Open Collections

UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

Investigation into the role of Y-box binding protein-1 (YB-1) in childhood sarcomas EL-Naggar, Amal Mohammad 2013

Your browser doesn't seem to have a PDF viewer, please download the PDF to view this item.

Item Metadata

Download

Media
24-ubc_2014_spring_elnaggar_amal.pdf [ 9.03MB ]
Metadata
JSON: 24-1.0165716.json
JSON-LD: 24-1.0165716-ld.json
RDF/XML (Pretty): 24-1.0165716-rdf.xml
RDF/JSON: 24-1.0165716-rdf.json
Turtle: 24-1.0165716-turtle.txt
N-Triples: 24-1.0165716-rdf-ntriples.txt
Original Record: 24-1.0165716-source.json
Full Text
24-1.0165716-fulltext.txt
Citation
24-1.0165716.ris

Full Text

INVESTIGATION INTO THE ROLE OF Y-BOX BINDING PROTEIN-1 (YB-1) IN CHILDHOOD SARCOMAS  by AMAL MOHAMMAD EL-NAGGAR MBChB, Menoufia University, 1999 MSc Pathology, Menoufia University, 2005 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF  DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE AND POSTDOCTORAL STUDIES (Pathology & Laboratory Medicine)  THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver)  December 2013  ? AMAL MOHAMMAD EL-NAGGAR, 2013    ii  Abstract  Objective: To identify the potential roles played by YB-1 in childhood sarcoma progression.  Background: Sarcomas are mesenchymal-derived malignant neoplasms that are characterized by early metastatic spread, and poor prognosis. YB-1 is a member of the highly conserved CSD-containing family of proteins known to regulate transcription and translation of a multitude of genes. Importantly, YB-1 promotes an epithelial-to-mesenchymal transition (EMT) in non-invasive breast epithelial cells. In spite of its role in EMT, comprehensive investigations into the role of YB-1 in the progression of childhood sarcomas are currently lacking.  Methods: To study the potential role of YB-1 in childhood sarcomatogenesis, we used MNNG and MG63 (osteosarcoma), TC32 and TC71 (Ewing sarcoma), and Rh30 and Rh18 (rhabdomyosarcoma) tumour cell lines, and performed transient and stable YB-1 knockdown (kd) in each cell line. Then, cells were subjected to different assays.  Results: Using in vitro cell motility, invasion, and proliferation assays, we found that YB-1 kd significantly reduced migration and invasion of each of these cell lines and this was associated with enhanced proliferative capacity of childhood sarcoma cells. YB-1 kd also profoundly inhibited migration and metastasis of human sarcoma cell lines xenotransplanted into either the yolk sacs of zebrafish embryos or under the kidney capsule of NOD/SCID mice, a model previously utilized for epithelial-derived tumours. We then assessed potential mechanisms, and found that YB-1 directly bound and robustly activated the translation of HIF1? mRNAs, while it had no effect upon HIF1? transcription. YB-1 itself was robustly induced by hypoxia, and blocking this induction blocked HIF1? protein levels. HIF1? kd blocked YB-1 mediated induction of sarcoma cell migration and invasion, and ectopic expression rescued the effects of iii  YB-1 kd under the same parameters in vitro and in vivo. Notably, tumours with YB-1 kd exhibited extensive levels of haemorrhaging and necrosis compared to the control tumours, and this correlated significantly with reduced mean microvessel density and VEGF production.  Conclusions: YB-1 promotes childhood sarcoma cell metastasis through translational activation of HIF1?, underscoring the potential impact of YB-1 on sarcoma angiogenesis. Importantly, targeting YB-1 or its downstream effectors represents a promising strategy in the treatment of childhood sarcomas.     iv  Preface  Ethics approvals  The study of IHC evaluation of YB-1 and HIF1? expressions in childhood sarcomas was carried out under the University Of British Columbia?s Research Ethics Board (UBC REB Number H02-61375).   The studies using the murine renal subcapsular assay xenotransplantation model were carried out under the UBC animal care certificate # A10-0200.   Conducted studies  A portion of chapter 5 has been published. Expression and stability of hypoxia inducible factor 1? in osteosarcoma. El Naggar A, Clarkson P, Zhang F, Mathers J, Tognon C, Sorensen PH. Pediatr Blood Cancer; 59(7):1215-22, Copyright ? 1999?2013 John Wiley & Sons, Inc. 2012. http://onlinelibrary.wiley.com/doi/10.1002/pbc.24191/abstract The majority of the study was designed and conducted by myself with acknowledged contributions made by Dr Fan Zhang. I performed and analyzed approximately 80% of the work described. The first version of the manuscript was written and prepared by me. Cristina Tognon assisted in editing and with revisions of the manuscript. The figures derived from this work and incorporated into the thesis (Figures 5.2, 5.3, 5.4, 5.5, and 5.6) are comprised of experiments entirely conducted by me.  v  Contribution of co-authors: ? Paul Clarkson isolated the human osteoblasts and established them in culture.  ? Fan Zhang helped in revising the final design and assisted in editing the manuscript. ? Joan Mathers provided technical assistance. ? Cristina Tognon assisted in editing and revisions of the manuscript. ? Poul Sorensen supervised the project, and did the final revision of the manuscript.   A manuscript based on versions of chapter 3, chapter 4, and chapter 5 has been submitted. YB-1 facilitates metastasis of high-risk childhood sarcomas through translational activation of HIF1?. Amal M El-Naggar, Chansey J Veinotte, Cristina E Tognon, Dale P Corkery, Hongwei Cheng, Franck Tirode, Thomas Grunewald, Alastair H Kyle, Joan Mathers, Syam P Somasekharan, Steven McKinney, Andrew I Minchinton, Olivier Delattre, Yuzhuo Wang, Graham Dellaire, Jason N Berman, and Poul H Sorensen. Poul Sorensen, Cristina Tognon, and I participated in the design of the study. I performed and analyzed approximately 50% of the work described. The first version of the manuscript was written and prepared by me and then edited by Poul Sorensen.  Contribution of co-authors: ? Chansey J Veinotte and Dale P Corkery under supervision of Graham Dellaire and Jason N Berman performed the zebrafish xenotransplantation model (Figure 4.4A-B). ? Cristina Tognon provided skillful supervision, participated in design of the study, assisted in editing and revision of the manuscript and performed (Figure 5.7B).  ? Hongwei Cheng under supervision of Yuzhuo Wang performed the murine renal subcapsular assay (RSA) xenotransplantation model (Figure 4.5A). vi  ? Franck Tirode and Thomas Grunewald under supervision of Olivier Delattre performed bioinformatic analysis of YB-1 mRNA expression in sarcomas and its correlation with the outcome data of Ewing sarcoma (ES) patients.  ? Alastair H Kyle under supervision of Andrew I Minchinton performed the multilayered 3-D cell culture model (Figures 5.7E, 5.8A-B). ? Joan Mathers provided technical assistance. ? Syam P Somasekharan helped to conduct RNP IP experiment (Figure 5.10D). ? Steven McKinney helped in statistical analysis (Tables 5.3 and 5.4) and provided (Figure 5.9A).  ? Poul H Sorensen participated in the design of the study, supervised the project, and supervised the editing and revising of the manuscript.   Selected Presentation  AACR-JCA Joint Conference. Maui, HI, Feb.2013. Poster presentation on YB-1 contributes to sarcoma metastasis via translational activation of HIF1?. Amal El-Naggar, Chansey Veinotte, Cristina Tognon, Hongwei Cheng, Syam Somasekharan, Yuzhuo Wang, Jason Berman, and Poul H Sorensen.      vii  Table of Contents  Abstract .......................................................................................................................................... ii?Preface ........................................................................................................................................... iv?Table of Contents ........................................................................................................................ vii?List of Tables .............................................................................................................................. xiii?List of Figures ............................................................................................................................. xiv?List of Symbols .......................................................................................................................... xvii?List of Abbreviations ............................................................................................................... xviii?Acknowledgements ....................................................................................................................xxv?Dedication ................................................................................................................................ xxvii?Chapter  1: Background ................................................................................................................1?1.1? Cancer metastasis: A preamble ....................................................................................... 1?1.1.1? The classic metastatic cascade .................................................................................... 3?1.1.1.1? Cell motility: A crucial component of the metastatic process ............................ 3?1.1.1.1.1? Single cell motility patterns ........................................................................... 4?1.1.1.1.2? Collective cell motility .................................................................................. 5?1.1.1.1.3? Tumour cell motility: A distinct paradigm of movement plasticity .............. 5?1.1.1.2? The distinctive role of tumour cell adhesion in metastasis ................................. 6?1.1.1.2.1? Cadherins ....................................................................................................... 6?1.1.1.2.2? Integrins ......................................................................................................... 7?1.1.1.3? The role of proteolysis in cancer metastasis ....................................................... 9?1.1.1.4? Angiogenesis and cancer metastasis ................................................................... 9?viii  1.2? HIF1?: The master regulator of the hypoxic response ................................................. 12?1.3? Y box binding protein 1 (YB-1).................................................................................... 16?1.3.1? YB-1 protein structure .............................................................................................. 16?1.3.2? The role of YB-1 in DNA transcription .................................................................... 17?1.3.3? The role of YB-1 in mRNA translation .................................................................... 18?1.3.4? YB-1 and cancer ....................................................................................................... 21?1.3.4.1? YB-1, stress granules, and cancer ..................................................................... 22?1.4? Sarcomas ....................................................................................................................... 24?1.4.1? Sarcoma genetics ...................................................................................................... 25?1.4.1.1? Simple karyotypic defect- based sarcomas ....................................................... 25?1.4.1.2? Complex karyotypic defect- based sarcomas .................................................... 27?1.4.2? Molecular diagnosis of sarcomas .............................................................................. 27?1.4.3? Sarcomas subtypes .................................................................................................... 28?1.4.3.1? Osteosarcoma (OS) ........................................................................................... 28?1.4.3.2? Ewing sarcoma (ES) ......................................................................................... 29?1.4.3.3? Rhabdomyosarcoma (RMS).............................................................................. 31?1.5? Figures........................................................................................................................... 32?Chapter  2: Hypothesis and study objectives ............................................................................34?Chapter  3: YB-1 potentially contributes to the growth and phenotypic characteristics of childhood sarcoma cells ...............................................................................................................36?3.1? Rationale ....................................................................................................................... 36?3.2? Material and methods .................................................................................................... 36?3.2.1? Cell lines and culture conditions ............................................................................... 36?ix  3.2.2? Transient transfection ................................................................................................ 38?3.2.3? Stable YB-1 knockdown (kd) ................................................................................... 38?3.2.4? Protein extraction, western blotting, and antibodies ................................................. 39?3.2.5? Immunohistochemistery (IHC) for YB-1 expression in osteosarcoma ..................... 40?3.2.6? Phalloidin staining and quantitation of stress fibers ................................................. 42?Sucrose gradient and polysomal fractionation ...................................................................... 42?3.2.7? Anchorage-independent cell growth assay ............................................................... 43?3.2.8? Cell growth and BrdU incorporation ........................................................................ 43?3.2.9? Indirect immunofluorescence .................................................................................... 43?3.2.10? RNA isolation and quantitative real-time polymerase chain reaction (qRT-PCR)44?3.2.11? Statistical analysis ................................................................................................. 45?3.3? Results ........................................................................................................................... 45?3.3.1? YB-1 is highly expressed in sarcomas ...................................................................... 45?3.3.2? YB-1 knockdown affects the mesenchymal phenotype of sarcoma cells ................. 46?3.3.3? YB-1 retards sarcoma cell proliferation .................................................................... 47?3.3.4? YB-1 knockdown inhibits anchorage-independent growth of sarcoma cells in soft agar 47?3.3.5? Elevated YB-1 expression levels observed in osteosarcoma correlates with high grade disease ......................................................................................................................... 48?3.4? Discussion ..................................................................................................................... 49?3.5? Tables ............................................................................................................................ 52?3.6? Figures........................................................................................................................... 52?x  Chapter  4: YB-1 is a major contributor to sarcoma cell motility, invasion, and metastatic dissemination in vitro & in vivo ...................................................................................................62?4.1? Rationale ....................................................................................................................... 62?4.2? Material and methods .................................................................................................... 62?4.2.1? The murine renal subcapsular xenotransplantation model ........................................ 62?4.2.2? Zebrafish xenotransplantation ................................................................................... 63?4.2.2.1? Zebrafish husbandry .......................................................................................... 63?4.2.2.2? Cell staining, injection and screening ............................................................... 63?4.2.2.3? Live cell microscopy, and migration assay ....................................................... 64?4.2.3? Histopathological evaluation of xenografts .............................................................. 65?4.2.4? Quantitation of angiogenesis by assessing microvessel density ............................... 65?4.2.5? Transwell migration assay (Boyden chamber migration assay) ............................... 66?4.2.6? Transwell invasion assay .......................................................................................... 66?4.2.7? Matrigel 3-dimensional colony assay ....................................................................... 66?4.2.8? Wound healing assay ................................................................................................ 67?4.2.9? Statistical analysis ..................................................................................................... 67?4.3? Results ........................................................................................................................... 68?4.3.1? YB-1 increases childhood sarcoma cell motility in vitro and in vivo ....................... 68?4.3.2? YB-1 drives childhood sarcoma cell invasion and metastasis in vivo ...................... 69?4.4? Discussion ..................................................................................................................... 71?4.5? Figures........................................................................................................................... 74?Chapter  5: Translation activation of HIF1? by YB-1 is a novel mechanism contributing to childhood sarcoma metastasis .....................................................................................................91?xi  5.1? Rationale ....................................................................................................................... 91?5.2? Material and methods .................................................................................................... 91?5.2.1? Animal studies .......................................................................................................... 91?5.2.2? Histological evaluation and IHC studies of tumour xenografts ................................ 92?5.2.3? Hypoxia treatment ..................................................................................................... 92?5.2.4? RNA isolation, quantitative RT-PCR, and primers .................................................. 92?5.2.5? Antibodies ................................................................................................................. 93?5.2.6? In Vitro multilayered cell culture (3-dimensional tissue culture model) .................. 94?5.2.6.1? Cell culture ........................................................................................................ 94?5.2.6.2? IHC for BrdU, pimonidazole and HIF1? .......................................................... 94?5.2.6.3? IHC for YB-1 .................................................................................................... 95?5.2.7? Image acquisition ...................................................................................................... 95?5.2.8? In vitro transcription/translation ............................................................................... 95?5.2.9? Immunohistochemistry (IHC) for HIF1? in osteosarcoma ....................................... 96?5.2.10? IHC for YB-1 and HIF1? expressions in paediatric cancer tissue microarray ..... 96?5.2.11? HRE-GFP reporter assays ..................................................................................... 97?5.2.12? Luciferase assays .................................................................................................. 97?5.2.13? Transient HIF1? transfection ................................................................................ 98?5.2.14? HIF1? and HIF2? silencing .................................................................................. 98?5.2.15? HIF1? and HIF2? decay assay .............................................................................. 99?5.2.16? RNA immunoprecipitation.................................................................................... 99?5.2.17? Statistical analysis ............................................................................................... 100?5.3? Results ......................................................................................................................... 100?xii  5.3.1? YB-1 kd affects the expression of stress mitigating proteins including HIF1? ...... 100?5.3.2? HIF1? rather than HIF2? plays essential roles in sarcomas ................................... 102?5.3.3? HIF1? exhibited enhanced protein stability in osteosarcoma cells ......................... 103?5.3.4? Elevated HIF1? protein levels are associated with high grade osteosarcoma ........ 104?5.3.5? YB-1 is a survival factor induced under hypoxia ................................................... 105?5.3.6? YB-1-induced HIF1? is biologically active ............................................................ 105?5.3.7? Co-expression of YB-1 and HIF1? in paediatric sarcomas .................................... 106?5.3.8? YB-1 translationally activates HIF1? transcripts in childhood sarcoma cells ........ 106?5.3.9? YB-1 enhances HIF1? translational efficiency in vivo & in vitro .......................... 107?5.3.10? YB-1 contributes to HIF1? stability in sarcoma cells ......................................... 109?5.3.11? HIF1? is a potential effector for YB-1-mediated metastasis in vitro and in vivo 109?5.4? Discussion ................................................................................................................... 111?5.5? Tables .......................................................................................................................... 114?5.6? Figures......................................................................................................................... 117?Chapter  6: Conclusions and closing remarks .........................................................................150?References ...................................................................................................................................156? xiii  List of Tables  Table  3.1    Correlation of YB-1 expression with tumour grade in osteosarcoma ....................... 52?Table 5.1    Immunohistochemical characterization of HIF1? in osteosarcoma ........................ 115?Table 5.2    Correlation of HIF1? expression with tumour grade in osteosarcoma .................... 115?Table 5.3    Correlation of YB-1 and HIF1? expression in paediatric sarcoma subtypes .......... 116?Table 5.4    Association between YB1 staining intensity and HIF1? staining intensity within paediatric sarcoma subtypes ....................................................................................................... 116? xiv  List of Figures  Figure 1.1    The domain structure of HIF family of proteins ...................................................... 32?Figure 1.2   The domain structure of YB-1 protein and its functions ........................................... 33?Figure  3.1    YB-1 expression in sarcomas ................................................................................... 53?Figure  3.2    Inhibiting YB-1 expression affects sarcoma cell phenotype .................................... 54?Figure  3.3    YB-1 contributes to the formation of actin stress fibers ........................................... 56?Figure  3.4    YB-1-mediated effects on sarcoma cell proliferation .............................................. 59?Figure  3.5    YB-1-mediated effects on anchorage independent growth of sarcoma cells ........... 60?Figure  3.6    Examples of YB-1 immunohistochemical staining of osteosarcoma cases ............. 61?Figure 4.1    YB-1 contributes to the transmigratory ability of sarcoma cells in vitro ................. 75?Figure 4.2    YB-1 empowers the highly motile phenotype of sarcoma cells in vitro .................. 77?Figure 4.3    YB-1 promotes sarcoma cell invasion in vitro ......................................................... 79?Figure 4.4    YB-1 promotes sarcoma metastasis: Zebrafish xenotransplantation model ............. 80?Figure 4.5    The murine renal subcapsular assay for studying the metastatic capacity of +/- YB-1 kd cells in vivo .............................................................................................................................. 82?Figure 4.6    Characteristics of primary tumour xenografts  +/-YB-1 knockdown ....................... 84?Figure 4.7    Inhibiting YB-1 expression induces loss of tumour cell viability and inhibition of its local invasiveness capacity in vivo ............................................................................................... 86?Figure 4.8    YB-1 is a major contributor to sarcoma cell metastasis in vivo ............................... 87?Figure 4.9    YB-1 expression in pulmonary metastases ............................................................... 89?Figure 4.10    YB-1-mediated angiogenesis .................................................................................. 90?xv  Figure 5.1    Inhibiting YB-1 expression has a marked effect on HIF1? expression among other EMT related proteins .................................................................................................................. 118?Figure 5.2    HIF1? and HIF2? proteins levels under normoxia and hypoxia ............................ 119?Figure 5.3    Transcriptional effects of HIF1? and HIF2? in osteoblasts and osteosarcoma cell lines ............................................................................................................................................. 120?Figure 5.4  Comparison of HIF1? and HIF2? protein stability in osteoblast and osteosarcoma cell lines under normoxia and hypoxia .............................................................................................. 123?Figure 5.5  HIF1? and HIF2? protein stability in individual osteoblast and osteosarcoma cell lines under normoxia and hypoxia .............................................................................................. 125?Figure 5.6    Examples of HIF1? immunohistochemical staining of osteosarcoma cases .......... 126?Figure 5.7    Hypoxia-induced YB-1 protects against cell death ................................................ 129?Figure 5.8    YB-1 induces expression of biologically active HIF1? ......................................... 130?Figure 5.9    Co-expression of YB-1 and HIF1? in childhood sarcomas ................................... 133?Figure 5.10    YB-1-mediated HIF1? translational activation in sarcoma cells ......................... 134?Figure 5.11    YB-1 enhances HIF1? translational efficiency in vivo & in vitro ........................ 137?Figure 5.12    The predicted secondary structure of 5?UTR element of HIF1? ......................... 138?Figure 5.13    HIF1? protein stability in +/- YB-1 knockdown cells under hypoxia .................. 139?Figure 5.14    HIF1? is a potential YB-1 target mediating sarcoma cell invasive phenotype in vitro ............................................................................................................................................. 141?Figure 5.15    HIF1? is a potential YB-1 target mediating sarcoma cell metastasis in vivo ....... 143?Figure 5.16    YB-1-HIF1? axis potentially contributes to tumour angiogenesis ....................... 145?Figure 5.17    Total YB-1 rather than pYB-1 is induced under hypoxia .................................... 147?xvi  Figure 5.18    HIF1? expression in lung metastases of mice with +/- YB-1 knockdown xenografts..................................................................................................................................................... 148?Figure 5.19    The proposed mechanism of YB-1 mediated sarcoma metastasis ....................... 149?   xvii  List of Symbols  ? Alpha ? Beta ? Gamma                 xviii  List of Abbreviations  The following abbreviations have been used in the manuscript.   3' UTR  Three prime untranslated region 3D   3 dimensional 5' UTR  Five prime untranslated region A   Adenine Ab   Antibody ABC   Avidin/Biotin Complex ACC   Animal Care Committee ALDA   Aldolase A  ALK   Anaplastic lymphoma kinase  ARMS   Alveolar rhabdomyosarcoma ARNT   Aryl hydrocarbon receptor nuclear translocator  BCL2   B-cell lymphoma 2 C   Cytosine CAIX   Carbonic anhydrase IX CBP   CREB (cAMP response element-binding protein)-binding protein  CD44   Cluster of differentiation 44 CDK   Cyclin-dependent kinase CHX    Cyclohexamide  xix  COL1A1  Collagen, type I, alpha 1 CREB   cAMP response element-binding protein CRS   Cytoplasmic retention site  CSD   Cold shock domain CTRL   Control DAPI   4',6-diamidino-2-phenylindole DBPB   DNA-binding protein B   DFSP   Dermatofibrosarcoma protuberans DMEM  Dulbecco's Modified Eagle Media DNA   Deoxyribonucleic acid ECM   Extracellular matrix EDTA   Ethylenediaminetetraacetic acid EGF   Epidermal growth factor EGFR   Epidermal growth factor receptor  eIF4G   Eukaryotic initiation factors 4G elF4E   Eukaryotic initiation factors 4E EMT   Epithelial-to-mesenchymal transition ErbB2   V-erb-a erythroblastic leukemia viral oncogene homolog 2 ErbB4   V-erb-a erythroblastic leukemia viral oncogene homolog 4 EREG   Epiregulin  ERMS   Embryonal rhabdomyosarcoma ES   Ewing sarcoma ETS   E-twenty six xx  ETV6   ETS variant gene 6 EWSR1  Ewing sarcoma breakpoint region 1 FAK   Focal adhesion kinase FBS   Fetal bovine serum FGF   Fibroblast growth factor  FGFRs   Fibroblast growth factor receptors FKHR   Forkhead homolog 1 rhabdomyosarcoma  FLI1   Friend leukaemia virus integration 1 FOXO1  Forkhead box protein O1 FUS   Fused in Sarcoma G   Guanine G3BP   RasGAP SH3-binding protein GAPDH  Glyceraldehydes-3-phosphate dehydrogenase  GIST   Gastrointestinal stromal tumour  Glut-1   Glucose transporter 1  GTPase  Guanosine triphosphate hydrolase H&E   Hematoxylin & Eosin HA   Hyaluronan HB-EGF  Heparin-binding epidermal-growth-factor-like growth factor HIF1?   Hypoxia inducible factor 1 alpha HIF2?   Hypoxia inducible factor 2 alpha HRE   Hypoxia response element HuR   Human antigen R      xxi  IGF-1   Insulin like growth factor 1 IGF-1R  Insulin like growth factor 1 receptor  IHC   Immunohistochemistry IL6   Interleukin 6 iNOS   Inducible nitric oxide synthase  IPs   Immunoprecipitations IRES   Internal ribosome entry site Kb   Kilobase  Kd    Knockdown kDa    kilodalton  KO   Knock out LCL   Lower confidence limit LDHA   Lactate dehydrogenase A  Lin28   Abnormal cell lineage 28 Luc   Luciferase MAPK   Mitogen-activated protein kinases MCC   Multilayered cell culture MDM2  Murine double minute 2 MDR1   Multidrug resistance 1  MHC   Major histocompatability complex miRNA  microRNA MMP1   Matrix metalloproteinase 1 mRNA    Messenger ribonuceloprotein xxii  MSC   Mesenchymal stem cells  NBF   Neutral buffered formalin  NLS   Nuclear localization signals NOD-SCID  Nonobese diabetic-severe combined immunodeficient NSCLC  Non small cell lung cancer NSEP1  Nuclease sensitive element binding protein 1 NTRK3  Neutrophilic tyrosine kinase receptor, type3 O/N   Over night  OBs   Osteoblasts  OS   Osteosarcoma PARP   Poly (ADP-ribose) polymerase PBS   Phosphate-buffered saline  PCNA   Proliferating cell nuclear antigen  PCR   Polymerase chain reaction  PDGFRA   Platelet-derived growth factor receptor alpha PDGF?  Platelet-derived growth factor beta PGK-1   Phosphoglycerate kinase 1  PHD   Prolyl hydroxylase domain PI3K   Phosphatidylinositol 3' -kinase PKB   Protein Kinase B PTB   Polypyrimidine tract-binding protein  QRT-PCR  Quantitative real-time polymerase chain reaction  RAS   Rat sarcoma xxiii  RB1   Retinoblastoma 1 RIP   Ribonucleoprotein immunoprecipitation RMS   Rhabdomyosarcoma RNA   Ribonucleic acid RNP    Ribonucleoprotein ROS   Reactive oxygen species RPM   Revolutions per minute RPMI   Roswell Park Memorial Institute medium RSA   Renal subcapsular assay RT   Room temperature  SDS   Sodium dodecyl sulfate T   Thymine TAFII68  TATA-binding protein (TBP)-associated factor II68 TBS   Tris-buffered saline  TBST    Tris-buffered saline tween TET  Translocated in liposarcoma, Ewing sarcoma and TATA-binding  protein-associated factor 15 TFs Transcription factors TGF?   Transforming growth factor receptor beta TIA-1   T-cell internal antigen-1 TLS   Translocated in liposarcoma  TNF?   Tumour necrosis factor alpha TP53   Tumour protein 53  xxiv  UCL   Upper confidence limit VEGF   Vascular endothelial growth factor VHL    Von-Hippel Lindau xg   Times gravity YBX1 (YB-1)  Y box binding protein 1                 xxv  Acknowledgements  I am grateful for all the values my parents taught me which have always help me to pass through life events, and have supplied me with the strength to always proceed.  It is such a great honour to express my deep gratitude to Dr Poul Sorensen, my supervisor, for his wise guidance throughout this work, meticulous skilful supervision, and for his belief in me and my work. Also, I wish to express my sincere appreciation, and heartful thanks to my Supervisory Committee members, Dr Mladen Korbelik (Chair), Dr Torsten Nielsen, and Dr T. Michael Underhill (members) as well as Dr Colin Fyfe (former chair), for sharing their time and experience with me and for their help throughout this work.   No words can express my utmost thanks, respect, and gratitude to Dr Cristina Tognon, the senior lab scientist for her magnificent help, valuable advice, continuous encouragement, and never-failing enthusiasm. Further, I express my gratitude to all members of the Sorensen lab, notably Joan Mathers, Amy Li, Gabriel Leprivier, and Syam Somasekharan among many others whose support and experience were of great help.  I would like to express my deepest thanks to the Ministry of Higher Education, Egypt for supporting me with a generous 4-year scholarship.  I am forever indebted to my God father Dr Hesham EL-Naggar and to Dr Osama, Hany, and Ghada for their infinite sacrifices, inexhaustible giving flow and everlasting love. xxvi  I am always grateful to my son  Abdalrahman whose love and support have never let me down, and never allow me to give up.     