioma/fibrosarcoma KS3 DNA transfection studies Kaposi's sarcoma HST DNA transfection studies Stomach carcinoma Tyrosine Kinases: Integral Membrane Proteins, Growth Factor Receptors EGFR Amplification Squamous cell carcinoma v-fins Sarcoma v-kit Sarcoma v-ros Sarcoma TRK Rearrangement Colon carcinoma NEU Point mutation Neuroblastoma Amplification Carcinoma of breast Tyrosine Kinases: Non-receptor SRC Colon carcinoma v-yes Sarcoma v-fgr Sarcoma v-fes Sarcoma BCR/ABL Chromosome translocation Chronic myelogenous leukemia Membrane Associated G Proteins H-RAS Point mutation Colon, lung, pancreas carcinoma K-RAS Point mutation AML, thyroid carcinoma, melanoma N-RAS Point mutation Carcinoma, melanoma GEF Family of Proteins Dbl Rearrangement Diffuse B-cell lymphoma Ost Osteosarcomas Serine/Threonine Kinases: Cytoplasmic v-mos Sarcoma v-RAF Sarcoma Pim-1 Proviral insertion T-cell lymphoma Nuclear Protein Family v-myc Carcinoma myelocytomatosis N-MYC Gene amplification Neuroblastoma: Lung carcinoma L-MYC Gene amplification Carcinoma of lung v-myb Myeloblastosis v-fos Osteosarcoma v-jun Sarcoma v-ski Carcinoma v-rel Lymphatic leukemia v-ets Myeloblastosis v-erbA Erythroblastosis 31 [187-192]. Patients wi th germline mutations i n one allele of RBI are predisposed to other malignancies including osteosarcomas, soft tissue sarcomas and melanoma later i n life [90]. Lohmann et al. investigated a series of isolated unilateral retinoblastomas from 119 patients for the frequency and nature of germline RBI gene mutations [193]. Of the 119 patients studied, 99 (83%) contained mutations for the RBI gene. The types of mutations found included large deletions (15%), translocations (26%), and base substitutions (42%). p53 The p53 protein exists as a tetramer and acts as a tumor suppressor gene because it can inhibit tumor growth when introduced into a variety of transformed cells by blocking cells from entering the S phase of the cell cycle [194-197]. Other evidence has shown that p53 also blocks the transition from G2 to M via binding to CIP1 [128,198-200]. Mutations in the p53 gene represent the most frequently encountered genetic aberrations i n human malignancies [90, 201-204], Vogelstein and Kinzler outlined 5 mechanisms for p53 inactivation [109]. Deletion of one or both p53 alleles reduces the expression of tetramers, resulting in decreased expression of growth inhibi tory genes such as CIP1. Nonsense or splice site mutations that result i n truncation of the protein inhibit oligomerization, thus resulting in a similar reduction of p53 tetramers. Mutations of this type are fairly common i n lung [205], esophagus [206], and other cancers [207]. A third mechanism involves missense mutations resulting i n dominant-negative effects wi th an even greater reduction of functionally active tetramers. Such missense mutations are common i n colon [208, 209], brain [210], 32 lung [205], breast [207], skin [211, 212], and bladder cancers [213]. A fourth mechanism by which p53 is involved i n oncogenesis is common i n cervical cancers where the expression of the E6 gene of human papillomavirus (HPV) results i n the functional inactivation of p53 through binding and degradation [214]. Patients w i t h germline p53 mutations are predisposed to breast cancers, sarcomas, brain tumors, lymphomas and Li-Fraumeni syndrome [215-217]. The p53 pathway may also be disrupted by alteration of its negative regulator, M D M 2 (see above). This gene was originally identified by virtue of its amplification i n a spontaneously transformed mouse cell line [218]. The MDM2 gene is amplified (seen cytogenetically as HSRs and dmins) i n a significant fraction of the most common human sarcomas and the consequent overexpression of M D M 2 is likely to interfere wi th p53 activity [219]. Table 2 summarizes the tumor suppressor genes discussed above as wel l as a few others including their function and localization wi th in cells. 1.5 G E N E T I C A S P E C T S OF PEDIATRIC SOLID T U M O R S The presence of activated oncogenes and the deletion of tumor suppressor genes as discussed above has been reported i n many pediatric solid tumors. Pediatric solid tumors are known to contain various chromosomal aberrations, many of which can be detected by conventional cytogenetics. These aberrations include whole chromosome losses and gains (aneuploidy), translocations, HSRs and dmins (discussed above), deletions, duplications, ring chromosomes, invers ions and marker chromosomes. Aneuploidy, amplifications (dmins and HSRs) and 33 Table 2. Tumor suppressors and the tumors affected by their loss. From Vogelstein, 1998 [90]. SYNDROME GENE TUMORS LOCALIZATION FUNCTION Retinoblastoma RBI Ret, Ost Nucleus TF /ce l l cycle control L i -Fraumeni p53 Sar, breast and Nucleus T F brain tumors F a m i l i a l APC Adenomatous Cytoplasm Possible B-adenomatous polyps, C C catenin polyposis regulator N F Type I NF1 Neurofibromas, sar, gli Nucleus p21RAS-GTPase activator N F Type II NF2 Schwannomas, meningiomas Cytoplasm, Cytoskeleton membrane l i n k Famil ial breast BRCA1 Breast and ?Nucleus D N A repair cancer ovaries BRCA2 Breast and ?Nucleus D N A repair ?other W i l m s ' tumor WT1 Nephroblastoma Nucleus T F Abbreviations. Ret; retinoblastoma, Ost; osteosarcoma, TF; transcription factor, sar; sarcoma, g l i ; glioma, N F ; neurofibromatosis, C C ; colon cancer, ?= not known. 34 translocations are the most common recurrent abnormalities associated w i t h pediatric soft tissue tumors. Since gene amplification was discussed i n section above, the following discussion w i l l concentrate on aneuploidy and translocations i n pediatric solid tumors. Table 3 summarizes the common recurring abnormalities found i n various soft tissue tumors. 1.5.1 A n e u p l o i d y Every cell i n the human body (except for sperm and ova) contains 46 chromosomes. There are 22 autosomes (1 through 22) and two sex chromosomes (X and Y). Every normal cell contains two copies of each of the autosomes (44 chromosomes) and either a pair of X chromosomes (female) or an X and a Y chromosome (male). Since there are two copies of each chromosome (except i n male individuals who have only one X and one Y chromosome) the D N A content is said to be d ip lo id or 2n (n equals the haploid content of the cell). Aneup lo idy refers to the abnormal amount of genetic material on the chromosome level [90, 144]. Whole chromosomes or their arms may be lost or gained and this is easily detected using conventional cytogenetics. Some examples of soft tissue tumors that display aneuploidy are embryonal rhabdomyosarcomas (gains i n chromosomes 2, 8, 12, 13 and 20 as wel l as a loss of material from Hpl5.5) [220], leiomyosarcomas (frequent losses i n lOq and 13q and frequent gains i n 17p) [221], mesotheliomas (frequent deletions of specific regions wi th in chromosome arms l p , 3p, 6q, 9p, 15q and 22q) [222], prognostically poor neuroblastomas (deletions i n the short arm of 35 T A B L E 3. Summary of the various recurring chromosomal abnormalities found in pediatric soft tissue tumors. Adapted from Enzinger, 1988 [1]. H I S T O L O G Y C Y T O G E N E T I C S Clear cell sarcoma t(12;22)(ql3;ql2) /t(7;18)(pll.2;q21.3), +der(7)t(7;18)(pll.2;q21.3) / +8, +der (8;17)(ql0;ql0), t(12;22)(ql3;ql2.2-12.3) Dermatofibrosarcoma protuberans t(17;22)(q22;ql3), ring derived from t(17;22) Ewing sarcoma t(ll ;22)(q24;ql2) / t(l ;16)(qll;qll . l) / t(21;22)(q22;ql2)/ t(7;22)(p22;ql2) Extraskeletal myxoid chondrosarcoma t(9;22)q(22-31;qll-12), -Y Infantile fibrosarcoma +2, +8, +11, +17, +20 Hemangiopericytoma Translocation at 12ql3 Intraabdominal desmoplastic small round t(ll;22)(pl3;ql2) cell tumor Leiomyosarcoma Deletion of l p Malignant fibrous histiocytoma H i g h grade Complex M y x o i d Ring Chromosomes Malignant peripheral nerve sheath tumor Complex Neuroblastoma del(l)(p32-36), der(l)t(l;17)(p36;?), dmins and HSRs (amplification of NMYC) Rhabdomyosarcoma Alveolar t(2;13)(q35;q34), t(l;13)(p36;ql4) Embryonal +2q, +8, +20 Schwannoma -22 Synovial Sarcoma t(X;18)(pll;qll) Desmoid tumor +8, +20 deletion of 5q Lipoblastoma t(7;8)(q31;ql3) L i p o m a t(l;12)(p33-34;ql3-15), t(2;12)(p22-23;ql3-15), t(3;12)(q27-28;ql3-15), t(5;12)(q33;ql3-15), t(ll,T2)(ql3;ql3-15),t(12;21)(ql3-15;q21), t(ll;22)(q24;ql2), del(12)(ql3ql5), del(13)(ql2-q22) Liposarcoma (myxoid) t(12;16)(ql3;pll) Uterine leiomyoma t(12;14)(ql5;q24), deletion of 7q, +20 Abbreviations. Del= deletion and der= derivative. 36 chromosome 1), desmoid tumors (gains i n chromosomes 8 and 20) [27-29] and uterine leiomyomas (deletions of the long arm of chromosome 7) [223]. One possible role of an extra copy of a chromosome is to introduce an extra copy of a growth related gene (growth factors or their receptors) or oncogenes (such as RAS, PDGF, and MYC), leading to an increase i n the proliferative rate of the cell. Loss of a chromosome or a chromosome region could lead to a proliferative advantage for a cell if tumor suppressor genes (such as APC, p53, and RBI) were deleted. In addition to the examples provided above, there are other tumors that may have many extra copies of several chromosomes. A n example of this is uterine leiomyosarcoma which can have up to 8 copies (8n) of almost every chromosome i n contrast to two copies (2n) in normal cells [224]. 1.5.2 Tumor Specific Translocations A translocation refers to the exchange of chromosomal material between two chromosomes. A translocation can involve either a part of a chromosome arm or the entire arm itself. Robertsonian translocations result i n the fusion of the long arms of two acrocentric chromosomes (chromosomes 13-15 and 21 and 22) wi th the subsequent loss of the short arms [144]. Translocations can occur in either transcriptionally active or inactive regions. The translocation results in the formation of two derivative chromosomes (if the translocation is reciprocal). A derivative chromosome is a structurally rearranged chromosome generated either by a rearrangement invo lv ing two or more chromosomes or by multiple aberrations wi th in a single chromosome. 37 The majority of reciprocal translocations result in derivative chromosomes that give rise to no visible phenotypic change [225]. This can be explained by the fact that the breakpoint may fall wi th in D N A that does not contain any genes or is not expressing any genes. The expression of the genes around the breakpoint therefore, are not affected. Alternatively, there are two possibilities where the translocation can give rise to derivative chromosomes associated wi th a phenotypic change. One possibility involves the breakpoint occurring i n transcriptionally inactive D N A , as above, except that this region is responsible for the expression of nearby genes. The derivative chromosomes now contain a part of chromosome ' A ' fused to a part of chromosome 'B ' . Since the translocation w i l l affect the expression of nearby genes, the function of these genes w i l l determine the viabili ty of this rearrangement. For example, if the translocation results in the overexpression of nearby genes and one of these genes is an oncogene (e.g., MYC, PDGF or RAS) then the cell w i l l most likely have an increased proliferative rate. O n the other hand, i f the expression of the genes is suppressed and one of the genes involved is a tumor suppressor gene (e.g., ARC, pl6 or p53), then the proliferative rate may increase as above. If the gene(s) involved in the latter case is involved i n cell survival such as the glucose-6-phosphate molecule (necessary for glucose metabolism), then the phenotype may be lethal (see Fig. 4a). The most interesting result of a translocation occurs when the breakpoints occur wi th in expressed sequences (see Fig. 4b). This type of translocation is relatively common i n soft tissue tumors [90, 224, 226, 227], where a chimeric gene fusion is formed due to the translocation and w i l l now be discussed in more detail below. 38 FIGURE 4. The possible outcomes of chromosomal translocations. a) Translocations can occur in either transcriptionally inactive or active DNA. The phenotype of the former is usually nothing, unless the transcriptionally inactive region is involved in regulating the expression of nearby genes. The latter possibility can result in a deletion of a gene, the introduction of a stop codon, a non functional protein or a functional chimeric protein which fuses part of gene one to part of gene 2. b) Chimeric gene fusions can form as a result of an inter-exonic gene fusion or an intra-exonic gene fusion. The fusion gene can be oncogenic if the regulation of specific domains involved in the fusion are lost. 3 9 41 1.5.3 Tumor Specific Translocations Result in Functional Gene Fusions in Solid Tumors Hematopoietic tumors helped pave the way to understanding chimeric genes as a result of specific chromosomal translocations [228-230]. These translocations almost always result in a proliferative advantage for the malignant cell and this is accomplished by one of two mechanisms. The first possibility is that the translocation splices an oncogene to a positive regulatory element of the partner gene. The result is overexpression of the oncogene leading to an increased growth rate for the malignant cell. A n example of this is seen i n Burkitt's lymphoma due to the t(8;14) which places the M Y C oncogene under the control of the IgH locus promoter [229, 231, 232]. A s imilar situation exists i n the solid tissue tumor dermatofibrosarcoma protuberans (DFSP). In this tumor the expression of the PDGF-B chain molecule is placed under the control of the COL1A1 gene promoter [233]. The COL1A1 product is a major constituent of the connective tissue matrix. The increased expression of PDGF-B contributes to tumorigenesis since it has transforming activity and is a potent mitogen for a number of cell types [234-237]. Its role i n the oncogenic process however is not fully understood. The second possibility involves the juxtaposition of a portion of one gene to a portion of another gene. The breakpoints can occur wi th in an intron or an exon, but the end result is usually the i n frame fusion between the exons of two genes. These chimeric gene fusions contain functional domains from both genes and are largely responsible for the increased proliferation seen i n many soft tissue tumors 42 and leukemias [238]. Many sarcomas express chimeric transcription factors resulting from gene fusions [229]. Since our studies have identified a gene fusion i n congenital fibrosarcoma as a result of a chromosomal translocation, the remainder of this chapter w i l l concentrate on a few examples of solid tumors and their associated gene fusions. Ewing's Sarcoma Family of Peripheral Primit ive Neuroecto-Dermal Tumors Ewing sarcoma, peripheral neuroepithelioma and A s k i n tumor are a group of malignancies which are poorly differentiated and belong to the family of peripheral Primitive Neuroectodermal Tumors (pPNETs) [5, 239]. Ewings sarcomas (ES) and peripheral neuroepithelioma contain a t(ll;22)(ql2;q24) which juxtaposes the FLU gene from chromosome l l q l 2 to the EWS gene from chromosome 22q24 i n approximately 85% of cases [240-246]. The FLI1 gene is a member of the ETS family of transcription factors and is known to contain at least two functional domains [247, 248]. One of the domains, a helix loop helix domain ( H L H D ) , is responsible for protein-protein dimerization while the other domain, the ETS D N A binding domain, is responsible for binding to D N A . The EWS gene contains an R N A binding domain and a transcriptional activation domain [249, 250]. The translocation results in der(22) giving rise to a fusion gene containing the transcriptional activation domain from EWS as the 5'end of the fusion gene and the ETS D N A binding domain from the FLI1 gene as the 3' end (see Fig. 5) [244, 251]. Studies have shown the fusion product to be a transcriptional activator due to the swapping of the R N A binding domain from the EWS gene wi th the D N A binding 43 Q CQ Q H ttJ DC CA CU J3 i •rt H cu H S S c S S fi Q rou.c^uxs^g rocS-&£w>c^ c r o g r o r o f i - S ^ u o " Ji ^ M * £ b p | £ § < | c cn s ' y £ H n O j_ l ie | H a 11 roo3~(tla»'>ro'ii o _ , r o - t c y j j < 2 2 <y 2 £ u t s CO , ° fi -3 O WO n fi <u o o v ro I £ M •§ 3 v 1 o w c n H w c n H w c n -a vcu w ro LO ^ C O o o .fi £Txs .2 X JB rM £ 53 fi •Ti ro , ^ - ; H y ^ ro ro g cu •a S o . fi y -2 fi o S S a S § ro 44 domain from the FLU gene [252]. This places the D N A binding domain from F L U under the promoter elements of the EWS gene, which effectively disrupts the normal activity of the D N A binding domain. It is therefore thought that this altered D N A binding activity somehow gives rise to an increased proliferative rate. One possibility is that the chimeric fusion protein may be able to activate c - M Y C , since both E W S / F L I 1 and F L U are capable of activating the c - M Y C promoter [253]. The elucidation of the exact mechanism of pathogenesis, however, w i l l require further investigation. The majority of the remaining 15% of pPNETs appear to harbor a variant t(21;22)(q22;ql2), which has been shown to express an EWS-ERG chimeric transcript [247, 254, 255]. The fusion protein acts as a transcriptional activator and requires the amino terminal domain of EWS [256]. In addition, since the clinical behavior of the pPNETs wi th an EWS-FLI1 or an EWS-ERG fusion appear similar [254, 257], it is thought that the fusion proteins may be acting along similar oncogenic pathways. Another rare variant translocation has been discovered i n Ewing sarcoma where a t(7;22)(p22;ql2) results in the fusion of the 5' portion of the EWS gene to the 3' D N A binding domain (ETS domain) of the ETV1 gene, another member of the ETS family of transcription factors. It is still not known whether this variant functions i n similar ways as do the EWS-FLI1 and EWS-ERG chimeras. The EWS gene has been involved i n at least two other translocations invo lv ing the ATF1 and WT1 genes, however these fusions occur in malignant melanoma of soft parts and intra-abdominal desmoplastic small round cell tumor, respectively. 