I would also like to express my admiration to all clinicians & scientists throughout the human history-whose efforts were, are, and will be always illuminating the road in front of medicine in its difficult struggle to diagnose diseases, treat patients, and save lives. Amal EL-Naggar xxvii  Dedication       DEDICATED TO THE SOULS OF MY FATHER & MY MOTHER 1  Chapter  1: Background  1.1 Cancer metastasis: A preamble It is estimated that distant tumour metastasis is responsible for over ninety percent of cancer deaths1. Therefore, identifying factors that contribute to metastatic spread - and that can be targeted therapeutically- would have a tremendous impact on survival outcomes, and possibly reduce the toxicity of current chemotherapy regimens. It has been shown2, 3 that only a few overt metastases have the potential to develop out of the large number of cancer cells found in the blood stream. Therefore the process of metastasis is considered to be an inefficient process, particularly during the late steps of metastasis, including the initiation, growth, and formation of overt metastases at secondary organ sites. Dissemination of tumour cells takes place through three major routes including: spread via lymphatic vessels, a favoured route of most carcinomas; spread via blood vessels which is favoured by sarcomas; and spread via serosal surfaces as seen by a small group of tumours including mesothelioma and ovarian carcinomas4. Of interest, the drivers of oncogenic transformation, tumour initiation and survival of the metastatic tumour cells which may exhibit addiction to these genetic alterations5, are by themselves insufficient to establish metastatic lesions. Therefore, acquisition of additional genetic changes to allow cells to survive harsh local and distant microenvironmental conditions confronting tumour cells during the process of metastasis appears to be warranted6-8.  The metastatic process can be divided into distinct stages; initiation, progression, and homing with each stage is controlled by a specific subset of genes8, 9. For example the initiation phase is driven by genes that function to promote epithelial to mesenchymal transition (EMT), such as 2  Snail1, and Twist1 thus enhancing cell motility8, 10. While the progression phase, which is essential for tumour cell survival in the blood stream and for invading distant parenchyma, is driven by other distinct sets of mediators such as EREG, and MMP1 among many others8, 11, 12. The final stage of metastasis which is homing of the tumour cells to specific organs may be attributed to other factors such as IL6, and TNF?13. Extensive studies have demonstrated that there are many genes that contribute to more than one stage of metastasis progression and functional overlap is frequently encountered. For instance, MMPs were found to play a pivotal role not only in facilitating intravasation and extravasation, but also in affecting the ability of cancer cells to grow at secondary sites14, 15. Another example is exemplified by Snail1, and Twist1, two major players in the metastatic process, both of which play important roles at every stage of the metastatic process. For example, they enhance the early steps of metastasis, by promoting an epithelial to mesenchymal transition (EMT) resulting in local invasion, they also promote intravasation, transportation in the circulation, and extravasation. They are also important drivers of the late stages of metastasis, through their ability to promote the survival and proliferation of cancer cells at secondary organ sites, and formation of overt metastatic lesions16, 17. In addition, they stimulate angiogenesis8, 10.   Despite our enhanced understanding of the metastatic process, it is by no means fully understood and many questions remain to be answered. This gap in knowledge may be attributed to the difficulty in identifying and isolating metastatic lesions. Other factors that can also add to the complicated nature of the metastatic process include the interplay between tumour cells, and their surrounding microenvironment comprising the extracellular matrix components and stromal 3  cells which are considered to play an important role in a given tumour?s metastatic potential8, 18, 19.  1.1.1 The classic metastatic cascade  Cancer invasion is currently envisioned as an adaptive process in which tumour cells and their surrounding microenvironment influence one another in a reciprocal manner resulting in profound changes in cytoskeletal dynamics in tumour cells, cell adhesions, and mechanotransduction, and that eventually leads to cell migration/invasion into surrounding tissue. Adhesion and proteolysis play the primary role in coordination of these changes4. The individual cell-based sequential steps that together constitute the classic metastatic cascade are: (a) detachment of tumour cells from their primary mass, (b) invasion locally into the surrounding tissue, (c) intravasation into either lymphatic or blood vessels, survival in  the vascular system, and arrest at distant tissue/organs, (d) extravasation across the endothelium of the vascular system into the surrounding new parenchyma, and e) thriving in the new metastatic loci by adapting to the new microenvironment and/or subverting the new milieu to serve their needs20. Given the importance of tumour cell migration in the initiation of the metastatic cascade as well as the fundamental roles of adhesion, proteolysis and angiogenesis in metastasis, we will shed light on these aspects in the following sections.  1.1.1.1 Cell motility: A crucial component of the metastatic process Cell migration/and or invasion is an intricate multistep process, is usually provoked by a variety of stimuli, often extracellular, arising from nearby cells and/ or extracellular matrix (ECM), in addition to other diffusible factors. It has five distinct stages involving dynamic changes to 4  essential components; namely, cytoskeleton, cell-substrate adhesions and the extracellular matrix components. Further, the process of cell motility can be broken down into five distinct stages, including: 1) the formation of lamellipodial extension at the leading edge, 2) formation of new focal adhesions complexes, 3) ECM degradation via proteases, 4) actomyosin complex-induced cell body contraction, and finally 5) tail detachment4, 21. Different patterns of human cell motion have been reported that mediate tumour cells invasion into the surrounding microenvironment. The first is single cell motility which can be further subdivided into mesenchymal-type movement and amoeboid movement. The second is multicellular motility and includes collective migration patterns. Determining the type of cell motion is dependent on many variables such as extracellular protease activities, integrin-mediated cell-matrix adhesion, cadherin-mediated cell-cell adhesion, cell polarity and cytoskeletal arrangement. Cancer cells, owing to their plasticity and adaptability to microenvironmental changes, can switch between different kinds of motion21.   1.1.1.1.1 Single cell motility patterns Two types of single cell motility patterns have been described; mesenchymal and amoeboid motions. The mesenchymal-type motion is relatively slow kind of cell motion with average speeds of ~ 0.1-1 ?m/minute. It involves ECM degradation to create a path for the cell and is characterized by an elongated, spindle-shaped cell morphology like that observed for sarcoma cells.  Upon adequate stimulation, a cascade of events takes place to ensure coordinated actin polymerization at the front of the cell with microtubule attachment and alignment, eventually leading to formation of actin-rich protrusions with small integrin-dependent focal complexes that are formed, attaching the new protrusion to the ECM. The actinomyosin cellular contractility is transmitted to the ECM via focal adhesions accompanied with retraction of the lagging tail. 5  While mesenchymal cell motility exhibits degradation of the ECM and regulated extracellular proteolysis, amoeboid-type motion generally represents a non-proteolytic migratory pattern of movement. Cells displayed amoeboid motility are characterized by their epithelioid rounded morphology, and by their ability to squeeze themselves into gabs in ECM rather than degrading it21-23. This style of movement is largely independent of protease activity or integrin function and some carcinoma cells such as LS174T colon or A375m2 melanoma cells are known to use this movement style for their motility24.  1.1.1.1.2 Collective cell motility As the name indicates, this type of motion is executed by a group of cells maintaining their adherent junctions and they can maintain their connection to their tissue of origin or separated from it. Like single cell mesenchymal motion, these cells are dependent on protease-mediated ECM degradation and focal adhesions for their motility. Collective motility is a characteristic of specific tumour types such as lobular breast carcinoma, melanoma, and rhabdomyosarcoma23.   1.1.1.1.3 Tumour cell motility: A distinct paradigm of movement plasticity Tumour cells are characterized by their plasticity and as previously mentioned can shift from one type of movement to another. Further, upon exposure to microenvironmental cues, tumour cells can acquire de novo motility as a part of their adaptation strategy. As will be discussed later, epithelial-to-mesenchymal transition (EMT) is a program initiated by the invading/metastasizing carcinoma cells in which tumour cells lose their epithelial-like morphology and adopt a mesenchymal cell phenotype with a more migratory and  invasive nature, permitting them to invade through the ECM. These changes are driven by a series of transcription factors such as 6  Snail-1 and Snail-2 (Slug), Twist, ZEB-1-2, and YB-1. On contrast, tumour cells can switch from mesenchymal type motion to amoeboid motion in response to inhibition of integrin or proteases such as MMP thus sustaining the dissemination of tumour cells and this has been shown in fibrosarcoma and melanoma cells25, 26.   1.1.1.2 The distinctive role of tumour cell adhesion in metastasis Cell adhesion molecules contributing to cell-cell interaction or cell-extracellular matrix (ECM) interaction play a fundamental role in cancer metastasis. For initiating metastasis, tumour cells must first break free of the neighbouring cells. In normal tissue, cells are held together with a family of intercellular adhesion protein molecules called cadherins. Decreased cell adhesion observed in malignant cells is a prerequisite for successful metastasis.  While cell-cell interaction is mediated by cadherin family of proteins, cell-ECM interaction is mediated by the members of integrin family27.   1.1.1.2.1 Cadherins Cadherin family of proteins mediates cell adhesion through binding to one another, a process known as homotypic binding. E-cadherin and  N-cadherin are the most renowned members of the cadherin family. E-cadherin is the primary constituent of adherens junctions in epithelial cells. E-cadherin is characterized by a potent ability to recruit ?-catenin which at the plasma membrane, functions by linking E-cadherin to the cytoskeleton and stabilizing the adherens junctions. In addition, E-cadherin prevents ?-catenin nuclear localization and transactivation28. Extensive studies conducted in vitro and in vivo demonstrated that inhibition of E-cadherin in epithelial-derived malignant cells promoted their metastatic behaviour while enhanced 7  expression of E-cadherin led to a reduced invasive phenotype. Loss of E-cadherin expression/function may be attributed to several mechanisms including: 1) transcriptional repression by several factors that control E-cadherin expression such as Snail and Twist, 2)  disruption of cytoskeletal connections as a result of mutation in ?-catenin, 3) MMPs-mediated cleavage of E-cadherin extracellular domain4. Of interest, loss of E-cadherin is often accompanied by a gain of another cadherin; N-cadherin, in a process known as cadherin switch which is commonly observed in the process of epithelial-to-mesenchymal transition, a  mechanism by which epithelial cells acquire a more motile, fibroblast-like phenotype29. While E-cadherin inhibits the spread of tumour cells, N-cadherin represents a prominent contributor to invasion and metastasis. It functions by mediating interaction of N-cadherin expressing cells; tumour and stromal cells. In addition, interactions between N-cadherin and fibroblast growth factor receptors (FGFRs) contribute to activation of FGFR downstream signalling pathways implicated in tumour progression like phosphatidylinositol 3-kinase (PI3K) and mitogen-activated protein kinase (MAPK)30. Further, intracellular ?-secretase-mediated cleavage of N-cad leads to nuclear translocation of its carboxy terminus that binds to CBP (the cyclic AMP response element binding [CREB] binding protein) resulting in repression of CREB-binding protein-mediated transcription31.  1.1.1.2.2 Integrins Integrins are heterodimeric transmembrane adhesion receptors, composed of ? and ? subunits that anchor the cell to the ECM or other cells. Integrins are ubiquitously expressed on tumour cells, blood elements, and stromal cells suggesting a potential contribution to metastasis32.  Integrins play fundamental roles in cellular proliferation, migration, and invasion and numerous 8  studies have demonstrated that high expression levels of ?v?3, ?v?5, ?5?1, and ?6?4 integrins are correlated with metastatic progression and poor patients' survival33.   In mammals, eighteen ? and eight ? integrin subtypes have been recognized which associate to give rise to twenty-four different combination of integrin molecules The ? subunit determines ligand specificity while the ? subunit initiates intracellular signalling events. Upon ligand stimulation, integrins aggregate and bind to the actin cytoskeleton via adapter proteins such as talin and vinculin, and this forms the basis of focal adhesions which activate members of protein tyrosine kinases implicated in cellular adhesion and cellular migration, such as focal adhesion kinase (FAK) and Src kinase which in turn activate Rho GTPases that promote actin polymerization and formation of filopodia and lamellipodia27.   Indeed, integrins can associate with the epidermal growth factor receptors (EGFRs), ErbB2, and Met to enhance the growth of carcinomas. Further, EGF- and met-mediated ?4 phosphorylation result in disruption of hemidesmosomes and increased migration/invasion properties of malignant cells4. Moreover, integrin signalling disturbs adherens junctions via Snail/Slug mediated suppression of E-cadherin expression34.   Analogous to ?1 integrin function, the cell surface hyaluronan (HA) receptor CD44 promotes tumour cell adhesion and migration.  CD44 is ubiquitously expressed on the surface of many cell types with mesodermal and hematopoietic origin35. In sarcoma, a mesenchymal malignant neoplasm, CD44 plays an essential role in migration, invasion, and metastasis36. CD44 acts as a 9  cell surface scaffold for the assembly of various molecules, and it coordinates protease activity by localizing MMPs and their substrates on the cell surface37.    1.1.1.3 The role of proteolysis in cancer metastasis Degradation of natural barriers including basement membrane (BM) and ECM is virtually essential to the metastatic process, and is primarily conducted by different proteolytic enzymes produced by the invading tumour cells or by the surrounding stroma. MMPs constitute a major group of proteolytic enzymes that play crucial roles in metastasis. Given the structure and substrate specificity, MMPs are categorized into five groups: collagenases, gelatinases, stromelysins, matrilysins and membrane MMPs38. The BM is primarily composed of collagen IV which is only degraded by the transmembrane MMPs (MMP-14, MMP-15, and MMP-16), frequently upregulated in invading cancer cells, owing to their collagenase activity. In addition to the metastasis-promoting role, MMPs confer malignant cells with a more replicative capacity in vitro and in vivo, and stimulate angiogenesis via degrading the fibrin mesh-work encompassing the newly formed blood vessels39 and thus facilitating the entry of tumour cells into vascular spaces and vice versa. In addition to the direct metastasis-promoting role, MMPs interact with various adhesion molecules expressed on the cell surface, and form complexes with other crucial partners (e.g. CD44/MMP-9/TGF-?, HB-EGF/MMP7/ErbB4), thereby contributing to essential signalling pathways involved in survival, angiogenesis, and metastasis of tumour cells 4.  1.1.1.4 Angiogenesis and cancer metastasis Dynamic reciprocal interactions between tumour cells and their surrounding microenvironment play a substantial role in conferring metastatic ability. Tumour cells are distinctly characterized 10  by uncontrolled growth and high metabolic rates due to aberrant genetic events that lead to dysregulated growth. Indeed, they depend on their local microenvironment which supports their growth prior to the onset of metastatic spread. As a result of tumour progression, tumour cells will eventually outgrow their local blood supply and the immediate environment of cancer cells often becomes heterogeneous with areas of reduced oxygen levels, hypoxia, and nutrients. Exposure of tumour cells to these adverse conditions, notably hypoxia, can markedly influence their metastatic potential and their therapeutic response40-44. Tumour cells can respond to these metabolic stresses either by increasing supply, via promoting new tumour blood vessels formation/angiogenesis, or by acclimatizing to or tolerating the insufficiency45-48.  Angiogenesis is the process of recruiting new blood vessels to the expanding tumour tissue with the rate of proliferation of vascular endothelium is 20-2000 times faster in host-induced tumour endothelium than in normal tissue endothelium. Angiogenesis occurs in response to plenty of angiogenic factors released from the tumour or stromal cells and it is an essential component of tumour metastasis49. Besides being an indispensable source of nutrients and O2 for the rapidly dividing tumour cells to cope with the high growth and metabolic rates, and scavengers of their waste products, the newly formed blood vessels also provide an efficient route of entry into blood stream facilitated by basement membrane degradation and fewer functional intracellular connections50-52. Therefore, tumour vascular density is thought to provide a good prognostic indicator of metastatic potential for many solid malignancies53-57 and extensive experimental evidence has helped to prove a positive correlation between angiogenesis and tumour metastasis58-60 . Tumour vasculature is distinctly characterized by irregularity, heterogeneity in cell composition and increased leakiness denoting destruction of vascular integrity. Vascular 11  endothelial growth factor (VEGF) family members, and members of fibroblast growth factor (FGF) among other factors, either produced by the tumour cells or the host cells, provide potent stimuli triggering tumour angiogenesis56, 61. Angiogenesis in not only essential for initiating metastasis, but also for promoting metastatic colony growth, a finding largely supported by extensive in vivo studies which revealed that small diameter micrometastases are virtually avascular and once angiogenesis is turned on neovascularization takes place and tumour growth recovers56, 62, 63.   Of similar importance to angiogenic stimulators are angiogenic inhibitors produced by tumour cells such as angiostatins60 or naturally produced by host cells such as thrombospondin64. The angiogenic balance of stimulators and inhibitors plays essential role in controlling angiogenesis, and once the inhibitory signals are lost, the dormant metastatic colonies become vascularized and grow rapidly60, 65. It is therefore not surprising that the use of anti-angiogenic therapy in experimental models was found to potentially inhibit the progression of metastatic lesions66, and nowadays some anti-angiogenic therapies are successfully used in the clinical setting in conjunction with  conventional chemotherapeutic regimen such as Bevacizumab used for advanced non-small cell lung (NSCL) cancer67.  Given the fundamental role of the hypoxic tumour microenvironment in eliciting angiogenesis and enhancing the metastatic potential of most of solid tumours68, 69, it is essential to highlight the master transcriptional regulator of the adaptive response to hypoxia, hypoxia inducible factor1? (HIF1?), which represents one of the main proteins I have studied in my research project.   12  1.2 HIF1?: The master regulator of the hypoxic response Oxygen, aside from being vital to biological processes, represents an important regulator of signalling pathways that control the expression of specific genetic programs. Hypoxia is a state of low oxygen tension and cells adapt to hypoxia using mediators such as hypoxia inducible factors (HIFs). HIFs are heterodimeric transcription factors consisting of ? and ? subunits. To date, three ? subunits (HIF1?, HIF2?, and HIF3? ) and one ? subunit (HIF1?). These proteins belong to the basic helix-loop-helix-Per-ARNT-Sim (bHLH-PAS) protein family. In addition, they harbour two transactivation domains, N-terminal (N-TAD) and C-terminal (C-TAD). Of interest, HIF3? retains the N-terminal transactivation domain (NTAD) while lacking the C-terminal transactivation domain (CTAD) resulting in a weaker transcriptional activity than that of HIF1? and HIF2?, and therefore, it is considered as a competitor against HIF1? or HIF2?  by recruiting a common partner, HIF1?70. Schematic illustration of HIF family member protein domains is shown in (Fig. 1.1).    Hypoxia induces stabilization of the HIFs, which direct responses central to the survival and expansion of the malignant cell population. Hypoxia-inducible factor 1 (HIF1) is a heterodimeric transcription factor complex that is largely stabilized in malignant neoplasms due to intratumoral hypoxia. It is composed of two subunits, a regulatory hypoxia-inducible alpha subunit and a constitutively expressed beta subunit. HIF1 activation depends primarily on the accumulation of HIF1? in response to hypoxia, while HIF1? levels are often unaffected by hypoxia71. Under normoxic conditions (21% O2) HIF1? is hydroxylated by dioxygenase members; prolyl hydroxylases, namely PHD1, PHD2, and PHD3. Following hydroxylation HIF1? is subjected to ubiquitination and proteasomal degradation by a pVHL containing E3 ubiquitin ligase72, 73 which 13  recognizes two hydroxylated proline residues of HIF1? (P402 and P564)74, 75. Furthermore, asparaginyl hydroxylation takes place at N803 within the HIF1? C-terminal transactivation domain and prevents recruitment of the transcriptional co-activator p300/CBP, thus inhibiting HIF1? transactivation76. On the contrary, under hypoxic conditions, the hydroxylation process is inhibited due to limited oxygen availability resulting in HIF1? stabilization which initiates the HIF1? activation cascade involving dimerization with HIF1?, also known as aryl hydrocarbon receptor nuclear translocator (ARNT), recruitment of p300/CBP, and binding to specific hypoxia-responsive elements (HREs) within promoters of target genes which in turn activate the transcription of various genes needed to respond to hypoxic conditions77.   HIF2? is closely related to HIF1?. Both of them share similar functional domains, substantial sequence homology, and some overlapping functions; however, each has its own gene expression profile, and certain target genes are uniquely regulated by either HIF1? or HIF2?78.  Hypoxia has been shown to confer malignant cells with a more aggressive phenotype and resistance to therapeutic drugs and numerous studies have shown that HIF1? plays definite roles in tumorigenesis via regulating a multitude of genes whose functions contribute to cell proliferation, angiogenesis, invasion, and glycolysis79-81. Some well known HIF1 responsive genes include phosphoglycerate kinase 1 (PGK-1), glucose transporter 1 (Glut-1), lactate dehydrogenase A (LDHA), aldolase A (ALDA), glyceraldehydes-3-phosphate dehydrogenase (GAPDH), carbonic anhydrase IX (CA IX), vascular endothelial growth factor (VEGF) and inducible nitric oxide synthase (iNOS)82.   14  At the cellular level, the hypoxic response mediated through HIF1? can have two opposing effects: 1) it can serve as a stress factor inhibiting cell cycle progression and activating the caspase cascade leading to apoptosis and/or necrosis. This is usually seen with anoxia; complete lack of O2, or prolonged exposure to hypoxia, resulting in shutdown of the main metabolic pathways and activation of death machinery; or 2) it can function as an initiating factor for tumour progression and resistance to traditional treatment modalities68. Most studies have demonstrated that the second role is the predominant one. Hypoxia has also been shown to promote genetic instability leading to mutations in oncogenes and tumour suppressor genes in addition to facilitating local invasion and metastatic spread68, 69. Further, numerous studies have demonstrated the potential role of HIF1? in EMT induction as well as metastatic dissemination of tumour cells through various mechanisms83-86.  In the context of sarcomas, HIF1? was found to play important roles in sarcoma biology, and its elevated expression levels are associated with shorter overall and disease free survival87. Recently we have shown that HIF1? rather than HIF2? contributes to sarcoma progression in osteosarcoma. We have detected enhanced HIF1? expression in the malignant cells compared to non-malignant mesenchymal derived osteoblasts and that seems to be crucial for conferring resistance against cell death under both normoxic and hypoxic conditions. Further, we found a correlation between the enhanced HIF1? expression and the advanced disease grade, indicating a potential role of HIF1? in osteosarcoma progression88.     Indeed, enhanced HIF1? expression and activation of its signalling pathways during hypoxia is accompanied with reprogramming of the translational machinery, in which there is inhibition of cap-dependent translation and simultaneous activation of cap-independent translation of certain mRNAs including HIF1? itself, VEGF-A, and BCL2 whose function to support survival, inhibit 15  apoptosis, and facilitate the escape of tumour cells through angiogenesis89. One of the major general suppressor of cap-dependent translation under stress conditions 90, 91, is YB-1 whose downstream targets such as FOXO3a, TOB2, ZEB2 and Snail1 possess anti-proliferative and anti-apoptotic functions allowing cell survival in response to a variety of stresses including hypoxia92-96.   Although RNA-binding proteins including YB-1 are implicated in the hypoxic response97, however extensive investigation into the YB-1-hypoxia-HIF1? relationship and how this may possibly impact childhood sarcoma metastasis are currently lacking. Extensive evidences support a link between YB-1 and HIF1?. Indeed, both of them promote metastatic dissemination in a wide variety of cancers10, 98-101 potentially through the same downstream mediators, such as Twist10, 83.  Of note, YB-1 and HIF1? possess non-redundant roles in early mammalian development, and corresponding knockout (KO) mice show similar trends of progressive mortality and lethality at embryonic day E10.5 for YB-1 and E11 for HIF1?102-104.    Further, YB-1 was found to promote microtubule assembly105, which is intricately involved in orchestrating HIF1? translation106. Importantly, in our previous study of breast cancer, we found that HIF1? expression is increased in YB-1 expressing cells and this was associated with its enrichment in the polysomes10 and in the context of sarcomas, we found that inhibiting YB-1 expression has led to significant reduction of HIF1? protein levels, two crucial findings supporting a potential role of YB-1 in mediating HIF1? regulation, and hence contribution to the hypoxic response. In the following section, I will introduce YB-1, the main focus of my research and highlight its potential functions related to this project.  16  1.3 Y box binding protein 1 (YB-1) Y-box binding protein 1 (YB-1; also known as YBX-1, DBPB, or NSEP1), located on chromosome 1p34, is a member of the highly conserved DNA/RNA-binding family of cold-shock domain (CSD) -containing proteins that also includes the Let-7 miRNA inactivating proteins, Lin28 and Lin28B107-110. Consistent with its biologically fundamental functions, YB-1 disruption in mice causes severe developmental defects and embryonic lethality108. Structural analysis of the YB-1 gene revealed 8 exons spanning 19kb of genomic DNA transcribed into 1.5 kb long mRNA encoding a 43 kDa protein (324 amino acids)111, 112. Several transcription initiation sites as well as an extremely long 5' UTR seem to be involved in the regulation of gene expression111. YB-1 was initially isolated as a transcription factor binding to the consensus Y-box sequence, 5'-CTGATTGG-3', present in the promoter region of the MHC class II genes, and hence its name113. Other genes with the Y-box sequence in their cis-regulatory elements were identified including thymidine kinase, proliferating cell nuclear antigen (PCNA), DNA polymerase ?, epidermal growth factor receptor (EGFR), and multidrug resistance 1 (MDR1)114. Thus, YB-1 is involved in the activation and repression of many genes115.  1.3.1 YB-1 protein structure Mammalian YB-1 has three domains; highly conserved CSD flanked by N- and C- terminal domains (Fig. 1.2). The N-terminus, involved in trans-activation, is rich in alanine and proline residues and has an actin binding motif. The CSD, 80 amino acid long, is a five stranded ?-barrel structure recognizing both DNA and RNA and has two conserved RNP motifs believed to be essential for transport and translational control of mRNA109-111, 116. Indeed, CSD proteins, highly conserved across species from bacteria to human, are ubiquitously expressed and involved in 17  fundamental processes such as DNA repair, mRNA transcription, splicing, translation and stabilization. The vertebrate CSD shares over 45% identity with bacterial CSD107, 109, 110. The C-terminus region is formed of alternating clusters of basic and acidic amino acids potentially mediates protein-protein interaction and is involved in DNA/RNA binding as well111, 117. In addition, the C-terminus harbours nuclear localization signals (NLS) and a cytoplasmic retention site (CRS) for controlling YB-1 cellular localization118. YB-1 protein modifications involving phosphorylation, acetylation, and ubiquitylation are thought to regulate its biological activity111.    1.3.2 The role of YB-1 in DNA transcription YB-1 is a multifunctional protein involved in almost all vital cellular processes. Transcription is the first crucial step in gene expression. It has been long believed that YB-1 stimulates and inhibits the transcription of a multitude of genes including those contributing to cell growth, apoptosis, immune response, and stress response among many others107. Of interest, YB-1 binds to single- or double- stranded DNA either directly or through other DNA-binding proteins which serve as a bridge between YB-1 and its DNA target sequences. In double-stranded DNA, the consensus Y-box element; CCAAT, located in gene promoters is the binding site for YB-1, whereas it is not a prerequisite for YB-1 binding to single stranded DNA. Instead, it binds to pyrimidine- rich DNA sequences. There is a growing list of genes whose expression are transcriptionally regulated by YB-1, of which MDR1 has been thoroughly investigated107, 119, 120, 113, 121.   The MDR1 gene encodes P-glycoprotein responsible for outward transport of chemotherapeutic drug in resistant tumour cells. It has been shown that the promoter activity of MDR1 gene 18  increases in Y-box dependent manner in response to anticancer agents and ultraviolet irradiation122, 123. Surprisingly, however, some studies found that YB-1 is unable to interact with the double-stranded MDR1 Y-box oligonucleotide in nuclear extracts124, 125. Likewise, YB-1 binds the Y-box element located in the promoters of growth and proliferation related genes such as Cyclin A and Cyclin B1126, thus stimulating their expressions, and that YB-1 targeted disruption has led to defect in cell cycle and retarded growth127. Therefore, YB-1 is considered to be an oncogene and it is implicated in cancer progression. The expanding list of YB-1 regulated genes is reviewed in:107, 82.   YB-1 is no doubt a marker of cancer progression as supported by extensive evidence. However, the transcriptional activity of YB-1 becomes a matter of debate. Recently it has been shown that YB-1 does not bind to Y/CCAAT boxes in vivo128. Further, our previous study of breast cancer has supported the role of YB-1 as a major translational regulator rather than as a transcriptional factor. Recent studies highlight YB-1 as a novel translational target of mTORC1 signalling 129, and as a prominent translationally activated driver of mTORC1-mediated prostate cancer cell invasive capacity 130. These findings highlight the controversy surrounding YB-1?s role as a transcriptional activator.  1.3.3 The role of YB-1 in mRNA translation Gene expression is an essential process that requires DNA transcription to generate mRNAs which are then translated into proteins. While transcription coupled translation is the classical gene expression mechanism, under conditions that require immediate cellular response to specific environmental signals, waiting for transcriptional activation is not ideal. Thus, the 19  presence of ready-to-use competent mRNAs pools poised for translation is an efficient strategy utilized by cells to respond to different environmental cues. Microenvironmental stresses such as hypoxia, rapid phases of cell growth and division such as those found during development where rapid cell cycling may hinder transcriptional activity, or during growth phases where chromatin is inaccessible due to remodelling, are all common examples of circumstances that necessitate the presence of a large repository of translationally inactive/silent mRNA to provide a short cut for gene expression and allowing response to stimuli without transcriptional activity131. As an example, oocytes possess an accumulation of maternal mRNA for use during early embryogenesis132. Likewise, pachytene spermatocytes show accumulation of paternal mRNA in pachytene spermatocytes and haploid spermatids for use in spermatogenesis133.   In eukaryotes, translation of mRNAs takes place mainly through cap-dependent mechanisms in which the translation initiation step is the critical rate-limiting step that primarily relies upon availability of elF4E. A major fraction of the mRNA species, specifically growth-promoting and stress-inducible mRNAs with highly structured 5' UTR regions, are less amenable to translation initiation and are stored as translationally inactive mRNA particles (mRNPs) allowing for adequately prompt cellular response to environmental cues134, 135. YB-1 has been shown to be an integral component of mRNPs with the potential to bind up to 20% of the total mRNAs within a cell. Essentially, YB-1 competes with elF4E for binding to the capped 5' mRNA terminus thus inducing translational silencing91, 136. Indeed, YB-1 contributes to translational regulation either by stimulation or repression. These two mechanisms depend on the YB-1/mRNA ratio. Specifically, at a low ratio YB-1 enhances translation, likely via acting as chaperone. This is a characteristic feature of CSD proteins since they drive the melting and annealing of RNA 20  secondary structures137, 138,139, 140, or possibly, as also reported, through their association with RNA helicases whose purpose is to unwind nucleic acids141. While at higher ratios, YB-1 drives a protein synthesis shut down mechanism, potentially through competing with eIF4E and eIF4G for the association with the cap structure, therefore inhibiting cap-dependent protein synthesis at the very early initiation step142, 143.    