45 M y x o i d Liposarcoma Liposarcomas are malignant tumors of fat cells that primari ly occur i n the extremities and retroperitoneum of adults [1]. Of the various types of liposarcomas, myxoid liposarcoma (myxoid LPS) is the most common. Several independent groups characterized the t(12;16)(ql3;pll) i n myxoid LPS and found that the FUS gene from chromosome 1 6 p l l is fused to the CHOP gene from chromosome 12ql3 [258-262]. In addition, round cell liposarcoma, a poorly differentiated form of myxoid LPS, was also found to harbor the same gene fusion [263]. The FUS gene (also known as TLS (for "translocated i n /iposarcoma") is a nuclear R N A - b i n d i n g protein wi th extensive homology to EWS [259]. Moreover , the amino terminus of FUS appears to contain a strong transcriptional activation domain as found in EWS [264]. A m a n et al. found that the size of the FUS gene is 11 kb and consists of 15 exons [261]. The CHOP gene is a nuclear protein and acts as a dominant-negative transcription factor inhibitor for C / E B P and L A P [265]. CHOP stands for "C/EBP-/iomologous protein", but it has also been referred to as DDIT3 and GADD153 for " D N A damage-inducible transcript 3" and "growth arrest and D N A damage-inducible gene", respectively. C H O P inhibits the transcriptional activity of C / E B P and L A P by heterodimerizing wi th them through the leucine zipper domain, thus interfering wi th their D N A binding capabilities. This interaction is mainly seen in response to cellular stress, such as D N A damage [265]. In addition, C H O P is thought to function i n pathways of terminal differentiation and growth arrest i n fat cells [266]. 46 The t(12;16) results in the fusion of the FUS amino terminus (FAT) to the entire C H O P coding region [258, 259]. Transformation of NIH3T3 cells requires both the F A T portion as wel l as the entire C H O P region [264]. Furthermore, the carboxy terminal leucine zipper domain from C H O P was essential for transformation. Based on these results it would be reasonable to assume that the genes regulated by C / E B P and L A P are tumor suppressor genes and the constitutive inhibi t ion of their expression by F U S - C H O P could lead to oncogenesis. In addition to the FUS-CHOP fusion, the FUS gene was found to be rearranged i n an acute myeloid leukemia wi th a t(16:21)(pll;q22) [267, 268]. The fusion partner was identified as E R G , seen previously i n Ewing sarcoma as part of an E W S - E R G gene fusion. E R G expression i n both acute myeloid leukemia and i n Ewing sarcoma is therefore under the control of either the FUS or EWS promoter elements, respectively [269]. A m a n et al. states that although the N-terminal ends of FUS and EWS are different, they share extensive homology and are distinct f rom the N-terminal regions of other ribonucleoprotein (RNP)-carrying proteins [269]. Alveolar Rhabdomyosarcoma Rhabdomyosarcomas (RMS) are a heterogeneous group of malignant tumors and are the most common soft-tissue sarcoma of childhood [220]. There are three subtypes: embryonal R M S (ERMS) (~ 65% of R M S cases), alveolar R M S (ARMS) (~ 20% of R M S cases), and the less well-defined undifferentiated sarcomas (~15% of R M S cases) [5, 270]. Differentiation between these subtypes as wel l as other poorly defined sarcomas poses a problem to the pathologist, once again illustrating the need for more accurate tools for diagnosing these tumors. 47 Alveolar R M S cases harbor either a t(2;13)(q35;ql4) (-60% of A R M S cases) or a t(l;13)(p36;ql4) (-10-20% of A R M S cases) which fuses the paired box (PB) and homeobox (HB) D N A binding domains from either the PAX3 or PAX7 genes, respectively, to the acidic and proline rich domain of the FKHR gene [271-273]. The PAX3 and PAX7 genes are members of the P A X family of transcription factors w h i c h means the fusion gene is a chimeric transcription factor. The PAX genes appear to be important for myogenic differentiation during embryonic development [274-276]. FKHR belongs to another transcription factor family known as the forkhead family of transcription factors [277]. F K H R is known to contain a D N A binding domain as wel l as a carboxy terminal transactivation domain. The D N A binding domain of F K H R is of the winged-helix type. The fusion product between P A X 3 or P A X 7 and F K H R has the D N A binding domain from P A X 3 or P A X 7 attached to the transactivation domain from F K H R . The overexpression of PAX genes leads to oncogenic transformation [278]. This has lead researchers to hypothesize that the transcriptional disruption of normal PAX target genes due to the PAX-FKHR gene fusions plays a major role in A R M S oncogenesis. Synovial Sarcoma Synovial sarcoma is an aggressive soft tissue malignancy occurring predominantly i n the extremities of adolescents and young adults [1]. A recent case was misdiagnosed as a desmoplastic small round-cell tumor providing further evidence for the need of a molecular based approach to diagnosing morphological ly similar tumors [279]. The recently characterized t(X;18)(pll.2;qll.2) is found i n approximately 90% of human synovial sarcomas [280-282]. This results i n a der(X) 48 chromosome which gives rise to the fusion of the chromosome 18 SYT gene (also known as SSXT) located at 18qll.2, to either of 2 distinct genes (at least 2Mb apart from each other at the X p l l . 2 locus) from X p l l . 2 , SSX1 or SSX2 [283-286]. Recent studies have shown that the SYT protein contains a transcriptional co-activator domain [287]. The N-terminus of the SYT protein has a novel conserved 54 amino acid domain ( S N H domain) which has been observed in a wide variety of species. The C-terminal domain is rich in glutamine, proline, glycine and tyrosine (the Q P G Y domain), which harbors transcriptional activator sequences. Mutagenic analysis of the SYT gene has shown an increase in transcriptional activation along wi th the deletion of the S N H domain, suggesting that this domain acts as an inhibitor of the activation domain [287]. The mouse homologue of SYT, Syt, was isolated and sequenced i n full by de Bruijn et al. [288]. They found that during early embryogenesis, mouse Syt is ubiquitously expressed. Later, expression becomes confined to cartilage tissues, specific neuronal cells, and some epi thel ium-derived tissues and i n primary spermatocytes. The SSX1 and SSX2 proteins are 81% homologous and are r ich i n charged amino acids [289]. Due to the high homology between the molecules, it is l ikely that the function of SYT-SSX1 and SYT-SSX2 is similar. The SSX molecules contain a transcriptional co-repressor domain, known as the Kruppel-associated box ( K R A B ) , at their N-terminus [290-292]. K R A B domains have been previously identified only i n Kruppel-type zinc finger proteins, e.g., zinc finger protein-117 and -83 (ZNF117 and ZNF83). 49 The translocation i n synovial sarcomas fuses the transcriptional activating domain from the SYT gene to the transcriptional repressor domain from the SSX gene. Crew et al. found these transcripts i n 29 of 32 (91%) synovial sarcomas, thus providing pathologists wi th a useful diagnostic tool [286]. Further analysis of the breakpoints lead to the identification of 2 distinct fusion junctions for each of SYT-SSX2 and SYT-SSX1 fusion transcripts [286]. Since then, additional variants have been characterized, differing i n the placement of the breakpoint [293, 294]. In addition, subcellular localization studies have shown that SYT and SYT-SSX proteins co-localize wi th the human homologue of the SNF2/Brahma protein B R M i n the nucleus. The function of SNF2 i n mammals is unknown, but evidence i n S. cerevisiae and D. melanogaster suggests that it may act as a global activator of transcription [295]. In vitro studies have further shown that these molecules interact wi th each other [287]. This implies that the SYT-SSX fusion protein may have a role i n activating or inhibiting the normal role of human homologue of S N F 2 . 1.6 A I M S A N D OBJECTIVES We focussed on the genetics of congenital fibrosarcoma (CFS) and adult-type fibrosarcoma (ATFS) for three reasons. Firstly, CFS and A T F S are considered to be malignant lesions. Secondly, CFS and A T F S (but particularly CFS) are very difficult to differentiate from benign fibroblastic proliferations of childhood such as infantile fibromatosis (IFB) and aggressive fibromatosis (AFB) due to significant phenotypic overlap and similar age distributions. Lastly, they appear to differ markedly f rom 50 each other i n behavior and response to therapy, making a reliable marker for their distinction extremely important from a clinical perspective. W i t h this i n mind, our goal was to identify and characterize recurrent genetic alterations i n cellular fibroblastic tumours of childhood that would be useful for the pathologic and prognostic classification of members of this tumor subgroup. Cytogenetic analysis of CFS cases to date have only shown non random abnormalities invo lv ing whole chromosomes, such as trisomy of chromosome 11. The cytogeneticists at B.C. Children's Hospital identified an apparently non-random abnormality i n v o l v i n g the chromosomal regions 12pl3 and 15q25. The specific objectives of the work described i n this thesis were to: 1. Characterize, at the molecular cytogenetic level, the consequences of the 12pl3 and 15q25 aberrations by fluorescence i n situ hybridization. 2. Clone the chromosomal breakpoints and characterize the genes invo lved . 3. Characterize the ETV6-NTRK3 gene fusion at the c D N A level. 4. Characterize the E T V 6 - N T R K 3 protein product (molecular weight, hetero- and homodimerization status, and phosphorylation status) 5. Elucidate the downstream protein interactions of E T V 6 - N T R K 3 . 51 CHAPTER II MATERIALS AND METHODS 2.1 C L I N I C A L F E A T U R E S O F I N D E X C A S E A term baby girl (TB) was found at birth to display a quarter-sized discoloration i n the left lumbar region. Over the course of four weeks the mass grew in size to 7 x 5 cm. Initial biopsy was diagnosed as a congenital fibrosarcoma by a dermatologist. This diagnosis was confirmed by pathologist review. A CT scan and ultrasound confirmed the absence of any deep tissue extension. Surgery revealed a fleshy grey colored mass which was resected wi th overlying b lu i sh tinged skin. The deep margin was at the posterior superior iliac spine and was negative for tumor. There was no further treatment administered post surgery. T B was followed yearly and has since shown no recurrence at the local site and chest X -rays have been clear. 2.1.1 Patho logy of Index Case The gross specimen consisted of a large tan-brown mass wi th m i n i m a l evidence of hemorrhage or necrosis. Histologic analysis revealed a cellular spindle cell lesion in which there was nuclear pleomorphism and moderate mitotic activity. Cells showed no obvious evidence of specific differentiation other than having the appearance of possible fibroblastic origin. There was positive immunohistochemical staining for vimentin, but not for muscle specific actin, desmin, S100, histiocytic markers, or endothelial markers. Ultrastructural analysis 52 showed no rhabdomyoblastic differentiation, but also suggested a possible fibroblastic origin. Based on these features, the specimen was diagnosed as a congenital fibrosarcoma (see Fig. 6a). This appearance is contrasted wi th infantile fibromatosis (see Fig. 6b), which shows similar, but less pleomorphic cells, lower mitotic activity and increased cellular matrix between cells. 2.1.2 Cytogenetic Analysis of Index Case Cytogenetic analysis of cultured tumor tissue of the init ial CFS case showed an abnormal karyotype i n all cells examined as indicated i n Figure 7. A l l cells examined showed additional copies of chromosome 11, wi th a few cells inc lud ing chromosomes +20 and +2. There were structural abnormalities inc lud ing additional material on the long arm of chromosome 1, apparent deletion of the distal portion of the short arm of chromosome 12, and a rearrangement of the long arm of chromosome 15. The final cytogenetic assessment of 24 metaphases of this case was as follows: 46-49, X X , - X , add(l)(q43), +2, +11, del(12)(pl3), add(15)(q25), +20. 2.2 C L I N I C A L S A M P L E S . TISSUE C U L T U R E T E C H N I Q U E S A N D C Y T O G E N E T I C A N A L Y S I S A l l CFS, C M N , ATFS , and IFB cases analyzed i n this study were collected from either British Columbia's Children's Hospital, Childrens Hospital of Los Angeles, National W i l m s ' Tumor Study Group (NWTSG) tumor bank, or Cooperative H u m a n Tissue Network (CCHTN) at Columbus Children's Hospital , Columbus, Ohio and presented during the period 1988 to the present. The cases 53 F I G U R E 6. Histologic analysis of CFS and IFB. A, 40X magnification of a CFS case stained wi th hematoxylin and eosin (H&E). Note the characteristic highly cellular spindle morphology (bluish-purple), mitotic figures and nuclear pleomorphism. B, 40X magnification of an IFB case stained wi th H & E . Note the lower density of spindle cells (bluish-purple) and an increase i n the amount of extracellular collagenous matrix (pink) relative to CFS. 55 F I G U R E 7. G-banded karyotype of index case. A total of 24 metaphases were analyzed by B.C. Children's Hospital's cytogeneticists. The following karyotype displays 48 chromosomes, X X , add(l)(q43), +11, del(12)(pl3), add(15)(q25), and +20. Three of four CFS cases analyzed showed recurring anomalies including addition of chromosome 11 (vertical arrow), apparent deletion of 12pl3 (angled arrow), and a rearrangement of 15q25-qter (horizontal arrow). 57 were analyzed by short term culturing and cytogenetic analysis by cytogeneticists at either B.C. Children's Hospital or Childrens Hospital of L . A . according to established protocols [296, 297]. Briefly, excised tumor tissue was minced i n collagenase (200 un i t s /ml , Sigma) and incubated for 2 hours. Washed cells were incubated i n 60 m m plastic petri dishes i n R P M I 1640 medium wi th L-glutamine (Gibco BRL) supplemented wi th 15 or 20% fetal bovine serum (FBS, Sigma), 5% antibiotic-antimycotic solution (Gibco BRL), and maintained i n this medium at i 37°C i n a 5% C 0 2 incubator. Short-term cultures used for cytogenetic analysis were arrested i n metaphase wi th Colcemid (1 n g / m l final concentration, Gibco BRL) for 3 to 4 hours prior to harvesting. After swelling the cells i n a hypotonic solution, the cells were fixed i n a 3:1 solution of methanol to acetic acid and dropped onto glass slides. G-banding techniques were used to stain metaphases previously fixed, dried, and treated overnight at 60°C on the petri dish surface. Cells were harvested at various passages and were used for cytogenetic analysis and as a source of D N A or R N A . Frozen primary tumor specimens for molecular studies were stored at -70°C prior to analysis. Karyotypes are described in accordance wi th the International System for H u m a n Cytogenetic Nomenclature, 1995. Karyotypes and cl inical features for the 4 CFS cases initially analyzed cytogenetically are summarized i n Table 4. Cel l lines expressing N T R K 3 (kindly provided by Drs. B. N e l k i n and D. Kaplan) were grown i n R P M I 1640 supplemented as above wi th the addition of 200 58 T A B L E 4. Summary of cytogenetic analysis of initial B C C H CFS cases. Ini t ial karyotype refers to the initial cytogenetic assessment of the cases while f inal karyotype refers to the cytogenetic interpretation after FISH analysis. A final cytogenetic assessment on case 2 was not possible due to the absence of material for FISH. C A S E INITIAL C Y T O G E N E T I C S F I N A L C Y T O G E N E T I C S 1 46-49, X X , - X , +11, +20, +2, 46-49, X X , - X , +11, +20, +2, add(l)(q43), del(12)(pl3), t(l;12;15)(q44;pl3;q25) add(15)(q25) [cp 24] 2 48, X Y , +11, add(15)(q26), +21 [cp 16] N A / 48, X Y , +8, +11, add(15)(q26) [cp2] / 47, X Y , +11, add(15)(q26) [cp 2] 3 48, X Y , +11, +8, t(4;12)(pl0;ql0), add 48, X Y , +11, +8, (15)(q25), t(12;15)(pl3;q26) [cp 21] t(4;12;15)(ql2;pl3;q25) Abbreviations. cp= number of cells evaluated, N A = not available, add= addition, del= deletion. 59 u g / m l of G418 (Gibco/BRL). 2.3 Y A C A N D C O S M I D PROBES Yeast artificial chromosomes (YACs), mapping wi th in specific areas of interest were kindly provided by Dr. S. Scherer from the Canadian Genome Centre Y A C Core Facility at the Hospital for Sick Children, Toronto, Ontario. These included 890_E_3, 854_E_6, 924_H_12, 817_H_1, 954_G_10, 738_B_11 f rom chromosome 12pl3, and 882_H_8, 895_H_10, 932_F_12 (chimeric), and 802_B_4 from chromosome 15q25-26. A l l Y A C s were either previously confirmed to be n o n -chimeric [298, 299], or were confirmed to be non-chimeric by FISH analysis of normal metaphases in our laboratory. Cosmid contig probes spanning the ETV6 locus, including 179A6, 171H6, 45E12, 163E7, 54D5, and 148B6 [300], were generous gifts of Dr. Peter Marynen, University of Leuven. Y A C s and cosmids were grown and maintained i n our laboratory using standard methods [301]. Probes were labeled wi th either biotin or digoxygenin using a commercially available kit according to the manufacturer's instructions (Gibco/BRL). 2.4 D N A A N D R N A I S O L A T I O N Primary CFS, IFB, or C M N tumor tissue, normal fibroblasts and NIH3T3 cells infected wi th ETV6-NTRK3 retroviral constructs (kindly provided by Daniel W a i i n our laboratory) were used as sources of D N A or R N A . Several other previously established cell lines were used as either positive or negative controls including the leukemia cell lines K562 [302] and Jurkat [303], the neuroblastoma cell line SAN2 [304], a Ewing tumor cell line TC-71 [305], and the rhabdomyosarcoma cell line Birch 60 (established at St. Jude's Hospital, Memphis , T N , Piper S, unpublished data). D N A was extracted from these cells using standard methods and an App l i ed Biosystems D N A Extractor, Mode l 340A [306]. Total R N A was extracted using the acid guanidium thiocyanate phenol/chloroform method [307]. D N A used for FISH was isolated from either Y A C or cosmid clones (see section above). D N A from cosmids was extracted using a Plasmid M i d i K i t (Qiagen) according to the manufacturer's instructions. D N A from Y A C s was isolated by previously established methods (D. Ward , personal communications). Briefly, an A H C plate was innoculated wi th a Y A C clone and allowed to grow at 30°C for at least two days or unt i l colonies were visible. One red colony (the red colony indicates the presence of insert wi thin the Y A C ) was then used to innoculate either 50 m l of minimal media or yeast extract, peptone, dextrose (YPD) media, which was subsequently shaken overnight at 30°C at 225 rpm. Cells were then centrifuged at 2000 rpm for 3 minutes and the pellet was resuspended i n 0.5 m l of I M sorbitol (Difco)/0.1M Na2EDTA (pH 8) and 20 u l of Sigma Lyticase (Sigma)(12.5 m g / m l of Sigma Lyticase, prepared fresh, i n I M sorbi tol /O. lM Na2EDTA (pH 8)). The solution was then transferred to an eppendorf tube, incubated at 37°C for 60 m i n , and centrifuged for 1 minute. The pellet was resuspended (gently wi th a pipet) i n 0.5 m l 50 m M Tris-Cl (pH 7.4)/20 m M N a 2 E D T A (pH 8) before the addition of 50 u l 10% SDS. This mixture was incubated at 65°C for 30 minutes. The SDS was removed by adding 0.2 ml 5 M potassium acetate, incubating on ice for 60 minutes and then centrifuging at 14,000 rpm at 4°C for 5 minutes. The supernatant was then transferred to a fresh eppendorf tube and the D N A was precipitated w i t h 61 isopropanol according to previously established methods [306]. The D N A was RNase treated prior to hapten labeling for use as a FISH probe 2.5 S O U T H E R N A N D N O R T H E R N B L O T A N A L Y S I S Southern and Northern blot analyses were performed as previously described [306]. For Southern analysis, lOug of genomic D N A from each case was digested wi th Hindlll, BamHI, EcoRI, or Xhol. For Northern analysis, at least 20ug total R N A was used. Briefly, a 0.8% agarose gel was used for Southern analysis, while a 1.2% formaldehyde-agarose gel was used for Northern analysis. D N A or R N A was transferred onto nylon membrane filters ( H Y B O N D ) by capillary blotting and fixed to the membrane by baking at 80°C for 2 hours. Probes included: fu l l length ETV6 c D N A (a generous gift of Dr. Peter Marynen, Universi ty of Leuven); ETV6-5/6 , consisting of ETV6 exons 5 and 6 (nt 825-1137) and generated by digesting ETV6 full length c D N A wi th BamHI and Pvul); full length NTRK3 c D N A (a generous gift of Dr. Barry Nelk in , Johns Hopkins University); and NTRK3-PTK, consisting of NTRK3 nt 1740-2715 (including the P T K region), and generated by Xbal digestion of full length NTRK3 c D N A . Probes were radiolabeled wi th [a- 3 2P]dCTP (50uCi) using random primer extension (Oligo Labeling Ki t , P H A R M A C I A ) followed by nick-column purification ( P H A R M A C I A ) . The D N A and R N A membranes were hybridized overnight wi th the radiolabeled probes after w h i c h they were washed and autoradiographed at -70°C using standard methods [306]. To control for equal loading of R N A for Northern analysis, membranes were stripped and reprobed wi th a fi-actin D N A probe. 62 2.6 F L U O R E S C E N C E IN SITU H Y B R I D I Z A T I O N (FISH) STUDIES Cel l suspensions of normal cultured lymphocytes, fibroblasts, and primary CFS tumor cells were processed according to standard cytogenetic procedures [296] and stored at -20°C i n methanol/acetic acid fixative (3:1) unti l used. Metaphase chromosome spreads and interphase nuclei were prepared on glass microscope slides [296]. Slides for FISH were prepared by applying a drop of fixed cells onto a slide from approximately 1 foot above the slide, which was held at an angle of 45°. The slide was allowed to air dry and then aged by storing the slide i n a dessicating chamber overnight at room temperature. Prior to use, the slides were passed through a series of room temperature ethanol washes (2 minutes each i n 70%, 90% and 100% to dehydrate the slides). The chromosomes were denatured by submerging them i n 70% Formamide (Sigma)/2X SSC (pH 7.0) for 2 minutes. The slides were then immediately run through a -20°C ethanol series as above. The slides were then allowed to air dry before the probe was applied. The D N A probes for FISH ( Y A C , cosmid, and/or a-centromeric) were labeled wi th either biotin-14-dATP (Gibco/BRL) or d igoxigenin- l l -dUTP (Boehringer Mannheim) using BioNick Labeling K i t (Gibco/BRL) and purified by ethanol precipitation according to the manufacturer's instructions, a-centromeric probes were purchased prelabeled wi th biotin or digoxigenin (Oncor). The addition of 5 Units of D N A Polymerase I (New England Biolabs) per labeling reaction was necessary for proper labeling of Y A C and cosmid D N A . The purified probe (500 ug of labeled Y A C or 50 ng of cosmid probe) was then dissolved in Hybrisol VII (Oncor), denatured at 75°C for 10 minutes and allowed to preanneal at 37°C for 30 63 minutes (for cosmids) or 1 hour (for YACs) . The preannealed probe was then applied directly to the denatured slides, sealed wi th a coverslip and rubber cement (Canadian Tire) and allowed to hybridize for at least 16 hours at 37°C. After hybridization, the slides were washed i n 50% formamide/2X SSC (pH 7.0) at 45°C for 5 minutes, and another wash i n 2X SSC at 45°C for 5 minutes. Detection of the signal then proceeded according to the manufacturer's instructions (Oncor). The chromosomes were counter-stained i n diamidino-2-phenylindole dihydrochloride hydrate (DAPI) (2ul of a 200mg/ul stock i n 200ul of antifade; 20ul of this solution was applied to each slide) and visualized using a 100X o i l immersion objective through a Zeiss Axioplan Universal epifluorescence microscope. Images were captured wi th a C O H U H i g h Performance camera w i t h PSI Scientific Systems software (League City, TX). Images were converted to TIFF files and then processed through Adobe Photoshop 5.0 prior to printing. 2.7 3 ' A N D 5 ' R A P I D A M P L I F I C A T I O N O F c D N A E N D S (RACE) Two micrograms of total R N A were used as starting material for 3 ' -RACE. ETV6 primers 541 and 701 [308] were used sequentially as sense primers i n 3 ' - R A C E experiments performed according to the manufacturer's instructions (3 ' -RACE System; Gibco /BRL) . P C R conditions for both primers were as follows: 94°C for 1.5 minutes followed by 34 cycles of 94°C for 45 seconds, 58°C for 2 minutes, and 72 °C for 3 minutes, and a final extension of 72°C for 10 minutes. Products were analyzed on agarose gels, cloned using the Invitrogen T A Cloning System (Version 1.3), and 64 sequenced on an A B I Appl ied Biosystems 373A D N A Sequencer. Sequences were analyzed using D N A S T A R Sequence Analysis software. To determine if the 3' end of the ETV6 gene (the ETS D N A binding domain) was involved i n a gene fusion, 5 ' -RACE was utilized using five micrograms of R N A as starting material. Primers used included: T E L O U T : 5 ' - G C T G G G T A G T T T G T C T A A G G T G C - 3 ' T E L M I D : 5 ' - T G G T C T G C A A G A G A A G T G T C C C T - 3 ' T E L I N : 5 ' - C A G G G C T C T G G A C A T T T T C T C A T A - 3 ' Products were analyzed as described above. To determine whether the 5' end of the NTRK3 gene (extracellular l igand binding domain including the transmembrane domain) was involved i n a gene fusion, 3 ' - R A C E was utilized using 2 ug of R N A as starting material and primers: TRKC1044: 5 ' - G G A G T C C A A G A T C A T C C A T G T G G - 3 ' TRKC1329: 5 ' - T G C T G C T T T T G C C T G T G T C C T G - 3 ' Products were analyzed as described above. 2.8 R T - P C R A N A L Y S I S OF T U M O R S A M P L E S Total R N A (2ug) was isolated from primary tumor samples and used to make c D N A as previously described [309]. Olignucleotide primers for P C R included: ETV6 primers 541 and 701 [308] 352 ( 5 ' - G G T G A T G T G C T C T A T G A A C T C C - 3 ' ) 199 ( 5 * - A T T T A C T G G A G C A G G G A T G A C - 3 ' ) 65 used i n combination wi th NTRK3 primer: NTRK3-2 (TRKC nt 1816-1838: 5 - C C G C A C A C T C C A T A G A A C T T G A C - 3 ' ) . P C R conditions were as follows: 94°C for 1.5 minutes, followed by 33 cycles of 94°C for 45 seconds, 60°C for 1 minute and 72°C for 1 minute and a final extension of 72°C for 10 minutes. Products were analyzed as described above on agarose gels. The presence of amplifiable R N A i n all samples was confirmed by R T - P C R using (3-Ac t in primers as a control. A l l samples were confirmed for the presence of the breakpoint by Southern blot analysis as described above using a D N A oligo spanning the breakpoint: 5 ' - G G G A G A A T A G C A G A T G T G C A G C A C - 3 ' . 2.9 PREPARATION OF PROTEIN LYSATES FOR IMMUNOPRECIPITATION A N D IMMUNOBLOTTING Cells infected wi th the various retroviral constructs including ETV6-NTRK3, along wi th the individual mutants (see Table 5) were grown until 80-90% confluent at which point the media was decanted and the cells were rinsed twice wi th ice-cold PBS. Briefly, 1ml of Lysis Buffer (1.5 m M M g C l 2 (Fisher), 150 m M N a C l (Fisher), 50 m M Hepes (Sigma), 10 m M NaF (Sigma), 10 m M N a 4 P 2 0 7 (Sigma), 2 m M N a 3 V 0 4 (Sigma), 2 m M ethylene-diamine-tetraacetic acid (EDTA) (Fisher), 2 m M N a M o 0 4 2PLO (Sigma), 10% Glycerol (Fisher), 0.5-1.0% Nonidet P-40 (Fisher), Leupeptin (1:1000 dilution of 2 m g / m l stock made in H 2 0)(Sigma), Apro t in in (1:1000 d i lu t i on of l O m g / m L stock made in H 2Q)(Sigma), Phenylmethylsulfonyl Fluoride (PMSF) 66 T A B L E 5. Summary of the various constructs used to transfect NIH3T3 cells. The vector used to make these constructs was MSCVpac and contained the var ious mutants listed below. Many of the mutations involved replacing a tyrosine residue (Y) wi th a phenylalanine (F). The Kinase Dead mutant (KD) consisted of a mutation replacing a lysine residue to an arginine. The A H L H mutant contained a deletion encompassing nucleotides 191 - 347 of E T V 6 - N T R K 3 . Corresponding residues i n N T R K 3 are given for comparison. C O N S T R U C T M U T A T I O N IN ETV6-N T R K 3 C O R R E S P O N D I N G R E S I D U E IN NTRIC3 ETV6-NTRK3 - -A H L H A191-347 -A L D Y513F, Y517F, Y518F Y705, Y709, Y710 K D K380N K572 PLCyQ Y628Q Y820 PLOyT Y628T Y820 PLOyE Y628E Y820 Abbreviations. A= deletion, Y= tyrosine, F= phenylalanine, K= lysine, N= asparagine, Q= glutamine, T= threonine, and E= glutamate. 67 (1:200 dilution of a lOOmM solution made in dimethyl sulfoxide)(Sigma)) was then added to the rinsed cells and incubated for 15 minutes on ice [310, 311]. Lysates were then centrifuged at 12000 rpm for 10 minutes, at which point the supernatant was transferred into a fresh tube for further analysis. 2.10 I M M U N O P R E C I P I T A T I O N One mill i l i ter of lysate was incubated wi th gentle agitation for two hours at 4°C wi th either N T R K 3 antibody (20ul) (Santa Cruz Biotechnology) or a - E T V 6 : H L H (3ul) (generous gift of Dr. P. Marynen) along wi th lOul of Protein A-Sepharose (Pharmacia). The tubes were centrifuged at 2500 rpm for 5 minutes and the supernatant discarded. The pellet was washed 2 to 3 times i n Lysis Buffer (except the concentration of Nonidet P-40 was 0.1% instead of 1%), boiled i n L a e m m l i buffer [306], loaded onto a Protean E / x i Cell electrophoresis system (Bio-Rad) and electrophoresed on a 7.5, 10 or 15% polyacrylamide gel overnight at 70 - 100 Vol t s according to standard methods [306]. 2.11 I M M U N O B L O T T I N G Transfer of the proteins from the gel to Immobilon-P (Millipore) was accomplished wi th the Bio-Rad Trans-Blot SD Semi-Dry Transfer cell at 25 volts for 45 minutes using Towbin Transfer Buffer (25 m M Tris (Fisher), 192 m M glycine (Fisher), 20% methanol (Fisher). The membranes were blocked wi th Blocking Buffer ( IX TBS, 5% Sk im M i l k Powder (Safeway), 0.05% Tween-20 (Fisher)) or (IX TBS, 1% BSA, 0.05% Tween-20) (for the RC-20 anti-phosphotyrosine antibody 68 (Transduction Laboratories)) for one hour at room temperature wi th gentle agitation. The membrane was then incubated wi th one of the following antibodies: anti-TrkC (C14) [ lug /ml] (Santa Cruz Biotechnology), RC20-Horse Radish Peroxidase conjugated [1:2500], anti-SHC [1:250], anti-PI3-K [1:5000], anti-GRB2 [1:5000], anti-PLCy [1:1000] (Transduction Laboratories) (see Table 6). The membrane was washed three times for 5 minute intervals i n TBS/0.05% Tween-20 and then incubated in the secondary antibody: anti-mouse-horse radish peroxidase conjugated [1:7000] or anti-rabbit-horse radish peroxidase conjugated [1:7000] (Transduction Laboratories) for one hour wi th gentle agitation. The blot was then washed as above prior to visualization by enzymatic chemiluminescence (ECL) (Amersham/Pharmacia) according to the manufacturer's instructions. After E C L detection, the membrane was placed i n between two individual Saran wrap sheets and placed into an X-ray cassette and exposed to a X A R - 5 film for 10 seconds up to 20 minutes. 2.12 G E N E R A T I O N O F G S T - E T V 6 - N T R K 3 F U S I O N P R O T E I N S The BaculoGold Starter Package (Pharmingen) was used for the production and purification of recombinant virus encoding the fusion GST-ETV6-NTRK3 gene and subsequent infection of SF9 cells for the production of large quantities of recombinant protein. A l l materials mentioned were supplied in the kit unless otherwise stated. The recombinant protein was purified by affinity chromatography. Briefly, the lysate (prepared according to the manufacturer's instructions) was applied to a 0.8 x 4 cm Poly-Prep Chromatography C o l u m n (Bio-Rad) equipped w i t h a 2-way Stopcock (Bio-Rad) containing 1 m l of glutathione beads. The beads were 69 T A B L E 6. Summary of various antibodies used for immunoblotting, their source and required concentrations. A N T I B O D Y M A N U F A C T U R E R F A C T O R A n t i - E T V 6 - H L H Dr. Peter Marynen 1:5000 Ant i -TrkC (C14) SCBT 2.0 u g / m l A n t i - T r k C Dr. David Kaplan 1:2500 RC20-HRPO T L 1:2500 Ant i -Rabbi t -HRPO T L 1:7500 A n t i - M o u s e - H R P O T L 1:7500 A n t i - G R B 2 T L 1:5000 Ant i -PI3K T L 1:5000 A n t i - S H C T L 1:250 A n t i - P L C y T L 1:1000 Anti-SH2BfJ Dr. Lyiangyou Rui 1:15000 Abbreviations. SCBT= Santa Cruz Biotechnology, TL= Transduction Laboratories, Inc., HRPO= horse radish peroxidase. 