Mechanistically, extensive studies have showed that the silent species of competent mRNAs are commonly bound by CSD containing RNPs144 through stacking of aromatic side chain with particular RNA bases and through extensive hydrogen bonding145, 146 thus protecting them from the cellular degradation machinery. Under stress conditions including hypoxia, YB-1 globally suppresses  cap-dependent translation to block cell growth and proliferation, a crucial energy conserving response.  This allows for the appropriate cellular adaptive response to prevail90, 91. The non-translating mRNAs are temporarily stored in cytoplasmic structures called stress granules (SGs), which will be discussed later, for future storage, degradation, or reinitiation after stress termination and resuming the regular cell translational activity147-149. YB-1?s downstream targets are known to play crucial roles in cellular adaptive responses to various stresses92-96, 134. Resuming cap-dependent translational activity after surmounting various microenvironmental cues is an additional, equally important function led by YB-1. YB-1 acts as a safeguard of repressed mRNAs, maintaining their competence and availability, allowing for restoration of their cap-dependent translation under more suitable conditions. YB-1 phosphorylation by the serine/threonine protein kinase AKT/PKB at the Ser-102 residue provides a potential mechanism for releasing the stored mRNAs from its association with YB-1136, 150. In conclusion, YB-1 potentially competes with eIF4E under stress conditions, providing protection of mRNAs subsets 21  via silencing and storage, and finally ensuring their availability in response to activation of the PI3K-AKT pathway134.  1.3.4 YB-1 and cancer YB-1 is expressed in a wide variety of normal tissues. Despite being crucial for survival and other biological processes at low levels, enhanced YB-1 expression has been shown to play an important role in malignant transformation, cell invasion, and drug resistance in wide variety of cancers such as breast, prostate, glioblastoma multiforme, osteosarcoma, and synovial sarcoma10, 151-154. The localization of YB-1 protein within a cell varies and this significantly impacts its functions. While nuclear localization of YB-1 is associated with transcriptional activation of proliferation-related genes through binding to their Y-box promoter elements, cytosolic YB-1 binds growth-related messages thus conferring translational silencing150, 155, 156. Of interest, YB-1 is linked to all almost all hallmarks of cancer proposed by Hanahan and Weinberg157 including limitless replicative potential, insensitivity to anti-growth signalling, evasion of apoptosis, sustained angiogenesis, invasion and metastasis, deregulated metabolic pathways, evasion immune destruction, as well as enabling genomic instability and promoting inflammation (reviewed in120). In this review, YB-1, a bona fide oncoprotein, has been described as a master regulator of cancer cell biology through its contribution to the fundamental criteria of tumour cells, thus providing a very attractive therapeutic target.  Our group recently showed that increased YB-1 expression in H-Ras transformed breast cancer cells reduces cell growth by repressing growth-related mRNAs, while simultaneously inducing an EMT and increasing their metastatic ability through translational activation of Snail1 and 22  Twist  mRNAs10.  While these studies on YB-1 in epithelial-derived malignancies have revealed many of its fundamental roles 10, 98, 151, 158, extensive investigation into YB-1?s potential contribution to childhood sarcomas are currently lacking, with only few reports linking upregulated YB-1 expression to drug resistance and poor prognosis153, 154. Of interest, both of physiological processes such as morphogenesis and pathological ones like EMT require translational reprogramming entailing inhibition of growth and proliferation related mRNAs and enhancing stress related mRNAs. Repression of cell growth and proliferation related mRNAs provide a selective advantage during these processes92, 159. Energy conservation in migrating/invading cells is related to the anabolic function of protein synthesis, conferring anoikis resistance during detachment from the extracellular matrix160, and eliminating the possible detrimental effect of improper cell division which are all major advantages afforded by translational reprogramming during EMT134.  1.3.4.1 YB-1, stress granules, and cancer Stress granules (SGs) are distinct large nonmembranous cytoplasmic aggregates formed primarily of ribonucleoprotein complexes that are assembled, both in vitro and in vivo, in cells exposed to various types of stress such as hypoxia, oxidative stress, and UV irradiation, all of which inhibit protein synthesis at the initiation phase147. In order to minimize stress-mediated mRNA damage, and to conserve energy for initiating prompt repair strategies to alleviate the deleterious molecular damage of such environmental cues, the cap-dependent translation gets compromised, and only a subset of mRNAs are selectively translated whose protein products are required for the repair process such as DNA-repair proteins and chaperone proteins, whereas the non-translatable mRNAs are temporarily stored in the SGs for further processing147-149, as 23  previously mentioned. SG formation is a highly conserved cellular response often triggered by phosphorylation of eIF2?, a component of the ternary complex involved in initiation codon recognition. However, SGs assembly can also occur independently of eIF2? phosphorylation. Extensive studies and compelling evidences have demonstrated that SGs protein components are highly dynamic, shuttle between the cytoplasm and SGs, a process highly orchestrated by the microtubules which in addition plays major roles in the SGs assembly, coalescence, and disassembly147, 161. The cytoprotective effect of SGs is supported by numerous studies which demonstrated that of proteins sequestered within the SGs are those involved in inflammation and apoptotic signaling such as RACK1, thus SGs promote cell survival162. In addition, interference with SGs assembly has invariably led to enhanced stress-induced cell death163, 164. Indeed, compelling evidences have demonstrated that SGs are implicated in cancer biology, and it confers survival advantage and chemotherapeutic resistance to the tumour cells, notably in hypoxic tumour cells which represent the main pool of metastatic colonies162, 165.   YB-1 has been previously reported in the stress granules166, 167 and established as a prominent SGs marker168. Of interest, our group recently found that YB-1 is not only a prerequisite for the SG assembly but also it regulates the expression of a well-known SG proteins TIA-1 and G3BP169, 170 which potentially contribute to the assembly, composition, and function of the SGs (manuscript under preparation). Taken together, these findings provide strong evidence of  multiple fundamental roles for YB-1 in cancer progression.  In the current study, we provide a detailed in-depth investigation into the potential contribution of YB-1 in sarcomatogenesis, notably to the metastatic phenotype, using the three dominant 24  childhood sarcoma subtypes, rhabdomyosarcoma, osteosarcoma and Ewing sarcoma in our study model and demonstrate how YB-1 contributes to the cellular hypoxic response that affects the metastatic phenotype in order to gain a better understanding of the role of YB-1 plays in childhood sarcoma progression.   In the following section, I will briefly discuss sarcomas, the cancer model utilized in my research project, with emphasis on the three dominant childhood sarcoma subtypes.   1.4 Sarcomas Sarcomas comprise a heterogeneous group of malignancies of mesenchymal origin that affect bone and soft tissues and often display highly aggressive behaviour with a propensity for early haematogenous metastasis. To date, sarcomas remain a challenging malignancy to treat. They are relatively rare, accounting for 1% of all cancers and are categorized as tumours arising primarily from the bone such as osteosarcoma (OS) and Ewing sarcoma (ES) and those that arise from soft tissues such as rhabdomyosarcoma (RMS). Despite occurring throughout the life spectrum, these tumours are proportionally more common in paediatric compared to adult age groups as they account for up to 20% of all paediatric malignancies171, 172.   Indeed, many etiological factors and genetic aberrations may play a role in sarcoma development, and as a result of rapidly evolved genomic and proteomic technologies, our understanding of the oncogenic mechanisms underlying sarcomatogenesis is being markedly improved. The majority of sarcomas are sporadic with unknown aetiology. However, certain hereditary syndromes such as Familial Retinoblastoma, as well as environmental factors such as 25  ionising radiation play a role in specific types of sarcomas173, 174. In general, the management strategies for sarcomas involve chemotherapy followed by surgery and/or radiation. Modern multi-agent chemotherapy regimens have made tremendous improvements to the outcomes of patients with localized high-risk disease such as RMS. However, the prognosis for patients with metastatic disease remains dismal175-178. Therefore a better understanding of the signalling pathways that impact metastatic spread has tremendous potential to reduce disease burden in childhood sarcoma.  1.4.1 Sarcoma genetics  The precise cellular origin of sarcomas remains largely unknown. However, it is widely believed that sarcomas are developed as a result of genetic mutations in mesenchymal progenitor/stem cells179. Compelling evidences suggest that sarcomas can be categorized into two distinct genetic groups: (1) those with simple karyotypic defects, and (2) those with complex karyotypic defects. ES represents the prototype of the first group while osteosarcoma represents the prototype of the second group180, 181.   1.4.1.1 Simple karyotypic defect- based sarcomas Indeed, simple karyotypic defects include recurrent chromosomal translocations, chromosomal amplifications, and specific oncogenic mutations. The outcome of these genetic alterations may vary from production of novel chimeric transcription factors, increased gene expression, or the alteration of signalling pathway by affecting the function of specific signalling proteins180. Typically, recurrent disease-specific chromosomal translocations, often occurring singly in an otherwise chromosomally normal background, are the prototype of the simple karyotypic defect. 26  It results in the joining of parts of two different genes to produce a chimeric fusion that encodes aberrant transcription factor harbouring both a powerful transactivating domain and DNA binding, and drives cellular transformation by altering the gene expression profile of susceptible cells. By far, this is the most dominantly observed genetic event. The prototype of this category is ES in which the transcriptional regulatory domain of EWSR1 fuses to the DNA-binding domain of FLI1 to generate a powerful transcription factor; the EWSR1-FLI1 chimera182.   Using heterologous system to study functions of oncoproteins has provided insights into their roles in cellular transformation and trans-differentiation179.  Indeed, it is believed that TET family including EWSR1, TLS/FUS, and TAFII68 contributes to over fifty percent of the chimeric fusions179. The recurrent genetic events represent a powerful diagnostic tool in many instances as they are considered to be unique to a particular subtype of sarcoma such as ETV6-NTRK3  which is found in congenital fibrosarcoma183, and SS18-SSX which is found in synovial sarcoma184. Furthermore, some of the chromosomal specific translocations may have prognostic value as well, such as PAX7-FOXO1A fusion that has been reported to be associated with better prognosis compared to PAX3-FOXO1A in ARMS185, 186.   Less frequently, the fusion oncoprotein may result in growth factor overexpression such as PDGF? observed in dermatofibrosarcoma protuberans (DFSP) as a result of fusion of the COL1A1 promoter with the PDGF? coding sequence187. Additionally, chromosomal translocations may affect signalling pathways as in inflammatory myofibroblastic tumours which display constitutive activation of proliferation and survival pathways as a result of a chimeric fusion that encodes a functional ALK receptor tyrosine kinase188, 189. Specific oncogenic 27  mutations have been reported in certain sarcoma subtypes such as GIST with activating mutations in the KIT growth factor receptor or PDGFRA190. Chromosomal amplification has also been reported in some kinds of sarcomas such as well-differentiated liposarcoma191, 192. In such cases, there is amplification of q14~15 of chromosome 12 resulting in overexpression of certain gene loci, including MDM2 and CDK4191.    1.4.1.2 Complex karyotypic defect- based sarcomas This group of sarcomas is characterized by high genomic instability featuring multiple chromosomal duplication, deletions, and complex rearrangements180. P53 mutations are frequently observed with this group as well as loss of telomeres which is believed to be responsible for repeated chromosome breakage and fusion during mitosis193. Additional genetic events have been identified including mutation in oncogenes such as RAS or tumour suppressor genes including RB1, however, their diagnostic utility remains questionable194.   1.4.2 Molecular diagnosis of sarcomas  There is an ever expanding array of molecular tests used for their diagnostic utility and prognostic value in sarcomas, and can be easily conducted on fresh or formalin fixed, paraffin embedded tissue biopsies. Over the years, the classic cytogenetic analysis has been used to identify the recurrent chromosomal translocation. However, significant limitations of this technique were encountered. Later, the introduction of new molecular diagnostic techniques such as fluorescence in-situ hybridization (FISH) and PCR-based methods has solved many of the previously encountered limitations and enabled identification of specific molecular aberrations on gene level195, 196.   28  1.4.3 Sarcomas subtypes While sarcomas comprises over 50 histologic subtypes, ES, OS, and RMS are the most common sarcomas of childhood and adolescence197, 198. We will focus on these three dominant sarcoma subtypes representing our study model.  1.4.3.1 Osteosarcoma (OS) OS is the most common primary malignancy of bone, and comprises ~35% of all bone tumours. It is predominantly a disease of adolescence and young adulthood, with 60% of patients aged less than 25 years at diagnosis; however there is a second peak of incidence in later life, with 30% of patients being over 40 years of age. Typically, OS arises from fast-growing bone plates such as metaphysis of long bones. OS have a spectrum of gross appearance, ranging from well circumscribed to infiltrate, and display areas of dense yellow-white sclerosis to gray-tan pumice-like granularity199, 200. The histologic appearance is similarly wide ranging. There is a pleomorphic composition of cells which appear small and round, clear, multinucleated, spindled, epithelioid, plasmacytoid, or fusiform. Histopathologic diagnosis of OS relies on the presence of osteoid, which is comprised of homogenous pink, structureless extracellular material produced by the tumour cells. As can be expected from their heterogeneous appearance, OS have no consistent genetic abnormalities201. There are several sub-types of OS described, which have in common the production of osteoid by the malignant cells and the potential for systemic metastasis202.   The aetiology of OS is poorly understood, and most cases are sporadic203, but certain environmental and hereditary factors are associated with OS204, 205. Admittedly, RB1 and TP53 29  are two well-characterized genes implicated in OS as demonstrated by extensive studies. TP53 is commonly altered in OS either by allelic loss, point mutations or gene rearrangements206-208. Further, RB1, located at chromosome 13q14 and producing a110 kDa protein, negatively regulates cell cycle progression from G0/G1 to the S phase209, 210. Up to 60% of high grade OS show TP53 mutations, compared with 1% of low grade OS211, 212. The current treatment strategy includes surgery combined with pre- and postoperative chemotherapy. With this multidisciplinary approach, long-term survival has increased to 70%, but patients who have recurrent disease or metastatic lesions (typically to lungs) at diagnosis have a lower survival rate of 20%213, 214.   1.4.3.2 Ewing sarcoma (ES) ES is a malignant cancer of soft tissue or bone predominantly affecting children, adolescents and young adults with ~ 70% of cases present before the age of 20. It is ranked the second most common tumour in childhood and represents 3% of all paediatric malignancies215, 216. The exact cell of origin for ES is uncertain but there is some evidence to suggest the possibility that it may originate from primitive neuroectodermal or mesodermal cells, or marrow mesenchymal stem cells (MSC)217-220. ES typically presents with localized pain and swelling with an associated soft tissue mass221. Several factors have been identified that are associated with prognosis. The most important negative prognostic factor in ES is the presence of metastatic disease. Other negative prognostic factors include tumours of the axial skeleton, especially the pelvic bones, large primary tumours (>8 cm in greatest diameter and >200 mL volume), elevated lactate dehydrogenase, and older age. Even though only 25% of patients with ES present with overt metastatic disease, most will ultimately fail therapy if not treated with systemic agents222.  30  ES is characterized by etiologic gene fusions of EWS to different members of the ETS transcription factor family, and the expression of these chimeric EWS-ETS fusion proteins is pathognomonic of the disease223-225. Moreover, numerous studies have dissected how EWS-ETS fusion proteins impart malignant behaviour to transformed cells226-228.   ES is a highly invasive tumour with nearly twenty five percent of cases presenting with metastatic spread to the lung, bone marrow and other tissues at the time of diagnosis229. As for other sarcomas, the most unfavourable prognostic factor for ES is the presence of distant metastases at the time of initial diagnosis, or their subsequent development230. While revealing important insights into disease pathogenesis and establishing new molecular tools for diagnosis, these findings have not impacted upon treatment and outcome. Firstly, as chimeric transcription factors, EWS-ETS chimeras are challenging to target, although several recent reports have employed several approaches to reveal potential downstream pathways, which may be more promising231-233. Secondly, translocations are found in both localized and metastatic disease, and thus do not on their own account for metastatic behaviour230, 234.   The treatment modality combines induction systemic multi-agent chemotherapy, followed by local treatment and then further chemotherapy. Surgery, radiotherapy or both are used for local control of the primary tumour. Weighting the benefits versus risks determines the best approach for the treatment strategy222. Of interest, insulin like growth factor 1 receptor (IGF-1R) has been explored as a therapeutic target in ES and IGF-1R inhibitors are currently undergoing evaluation in clinical trials235. The 5-year event-free survival rate for patients with localized ES is 50% to 75%, with disease-free survival reflecting the initial disease characteristics at diagnosis. The 5-31  year disease-free survival rate is 20% to 25% for patients with metastatic disease at diagnosis236, 237.   1.4.3.3 Rhabdomyosarcoma (RMS) RMS is the most common STS of childhood, arising in limbs, the central axis, or head and neck areas, and comprising >50% of STSs in children. Two thirds of cases occur before the age of 6 years, with a second peak occurring during mid-adolescence. RMS has different histologic variants; embryonal and alveolar subtypes are the most common while botryoid and pleomorphic subtypes are less frequent. The embryonal subtype (~70% of RMS cases) tends to affect infants and toddlers, whereas the alveolar subtype (~20% of RMS cases) affects all age groups. The botryoid and pleomorphic subtypes (~10% of RMS cases) commonly affect adults198, 238. The genetic mechanism underlying the pathogenesis of the alveolar subtype is the characteristic chromosomal translocations: t(2;13)(q35-37;q14) and t(1;13)(p36;q14), producing PAX3-FKHR and PAX7-FKHR gene fusions, respectively239-241.   Although RMS most commonly occurs as a mass, the presenting signs and symptoms are related to the anatomic site of the primary tumour. The most common locations of primary disease are the head and neck region, genitourinary tract, and extremities 242, 243, and survival is poor for patients with distant tumour spread 244, 245. Of note, a common feature of the previously described childhood sarcoma subtypes is that metastatic spread is the single-most powerful predictor of poor outcome in each tumour type.   32  1.5 Figures   Figure 1.1    The domain structure of HIF family of proteins Schematic representation of HIF family member protein domains. The bHLH: basic helix-loop helix; PAS: Per-ARNT-Sim; ODD: oxygen-dependent degradation domain; TAD: transactivation domain; N: N-terminus; C: C-terminus, P402 and P564: proline residue 402 and 564, respectively; N803: Asparaginyl residue 803.   33    Figure 1.2   The domain structure of YB-1 protein and its functions Schematic illustration of different YB-1 protein domains and their contribution to major cellular processes.  34  Chapter  2: Hypothesis and study objectives  While sarcomas are rare tumours in adults, they are rather prevalent in children and extensive efforts are directed towards understanding disease pathogenesis and improving its outcome. Very few studies have shown a role for YB-1 in sarcomas. More specifically, it has been linked to poor prognosis and drug resistance. In addition, high levels of YB-1 were observed in some childhood sarcomas. More recently, YB-1 has been described as an excellent marker of cancer progression. Therefore we were interested to study the possible potential contribution of YB-1 to paediatric sarcoma progression.   Given that YB-1 promotes EMT, a process by which epithelial-derived malignant cells acquire a more mesenchymal phenotype with enhanced metastatic ability and reduced growth potential through translational upregulation of mesenchymal related messages and simultaneous downregulation of epithelial and growth related messages, I  hypothesize that YB-1 may play a similar role in sarcomas.    I will address my hypothesis in multiple ways. First, I will investigate YB-1 expression across a wide range of sarcoma cell lines encompassing the different sarcoma subtypes. In addition, I  will assess its expression in formalin fixed paraffin embedded (FFPE) tissue blocks of human  sarcoma tissues. I  will include mesenchymal-derived, non-tumorous cell lines or tissues to serve as normal control.   35  Secondly, I  will address the contribution of YB-1 to distinctive features observed in sarcoma cells, including but not limited to their proliferation and metastatic capacity in vitro and in vivo using different kinds of in vitro growth and motility assays. In addition, with our collaborators, I will use two novel animal models for studying cancer progression in vivo; the zebrafish xenotransplantation model, and the renal subcapsular assay (RSA) xenotransplantation model.   Third, I  will determine the mechanisms by which YB-1 promotes sarcoma cell metastasis. My central hypothesis can be broken down into three sub-hypotheses.  1. YB-1 is highly expressed in childhood sarcomas and is required for the mesenchymal phenotype and the growth potential of sarcoma cells. 2. YB-1 is a major contributor to sarcoma cell invasion and metastatic dissemination in vitro and in vivo. 3. YB-1 promotes sarcoma cell invasion and metastasis through translational activation of stress mitigating proteins such as Snail1, Twist, and HIF1? whose function is to promote EMT.    I  believe that my study will provide new insights into our understanding of the processes of sarcoma metastasis. Assessing the role of the YB-1 in sarcoma metastasis and detecting its downstream effectors will aid in a better understanding of this clinically important process, as it is the main challenge facing clinicians treating cancer patients, and hope this further understanding that may lead to the discovery of novel therapies for the treatment of sarcomas.  36  Chapter  3: YB-1 potentially contributes to the growth and phenotypic characteristics of childhood sarcoma cells  3.1 Rationale Overexpression of YB-1 has been reported in various tumours including some sarcoma subtypes246 and aggressive nonsarcomatous tumours with sarcoma-like features247. Our group has found that elevated YB-1 expression levels confer a more metastatic phenotype, microscopically resembling sarcoma cell morphology, at the expense of cell growth in breast cancer models10. Further, we have recently shown that inhibiting YB-1 expression in sarcoma cells has led to enhanced protein translation involving many messages contributing to cell growth and DNA replication. The YB-1 deficiency-mediated enhanced translation was accompanied by overwhelming cell death248. Since highly glycolytic cells such as tumour cells are more sensitive to cell death249 and given that YB-1 has been implicated as a cell survival factor108, I  hypothesize that in paediatric sarcomas; mesenchymal-derived, highly metastatic, and highly proliferative tumours, YB-1 contributes to sarcomatogenesis retarding the uncontrolled sarcoma cell proliferation which is potentially protective  against cell death.   3.2 Material and methods 3.2.1 Cell lines and culture conditions  OBB human osteoblasts from joint replacement patients were kindly provided by Dr. Marianne Sadar (University of British Columbia), OB3 human osteoblasts from scoliosis patients were developed in Dr. Poul Sorensen?s lab and previously described88. Briefly, scoliosis is a spine 37  disorder that typically affects individuals found within a similar age range as those with adolescent osteosarcoma. The mainstay of treatment for progressive scoliosis is surgical stabilization of the spine using bone graft and internal fixation devices250. Ethics approval was obtained from the Research Ethics Board of British Columbia and all patients provided informed consent along with parental consent as required. At the time of surgery excess bone graft material from the posterior spinous processes were fragmented, washed with PBS, and cultured in MEM with 20% FBS and 1% antibiotic?antimycotic for 4?8 weeks. Media was changed weekly. Cells migrating from the bone fragments were harvested using trypsin and the bone fragments discarded. The cells were then serially passaged and differentiated using bone morphogenic protein (BMP2/BMP7 heterodimer; R&D Systems, Minneapolis, MN). Individual cell lines were characterized for osteogenic potential. We were able to successfully propagate six out of seven cell lines. OBB and OB3 were maintained in modified Eagle?s medium (MEM; Invitrogen) supplemented with 20% fetal bovine serum (FBS; Invitrogen) and 1% antibiotic-antimycotic (Invitrogen).   MG63 and MNNG osteosarcoma cell lines were obtained from the American Type Culture Collection (ATCC, catalog numbers CRL-1427, CRL-1547), respectively, and were grown in minimal essential medium (MEM) supplemented with 10% FBS. ES cell lines; CHLA258 grown in Roswell Park Memorial Institute medium (RPMI; Invitrogen) supplemented with 10% FBS, and both of CHLA9 and CHLA10 grown in Iscove's Modified Dulbecco's Medium (IMDM; Invitrogen) supplemented with 20% FBS, 1% Insulin-Transferrin-Selenium (ITS, Invitrogen) were kindly provided by Dr. Patrick Reynolds (Children?s Oncology Group/COG) while TC71, TC32 ES cell lines, and Rh30 rhabdomyosarcoma cell lines, grown in RPMI media 38  supplemented with 10% FBS, were  kind gift from Dr. Timothy Triche, Childrens Hospital Los Angeles, University of Southern California. The Rh18 rhabdomyosarcoma cell line grown in RPMI medium supplemented with 10% FBS was kindly provided by Dr. Catherine Pallen (University of British Columbia). All media were additionally supplemented with 1% antibiotic-antimycotic.    3.2.2 Transient transfection  For transient inhibition of YB-1 expression in sarcoma cell lines, cells were grown to ~40% confluence and then cell populations were transfected with two single siRNAs (siRNA 1: UGACACCAAGGAAGAUGUA; siRNA 2: GUGAGAGUGGGGAAAAGAA) targeting YB-1 as described10, or non-targeting siRNA (D-001210-03-20, Dharmacon) at a final concentration of 20 nM using siLentFect (Bio-Rad) reagent, according to the manufacturer?s instructions. Four days post-transfection, cells were examined by Western blotting for YB-1 downregulation and this was considered day 0.    3.2.3 Stable YB-1 knockdown (kd)  Cells were plated 24 hours prior to transfection in 12-well plates. On second day, and with ~50% confluence, cells were transduced with either control ready-to-use shRNA lentiviral particles (Santa Cruz Biotech., sc-108080), or ready-to-use YB-1 shRNA human lentiviral particles (Santa Cruz Biotech., sc-38634-V) following the manufacturer recommendations. Selection of polyclonal population of homogeneously transduced cells was performed using Puromycin dihydrochloride (Santa Cruz Biotech., sc-108071). Occasionally, two rounds of transduction were conducted for efficient YB-1 knockdown. 39  3.2.4 Protein extraction, western blotting, and antibodies  Cells were lysed with Nonidet P-40 lysis buffer (20mM HEPES-KOH [pH 7.6], 100 mM KCl, 5mM MgCl2, 2 mM dithiothreitol, 0.25% Nonidet P-40, 2 ?g/ml leupeptin, 2 ?g/ml pepstatin, and 10 ?g/ml cycloheximide. Protein concentrations were determined using Bradford protein assay kit (Bio-Rad) according to the instructions of the supplier. Twenty micrograms of lysate protein were prepared for loading into gels by admixing with loading buffer with the anionic denaturing detergent sodium dodecyl sulfate (SDS), previously described251 in 1:1 ratio. The mixture was boiled at 95-100?C for 5 minutes. Proteins were separated by SDS-PAGE under reducing conditions and blotted onto nitrocellulose membranes. Membranes were incubated with the primary at 4?C over night with gentle shaking and then with 0.2 ?g/ml horseradish peroxidase (HRP)-conjugated secondary Ab and revealed with enhanced chemiluminescence (ECL) light substrate (Thermo Scientific Pierce) for detection of HRP enzyme activity. The working solutions of the substrates were prepared according to the manufacturers? instructions and added to the membranes for 1 minute. The membranes were removed from the substrates and placed in plastic sheet protectors. Each membrane was exposed to high performance, radiography films (VWR). All western blots were repeated at least twice. Total Akt and Grb2 were used to demonstrate equal loading as their protein levels were relatively stable in +/- YB-1 knockdown cells, and this has been previously reported for Akt102. The antibodies used in the current study are described below.  Antibodies Company Catalogue No. Dilution  Actin (I-19): goat polyclonal Bromodeoxyuridine: mouse monoclonal  Cyclin D1: mouse monoclonal Santa Cruz  Cedarlane Cell Signaling sc-1616 11170376001 2926 1:1000 1:250 1:500 40  Cyclin E: mouse monoclonal Grb2: mouse monoclonal N-cad: rabbit polyclonal Snail1: rabbit polyclonal Twist: rabbit polyclonal YB-1 (59-Q): mouse monoclonal YB-1 (D299): rabbit polyclonal pYB-1: rabbit monoclonal Upstates BD Biosciences Cell Signaling Abcam Santa Cruz Biotech. Santa Cruz Biotech. Cell Signaling Cell Signaling 05-363 610111 4061 Ab63371 sc-15393 sc-101198 4202 2900 1:1000 1:1000 1:500 1:500 1:500 1:1000 1:500 1:1000   3.2.5 Immunohistochemistery (IHC) for YB-1 expression in osteosarcoma In the current study, twenty five OS cases with minimal tissue decalcification treatment effect were retrospectively collected. OS cases were assigned grades252 as follows: ? Parosteal osteosarcoma, and well differentiated intramedullary (fibrous dysplasia-like) osteosarcoma (Grade 1) ? Periosteal osteosarcoma (Grade 2) ? All osteosarcoma cases except the previously mentioned variants are (Grade 3)  With kind assistance from Dr Nielson, histological slides obtained from the paraffin blocks of OS cases were reviewed to ensure that adequate quality and quantity of tumour and the presence of normal tissue adjacent to the tumour to serve as internal control. Four ?m thick sections were cut from each available paraffin blocks. The IHC protocol used in this study in formalin-fixed, paraffin embedded (FFPE) tissues was previously described253. Briefly, after deparrafinization, the slides were treated with 0.3% hydrogen peroxide to inactivate endogenous peroxidase, washed in phosphate-buffered-saline (PBS), and antigen retrieval has been conducted for 30 41  minutes by incubating the slides in a steamer containing citrate buffer (10 minute, pH 6.0) warmed to 95oC. Twenty minutes after cooling down, the slides were washed three times in PBS. After washing in PBS and blocking in 10% normal rabbit serum, the sections were incubated in a 20 ?l of the YB-1 antibody (Cell Signaling) at 1:25 dilution and incubated overnight at 4oC (additional confirmatory staining was conducted using YB-1 antibody (Santa Cruz Biotech.) at 1:25 dilution). On next morning, excess reagent was thrown off and slides were rinsed three times in PBS-Tween (PBST), 5 minutes each. After removal of excess buffer, the samples were incubated in streptaviden-horseradish peroxidase and then placed in a humidity chamber at room temperature for 30 minutes. Then, the slides were rinsed again three times in PBST, 5 minutes each. Distribution and intensity of YB-1 reactivity was visualized of the stain was conducted using 3, 3?diaminobenzadine (DAKO cat# K 3468) as a substrate and the sections were counterstained with Gills solution.   The slides were then examined and analyzed with an Axioplan2 fluorescence microscope (Zeiss) by Dr Poul Sorensen, and Amal EL-Naggar.  For each case, the percent of cells positively stained for YB1 and staining intensity were evaluated. For the percentage of positive cells, the staining patterns of tumour cells were divided into two categories: (1) positive (whether cytoplasmic, nuclear, or nucleo-cytoplasmic), and (2) negative (with no detectable staining). Based on the mean value of YB-1 expression in the 25 OS cases examined, 11 (44%) showed <40% reactivity mild to moderate cytoplasmic YB-1 immunoreactivity while the remaining 14 (56%) cases exhibited showed > 40% reactivity with moderate to strong predominantly cytoplasmic YB-1 immunoreactivity. For staining intensity, we used a 4-point scale (0-3+); no staining = 0, weak staining = 1, moderate staining = 2, strong staining = 3, as previously 42  described254. Strong YB-1 immunoreactivity was usually noted with cases showing higher percentage of positive cells (> 40%).  3.2.6 Phalloidin staining and quantitation of stress fibers Cells were fixed in 4% paraformaldehyde for 15 minutes at room temperature (RT), after brief rinsing into PBS. Cells were then washed three times with PBS, 1 minute each and permeabilized in 0.1% Triton-X100 in PBS for 1 min followed by incubation in Rhodamine-phalloidin stain (Cytoskeleton) diluted 1:1000 in PBS for 15 min. Final rinse 3 times in PBS, 5 min/wash was conducted. Cells were mounted with VECTASHIELD Mounting Medium with 4',6-diamidino-2-phenylindole (DAPI) (Vector Labs). Images were acquired with an Axioplan2 fluorescence microscope (Zeiss). Quantitation of stress fibres was conducted as previously described255. In brief, ten random low power fields were examined. For each field, cells displaying stress fibres were counted and changed into percentage. The average percentage of the total examined fields were displayed + SD.   Sucrose gradient and polysomal fractionation Cells lysis was performed using Nonidet P-40 lysis buffer. Polysomal fractionation to categorize translationally active transcripts was performed as previously described136, 256 with minor modification. Following cell lysis, nuclei and cell debris were cleared by centrifugation at 13,000 rpm, 20min at 4oC. The resulting supernatant was loaded onto a 30% (w/v) sucrose gradient which was further centrifuged at 48,000 rpm for 15-20 min in a Beckman coulter T1-14 centrifuge rotor at 4oC. The polysomes precipitated on the wall of the tube as a transparent pellet. 43  The pellet was dissolved in NP40 lysis buffer. This was followed by RNA extraction and cDNA synthesis for conducting real-time PCR.  3.2.7 Anchorage-independent cell growth assay  Soft agar assays were conducted in 6-well plates where the bottom layer was constructed using 0.4% agar in DMEM/FBS. Then cells suspended in 0.25% agar in DMEM/FBS were plated at a concentration of 8000 cells/well. Cells were incubated at 37?C and supplemented with two drops of media every three days until colonies appeared. Single cells as well as colonies per high power field were counted and the results formulated as a percentage of macroscopic (>0.1-mm) colonies formed/total number of cells plated.  3.2.8 Cell growth and BrdU incorporation For assessing cell growth, 15,000 cells transduced with either scrambled (shCTRL) or shYB-1 were plated followed by subsequent harvesting every 24 hours for 5 days. Cells were counted using the Cellometer Auto T4 automated cell counter (Nexcelom Biosciences). To assess cell proliferation, we used BrdU incorporation assay. Cells were grown on cover slips and incubated in fresh MEM containing 10%FBS and 50?M 5-bromo-2?-deoxybromouridine (BrdU) (Roche) for 1 hour minutes at 37oC. Cells were then washed, fixed and stained using a BrdU labeling and detection kit (Rohe).     3.2.9 Indirect immunofluorescence Cells were grown on cover slips to 80% confluence, then fixed for 30 minutes at RT with 3.5% paraformaldehyde solution and subsequently permeabilized with 0.1% Triton X-100 in PBS for 44  10 minutes at RT.  To reduce background interference, cells were incubated with blocking buffer of 5% solution of non fat powdered milk in Tris-buffered saline (TBS) for 30 minutes at RT. Then, incubation with the primary monoclonal antibody against YB-1 (Santa Cruz Biotech); 1/250 in 0.5% milk in TBS was done in humidified chamber, over night (O/N) at 4oC. On second day, cells were washed 3 times with TBS-Tween (TBST), and then incubated with the fluorophore-conjugated secondary antibody; Alexa 488 anti-mouse, 1/250 in 0.5% milk TBST at RT in dark for 30 minutes. Later, cells were washed 3 times with TBST. Finally, 5 ul of mounting medium with DAPI (Vector Shield; Vector Laboratories) was applied to each glass slides and covered with the cover slips and examined under microscope.  Slides were then mounted with mounting medium and analyzed with a Zeiss confocal microscope.  3.2.10 RNA isolation and quantitative real-time polymerase chain reaction (qRT-PCR)  RNA extraction was performed using Trizol? reagent (Invitrogen, cat # 15596-018) extraction method, previously described257. Then, samples were harvested, vortexed and subjected to 15 minutes centrifugation at 4oC. The aqueous layer containing the RNA was transferred to a new tube and the RNA precipitated with isopropanol. After further centrifugation the isopropanol was removed and the RNA pellet washed with 75% ethanol and then re-suspended in 20-30?l of RNAase-free water. RNA integrity and concentration were assessed by NanoDrop spectrophotometer (Thermo Scientific) and gel electrophoresis, and subsequently converted into cDNA. QRT-PCR was performed using the ABI PRISM 7900 HT Sequence Detection System (Applied Biosystems) according to the manufacturer?s protocol, and as previously described258. The human primers used in this study are listed below.  45   The Taqman primers used in the current study are listed below. Gene Assay ID CCND1(homo sapiens) GAPDH (homo sapiens) YBX1 (homo sapiens) Hs00277039_m1 Hs02786624_g1 Hs02742755_g1 The q-PCR cycling conditions for Applied Biosystems predetermined assays consisted of incubation at 50oC for 2 minutes, followed by 40 cycles of denaturation (10 minutes at 95oC), annealing (1 minute at 94oC) and extension (15 sec at 60oC). Reactions were performed in triplicate, and each experiment repeated twice. The relative expression levels of the specified gene was determined using the 2-delta delta Ct analysis method259, using GAPDH as an endogenous control.  3.2.11 Statistical analysis A two-tailed student?s T test was used to analyze the data, unless otherwise indicated, with p value <0.05 indicating statistically significant differences.  3.3 Results 3.3.1 YB-1 is highly expressed in sarcomas To investigate into the potential roles, if any, of YB-1 in sarcomas, we first looked at YB-1 expression in a wide variety of sarcoma cell lines. Interestingly, YB-1 is highly expressed in all sarcoma cell lines compared to non-transformed mesenchymal derived bone cells; osteoblasts (OBs) (Fig. 3.1A) and is mainly localized to the cytoplasmic compartment (Fig. 3.1B). Furthermore, IHC evaluation for YB-1 expression in human sarcoma tissue samples showed 46  strong cytoplasmic expression in the malignant counterparts; OS and RMS compared to mild cytoplasmic expression in the benign counterparts; osteoblasts and skeletal muscles, respectively (Fig. 3.1C). Two YB-1 antibodies from Cell signalling and Santa Cruz companies were used and similar results were obtained.   3.3.2 YB-1 knockdown affects the mesenchymal phenotype of sarcoma cells To further elucidate the role of YB-1 in sarcomas, sarcoma cells with transient or stable YB-1 knockdown (kd) were generated (Fig. 3.2A) and the impact of YB-1 downregulation on sarcoma cells criteria was evaluated using different in vitro assays. Generally, sarcoma cells are characterized by elongated, spindle-shaped, fibroblast like morphology260. Interestingly, inhibiting YB-1 expression led to significant impact on their morphological characteristics manifested in loss of the spindle-shaped morphology, increase in cell size, acquisition of a more flattened morphology, and tendency for cohesive cluster formation (Fig. 3.2B) and this was accompanied by downregulation of some known mesenchymal markers such as N-cad, Snail1, and Twist (Fig. 3.2C). However, the expression levels of crucial mesenchymal marker such as vimentin did not change in YB-1 kd cells. Further, none of  the examined  +/- YB-1 kd cells showed enhanced  E-cad expression upon YB-1 kd (Fig. 3.2D), thus preventing the claim of classical mesenchymal-to-epithelial (MET) conversion. Of interest, loss of the sarcomatous phenotype was simultaneously accompanied by cytoskeletal changes with a marked downregulation of actin stress fibres as detected by phalloidin staining (Fig. 3.3A and 3.3B), which may explain the impact of YB-1 downregulation on sarcoma cell morphological appearance. Our results are consistent with other reports showing the crucial contribution of YB-1 to stress fibres formation102, 261.  47  3.3.3 YB-1 retards sarcoma cell proliferation It was reported that cytosolic YB-1 suppresses the proliferative capacity of cells potentially through binding and translational silencing of growth-related transcripts150, 155, 156. Therefore, we looked at the impact of YB-1 downregulation on growth ability of sarcoma cells. We found that YB-1 was able to affect sarcoma cell proliferation, its inhibition has led to enhanced proliferation (Fig. 3.3A). However, the difference was not statistically significant. Indeed, this enhanced trend of proliferation is correlated with an increase in expression of certain important cell cycle regulators such as cyclin D1 and cyclin E (Fig. 3.3B). The role of YB-1 in controlling sarcoma cell growth can be attributed to its ability to inhibit the translation of growth-related messages (Fig. 3.3C) and this is consistent with our results in mammary cells10. In addition, some cell lines showed enhanced phospho-YB-1 expression upon knocking down YB-1, an interesting finding that may partially explain the enhanced proliferation of these cells as well (Fig. 3.3B). Further, cell proliferation assays using BrdU incorporation showed significantly higher numbers of S-phase cells in cells with YB-1 kd (Fig. 3.3D). Interestingly, in human clinical samples, mitotically active sarcoma cells almost always showed lower YB-1 expression levels compared to the surrounding non-dividing tumour cells (Fig. 3.3E) which further support our previous findings.   3.3.4 YB-1 knockdown inhibits anchorage-independent growth of sarcoma cells in soft agar We next examined the effects of YB-1downregulation on anchorage-independent cell growth; we found that inhibiting YB-1 expression has lead to remarkable inhibition of colony formation in soft agar (Fig. 3.4A-3.4B) as previously reported for SF188 paediatric glioblastoma cells, 48  MDA-MB-453, BT474-m1, and HCC1937152, 262-264. Of interest, the formed colonies of YB-1 deficient cells were much larger compared to that of YB-1 competent cells (Fig. 3.4A), reflecting the enhanced proliferation of YB-1 deficient cells. These findings potentially reflect the alteration of sarcomatogenesis activity upon YB-1 inhibition and are consistent with a pro-oncogenic role for YB-1 in sarcoma cells.   3.3.5 Elevated YB-1 expression levels observed in osteosarcoma correlates with high grade disease We next used immunohistochemistry to assess YB-1 expression in 25 formalin-fixed, paraffin-embedded (FFPE) OS cases selected for the presence of both malignant tissue together with adjacent normal bone as an internal control (osteoblasts). Osteoblasts, which line normal bone trabeculae, exhibited YB-1 cytoplasmic immunoreactivity with mild intensity (Fig. 3.5A). In malignant cells, YB-1 demonstrated a predominantly high cytoplasmic staining, (Fig. 3.5B-3.5F). YB-1 immunoreactivity in osteosarcoma samples was estimated by counting the number of positive cells per 1000 tumour cells. The YB-1 mean expression value was 40%. Of 25 osteosarcoma cases examined, 11 (44%) showed less than 40% expression levels with mild to moderate cytoplasmic YB-1  immunoreactivity while the remaining 14 (56%) cases exhibited equal or higher than 40% expression levels with predominantly strong cytoplasmic YB-1 immunoreactivity (Table 3.1, Fig. 3.5B-3.5F). Pathological data was then examined for correlations with YB-1 expression and we found that enhanced YB-1 expression is associated with high grade osteosarcoma Table 3.1, potentially suggestive of a role for YB-1 in sarcoma progression.   49  3.4 Discussion Tumour cells are distinctly characterized by uncontrolled growth and high metabolic rates due to aberrant genetic events that lead to dysregulated growth40, 43, a biological property of tumour cells which has been exploited therapeutically through the development of anticancer-drugs that target either the cell cycle or rapidly dividing cells265. Indeed, unchecked proliferation may be disadvantageous to tumour cells encountering harsh microenvironmental conditions or during their attempt to metastasize and this explains why disseminated tumour cells rarely express proliferation-associated markers266 and that metastatic tumour cells can remain in a quiescent state called dormancy for years prior to developing micrometastatic colonies267, a mechanism that appears to be crucial for their survival and progression as well as for escaping the effects of conventional chemotherapeutic drugs. An interesting mediator recently shown to enhance tumour cell metastatic spread at the expense of cell growth is YB-1.  Y-box binding protein 1 (YB-1) is member of the CSD-containing family of proteins that are ubiquitously expressed and involved in fundamental processes such as DNA repair, mRNA transcription, splicing, translation and stabilization. Extensive literature links YB-1 up-regulation to tumour aggressiveness in both epithelial and mesenchymal malignancies10, 92, 98, 154, 268, 269. YB-1 is described as both a transcription and a translation factor, and while data exists for both functions, the relative role of transcription versus translation in YB-1 functions remains controversial107. However, our work10 and that of many others strongly argue for a prominent role of translational regulation in YB-1 activity270, 271. Our group recently found that increased YB-1 expression in breast cancer cells reduces cell growth by repressing growth-related mRNAs, while simultaneously increasing their metastatic ability. Specifically, YB-1 translationally 50  represses growth-related messages such as cyclin D1 and cyclin E as well as several cyclin-dependent kinases (CDKs) to block cell proliferation, while translationally activating Snail1 and Twist EMT-associated messages which possess anti-proliferative properties as well10. Given these observations we wondered whether YB-1 could play a similar role in sarcoma development.    In the current study we found that YB-1 is highly expressed in sarcomas. Inhibiting YB-1 expression in sarcoma cells affected their mesenchymal phenotype and this was associated with loss of expression of crucial mesenchymal markers such as N-cad, Twist, and Snail1 which is consistent with a previous study10.     Further, YB-1 downregulation is significantly associated with downregulation of actin stress fibres272. Admittedly, actin organization has a substantial role in promoting the migratory phenotype of cells273. The consequences of YB-1 downregulation on sarcoma cells motility will be extensively discussed in Chapter 4.  Moreover, YB-1 downregulation was associated with an enhanced proliferative capacity and an inhibition of colony formation in vitro. Recently, our group has shown that YB-1 downregulation in sarcoma cell model leads to an increase in global protein synthesis in which many messages involved in cell growth and DNA replication were induced jointly accompanied with overwhelming cell death248, underscoring the adverse effect of enhanced cell growth on cell survival. Of interest, inhibiting YB-1 expression in our cells led to enhanced expression of phospho-YB-1 in a subset of the cells. A similar finding has been described by another group in 51  glioblastoma cells where inhibiting YB-1 expression led to enhanced expression of pYB-1 which was associated with enhanced proliferation274.  Many reports have shown that loss of YB-1 expression leads to growth inhibition in different epithelial systems275-277. However, in sarcomas the role of YB-1 in inhibiting cell growth was highlighted and we believe this property is due to YB-1?s dominant cytoplasmic localization and translational silencing of growth-related messages as previously described150, 155, 156.  Further, IHC studies performed to examine YB-1 expression in human tissues has revealed that YB-1 is highly expressed in malignant sarcomas compared to benign mesenchymal-derived tissues and we showed that enhanced YB-1 expression is correlated with advanced histological grade in OS tissue specimens, highly indicative of the potential role of YB-1 in sarcoma progression. Additional investigation using a larger number of cases is obviously warranted.  Finally, it is worth mentioning that in our current study the level of YB-1 inhibition achieved was 90% or more of its basal expression and this level of knockdown was consistently achieved. Of interest, in one of our tested cells A673 ES cell lines, we observed growth retardation upon inhibiting YB-1 expression to ~ 50% of its basal expression level. This finding could be a cell-type specific response or the different concentrations of YB-1 may impact its function, and this is consistent with other reports revealing that small changes in protein abundance can have large functional effects278. Based on these findings, we conclude that YB-1 plays a potential role in sarcoma. YB-1 contributes to the mesenchymal phenotype of sarcoma cells and limits the unchecked proliferation of sarcoma cells through translational repression of growth-related 52  messages. Given that sarcomas are highly metastatic tumours279., and reduced proliferation is usually associated with enhanced motility as has been previously described10,92, we  wondered whether YB-1 potentially contributes to the highly metastatic phenotype of sarcoma cells, and this is to be discussed in Chapter 4.   3.5 Tables Table 3.1    Correlation of YB-1 expression with tumour grade in osteosarcoma  Variable Osteosarcoma (25cases) Test of significance P value <40 % (11/25, 44%) >40 % (14/25, 56%) Grade I II III  6(24%) 1(4%) 4(16%)  1(4%) 2(8%) 11(44%)    chi-square   (??)    0.04    3.6 Figures  53    Figure 3.1    YB-1 expression in sarcomas A. Immunoblot showing YB-1 expression levels in a wide variety of sarcoma cell lines compared to non-transformed mesenchymal derived osteoblasts. Akt was used as loading control.  B. Endogenous YB-1 expression and its localization in the indicated sarcoma cell lines as detected by fluorescent microscopy. Scale bars 10 ?m.        C. Representative high-powered magnification (400x) of YB-1 staining in normal mesenchymal derived tissues (OBs and skeletal muscle) versus sarcoma tissue specimens (OS and RMS). OBs: osteoblasts; OS: osteosarcoma; ES: Ewing sarcoma; and RMS: rhabdomyosarcoma.  54    Figure 3.2    Inhibiting YB-1 expression affects sarcoma cell phenotype A. The indicated sarcoma cells were either transfected with YB-1 siRNAs (MG63, CHLA258) or transduced with YB-1 shRNA (MNNG, TC71, TC32, CHLA10, and Rh30). Scrambled siRNA or shRNA were used as experimental controls, respectively. Cell lysates were collected and examined by Western blot for YB-1 downregulation. Grb2 was used as loading control.  B. Effects of shRNA-mediated YB-1 knockdown on the morphology of the indicated sarcoma cells as detected by phase contrast microscopy. Scale bars: 10?m.  C. Western blot showing the effects of siRNA-mediated YB-1 knockdown on the expression levels of common mesenchymal markers (N-cad, snail1, and twist). Actin was used as loading control.  55  D. Western blot showing the effects of siRNA-mediated YB-1 knockdown on the expression levels of E-cad, and vimentin). Grb2 was used as loading control.                      56    Figure 3.3    YB-1 contributes to the formation of actin stress fibers A. The Effects of shRNA-mediated YB-1 knockdown on actin stress fibres in the indicated sarcoma cells were examined by rhodamine-phalloidin stain and fluorescent microscopy. Scale bars: 25?m.  57  B. Quantitation of stress fibres observed in D.  The percentage of +/- YB-1 knockdown sarcoma cells displaying stress fibres was evaluated in 10 low power fields, and the average percent (%) was calculated. Error bars represent + SD. *** = p<0.0005.   58   Figure legend starts next page 59  Figure 3.4    YB-1-mediated effects on sarcoma cell proliferation A. Growth curves of the indicated sarcoma cells +/- shRNA-mediated YB-1 knockdown. Each data point represents the mean + SD from two independent experiments.  B. Immunoblot showing the effects of siRNA-mediated YB-1 knockdown on the expression levels of common cell cycle regulators at the fifth day post-transfection. Grb2 was used as loading control.  C. Effects of siRNA-mediated YB-1 knockdown on the translational activity of cyclin D1 at the fifth day post-transfection. Relative levels of cyclin D1 mRNA in the indicated sarcoma cell lines +/- YB-1 kd compared to HeLa cells as measured by quantitative RT-PCR of total RNA or polysomal fractionated RNA. Data were normalized to endogenous GAPDH from two runs performed in triplicate, analyzed with a two-tailed student?s T test, and presented as means + SD. D. BrdU incorporation analysis of MG63 OS cells +/- shRNA-mediated YB-1 knockdown. Left panel: cells were serum-starved for 18 hrs and then  restimulated with complete medium for 4 hrs and labelled with BrdU for visualizing cells entering S phase (green nuclei). Representative fields are shown. Scale bars 25 ?m. Right panel: percentage of BrdU positive (green) cells were calculated relative to the total cell population (blue) in five random fields of view in MG63 cells +/- YB-1 kd using cell profiler imaging analysis software (MATLAB version) with in-home modifications. Results of two independent experiments are represented as means + SD. E. IHC for YB-1 expression in sarcoma tissue specimens showing low expression levels in mitotically active sarcoma cells (arrows) compared to the surrounding non-dividing (resting) malignant cells. Scale bars 25?m. Where shown *=p<0.05, **=p<0.005.   60    Figure 3.5    YB-1-mediated effects on anchorage independent growth of sarcoma cells A. YB-1 knockdown inhibits sarcoma cell colony formation in vitro. Colonies were counted under the phase contrast light microscope 14 days after being plated. Scale bars 100 ?m. B. Charts represent percent quantitation of the number of colonies formed after 14 days. Averaged values of two independent experiments counted in triplicates were presented + SD.  Where shown * = p<0.05; **=p<0.005; *** = p<0.0005.    61   Figure 3.6    Examples of YB-1 immunohistochemical staining of osteosarcoma cases A. Osteoblasts (normal bone cells) exhibiting mild cytoplasmic immunoreactivity; B. Grade I osteosarcoma exhibiting moderate cytoplasmic immunoreactivity; C. Grade II osteosarcoma showing moderate cytoplasmic immunoreactivity; D. Grade III osteosarcoma showing strong cytoplasmic immunoreactivity. E, and F. Grade III osteosarcoma showing strong cytoplasmic immunoreactivity Scale bars, 50 ?m.          62  Chapter  4: YB-1 is a major contributor to sarcoma cell motility, invasion, and metastatic dissemination in vitro & in vivo  4.1 Rationale YB-1 contributes to aberrant activation of the EMT developmental program which can endow cancer cells with migratory and invasive capabilities required for metastatic competence280 and many studies have supported the role of elevated levels of YB-1 in cancer metastasis10, 281, mostly at the expense of cell growth92. Since YB-1 is highly expressed in childhood sarcoma cells and it contributes to its mesenchymal phenotype as shown in Chapter 3 and also since its inhibition is associated with enhanced cell growth and loss of stress fibres involved in cell motility we hypothesized that YB-1 may serve as a substantial metastatic marker in childhood sarcomas.   4.2 Material and methods 4.2.1 The murine renal subcapsular xenotransplantation model  In collaboration with Dr Yuzhuo Wang at the BC Cancer Research Centre, we established the use of murine renal subcapsular assay (RSA) to monitor growth and dissemination of human childhood sarcoma cells in vivo. Dr Yuzhuo Wang and his group have developed and had previous success in using this model282-285. This technique involves two steps, namely preparing xenograft cell blocks and then performing the grafting procedure which was conducted by Dr Hongwei Cheng. Briefly, for xenograft cell blocks 1x106 luciferase- labelled cells (for the TC71 and TC32 cell lines) or 0.5x106 cells (for the CHLA10 cell line) were well-mixed with 50ul of 63  5X DMEM-tail collagen gel, seeded in cell culture plates, and incubated for 30 minutes. For the grafting procedure, 6-8 week-old NOD-SCID male mice (8 per condition for TC71 and 10 per condition for TC32 & CHLA10) were engrafted and maintained according to UBC Animal Care Committee (ACC) regulations. A 0.5 cm incision was made in the mouse body wall to exteriorize the kidney which was then removed and laid on the body wall. A 2-4 mm incision was made in the kidney capsule using fine spring-loaded scissors to open the kidney capsule. The xenograft cell block was transferred to the surface of the kidney and inserted into the pocket under the capsule. Once the grafting procedure was completed, the kidney was gently eased back into the body cavity, and the skin edges were then sutured closed. Mice were subsequently monitored for tumour growth using both clinical monitoring and In Vitro Imaging System (IVIS)for 8 weeks in for TC71 and TC32 and 10 weeks for CHLA10.  4.2.2 Zebrafish xenotransplantation 4.2.2.1 Zebrafish husbandry In collaboration with Dalhousie/IWK Health Centre paediatric haematologist Dr. Jason Berman, we used zebrafish xenotransplantation model to monitor dissemination of +/- YB-1 kd human childhood sarcoma cells in vivo.   This work was conducted and described by Chansey Veinotte and Dale Corkery. The transparent Casper strain of zebrafish used in the current study was maintained according to standard protocols286. Embryos were dechorionated using 10 mg/ml pronase (Roche Applied Science).   4.2.2.2 Cell staining, injection and screening For in vivo zebrafish studies TC32 cells with or without stable shRNA knockdown of YB-1 were 64  stained with CM-DiI (red fluorescence, Invitrogen). Cells were grown to confluence and trypsinized with EDTA. Cells were washed with RPMI Media 1640 (Gibco), transferred to 15 ml Falcon tubes and centrifuged for 5 minutes  at 1200 rpm or 100 X G. Cells were re-suspended, at a concentration of 10 million cells per mL, in 1X PBS (Gibco) with a 5 ?g/mL final concentration of CM-DiI. The suspension was incubated for 4 minutes at 37?C and then 15 minutes at 4?C as previously described287. Cells were washed once in 1X PBS before being suspended in RPMI for injection into embryos. All centrifugation was performed using a Thermo IEC Centra CL2 centrifuge. Forty-eight hours post-fertilization (hpf) and prior to injection, Casper embryos were dechorionated and anaesthetized with 200 ?g/mL tricaine (Sigma). A manual PLI-100 microinjector (Medical Systems Corp, Greenvale, NY) was used to load the cell suspension into a pulled capillary needle for embryo injection. Approximately 100-150 TC32 cells were injected into the yolk sac of each embryo. Following injection, embryos were kept at 28?C for 30 minutes and at 35?C for the duration of the experiments. At 12-24 hrs post-injection (hpi), embryos were screened for the presence of a fluorescent cell mass within the yolk site. Positive embryos were isolated for experiments.  4.2.2.3 Live cell microscopy, and migration assay Every 24 hrs for ~7 days, 4-6 embryos were imaged and analyzed for cellular interactions within the zebrafish embryonic microenvironment. An inverted Axio Observer Z1 microscope equipped with a Colibri LED light source (Carl Zeiss, Westlar, Germany) and an Axiocam Rev 3.0 CCD camera and Axiovision Rel 4.0 software (Carl Zeiss Microimaging Inc.) was used to screen, observe, and capture images of injected embryos. To quantify cellular migration in zebrafish, groups of 30 embryos were followed every 24 hrs for 144 hrs using live cell microscopy of the 65  tail region. Embryos were scored based on the presence or absence of fluorescent cells from the cloaca to the tip of the tail. Embryos displaying 6 or more cells within the tail region were scored positively for migration. TC32 cells fixed in 4% paraformaldehyde and TetraspeckTM microspheres were employed as negative migratory controls.  4.2.3 Histopathological evaluation of xenografts  Four ?m-thick tissue sections of FFPE tissue blocks were stained with H&E stain and examined using Axioplan2 fluorescence microscope (Zeiss). IHC for YB-1 was performed, after de-parafinization and antigen retrieval, using YB-1 antibody (Cell Signaling) at 1:25 dilution using the Ventana discovery XT system. Antigen retrieval was performed using Ventana CC1. Detection was carried out using Ultramap anti-Rabbit HRP (Ventana).  Additional IHC for YB-1 expression was conducted using another YB-1 antibody (Santa Cruz Biotech.) at 1:25 dilution using alkaline-phosphatase- labelled streptaviden-biotin ABC kit (Vector Labs). Sections were counterstained with haematoxylin (Vector Labs).  Images were acquired with an Axioplan2 fluorescence microscope (Zeiss). The detailed procedure was described in Chapter 3. Further IHC on the tissue sections was conducted using the Ventana Discover XT system with the following additional antibodies used in the current study; CD31 (Epitomics) at dilution of 1:400, and CD99 (Abcam) at dilution of 1:400.  4.2.4 Quantitation of angiogenesis by assessing microvessel density Microvessel density assessment was performed as previously described55, 288 using anti-CD31 antibody. Tissue sections were scanned for identification areas with the highest microvessel density. These areas were then used for counting microvessels in 10 high-power fields. 66  Microvessel density was calculated using the mean vessel number counted in 10 high-power fields. Only those vessels clearly demarcated by positively stained individual endothelial cells or clusters of stained endothelial cells were taken into consideration.  4.2.5 Transwell migration assay (Boyden chamber migration assay) Cells were starved in serum-free media for 24hrs then added into the top chamber of 24-well Transwell plates (8 ?m; Trevigen; 100,000 cells/well). The bottom chambers were filled with medium supplemented with either 10% fetal bovine serum (FBS), 20 ng/ml insulin, or 100 ng/ml insulin like growth factor 1 (IGF-1). After 24 hrs of culturing, the migrating cells in the bottom chamber were collected in cell dissociation solution (Trevigen) containing 1 ?M of Calcein-AM. The percentage of migrated cells was calculated according to the standard curves previously created for each cell line.  4.2.6 Transwell invasion assay  Assays for cell invasion into basement membrane were conducted using Culturex Coated? 24 Well BME-Coated Cell Invasion platform (Cat# 3480-024-k; Cedarlane; 100,000 cells/well) according to manufacturer recommendations. Briefly, initial rehydration of the membranes was performed and followed by similarly ordered steps as described in the previous migration studies.  4.2.7 Matrigel 3-dimensional colony assay The assays were conducted in 6-well plate as previously described289. A bottom layer was created by applying 500?L of Growth factor reduced matrigel (BD Biosciences, cat# 354230) to 67  the wells and allowing for solidification in the incubator at 37 ?C. Then cells were prepared, after being washed, and harvested by re-suspending in the assay media (DMEM/F10 supplemented with 10 ?g/ml Epidermal Growth Factor, 5% Hoarse Serum, and 1% Penicillin-Streptomycin-Fungicide) in a final concentration of 20,000 cells/ml. Another preparation of the assay medium containing 4% matrigel was prepared and mixed with the cell-containing suspension in a 1:1 ratio and 500 ?l were added to each well. The culture was assessed to ensure single suspension and to exclude clump formation. Cells were then fed with 3-4 drops assay medium containing 2% matrigel every 3-5 days. The cultures were examined with phase-contrast microscopy every 2 days and imaged for detecting phenotypic changes. The experiment was conducted two times in triplicate for each cell line.  4.2.8 Wound healing assay Cells were grown to confluence then scratched in the middle followed by imaging over a 20-hr period at 1 hr interval to monitor their attempt to close the wound edge using an Axioplan2 fluorescence microscope (Zeiss) fitted with O2/CO2/temperature modules. Images were then analyzed using Volocity software. Eight cells per condition were randomly chosen from the wound edge and their paths were tracked with x- and y-axis migration plots of each cell were generated as well as the average total length of tracks for each cell line.   4.2.9 Statistical analysis Two- tailed student t test was used to analyze the data, unless otherwise indicated, with p value <0.05 indicating statistically significant differences.  68  4.3 Results 4.3.1 YB-1 increases childhood sarcoma cell motility in vitro and in vivo The ability of tumour cells to migrate and colonize nutrient-rich areas is an inherent feature of the metastatic process18. Given that YB-1 promotes EMT and elevated YB-1 levels are associated with increased invasiveness and metastatic capacity of breast cancer cells10, we hypothesized that YB-1 might also drive the metastatic progression of childhood sarcomas.  We therefore used different in vitro assay to study the impact of YB-1 downregulation on sarcoma cell migration. Using Boyden chamber transwell-migration assay, we found that serum-starved YB-1-competent cells exhibited a strong tendency to move towards different growth factors, including serum, insulin, and IGF1 (Fig. 4.1A). We then tested several sarcoma cell lines with YB-1 kd and consistently demonstrated a significant reduction in their ability to migrate towards serum (Fig. 4.1B). Using another in vitro assay, time-lapse imaging during wound-healing, we observed a marked reduction in motility by YB-1 deficient cells compared to the swift and erratic movement exhibited by YB-1 competent cells (Fig. 4.2A-B). The decrease in rate of movement of YB-1 kd cells in vitro may represent a potential indicator of inhibited metastatic ability in vivo. Moreover, data obtained from in vitro invasion assays revealed a significant reduction in the invasiveness capacity of YB-1 kd cells, shown for MG63 OS cells (Fig. 4.3A). Interestingly, matrigel 3-dimensional colony assays revealed a branching invasive phenotype for YB-1 competent cells versus the spheroid colonies displayed by YB-1 kd cells (Fig. 4.3B). Taken together, these data support a role for YB-1 in sarcoma cell movement and invasion which may in turn reflect a potential role for YB-1 in sarcoma metastasis.  To confirm our in vitro observations in vivo, we collaborated with Dr Jason Berman and his group and employed the zebrafish model which is highly efficient system for studying human 69  cancer development290. Their group has previously xenografted human leukemia cell lines into 48 hour zebrafish embryos to evaluate responses to targeted therapeutic agents287. We therefore adapted this system to childhood sarcoma cells and successfully xenotransplanted TC32 ES cells into zebrafish embryos. This demonstrated that TC32 cells survive in the yolk sac and then rapidly migrate to the tails of embryos within 120 hrs (Fig. 4.4A-B). In contrast to CTRL TC32 cells, YB-1 kd cells demonstrated a significantly reduced ability to migrate into embryo tails, supporting the above in vitro findings (Fig. 4.4A-B). Together, these data provide strong evidence that in childhood sarcoma cells, YB-1 strongly enhances their in vitro and in vivo migratory activity.  