70 washed wi th washing buffer and the protein was eluted wi th 3 m l of elution buffer (both of which are supplied i n the kit). The concentration of protein was determined using the BioRad Protein Determination Assay Ki t according to the manufacturer's instructions. The fusion protein was then aliquoted and frozen at -20°C until use. 2.13 IN VITRO P R O T E I N A S S O C I A T I O N STUDIES To determine whether there is heterodimerization between E T V 6 - N T R K 3 and w i l d type ETV6, both ETV6 and ETV6-NTRK3 were co-translated wi th the T N T T7/T3 Coupled Reticulocyte Lysate System (Promega). The ETV6-NTRK3 construct was contained wi th in pBluescript II K S (kindly provided by Daniel W a i i n the laboratory) and required the T3 promoter for the production of m R N A , while the ETV6 construct was contained wi th in the pQE9 vector (generous gift of Dr. P. Marynen) and requiring the addition of 1 Uni t of R N A Polymerase, E. col i (Boehringer Mannheim) for proper m R N A production. The in vitro translated materials were then immunoprecipitated wi th the N T R K 3 (C-14) antibody (Santa Cruz Biotechnology), electrophoresed on 10% polyacrylamide gels, blotted onto Immobilon-P and immunoblotted wi th the E T V 6 : H L H antibody, as described above. To determine if the E T V 6 - N T R K 3 protein had homodimer iza t ion properties, 5ug of the GST-ETV6-NTRK3 recombinant protein (see above) was mixed wi th 3 5 S-Methionine (Amersham) labeled in vitro translated E T V 6 - N T R K 3 (labeled according to the instructions provided wi th the T N T T7/T3 Coupled 71 Reticulocyte Lysate System), immunoprecipitated wi th glutathione beads, and electrophoresed as described above. The resolving gel was then dried using a Ge l Drier (Labconco) for 1.5 hours prior to exposing the gel to X A R - 5 f i lm for 4 to 24 hours. 2.14 S U B C E L L U L A R L O C A L I Z A T I O N B Y I M M U N O F L U O R E S C E N C E Green fluorescent protein constructs of E T V 6 - N T R K 3 , H L H , and K D were made using the pEGFP-N3 vector (Clonetech). Since the GFP portion was placed o n the 3' end of the various constructs, the stop codon was replaced wi th a glycine codon to allow for the continued translation of GFP. This was accomplished using the Q u i k C h a n g e ™ Site-directed mutagenesis kit (Stratagene) according to the manufacturer's instructions. Primers used to mutate the stop codon (TAG) to a glycine (GGG) for the various constructs were: 5 ' - G A C A T T CTT G G C G G G T G G T G G C T G G T G GTC-3 ' (forward) 5 ' - G A C C A C C A G C C A C C A C C C G C C A A G A A T GTC-3 ' (reverse) NIH3T3 cells were then transfected wi th 2 ug of the purified construct and LipofectAMINE™ (Gibco/BRL) according to the manufacturer's instructions. During transfection, the cells were grown in Opt iMEM™ I Reduced Serum Media (Gibco/BRL). Two days post-transfection, the cells were passaged 1:10 into selective medium (RPMI 1640 wi th L-Glutamine, 10% calf serum, lxPSF, 400 ug Genet ic in (Gibco/BRL)) and grown in this medium for 5 days. The cells were then seeded onto Fisherbrand Superfrost/Plus (Fisher) glass slides and allowed to grow i n regular growth medium (RPMI 1640 wi th L-Glutamine, 10% calf serum and lxPSF) 72 until 50-80% confluent. The slides were then quickly rinsed wi th PBS and fixed i n 4% paraformaldehyde (pH 7.4) (Sigma) in PBS for 10 minutes at room temperature. The slides were then washed in PBS (3 x 5), counterstained wi th D A P I (as above for FISH studies) and briefly rinsed in PBS. One drop of VectaShield™ was then applied and the slide was covered wi th a coverslip and visualized by confocal microscopy using a Bio-Rad MRC-600 Laser Scanning Confocal Microscope. In addition, NIH3T3 cells infected wi th the various constructs inc lud ing NIH3T3 cells transfected wi th vector alone (for use as a negative control) (provided by D. W a i i n our lab) were analyzed by immunofluorescence. Cells were grown o n Fisherbrand Superfrost/Plus (Fisher) glass slides as described above until the confluency reached approximately 80%. The slides were then rinsed quickly w i t h PBS and fixed i n 4% paraformaldehyde (pH 7.4) i n PBS for 10 minutes at r o o m temperature and permeabilized wi th 0.1% TritonX-100 (Sigma) for an additional 10 minutes at room temperature. A n antibody towards the carboxy terminal por t ion of N T R K 3 was used as a primary antibody [1:2500] (a generous gift of Dr. D. Kaplan). The slide was incubated wi th the primary antibody for 1 hour at 37°C, at w h i c h point the slide was run through a series of four 5 minute washes i n I X phosphate-buffered saline (PBS). Rhodamine anti-rabbit (Boehringer Mannheim) was used as the secondary antibody at a concentration of 30 u g / m l and the slide was incubated at 37°C for 30 minutes. The slides were then run through another PBS series, counterstained, mounted and visualized by confocal microscopy as described above. 73 CHAPTER III A NOVEL t(12:15)(pl3: q25) IN CONGENITAL FIBROSARCOMA 3.1 I N T R O D U C T I O N Subtle chromosomal translocations, inversions, and other rearrangements are often missed by conventional cytogenetics [312]. Molecular cytogenetics has overcome these barriers by providing more sensitive techniques offering higher resolution [297]. Fluorescence in situ hybridization (FISH), for example, is a powerful molecular tool, which can be used to determine if a certain chromosomal region has been deleted or rearranged or to finely map a newly discovered gene. Conventional cytogenetics along wi th molecular cytogenetics has, therefore, permitted scientists to investigate genetic alterations i n cancer cells, thus a l lowing for the detection of recurrent genetic abnormalities i n certain tumors [238, 313]. The diagnosis of CFS has historically been very challenging for the pathologist due to its morphologic overlap wi th other childhood spindle cell tumors including ATFS, IFB, myofibromatosis (MFB), and A F B [314, 315]. W e therefore performed cytogenetic analysis on a group of CFS cases at B C C H , as wel l as a series of A T F S , IFB, M F B , and A F B cases i n an attempt to identify recurrent genetic markers of these entities. Of these cases, only the CFS cases displayed a recurring chromosomal abnormality involv ing chromosome 12pl3 and 15q25. To determine if this aberration was a translocation, whole chromosome FISH of a C F S case using a chromosome 12 painting probe was performed. This revealed a 74 portion of chromosome 12 on a smaller acrocentric chromosome. We therefore wanted to elucidate the exact consequences of this rearrangement. 3.2 RESULTS 3.2.1 Cytogenetic Analysis To screen for recurrent genetic markers of CFS, cytogenetic analysis was performed on a series of CFS (n=4), IFB (n=15) and A T F S (n=2) cases diagnosed i n patients either from B C C H or C H L A . CFS Cases 1-3 each demonstrated abnormal metaphases wi th a subtle rearrangement of chromosome 15q25-26. Two of these cases had additional abnormalities of chromosome 12pl3. Cytogenetic analysis of A T F S (n=2) and IFB (n=15) cases showed no rearrangements invo lv ing either chromosome 12 nor 15 (see Fig. 7 (chapter 2)). 3.2.2 Fish Analysis Identifies a Common Derivative Chromosome We next performed fluorescence in situ hybridization (FISH) analysis of 12pl3 and 15q25-26 alterations using the FISH probes depicted i n Fig. 8. Only CFS cases 1 and 2 had tumor metaphases available for FISH. To map each breakpoint, we screened CFS metaphases using a series of non-chimeric 12pl3 and 15q25-26 yeast artificial chromosomes (YACs) to identify those spanning the breakpoints. Initially, we started wi th Y A C s representing the telomeric portions of 12p and 15q. To test for a translocation of 12p material to chromosome 15q, we used either Y A C 890_E_3 or 854_E_6 (from 12p telomere) along wi th an oc-centromeric chromosome 15 probe. Conversely, we tested either Y A C 882_H_8 or 895_H_10 75 12pl3 D12S380E D12S99 D12S89 D12S358 Tel. D12S381E D12S1275 D12S308 Cen. 12q 890E3 854E6 924H12 954G10 738B11 817H1 TEL locus r 40 kb D12S89 1A D12S98 IB 3 4 5 6 7 8 179A6 163E7 148B6 45E12 171H6 54D5 802B4 932F12 895H10 882H8 Cen. D15S151 D15S202 D15S963 D15S120 CHLC.ATA28G05 D15S157 D15S203 15q25-26 F I G U R E 8. Mapping of chromosome 12pl3 and 15q25-26 breakpoints in CFS. Chromosomes 12pl3 and 15q25-26 are schematically represented at the top and bottom, respectively, of the diagram. The positions of the ETV6 (TEL) and KIP1 loci are indicated by boxes. Relative YAC positions are indicated by solid lines; neither chromosome or YAC sizes are to scale. The ETV6 locus is drawn to scale, and exons are indicated by numbers and letters above the solid line. Cosmid positions are indicated by open lines. The position of the NTRK3 (TRKQ locus relative to 802B4 is based on the present study. 76 (from 15q telomere) along wi th an a-centromeric chromosome 12 probe. When the telomeric 12p Y A C 890_E_3 and the chromosome 15 a-centromeric probe were used i n a dual colored metaphase preparation, one of the chromosomes hybridized both probes (see Fig. 9a). This study demonstrated that the rearrangement involving 12p represented a translocation of material to 15qter. O n the other hand, material from 15q25 did not translocate back to 12pter, but rather to another chromosome (later identified as chromosome 1 by D A P I banding) (see Fig. 9b). FISH using the above Y A C s together wi th a series of chromosome-specific probes demonstrated complex three-way translocations for both cases i n which material from distal 12p was translocated to distal 15q and material from distal 15q was translocated to either lq43 or 4ql0, respectively. 3.2.3 Identification of the Breakpoint Region by Y A C M a p p i n g To narrow down the breakpoint, we "walked" along chromosome 12p and 15q using various Y A C clones until we found one which spanned the breakpoint. We found that the 12pl3 Y A C , 817_H_1, and the 15q25-26 Y A C , 802_B_4, were each split i n CFS cells, giving 3 signals as opposed to only 2 i n IFB and other controls. When these Y A C s were used together in dual-coloured FISH, a derivative chromosome hybridizing a fusion signal was detected i n both CFS cases (see Fig. 10a). This derivative was shown to represent a der(15)t(12;15) as it s imultaneously hybridized both an a-centromeric chromosome 15 probe and 817_H_1 by dual-color FISH (data not shown). Similar experiments on 3 ATFS cases and 6 IFB cases 77 F I G U R E 9. Dual-coloured FISH of CFS. A, A metaphase from a CFS case was used i n a dual colored FISH experiment using the 12p telomeric Y A C 890_E_3 (green), and a 15 a-centromere (red). A der(15) chromosome hybridizing both probes shows the translocation of material from 12pter to 15qter. B, Another metaphase preparation from the same CFS case was subjected to dual colored FISH using the 15qter Y A C , 882_H_8 (green), along wi th a 12 a-centromere (red), however , material from 15qter, as seen by the 15qter Y A C , was translocated to another chromosome, later identified as chromosome 1 by computer generated D A P I banding. 78 79 FIGURE 10. FISH analysis for CFS breakpoints. A, Dual-coloured FISH of CFS case 2 metaphase using the 12pl3 breakpoint-spanning Y A C , 817_H_1 (green), and the 15q25-26 breakpoint-spanning Y A C , 802_B_4 (red). The arrowhead shows a yel low fusion signal indicating the der(15)t(12;15)(pl3;'q25-26). B, Dual-coloured FISH of CFS case 1 interphase cell using ETV6 exon 1-containing cosmid 179A6 (green) and ETV6 exon 8-containing cosmid 148B6. The arrowhead shows a fusion signal representing one copy of normal chromosome 12 i n each cell, while the arrows show separate signals indicating disruption of the ETV6 gene. 80 81 revealed normal FISH patterns only with these probes. FISH using 802_B_4 together with an a-centromeric 12 probe failed to detect reciprocal der(12)t(12;15) chromosomes in either case 1 or 2 (data not shown). In each case, FISH identified derivative chromosomes that had not been previously detected cytogenetically (see Table 7). 3.2.4 Micromapping the Breakpoint with Cosmid Probes The 12pl3 breakpoint was narrowed to the telomeric end of 817_H_1 as the distal overlapping 12pl3 Y A C , 924_H_12, was also split in CFS (data not shown). This region contains the ETV6 gene (TEL), a member of the ETS family of transcription factors and has been involved in a varietv of translocations which have given rise J to gene fusions in human leukemias [308, 316-320]. To test for the involvement of ETV6 in CFS, we performed dual-coloured FISH using 802_B_4 together with either cosmid 179A6, which contains ETV6 exon 1, or cosmid 148B6; containing ETV6 exon 8 [300]. This revealed a fusion signal with 179A6 but not with 148B6 (data not shown). Dual-coloured FISH of CFS using 179A6 and 148B6 together revealed one fusion signal as expected for normal chromosome 12, as well as two widely separated signals (see Fig. 10b). This indicates that the breakpoint lies between exon 1 and 8 of ETV6 in CFS, and that the telomeric portion of ETV6 is translocated to chromosome 15q25-26. Further mapping using cosmids 171H6, 45E12, 163E7, and 54D5 (see Fig. 8), localized the ETV6 breakpoint to the region between exons 5 and 7 of ETV6. Fusion signals were not detected by FISH using 82 Table 7. Summary of ETV6 rearrangements in human neoplasia. Gene Fusion Translocation Protein (ETV6-Gcne) - Partner Phenotype Ref. E TV6-MDS/E Vll t(3;12)(p26;pl3) N F D - N D M D S [319] ETV6-JAK2 t(9;12)(p24;pl3) H L H - PTK preB c A L L , aCML [320,321] ETV6-PDGF/3R t(5;12)(q33;pl3) H L H - PTK C M M L [316] ETV6-STL t(6;12)(q23;pl3) H L H , D B D - N D B-cell A L L cell line [322] ETV6-ABL t(9;12)(q34;pl3) H L H - PTK A M L , A L L , aCML [318] ETV6-CBFA2 t(12;21)(pl3;q22) H L H - DBD, cALL [308,323-(AML1) T A D 326] ETV6-MN1 t(12;22)(pl3;q22) D B D - N D A M L and M D S [317] ETV6-CDX2 t(12;13)(pl3;ql2) N F D - N D A M L [327,328] ETV6-ACS2 t(5;12)(q31;pl3) N D R A E B , A M L , A E L [329] ETV6-BTL t(4;12)(qll-ql2;pl3) N D A M L [330] ETV6-ARG t(l;12)(q25;pl3) HLH-SH2, SH3, A M L P T K [331] ETV6-? t(7;12)(q22;pl3) N D M D S [332] ETV6-? t(7;12)(q36;pl3) N D A M L [332] • ETV6-? t(9;12)(qll;pl3) N D A L L [332] ETV6-? t(10;12)(q24;pl3) N D M D S [333] ' ETV6-? t(6;12;17)(p21;pl3;q25) N D A M L [328] ETV6-? t(7;12)(pl4;pl3) N D A M L [328] ETV6-? t(12;17)(pl3;qll) N D B - N H L [328] ETV6-? t(7;12)(pl2;pl3) N D A M L [328] ETV6-? t(X;12)(q28;pl3) N D M D S [334] ETV6-? t(l;12)(q21;pl3) N D A M L [334] ETV6-? t(9;12)(p23-24;pl3) N D A L L [334] ETV6-? t(12;14)(pl3;qll) N D A L L [335] ETV6-? t(7;12)(q35;pl3) N D A L L [335] Abbreviations. AML= acute myelogenous leukemia, CMML= chronic myelomonocytic leukemia, B-NHL= B-cell non-Hodgkin's lymphoma, SH2(3)= Src homology 2(3), MDS= myelodysplastic syndrome, ALL= acute lymphoblastic leukemia, cALL= childhood acute lymphoblastic leukemia, aCML= atypical chronic myeloid leukemia, ? or ND= not determined, NFD= no functional domain, HLH= helix loop helix domain, PTK= protein tyrosine kinase domain, TAD= transactivation domain, DBD= ETS DNA binding domain, RAEB= refractory anemia with excess blasts (with basophilia), AEL= acute eosinophilic leukemia. 83 802_B_4 in conjunction with 148B6, confirming the absence of the der(12) t(12;15)(pl3;q25-26) in tumor cells. 3.3 DISCUSSION In this report we describe for the first time the association of a t(12;15)(pl3;q25-26) translocation with congenital fibrosarcoma. All CFS cases with abnormal karyotypes in this study demonstrated alterations of the distal long arm of chromosome 15 (15q25-26), while 2 also showed subtle alterations involving chromosome 12pl3. The normal karyotype of case 4 likely represents in vitro overgrowth by normal fibroblasts. In contrast, similar abnormalities were not detected in a series of IFB, MFB, or AFB cases. FISH analysis using region-specific YAC probes confirmed the presence of a der(15)t(12;15)(pl3;q25-26) in 2 of 2 CFS cases with metaphases available for FISH studies. Moreover, when we screened with a series of 12pl3 and 15q25-26 specific YACs against CFS metaphases, we found that the same region-specific YAC was split in both CFS cases, i.e. YAC 817_H_1 spanning the chromosome 12pl3 breakpoint and YAC 802_B_4 spanning the 15q25-26 breakpoint. Therefore the breakpoints in each chromosome are contained within identical region-specific YACs in both of the CFS cases tested, providing further evidence that this represents a non-random rearrangement in CFS. We also tested 3 ATFS, 3 IFB, and 3 AFB cases using the identical probes. None of the 9 non-CFS cases demonstrated similar findings, suggesting that the der(15)t(12;15)(pl3;q25-26) is specific for CFS. We are currently accumulating and 84 testing additional cases of CFS, ATFS, IFB, and A F B to more rigorously test this hypothesis. Finer FISH mapping of the breakpoint using cosmid probes localized the breakpoint within the ETV6 gene. This is the first known involvement of the ETV6 gene fusion in solid tumors, as such rearrangements were previously observed only in leukemias [308, 316-320, 333]. The ETV6 (ETS variant gene 6) gene is located on chromosome 12pl3 and was cloned as a result of a t(5; 12)(q33; pl3), fusing it to the platelet-derived growth factor B receptor (PDGFfiR) gene in chronic myelomonocytic leukemia ( C M M L ) [316]. ETV6 (also known as TEL: translocation ETS /eukemia) is a member of the large family of transcription factors known as the E26 transformation-specific (ETS) transcription factors first discovered as part of the E26 avian erythroblastosis virus genome [336]. The ETS family of transcription factors recognize the core motif C / A G G A A / T [337]. The ETV6 gene is approximately 240 kb in size and is made up of 8 exons (with one alternatively spliced exon) which are oriented from telomere to centromere (see Fig. 8) [300]. It encodes a nuclear phosphoprotein with a helix loop helix dimerizing domain (HLHD) encoded within exons 3 and 4 and a D N A binding domain (DBD) encoded within exons 6-8 [300, 338]. The ETS D N A binding domain is known to contain the nuclear localization signal and sequence specific D N A binding activity [339]. The m R N A encoding the ETV6 protein contains two possible initiation sites and results in two species of transcripts. One of the ETV6 proteins migrates around 50 kDa and the other around 57 kDa. Both contain the H L H D and 85 the ETS D B D , but only the full length species encodes a M A P K consensus site (amino acids 20-23), which is phosphorylated in v ivo [338]. This suggests that the regulation of expression of these two proteins are different. Poirel et al. noted that most of the cell lines they examined showed greater expression of the higher molecular weight ETV6 species [338]. Since its discovery i n 1994, the ETV6 gene has been implicated i n a large number of hematopoietic malignancies. Fluorescence in situ hybridization (FISH) analysis of the 12p chromosomal region has shown numerous translocations invo lv ing the ETV6 gene, wi th some cases showing a deletion in addition to the translocation [308, 324, 340-343]. Approximately 50% of the rearrangements invo lv ing the ETV6 gene wi th in these neoplasms have been characterized (see Table 7). Cytogenetic and molecular genetic analysis of these rearrangements has led to the discovery of chimeric gene fusions involv ing specific exons from the ETV6 gene as a result of chromosomal translocations. In C M M L , for example, the helix loop helix dimerization domain (HLH) from the ETV6 gene is fused to the protein tyrosine kinase domain (PTK) from the PDGF PR gene [316]. The H L H domain acts as a protein-protein dimerizing domain and constitutively activates the P T K domain by ligand-independent dimerization. The t(12;22)(pl3;qll) i n myeloproliferative disorders (MDS) results in the fusion of nearly the entire M N 1 protein to the ETS D N A binding domain from the ETV6 gene [317]. In this case, it is thought that oncogenesis is due to the altered transcriptional activity of the ETV6 gene. The ETV6-CDX2 and the ETV6-STL gene fusions i n A M L and a B-cell A L L cell-line, respectively, only contain the first two exons from the ETV6 gene fused to 86 unknown 3' sequences from the partner gene [322, 327]. There are no k n o w n functional domains in the first two exons of ETV6 and it is thought that the STL and CDX2 genes are under transcriptional control from the ETV6 promoter. The wel l characterized ETV6-CBFA2 (TEL-AML1) is usually accompanied by a deletion of the residual ETV6 gene on the normal chromosome [308]. It is possible that the normal ETV6 protein dimerizes with the ETV6-CBFA2 protein interfering wi th its oncogenic potential or that ETV6-CBFA2 functions as a dominant negative inhibitor of normal ETV6 function. Protein analysis led to the discovery of up to 5 different species due to alternative translational initiation and postranslational modification [338, 344]. Other ETS family members are known to be phosphorylated and it has been shown that these postranslational modifications play an important role i n the function of the protein [345-348]. The mechanism by which a chromosome participates i n a translocation is still unclear. In a recent study, however, the ETV6-CBFA2 breakpoint was cloned at the genomic level and almost all breakpoints i n the ETV6 gene were found near a pur ine /pyr imidine rich sequence wi th in intron 5 of E T V 6 (most breakpoints invo lv ing ETV6 occur i n intron 5) [349]. This suggests that this region may be susceptible to D N A breakage and re-ligation, including translocations. Figure 11 summarizes the known breakpoints wi th in the ETV6 gene as we l l as the corresponding fusion genes. ETV6 expression is seen in almost all tissues during development and in the adult. ETV6 has recently been shown to be important i n angiogenesis and its transcription is downregulated by an angiogenic growth factor, vascular endothelic 87 F I G U R E 11. Schematic representation of the c D N A for ETV6 as wel l as some of the more common rearrangements involving the ETV6 gene. The ETV6 gene is located on chromosome 12pl3 and consists of 8 exons. The top panel shows the coding sequence (cDNA) of ETV6 along wi th the nucleotide position of each exon (exons are separated by a vertical line wi th the nucleotide position shown below). A number of translocation breakpoints have been found i n the E T V 6 gene resulting i n its i n frame fusion to other genes such as PDGFfiR (in C M M L ) , ABL (in A M L , A L L , and aCML) , AML1 (in cALL) and MN1 (in A M L and MDS) . The fusion proteins generated juxtapose either the H L H dimerization domain from exons 3 and 4 of ETV6 to the protein tyrosine kinase (PTK) domain of PDGFBR, A B L and the runt and transactivation domain (TAD) of the A M L 1 gene or the ETS D N A binding domain (ETS domain) from exons 6, 7 and 8 to the M N 1 region of the M N 1 gene (shown as protein structures i n the bottom 5 panels). The location of some of the more common breakpoints found in various hematologic malignancies are shown i n the c D N A sequence (arrows) (see Table 7 for more information). 89 growth factor (VEGF) [350]. Angiogenesis refers to the process by which new vascular elements emerge from pre-existing vasculature. This group, however, found that only the lower molecular weight species was expressed i n h u m a n umbilical vein endothelial cells (HUVE) . In addition, they were able to demonstrate the loss of one of the m R N A ETV6 species upon treatment of the cells w i th V E G F . The precise role of ETV6 in blood vessel formation and maturation is presently under investigation. It has been suggested that the ETV6 protein may act as a transcriptional repressor (Golub et al., unpublished data). Since V E G F promotes vascular growth and inhibits ETV6 transcription, it is possible that the transcription of genes inhibited by ETV6, are involved i n blood vessel formation and maturation. ETV6 knockout mice showed embryonic lethality and failed to maintain yolk sac blood vessel formation. Furthermore, studies have shown ETV6 to be necessary for bone marrow hematopoiesis but not essential for liver hematopoiesis [351]. We observed complex three-way translocations i n both CFS cases studied by FISH (see Table 4 (chapter 2)), suggesting that these alterations might be favored i n CFS. Three-way translocations generating typical EWS-FLI1 gene fusions are described i n Ewing tumors [352, 353], as are complex translocations i n ETV6-CBFA2 positive leukemia cell lines [354]. Moreover, ETV6-CBFA2 positive cases wi th the t(12;21) show a high frequency of variant derivative 21 chromosomes and absence of CBFA2-ETV6 expression, while other ETV6-CBFA2 positive cases commonly delete the normal ETV6 allele [355]. This suggests that reciprocal fusion products or normal ETV6 itself may inhibit functional ETV6 chimeric oncoproteins. Further 90 rearrangements of the der(12)t(12;15) in CFS may therefore be selected for as a mechanism of eliminating expression of an inhibitory N T R K 3 - E T V 6 molecule. In summary, cytogenetic analysis coupled wi th detailed FISH mapping of two CFS cases wi th available metaphases resulted i n the identification of a complex three-way rearrangements for both cases, interpreted as t(l;12;15)(q44;pl3;q25-26) and t(4;12;15)(ql0;pl3;q25-26), respectively [356]. We now report an apparently n o n -random association between the presence of a der(15)t(12;15)(pl3;q25-26) chromosome and the diagnosis of CFS. This rearrangement was not detected i n cases of A T F S , IFB, M F B nor in A F B , and therefore may be useful in the differentiation of these entities from CFS. Furthermore, our findings provide additional evidence that CFS is biologically distinct from fibrosarcomas occurring i n older children. These data suggest that CFS may be characterized by an ETV6 gene fusion; the identification of the partner gene is the topic of the next chapter. 91 CHAPTER IV CLONING AND CHARACTERIZATION OF THE t(12:15) IN CFS 4.1 INTRODUCTION Translocations leading to specific gene fusions are a common event in cancer [238]. Molecular analysis of these gene fusions has led to the discovery of chimeric oncogenes. Most of these chimeras appear to act as aberrant transcription factors, l ikely functioning i n transformation by dysregulating, ablating or introducing new gene expression profiles wi th in the cell [238]. Bone and soft tissue sarcomas of childhood have provided an abundant resource for studying various types of gene fusions [238, 357]. The detection of these fusion transcripts i n tumor specimens, specific for given tumor subtypes, has become an extremely useful diagnostic tool for the pathologist. Chi ldhood sarcomas tend to be extremely primit ive i n appearance and therefore very difficult to differentiate from each other morphologically [5]. Accurate diagnosis of two morphologically similar, yet distinct, tumors is extremely important since initial diagnosis often determines which treatment protocol a patient is enrolled in. Accurate pathologic classification is, therefore, a critical prognostic factor for these patients. The identification of the breakpoint wi th in the ETV6 gene led us to believe there may be a gene fusion involv ing this gene i n CFS. The ETV6 gene has only been rearranged i n leukemias to date (see Table 7 in Chapter 3), but this does not rule out its potential involvement i n a solid tumor as well . ERG and FLU are two other ETS family members found to be fused wi th EWS or TLS/FUS as a result of 92 chromosome translocations i n human solid tumors and leukemias [254, 258, 259, 268]. We therefore hypothesized that the t(12;15) rearrangement may s imilar ly give rise to an oncogenic gene fusion in CFS. Because the der(15)t(12;15)(pl3;q25-26) was common to both CFS cases analyzed by molecular cytogenetics, we further reasoned that a functional gene fusion, if present, might be expected to be expressed from this derivative chromosome and would involve the ETV6 gene. 4.2 R E S U L T S 4.2.1 Clon ing the t(12;15) Breakpoint i n CFS The 8 exon ETV6 locus is oriented in a telomere to centromere direction, wi th exons 3 and 4 encoding a helix-loop-helix (HLH) protein dimerization d o m a i n and exons 6-8 contributing to the ETV6 ETS D N A binding domain [300, 316]. Because the ETV6 5 ' - H L H region is fused to other partner genes i n h u m a n leukemias [308, 316-320], we performed 3'-rapid amplification of c D N A ends (RACE) using ETV6 exon 5 primers 541 and 701 along wi th a poly-dT-linked primer (see Chapter 2). R A C E using primer 701 generated similar ~1.5 kb fragments i n 3/3 CFS cases but not in 3 ATFS, 3 IFB, or other control cases. Cloning and sequencing of these fragments revealed that 333 bp of ETV6 sequence were fused in-frame to 1115 bp of unknown sequence. Database analysis revealed this to represent the terminal 1115 bp of the human NTRK3 gene encoding the neurotrophin-3 surface receptor [358-360] (see Fig. 12a). The fusion point in all 3 cases was after nt 1033 of ETV6, 93 ETV6 (nt 1033) ««- > N T R K 3 (nt 1601) tec ccg cct gaa gag cac gec atg ccc att ggg aga ata gca gat gtg cag cac art aag agg aga gac ate gtg ctg aag cga S P P E E H A M P I G R I A D V Q H I K R R D I V L K R B M 1 2 3 4 5 6 7 8 9 10 872 bp 603 bp 310 bp F I G U R E 12. ETV6-NTRK3 gene fusions in CFS. A, Junctional nucleotide (small case) and single letter amino acid sequence of PCR fragments generated by 3 ' -RACE of c D N A from CFS cases 1-3, using sense primers 541 or 701 from ETV6 exon 5 i n combination wi th a poly-dT primer. Sequence analysis revealed an in-frame fusion after ETV6 nt 1033 wi th nt 1601-2715 of the human NTRK3 gene. B, RT-PCR using ETV6 primer 541 and primer NTRK3-2 demonstrates a 731 bp fragment in CFS (lanes 3-5) but not in normal fibroblasts (lanes 1 and 2), IFB (lanes 6-8), A T F S (lane 9), or the Jurkat leukemia cell line (lane 10). 94 which is the last nt of ETV6 exon 5 [300]. The ETV6 breakpoints therefore appear to be localized to intron 5. The NTRK3 portion originated at NTRK3 nt 1601 and included the entire protein tyrosine kinase (PTK) domain and remaining C-terminus of N T R K 3 [358-360]. 4.2.2 Reciprocal Fusions were not Detected 5 ' -RACE wi th 5'-NTRK3 and 3 ' -RACE wi th 3'-ETV6 ETS region primers failed to detect fusion transcripts that might be encoded by functional der(12)t(l;12) or der(l)t(l;15) chromosomes, thus rul ing out additional ETV6 or NTRK3 gene fusions involving the 3'-ETS region and the 5 ' -NTRK3 extracellular ligand binding domain, respectively (data not shown). 4.2.3 R T - P C R Analysis of CFS and Other Morphological ly Simi lar Tumors RT-PCR using ETV6 primer 541 and the TRKC-2 primer from the NTRK3 P T K region amplified the expected 731 bp ETV6-NTRK3 fusion transcripts i n all 3 CFS cases, while ATFS , IFB, and other controls were negative (see Fig. 12b). R T -P C R using ETV6 primer 114 located 5' to the H L H region, together wi th T R K C - 2 generated the expected 1158 bp product only in CFS samples, and sequencing of this product confirmed the presence of the entire ETV6 H L H region i n fusion transcripts (data not shown). 95 4.2.4 Northern and Southern Blot Analysis Northern blot analysis using a full length c D N A probe for ETV6 hybridized to three transcripts wi th sizes of 6.2, 4.3, and 2.4 kb and was found to be ubiquitously expressed i n CFS, ATFS, IFB, and other control cells. W h e n we used a NTRK3 c D N A or a partial c D N A probe including the NTRK3 P T K motif, only the 4.3 kb transcript i n CFS cells hybridized (see Fig. 13). Southern blot analysis of genomic D N A isolated from CFS primary tumor tissue showed the disruption of both ETV6 and NTRK3 genes when hybridized with the ETV6 5/6 and NTRK3-PTK probes (see Fig. 14). The multiple bands seen i n the CFS lane are due to the chromosomal rearrangement involving the ETV6 gene. 4.3 D I S C U S S I O N By cloning the chromosome breakpoints we show that the rearrangement fuses the ETV6 (TEL) gene from 12pl3 wi th the 15q25 NTRK3 neurotrophin-3 receptor gene (TRKC). Analysis of m R N A revealed the expression of ETV6-NTRK3 chimeric transcripts in 3 of 3 CFS tumors. These were not detected i n A T F S or infantile fibromatosis (IFB), a histologically similar but benign fibroblastic proliferation occurring in the same age group as CFS. The NTRK3 gene (also known as TRKC and neurotrophin 3 receptor) is the third member of the T R K family of tyrosine kinase receptors. Lamballe et al. found the NTRK3 gene to contain up to 97 and 98% homology to the rat and porcine Trk sequences, respectively [358, 359]. The human NTRK3 gene was cloned and 96 TEL TRKC p-Actin FIGURE 13. Northern analysis of CFS cases. Blots were probed sequentially w i t h full length ETV6 c D N A (top panel), a 3' NTRK3 partial c D N A probe encoding the P T K domain (NTRK3-PTK; middle panel), and a p-actin c D N A probe (bottom panel) to test for equal loading. The ETV6 c D N A probe detected previously described 6.2, 4.3, and 2.4 kb transcripts in multiple samples (9), including CFS, while NTRK3-PTK detected a 4.3 kb species only in CFS cases (lanes 3 and 4; arrow). Identical results were obtained using full length NTRK3 c D N A (data not shown). Lanes 1 and 2, normal fibroblasts; 5-7, three IFB cases; 8 and 9, leukemia cell lines K562 and fnrkat, respectively; 10, neuroblastoma cell line SAN2; 11, rhabdomyosarcoma cell line Birch. 97 FIGURE 14. Southern analysis of CFS cases. Hindlll digests probed wi th ETV6-5/6 (left panel) and NTRK3-PTK (right panel) revealed rearranged bands in CFS case 3 and 1 (lanes 1 and 2, respectively), indicated by arrows. Germline bands only were observed in IFB (lane 3), SAN2 (lane 4), Birch (lane 5), and normal fibroblasts (lane 6). 