4.3.2 YB-1 drives childhood sarcoma cell invasion and metastasis in vivo We have used murine renal subcapsular xenotransplantation into NOD/SCID mice to assess the metastatic ability of +/- YB-1 kd cells in vivo. Initially, we tested luciferase- labelled TC71, and TC32, ES cell lines (Fig. 4.5A) for their ability to grow and metastasize. In vivo monitoring of the luciferase positive cell lines confirmed that they grew well under the renal capsule and that a proportion of the cells metastasized to the lungs (Fig. 4.5B), a predominant location for sarcoma spread12. YB-1 kd groups showed marked inhibition of their metastatic abilities (Fig. 4.5B). We also assessed YB-1 expression in the primary tumour xenografts to confirm its downregulation (Fig. 4.5C). Consistent with our findings in Chapter 3, primary tumour xenografts of YB-1 kd TC71 and TC32 cells showed enhanced growth compared to that of the control group (Fig. 4.6), however the difference did not reach statistical significance which may be attributed to the impact of tumour microenvironment, that can modulate the malignant behaviour of tumour cells in vivo.   70  Unexpectedly, YB-1 kd primary implantation site tumours demonstrated extensive regions of haemorrhage and necrosis compared with CTRL tumours, which was evident grossly (Fig. 4.7A, see arrows), microscopically (Fig. 4.7B; see arrows), and quantitatively (Fig. 4.7C). Moreover, as shown in Figure 4.7D for CHLA-10 and TC71 cells, CTRL tumours showed highly infiltrative borders with direct invasion into adjacent normal kidney, whereas YB-1 kd altered the growth pattern to non-invasive ?pushing? borders abutting neighbouring normal kidney. This provides direct evidence that YB-1 confers invasive behaviour to sarcoma cells in vivo. We then used morphology (Fig. 4.5B) and CD99 (an ES marker222) immunostaining (Fig. 4.8A) to screen for microscopic evidence of lung metastases. As shown in Fig. 4.8B-4.8D, YB-1 kd significantly reduced metastatic spread to lungs in all three ES cell lines tested using the murine renal subcapsular assay in terms of total numbers of mice with lung metastases (Fig. 4.8B) or the number of metastases in positive cases (Fig. 4.8D). For example, YB-1 kd completely eliminated the morphologic evidence of metastasis in the CHLA-10 ES cells (Fig. 4.8C & 4.8D, see right panel). The total numbers of mice with lung metastases for each cell line are illustrated in Figure 4.8C.  Interestingly, pulmonary metastases developed in mice harbouring YB-1 competent cells or YB-1 deficient cells (calculated for the few animals with lung metastases) revealed similarly high expression levels of YB-1 (Fig. 4.9) as detected by IHC. These metastatic cells could represent a pre-existing subset of most aggressive tumour cells found in the heterogeneous population of cells within the primary tumour that initially expressed high levels of YB-1291, 292. These results suggest that YB-1 expressing tumour cells are the more aggressive cells since they are able to invade, survive in the circulation, extravasate and colonize distant organs. Together, these 71  findings provide compelling evidence that YB-1 confers invasive and metastatic capacity to childhood sarcoma cells in vivo.  Since YB-1-expressing tumour cells have recently been shown to associate with endothelial cells293 and it is well established that angiogenesis is critical for tumour invasion and metastasis55, we assessed whether YB-1 kd would affect tumour vascularization as it could provide be targeted in order to inhibit metastatic spread. We assessed tumour blood vessels in +/-YB-1 kd primary tumour xenografts using IHC for the endothelial cell marker CD31294. We observed significant reduction of CD31 staining in the YB-1 kd primary tumour xenografts, suggesting a potential role for YB-1 in tumour angiogenesis (Fig. 4.10A & 4.10B). Taken together, these data provide strong evidence that YB-1 enhances the in vitro and in vivo migratory characteristics of childhood sarcoma cells.   4.4 Discussion Metastatic dissemination of tumour cells is the leading cause of death in cancer patients1, and this is especially true for sarcomas. Indeed, modern multi-agent chemotherapy regimens have made tremendous improvements to the outcomes of patients with localized high-risk childhood sarcomas such as ES, RMS, and OS. However, the prognosis for patients with metastatic disease remains dismal and still the presence of metastatic disease is the single-most powerful predictor of outcome in childhood sarcomas175, 177, 213, 245, 295, 296. While genetic lesions such as EWS-ETS gene fusions arising from translocations in ES297,6, and PAX3-FKHR and PAX7-FKHR gene fusions in alveolar RMS7-9, these alterations are present in both localized and widespread disease. They alone cannot therefore account for metastatic behaviour10, and many similar agents 72  are used for both localized and metastatic childhood sarcomas. While intensifying chemotherapy has improved the outcome in localized lesions, it has done little to impact the progression of metastatic disease. Thus, a prompting need to investigate and identify novel mediators that may serve as potential therapeutic targets is fundamental.   Of the crucial factors believed to play a role in sarcoma progression is the transcription/translation factor YB-1. Extensive literature underscored the oncogenic function of YB-1 and its remarkable contribution to tumour aggressiveness10, 92, 98, 154, 268, 269, however the potential contributions of YB-1 to childhood sarcomas are not fully elucidated yet. Our current study identifies important aspects of the mechanisms underlying sarcoma cell metastasis. We have now found that YB-1 protein levels are markedly elevated across virtually all ES, RMS, and OS cell lines tested, as well as in a subset of primary tumours as shown in Chapter 3. YB-1 promotes the motility of ES, RMS, and OS cell lines and YB-1 kd dramatically reduced sarcoma cell invasion in vitro. Conferring these characteristics on sarcoma cells is predicted to render them more likely to spread to distant sites and less likely to respond to conventional chemotherapy agents. While in vitro results demonstrating that YB-1 contributes to the invasion and migratory phenotype of sarcoma cells support a role for YB-1 in sarcoma metastasis, these assays do not take the tumour microenvironment into account. Studying the interactions of tumour cells with their surrounding microenvironment is necessary in order to more closely recapitulate what may occur in human patients with sarcomas.   To determine the effects of YB-1 expression on sarcoma cell metastasis in vivo, we have used zebrafish xenotransplantation. The zebrafish transplantation model has emerged as a highly 73  efficient model system for studying human cancer development291, 298, 299. Many oncogenes and tumour suppressor genes that play important roles in human malignancies have zebrafish homologues, and critical pathways regulating cell growth, proliferation, apoptosis, and differentiation are well-conserved 300-302. Large numbers of offspring, external fertilization, rapid embryonic development and optical clarity 302-304 are additional advantages of zebrafish models, and make them an attractive tool as a cancer model. Zebrafish xenotransplantation model has characterized YB-1 as a potent migratory/metastatic marker in childhood sarcomas. For our studies we also successfully used a unique murine renal subcapsular assay (RSA) entailing implantation of tumour cells under the kidney capsule, subsequently followed by monitoring using IVIS. The RSA has distinct advantages over subcutaneous or orthotopic injection models, due to the high level of vascularization within the sub-renal capsule graft site, which facilitates engraftment as well as tumour cell extravasation. Using this model, many human tumour cell lines exhibit predictable macroscopic, high-volume metastases283, 305, 306. Of the fundamental findings uncovered through this work is the link between YB-1 and angiogenesis. Tumour blood vessels play an important role in promoting tumour cell invasion and metastasis307. Therefore, it is not surprising that targeting proliferating endothelial cells of tumour blood vessels, through the use of anti-angiogenic drugs, is important aspect of a successful cancer treatment regimen308. Some anti-angiogenic drugs have been found to improve clinical outcome with various cancers309, 310. For example, the humanized monoclonal antibody to VEGF, bevacizumab (Avastin), in combination with conventional chemotherapy has achieved some success in treating advanced colon cancer311. In our study we also found that inhibiting YB-1 expression led to significant reduction in tumour blood vessel density and resulted in enhanced necrosis. These findings may have identified a new potential role for YB-1 in 74  metastatic dissemination of malignant cells. Our results have defined YB-1 as a promising new target to be considered for cancer therapy. Taken altogether, these results support a dual role for YB-1 in sarcoma metastasis, first through YB-1?s ability to enhance migration and invasion of sarcoma cells, and second through its ability to enhance recruitment of new blood vessels to the tumour, providing a mean for it to access nutrients as well as a way to enter the circulation, both of which are indispensable for metastatic spread.   To demonstrate whether our results have relevance to the clinical setting, Dr Olivier Delattre at the Institut Curie, France, and his group specifically analyzed a publically available ES database (GSE34620), comprised of 117 cases linked to patient outcome data 312, which revealed significantly higher YB-1 transcript expression in metastatic compared to localized ES. They found that elevated YB-1 expression levels also correlated significantly with higher mortality in ES, supporting a role for YB-1 as a predictive marker of poor survival in this disease. Further, elevated YB-1 expression in localized or metastatic ES at diagnosis also correlated significantly with reduced survival time and higher mortality rates.  Our findings therefore undoubtedly support a crucial role for YB-1 in metastatic spread and poor survival of childhood sarcomas. As mentioned, our group previously found that YB-1 promotes metastatic spread of epithelial-derived  mammary carcinoma cells via translational upregulation of Snail1 and Twist mRNAs10, so we wondered whether a similar mechanism is involved in sarcoma metastasis, and this is to be discussed in Chapter 5.   4.5 Figures   75   Figure 4.1    YB-1 contributes to the transmigratory ability of sarcoma cells in vitro  A. Culturex cell migration assays of the indicated sarcoma cell lines, transfected with 20 nM individual YB-1 siRNAs or scrambled siRNA. The experiment was conducted at the fifth day post-transfection. The results are displayed as the percentage of cells migrating to chambers containing no serum. i.e. serum starved (SS), 10% fetal bovine serum (FBS), 100 nM insulin, or 76  100 ng/ml insulin-like growth factor 1 (IGF1) as indicated. The result of three independent experiments, each performed in triplicate, are presented as mean + SD. B. Migration of different sarcoma cells +/- siRNA-mediated YB-1 kd at the fifth day post-transfection  towards serum. Serum starved (SS) media was used as negative control. The results of three independent experiments counted in triplicate are represented as a mean + SD.   Where shown * = p<0.05; ** = p <0.01.                  77   Figure 4.2    YB-1 empowers the highly motile phenotype of sarcoma cells in vitro A. Wound healing assay. The migration pattern of individual cells was determined by time-lapse imaging. The migration path of eight cells taken randomly from the wound edge for each of +/- YB-1 knockdown treated cell lines.  78  B. Charts represent Comparison of the average distance travelled by +/- YB-1 knockdown sarcoma cells. Data represent the mean of two independent experiments counted in triplicate + SD for eight cells tracked for each cell line.  Where shown * = p<0.05, ** = p<0.005,                79   Figure 4.3    YB-1 promotes sarcoma cell invasion in vitro A. In vitro invasion of MG63 cells +/- shRNA-mediated YB-1 knockdown into a basement membrane containing matrix towards 10% FBS. The results of two independent experiments performed in triplicate are shown as mean + SD.   B. Invasive capacity measured using Matrigel 3-dimensional colony assays. The TC71 ES cell line +/- shRNA-mediated YB-1 knockdown was grown in Matrigel for 10 days. Three-dimensional structures were then photographed using phase-contrast microscopy. Scale bars: 25 ?m.  Where shown *** = p <0.001.     80   Figure 4.4    YB-1 promotes sarcoma metastasis: Zebrafish xenotransplantation model A. TC32 ES cells +/- shRNA-mediated YB-1 knockdown were labelled with Cm-DiI (white coloration) and injected into 48 hour post-fertilization (hpf) casper zebrafish embryos. Bright-field (Bf) and fluorescent (Fl) live lateral images of the head (anterior) and tail (posterior) of embryos at 24 and 120 hours post?injection (hpi) are shown (n = 15-20 embryos). Insets show representative high power magnification of fluorescent TC32 cells in embryo tail regions at 120 hpi. 81  B. Quantification of sarcoma cell migration to the tail regions of xenotransplanted casper embryos at the indicated time points.  Where shown * = p <0.05; hpi: hours post-injection.             82    Figure 4.5    The murine renal subcapsular assay for studying the metastatic capacity of +/- YB-1 kd cells in vivo A. Effects of YB-1 kd on the dissemination of luciferase- labelled TC71 and TC32 ES cells in vivo. Luciferase- labelled cells +/- shRNA-mediated YB-1 knockdown were subjected to renal subcapsular implantation in immunocompromised mice and monitored for tumour formation using an in vivo imaging system (IVIS) at 6 weeks post-implantation. B. Low power photomicrographs showing H&E staining of metastatic pulmonary lesions (arrows) in mice with renal subcapsular tumour xenografts of the indicated ES cell lines +/- YB-1 kd. Scale bars: 100 ?m. 83  C. IHC staining of YB-1 in primary tumour xenografts established from the indicated ES cell lines +/- YB-1 kd. Scale bars: 25 ?m.  ?              84   Figure 4.6    Characteristics of primary tumour xenografts  +/-YB-1 knockdown  Representative pictures of the +/- YB-1 knockdown primary tumour xenografts which were formed after renal subcapsular implantation in immunocompromised mice.          85   Figure legend starts next page 86  Figure 4.7    Inhibiting YB-1 expression induces loss of tumour cell viability and inhibition of its local invasiveness capacity in vivo A. Representative pictures of primary tumour xenografts of the indicated cell lines +/- shRNA-mediated YB-1 knockdown formed after renal subcapsular implantation in immunocompromised mice. Arrows indicate areas with extensive grossly evident haemorrhage and necrosis. B. TC71 and TC32 ES cell lines +/- shRNA-mediated YB-1 knockdown were implanted under the renal capsules of immunocompromised mice as described in the Extended Experimental Procedures. Photomicrographs show haematoxylin & eosin (H&E) stained sections of primary tumour xenografts of the indicated cell lines. Arrows show areas of necrosis. Scale bars: 100 ?m.  C. Left panels: Gross photos showing H&E staining of whole mount tissue sections from TC71 primary renal subcapsular tumour xenografts. Black lines are drawn around necrotic areas (pink). Right panel: Quantification of necrotic areas from renal subcapsular tumour xenografts of TC71 and TC32 cell lines +/- YB-1 knockdown, as assessed by H&E staining. Results are expressed as the percentage of necrotic areas compared to total sectioned areas of tumours. Error bars indicate SD for n = 3. D. Photomicrographs of H&E stained sections of the tumour-normal kidney interfaces of renal subcapsular primary tumour xenografts of CHLA10 and TC71 cell lines +/- YB-1 knockdown. Arrows in control tumours (CTRL) show highly infiltrative borders with direct invasion into adjacent normal kidney, whereas arrows in YB-1 knockdown tumours show so-called ?pushing? non-invasive borders. Scale bars: 100 ?m. Where shown * = p<0.05, ** = p<0.01.   87    Figure 4.8    YB-1 is a major contributor to sarcoma cell metastasis in vivo A. Left panels: Immunohistochemical detection of CD99 surface expression in primary tumour xenografts of TC32 cells using anti-CD99 antibodies. Right panels: IHC staining of the ES marker CD99 in lungs of mice with renal subcapsular tumour xenografts of TC32 ES cells +/- YB-1 knockdown. Scale bars: 100?m. 88  B. Graph comparing the total numbers of mice bearing renal subcapsular tumour xenografts of TC71, TC32, and CHLA10 cell lines +/- YB-1 knockdown that developed pulmonary metastases determined using a Fisher?s exact test. C. Graph showing the total number of mice that developed pulmonary metastases (Mets) 10-12 weeks following renal subcapsular implantation of TC71, TC32, and CHLA10 ES cell lines +/- YB-1 kd in immunocompromised mice. D. Comparison of the number of pulmonary metastases in mice bearing renal subcapsular tumour xenografts of TC71, TC32, and CHLA10 cell lines +/- YB-1 knockdown determined using a Wilcoxon two-sided rank sum test. Where shown * = p<0.05; ***=P<0.001.               89    Figure 4.9    YB-1 expression in pulmonary metastases IHC staining for YB-1 in lung metastases in mice bearing renal subcapsular tumour xenografts of TC71, and TC32 cell lines +/- YB-1 kd. Scale bars: 100 ?m.          90   Figure 4.10    YB-1-mediated angiogenesis A. Tumour blood vessel development in primary implantation site tumours of TC71, TC32, and CHLA10 ES cell lines +/- YB-1 knockdown, as detected by CD31 IHC on formalin-fixed, paraffin embedded tissue sections. Scale bars: 50 ?m. b. Quantification of microvessel density from (A) as determined using a two-tailed student?s T test. Where shown *=<0.05; ***, = p<0.001.          91  Chapter  5: Translation activation of HIF1? by YB-1 is a novel mechanism contributing to childhood sarcoma metastasis   5.1 Rationale In Chapter 4, we established YB-1 as a metastatic marker in childhood sarcomas. Metastasis-associated genes have several features in common, the most important of which is their ability to initiate response to environmental stresses313. Given that YB-1 mediates increased invasiveness and metastatic capacity of breast cancer cells through the translational up-regulation of Snail1 and Twist  mRNAs10, two crucial molecules that play integral roles in the stress response314, we wondered if a similar mechanism could explain the effects of YB-1 on sarcoma cell invasiveness. As YB-1 has been established as a translational regulator91, 315, we hypothesized that YB-1 promotes invasion and metastatic dissemination of ES, RMS, and OS through translational up-regulation of stress-mitigating proteins, and that this is mechanistically related to sarcoma metastasis.  5.2 Material and methods 5.2.1 Animal studies We have used the murine renal subcapsular assay as previously described in Chapter 4. To prepare xenograft cell blocks 1x106 cells (for CHLA10 or MNNG cell lines) per block were used in this study. For the grafting procedure, 6-8 week-old NOD-SCID male mice (6 per condition for the CHLA10 and MNNG cell lines) were used and maintained according to UBC Animal 92  Care Committee (ACC) regulations. Mice were monitored for tumour growth for 8 weeks for CHLA10 and 12 weeks in for MNNG.   5.2.2 Histological evaluation and IHC studies of tumour xenografts Histological evaluation and IHC was conducted as previously described in Chapters 3 and 4. HIF1? IHC staining was performed using HIF1? antibody (Novus;1:150 dilution), while VEGF was provided in a pre-diluted, ready-to-use format. IHC was conducted using the Ventana discovery XT system. Antigen retrieval was performed using the Ventana CC1. Detection was carried out using Ultramap anti-Rabbit HRP (Ventana). Images were acquired with an Axioplan2 fluorescence microscope (Zeiss).  5.2.3 Hypoxia treatment A hypoxic incubation chamber (COY, Laboratory Products Inc. Grass Lake, MI) was employed to maintain the cells at 37oC in a 1% O2, 5% CO2, 95% N2-containing atmosphere.  5.2.4 RNA isolation, quantitative RT-PCR, and primers The procedures were previously described in Chapter 3. The human primers used in this study are listed below. Gene    Primer sequences HIF1?    forward: 5'-TGATGACCAGCAACTTGAGG-3'               reverse: 5'-CTGGGGCATGGTAAAAGAAA-3' Probe is: HIF1? 5'-AATTTGGCAATGTCTCC-3 HIF2? (SYBR Green)  forward:5'-GTGCATCATGTGTGTCAACTACG-3' reverse:  5'-GGGCTTGAACAGGGATTCAGTC-3' 93  Gene Primer Sequences L 32  VEGF (SYBR Green)  XIAP (SYBR Green) forward: 5'-GGCGGAAACCCAGAGGCATTGA-3' reverse: 5'-CCTGGCGTTGGGATTGGTGACTCT-3' forward: 5'-AGGCCAGCACATAGGAGAGA-3' reverse: 5'-GCGAGTCTGTGTTTTTGCAG-3' forward: 5'-GACAGTATGCAAGATGAGTCAAGTCA-3' reverse: 5'-GCAAAGCTTCTCCTCTTGCAG-3'  List of Taqman Primers used for qRT-PCR  Gene Catalogue No. GAPDH (homo sapiens) YBX1 (homo sapiens)  Hs02786624_g1 Hs02742755_g1   For HIF1? and SYBR Green assays, 45 cycles of denaturation, annealing and extension were carried out.  The q-PCR cycling conditions were [2 minutes at 50oC, 10 minutes at 95oC, 1 minute at 94oC, 45 sec at 60 oC, 45 sec at 72 oC, 7.00 minutes. (Hold)]. Dissociation curves are carried out at the end of a qPCR experiment for SYBR Green assays. The q-PCR cycling conditions for Applied Biosystems predetermined assays were previously described in Chapter 3. 5.2.5 Antibodies Additional antibodies used in this study are as listed below. Antibodies Company Catalogue No. Dilution Akt (total): rabbit polyclonal HIF1a: rabbit polyclonal HIF2?: rabbit polyclonal VEGF: rabbit polyclonal NEB Canada Cayman Sigma IHC World 9272 610111 E5408 IW-PA1080 1:1000 1:1000 1:1000 Predilute 94  5.2.6 In Vitro multilayered cell culture (3-dimensional tissue culture model) 5.2.6.1 Cell culture In collaboration with Dr. Andrew Minchinton and his group at BC Cancer Research Center, we employed a three dimensional (3D) tissue culture model to study the relationship between YB-1 and HIF1?. 3-D tissue discs were grown by seeding 1 or 4 ?l of cells +/- YB-1 kd into collagen coated tissue culture inserts (CM 12 mm, pore size 0.4?m, Millipore, Nepean ON). Seeded inserts were then incubated for 16 hours prior to transfer to stirred growth vessels. The tissue was then grown for 5 days under 5% O2, 5% CO2 at 37?C. On day 5 cultures were incubated with 100 ?M BrdU (Sigma) and 50 ?M pimonidazole (Hypoxyprobe, Inc. USA) for 2 hours to label proliferating cells and hypoxic areas. The work described below was performed by Dr. Alastair Kyle.  5.2.6.2 IHC for BrdU, pimonidazole and HIF1? Ten micron thick cryosections taken from the tissue discs were dried overnight and then fixed in a 1:1 mixture of acetone-methanol for 10 minutes at room temperature. Sections were then stained for 2 hours with a 1:200 dilution of mouse-anti-pimonidazole (Hypoxyprobe, Inc. USA) and 1:500 rabbit-anti- HIF1?. Secondary detection was carried out using 1:200 anti-mouse Alexa 488 and anti-rabbit-546. Slides were imaged for fluorescence and then transferred to distilled water for 10 minutes and then treated with 2 M HCl at room temperature for 1 hour followed by neutralization for 5 minutes in 0.1 M sodium borate. Slides were then washed in PBS. BrdU incorporated into DNA was detected using a 1:500 dilution of monoclonal rat anti-BrdU (clone BU1/75, Abcam) followed by 1:200 dilution of Alexa-anti-mouse 647 and imaged. Slides were 95  then counterstained with haematoxylin, dehydrated and mounted using Permount (Fisher Scientific, Fair Lawn, NJ, USA).  5.2.6.3 IHC for YB-1 Ten micron thick cryosections taken from the tissue discs, were dried overnight and then fixed in neutral buffered formalin (NBF) for 15 minutes and then washed in PBS with 0.1% Tween 20 (used for all washes and dilutions). Slides were immunostained with either 1:200 mouse-anti YB-1 (101198, Santa cruz Biotech.) or 1:100 rabbit-anti-YB-1 (D299, cell signaling). Secondary detection was carried out using either 1:200 anti-mouse Alexa 546 or anti-rabbit Alexa 546. Slides were then imaged for fluorescence.  5.2.7 Image acquisition The imaging system consists of a robotic fluorescence microscope (Zeiss Axioimager Z1, Oberkochen, Germany), a cooled, monochrome CCD camera (Retiga 4000R, QImaging, Vancouver, BC, Canada), a motorized slide loader and x-y stage (Ludl Electronic Products, Hawthorne, NY, USA) and customized ImageJ software (public domain program developed at the U.S. National Institutes of Health, available at http://rsb.info.nih.gov/ij/) running on a Macintosh computer (Apple, Cupertino, CA, USA).   5.2.8  In vitro transcription/translation HIF1? 5?UTR-LUC vector harbouring the entire HIF1? 5?UTR sequence was a kind gift from Dr. Gregory Goodall (University of Adelaide) and was previously described 316 while the ?-96  globin 5?UTR-LUCvector coding for the 5?UTR ?-globin-LUC was kindly provided by Dr. Valentina Evdokimova (Sunnybrook Health Sciences Centre) and previously described10.   In vitro transcription/translation reactions were performed using TNT? SP6 Quick Coupled Transcription/Translation System (L2080, Promega) according to the manufacturer?s recommendations. Two ?g of DNA constructs were added to TNT? Quick Master Mix in the absence or presence of increasing amounts of YB-1 recombinant protein (H00004904-P01, Abnova), previously described317, and incubated in a 25?l reaction volume for 90 minutes at 30?C. The expression reaction was assayed for luciferase activity. The results of two independent experiments counted in triplicate was presented as mean + SD.  5.2.9 Immunohistochemistry (IHC) for HIF1? in osteosarcoma IHC for HIF1? in osteosarcoma was conducted as previously described in Chapter 3.   5.2.10 IHC for YB-1 and HIF1? expressions in paediatric cancer tissue microarray  Tissue microarray (TMA) of paediatric tumours, kindly provided by Dr. Catherine Pallen (University of British Columbia), was constructed from ES (20 cases) and RMS (41 cases) formalin-fixed, paraffin-embedded (FFPE) tissue samples. IHC was performed as described above using YB-1 or HIF1? antibodies. Two cores for each case were used. YB-1 IHC staining was performed using the YB-1 antibody (Cell Signaling; 1:25 dilution) while HIF1? IHC staining was performed using the HIF1? antibody (Novus; 1:150 dilution). For each case, the percent of cells positively stained for YB1 and HIF1? and staining intensity were evaluated as previously described in Chapter 3. Scoring of the TMA was carried out by Dr. Poul Sorensen, and Amal EL-Naggar and correlation between YB-1 and HIF1? percent 97  positive cells stained within sarcoma subtypes was statistically evaluated using a cor. test. While association associations between YB1 and HIF1? staining intensity was statistically evaluated using Fisher's exact test. Statistical analysis was conducted by Dr. Steven McKinney of the BC Cancer Research Center.   5.2.11 HRE-GFP reporter assays   Sarcoma cells +/- YB-1 kd were transfected with a GFP-HRE construct (a kind gift from Dr. Peggy Olive, BC Cancer Research Center) at a final concentration of 1?g/well using the siLentFect transfection reagent and further incubated either under 21% O2 or 1% O2 for 16hrs. This was followed by fixation of the cells using 4% paraformaldehyde. GFP expression (green) was observed using a fluorescence microscope. DAPI was the nuclear counterstain. pMAX-GFP construct (Amaxa GmbH, Cologne, Germany) was used as positive control. For GFP-HRE transfection into +/- HIF1? or HIF2? kd cells, cells were first transfected with HIF1? or HIF2? siRNAs and 2 days later were further transfected with GFP-HRE construct. The following experimental steps were conducted as previously described above.   5.2.12 Luciferase assays The pRF-hHIF1? vector harbouring the 5'-UTR of human HIF1? (gift from Dr. Gregory Goodall), previously described316 or the pRF control bicistronic vectors were transiently transfected into +/- YB-1 kd sarcoma cells using siLentFect (Bio-rad). 500 ng of each of the indicated vectors was transfected per well in 12-well plates (Costar). 48 hrs post-transfection, cells were lysed and analyzed for Rluc and Fluc activity using the Dual Luciferase Reporter Assay System (Promega). Luciferase activity was expressed as the ratio of Fluc HIF1? 98  containing vector/ Fluc control vector. Results of two independent experiments counted in triplicate were presented as means + SD. For HRE-Luc assay in +/- HIF1? or HIF2? kd cells, cells were transfected with a PGL2-HRE-firefly luciferase reporter construct (Addgene, Cambridge, MA, USA) and the control renilla luciferase plasmid (Addgene) together with isoform-specific HIF siRNA using Lipofectamine 2000 (Invitrogen). Twenty-four hours post-transfection cells were exposed to normoxic or hypoxic conditions for additional 24 hrs then lysed in passive lysis buffer (Promega, Madison, USA). Firefly and renilla luciferase were assayed using the Dual-Luciferase Reporter Assay System (Promega). Firefly luciferase values were normalized to the renilla transfection control. The experiment was repeated three times and error bars represent the average + SD.   5.2.13 Transient HIF1? transfection Wild type HIF1? or mutant form (resistant to degradation), kind gift from Dr Thailo Hagen (University of Singapore) were transfected into +/- YB-1 kd sarcoma cells using siLentFect. 500 ng of wild type HIF1?, mutant vector, or empty vector were transfected per well in 12-well plates. One day post-transfection, cells were incubated under normoxia (21%O2) or hypoxia (1%O2) for 40 minutes. Then, cells were lysed and analyzed for HIF1? expression.  5.2.14 HIF1? and HIF2? silencing  Transfection was performed using siLentFect (Bio-Rad, Hercules, CA, USA). The cells were plated at 60% confluence then transfected either with scrambled (siCTRL) (Dharmacon, Lafayette, CO, USA), HIF1?-specific (siHIF1?) siRNA pools (Dharmacon) or HIF2?-specific (siHIF2?) 27 mer siRNA duplexes (OriGene, Rockville, MD, USA) at a final concentration of 99  25nM. One day later, the cells were incubated under either normoxic or hypoxic conditions for 40 minutes. The samples were then collected for Western blot analysis.     5.2.15 HIF1? and HIF2? decay assay  Cells were exposed to normoxic or hypoxic conditions for two hours to allow HIF1? protein accumulation. Cyclohexamide was then added to the cultured cells at a final concentration of 100ug/ml to inhibit the translation process. Samples were collected at different time points (0, 20, 40, and 60 minutes) both under normoxia and hypoxia to assess the rate of decay of HIF1? and HIF2?.  5.2.16 RNA immunoprecipitation Direct interaction between HIF1? transcript and YB-1 protein was confirmed by pull-down experiments using RIP-Assay Starter Kit YBX1 (MBL International Corporation, Cederlane), previously described318-320, following manufacturer instructions. All steps were performed on ice, with ice-cold reagents unless otherwise specified. 1.5x107 cells were washed and resuspended in 500 ?L ice-cold lysis buffer containing protease inhibitor, RNase inhibitors, and Dithiothreitol (DTT). Following cell lysis, nuclei and cell debris were cleared by centrifugation at 12,000 x g, 5 minutes, 4oC. The isolated supernatant from the previous step was applied to 25 ?L protein A agarose beads slurry resuspended in lysis buffer and incubated at 4oC with rotating for 1 hour followed by centrifugation at 2,000 x g for 1 minute at 4oC and the resultant supernatant was transferred to a new tube. Thereafter, 25 ?L of 50% protein A agarose beads slurry resuspended in lysis buffer was incubated with either Normal Rabbit IgG (NRS) or anti-YBX-1 antibody with gentle agitation for 1 hour at 4oC. Thereafter, beads were washed once with ice-cold lysis buffer, 100  centrifuged at 2,000 x g for 1 minute, and then incubated with 500 ?L of cell lysate with gentle agitation for 3 hours at 4oC. Then, beads were washed 4 times with wash buffer and centrifuged at 2,000 x g for 1 minute. Then, 400 ?L of Master mix solution was added to the beads followed by thorough vortexing and spinning down. Following the previous step, 250 ?L of solution III was added followed by thorough vortexing and spinning down. Centrifugation at 2,000 x g for 2 minutes was conducted and the supernatant was transferred to a new tube containing 2 ?L of solution IV. Thereafter, 600 ?L of ice-cold 2-propanol was added and sample was placed at -20 oC for 20 minutes followed by centrifugation at 12,000 x g for 10 minutes. The pellet washed twice with 0.5 mL of ice-cold 70% Ethonol. Then, we allowed the pellet to dry up for 5-15 minutes. Finally, the pellet was dissolved in nuclease-free water. RNA was quantified with NanoDrop (Thermo Fisher Scientific Inc.) with optical density (OD) A260/A280=2. Equal amounts of cDNA were synthesized using the QuantiTect Reverse Transcription Kit (Qiagen) which was subsequently used for conducting quantitative real time RT-PCR.  5.2.17 Statistical analysis Two- tailed student t test was used to analyze the data, unless otherwise indicated, with p value <0.05 indicating statistically significant differences.   5.3 Results 5.3.1 YB-1 kd affects the expression of stress mitigating proteins including HIF1?  To address whether YB-1 promotes childhood sarcoma metastasis through a similar mechanism as was found in our breast cancer study10, we checked the expression of common mesenchymal related proteins involved in EMT activation and previously shown to be regulated by YB-1. 101  Consistent with our previous findings321 and confirming our results displayed in Chapter 3, YB-1 kd negatively impacts the expression of common EMT-related proteins such as N-cad, Twist, and Snail1 (Fig. 5.1A & 5.1B). However, we found that Snail1 and Twist are only weakly or not consistently expressed across the childhood sarcoma line panel used in this study, although YB-1 kd did reduce Snail1 and Twist protein levels in several sarcoma cell lines with detectable expression. We therefore explored other EMT-associated transcription factors (TFs) potentially regulated by YB-1 in breast cancer10. As mentioned, in the RSA model YB-1 kd primary implantation site tumours exhibited extensive haemorrhage and necrosis compared to CTRL tumours, both grossly and histologically (See Chapter 4). This was similar to what has previously been observed in tumours with reduced HIF1? levels322, and so we chose to focus on the pro-angiogenic protein, hypoxia inducible factor 1? (HIF1?). Roles for HIF1? in tumour angiogenesis, cancer progression, and drug resistance are well documented101, 323, 324. We found that protein levels of HIF1? were markedly reduced by YB-1 kd across all childhood sarcoma cell lines tested, under both normoxia and hypoxia (Fig. 5.1C & 5.1D), respectively. This finding was not observed for HIF2? (Fig. 5.1A). Of interest, both HIF1? and YB-1 showed induction, observed only in YB-1 competent cells, over time-course of hypoxia (Fig. 5.1E).  In the breast cancer system, we observed increased HIF1? expression in cells with upregulated YB-1 protein levels10. In the CHLA-9 and CHLA-10 ES cell line pair derived from the diagnostic specimen (CHLA9) or from the recurrent tumour after four rounds of chemotherapy (CHLA10)325, we observed that the increased HIF1? protein levels mirrored the elevated YB-1 expression in CHLA-10 cells (Fig. 5.1F).  102  5.3.2 HIF1? rather than HIF2? plays essential roles in sarcomas To address whether HIF1? plays crucial roles in our system, we investigated HIF1? expression in osteosarcoma as a representative example of our model system. Osteoblasts; normal mesenchymal-derived bone cells, were used as the experimental control. We looked at relative HIF1? and HIF2? protein levels in osteoblasts (OB3, OB5) and osteosarcoma (MG63, MNNG) cell lines by Western blotting. HIF1? and HIF2? tend to be expressed at detectable levels in osteosarcoma cell lines, even under normoxic condition (Fig. 5.2; left panels). As expected, HIF1? and HIF2? were induced under hypoxia in all cell lines. Increased induction of HIF1? and HIF2? was observed in osteosarcoma compared to osteoblast cell lines and was associated with reduced cell death under hypoxia, as measured by cleaved caspase-3 levels and PARP cleavage (Fig. 5.2; left panels). To determine if this was due to increased expression of HIF1? or HIF2?, or both, we used specific versus scrambled control siRNAs to knock down HIF1? or HIF2? (Fig. 5.2; middle and right panels, respectively). After 24 hrs, cells were placed under normoxia or hypoxia for 40 minutes. The most obvious effects were observed with HIF1? knockdown under hypoxia, leading to a pronounced increase in the cleavage of caspase-3 and PARP mainly observed in the osteosarcoma cell lines (Fig. 5.2; middle and right panels). Only moderate effects were observed with HIF2? siRNA treatment. These data point to a crucial role of HIF1? in osteosarcoma.  