98 mapped to chromosome 15q25 by McGregor et al. [360]. Expression of NTRK3 i n adult tissues is predominantly restricted to the central and peripheral nervous systems, but detection of transcripts i n non-neural cells including intestinal glandular cells, adrenal medullary cells, ovarian granulosa and thecal cells, kidney tubular cells, as wel l as skeletal muscle, lung, testis, prostate, and heart has also been reported [361, 362]. Fetal tissues show strong expression in brain, kidney, lung , and heart tissues; however, the role of N T R K 3 i n non-neural tissues is not presently known. Studies of NT-3 knockout mice showed multiple heart defects as wel l as the ablation of proliferation and survival of neural crest cells suggesting an important role for NT-3 mediated pathways i n cardiogenesis and neurogenesis [363]. Addi t ional studies have shown that mechanical injury to the hippocampus results i n the increased expression of N T R K 3 and inducible transcription factors (ITFs) such as Fos, c-Jun, and Krox-24 [364, 365]. The results of this study suggest that the expression of N T R K 3 may be controlled in part by ITFs. The human NTRK3 gene is organized wi th its 20 exons from telomere to centromere [366]. The extracellular domain consists of a signal peptide, 2 cysteine clusters, a leucine r ich motif, and 2 Ig-like domains. These structures are encoded by exons 1 through 8. The transmembrane domain is encoded by exons 11-13 and the protein tyrosine kinase domain (intracellular domain) by exons 13-18. Exons 9 and 16 encode the alternative inserts found i n the extracellular and kinase domains and exons 13b and 14b encode the terminal domain of the truncated isoform [361]. The truncated and full length isoforms have a carboxy terminal tail and a 3' U T R which are encoded by exons 14b and 18, respectively. The isoform without any 99 inserts represents the active tyrosine kinase receptor, while the isoforms which are truncated or have inserts wi th in the kinase domain are inactive tyrosine kinase receptors [367-369]. The function of the inserts is not yet known, however, there is evidence that the truncated N T R K 3 receptors are important i n the modulatory development of certain neural cell populations [370]. Palko et al. found that overexpression of a truncated form of the N T R K 3 receptor lacking the tyrosine kinase domain, resulted i n a phenotype which closely resembled NT-3 deficient cells, suggesting that the truncated isoforms serve a role as NT-3 sequestering molecules preventing the activation of the functional N T R K 3 isoform [370]. Our studies showed no evidence of truncated forms of N T R K 3 nor of inserts i n the N T R K 3 portion of E T V 6 - N T R K 3 . Studies of knockout mice defective for the NTRK3 gene displayed numerous anomalies i n their neuroanatomical development [371, 372]. There was a 20% loss of neural cells from dorsal root ganglia, 100% loss of Ia muscle afferents from the spinal cord, 50% loss of myelinated axons from the spinal cord/dorsal roots and 30% loss of various fibers from the spinal cord/ventral roots. In addition, these mice displayed highly unusual behavioral characteristics mainly invo lv ing the positioning of their limbs in relation to their trunk. This is suggestive of a faulty development i n proprioception and can be mostly attributed to the faulty development of the nervous system. Finally, these mice have a relatively short lifespan (most die by the third week after birth (P21)) suggesting that these mice may have additional neural defects. U p o n NT-3 activation, N T R K 3 molecules dimerize wi th each other w h i c h 100 leads to the autophosphorylation of tyrosine moieties wi th in the intracellular domain [372-374]. The phosphotyrosines flanking the tyrosine kinase domain act as anchors for downstream signaling molecules including SNT, S H C , P L C - y l , r A P S , and SH2BB. These molecules, in turn, activate other molecules ultimately leading to the activation or suppression of certain genes wi th in the nucleus. These signaling pathways are blocked by the binding of monoamine activated a-2 Macroglobulin (MA-oc 2 M) to N T R K 1 , N T R K 2 or N T R K 3 [375]. M A - a 2 M is a ubiquitously expressed glycoprotein and may be involved i n neuronal regulation and certain neuropathologic conditions. NTRK1 and NTRK3 have been the only two members of the N T R K family found to be involved i n a variety of human cancers. N T R K 1 is activated and has been implicated as a causative factor in human prostate [376, 377], breast [378], thyroid [379-381], and colon cancer [382-385]. In colon cancer, for example, the N T R K 1 molecule is activated due to a chromosomal rearrangement fusing the coiled coil domain from the tropomyosin 3 (TPM3) gene from chromosome lq22 to the N T R K 1 kinase domain [385]. Papillary thyroid carcinomas, however, have been found to contain rearrangements of NTRK1 resulting in its fusion of the kinase domains to either TPR on chromosome lq25 [381, 386], or TPM3 [387]. Alternatively, expression of either NTRK1 or NTRK3 is a marker of favorable prognosis marker in neuroblastomas and medulloblastomas (reviewed i n [388]). The predicted E T V 6 - N T R K 3 chimeric protein consists of the ETV6 H L H protein dimerization domain fused to the P T K domain of the N T R K 3 nerve 101 growth factor receptor (see Fig. 15). The ETV6-NTRK3 fusion is similar to the T P R -N T R K 1 fusion where the coiled coil protein dimerization domain from TPR is fused to the protein tyrosine kinase domain of N T R K 1 [381]. In human leukemias, the ETV6 H L H domain is fused to the P T K domains of PDGFf} receptor, A B L , and JAK2 [316, 318-320]. Resulting chimeric proteins have constitutively active P T K domains that stimulate corresponding PTK-mediated signal transduction pathways i n leukemic cells [318, 321]. Receptor PTKs, including N T R K 3 , require l igand-mediated cell surface oligomerization leading to autophosphorylation of cytoplasmic tyrosine residues and consequent kinase activation [37]. In E T V 6 -N T R K 3 fusions, the N T R K 3 ligand binding domain is replaced by the ETV6 H L H domain, potentially resulting i n HLH-mediated dimerization and l igand-independent activation of the N T R K 3 protein tyrosine kinase domain. Also , since ETV6 is widely expressed i n mammalian tissues while NTRK3 expression is primarily restricted to neuronal cells, an additional role of ETV6 may be to provide an active promoter driving ectopic expression of NTRK3- induced signal transduction. In fact, while ETV6 was expressed i n normal fibroblasts, there was no evidence of NTRK3 expression in these cells (see Fig. 13). Our studies indicate that a chimeric P T K is expressed i n CFS that may contribute to oncogenesis by dysregulation of N T R K 3 signal transduction pathways. We have identified previously unrecognized rearrangements giving rise to CFS-specific ETV6-NTRK3 gene fusions. This is the first known involvement of the N T R K receptor family i n human oncogenesis. It is also the first ETV6 gene fusion described i n solid tumors, as such rearrangements were previously observed only i n leukemias. These data 102 n top TO re re TO TO 5 53 £ •xi S TO TO w-T c -~ > ^ h m g m w .a o cn .S o in p ^ 103 therefore provide a new example of a fusion gene partner implicated i n both leukemogenesis and solid tumor formation. Our data support the notion that CFS is a biologically distinct entity, and ETV6-NTRK3 detection provides a diagnostic screening tool potentially useful i n the clinical evaluation of children wi th spindle cell tumors. 104 CHAPTER V ETV6-NTRK3 GENE FUSIONS AND TRISOMY 11 ESTABLISH A HISTOGENETIC LINK BETWEEN MESOBLASTIC NEPHROMA AND CONGENITAL FIBROSARCOMA 5.1 I N T R O D U C T I O N Congenital mesoblastic nephroma ( C M N ) is a renal spindle cell tumor that occurs predominantly in newborns and very young infants, wi th most cases being diagnosed before three months of age [224, 389]. This tumor is subdivided into so-called classical and cellular forms based on histologic features. Classical C M N consists of a moderately cellular proliferation of loosely arranged bland fibroblastic cells, while cellular (or atypical) C M N is characterized by high cellularity, numerous mitoses, and cellular pleomorphism [224]. Mixed forms are also k n o w n to occur, and it has been suggested that cellular C M N may arise from classical C M N . Despite the infiltrative growth patterns seen in all forms of C M N , these tumors are generally thought to have an excellent prognosis wi th surgery alone being curative [390]. However, there are several reports of local recurrences and metastatic spread, and these are almost exclusively associated wi th the cellular variant [391, 392]. It therefore remains to be determined whether cellular morphology is predictive of a more aggressive course. The histogenesis of C M N is unknown. Several lines of evidence point to a derivation from primitive nephrogenic mesenchyme and a possible relationship to 105 other pediatric kidney tumors [393]. A link to W i l m s ' tumor (WT) has been proposed based on similar patterns of loss of heterozygosity (LOH) i n v o l v i n g chromosome l l p l 3 - 1 5 i n W T and C M N [394, 395]. However, other studies failed to detect L O H of this region in C M N [396]. Moreover, the observed pattern i n C M N of abundant expression of insulin-like growth factor II (IGFII) coupled wi th lack of Wi lms ' tumor gene 1 (WT1) expression is distinct from the documented expression of both transcripts in W T [396, 397]. In fact, the pattern of expression of these genes i n C M N is reminiscent of that observed i n clear cell sarcoma of the kidney (CCSK), a highly aggressive pediatric renal neoplasm [398] and it has been proposed that C C S K may be the malignant counterpart of C M N [224]. Cytogenetic analysis of classical and cellular C M N has led to an alternate hypothesis for the derivation of these tumors. The most consistent non-random karyotypic finding in C M N is trisomy 11, wi th additional copies of chromosomes 8, 10, 17, and 20 being less commonly reported [34, 395, 399-401]. Moreover, trisomy 11 appears to correlate wi th the cellular phenotype [34, 400, 401], whereas classical C M N cases are only rarely associated wi th this finding [34, 400]. This is h ighly reminiscent of the pattern of trisomy 11 and other trisomies i n congenital fibrosarcoma (CFS), a malignant tumor of fibroblasts that occurs i n patients aged 2 years or younger that has striking morphologic similarity to cellular C M N [402]. CFS is characterized by local recurrence but, like cellular C M N , has an excellent prognosis and a very low metastatic rate [402]. Its benign counterpart, infantile fibromatosis (IFB), occurs i n the same age group as CFS but, like classical C M N , lacks trisomy 11 [6]. This, together wi th ultrastructural similarities, has led to the 106 proposal that classical and cellular C M N are the renal counterparts of IFB and CFS, respectively [403]. As described in chapters 3 and 4, we have recently identified a n o v e l t(12;15)(pl3;q25) translocation in CFS, and have shown that this rearrangement fuses the ETV6 (TEL) gene from 12pl3 wi th the 15q25 neurotrophin-3 receptor gene, NTRK3 (TRKC) [356]. ETV6-NTRK3 fusion transcripts encoding the helix-loop-helix (HLH) protein dimerization domain of ETV6 fused to the protein tyrosine kinase (PTK) domain of N T R K 3 were identified in CFS tumors but not i n adult-type fibrosarcoma or IFB. The CFS cases studied also showed trisomy 11 [404]. Several previous reports have described alterations of chromosomes 12 and/or 15 i n C M N [399, 401, 405], including a t(12;15)(pl3;q25) [401]. We therefore screened a series of classical and cellular C M N cases for both ETV6-NTRK3 gene fusions and trisomy 11. We found that cellular C M N was strongly correlated wi th ETV6-NTRK3 expression and trisomy 11, but that classical C M N was negative for both findings. These results suggest that cellular C M N is distinct from classical C M N and is histogenetically related to CFS. 5.2 R E S U L T S 5.2.1 Cl in ica l History and Cytogenetics The clinical features of the 15 C M N cases analyzed i n this study are summarized i n Table 8. These included 9 cellular C M N s , 2 mixed C M N s , and 4 classical C M N s i n 9 males and 6 females. The diagnosis for each case was based o n 107 Table 8. Clinical characteristics and molecular genetic findings i n C M N cases. Case C M N Subtype Age (months) Sex ETV6-NTRK3 (RT-PCR) Trisomy 11 (FISH) 1 cellular 2 F + + 2 cellular 16 M + + 3 cellular 1 F + + 4 cellular 2.5 M + + 5 cellular 14 days M + + 6 cellular 1 F + + 7 cellular 2 F + + 8 cellular 1 M + N D 9 cellular 9 F - -10 mixed 5 days M + + 11 mixed 7 days M + + 12 classical 36 F - -13 classical 1 day M - -14 classical 2 days M - -15 classical 2 F - -fl+, present; -, absent; N D , not determined 108 standard pathologic criteria [224] and was confirmed by N W T S G or C H T N pathologic review. A l l cases were in patients 3 years of age or younger, and the majority were i n patients younger than 3 months as expected for C M N [224]. Two of the cellular C M N cases from this study (case 1 and 2 i n Table 8) had previous cytogenetic analysis performed on tumor metaphases. Case 1 was previously published as having a t(12;15)(pl3;q25) i n addition to trisomy 11 and other trisomies [401]. Case 2 had a similar karyotype, wi th a t(12;15)(pl3;q24.1), trisomy 11, and other trisomies (data not shown). These findings, coupled w i t h known morphologic similarities between cellular C M N and CFS, prompted us to screen the cohort of C M N cases for CFS-associated ETV6-NTRK3 gene fusions [404]. 5.2.2 R T - P C R Analysis of C M N Cases We performed RT-PCR to detect ETV6-NTRK3 fusion transcripts using a previously described assay [356]. As shown i n Fig. 16, 8/9 cellular C M N s and 2/2 mixed C M N s were positive for the expected 731 bp ETV6-NTRK3 fusion transcript, while all 4 classical C M N s were negative. Sequencing of the amplification products demonstrated identical fusion sequences as those described for CFS ([356]; data not shown). We also screened primary tumor tissue from 12 cases of C C S K as wel l as one case of predominantly spindle cell monomorphic W T i n a 16 month chi ld . These cases were uniformly negative for identical ETV6-NTRK3 fusion transcripts (data not shown). 109 £-1 u 2 « •5? v 1 N o fi QJ ro g LO ^ eft OJ g I • 1"( 1—I >H S ' P H cn feu .£ .* 3 g C N QJ c4 uPH IH C N c ra C J < N .g OJ _ fi fi O cn CN m m o m oo u QJ -*-» 0) CO S* B H E-H I vo W VO 01 JH 3 60 U VO i ro 0 u ON QJ > CO g ro bO QJ fi fi QJ 6 ro 110 5.2.3 Northern Blot Analys is To confirm our results, we performed Northern blot analysis of a cellular and classical C M N using ETV6 and NTRK3 probes. Both samples demonstrated 6.2-, 4.3-, and 2.4-kb ETV6 transcripts (data not shown), as expected for this ubiquitously expressed gene [316]. However, only the cellular C M N expressed a 4.3-kb transcript also hybridizing either a full length NTRK3 c D N A probe or a probe for the NTRK3 P T K region (see Fig. 17), as is observed for CFS [356]. These data indicate that cellular C M N , but not classical C M N , C C S K , or W T , expresses identical ETV6-NTRK3 fusion transcripts as those detected i n CFS. 5.2.4 F I S H Analysis We next wanted to determine whether there was a correlation i n C M N between the expression of the ETV6-NTRK3 gene fusion and trisomy 11 as we had previously observed for CFS. We therefore prepared touch preparations of each C M N case and probed them wi th an a-centromeric chromosome 11 probe. A s shown i n a representative example in Fig. 18, trisomy for chromosome 11 was observed i n every case which expressed ETV6-NTRK3 fusion transcripts. Tr isomy 11 was never observed i n C M N cases lacking this gene fusion (see Table 8), including the cellular C M N case which was RT-PCR negative. 5.3 DISCUSSION Congenital mesoblastic nephroma (CMN) is a renal, spindle cell tumor of infancy which is subdivided into a cellular, mixed, and classical forms based on I l l 28S - | 18S - • (3-Actin Figure 17. Northern analysis of C M N cases. Blots were probed wi th a 3' NTRK3 partial c D N A probe encoding the P T K domain (NTRK3-PTK; top panel), and a (3-actin c D N A probe (bottom panel) to test for equal loading. The ETV6 c D N A probe detected previously described 6.2, 4.3, and 2.4 kb transcripts in multiple samples (data not shown), including CFS and C M N cases, while NTRK3-PTK detected a 4.3 kb species only in CFS and cellular C M N cases (lanes 1 and 3; arrow). Lane 2, classical C M N ; 4, Ewing's TC71; 5, SAN-2; 6, human brain R N A . 112 Figure 18. FISH analysis for trisomy 11. The presence of an extra copy of chromosome 11 was determined by FISH analysis on touch preparations made from primary tissue specimens. A n a-centromeric 11 probe was used to probe touch preparations of all C M N cases. Shown above is a cellular C M N case w i t h three copies of chromosome 11. 113 mitotic activity and degree of cellularity. Histologic and cytogenetic evidence has suggested that C M N and CFS are histogenetically related. This prompted us to screen C M N cases for the t(12;15)(pl3;q25)-associated ETV6-NTRK3 gene fusion previously reported i n CFS. Two of two mixed and 8 of 9 cellular C M N s were positive for the ETV6-NTRK3 gene fusion while all 4 classical C M N cases tested were negative for this alteration. We also found a striking correlation between trisomy 11 and fusion gene expression, wi th all C M N cases harboring the ETV6-NTRK3 gene fusion displaying an extra copy of chromosome 11 by FISH. This included two cases (cases 1 and 2, Table 8) wi th cytogenetically proven extra copies of chromosome 11. Our findings strongly support the notion that cellular C M N and CFS are histogenetically related. The data do not support a relationship wi th C C S K or W T as has been previously proposed [224,394,395]. Molecular testing for ETV6-NTRK3 gene fusions therefore provides a potential modality for the diagnosis of cellular C M N . Our data also suggest that classical and cellular C M N are genetically distinct entities, as no cases wi th classical morphology displayed either ETV6-NTRK3 gene fusions or trisomy 11. It is tempting to speculate, as have others [403], that cellular and classical C M N represent the renal counterparts of CFS and IFB, respectively, particularly given the overlapping age ranges of these lesions. The fact that both mixed C M N cases tested in this study expressed ETV6-NTRK3 fusion transcripts lends support to the intriguing possibility that the mixed form represents a transitional stage i n which distinct regions wi th in classical C M N have acquired the 114 chromosomal aberrations found i n cellular C M N . Tissue microdissection experiments may be useful to address this question. It remains unclear as to how ETV6-NTRK3 expression confers a proliferative advantage to tumor cells. The gene fusion links the H L H dimerization domain of the ETV6 ETS family transcription factor to the P T K domain of N T R K 3 [356]. NTRK3 is a member of the N T R K family of receptor PTKs and binds neurotrophin-3 (NT-3) wi th high affinity [358, 359]. NT-3 binding induces receptor d imer iza t ion and autophosphorylation of P T K tyrosine residues. These residues serve as anchors for downstream signal transduction molecules such as S H C , phospholipase C y l (PLCyl), and PI-3K [358, 359]. We have hypothesized that the ETV6 H L H domain induces ligand-independent dimerization and constitutive activation of N T R K 3 signaling. The finding that all fusion positive C M N and CFS cases demonstrate trisomy 11 suggests that this alteration also contributes to tumorigenesis. The IGFII gene, a paternally expressed member of a cluster of imprinted genes localized to chromosome H p l 5 . 5 , encodes an insulin-like growth factor expressed i n certain human tumors and overgrowth syndromes [406]. It is therefore possible that some form of complementarity or synergism occurs between E T V 6 - N T R K 3 and IGFII signaling pathways that is required for CFS or C M N tumor cells to proliferate, as has been observed for other oncogenes [407]. Further studies w i l l be necessary to elucidate the comparative roles of these alterations i n oncogenesis and to determine if this relationship is unique to tumors of very young children. 115 CHAPTER VI M O L E C U L A R STUDIES OF THE E T V 6 - N T R K 3 FUSION PROTEIN 6.1 I N T R O D U C T I O N Tyrosine kinase receptors are activated through a process k n o w n as l igand mediated receptor dimerization. Briefly, after a ligand, such as a growth factor, has attached itself to the extracellular ligand binding domain of a tyrosine kinase receptor, the receptor undergoes a conformational change favoring its interaction wi th another similar receptor [37]. This interaction induces the cross phosphorylation of certain tyrosine moieties wi th in the intracellular domain, thus activating the receptor-dimer complex leading to further interactions w i t h cytoplasmic substrate proteins. The deactivation of the receptor-dimer complex by specific protein tyrosine phosphatases (PTPs) is equally important i n the regulation of signal transduction [408]. When a cell acquires a chromosomal translocation, a rearrangement may be generated which produces a fusion gene (discussed in the previous chapters). This fusion gene consists of part of one gene fused to a part of another gene. In the case of ETV6 gene fusions, we saw how a translocation can result i n the fusion of the H L H domain from ETV6 to the protein tyrosine kinase domain from either PDGF0R, A B L or JAK2 [316, 318-320, 409]. These fusion proteins lack the extracellular ligand binding (regulatory) domains for the tyrosine kinase receptors; instead, these domains are replaced by the H L H domain encoded by the ETV6 gene. 116 The H L H domain is known to induce dimerization, and therefore, acts to ablate the necessity for ligand induced activation resulting in the constitutive activation of the tyrosine kinase domain [339, 410, 411]. The downstream pathways affected by the activated tyrosine kinase domain are, therefore, constantly activated. Other studies have shown fusion genes invo lv ing the ETV6 gene are able to heterodimerize wi th normal ETV6 as wel l as the ability to homodimerize g iv ing rise to the concept that the introduction of constitutively activated signaling pathways or the disruption of normal ETV6 function, or a combination of both may be the source of oncogenesis [321,412,413]. We therefore wanted to determine the specific characteristics of the E T V 6 -NTRK3 gene product, including downstream interactions (similar to the ones that interact wi th NTRK3) , dimerization status (homo- and heterodimerization) and phosphorylation status. 6.2 RESULTS 6.2.1 Expression and Phosphorylation Status of ETV6-NTRK3 and ETV6-NTRK3 Mutant Proteins in NIH3T3 Cells Analysis of the ETV6-NTRK3 nucleotide sequence using Lasergene Navigator software ( D N A S T A R ) estimated the molecular weight of the fusion protein to be approximately 74,300 Da. Immunoprecipitation of lysates derived from primary CFS tumor cells grown i n culture, in vitro translated E T V 6 - N T R K 3 as wel l as NIH3T3 cells infected wi th an ETV6-NTRK3 retroviral construct (supplied by D. Wai in our lab) wi th either the a - E T V 6 : H L H or a - N T R K antibodies 117 followed by immunoblott ing analysis using the opposite antibody detected a doublet (due to the presence of two initiation sites wi th in ETV6) i n the 70-80 kDa range, confirming the presence of ETV6-NTRK3 proteins (see Fig. 19). We were interested in determining which domains of the E T V 6 - N T R K 3 protein ( H L H domain, P T K domain, A T P binding domain, etc.) were important for transformation and what these domains were responsible for. We therefore generated a series of constructs (see Table 5 i n chapter 2) which were used to infect NIH3T3 cells including: E T V 6 - N T R K 3 (as the positive control), Vector (NIH3T3 cells transfected wi th MSCVpac vector alone containing no insert, as the negative control), A H L H (we deleted the H L H dimerization domain i n E T V 6 - N T R K 3 to test for its significance i n oncogenesis), PLGyQ, PLCyT, PLCyE (the tyrosine residue specific for P L C y l binding was replaced wi th a glutamine (Q), threonine (T), or a glutamate (E) residue, respectively, to ablate the ability of P L C y l to bind to E T V 6 -N T R K 3 and test for its significance i n oncogenesis), Act ivat ion Loop Dead (ALD) (the three tyrosines known to be essential for autophosphorylation of N T R K 3 were mutated i n order to determine their importance i n the oncogenic process), and KinaseDead (KD) (the A T P binding site was mutated so that tyrosine phosphorylation of the E T V 6 - N T R K 3 protein would be ablated i n order to determine the significance of tyrosine phosphorylation i n the oncogenic process) [414, 415]. Of these constructs, only E T V 6 - N T R K 3 and the PLCyQ, PLCyT, PLCyE mutants were able to transform NIH3T3 cells while cells infected wi th Vector, 118 CO CN i Q ON co ai ^ S 2 Q LO CU 2 (3 fi fi CS V fi£ g3 ^ S H O ' f i rc a> g PH -3 < > f—1 .5 U CN QJ * fi P_CN $ a; cov v fi M 4S 3 "a3 2 d ^ r - 1 < !> 1 — 1 pq Z fi * H MH <U CO CO ^ ro i fi M CU -!-> CO I ON rH W D a l-H PH OJ c ro OH fi -fi ro bp HH • OJ I X t—1 QJ l a u 119 A H L H , A L D and K D constructs appeared morphologically normal. These cells were subsequently analyzed by immunoprecipitation and immunoblott ing, as described above for cells expressing ETV6-NTRK3 (see Fig. 19). To determine the tyrosine phosphorylation status of the E T V 6 - N T R K 3 protein and the various mutants, we immunoprecipitated lysates from var ious NIH3T3 cells expressing either E T V 6 - N T R K 3 or one of the mutant constructs w i t h ant i -NTRK3 antibody and subsequently immunoblotted wi th the anti-phosphotyrosine antibody, RC-20. We were able to show tyrosine phosphorylat ion for E T V 6 - N T R K 3 , A L D , and PLCyQ, P L C Y T , PLCyE, while Vector, A H L H , and K D failed to show any signs of tyrosine phosphorylation (see Fig. 20). 6.2.2 E T V 6 - N T R K 3 Homodimerizes and Heterodimerizes w i t h E T V 6 To examine the possibility that E T V 6 - N T R K 3 is capable of homodimerization, we took advantage of the high affinity between glutathione-S-transferase (GST) and glutathione-agarose beads [416]. A vector coding for a 5'-GST protein was used to generate an GST-ETV6-NTRK3 construct, which was used to transfect SF9 insect cells for the production and subsequent purification of GST-E T V 6 - N T R K 3 protein. We checked for homodimerization by mixing purified G S T - E T V 6 - N T R K 3 fusion protein wi th in vitro translated E T V 6 - N T R K 3 radiolabeled wi th 3 5 S -Methionine and glutathione beads. E T V 6 - N T R K 3 was pulled down wi th the GST-E T V 6 - N T R K 3 protein by the addition of glutathione beads, but not w i t h glutathione beads alone suggesting that ETV6-NTRK3 is able to homodimerize (see 00 i n 01 '53 'Z, c re > re > 5 CQ CO co TO < PQ 121 Fig. 21a). Since dimerization is thought to act through the H L H domain we tested the ability of the A H L H mutant (ETV6-NTRK3 lacking the H L H domain) to dimerize wi th either ETV6 or E T V 6 - N T R K 3 . The A H L H mutant d id not dimerize wi th ETV6 nor the E T V 6 - N T R K 3 protein (see Fig. 21b). To determine if E T V 6 -N T R K 3 was able to heterodimerize wi th ETV6, we co-m vitro translated E T V 6 -N T R K 3 and ETV6, immunoprecipitated wi th an t i -NTRK3 antibody and immunoblotted wi th an t i -ETV6:HLH antibody. Figure 21c shows E T V 6 coimmunoprecipitating along wi th E T V 6 - N T R K 3 protein suggesting that the E T V 6 - N T R K 3 is able to heterodimerize wi th ETV6. 6.2.3 Downstream Interactors Affected by the E T V 6 - N T R K 3 Molecule N T R K 3 is known to interact wi th specific cytoplasmic tyrosine kinases including S H C , GRB2, SH2Bp, rAPS, PI3K, and P L C y l [310, 311, 417, 418]. We were interested i n determining if these molecules were able to interact wi th the E T V 6 -N T R K 3 fusion protein. We were interested i n determining which regions of the E T V 6 - N T R K 3 protein were important i n downstream interactions. Sequence analysis of the E T V 6 - N T R K 3 chimera determined that the S H C and PI3K interaction site were lost as a result of the position of the breakpoint, but the P L C y l site was retained [356]. We therefore wished to elucidate the downstream interactors wi th E T V 6 - N T R K 3 by testing S H C , SH2Bp\ GRB2, PI3K, and P L C y l (rAPS was not included due to the lack of antibody). We lysed cells expressing the E T V 6 - N T R K 3 protein, immunoprecipitated wi th ant i -NTRK3 antibody, and subsequently immunoblotted wi th antibodies toward either S H C , GRB2, 122 m CN II ro J3 ro ro ro cu 3 ro c n PQ CO CN ro T3 ro -sa ro a; g jo re ro ro ro ro si ; x s .a « 5 a ro i l l s 123 SH2BP (data not shown), PI3K or P L C y l (see Fig. 22). Of these molecules, only P L C y l coimmunoprecipitated wi th E T V 6 - N T R K 3 as wel l as wi th the A L D mutant. Similar analysis of the other E T V 6 - N T R K 3 mutants, including Vector, PLCyQ, PLCyT, PLCyE, A H L H , and K D failed to coimmunoprecipitate P L C y l (see Fig. 23). None of the constructs, including ETV6-NTRK3, were able to coimmunoprecipitate S H C , GRB2, PI3K or SH2Bf3. 6.2.4 Subcellular Localization We were interested i n the subcellular localization of the E T V 6 - N T R K 3 fusion protein as this wou ld help explain its mechanism of action as an oncogenic molecule wi th in the cell. Cells infected wi th E T V 6 - N T R K 3 , A H L H , K D , or Vector constructs were fixed in paraformaldehyde, incubated wi th an t i -NTRK3 antibody followed by another incubation wi th rhodamine-anti-rabbit and subsequently analyzed by confocal microscopy. Our preliminary results suggest that E T V 6 -N T R K 3 is mainly localized within the cytoplasm wi th low amounts i n the nucleus. Similar results were obtained for A H L H and K D , while Vector showed relatively little fluorescence and was used as the negative control (see Fig. 24). 6.3 DISCUSSION The ETV6 gene has been shown to be involved in numerous translocations giving rise to various gene fusions in human leukemias (see Table 7, Chapter 3). 1 2 3 4 GRB2>-F I G U R E 22. ETV6-NTRK3 interacts with PLCybu t not wi th SHC, GRB2, or PI-3K p85 subunit. Whole cell lysates were prepared from a human medullary thyroid carcinoma cell line overexpressing wild-type N T R K 3 (lane 1), as wel l as from NIH3T3 cells expressing ETV6-NTRK3 (lane 2), Vector (lane 3), or K D (lane 4). Immunoprecipitation was performed with antibodies against the N T R K 3 P T K domain followed by immunoblotting with antibodies directed against SHC, GRB2, PI-3K, or P L C y l as indicated. Only wild-type N T R K 3 was found to associate with SHC, GRB2, and PI-3K (Lanes 1 of top three panels), while both N T R K 3 and ETV6-NTRK3 bound PLCy (lanes 1 and 2 of bottom panel). 125 O N OO co CN fN M 5 2 3 ^ •2 T3 CO CO p $ QJ 126 F I G U R E 24. Confocal microscopy of ETV6-NTRK3 and A H L H expressing NIH3T3 cells. NIH3T3 cells expressing either ETV6-NTRK3 (panels A and B) and A H L H (panels C and D) were probed with either a -NRTK3 antibody (panels A and C) or with a - E T V 6 : H L H antibody (panels B and D). When probed with a -NTRK3 antibody, confocal microscopy showed localization of ETV6-NTRK3 and A H L H to the cytoplasm with relatively low amounts in the nucleus. Probing with a - E T V 6 : H L H antibody detected w i l d type ETV6 in addition to ETV6-NTRK3 and as a result showed more intense nuclear staining. 127 Most of the gene fusions encoding the H L H domain of ETV6 as the 5' end, possess sequences encoding a tyrosine kinase domain as the 3' end [320, 409, 419]. This is expected to result in constitutive activation of the P T K domain due to the l igand independent dimerizing capabilities of the H L H domain. The recently characterized ETV6-NTRK3 gene fusion i n CFS and cellular C M N was identified i n our studies predominantly as a protein doublet i n lysates derived from NIH3T3 cells infected wi th either an E T V 6 - N T R K 3 construct or one of the mutants. This can, i n part, be explained by the fact that the ETV6 gene has two transcription initiation sites. Fol lowing the translation of these two species, post-translational modifications (primarily tyrosine phosphorylation of the N T R K 3 portion) can further divide each of the two bands producing a total of four bands (two unphosphorylated and two phosphorylated). A study analyzing the ETV6 and E T V 6 - C B F A 2 fusion protein identified this fusion protein as several different species due to alternative initiation sites in the ETV6 gene i n addition to the post-translational modification of these proteins [338, 344]. Further studies are st i l l needed to clarify the role of each species i n the oncogenic process. The A H L H and K D mutants were unable to transform NIH3T3 cells and d id not show any sign of being tyrosine phosphorylated. The only difference between K D and ETV6-NTRK3 is a single amino acid (lysine 380 to an arginine), that blocks A T P binding which is crucial for P T K activity. Previous attempts to inactivate the A T P binding site i n N T R K molecules showed similar results as the N T R K molecule was no longer able to tyrosine phosphorylate (even after l igand-induced dimerization) and interact wi th downstream molecules [420]. This indicates that an 128 intact P T K domain is essential for transformation. The A H L H mutant lacks most of the helix loop helix domain from exons 3 and 4, which has been shown to be important i n dimerization. Dimerization is essential for bringing the tyrosine kinase domains i n close proximity so that they can phosphorylate each other, suggesting that the H L H domain is critical in the process of activation of the tyrosine kinase domain i n the E T V 6 - N T R K 3 fusion. The A H L H and K D mutants, therefore, provide evidence that dimerization and tyrosine phosphorylation are both required for transformation of NIH3T3 cells. We were able to show that the E T V 6 - N T R K 3 can homodimerize as wel l as heterodimerize wi th ETV6. E T V 6 - N T R K 3 heterodimerization wi th ETV6 might ablate the normal function of ETV6 by interfering wi th its D N A binding potential. There has been some evidence suggesting that ETV6 may actually be a tumor suppressor gene [421-423]. Furthermore, we were able to show that the E T V 6 -N T R K 3 fusion protein was predominantly localized wi th in the cytoplasm w i t h lower amounts i n the nucleus. Further studies are necessary to determine the role of dimerization and oncogenesis in these tumors. Our results suggest that most of the known signaling interactors w i t h N T R K 3 (namely, S H C , GRB2, PI3-K, and SH2BB) do not associate wi th the E T V 6 -N T R K 3 molecule. Phospholipase C-y l (PLCyl) was, however, able to interact w i t h the E T V 6 - N T R K 3 fusion protein. P L C y l is a member of the family of inosi to l phospholipid phosphodiesterases [424]. There are a total of