Next, we assessed HIF1? & HIF2? transcriptional activity using a GFP reporter driven by a hypoxia responsive element (HRE)-containing promoter (GFP-HRE). Consistent with the above findings, MG63 and MNNG cell lines exhibited increased HRE-driven GFP fluorescence relative to osteoblast cell lines under both normoxia and hypoxia. HRE-GPF expression was blocked when the osteosarcoma cell lines were pre-treated with HIF1?-specific siRNA suggesting that 103  HIF1? plays a predominant role in the hypoxic response in these cells (Fig. 5.3A). Similar patterns of expression were observed in the positive controls, i.e., osteoblasts and osteosarcoma cells transfected with pCMV promoter-GFP construct (pMAX-GFP) (Fig. 5.3B).  To further confirm the transcriptional activity of HIF1? and HIF2?, cells were co-transfected with a renilla-expressing vector and a luciferase reporter plasmid containing the human HRE element, together with scrambled siRNA controls or siRNAs specific for either HIF1? or HIF2?. One day post-transfection, cells were exposed to normoxic or hypoxic conditions for 24 hrs. As shown in Fig. 5.3C, luciferase activity in all cell lines transfected with HIF1? or HIF2? siRNAs showed significant decreases in activity mainly under hypoxic conditions; however, effects of blocking HIF1? was found to be much more robust than HIF2? knockdown, further supporting a predominant role for HIF1? in the hypoxic response of MG63 and MNNG osteosarcoma cell lines.   5.3.3 HIF1? exhibited enhanced protein stability in osteosarcoma cells  Given that HIF1? is rapidly degraded under normoxia326, it was interesting to detect elevated expression under normoxic conditions in osteosarcoma cells compared to the normal osteoblasts (Fig. 5.2, left panel), so we wondered if a difference in protein stability could play a role in this finding. We therefore assessed the rates of HIF1? protein degradation in osteoblast and osteosarcoma cell lines in the presence of cycloheximide under normoxia and hypoxia by Western blotting (Fig. 5.4A). HIF1? protein levels were quantified at varying time points, normalized to loading controls, and graphically displayed (Fig. 5.4B). As shown in Fig. 5.4B, when these values were averaged for osteoblasts and osteosarcoma cell lines, we observed that 104  HIF1? was more rapidly degraded in osteoblast compared to osteosacoma cell lines. As shown in Fig. 5.4B and 5.5A, the half-life of HIF1? in OB3, OB5 osteoblast cell lines was approximately10-12 minutes under normoxia while under hypoxia; the protein half-life was approximately 16-27 minutes. In MNNG and MG63 cells, the half-life of HIF1? was approximately 18-20 minutes under normoxia while under hypoxia; the protein half-life was approximately 19-36 minutes. Similar to HIF1?, HIF2? was more stable in all cell lines under hypoxia than under normoxia, and more stable in osteosarcoma compared to osteoblast cell lines under both conditions (Fig. 5.4A and 5.4B). HIF2? was also more stable than HIF1? under all conditions. As shown in Fig. 5.4B and 5.5B, the half-life of HIF2? under normoxia in OB3 and OB5 was approximately 35-40 minutes, while under hypoxia it was > 60 minutes. For MNNG and MG63, the half-life of HIF2? was > 60 minutes) under both conditions. Taken together, these data suggested that changes in HIF1? protein levels may play a role in sarcoma progression.  5.3.4 Elevated HIF1? protein levels are associated with high grade osteosarcoma We next used IHC to assess HIF1? expression in the same osteosarcoma cases used for assessing YB-1 expression, Chapter 3. We found that osteoblasts lining the normal bone trabeculae exhibited HIF1? cytoplasmic immunoreactivity with mild to moderate intensity. In addition, HIF1? showed variable expression in the cytosol of endothelial cells and scattered stromal cells (Fig. 5.6Ai). In malignant cells, HIF1? showed various staining patterns (Fig. 5.6Aii-v). Indeed, similar patterns of HIF1? expression were observed in osteoblasts and osteosarcoma cell lines (Fig. 5.6B-C). HIF1? immunoreactivity in the clinical samples was estimated by counting the number of positive cells per 1000 tumour cells. Of the 25 osteosarcoma cases examined, 12 105  (48%) showed mild to moderate cytoplasmic HIF1? immunoreactivity while the remaining 13 (52%) cases exhibited moderate to strong predominantly nuclear HIF1? immunoreactivity, (Fig. 5.6Aii-v, Table 5.1), based on the median value of HIF1? expression. Pathological data was then examined for correlations with HIF1? expression, and similar to YB-1, increased HIF1? expression significantly correlated with advanced grade (Table 5.2).  5.3.5 YB-1 is a survival factor induced under hypoxia  To further investigate the potential relationship between YB-1, hypoxia and HIF1?. We checked YB-1 expression under hypoxia. YB-1 itself was upregulated under hypoxia in each cell line, shown for MNNG, TC71, and Rh18 cells in Fig. 5.7A & 5.7B. These results suggest that YB-1 is a hypoxia-induced factor, and in TC71 cells its induction clearly preceded that of HIF1? (Fig. 5.7A). YB-1 has a protective effect on sarcoma cells under hypoxia, as YB-1 kd led to enhanced cell death when cells were grown at 1% O2 (Fig. 5.7C & 5.7D), similar to the observed effect of HIF1? kd (Fig. 5.2). We then used the previously established multilayered cell culture (MCC), a three-dimensional tissue culture model of solid tumours for analyzing HIF1? expression, in which an O2 gradient is generated across 3-D cultures of tumour cell lines327.  Using pimonidazole to highlight hypoxic regions (Fig. 5.7E; green labeling in left panel); YB-1 was specifically induced within central hypoxic areas of CTRL sarcoma cell line cultures in this model (Fig. 5.7E; right panel).  5.3.6 YB-1-induced HIF1? is biologically active To further demonstrate the impact of YB-1 on HIF1? protein expression, we used 3D cell culture systems to complement results obtained using traditional monolayer culture systems as they 106  permitted cell-cell and cell?matrix interactions which help to more closely recapitulate the in vivo tumour than 2D monolayer cultures. While HIF1? was also highly induced in the hypoxic zones found in the CTRL sarcoma cells (Fig. 5.8A), this was significantly reduced whenYB-1was knocked down in the same cells (Fig. 5.8A & 5.8B). To assess whether HIF1? induced by YB-1 is biologically active, we used a hypoxia response element (HRE) reporter construct linked to GFP328 and monitored its activity in sarcoma cells +/- YB-1 kd. This revealed significantly higher levels of HRE activity in CTRL compared to Rh30 YB-1 kd cells under both normoxia and hypoxia (Fig. 5.8C). Together, these data provide strong evidence to support a role for YB-1 in controlling HIF1? expression across diverse childhood sarcoma cell lines, particularly under hypoxia.  5.3.7 Co-expression of YB-1 and HIF1? in paediatric sarcomas  To investigate whether YB-1 and HIF1? are co-expressed in sarcoma specimens, tissue microarrays (TMA), kindly provided by Dr Catherine Pallen (University of British Columbia), were constructed from ES (20 cases) and RMS (41 cases) and stained for YB-1 and HIF1?. YB-1 expression strongly correlated with HIF1? immunstaining in both ES and RMS cases (Tables 5.3-4, Fig. 5.9A & 5.9B), consistent with our in vitro assays, supporting a potential role for YB-1 in regulating HIF1? in paediatric sarcomas. Unfortunately, survival data were unavailable for these cases.  5.3.8 YB-1 translationally activates HIF1? transcripts in childhood sarcoma cells We next sought to determine the mechanism by which YB-1 regulates HIF1? expression. YB-1 is known to act through both transcriptional and translational mechanisms107.  To distinguish 107  between these processes we first compared HIF1? total and translationally active polysome-bound mRNA levels in cells with or without YB-1 kd, using quantitative RT-PCR and HIF1? specific primers. Total transcript levels were consistently not induced by YB-1. In fact in Rh30 and MNNG YB-1 kd sarcoma cells, HIF1? transcripts either increased or stayed the same as CTRL cells, thus failing to support YB-1-mediated transcriptional regulation of HIF1?. In contrast, we observed strong enrichment of HIF1? transcripts in polysomal fractions under normoxia, which was significantly reduced by YB-1 kd (Fig. 5.10A). This was markedly enhanced under hypoxia, with increased HIF1? polysomal distribution compared to YB-1 kd cells (Fig. 5.10B) while HIF2? transcript levels were unaffected under identical conditions (Fig. 5.10C). These data indicate that, similar to translational activation of Snail1 and Twist mRNAs in breast cancer10, YB-1 specifically enhances the proportion of HIF1? mRNAs undergoing active translation in childhood sarcoma cells. To further validate this, we performed ribonucleoprotein (RNP) immunoprecipitation (RIP) with anti-YB-1 antibodies, similarly reported136. We found that YB-1 directly binds HIF1? transcripts in sarcoma cells (shown for MNNG cells in Fig. 5.10D). YB-1 and CCND1 transcripts, previously shown to be bound by YB-1136, 329, served as positive controls, while L32 served as a negative control 136.   5.3.9 YB-1 enhances HIF1? translational efficiency in vivo & in vitro It is plausible that hypoxia promotes a switch to cap-independent translation of a limited subset of essential mRNAs that contain IRESs in their 5? UTRs, including HIF1?, while at the same time repressing most protein synthesis330. To address whether YB-1 may promote HIF1? translation through its 5? UTR, we used a bicistronic reporter construct harbouring the entire HIF1? 5? UTR region cloned in between the Renilla and firefly luciferase (LUC), and driven by 108  simian virus 40 (SV40) promoter as previously described316, to assess the impact of this 5?-UTR on YB-1-driven mRNA translation efficiency in vivo. This revealed significantly higher LUC activity in MNNG CTRL versus YB-1 kd cells (Fig. 5.11A), indicating that in living cells, YB-1 is able to facilitate translation of transcripts containing the HIF1? 5? UTR. To directly address this, we compared in vitro translation efficiencies of a LUC reporter mRNA linked to the 5?-UTR of either HIF1? (5?-HIF1?-LUC)316 or 5?-?-globin (5?-?-globin-LUC) in SP6 vector, previously described316. As shown in Figure 5.11B, YB-1 increased the translation efficiency of 5?-HIF1?-LUC at lower YB-1 concentration while having no effect or lowering translation of 5?-?-globin-LUC. These results are very similar to the observed effects of YB-1 on Snail1 mRNA expression in breast cancer cells10, and strongly point to a role for YB-1 in controlling HIF1? translation. Indeed, cross-species analysis reveals numerous conserved G-C rich sequences in the 5?-UTR of HIF1? (Fig. 5.11C), and several of these sequences are conserved between 5? UTR regions of HIF1? and Snail1 (Fig. 5.11D). As with the Snail15?-UTR10, the HIF1? 5?UTR is also predicted to form highly stable stem loop structures (Fig. 5.12), and thus to be susceptible to YB-1-mediated cap-independent translational regulation.  Furthermore, we wondered if YB-1 promotes enhanced expression of HIF1? by contributing to its stability as well. Treatment of YB-1 kd cells with the proteasome inhibitor MG132 only partially restored HIF1? protein levels and not to same levels as in CTRL cells suggesting that proteasome degradation was also not involved (Fig. 5.13A). Then, we assessed the rates of HIF1? protein degradation in +/- YB-1 kd sarcoma cells using cyclohexamide decay assay under hypoxia by Western blotting (Fig. 5.13B, top panel). HIF1? protein levels were quantified at varying time points, normalized to loading controls, and graphically displayed (Fig. 5.13B, 109  lower panel). In addition, we assessed the major components involved in HIF1? degradation in +/- YB-1 kd sarcoma cell (Fig. 5.13C). Indeed, there is no strong evidence suggestive of a role for YB-1 in HIF1? protein stability as there are no significant differences between +/- YB-1 kd sarcoma cells. However, further investigations involving different cell lines are highly required.    5.3.10 YB-1 contributes to HIF1? stability in sarcoma cells Given the observed enhanced stability of HIF1? in ostesarcoma cells (Fig. 5.4 and 5.5), we wondered if YB-1 promotes enhanced expression of HIF1? by contributing to its stability as well. We therefore assessed the rates of HIF1? protein degradation in +/- YB-1 kd sarcoma cells using cyclohexamide decay assay under hypoxia by Western blotting (Fig. 5.13A). HIF1? protein levels were quantified at varying time points, normalized to loading controls, and graphically displayed after normalizing to control cells. As shown in Fig. 5.13B-C, we observed that HIF1? was more rapidly degraded in YB-1 deficient cells. As shown in Fig. 5.13C, the half-life of HIF1? in YB-1 competent cells was approximately 35 minutes under hypoxia, while the protein half-life in YB-1 deficient cells was approximately 28 minutes under the same condition, possibly suggestive of a role for YB-1 in HIF1? stability.  5.3.11 HIF1? is a potential effector for YB-1-mediated metastasis in vitro and in vivo To determine whether HIF1? acts downstream of YB-1 to promote paediatric sarcoma metastasis, we inhibited HIF1? expression using siRNAs in YB-1-competent sarcoma cells, and performed in vitro invasion assays under normoxic and short-term hypoxic conditions.  HIF1? kd led to a significant inhibition of sarcoma cell invasion under both normoxic and short term hypoxic conditions, similar to what was observed when YB-1 was knocked down (Fig. 5.14A & 5.14B). Further, we transfected YB-1 kd cells with HIF1?-expressing vectors and then assessed 110  cell invasion under normoxic and short term hypoxic conditions. HIF1? expression reinstated the invasive capacity of YB-1 kd cells (Fig. 5.14C & 5.14D), underscoring the importance of HIF1? as a YB-1 downstream target mediating sarcoma cell invasion.   To address the impact of HIF1? restoration on the metastatic capacity of YB-1kd cells in vivo, we tested two cell lines, CHLA10 and MNNG, using the murine RSA model. The primary tumour xenografts successfully grew and were harvested. We examined them histopathologically with H&E stain as well as with a panel of IHC antibodies to ensure successful YB-1 kd and HIF1? overexpression and to assess pulmonary metastases (Fig. 5.15A). Consistent with our in vitro results, HIF1? re-introduction largely restored the overall metastatic capacity of YB-1 kd sarcoma cells (Fig. 5.15B), suggesting that HIF1?is a potential YB-1 target an important role in sarcoma metastasis. The results of individual cell lines were shown in Fig. 5.15C.  In Chapter 4, we highlighted the role of YB-1 in tumour angiogenesis as a potential mechanism for it to contribute to metastasis. Since pro-angiogenic vascular endothelial growth factor (VEGFA) is a major HIF1?transcriptional target, VEGF transcripts were found to be significantly downregulated in YB-1 deficient cells (Fig. 5.16A). Moreover, YB-1 kd primary implantation site tumour xenografts showed a marked reduction of both HIF1? and VEGF protein levels (Fig. 5.16B). Further, restoration of HIF1? in YB-1 deficient cells demonstrably enhanced the expression of both VEGF and CD31 as detected by IHC (Fig. 5.16C) in RSA primary implantation site xenografts and significantly increased the mean vascular density as well (Fig. 5.16D). Taken together, these findings provide strong evidence for a novel axis involving YB-1, HIF1?, and VEGF in sarcoma metastasis. 111  5.4 Discussion The hypoxic response, involving HIF1?, plays a dominant role in tumour invasion and metastasis47, 101. Translational regulation of HIF1? expression represents a major regulatory step, accounting for ~ 40-50% of the elevated protein levels under hypoxia331.  In addition, modulation of HIF1? expression by numerous stimuli has been reported under normoxia (reviewed in332, 333) and is indicative of the critical role it plays under both normoxia and hypoxia.   RNA-binding proteins such as polypyrimidine tract-binding protein (PTB) and human antigen R (HuR) play crucial roles in the hypoxic response through the translational activation of HIF1?97. However, the contribution of YB-1, a major RNA-binding protein, to the hypoxic response has not yet been studied. Growing evidence highly supports the notion that it is the cytoplasmic fraction of YB-1 rather than nuclear pYB-1 that confers different tumours, such as multiple myeloma247, 334, and breast cancer335, 336, with an aggressive phenotype. This effect of cytoplasmic YB-1 is thought to be mediated through its ability to bind mRNA which drives translation of crucial transcripts involved in tumour progression337,334.   Extensive evidence from literature point indirectly to a link between YB-1 and HIF1?. Most notably, YB-1 and HIF1? both promote metastatic dissemination in a wide variety of cancers 10, 98-101 potentially through the same downstream mediators, such as Twist 10, 83.  However, a link between YB-1 and HIF1? has to date not been described. Of note, YB-1 and HIF1? possess non-redundant roles in early mammalian development, and corresponding knockout (KO) mice show similar trends of progressive mortality and lethality at embryonic day E10.5 for YB-1 and E11 for HIF1?102-104. In addition, the Multidrug Resistance Gene (MDR1), a crucial YB-1 target 112  mediating tumour cell resistance to chemotherapy338, has been shown to be regulated by HIF1?339. Moreover, PTB, a known HIF1? transcript translational activator340, 341, exhibits a strong correlation with YB-1 in some tumours342. Indeed, both YB-1 and PTB are part of a complex that binds the VEGF mRNA 5'- and 3'-UTRs, and perform stabilizing functions343. In addition, compelling indirect evidence support a role for YB-1 in HIF1? mRNA translational regulation. YB-1 was found to promote microtubule assembly105, which is intricately involved in orchestrating HIF1? translation106. In addition, YB-1 is a survival factor mediating many stress-related activities108, 344, 345. Importantly, in our previous study in breast cancer, we found that HIF1? expression is increased in YB-1 expressing cells and this was associated with its enrichment in the polysomes10. In our current work we found that inhibiting YB-1 expression led to a significant reduction in HIF1? protein expression. These two crucial findings support a link between the effects mediated by YB-1 and HIF1?. In Chapter 3 we described that YB-1 kd primary implantation site tumours xenografts exhibited extensive haemorrhage and necrosis, both grossly and histologically, compared to CTRL tumours. This was similar to what has previously been observed in tumours with reduced HIF1? levels322. Given all these data, we felt it was crucial to investigate the precise relationship between YB-1-HIF1? and to determine how it could impact and contribute to sarcoma progression.   In our current study, we found that both HIF1? and YB-1 play important and very similar roles in sarcoma progression. The highly expressed cytoplasmic pool of YB-1 operates upstream of HIF1? allowing tumour cell adaptation to hostile environments, thereby promoting survival, and eventually metastatic spread. YB-1 is a major enhancer of HIF1? translation and HIF1? mRNA is a putative target of YB-1. Re-expressing HIF1? in YB-1 deficient sarcoma cells restored their 113  invasiveness capacity in vitro and in vivo highly underscoring HIF1? as a novel YB-1 candidate mediating metastasis.  While we found that YB-1 is a major translational activator of HIF1? the mechanism involved in this regulation remains to be resolved.   While hypoxia inhibits general protein synthesis translational activation of HIF1? takes place, how this process takes place remains incompletely understood331, 340, 346. The presence of an internal ribosome-entry-site (IRES) element in the 5? UTR of HIF1? seems to be highly suggestive of a translational mechanism behind enhanced HIF1? during hypoxia341, 347. However, the presence of IRES elements remains a matter of controversy notably following recent work that convincingly argue against it 316, 348. Additionally, other reports pointed to the contribution of 3? UTR and 5? UTR of HIF1? to its translational activation340, 341. Recent work on breast cancer showed the enhanced cap-independent translation over cap-dependent translation of HIF1?, proangiogenic molecules, and survival factors349.   In the current study, we found that recombinant YB-1 protein enhances the activity of 5? UTR of HIF1?. The presence of 5?UTR HIF1?- conserved G-C rich sequences across species, the presence of highly conserved sequences between of 5?UTR HIF1? and 5?UTR Snail1 which were previously shown to be translated through a YB-1-mediated-cap-independent mechanism, and  the predicted highly stable stem loop structure found in HIF1?, all point towards a direct cap-independent mechanism involved in YB-1-mediated HIF1? translation.   Of the numerous HIF1? targets, VEGF is of interest as it mediates angiogenesis and new blood vessel formation to promote tumour cell invasion and dissemination307. Both VEGF protein 114  levels and tumour vasculature are significantly downregulated in YB-1 kd xenografts. In our study, we did not recognize a major role for phospho-YB-1 as YB-1 kd cells showed a similar or even occasionally enhanced pYB-1 expression (see Chapter 2), similar findings by another group have been found in glioblastoma cell lines274. Further, we could not detect significant changes affecting pYB-1 under hypoxia (Fig. 5.17).   YB-1 may represent an exciting novel target for therapeutic intervention in sarcomas. Consistent with our finding in Chapter 4, pulmonary metastases developed in mice harbouring +/- YB-1 kd xenografts revealed similarly high expression levels of both YB-1 and HIF1? (Fig. 5.18), highly indicative of clonal selection as the most competent metastatic cells are able to invade, survive in the circulation,  extravasate and colonize distant organs. Our finding that YB-1 directly activates HIF1? translation, which in turn mediates sarcoma cell metastatic dissemination through enhanced neovascularization via VEGF, illustrates a novel mechanism for HIF1? regulation.   Further, we characterize a mechanism by which YB-1 may contribute to human malignancies. YB-1 has been reported to enhance VEGF mRNA stability and thus may impact VEGF directly343 or alternatively act through HIF1?350, to enhance neovascularization and  metastatic spread of sarcoma cells (Fig. 5.19). However, YB-1 regulates many other mediators contributing to metastasis10, 99, 351 which may explain the relatively higher trend of YB-1 competent cells for metastatic spread (Fig. 5.15B-C) compared to HIF1? over-expressing cells.   5.5 Tables  115  Table 5.1    Immunohistochemical characterization of HIF1? in osteosarcoma  Criteria HIF1? Immunoreactivity Localization Cytoplasmic Nucleo-cytoplasmic Number of cases 12 13 % of positive malignant cells 5%-80% 55%-100% Median 30% 70% Mean 35% 82%   Table 5.2    Correlation of HIF1? expression with tumour grade in osteosarcoma  P value Test of Significance Total (25) Osteosarcoma (25 cases) Variable Nucleo-cytoplasmic immunoreactivity (13 cases) Cytoplasmic immunoreactivity   (12 cases)   0.009   Chi square test  (X2)   7 3 15  0 1 12  7 2 3 Grade I II III     116  Table 5.3    Correlation of YB-1 and HIF1? expression in paediatric sarcoma subtypes Sarcoma Subtype Correlation between YB1 and HIF1? percent positive cells Correlation 95% Lower Confidence Limit (LCL) Correlation  95% Upper Confidence Limit (UCL) p-value Benjamini-Hochberg adjusted p-value ES 0.674358 0.330448 0.860184 0.0011 0.0078 RMS 0.428018 0.138621 0.650067 0.0052 0.018 Data analyzed using cor. test. P-values adjusted for multiple comparisons shown in rightmost column.   Table 5.4    Association between YB1 staining intensity and HIF1? staining intensity within paediatric sarcoma subtypes    Sarcoma Subtype p-value Benjamini-Hochberg adjusted p-value ES 0.031 0.11 RMS 0.0055 0.038 Data analyzed using Fisher?s exact test. P-values adjusted for multiple comparisons shown in rightmost column.    117  5.6 Figures   Figure legend next page  118  Figure 5.1    Inhibiting YB-1 expression has a marked effect on HIF1? expression among other EMT related proteins A. Immunoblot showing the impact of YB-1 downregulation on the protein levels of N-cadherin, HIF2?, and Twist in MNNG OS and Rh30 RMS cells transduced with scrambled shRNA or YB-1 shRNA lentiviral particles. Total AKT was used as loading control.  B. Immunoblot showing Snail1 protein expression levels in Rh30 RMS cells transduced with scrambled shRNA or YB-1 shRNA lentiviral particles. Grb2 was used as a loading control.  C. Western blot showing effects of siRNA-mediated knockdown of YB-1 on HIF1? protein levels under normoxia in TC71 and MNNG cell lines. Grb2 was used as loading control. D. Western blot showing HIF1? expression in TC32, CHLA10, and Rh30 cell lines transfected with non-targeting siRNA or YB-1 siRNA and grown under hypoxia (1% O2) for 40 minutes. Akt was used as a loading control.  E. Western blot showing HIF1? and YB-1 expression in MNNG cells transduced with scrambled shRNA or YB-1 shRNA lentiviral particles and grown under hypoxia for the indicated times. Akt was used as a loading control. * denotes a non-specific band. F. Western blotting showing the expression protein levels of YB-1 and HIF1? in the CHLA-9 and CHLA-10 ES cell line pair derived from the diagnostic specimen (CHLA9) or from the recurrent tumour after four rounds of chemotherapy (CHLA10). Actin was used as loading control.     119    Figure 5.2    HIF1? and HIF2? proteins levels under normoxia and hypoxia  HIF1? and HIF2? protein levels, cleavage of PARP and caspase 3 in osteoblast and osteosarcoma cell lines under normoxia and hypoxia. Immunoblots showing the protein levels of HIF1? and HIF2?, as well as cleaved PARP and cleaved caspase-3, in siCTRL (left panel) , siHIF1? siRNA  (middle panel) and siHIF2? siRNA (right panel) treated cell lines under both normoxic (21% O2) and hypoxic (1% O2) conditions in the osteoblast cell lines OB3 and OB5 and the osteosarcoma cell lines MG63 and MNNG.  Actin was used a loading control.          120    Figure 5.3    Transcriptional effects of HIF1? and HIF2? in osteoblasts and osteosarcoma cell lines  A. HRE-GFP activity in the presence of isoform-specific HIF siRNAs. The relative contribution of HIF1? and HIF2? to the HIF transcriptional response in osteoblast and osteosarcoma cell lines 121  was assessed under normoxic and hypoxic conditions using an HRE-GFP reporter construct. Under normoxic conditions HRE-GFP activity was equally reduced in osteoblast cell lines by both HIF1? and HIF2? siRNAs while only by HIF1? siRNA in osteosarcoma cell lines. Under hypoxic conditions, HRE-GFP activity was elevated in osteosarcoma cell lines and this was significantly reduced by HIF1? siRNA but much less so by HIF2? siRNA.  B. Osteoblasts and osteosarcoma cells were plated at 40% confluence on glass cover slips in a six-well plate. One day later, cells were transfected with pmax-GFP construct from AMAXA at a final concentration of 1?g/well which served as positive control for HRE-GFP construct. After 16 hours, cells were fixed with 3.5% PFA and analyzed by fluorescence microscopy.  C. HRE-Luc activity was also used to quantify HIF activity. Under hypoxia osteosarcoma cell lines (MNNG and MG63) exhibited higher luciferase activity compared to osteoblasts (OB3 and OB5). HIF1? or HIF2? siRNA treatment decreased the activity of HRE-Luc with a more robust impact of HIF1? siRNA in the osteosarcoma cell lines. Firefly luciferase values were normalized to the renilla transfection control and error bars represent + SD. Where shown; *=p<.05; **=p<.01; ***=p<.001. Scale bars, 50?m.         122   Figure legend next page 123  Figure 5.4  Comparison of HIF1? and HIF2? protein stability in osteoblast and osteosarcoma cell lines under normoxia and hypoxia A. Immunoblots showing HIF1? and HIF2? protein decay assays in osteoblast versus osteosarcoma cell lines under normoxia and hypoxia. Grb2 was used a loading control. CHX: Cyclohexamide.  B. Diagrammatic representation of the average values for HIF1? and HIF2? protein half lives in osteoblasts versus osteosarcoma cell lines under normoxic and hypoxic conditions. Data represents the mean of three independent experiments + SD.                124   Figure legend next page 125  Figure 5.5  HIF1? and HIF2? protein stability in individual osteoblast and osteosarcoma cell lines under normoxia and hypoxia A. HIF1? and B. HIF2? protein half life in osteoblast and osteosarcoma cell lines under normoxic and hypoxic conditions. Data represents the mean of three independent experiments + SD.    126    Figure 5.6    Examples of HIF1? immunohistochemical staining of osteosarcoma cases Ai. Osteoblasts (normal bone cells) exhibiting mild cytoplasmic immunoreactivity; Aii. Grade I osteosarcoma exhibiting mild cytoplasmic immunoreactivity; Aiii. Grade III osteosarcoma showing strong nuclear immunoreactivity; Aiv. Grade III osteosarcoma showing strong nucleo-127  cytoplasmic immunoreactivity; Av. Grade III chondroblastic osteosarcoma showing strong nucleo-cytoplasmic immunoreactivity.  B. Immunohistochemical analysis of HIF1? expression in cell pellets showing strong cytoplasmic immunoreactivity as well as scattered nuclear staining in MNNG and MG63 osteosarcoma cells contrary to the faint cytoplasmic staining observed in OB3 and OB5 osteoblasts cells.   C. Immunoflourescence analysis of HIF1? expression in MG63 osteosarcoma cell line and OB5 Osteoblast cell line displaying strong nucleo-cytoplasmic staining in MG63 osteosarcoma cell line contrary to mild cytoplasmic staining in OB5 osteoblast cell line.  Blue: DAPI (nuclear stain); green: HIF1?. Scale bars, 50?m.                128   Figure legend next page 129  Figure 5.7    Hypoxia-induced YB-1 protects against cell death A. Western blot showing induction of HIF1? and YB-1 expression under hypoxia in TC71 cells. Actin was used as a loading control. B. Left panels: Endogenous YB-1 protein induction under normoxic and hypoxic conditions (12 hours) as detected by immunofluorescence using fluorescein-conjugated anti-YB-1 antibodies (green) in the indicated cell lines. Representative fields are shown. Scale bars: 25?m. Right panel: Quantification of fluorescence intensity of the indicated cell lines +/- YB-1 kd. Results are expressed as the mean staining intensity of 50 cells + SD.  C. Effects of shRNA-mediated knockdown of YB-1 on cell morphology and viability of MNNG osteosarcoma cells following overnight (O/N) incubation under hypoxic conditions (1% O2) as detected by phase-contrast microscopy.  Scale bars: 50 ?m.  D. Left panel: Western blot showing the impact of YB-1 knockdown in MNNG osteosarcoma cells on PARP cleavage under hypoxic conditions (1% O2). Grb2 was used as loading control. Right panel: Quantification of PARP cleavage results in MNNG osteosarcoma cells +/- YB-1 kd. Data were normalized to endogenous Grb2 from two runs, analyzed with a two-tailed student?s T test, and presented as means + SD. E. Cryosections from TC71 and TC32 cells +/- YB-1 kd grown as 3-D discs (see Experimental Procedures) and examined by the indicated staining methods. Right side: IHC detection of BrdU incorporation to measure proliferation (outer zones; red color), Pimonidazole staining to detect hypoxic areas (inner zones; green color), and hematoxylin staining as a nuclear marker (blue color). Left panel: IHC to detect YB-1 expression using anti-YB-1 antibodies (black = positive staining). Scale bars: 150?m.  Where shown * = p<0.05. 130    Figure 5.8    YB-1 induces expression of biologically active HIF1? A. 3-D tissue discs of MNNG cells, transduced with scrambled shRNA or YB-1 shRNA, were grown on collagen-coated tissue culture inserts as described in the Experimental Procedures. Seeded inserts were then incubated for 16 hours prior to their transfer to stirred growth vessels. Cryosections from tissue discs were examined by IHC for expression of YB-1, the hypoxic marker pimonidazole, and HIF1?. Scale bars: 150 ?m.  B. Quantification of the staining intensity shown from (F) using customized ImageJ software.  131  C. Left panels: Hypoxia response element (HRE)-GFP activity in Rh30 RMS cells transduced with scrambled shRNA or YB-1 shRNA under normoxic and hypoxic conditions (24 hrs) as detected by fluorescence microscopy. Middle panels: GFP vector was used as positive control -. Scale bars: 25 ?m. Right panel: Quantification of the above fluorescence measurements. Results are expressed as mean staining intensity of five fields + SD. Where shown * = p<0.05; ***=p<0.001.                   132   Figure legend next page 133  Figure 5.9    Co-expression of YB-1 and HIF1? in childhood sarcomas A. Correlation between YB-1 and HIF1? protein expression in ES and alveolar RMS primary tumours by IHC on ES and RMS tissue microarrays. Correlation estimates are shown at the top of each graph (with 95% confidence intervals shown in brackets). Blue curves show functional forms of each association, and black lines show a linear regression model fit from which the correlation coefficients were estimated. B. Representative examples of IHC-based correlation between YB-1 and HIF1? expression in ES and RMS subtypes from (A). Scale bars: 25 ?m.                134    Figure 5.10    YB-1-mediated HIF1? translational activation in sarcoma cells A. Relative levels of HIF1? mRNA in Rh30 and MNNG cell lines transduced with scrambled shRNA or YB-1 shRNA was measured by quantitative real-time RT-PCR of total or polysomal 135  fractionated RNA. Values shown were normalized against GAPDH from two experiments in triplicate, reported relative to Hela cells, and presented as a mean + SD.  B. Relative levels of polysomal YB-1 and HIF1? mRNAs in MG63 cells transfected with control siRNA or YB-1 siRNA and grown under normoxic or hypoxic conditions (4 hrs) measured by quantitative real-time RT-PCR. Values shown were normalized against GAPDH from two runs performed in triplicate, and presented as means + SD. C. Relative HIF2? mRNA levels in MNNG OS and TC32 ES cell lines, transduced with scrambled shRNA or YB-1 shRNA, under normoxic conditions measured by quantitative RT-PCR of total RNA or polysomal fractionated RNA. Data were normalized to endogenous GAPDH from two runs performed in triplicate, analyzed with a two-tailed student?s T test, and presented as means + SD. D. Fold-change of HIF1? mRNA bound to YB-1 as measured by quantitative real-time RT-PCR after YB-1 immunoprecipitation. Controls include two known YB-1 bound transcripts, YB-1 itself and Cyclin D1, and one known non-YB-1 binding transcript, L32. Values shown were normalized against XIAP from two runs performed in triplicate and presented as mean + SD.  