three members to this family including B, y, and 8. PLCy is activated by the intracellular tyrosine kinase 129 domains of its respective receptors [425]. Activation of P L C y leads to the production of intracellular second messengers inositol (1, 4, 5)-triphosphate (InsP3) and sn 1, 2-diacylglycerol (DAG) [424]. A t the amino terminus of the P L C molecules is a structure k n o w n as the pleckstrin homology domain (PH) responsible for facilitating binding of PLCy to PtdInsP 2 and other inositol phosphates [426]. The P L C molecules also contain an EF Hand domain which is thought to bind C a + + , as these molecules are C a + + dependant enzymes [427]. Deletion of the EF Hand abrogates activity [428]. The two most highly conserved structures i n mammalian PLC molecules are the X and Y boxes. These structures are essential for activity and may also determine which substrates and subsequent reactions the molecule can support [429]. O n the tertiary leve l , these boxes form a structure known as a T I M barrel. Wi th in the T I M barrel are P H , SH2 and SH3 domains responsible for the specific interactions wi th substrate molecules such as PtdInsP 2 (SH2 domains were first discussed i n Chapter 1 as structures essential for the interaction wi th phosphorylated tyrosines) [430]. The last domain in PLCy (conserved and present i n PLC|3 and 8) is the C2 domain , which is C a + + dependant and seems to act as an interface between the EF Hand and the T I M barrel catalytic domain [431, 432]. P L C y l is a ubiquitously expressed tyrosine kinase substrate responsible for the control of intracellular levels of C a + + and D A G [433]. After tyrosine phosphorylation of P L C y l , the molecule translocates to the membrane where the P H domains recognize PI 4, 5-P2 and Ins 1, 4, 5-P3 as ligands. The deactivation by 130 phosphatases is evident, but unclear. In addition to tyrosine phosphorylation, the P L C y l molecule undergoes serine-threonine phosphorylation, but the kinases and phosphatases of this system have not been characterized yet. The main reaction catalyzed by P L C y l is the conversion of Ptdlns 4,5-P 2 to D A G and Ins 1,4,5-P 3. Ins 1, 4, 5-P 3 is responsible for stimulating the secretion of C a + + from the endoplasmic reticulum [433]. Ins 1, 4, 5-P 3 is responsible for tethering protein kinase C (PKC) to the membrane while D A G acts as a potent activator of P K C [433]. There has been some evidence suggesting that P K C then activates the M A P K pathway through R A F and can ultimately result in an increase in cellular proliferation ( in undifferentiated cells) and other cellular responses such as contraction, secretion and membrane conductance (in differentiated cells) [434, 435]. Our studies showed that there is no loss in the transformation capabilities of non -PLCyl binding E T V 6 - N T R K 3 mutants, suggesting that P L C y l signaling may not contribute to E T V 6 - N T R K 3 transformation. Our results failed to show an association between E T V 6 - N T R K 3 and known N T R K 3 interactors such as S H C , GRB2, PI3-K, and SH2BB. These results, however, do not rule out that these molecules are involved indirectly in E T V 6 - N T R K 3 transformation activity. Other adaptor molecules may link E T V 6 - N T R K 3 to known N T R K 3 signaling pathways. These molecules must be assayed directly to assess their possible roles i n E T V 6 -N T R K 3 signaling. Alternatively, a completely novel pathway may be i n v o l v e d . The increase i n proliferation and transformation seen i n E T V 6 - N T R K 3 infected NIH3T3 cells can be explained by the fact that some other molecule is able to 131 associate wi th E T V 6 - N T R K 3 and activate proliferative and/or cell survival signal transduction pathways. The possibility that the E T V 6 - N T R K 3 fusion is interacting wi th normal ETV6 and dysregulating it is unlikely, since the K D mutant is capable of dimerizing wi th normal ETV6 (data not shown), but is non-transforming. In view of this, the A H L H mutant is non-transforming because of its inability to dimerize and activate the tyrosine kinase domain and not because of its inability to dimerize and interfere wi th normal ETV6 function. Further studies such as yeast two hybrid screening w i l l be necessary (and are currently being performed in our laboratory) for characterizing new and essential downstream interactors wi th the N T R K 3 portion (and possibly the ETV6 portion) of the E T V 6 - N T R K 3 chimeric fusion. 132 CHAPTER VII SUMMARY AND CONCLUSIONS The dilemma in finding a cure for cancer is that there w i l l not be one cure for all cancers, but rather a specific conglomerate of approaches for each i n d i v i d u a l cancer. Developing treatment protocols for a specific cancer wou ld require a number of factors. The first requirement, which wou ld be crucial i n determining the course of action for a particular tumor, is the ability to accurately differentiate and diagnose that tumor from other tumors. Secondly, an in-depth knowledge of the molecular basis for tumorigenesis of the specific tumor is essential. Once the tumor has been characterized at the molecular level, the knowledge on how it has evolved and maintains itself needs to be integrated in order to develop a treatment strategy. The treatment protocol would need to target tumor cells while leaving normal cells unharmed. We have accomplished i n part the first two tasks wi th congenital fibrosarcoma (CFS), a soft tissue pediatric spindle cell lesion. CFS is difficult to diagnose because of its histologic similarity wi th adult type fibrosarcoma, aggressive fibromatosis and infantile fibromatosis. Cytogenetic analysis on a series of CFS, ATFS , A F B and IFB cases revealed a recurring rearrangement i n v o l v i n g chromosomes 12 and 15 only i n CFS. Whole chromosome FISH of a CFS case using a chromosome 12 painting probe revealed a portion of chromosome 12 on a smaller acrocentric chromosome. This rearrangement was not seen i n n o r m a l 133 fibroblastic tissues. This warranted the characterization of the rearrangement on a molecular level, i n hopes of finding a recurring molecular marker of CFS w h i c h would be an invaluable tool for the pathologist. 7.1 IDENTIFICATION OF A RECURRING t(12:15) in CONGENITAL FIBROSARCOMA Cytogenetic analysis revealed a rearrangement invo lv ing chromosomes 12pl3 and 15q25-q26. FISH analysis using a combination of a-centromeric chromosome specific probes and region specific Y A C probes identified a translocation of material from chromosome 12pl3 to chromosome 15q26. One of the Y A C s , namely 817_H_1, was found to be split, indicating that it spanned the breakpoint and contained the gene involved. This Y A C was known to harbor the ETV6 gene, which has been rearranged i n numerous hematopoietic malignancies. H i g h resolution FISH mapping using cosmids specific for certain exons of the ETV6 gene placed the breakpoint wi thin the ETV6 gene thus confirming its i nvo lvement . E T V 6 belongs to the ETS family of transcription factors and has been shown to be involved i n gene fusions in hematopoietic malignancies. The involvement of a gene in both hematopoietic malignancies and sarcomas has been described previously, as the ERG and FLU genes (other ETS family members) are found to be fused wi th two similar genes, EWS and TLS/FUS, as a result of chromosome translocations i n human solid tumors and leukemias. 134 7.2 THE ETV6-NTRK3 GENE FUSION CHARACTERIZES CONGENITAL FIBROSARCOMA We hypothesized that ETV6 was involved i n a gene fusion i n CFS. The ETV6 gene is known to be oriented in a telomere to centromere fashion o n chromosome 12pl3. Therefore the t(12;15) i n CFS is expected to result i n the translocation of 5' ETV6 material to chromosome 15. We therefore performed 3' rapid amplification of c D N A (3'RACE) using known ETV6 sequence primers to amplify and clone the breakpoint. Sequence analysis of R A C E products revealed 5' ETV6 sequence unti l nucleotide 1033, which corresponds to the last nucleotide i n ETV6 exon 5. The remaining sequence was compared to public databases and was 100% homologous to a portion of the NTRK3 gene. The rearrangement was confirmed by Southern analysis and the presence of an ETV6-NTRK3 gene fusion was confirmed by Northern analysis and RT-PCR. As this is one of two recurring chromosomal abnormalities along wi th trisomy 11, it was hypothesized that this fusion gene is etiologic i n CFS oncogenesis. 7.3 TRISOMY 11 A N D THE ETV6-NTRK3 GENE FUSION LINK CONGENITAL FIBROSARCOMA TO CONGENITAL MESOBLASTIC NEPHROMA Congenital mesoblastic nephroma ( C M N ) is an infantile spindle cell tumor of the kidney which has an excellent prognosis similar to that of CFS. C M N is subdivided into classic and cellular forms depending on the degree of cellularity and mitotic activity of the spindle cells. The cellular variant is vir tually identical histologically and cytogenetically to CFS, and this morphologic overlap has led to 135 the hypothesis that these tumors are histogenetically related. Cytogenetic studies have reported common trisomies i n CFS and cellular C M N , particularly of chromosome 11. We analyzed C M N cases and found that the t(12;15)(pl3;q25) rearrangement i n CFS is also present in cellular C M N and may underlie the distinctive biological properties of these two tumors. Analysis of m R N A revealed the expression of ETV6-NTRK3 chimeric transcripts i n 8 of 9 cellular C M N cases as we l l as i n 2 of 2 mixed C M N cases. Four of four classical C M N cases were negative for the ETV6-NTRK3. In addition, we found trisomy 11 to be strongly correlated wi th the presence of the ETV6-NTRK3 gene fusion. Our studies therefore indicate that congenital fibrosarcoma and cellular congenital mesoblastic nephroma are histogenetically related. Table 9 summarizes the results of RT-PCR analysis on a series of CFS, C M N , A T F S , IFB, and A F B primary tumor samples to date. Briefly, the E T V 6 - N T R K 3 fusion was found i n 100% of CFS cases (n=15), 90% of cellular C M N cases (n=10) and 100% of mixed C M N cases (n=2) analyzed. Fusion transcripts were not detected i n A T F S (n=10), IFB (n=12), and A F B (n=5) cases, nor i n classical C M N cases analyzed (n=4). Only one of ten cellular C M N cases was negative for the fusion transcript. 7.4 M O L E C U L A R STUDIES OF T H E E T V 6 - N T R K 3 F U S I O N P R O T E I N Understanding the oncogenic process requires the familiarization wi th the complex biochemical interactions wi th in the tumor cell. We generated a fu l l length E T V 6 - N T R K 3 construct encoding the helix-loop-helix ( H L H ) dimerization 136 TABLE 9. Summary of ETV6-NTRK3 (TEL-TRKC) Analysis. TUMOR NUMBER ETV6-NTRK3 ETV6-NTRK3 FUSION A N A L Y Z E D FUSION POSITIVE NEGATIVE CFS 12 12 0 ATFS 10 0 10 IFB 12 0 12 AFB 5 0 5 C M N 1. Classical 4 0 4 2 . Cellular 10 9 1 3. Mixed 2 2 0 Abbreviations. CFS, congenital fibrosarcoma; ATFS, adult-type fibrosarcoma; IFB, infantile fibrosarcoma; A F B , aggressive fibrosarcoma; C M N , congenital mesoblastic nephroma. 137 domain of ETV6 fused to the protein tyrosine kinase (PTK) domain of N T R K 3 . NIH3T3 cells were infected wi th recombinant retroviral viruses carrying either the full-length ETV6-NTRK3 c D N A or one of the mutants (kindly provided by Danie l W a i i n our laboratory). Cells expressing the ETV6-NTRK3 construct exhibited a transformed phenotype and formed macroscopic colonies i n soft agar. W e hypothesized that chimeric proteins mediate transformation by dysregulating N T R K 3 signal transduction pathways via ligand-independent dimerization and PTK-autophosphorylation. To test this hypothesis, a series of different mutants were generated to help determine which regions of the E T V 6 - N T R K 3 fusion protein were necessary for oncogenesis. We showed that E T V 6 - N T R K 3 homodimerizes and is capable of forming heterodimers wi th wild-type ETV6 in vitro. The H L H domain of ETV6 in the E T V 6 - N T R K 3 fusion was deleted i n order to investigate the role of protein dimerization in transformation ( A H L H mutant). The A H L H mutant was not able to associate wi th ETV6, nor wi th E T V 6 - N T R K 3 . Cells expressing this mutant protein were morphologically non-transformed and failed to grow i n soft agar. To investigate the role of the N T R K 3 P T K domain , NIH3T3 cells were transfected wi th a variety of E T V 6 - N T R K 3 mutants w i t h activation loop amino acid substitutions (ALD) as wel l as a kinase inactive mutant unable to bind A T P (KD). The three P T K activation-loop tyrosines mutated (ALD) to phenylalanines still became tyrosine phosphorylated but were unable to transform NIH3T3 cells. The K D mutant failed to autophosphorylate and lacked transformation ability. Of a series of signaling molecules wel l known to bind to wild-type N T R K 3 , only P L C y l was found to associate wi th and become tyrosine 138 phosphorylated by E T V 6 - N T R K 3 . Interestingly, several P L C y l binding mutants were unable to bind P L C y l , but were still capable of transforming NIH3T3 cells suggesting that another pathway is being activated and is responsible for the transforming abilities of E T V 6 - N T R K 3 . In addition, preliminary subcellular localization studies showed that the E T V 6 - N T R K 3 fusion protein localizes ma in ly i n the cytoplasm, wi th limited presence i n the nucleus. Our studies confirm that E T V 6 - N T R K 3 is a transforming protein that requires both an intact d imer iza t ion domain and a functional P T K domain for transformation activity. 7.5 G E N E R A L C O M M E N T S The discovery of the ETV6-NTRK3 gene fusion i n CFS and cellular C M N lead us to screen other cancers for the same gene fusion. Interestingly, we were able to detect the ETV6-NTRK3 gene fusion i n a breast carcinoma from a 6 year o ld patient. Cytogenetic analysis on this case confirmed the presence of the t(12;15)(pl3;q25) but failed to show further evidence of any other chromosomal abnormalities including trisomy chromosome 11. Our transfection studies have shown that the ETV6-NTRK3 gene fusion product has transformation ability, which supports the notion that the breast carcinoma may have arisen solely due to the gene fusion. Another group has recently identified the ETV6-NTRK3 gene fusion i n an adult acute myeloid leukemia [436]. The ETV6 gene has been implicated i n numerous hematopoietic rearrangements, but this is the first report of NTRK3 involvement wi th a leukemia. This makes the ETV6-NTRK3 gene fusion the only documented gene fusion to date which has been involved i n both a 139 solid tumor as wel l as a leukemia. The ETV6-NTRK3 gene fusion may therefore have a wider spectrum of involvement i n human malignancies. Further studies are required to test this possibility in detail. Recent evidence suggests that the ETV6 central domain (previously thought to contain no known domains) mediates transcriptional repression by associating wi th S M R T and mSin3A, while the ETV6 H L H domain represses gene transcription through a mechanism that is independent of known corepressors [437]. This domain is a part of the ETV6-NTRK3 gene fusion. Further studies are therefore needed to determine if there are other molecules interacting wi th E T V 6 -N T R K 3 which are responsible for oncogenesis. This is currently being explored i n our laboratory by the use of yeast-2-hybrid screening. Future studies, some of which are being explored currently i n our laboratory, include elucidating the signal transduction pathways which are being util ized by E T V 6 - N T R K 3 . To look for other interactors wi th E T V 6 - N T R K 3 , yeast-2-hybrid screening is currently being used to identify novel interactors. To help increase the » resolution of the yeast two hybrid approach, the E T V 6 - N T R K 3 has been d iv ided into two so that interactors wi th the ETV6 portion and the N T R K 3 portion can be studied independently. Specific inhibitors of signaling pathways (e.g., w o r t m a n n i n specifically inhibits PI-3K; PD98059 inhibits M E K 1 , and U73122 inhibits PLC molecules) should be tested to determine their impact on tumor growth and whether or not they can be used to treat CFS and cellular C M N i n patients. Similarly further research is needed to see if N T R K specific inhibitors could be used to treat CFS and cellular C M N (e.g., K252a and CEP-751 are effective N T R K tyrosine 140 kinase inhibitors [438, 439]). To explore the possibility that the d imer iza t ion domain of ETV6 is dimerizing wi th other molecules or contributing other functions to the oncogenic process, the 3'-portion of NTRK3 involved i n the CFS translocation has recently been fused to an inducible dimerization domain, F K B P 3 6 v [440, 441], i n our laboratory. This w i l l allow us to control dimerization and therefore the oncogenic activity of the N T R K 3 portion of E T V 6 - N T R K 3 i n NIH3T3 cells and w i l l provide valuable information on the proliferative and transformation process. 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