Normal rabbit serum (NRS) was used as a control antibody for immunoprecipitation. Where shown * = p<0.05; **=p<0.01.         136     137  Figure 5.11    YB-1 enhances HIF1? translational efficiency in vivo & in vitro A. A bi-cistronic mRNA reporter construct harboring the HIF1? 5? UTR region linked to firefly luciferase and renilla linked to SV40 promotor  was used to assess the translation in MNNG cells +/- YB-1 knockdown under normoxia using a dual Luciferase reporter system. Results are displayed as means + SD from two independent experiments performed in triplicate. B. In vitro translation assay in which constructs harbouring the 5? UTRs of either HIF1? or ?-globin linked to SP6 RNA polymerase promoter, were used for in vitro coupled transcription/translation in the presence of increasing amounts of YB-1 recombinant protein, and assessed for luciferase activity. The results are displayed as means + SD from two independent experiments performed in triplicate. C. Nucleotide sequence alignment of HIF1? 5? UTRs from different species using T-Coffee software as previously described 352, 353. Species: Homo sapiens (human, BC012527.2); Mus musculus (Mouse, NM_010431.2); Macaca mulatta (Rhesus monkey, XM_002805060.1); Sus scrofa (Pig, NM_001123124.1, Nomascus leucogenys (Northern white-cheeked gibbon, XM_003267779.1). Red lettering and asterisks denote sequence identify. D. Alignment of Homo sapiens HIF1? 5?UTR (BC012527.2) and Snail1 5? UTRs (NM_005985.3). Asterisks denote conserved nucleotides. Where shown ***=p<0.001.       138     Figure 5.12    The predicted secondary structure of 5?UTR element of HIF1? The predicted secondary structure of the Homo sapiens 5? UTR HIF1? mRNA using the Vienna RNA secondary structure prediction tool (http://rna.tbi.univie.ac.at/cgi-bin/RNAfold.cgi), as previously described 354, 355. Red asterisks (*) denote conserved nucleotides across Homo sapiens, Mus musculus, Macaca mulatta, Sus scrofa, and Nomascus leucogenys.  .      139    Figure 5.13    HIF1? protein stability in +/- YB-1 knockdown cells under hypoxia A. Immunoblot showing the impact of the proteosome inhibitor MG132 on HIF1? accumulation at different time points in the indicated +/- shRNA-mediated YB-1 knockdown cells under hypoxia. Grb2 was used as loading control. B. Cyclohexamide chasing assay. Top panel: Immunoblots showing HIF1? protein decay in MNNG cells +/- YB-1 knockdown at the indicated time points after cycloheximide (100 ug/ml) 140  added under hypoxia. Grb2 was used a loading control. CHX: Cyclohexamide. Lower panel: Diagrammatic representation of the average values for HIF1? protein half lives in +/- YB-1 knockdown cells under hypoxic conditions. Data represents the results of two independent experiments + SD. C.?Immunoblot showing the impact of shRNA-mediated YB-1 kd on components of the HIF1? degradation pathway. Grb2 was used as loading control.   141    Figure 5.14    HIF1? is a potential YB-1 target mediating sarcoma cell invasive phenotype in vitro A. MNNG osteosarcoma cells transfected with 25nM of control (siCTRL) or siRNAs targeting HIF1? (siHIF1? #1) or (siHIF1? #2) were incubated under hypoxia for 40 minutes, and then examined for HIF1?, HIF2?, and YB-1 protein expression. Akt was used as a loading control. 142  B. Effects of YB-1 versus HIF1? knockdown on invasive capacity of MNNG OS cells under normoxic and hypoxic conditions (8 hrs) as assessed by in vitro invasion using BME-coated cell invasion assays. Results are displayed as a mean + SD from two independent experiments counted in triplicate. C. MNNG osteosarcoma OS cells +/- YB-1 knockdown were co-transfected with either empty vector or wild type (wt) or degradation-resistant HIF1? mutant, in which Prolines 564 and 402 were mutated to alanine, expression vectors, incubated under hypoxia for 40 minutes, and then assessed by Western blot for HIF1? and YB-1 expression. Akt was used as a loading control. D. Effects of ectopic expression of wild type (wt) or degradation-resistant HIF1? mutant, in which Prolines 564 and 402 were mutated to alanine,?  on the invasive capacity of MNNG OS cells with YB-1 knockdown under normoxic and hypoxic conditions (8 hrs) as assessed by in vitro invasion using BME-coated cell invasion assays. Results are displayed as means + SD from two independent experiments counted in triplicate.  Where shown *=p<0.05, **=p<0.005, ***=p<0.0005.            143   Figure 5.15    HIF1? is a potential YB-1 target mediating sarcoma cell metastasis in vivo A. Photomicrographs of primary tumours and lungs from mice bearing renal subcapsular tumour xenografts of CHLA10 and MNNG cell lines +/- YB-1 knockdown and with or without ectopic 144  wt HIF1? expression. (Ai-vi) H&E staining of primary implantation site tumours of the indicated cell lines (Avii-xii) IHC of YB-1 in primary implantation site tumours. (Axiii-xviii) IHC of HIF1? in primary implantation site tumours. (Axix-xxiv). H&E staining of metastatic pulmonary lesions (arrows) in mice bearing renal subcapsular tumour xenografts of the indicated cell lines +/- YB-1 kd or YB-1 kd plus ectopic wt HIF1? expression. (Axxv-xxvii) IHC of the CD99 ES marker in pulmonary metastases of mice bearing renal subcapsular CHLA10 ES tumour xenografts. Scale bars: 50?m. B. Comparison of the total number of mice bearing renal subcapsular tumour xenografts of CHLA10 or MNNG cell lines +/- YB-1 knockdown versus YB-1 knockdown plus ectopic wt HIF1? that developed pulmonary metastases, determined using a Fisher's Exact Test. C. Immuncompromised mice were xenotransplanted with the MNNG OS or CHLA10 ES cell lines +/- YB-1 knockdown, or YB-1 knockdown cells co-transfected with a wt HIF1? expression construct (YB-1 kd + HIF1?) and then monitored for development of pulmonary metastases (Mets). The Y-axis shows the total numbers of mice with pulmonary metastases per each condition for the cell lines tested. Where shown *=p<0.05.         145    Figure 5.16    YB-1-HIF1? axis potentially contributes to tumour angiogenesis A. Relative expression of VEGF mRNA in TC32 cells transfected with scrambled siRNA or YB-1 siRNA as measured by quantitative RT-PCR. Data were normalized to endogenous GAPDH and reported relative to Hela cells from two experiments performed in triplicate, analyzed with a two-tailed student?s T test, and presented as means + SD. B. IHC of HIF1? (left panels) and VEGF (right panels) in primary implantation site tumours of mice transplanted with scrambled shRNA or YB-1 sh-RNA transduced TC71, TC32, and CHLA10 ES cells. Scale bars: 25 ?m.  146  C. IHC of VEGF and CD31 expression in primary implantation site tumours of mice transplanted with scrambled shRNA or YB-1 sh-RNA transduced MNNG OS cells versus YB-1 knockdown cells with ectopic wt HIF1? expression, showing the impact of ectopic HIF1? on VEGF levels and tumour vessel formation, respectively. Scale bars: 50 ?m. D. Quantification of tumour microvessel density in primary implantation site tumours from (C) determined using a two-tailed student?s T test. Where shown * = p<0.05.             147    Figure 5.17    Total YB-1 rather than pYB-1 is induced under hypoxia A. Immunoblot showing the expression levels of YB-1 and pYB-1 in Rh30 RMS cells under normoxic and hypoxic conditions. Total Akt was used as loading control. B. The bar graph shows pYB-1 protein levels relative to the total levels under normoxic and hypoxic conditions of 2 runs. Error bars represent + SD.           148     Figure 5.18    HIF1? expression in lung metastases of mice with +/- YB-1 knockdown xenografts IHC for HIF1? expression levels was conducted on lung tissues with metastases of mice xenotransplanted with scrambled shRNA or YB-1 sh-RNA transduced TC71 and TC32 ES cells. Strong immunoreactivity in the developed pulmonary metastases was detected irrespective of the YB-1 status of the primary tumour xenografts. Scale bars: 100?m.      149    Figure 5.19    The proposed mechanism of YB-1 mediated sarcoma metastasis  Schematic illustrating the proposed role of a novel YB-1-HIF1?-VEGF axis in promoting blood vessel formation and metastatic dissemination of childhood sarcoma cells.      150  Chapter  6: Conclusions and closing remarks  Our current studies represent the first comprehensive set of experiments that shed new light on a critical role for YB-1 in paediatric sarcomas, namely its role in metastatic progression. In this project we have tried to further understand the complexity underlying the metastatic process in sarcomas by tracking the metastasis promoting factor YB-1. Our lab previously found that YB-1 plays essential role in breast cancer metastasis through induction of EMT program, by switching off translation of epithelial and growth related messages such as CCDN1 and switching on translation of crucial mesenchymal related messages such as Snail, Twist, and HIF1?.  Interestingly, similar growth controlling effect were also identified for YB-1 in sarcomas. The similarities observed between breast cancer and sarcomas highlight an important point as we believe that growth of tumour cells and their metastatic ability are two sides of the same coin. Sustaining proliferative signalling is the first hallmark of cancer and sarcoma cells, by definition, are highly proliferative. However, unchecked proliferation may lead to mTOR-mediated energetic stress and eventual cell death, thereby representing an Achilles' heel for tumour cells. Therefore controlling this process would be expected to be highly advantageous to tumour cells. Firstly, if controlled tumour cells would not be exposed to the detrimental effects of energetic stress and secondly tumour cell could conserve the energy required to metastasize. It worth mentioning that efficient YB-1 downregulation was achieved ~ 5 days post-transfection with siRNAs. The effect of YB-1 on sarcoma cell growth has distinct phases. We consistently observed a period of initial growth arrest following the siRNA transfection which continued for a period of time (~1-4 days) based on the cell line. Then, there was a period of accelerated growth 151  that eventually lead to growth inhibition and death of the cells, indicative of a potential reprogramming of the transcriptional/translational machinery upon altering the levels of cellular YB-1. The growth studies described were conducted during the accelerated growth phase in which YB-1 expression levels were at their lowest.   We then went on to address YB-1?s contribution to sarcoma metastasis and established YB-1 as a potent metastatic marker in sarcomas using two novel animal models. Through my previous work on HIF1? in osteosarcoma356, HIF1? was found to be highly expressed and stable in malignant vs normal cells and its expression was associated with advanced disease grade, similar to our current results obtained with YB-1. By taking into account the extensive roles that hypoxia, HIF1?, and YB-1 play in metastasis of solid tumours10, 98-101, we decided to focus on the novel YB-1-hypoxia-HIF1? signalling axis. As YB-1 has an established role in translational regulation, we hypothesized that YB-1 could potentially regulate HIF1? expression via a similar mechanism. In this study, as we expected, we identified HIF1? as a crucial YB-1 downstream target and furthermore uncovered a mechanism whereby HIF1? is potentially expressed via a translational process that is dependent upon YB-1.  The fundamental conclusions from my doctoral studies are:  i. YB-1 is a crucial factor in mesenchymal-derived tumours and its elevated level of expression potentially contributes to sarcomatogenesis. ii. Cytoplasmic YB-1 rather than nuclear pYB-1 may serve as a potential prognostic marker in paediatric sarcomas. iii. YB-1 helps to regulate tumour cell proliferation to ensuring sarcoma cell survival.  152  iv. YB-1 is an indispensible survival factor under both ambient and stress conditions and its inhibition eventually leads to cell death. v. YB-1 is a major contributor to sarcoma cell motility, invasion, and metastatic dissemination in vitro and in vivo at the expense of cell growth. vi. HIF1? is a potential YB-1 downstream target promoting sarcoma metastasis. vii. YB-1-HIF1?-VEGF-angiogenesis is a novel axis involved in paediatric sarcoma metastasis which has the potential to be targeted therapeutically.   These conclusions underscore an essential role for YB-1 in sarcomatogenesis. Understanding the complex process of paediatric sarcoma metastasis is essential in order to design successful treatment strategies. It has been shown that YB-1 synthesis is regulated by the mTORC1 signalling pathway. The mTORC1 complex is a serine/threonine kinase lying downstream of the PI3K/AKT pathway. In keeping with this, deregulation of this pathway, such as through inactivation of the AKT inhibitor phosphatase and tensin homolog (PTEN), has been reported as a major mechanism implicated in intrinsic activation of the mTORC1 pathway in sarcomas130 357. Therefore, mTORC1 is a potentially promising therapeutic target in sarcomas. Published mTOR inhibitors such as temsirolimus (Torisel) and everolimus (Afinitor), are in clinical trials and showed potential benefits when used alone or in combination with other anticancer regimen for the treatment of RMS, reviewed in358, 359. Further, targeting YB-1 downstream mediator HIF1??by inhibiting its translation and/or transactivation, or by enhancing its degradation may represent another promising treatment strategy for the treatment of sarcoma patients.  153  Indeed, several agents inhibiting HIF1? protein expression have been identified, however, a few of them have been found to affect HIF1? expression in vivo. Interestingly, targeting the mTORC1 signalling pathway using agents such as CCI-779 or RAD001 was found to inhibit HIF1? translation. Further, the cytotoxic agent Topotecan, clinically approved for the treatment of lung and ovarian cancer, has been shown to inhibit HIF1? activity in a cell-based screen. In addition, Hsp90 inhibitors were found to enhance HIF1? degradation. Moreover, YC-1, another HIF1? inhibitor, was found  to decrease HIF1? levels in vitro and in vivo by accelerating HIF1?  degradation,  inhibiting HIF1? protein synthesis, and stimulating the factor inhibiting hypoxia (FIH)-dependent dissociation of p300 from HIF1?, reviewed in360.    It is worth mentioning that several features serve to make YB-1 unique when compared to other well-known metastasis promoting/EMT-inducing factors. Unlike most of these factors, such as Twist, that have been shown to be downregulated during the late phases of metastasis, at the time when metastatic colonies are established, YB-1 is required to trigger both the early metastatic phase such as EMT as well as the late phase of metastatic colonization. We observed that the pulmonary metastases in mice xenotransplanted with YB-1 deficient cell/overexpressing HIF1??are relatively small compared to metastases developed by YB-1 competent cells, potentially indicating an important role for YB-1 in the colonization stage. Given the substantial contributions of YB-1 to both cell survival and the cellular stress response, it is not unexpected that YB-1 expression would persist during the entirety of the metastatic process. A number of questions remain to be answered. First, what are the differentially expressed transcripts in +/- YB-1 kd xenografts? This could be addressed using similar animal studies as described in Chapter 4. With aid of extensive IVIS monitoring, RNA-seq could be performed on polysome-154  bound mRNA obtained at different time points during tumour progression. Profiling could be done to characterize the different stages of the metastatic process beginning during local invasion up until the final colonization stage by sacrificing animals at different time periods during tumour progression. Nevertheless, the main challenge of this approach is the limited capabilities of the current imaging modalities which make it hard to detect the early stages of tumour invasion. This challenge could potentially be overcome by using advanced imaging systems such as MRI and CT. At the same time, one could take advantage of additional animal models, such as the transparent Casper fish, which permits visualization of the very early stages of tumour progression, thereby allowing for detection and cellular profiling of each metastasis-related phase. Another hypothesis to test would be to address the effectiveness of the currently available YB-1 inhibitors on the invasive capacity of malignant cells and to perform additional large-scale screens to determine the sensitivity of tumour cells to chemotherapeutic agents in the presence of YB-1 inhibitors.   It will be important also to determine the hypoxic signature in YB-1 competent, HIF1? deficient cells as well as the hypoxic signature in YB-1 deficient, HIF1? expressing cell. It has been established that HIF1? is the master regulator of the cell?s adaptive response to hypoxia. Through our work we have identified a new player working upstream of HIF1? although much about how it contributes to the adaptive hypoxic response remain to be determined. By identifying differentially expressed transcripts and selectively polysome-enriched messages we will be able to determine the specific contributions YB-1 and HIF1? make toward modulating the hypoxic response and how this may impact tumour cell fate.   155  One of the important finding from this study is that in addition to the central role of YB-1 in activating HIF1? translation. This finding reveals a novel role for YB-1 in controlling HIF1? expression and perhaps other targets. It will be interesting to determine how generalized this phenomenon is; i.e. is YB-1-mediated HIF1? translation relevant to additional tumour types such as carcinomas, hematologic malignancies, and CNS tumours. Further, it will be important to know whether YB-1-mediated HIF1? translation is only observed in cancer cells (transformed cells), or it may also take place in non-transformed cells. Finally, in addition to these mechanistic insights, the novel xenograft systems we have developed through our work provide complementary robust preclinical models for the future evaluation of anti-angiogenic agents alone and in combination with other therapies to inhibit the spread of sarcoma cells mediated by YB-1.   In summary, the current studies highlight YB-1 as a potent metastatic driver in high-risk childhood sarcomas through enhanced HIF1? translation. YB-1 may therefore represent an exciting novel target for therapeutic intervention in sarcomas. Alternatively, targeting HIF1? itself, or neovascularization via VEG, may offer more tractable clinical strategies. Indeed, YB-1 has been reported to enhance VEGF mRNA stability and thus may impact VEGF directly 343. The novel xenograft systems we describe should provide complementary and robust preclinical models for future evaluation of antiangiogenic agents alone and in combination with other therapies to inhibit the spread of sarcoma cells mediated by YB-1. 156  References 1. Gupta, G.P. & Massague, J. Cancer metastasis: building a framework. Cell 127, 679-95 (2006). 2. Tarin, D., Price, J.E., Kettlewell, M.G., Souter, R.G., Vass, A.C. & Crossley, B. Mechanisms of human tumor metastasis studied in patients with peritoneovenous shunts. Cancer Res 44, 3584-92 (1984). 3. Weiss, L. Metastatic inefficiency. Adv Cancer Res 54, 159-211 (1990). 4. Bacac, M. & Stamenkovic, I. Metastatic cancer cell. Annu Rev Pathol 3, 221-47 (2008). 5. Slamon, D.J., Leyland-Jones, B., Shak, S., Fuchs, H., Paton, V., Bajamonde, A., Fleming, T., Eiermann, W., Wolter, J., Pegram, M., Baselga, J. & Norton, L. Use of chemotherapy plus a monoclonal antibody against HER2 for metastatic breast cancer that overexpresses HER2. N Engl J Med 344, 783-92 (2001). 6. Minna, J.D., Kurie, J.M. & Jacks, T. A big step in the study of small cell lung cancer. Cancer Cell 4, 163-6 (2003). 7. Klein, C.A. The systemic progression of human cancer: a focus on the individual disseminated cancer cell--the unit of selection. Adv Cancer Res 89, 35-67 (2003). 8. Nguyen, D.X., Bos, P.D. & Massague, J. Metastasis: from dissemination to organ-specific colonization. Nat Rev Cancer 9, 274-84 (2009). 9. Kraljevic Pavelic, S., Sedic, M., Bosnjak, H., Spaventi, S. & Pavelic, K. Metastasis: new perspectives on an old problem. Mol Cancer 10, 22 (2011). 10. Evdokimova, V., Tognon, C., Ng, T., Ruzanov, P., Melnyk, N., Fink, D., Sorokin, A., Ovchinnikov, L.P., Davicioni, E., Triche, T.J. & Sorensen, P.H. Translational activation 157  of snail1 and other developmentally regulated transcription factors by YB-1 promotes an epithelial-mesenchymal transition. Cancer Cell 15, 402-15 (2009). 11. Chiang, A.C. & Massague, J. Molecular basis of metastasis. N Engl J Med 359, 2814-23 (2008). 12. Nguyen, D.X. & Massague, J. Genetic determinants of cancer metastasis. Nat Rev Genet 8, 341-52 (2007). 13. Smith, H.A. & Kang, Y. The metastasis-promoting roles of tumor-associated immune cells. J Mol Med (Berl) 91, 411-29 (2013). 14. Chambers, A.F. & Matrisian, L.M. Changing views of the role of matrix metalloproteinases in metastasis. J Natl Cancer Inst 89, 1260-70 (1997). 15. Koop, S., Khokha, R., Schmidt, E.E., MacDonald, I.C., Morris, V.L., Chambers, A.F. & Groom, A.C. Overexpression of metalloproteinase inhibitor in B16F10 cells does not affect extravasation but reduces tumor growth. Cancer Res 54, 4791-7 (1994). 16. Welch, D.R., Steeg, P.S. & Rinker-Schaeffer, C.W. Molecular biology of breast cancer metastasis. Genetic regulation of human breast carcinoma metastasis. Breast Cancer Res 2, 408-16 (2000). 17. Fu, J., Qin, L., He, T., Qin, J., Hong, J., Wong, J., Liao, L. & Xu, J. The TWIST/Mi2/NuRD protein complex and its essential role in cancer metastasis. Cell Res 21, 275-89 (2011). 18. Wels, J., Kaplan, R.N., Rafii, S. & Lyden, D. Migratory neighbors and distant invaders: tumor-associated niche cells. Genes Dev 22, 559-74 (2008). 158  19. Talbot, L.J., Bhattacharya, S.D. & Kuo, P.C. Epithelial-mesenchymal transition, the tumor microenvironment, and metastatic behavior of epithelial malignancies. Int J Biochem Mol Biol 3, 117-36 (2012). 20. Jeon, J.S., Zervantonakis, I.K., Chung, S., Kamm, R.D. & Charest, J.L. In vitro model of tumor cell extravasation. PLoS One 8, e56910 (2013). 21. Parri, M. & Chiarugi, P. Rac and Rho GTPases in cancer cell motility control. Cell Commun Signal 8, 23 (2010). 22. Pathak, A. & Kumar, S. Biophysical regulation of tumor cell invasion: moving beyond matrix stiffness. Integr. Biol 3, 267?278 (2011). 23. Friedl, P. & Wolf, K. Tube travel: the role of proteases in individual and collective cancer cell invasion. Cancer Res 68, 7247-9 (2008). 24. Sahai, E. & Marshall, C.J. Differing modes of tumour cell invasion have distinct requirements for Rho/ROCK signalling and extracellular proteolysis. Nat Cell Biol 5, 711-9 (2003). 25. Friedl, P. & Wolf, K. Tumour-cell invasion and migration: diversity and escape mechanisms. Nat Rev Cancer 3, 362-74 (2003). 26. Yilmaz, M. & Christofori, G. Mechanisms of motility in metastasizing cells. Mol Cancer Res 8, 629-42 (2010). 27. Pavese, J.M., Farmer, R.L. & Bergan, R.C. Inhibition of cancer cell invasion and metastasis by genistein. Cancer Metastasis Rev 29, 465-82 (2010). 28. Fagotto, F. Looking beyond the Wnt pathway for the deep nature of beta-catenin. EMBO Rep 14, 422-33 (2013). 159  29. Niessen, C.M., Leckband, D. & Yap, A.S. Tissue organization by cadherin adhesion molecules: dynamic molecular and cellular mechanisms of morphogenetic regulation. Physiol Rev 91, 691-731 (2011). 30. Berx, G. & van Roy, F. Involvement of members of the cadherin superfamily in cancer. Cold Spring Harb Perspect Biol 1, a003129 (2009). 31. De Strooper, B., Iwatsubo, T. & Wolfe, M.S. Presenilins and gamma-secretase: structure, function, and role in Alzheimer Disease. Cold Spring Harb Perspect Med 2, a006304 (2012). 32. Bendas, G. & Borsig, L. Cancer cell adhesion and metastasis: selectins, integrins, and the inhibitory potential of heparins. Int J Cell Biol 2012, 676731 (2012). 33. Desgrosellier, J.S. & Cheresh, D.A. Integrins in cancer: biological implications and therapeutic opportunities. Nat Rev Cancer 10, 9-22 (2010). 34. Canel, M., Serrels, A., Frame, M.C. & Brunton, V.G. E-cadherin-integrin crosstalk in cancer invasion and metastasis. J Cell Sci 126, 393-401 (2013). 35. Jaggupilli, A. & Elkord, E. Significance of CD44 and CD24 as cancer stem cell markers: an enduring ambiguity. Clin Dev Immunol 2012, 708036 (2012). 36. Weber, G.F., Bronson, R.T., Ilagan, J., Cantor, H., Schmits, R. & Mak, T.W. Absence of the CD44 gene prevents sarcoma metastasis. Cancer Res 62, 2281-6 (2002). 37. Yu, W.H., Woessner, J.F., Jr., McNeish, J.D. & Stamenkovic, I. CD44 anchors the assembly of matrilysin/MMP-7 with heparin-binding epidermal growth factor precursor and ErbB4 and regulates female reproductive organ remodeling. Genes Dev 16, 307-23 (2002). 160  38. Liu, D., Guo, H., Li, Y., Xu, X., Yang, K. & Bai, Y. Association between polymorphisms in the promoter regions of matrix metalloproteinases (MMPs) and risk of cancer metastasis: a meta-analysis. PLoS One 7, e31251 (2012). 39. Arroyo, A.G. & Iruela-Arispe, M.L. Extracellular matrix, inflammation, and the angiogenic response. Cardiovasc Res 86, 226-35 (2010). 40. Hanahan, D. & Weinberg, R.A. The hallmarks of cancer. Cell 100, 57-70 (2000). 41. Rajendran, J.G., Mankoff, D.A., O'Sullivan, F., Peterson, L.M., Schwartz, D.L., Conrad, E.U., Spence, A.M., Muzi, M., Farwell, D.G. & Krohn, K.A. Hypoxia and glucose metabolism in malignant tumors: evaluation by [18F]fluoromisonidazole and [18F]fluorodeoxyglucose positron emission tomography imaging. Clin Cancer Res 10, 2245-52 (2004). 42. Tavaluc, R.T., Hart, L.S., Dicker, D.T. & El-Deiry, W.S. Effects of low confluency, serum starvation and hypoxia on the side population of cancer cell lines. Cell Cycle 6, 2554-62 (2007). 43. Deisboeck, T.S. & Wang, Z. A new concept for cancer therapy: out-competing the aggressor. Cancer Cell Int 8, 19 (2008). 44. Cuvier, C., Jang, A. & Hill, R.P. Exposure to hypoxia, glucose starvation and acidosis: effect on invasive capacity of murine tumor cells and correlation with cathepsin (L + B) secretion. Clin Exp Metastasis 15, 19-25 (1997). 45. Liu, R., Li, Z., Bai, S., Zhang, H., Tang, M., Lei, Y., Chen, L., Liang, S., Zhao, Y.L., Wei, Y. & Huang, C. Mechanism of cancer cell adaptation to metabolic stress: proteomics identification of a novel thyroid hormone-mediated gastric carcinogenic signaling pathway. Mol Cell Proteomics 8, 70-85 (2009). 161  46. Zhong, H., De Marzo, A.M., Laughner, E., Lim, M., Hilton, D.A., Zagzag, D., Buechler, P., Isaacs, W.B., Semenza, G.L. & Simons, J.W. Overexpression of hypoxia-inducible factor 1alpha in common human cancers and their metastases. Cancer Res 59, 5830-5 (1999). 47. Birner, P., Schindl, M., Obermair, A., Plank, C., Breitenecker, G. & Oberhuber, G. Overexpression of hypoxia-inducible factor 1alpha is a marker for an unfavorable prognosis in early-stage invasive cervical cancer. Cancer Res 60, 4693-6 (2000). 48. Izuishi, K., Kato, K., Ogura, T., Kinoshita, T. & Esumi, H. Remarkable tolerance of tumor cells to nutrient deprivation: possible new biochemical target for cancer therapy. Cancer Res 60, 6201-7 (2000). 49. Morgan-Parkes, J.H. Metastases: mechanisms, pathways, and cascades. AJR Am J Roentgenol 164, 1075-82 (1995). 50. Zhou, Z., Doi, M., Wang, J., Cao, R., Liu, B., Chan, K.M., Kortesmaa, J., Sorokin, L., Cao, Y. & Tryggvason, K. Deletion of laminin-8 results in increased tumor neovascularization and metastasis in mice. Cancer Res 64, 4059-63 (2004). 51. Moserle, L. & Casanovas, O. Anti-angiogenesis and metastasis: a tumour and stromal cell alliance. J Intern Med 273, 128-37 (2013). 52. Gupta, S.C., Kim, J.H., Prasad, S. & Aggarwal, B.B. Regulation of survival, proliferation, invasion, angiogenesis, and metastasis of tumor cells through modulation of inflammatory pathways by nutraceuticals. Cancer Metastasis Rev 29, 405-34 (2010). 53. Yamazaki, K., Abe, S., Takekawa, H., Sukoh, N., Watanabe, N., Ogura, S., Nakajima, I., Isobe, H., Inoue, K. & Kawakami, Y. Tumor angiogenesis in human lung adenocarcinoma. Cancer 74, 2245-50 (1994). 162  54. Weidner, N., Carroll, P.R., Flax, J., Blumenfeld, W. & Folkman, J. Tumor angiogenesis correlates with metastasis in invasive prostate carcinoma. Am J Pathol 143, 401-9 (1993). 55. Weidner, N., Semple, J.P., Welch, W.R. & Folkman, J. Tumor angiogenesis and metastasis--correlation in invasive breast carcinoma. N Engl J Med 324, 1-8 (1991). 56. Zetter, B.R. Angiogenesis and tumor metastasis. Annu Rev Med 49, 407-24 (1998). 57. Hollingsworth, H.C., Kohn, E.C., Steinberg, S.M., Rothenberg, M.L. & Merino, M.J. Tumor angiogenesis in advanced stage ovarian carcinoma. Am J Pathol 147, 33-41 (1995). 58. Taylor, S. & Folkman, J. Protamine is an inhibitor of angiogenesis. Nature 297, 307-12 (1982). 59. Mori, S., Ueda, T., Kuratsu, S., Hosono, N., Izawa, K. & Uchida, A. Suppression of pulmonary metastasis by angiogenesis inhibitor TNP-470 in murine osteosarcoma. Int J Cancer 61, 148-52 (1995). 60. O'Reilly, M.S., Holmgren, L., Shing, Y., Chen, C., Rosenthal, R.A., Moses, M., Lane, W.S., Cao, Y., Sage, E.H. & Folkman, J. Angiostatin: a novel angiogenesis inhibitor that mediates the suppression of metastases by a Lewis lung carcinoma. Cell 79, 315-28 (1994). 61. Carmeliet, P. & Jain, R.K. Angiogenesis in cancer and other diseases. Nature 407, 249-57 (2000). 62. Hanahan, D. & Folkman, J. Patterns and emerging mechanisms of the angiogenic switch during tumorigenesis. Cell 86, 353-64 (1996). 163  63. Holmgren, L., O'Reilly, M.S. & Folkman, J. Dormancy of micrometastases: balanced proliferation and apoptosis in the presence of angiogenesis suppression. Nat Med 1, 149-53 (1995). 64. Dameron, K.M., Volpert, O.V., Tainsky, M.A. & Bouck, N. The p53 tumor suppressor gene inhibits angiogenesis by stimulating the production of thrombospondin. Cold Spring Harb Symp Quant Biol 59, 483-9 (1994). 65. O'Reilly, M.S., Boehm, T., Shing, Y., Fukai, N., Vasios, G., Lane, W.S., Flynn, E., Birkhead, J.R., Olsen, B.R. & Folkman, J. Endostatin: an endogenous inhibitor of angiogenesis and tumor growth. Cell 88, 277-85 (1997). 66. Folkman, J. Seminars in Medicine of the Beth Israel Hospital, Boston. Clinical applications of research on angiogenesis. N Engl J Med 333, 1757-63 (1995). 67. Sandler, A., Gray, R., Perry, M.C., Brahmer, J., Schiller, J.H., Dowlati, A., Lilenbaum, R. & Johnson, D.H. Paclitaxel-carboplatin alone or with bevacizumab for non-small-cell lung cancer. N Engl J Med 355, 2542-50 (2006). 68. Vaupel, P., Thews, O. & Hoeckel, M. Treatment resistance of solid tumors: role of hypoxia and anemia. Med Oncol 18, 243-59 (2001). 69. Hockel, M. & Vaupel, P. Tumor hypoxia: definitions and current clinical, biologic, and molecular aspects. J Natl Cancer Inst 93, 266-76 (2001). 70. Rankin, E.B. & Giaccia, A.J. The role of hypoxia-inducible factors in tumorigenesis. Cell Death Differ 15, 678-85 (2008). 71. Huang, L.E., Gu, J., Schau, M. & Bunn, H.F. Regulation of hypoxia-inducible factor 1alpha is mediated by an O2-dependent degradation domain via the ubiquitin-proteasome pathway. Proc Natl Acad Sci U S A 95, 7987-92 (1998). 164  72. Tanimoto, K., Makino, Y., Pereira, T. & Poellinger, L. Mechanism of regulation of the hypoxia-inducible factor-1 alpha by the von Hippel-Lindau tumor suppressor protein. EMBO J 19, 4298-309 (2000). 73. Maxwell, P.H., Wiesener, M.S., Chang, G.W., Clifford, S.C., Vaux, E.C., Cockman, M.E., Wykoff, C.C., Pugh, C.W., Maher, E.R. & Ratcliffe, P.J. The tumour suppressor protein VHL targets hypoxia-inducible factors for oxygen-dependent proteolysis. Nature 399, 271-5 (1999). 74. Ivan, M., Kondo, K., Yang, H., Kim, W., Valiando, J., Ohh, M., Salic, A., Asara, J.M., Lane, W.S. & Kaelin, W.G., Jr. HIFalpha targeted for VHL-mediated destruction by proline hydroxylation: implications for O2 sensing. Science 292, 464-8 (2001). 75. Jaakkola, P., Mole, D.R., Tian, Y.M., Wilson, M.I., Gielbert, J., Gaskell, S.J., Kriegsheim, A., Hebestreit, H.F., Mukherji, M., Schofield, C.J., Maxwell, P.H., Pugh, C.W. & Ratcliffe, P.J. Targeting of HIF-alpha to the von Hippel-Lindau ubiquitylation complex by O2-regulated prolyl hydroxylation. Science 292, 468-72 (2001). 76. Lando, D., Peet, D.J., Whelan, D.A., Gorman, J.J. & Whitelaw, M.L. Asparagine hydroxylation of the HIF transactivation domain a hypoxic switch. Science 295, 858-61 (2002). 77. Ke, Q. & Costa, M. Hypoxia-inducible factor-1 (HIF-1). Mol Pharmacol 70, 1469-80 (2006). 78. Talks, K.L., Turley, H., Gatter, K.C., Maxwell, P.H., Pugh, C.W., Ratcliffe, P.J. & Harris, A.L. The expression and distribution of the hypoxia-inducible factors HIF-1alpha and HIF-2alpha in normal human tissues, cancers, and tumor-associated macrophages. Am J Pathol 157, 411-21 (2000). 165  79. Hockel, M. & Vaupel, P. Biological consequences of tumor hypoxia. Semin Oncol 28, 36-41 (2001). 80. Aryee, D.N., Niedan, S., Kauer, M., Schwentner, R., Bennani-Baiti, I.M., Ban, J., Muehlbacher, K., Kreppel, M., Walker, R.L., Meltzer, P., Poremba, C., Kofler, R. & Kovar, H. Hypoxia modulates EWS-FLI1 transcriptional signature and enhances the malignant properties of Ewing's sarcoma cells in vitro. Cancer Res 70, 4015-23 (2010). 81. Vaupel, P., Kelleher, D.K. & Hockel, M. Oxygen status of malignant tumors: pathogenesis of hypoxia and significance for tumor therapy. Semin Oncol 28, 29-35 (2001). 82. Marignol, L., Lawler, M., Coffey, M. & Hollywood, D. Achieving hypoxia-inducible gene expression in tumors. Cancer Biol Ther 4, 359-64 (2005). 83. Yang, M.H., Wu, M.Z., Chiou, S.H., Chen, P.M., Chang, S.Y., Liu, C.J., Teng, S.C. & Wu, K.J. Direct regulation of TWIST by HIF-1alpha promotes metastasis. Nat Cell Biol 10, 295-305 (2008). 84. Li, Y., Qiu, X., Zhang, S., Zhang, Q. & Wang, E. Hypoxia induced CCR7 expression via HIF-1alpha and HIF-2alpha correlates with migration and invasion in lung cancer cells. Cancer Biol Ther 8, 322-30 (2009). 85. Mendez, O., Zavadil, J., Esencay, M., Lukyanov, Y., Santovasi, D., Wang, S.C., Newcomb, E.W. & Zagzag, D. Knock down of HIF-1alpha in glioma cells reduces migration in vitro and invasion in vivo and impairs their ability to form tumor spheres. Mol Cancer 9, 133 (2010). 166  86. Choi, J.Y., Jang, Y.S., Min, S.Y. & Song, J.Y. Overexpression of MMP-9 and HIF-1alpha in Breast Cancer Cells under Hypoxic Conditions. J Breast Cancer 14, 88-95 (2011). 87. Huang, J.H., Lee, F.S., Pasha, T.L., Sammel, M.D., Karakousis, G., Xu, G., Fraker, D. & Zhang, P.J. Analysis of HIF-1alpha and its regulator, PHD2, in retroperitoneal sarcomas: Clinico-pathologic implications. Cancer Biol Ther 9 (2010). 88. El Naggar, A., Clarkson, P., Zhang, F., Mathers, J., Tognon, C. & Sorensen, P. Expression and stability of hypoxia inducible factor 1alpha in osteosarcoma. Pediatr Blood Cancer (2012). 89. Holcik, M. & Sonenberg, N. Translational control in stress and apoptosis. Nat Rev Mol Cell Biol 6, 318-27 (2005). 90. Richter, J.D. & Sonenberg, N. Regulation of cap-dependent translation by eIF4E inhibitory proteins. Nature 433, 477-80 (2005). 91. Evdokimova, V., Ovchinnikov, L.P. & Sorensen, P.H. Y-box binding protein 1: providing a new angle on translational regulation. Cell Cycle 5, 1143-7 (2006). 92. Evdokimova, V., Tognon, C., Ng, T. & Sorensen, P.H. Reduced proliferation and enhanced migration: two sides of the same coin? Molecular mechanisms of metastatic progression by YB-1. Cell Cycle 8, 2901-6 (2009). 93. Bakker, W.J., Harris, I.S. & Mak, T.W. FOXO3a is activated in response to hypoxic stress and inhibits HIF1-induced apoptosis via regulation of CITED2. Mol Cell 28, 941-53 (2007). 94. Ikematsu, N., Yoshida, Y., Kawamura-Tsuzuku, J., Ohsugi, M., Onda, M., Hirai, M., Fujimoto, J. & Yamamoto, T. Tob2, a novel anti-proliferative Tob/BTG1 family member, 167  associates with a component of the CCR4 transcriptional regulatory complex capable of binding cyclin-dependent kinases. Oncogene 18, 7432-41 (1999). 95. Vega, S., Morales, A.V., Ocana, O.H., Valdes, F., Fabregat, I. & Nieto, M.A. Snail blocks the cell cycle and confers resistance to cell death. Genes Dev 18, 1131-43 (2004). 96. Mejlvang, J., Kriajevska, M., Vandewalle, C., Chernova, T., Sayan, A.E., Berx, G., Mellon, J.K. & Tulchinsky, E. Direct repression of cyclin D1 by SIP1 attenuates cell cycle progression in cells undergoing an epithelial mesenchymal transition. Mol Biol Cell 18, 4615-24 (2007). 97. Masuda, K., Abdelmohsen, K. & Gorospe, M. RNA-binding proteins implicated in the hypoxic response. J Cell Mol Med 13, 2759-69 (2009). 98. Gluz, O., Mengele, K., Schmitt, M., Kates, R., Diallo-Danebrock, R., Neff, F., Royer, H.D., Eckstein, N., Mohrmann, S., Ting, E., Kiechle, M., Poremba, C., Nitz, U. & Harbeck, N. Y-box-binding protein YB-1 identifies high-risk patients with primary breast cancer benefiting from rapidly cycled tandem high-dose adjuvant chemotherapy. J Clin Oncol 27, 6144-51 (2009). 99. Lovett, D.H., Cheng, S., Cape, L., Pollock, A.S. & Mertens, P.R. YB-1 alters MT1-MMP trafficking and stimulates MCF-7 breast tumor invasion and metastasis. Biochem Biophys Res Commun 398, 482-8 (2010). 100. Rohwer, N., Lobitz, S., Daskalow, K., Jons, T., Vieth, M., Schlag, P.M., Kemmner, W., Wiedenmann, B., Cramer, T. & Hocker, M. HIF-1alpha determines the metastatic potential of gastric cancer cells. Br J Cancer 100, 772-81 (2009). 101. Lee, S.L., Rouhi, P., Dahl Jensen, L., Zhang, D., Ji, H., Hauptmann, G., Ingham, P. & Cao, Y. Hypoxia-induced pathological angiogenesis mediates tumor cell dissemination, 168  invasion, and metastasis in a zebrafish tumor model. Proc Natl Acad Sci U S A 106, 19485-90 (2009). 102. Uchiumi, T., Fotovati, A., Sasaguri, T., Shibahara, K., Shimada, T., Fukuda, T., Nakamura, T., Izumi, H., Tsuzuki, T., Kuwano, M. & Kohno, K. YB-1 is important for an early stage embryonic development: neural tube formation and cell proliferation. J Biol Chem 281, 40440-9 (2006). 103. Iyer, N.V., Kotch, L.E., Agani, F., Leung, S.W., Laughner, E., Wenger, R.H., Gassmann, M., Gearhart, J.D., Lawler, A.M., Yu, A.Y. & Semenza, G.L. Cellular and developmental control of O2 homeostasis by hypoxia-inducible factor 1 alpha. Genes Dev 12, 149-62 (1998). 104. Tomita, S., Ueno, M., Sakamoto, M., Kitahama, Y., Ueki, M., Maekawa, N., Sakamoto, H., Gassmann, M., Kageyama, R., Ueda, N., Gonzalez, F.J. & Takahama, Y. Defective brain development in mice lacking the Hif-1alpha gene in neural cells. Mol Cell Biol 23, 6739-49 (2003). 105. Chernov, K.G., Mechulam, A., Popova, N.V., Pastre, D., Nadezhdina, E.S., Skabkina, O.V., Shanina, N.A., Vasiliev, V.D., Tarrade, A., Melki, J., Joshi, V., Baconnais, S., Toma, F., Ovchinnikov, L.P. & Curmi, P.A. YB-1 promotes microtubule assembly in vitro through interaction with tubulin and microtubules. BMC Biochem 9, 23 (2008). 106. Carbonaro, M., O'Brate, A. & Giannakakou, P. Microtubule disruption targets HIF-1alpha mRNA to cytoplasmic P-bodies for translational repression. J Cell Biol 192, 83-99 (2011). 107. Eliseeva, I.A., Kim, E.R., Guryanov, S.G., Ovchinnikov, L.P. & Lyabin, D.N. Y-box-binding protein 1 (YB-1) and its functions. Biochemistry (Mosc) 76, 1402-33 (2011). 169  108. Lu, Z.H., Books, J.T. & Ley, T.J. YB-1 is important for late-stage embryonic development, optimal cellular stress responses, and the prevention of premature senescence. Mol Cell Biol 25, 4625-37 (2005). 109. Makino, Y., Ohga, T., Toh, S., Koike, K., Okumura, K., Wada, M., Kuwano, M. & Kohno, K. Structural and functional analysis of the human Y-box binding protein (YB-1) gene promoter. Nucleic Acids Res 24, 1873-8 (1996). 110. Wistow, G. Cold shock and DNA binding. Nature 344, 823-4 (1990). 111. Kohno, K., Izumi, H., Uchiumi, T., Ashizuka, M. & Kuwano, M. The pleiotropic functions of the Y-box-binding protein, YB-1. Bioessays 25, 691-8 (2003). 112. Toh, S., Nakamura, T., Ohga, T., Koike, K., Uchiumi, T., Wada, M., Kuwano, M. & Kohno, K. Genomic organization of the human Y-box protein (YB-1) gene. Gene 206, 93-7 (1998). 113. Didier, D.K., Schiffenbauer, J., Woulfe, S.L., Zacheis, M. & Schwartz, B.D. Characterization of the cDNA encoding a protein binding to the major histocompatibility complex class II Y box. Proc Natl Acad Sci U S A 85, 7322-6 (1988). 114. Kohno, K., Sato, S., Uchiumi, T., Takano, H., Kato, S. & Kuwano, M. Tissue-specific enhancer of the human multidrug-resistance (MDR1) gene. J Biol Chem 265, 19690-6 (1990). 115. Swamynathan, S.K., Nambiar, A. & Guntaka, R.V. Role of single-stranded DNA regions and Y-box proteins in transcriptional regulation of viral and cellular genes. FASEB J 12, 515-22 (1998). 170  116. Wu, J., Stratford, A.L., Astanehe, A. & Dunn, S.E. YB-1 is a Transcription/Translation Factor that Orchestrates the Oncogenome by Hardwiring Signal Transduction to Gene Expression. Translational Oncogenomics 2, 49 (2007). 117. Murray, M.T., Schiller, D.L. & Franke, W.W. Sequence analysis of cytoplasmic mRNA-binding proteins of Xenopus oocytes identifies a family of RNA-binding proteins. Proc Natl Acad Sci U S A 89, 11-5 (1992). 118. Pinto, D., de Haan, G.J., Carton, D., Bader, A., Witte, J., Peters, E., van Erp, M.G., Vandereyken, W., Boezeman, E.H., Boon, P., Halley, D.J., Koeleman, B.P. & Lindhout, D. Gene symbol: KCNQ2. Disease: Benign neonatal familial convulsion. Hum Genet 117, 300 (2005). 119. Brandt, S., Raffetseder, U., Djudjaj, S., Schreiter, A., Kadereit, B., Michele, M., Pabst, M., Zhu, C. & Mertens, P.R. Cold shock Y-box protein-1 participates in signaling circuits with auto-regulatory activities. Eur J Cell Biol 91, 464-71 (2012). 120. Lasham, A., Print, C.G., Woolley, A.G., Dunn, S.E. & Braithwaite, A.W. YB-1: oncoprotein, prognostic marker and therapeutic target? Biochem J 449, 11-23 (2013). 121. Goldsmith, M.E., Madden, M.J., Morrow, C.S. & Cowan, K.H. A Y-box consensus sequence is required for basal expression of the human multidrug resistance (mdr1) gene. J Biol Chem 268, 5856-60 (1993). 122. Uchiumi, T., Kohno, K., Tanimura, H., Matsuo, K., Sato, S., Uchida, Y. & Kuwano, M. Enhanced expression of the human multidrug resistance 1 gene in response to UV light irradiation. Cell Growth Differ 4, 147-57 (1993). 171  123. Kohno, K., Sato, S., Uchiumi, T., Takano, H., Tanimura, H., Miyazaki, M., Matsuo, K., Hidaka, K. & Kuwano, M. Activation of the human multidrug resistance-1 (mdr1) gene promoter in response to inhibitors of DNA topoisomerases. Int J Oncol 1, 73-7 (1992). 124. Hu, Z., Jin, S. & Scotto, K.W. Transcriptional activation of the MDR1 gene by UV irradiation. Role of NF-Y and Sp1. J Biol Chem 275, 2979-85 (2000). 125. Sundseth, R., MacDonald, G., Ting, J. & King, A.C. DNA elements recognizing NF-Y and Sp1 regulate the human multidrug-resistance gene promoter. Mol Pharmacol 51, 963-71 (1997). 126. Jurchott, K., Bergmann, S., Stein, U., Walther, W., Janz, M., Manni, I., Piaggio, G., Fietze, E., Dietel, M. & Royer, H.D. YB-1 as a cell cycle-regulated transcription factor facilitating cyclin A and cyclin B1 gene expression. J Biol Chem 278, 27988-96 (2003). 127. Swamynathan, S.K., Varma, B.R., Weber, K.T. & Guntaka, R.V. Targeted disruption of one allele of the Y-box protein gene, Chk-YB-1b, in DT40 cells results in major defects in cell cycle. Biochem Biophys Res Commun 296, 451-7 (2002). 128. Dolfini, D. & Mantovani, R. YB-1 (YBX1) does not bind to Y/CCAAT boxes in vivo. Oncogene (2012). 129. Thoreen, C.C., Chantranupong, L., Keys, H.R., Wang, T., Gray, N.S. & Sabatini, D.M. A unifying model for mTORC1-mediated regulation of mRNA translation. Nature 485, 109-13 (2012). 130. Hsieh, A.C., Liu, Y., Edlind, M.P., Ingolia, N.T., Janes, M.R., Sher, A., Shi, E.Y., Stumpf, C.R., Christensen, C., Bonham, M.J., Wang, S., Ren, P., Martin, M., Jessen, K., Feldman, M.E., Weissman, J.S., Shokat, K.M., Rommel, C. & Ruggero, D. The 172  translational landscape of mTOR signalling steers cancer initiation and metastasis. Nature 485, 55-61 (2012). 131. Sommerville, J. Activities of cold-shock domain proteins in translation control. Bioessays 21, 319-25 (1999). 132. Meric, F., Searfoss, A.M., Wormington, M. & Wolffe, A.P. Masking and unmasking maternal mRNA. The role of polyadenylation, transcription, splicing, and nuclear history. J Biol Chem 271, 30804-10 (1996). 133. Kleene, K.C. Patterns of translational regulation in the mammalian testis. Mol Reprod Dev 43, 268-81 (1996). 134. Evdokimova, V., Tognon, C.E. & Sorensen, P.H. On translational regulation and EMT. Semin Cancer Biol 22, 437-45 (2012). 135. Rajasekhar, V.K., Viale, A., Socci, N.D., Wiedmann, M., Hu, X. & Holland, E.C. Oncogenic Ras and Akt signaling contribute to glioblastoma formation by differential recruitment of existing mRNAs to polysomes. Mol Cell 12, 889-901 (2003). 136. Evdokimova, V., Ruzanov, P., Anglesio, M.S., Sorokin, A.V., Ovchinnikov, L.P., Buckley, J., Triche, T.J., Sonenberg, N. & Sorensen, P.H. Akt-mediated YB-1 phosphorylation activates translation of silent mRNA species. Mol Cell Biol 26, 277-92 (2006). 137. Skabkin, M.A., Evdokimova, V., Thomas, A.A. & Ovchinnikov, L.P. The major messenger ribonucleoprotein particle protein p50 (YB-1) promotes nucleic acid strand annealing. J Biol Chem 276, 44841-7 (2001). 138. Evdokimova, V.M., Kovrigina, E.A., Nashchekin, D.V., Davydova, E.K., Hershey, J.W. & Ovchinnikov, L.P. The major core protein of messenger ribonucleoprotein particles 173  (p50) promotes initiation of protein biosynthesis in vitro. J Biol Chem 273, 3574-81 (1998). 139. Jiang, W., Hou, Y. & Inouye, M. CspA, the major cold-shock protein of Escherichia coli, is an RNA chaperone. J Biol Chem 272, 196-202 (1997). 140. Ladomery, M. & Sommerville, J. Binding of Y-box proteins to RNA: involvement of different protein domains. Nucleic Acids Res 22, 5582-9 (1994). 141. Ladomery, M., Wade, E. & Sommerville, J. Xp54, the Xenopus homologue of human RNA helicase p54, is an integral component of stored mRNP particles in oocytes. Nucleic Acids Res 25, 965-73 (1997). 142. Evdokimova, V., Ruzanov, P., Imataka, H., Raught, B., Svitkin, Y., Ovchinnikov, L.P. & Sonenberg, N. The major mRNA-associated protein YB-1 is a potent 5' cap-dependent mRNA stabilizer. EMBO J 20, 5491-502 (2001). 143. Nekrasov, M.P., Ivshina, M.P., Chernov, K.G., Kovrigina, E.A., Evdokimova, V.M., Thomas, A.A., Hershey, J.W. & Ovchinnikov, L.P. The mRNA-binding protein YB-1 (p50) prevents association of the eukaryotic initiation factor eIF4G with mRNA and inhibits protein synthesis at the initiation stage. J Biol Chem 278, 13936-43 (2003). 144. Wolffe, A.P., Tafuri, S., Ranjan, M. & Familari, M. The Y-box factors: a family of nucleic acid binding proteins conserved from Escherichia coli to man. New Biol 4, 290-8 (1992). 145. Schindelin, H., Jiang, W., Inouye, M. & Heinemann, U. Crystal structure of CspA, the major cold shock protein of Escherichia coli. Proc Natl Acad Sci U S A 91, 5119-23 (1994). 174  146. Schroder, K., Graumann, P., Schnuchel, A., Holak, T.A. & Marahiel, M.A. Mutational analysis of the putative nucleic acid-binding surface of the cold-shock domain, CspB, revealed an essential role of aromatic and basic residues in binding of single-stranded DNA containing the Y-box motif. Mol Microbiol 16, 699-708 (1995). 147. Bartoli, K.M., Bishop, D.L. & Saunders, W.S. The role of molecular microtubule motors and the microtubule cytoskeleton in stress granule dynamics. Int J Cell Biol 2011, 939848 (2011). 148. Srivastava, S.P., Kumar, K.U. & Kaufman, R.J. Phosphorylation of eukaryotic translation initiation factor 2 mediates apoptosis in response to activation of the double-stranded RNA-dependent protein kinase. J Biol Chem 273, 2416-23 (1998). 149. van der Laan, A.M., van Gemert, A.M., Dirks, R.W., Noordermeer, J.N., Fradkin, L.G., Tanke, H.J. & Jost, C.R. mRNA cycles through hypoxia-induced stress granules in live Drosophila embryonic muscles. Int J Dev Biol 56, 701-9 (2012). 150. Bader, A.G. & Vogt, P.K. Phosphorylation by Akt disables the anti-oncogenic activity of YB-1. Oncogene 27, 1179-82 (2008). 151. Gimenez-Bonafe, P., Fedoruk, M.N., Whitmore, T.G., Akbari, M., Ralph, J.L., Ettinger, S., Gleave, M.E. & Nelson, C.C. YB-1 is upregulated during prostate cancer tumor progression and increases P-glycoprotein activity. Prostate 59, 337-49 (2004). 152. Gao, Y., Fotovati, A., Lee, C., Wang, M., Cote, G., Guns, E., Toyota, B., Faury, D., Jabado, N. & Dunn, S.E. Inhibition of Y-box binding protein-1 slows the growth of glioblastoma multiforme and sensitizes to temozolomide independent O6-methylguanine-DNA methyltransferase. Mol Cancer Ther 8, 3276-84 (2009). 175  153. Oda, Y., Sakamoto, A., Shinohara, N., Ohga, T., Uchiumi, T., Kohno, K., Tsuneyoshi, M., Kuwano, M. & Iwamoto, Y. Nuclear expression of YB-1 protein correlates with P-glycoprotein expression in human osteosarcoma. Clin Cancer Res 4, 2273-7 (1998). 154. Oda, Y., Ohishi, Y., Saito, T., Hinoshita, E., Uchiumi, T., Kinukawa, N., Iwamoto, Y., Kohno, K., Kuwano, M. & Tsuneyoshi, M. Nuclear expression of Y-box-binding protein-1 correlates with P-glycoprotein and topoisomerase II alpha expression, and with poor prognosis in synovial sarcoma. J Pathol 199, 251-8 (2003). 155. Bader, A.G. & Vogt, P.K. Inhibition of protein synthesis by Y box-binding protein 1 blocks oncogenic cell transformation. Mol Cell Biol 25, 2095-106 (2005). 156. Evdokimova, V.M. & Ovchinnikov, L.P. Translational regulation by Y-box transcription factor: involvement of the major mRNA-associated protein, p50. Int J Biochem Cell Biol 31, 139-49 (1999). 157. Hanahan, D. & Weinberg, R.A. Hallmarks of cancer: the next generation. Cell 144, 646-74 (2011). 158. Garand, C., Guay, D., Sereduk, C., Chow, D., Tsofack, S.P., Langlois, M., Perreault, E., Yin, H.H. & Lebel, M. An integrative approach to identify YB-1-interacting proteins required for cisplatin resistance in MCF7 and MDA-MB-231 breast cancer cells. Cancer Sci 102, 1410-7 (2011). 159. Peinado, H., Olmeda, D. & Cano, A. Snail, Zeb and bHLH factors in tumour progression: an alliance against the epithelial phenotype? Nat Rev Cancer 7, 415-28 (2007). 160. Ng, T.L., Leprivier, G., Robertson, M.D., Chow, C., Martin, M.J., Laderoute, K.R., Davicioni, E., Triche, T.J. & Sorensen, P.H. The AMPK stress response pathway 176  mediates anoikis resistance through inhibition of mTOR and suppression of protein synthesis. Cell Death Differ 19, 501-10 (2012). 161. Dang, Y., Kedersha, N., Low, W.K., Romo, D., Gorospe, M., Kaufman, R., Anderson, P. & Liu, J.O. Eukaryotic initiation factor 2alpha-independent pathway of stress granule induction by the natural product pateamine A. J Biol Chem 281, 32870-8 (2006). 162. Arimoto, K., Fukuda, H., Imajoh-Ohmi, S., Saito, H. & Takekawa, M. Formation of stress granules inhibits apoptosis by suppressing stress-responsive MAPK pathways. Nat Cell Biol 10, 1324-32 (2008). 163. Eisinger-Mathason, T.S., Andrade, J., Groehler, A.L., Clark, D.E., Muratore-Schroeder, T.L., Pasic, L., Smith, J.A., Shabanowitz, J., Hunt, D.F., Macara, I.G. & Lannigan, D.A. Codependent functions of RSK2 and the apoptosis-promoting factor TIA-1 in stress granule assembly and cell survival. Mol Cell 31, 722-36 (2008). 164. Kim, B., Cooke, H.J. & Rhee, K. DAZL is essential for stress granule formation implicated in germ cell survival upon heat stress. Development 139, 568-78 (2012). 165. Gardner, L.B. Hypoxic inhibition of nonsense-mediated RNA decay regulates gene expression and the integrated stress response. Mol Cell Biol 28, 3729-41 (2008). 166. Gallois-Montbrun, S., Kramer, B., Swanson, C.M., Byers, H., Lynham, S., Ward, M. & Malim, M.H. Antiviral protein APOBEC3G localizes to ribonucleoprotein complexes found in P bodies and stress granules. J Virol 81, 2165-78 (2007). 167. Goodier, J.L., Zhang, L., Vetter, M.R. & Kazazian, H.H., Jr. LINE-1 ORF1 protein localizes in stress granules with other RNA-binding proteins, including components of RNA interference RNA-induced silencing complex. Mol Cell Biol 27, 6469-83 (2007). 177  168. Kedersha, N. & Anderson, P. Mammalian stress granules and processing bodies. Methods Enzymol 431, 61-81 (2007). 169. Vanderweyde, T., Yu, H., Varnum, M., Liu-Yesucevitz, L., Citro, A., Ikezu, T., Duff, K. & Wolozin, B. Contrasting pathology of the stress granule proteins TIA-1 and G3BP in tauopathies. J Neurosci 32, 8270-83 (2012). 170. Xie, W. & Denman, R.B. Protein methylation and stress granules: posttranslational remodeler or innocent bystander? Mol Biol Int 2011, 137459 (2011). 171. SEER*Stat Database: Incidence - SEER 9 Regs Research Data, N.S.K.R.P.A.L.T.C.A.-T.U.S. 172. Burningham, Z., Hashibe, M., Spector, L. & Schiffman, J.D. The epidemiology of sarcoma. Clin Sarcoma Res 2, 14 (2012). 173. Kansara, M. & Thomas, D.M. Molecular pathogenesis of osteosarcoma. DNA Cell Biol 26, 1-18 (2007). 174. Post, S.M. Mouse models of sarcomas: critical tools in our understanding of the pathobiology. Clin Sarcoma Res 2, 20 (2012). 175. Bernstein, M.L., Devidas, M., Lafreniere, D., Souid, A.K., Meyers, P.A., Gebhardt, M., Stine, K., Nicholas, R., Perlman, E.J., Dubowy, R., Wainer, I.W., Dickman, P.S., Link, M.P., Goorin, A., Grier, H.E., Pediatric Oncology, G., Children's Cancer Group Phase, I.I.S. & Children's Oncology, G. Intensive therapy with growth factor support for patients with Ewing tumor metastatic at diagnosis: Pediatric Oncology Group/Children's Cancer Group Phase II Study 9457--a report from the Children's Oncology Group. J Clin Oncol 24, 152-9 (2006). 178  176. Granowetter, L., Womer, R., Devidas, M., Krailo, M., Wang, C., Bernstein, M., Marina, N., Leavey, P., Gebhardt, M., Healey, J., Shamberger, R.C., Goorin, A., Miser, J., Meyer, J., Arndt, C.A., Sailer, S., Marcus, K., Perlman, E., Dickman, P. & Grier, H.E. Dose-intensified compared with standard chemotherapy for nonmetastatic Ewing sarcoma family of tumors: a Children's Oncology Group Study. J Clin Oncol 27, 2536-41 (2009). 177. Walterhouse, D. & Watson, A. Optimal management strategies for rhabdomyosarcoma in children. Paediatr Drugs 9, 391-400 (2007). 178. Hegyi, M., Felne Semsei, A., Jakab, Z., Antal, I., Kiss, J., Szendroi, M., Csoka, M. & Kovacs, G. [Results of the treatment of pediatric osteosarcoma in the Hungarian population]. Magy Onkol 56, 30-7 (2012). 179. Riggi, N., Cironi, L., Suva, M.L. & Stamenkovic, I. Sarcomas: genetics, signalling, and cellular origins. Part 1: The fellowship of TET. J Pathol 213, 4-20 (2007). 180. Toguchida, J. & Nakayama, T. Molecular genetics of sarcomas: applications to diagnoses and therapy. Cancer Sci 100, 1573-80 (2009). 181. Mitelman, F., Johansson, B. & Mertens, F. The impact of translocations and gene fusions on cancer causation. Nat Rev Cancer 7, 233-45 (2007). 182. May, W.A., Lessnick, S.L., Braun, B.S., Klemsz, M., Lewis, B.C., Lunsford, L.B., Hromas, R. & Denny, C.T. The Ewing's sarcoma EWS/FLI-1 fusion gene encodes a more potent transcriptional activator and is a more powerful transforming gene than FLI-1. Mol Cell Biol 13, 7393-8 (1993). 183. Lannon, C.L. & Sorensen, P.H. ETV6-NTRK3: a chimeric protein tyrosine kinase with transformation activity in multiple cell lineages. Semin Cancer Biol 15, 215-23 (2005). 179  184. Su, L., Sampaio, A.V., Jones, K.B., Pacheco, M., Goytain, A., Lin, S., Poulin, N., Yi, L., Rossi, F.M., Kast, J., Capecchi, M.R., Underhill, T.M. & Nielsen, T.O. Deconstruction of the SS18-SSX fusion oncoprotein complex: insights into disease etiology and therapeutics. Cancer Cell 21, 333-47 (2012). 185. Kazanowska, B., Reich, A., Stegmaier, S., Bekassy, A.N., Leuschner, I., Chybicka, A. & Koscielniak, E. Pax3-fkhr and pax7-fkhr fusion genes impact outcome of alveolar rhabdomyosarcoma in children. Fetal Pediatr Pathol 26, 17-31 (2007). 186. Sorensen, P.H., Lynch, J.C., Qualman, S.J., Tirabosco, R., Lim, J.F., Maurer, H.M., Bridge, J.A., Crist, W.M., Triche, T.J. & Barr, F.G. PAX3-FKHR and PAX7-FKHR gene fusions are prognostic indicators in alveolar rhabdomyosarcoma: a report from the children's oncology group. J Clin Oncol 20, 2672-9 (2002). 187. McArthur, G. Dermatofibrosarcoma protuberans: recent clinical progress. Ann Surg Oncol 14, 2876-86 (2007). 188. Butrynski, J.E., D'Adamo, D.R., Hornick, J.L., Dal Cin, P., Antonescu, C.R., Jhanwar, S.C., Ladanyi, M., Capelletti, M., Rodig, S.J., Ramaiya, N., Kwak, E.L., Clark, J.W., Wilner, K.D., Christensen, J.G., Janne, P.A., Maki, R.G., Demetri, G.D. & Shapiro, G.I. Crizotinib in ALK-rearranged inflammatory myofibroblastic tumor. N Engl J Med 363, 1727-33 (2010). 189. Lawrence, B., Perez-Atayde, A., Hibbard, M.K., Rubin, B.P., Dal Cin, P., Pinkus, J.L., Pinkus, G.S., Xiao, S., Yi, E.S., Fletcher, C.D. & Fletcher, J.A. TPM3-ALK and TPM4-ALK oncogenes in inflammatory myofibroblastic tumors. Am J Pathol 157, 377-84 (2000). 180  190. Rubin, B.P., Blanke, C.D., Demetri, G.D., Dematteo, R.P., Fletcher, C.D., Goldblum, J.R., Lasota, J., Lazar, A., Maki, R.G., Miettinen, M., Noffsinger, A., Washington, M.K. & Krausz, T. Protocol for the examination of specimens from patients with gastrointestinal stromal tumor. Arch Pathol Lab Med 134, 165-70 (2010). 191. Pedeutour, F., Suijkerbuijk, R.F., Van Gaal, J., Van de Klundert, W., Coindre, J.M., Van Haelst, A., Collin, F., Huffermann, K. & Turc-Carel, C. Chromosome 12 origin in rings and giant markers in well-differentiated liposarcoma. Cancer Genet Cytogenet 66, 133-4 (1993). 192. Bovee, J.V. & Hogendoorn, P.C. Molecular pathology of sarcomas: concepts and clinical implications. Virchows Arch 456, 193-9 (2010). 193. Murnane, J.P. Telomere loss as a mechanism for chromosome instability in human cancer. Cancer Res 70, 4255-9 (2010). 194. Mahalingam, D., Mita, A., Sankhala, K., Swords, R., Kelly, K., Giles, F. & Mita, M.M. Targeting sarcomas: novel biological agents and future perspectives. Curr Drug Targets 10, 937-49 (2009). 195. Pawel, B. Recent advances in the molecular diagnosis of paediatric soft tissue sarcomas. Diagnostic Histopathology 17, 25-35 (2011). 196. Bridge, J.A. & Cushman-Vokoun, A.M. Molecular diagnostics of soft tissue tumors. Arch Pathol Lab Med 135, 588-601 (2011). 197. Thomas, D.M., Savage, S.A. & Bond, G.L. Hereditary and environmental epidemiology of sarcomas. Clin Sarcoma Res 2, 13 (2012). 181  198. David Parham, M., Robert G. Maki, MD, PhD, and Karen H. Albritton, MD. Pediatric and Adult Sarcomas: Pathologic and Management Issues. American Society of Clinical Oncology (2011). 199. Bielack, S., Carrle, D. & Casali, P.G. Osteosarcoma: ESMO clinical recommendations for diagnosis, treatment and follow-up. Ann Oncol 20 Suppl 4, 137-9 (2009). 200. Dorfman, H.D. & Czerniak, B. Bone cancers. Cancer 75, 203-10 (1995). 201. Julie M. Wu, M.a.E.M., MD. . Classification and Pathology of Childhood Sarcomas. Surgical Clinics of North America - Volume 88, Issue 3. (2008). 202. Marina, N., Gebhardt, M., Teot, L. & Gorlick, R. Biology and therapeutic advances for pediatric osteosarcoma. Oncologist 9, 422-41 (2004). 203. Fuchs, B. & Pritchard, D.J. Etiology of osteosarcoma. Clin Orthop Relat Res, 40-52 (2002). 204. Nellissery, M.J., Padalecki, S.S., Brkanac, Z., Singer, F.R., Roodman, G.D., Unni, K.K., Leach, R.J. & Hansen, M.F. Evidence for a novel osteosarcoma tumor-suppressor gene in the chromosome 18 region genetically linked with Paget disease of bone. Am J Hum Genet 63, 817-24 (1998). 205. Wick, M.R., Siegal, G.P., Unni, K.K., McLeod, R.A. & Greditzer, H.G., 3rd. Sarcomas of bone complicating osteitis deformans (Paget's disease): fifty years' experience. Am J Surg Pathol 5, 47-59 (1981). 206. Siddiqui, R., Onel, K., Facio, F., Nafa, K., Diaz, L.R., Kauff, N., Huang, H., Robson, M., Ellis, N. & Offit, K. The TP53 mutational spectrum and frequency of CHEK2*1100delC in Li-Fraumeni-like kindreds. Fam Cancer 4, 177-81 (2005). 182  207. Overholtzer, M., Rao, P.H., Favis, R., Lu, X.Y., Elowitz, M.B., Barany, F., Ladanyi, M., Gorlick, R. & Levine, A.J. The presence of p53 mutations in human osteosarcomas correlates with high levels of genomic instability. Proc Natl Acad Sci U S A 100, 11547-52 (2003). 208. Castresana, J.S., Rubio, M.P., Gomez, L., Kreicbergs, A., Zetterberg, A. & Barrios, C. Detection of TP53 gene mutations in human sarcomas. Eur J Cancer 31A, 735-8 (1995). 209. Grana, X., Garriga, J. & Mayol, X. Role of the retinoblastoma protein family, pRB, p107 and p130 in the negative control of cell growth. Oncogene 17, 3365-83 (1998). 210. Weinberg, R.A. The retinoblastoma protein and cell cycle control. Cell 81, 323-30 (1995). 211. Gokgoz, N., Wunder, J.S., Mousses, S., Eskandarian, S., Bell, R.S. & Andrulis, I.L. Comparison of p53 mutations in patients with localized osteosarcoma and metastatic osteosarcoma. Cancer 92, 2181-9 (2001). 212. Tsuchiya, T., Sekine, K., Hinohara, S., Namiki, T., Nobori, T. & Kaneko, Y. Analysis of the p16INK4, p14ARF, p15, TP53, and MDM2 genes and their prognostic implications in osteosarcoma and Ewing sarcoma. Cancer Genet Cytogenet 120, 91-8 (2000). 213. Bramer, J.A., van Linge, J.H., Grimer, R.J. & Scholten, R.J. Prognostic factors in localized extremity osteosarcoma: a systematic review. Eur J Surg Oncol 35, 1030-6 (2009). 214. Hegyi, M., Semsei, A.F., Jakab, Z., Antal, I., Kiss, J., Szendroi, M., Csoka, M. & Kovacs, G. Good prognosis of localized osteosarcoma in young patients treated with limb-salvage surgery and chemotherapy. Pediatr Blood Cancer 57, 415-22 (2011). 183  215. Jemal, A., Tiwari, R.C., Murray, T., Ghafoor, A., Samuels, A., Ward, E., Feuer, E.J., Thun, M.J. & American Cancer, S. Cancer statistics, 2004. CA Cancer J Clin 54, 8-29 (2004). 216. Denny, C.T. Ewing's sarcoma--a clinical enigma coming into focus. J Pediatr Hematol Oncol 20, 421-5 (1998). 217. Karosas, A.O. Ewing's sarcoma. Am J Health Syst Pharm 67, 1599-605 (2010). 218. Kovar, H. Downstream EWS/FLI1 - upstream Ewing's sarcoma. Genome Med 2, 8 (2010). 219. Mackintosh, C., Madoz-Gurpide, J., Ordonez, J.L., Osuna, D. & Herrero-Martin, D. The molecular pathogenesis of Ewing's sarcoma. Cancer Biol Ther 9, 655-67 (2010). 220. Lin, P.P., Wang, Y. & Lozano, G. Mesenchymal Stem Cells and the Origin of Ewing's Sarcoma. Sarcoma 2011 (2011). 221. Widhe, B. & Widhe, T. Initial symptoms and clinical features in osteosarcoma and Ewing sarcoma. J Bone Joint Surg Am 82, 667-74 (2000). 222. Bernstein, M., Kovar, H., Paulussen, M., Randall, R.L., Schuck, A., Teot, L.A. & Juergens, H. Ewing's sarcoma family of tumors: current management. Oncologist 11, 503-19 (2006). 223. Sorensen, P.H., Lessnick, S.L., Lopez-Terrada, D., Liu, X.F., Triche, T.J. & Denny, C.T. A second Ewing's sarcoma translocation, t(21;22), fuses the EWS gene to another ETS-family transcription factor, ERG. Nat Genet 6, 146-51 (1994). 224. Ordonez, J.L., Osuna, D., Herrero, D., de Alava, E. & Madoz-Gurpide, J. Advances in Ewing's sarcoma research: where are we now and what lies ahead? Cancer Res 69, 7140-50 (2009). 184  225. Arvand, A. & Denny, C.T. Biology of EWS/ETS fusions in Ewing's family tumors. Oncogene 20, 5747-54 (2001). 226. Erkizan, H.V., Uversky, V.N. & Toretsky, J.A. Oncogenic partnerships: EWS-FLI1 protein interactions initiate key pathways of Ewing's sarcoma. Clin Cancer Res 16, 4077-83 (2010). 227. Ouchida, M., Ohno, T., Fujimura, Y., Rao, V.N. & Reddy, E.S. Loss of tumorigenicity of Ewing's sarcoma cells expressing antisense RNA to EWS-fusion transcripts. Oncogene 11, 1049-54 (1995). 228. Tanaka, K., Iwakuma, T., Harimaya, K., Sato, H. & Iwamoto, Y. EWS-Fli1 antisense oligodeoxynucleotide inhibits proliferation of human Ewing's sarcoma and primitive neuroectodermal tumor cells. J Clin Invest 99, 239-47 (1997). 229. Balamuth, N.J. & Womer, R.B. Ewing's sarcoma. Lancet Oncol 11, 184-92 (2010). 230. Dubois SG, G.H., Lessnick SL. Ewing's sarcoma. In: Orkin SH, Fisher DE, Look AT, Lux SE, Ginsburg D, Nathan DG, eds. Oncology of Infancy and Childhood. Philadelphia: Saunders Elsevier; 2009:829-869. 231. Braunreiter, C.L., Hancock, J.D., Coffin, C.M., Boucher, K.M. & Lessnick, S.L. Expression of EWS-ETS fusions in NIH3T3 cells reveals significant differences to Ewing's sarcoma. Cell Cycle 5, 2753-9 (2006). 232. Prieur, A., Tirode, F., Cohen, P. & Delattre, O. EWS/FLI-1 silencing and gene profiling of Ewing cells reveal downstream oncogenic pathways and a crucial role for repression of insulin-like growth factor binding protein 3. Mol Cell Biol 24, 7275-83 (2004). 185  233. Smith, R., Owen, L.A., Trem, D.J., Wong, J.S., Whangbo, J.S., Golub, T.R. & Lessnick, S.L. Expression profiling of EWS/FLI identifies NKX2.2 as a critical target gene in Ewing's sarcoma. Cancer Cell 9, 405-16 (2006). 234. Ginsberg, J.P., de Alava, E., Ladanyi, M., Wexler, L.H., Kovar, H., Paulussen, M., Zoubek, A., Dockhorn-Dworniczak, B., Juergens, H., Wunder, J.S., Andrulis, I.L., Malik, R., Sorensen, P.H., Womer, R.B. & Barr, F.G. EWS-FLI1 and EWS-ERG gene fusions are associated with similar clinical phenotypes in Ewing's sarcoma. J Clin Oncol 17, 1809-14 (1999). 235. Olmos, D., Martins, A.S., Jones, R.L., Alam, S., Scurr, M. & Judson, I.R. Targeting the Insulin-Like Growth Factor 1 Receptor in Ewing's Sarcoma: Reality and Expectations. Sarcoma 2011, 402508 (2011). 236. Pinkerton, C.R., Bataillard, A., Guillo, S., Oberlin, O., Fervers, B. & Philip, T. Treatment strategies for metastatic Ewing's sarcoma. Eur J Cancer 37, 1338-44 (2001). 237. Paulussen, M., Ahrens, S., Burdach, S., Craft, A., Dockhorn-Dworniczak, B., Dunst, J., Frohlich, B., Winkelmann, W., Zoubek, A. & Jurgens, H. Primary metastatic (stage IV) Ewing tumor: survival analysis of 171 patients from the EICESS studies. European Intergroup Cooperative Ewing Sarcoma Studies. Ann Oncol 9, 275-81 (1998). 238. Parham, D.M. & Ellison, D.A. Rhabdomyosarcomas in adults and children: an update. Arch Pathol Lab Med 130, 1454-65 (2006). 239. Turc-Carel, C., Lizard-Nacol, S., Justrabo, E., Favrot, M., Philip, T. & Tabone, E. Consistent chromosomal translocation in alveolar rhabdomyosarcoma. Cancer Genet Cytogenet 19, 361-2 (1986). 186  240. Douglass, E.C., Valentine, M., Etcubanas, E., Parham, D., Webber, B.L., Houghton, P.J., Houghton, J.A. & Green, A.A. A specific chromosomal abnormality in rhabdomyosarcoma. Cytogenet Cell Genet 45, 148-55 (1987). 241. Whang-Peng, J., Knutsen, T., Theil, K., Horowitz, M.E. & Triche, T. Cytogenetic studies in subgroups of rhabdomyosarcoma. Genes Chromosomes Cancer 5, 299-310 (1992). 242. Dagher, R. & Helman, L. Rhabdomyosarcoma: an overview. Oncologist 4, 34-44 (1999). 243. Raney, R.B., Anderson, J.R., Barr, F.G., Donaldson, S.S., Pappo, A.S., Qualman, S.J., Wiener, E.S., Maurer, H.M. & Crist, W.M. Rhabdomyosarcoma and undifferentiated sarcoma in the first two decades of life: a selective review of intergroup rhabdomyosarcoma study group experience and rationale for Intergroup Rhabdomyosarcoma Study V. J Pediatr Hematol Oncol 23, 215-20 (2001). 244. Rodeberg, D. & Paidas, C. Childhood rhabdomyosarcoma. Semin Pediatr Surg 15, 57-62 (2006). 245. Dasgupta, R. & Rodeberg, D.A. Update on rhabdomyosarcoma. Semin Pediatr Surg 21, 68-78 (2012). 246. Kuwano, M., Uchiumi, T., Hayakawa, H., Ono, M., Wada, M., Izumi, H. & Kohno, K. The basic and clinical implications of ABC transporters, Y-box-binding protein-1 (YB-1) and angiogenesis-related factors in human malignancies. Cancer Sci 94, 9-14 (2003). 247. Chatterjee, M., Rancso, C., Stuhmer, T., Eckstein, N., Andrulis, M., Gerecke, C., Lorentz, H., Royer, H.D. & Bargou, R.C. The Y-box binding protein YB-1 is associated with progressive disease and mediates survival and drug resistance in multiple myeloma. Blood 111, 3714-22 (2008). 187  248. Somasekharan, S.P., Stoynov, N., Rotblat, B., Leprivier, G., Galpin, J.D., Ahern, C.A., Foster, L.J. & Sorensen, P.H. Identification and quantification of newly synthesized proteins translationally regulated by YB-1 using a novel Click-SILAC approach. J Proteomics 77, e1-e10 (2012). 249. Zong, W.X. & Thompson, C.B. Necrotic death as a cell fate. Genes Dev 20, 1-15 (2006). 250. Trobisch, P., Suess, O. & Schwab, F. Idiopathic scoliosis. Dtsch Arztebl Int 107, 875-83; quiz 884 (2010). 251. Laemmli, U.K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680-5 (1970). 252. Klein, M.J. & Siegal, G.P. Osteosarcoma: anatomic and histologic variants. Am J Clin Pathol 125, 555-81 (2006). 253. Liu, P., Patil, S., Rojas, M., Fong, A.M., Smyth, S.S. & Patel, D.D. CX3CR1 deficiency confers protection from intimal hyperplasia after arterial injury. Arterioscler Thromb Vasc Biol 26, 2056-62 (2006). 254. Koo, C.L., Kok, L.F., Lee, M.Y., Wu, T.S., Cheng, Y.W., Hsu, J.D., Ruan, A., Chao, K.C. & Han, C.P. Scoring mechanisms of p16INK4a immunohistochemistry based on either independent nucleic stain or mixed cytoplasmic with nucleic expression can significantly signal to distinguish between endocervical and endometrial adenocarcinomas in a tissue microarray study. J Transl Med 7, 25 (2009). 255. Rotblat, B., Prior, I.A., Muncke, C., Parton, R.G., Kloog, Y., Henis, Y.I. & Hancock, J.F. Three separable domains regulate GTP-dependent association of H-ras with the plasma membrane. Mol Cell Biol 24, 6799-810 (2004). 188  256. Sorokin, A.V., Selyutina, A.A., Skabkin, M.A., Guryanov, S.G., Nazimov, I.V., Richard, C., Th'ng, J., Yau, J., Sorensen, P.H., Ovchinnikov, L.P. & Evdokimova, V. Proteasome-mediated cleavage of the Y-box-binding protein 1 is linked to DNA-damage stress response. EMBO J 24, 3602-12 (2005). 257. Chomczynski, P. & Sacchi, N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 162, 156-9 (1987). 258. Mowat, F.M., Luhmann, U.F., Smith, A.J., Lange, C., Duran, Y., Harten, S., Shukla, D., Maxwell, P.H., Ali, R.R. & Bainbridge, J.W. HIF-1alpha and HIF-2alpha are differentially activated in distinct cell populations in retinal ischaemia. PLoS One 5, e11103 (2010). 259. Livak, K.J. & Schmittgen, T.D. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 25, 402-8 (2001). 260. Collini, P., Sorensen, P.H., Patel, S., Blay, J.Y., Issels, R.D., Maki, R.G., Eriksson, M. & del Muro, X.G. Sarcomas with spindle cell morphology. Semin Oncol 36, 324-37 (2009). 261. Frye, B.C., Halfter, S., Djudjaj, S., Muehlenberg, P., Weber, S., Raffetseder, U., En-Nia, A., Knott, H., Baron, J.M., Dooley, S., Bernhagen, J. & Mertens, P.R. Y-box protein-1 is actively secreted through a non-classical pathway and acts as an extracellular mitogen. EMBO Rep 10, 783-9 (2009). 262. Lee, C., Dhillon, J., Wang, M.Y., Gao, Y., Hu, K., Park, E., Astanehe, A., Hung, M.C., Eirew, P., Eaves, C.J. & Dunn, S.E. Targeting YB-1 in HER-2 overexpressing breast cancer cells induces apoptosis via the mTOR/STAT3 pathway and suppresses tumor growth in mice. Cancer Res 68, 8661-6 (2008). 189  263. Stratford, A.L., Habibi, G., Astanehe, A., Jiang, H., Hu, K., Park, E., Shadeo, A., Buys, T.P., Lam, W., Pugh, T., Marra, M., Nielsen, T.O., Klinge, U., Mertens, P.R., Aparicio, S. & Dunn, S.E. Epidermal growth factor receptor (EGFR) is transcriptionally induced by the Y-box binding protein-1 (YB-1) and can be inhibited with Iressa in basal-like breast cancer, providing a potential target for therapy. Breast Cancer Res 9, R61 (2007). 264. Fujii, T., Seki, N., Namoto-Matsubayashi, R., Takahashi, H., Inoue, Y., Toh, U., Kage, M. & Shirouzu, K. YB-1 prevents apoptosis via the mTOR/STAT3 pathway in HER-2-overexpressing breast cancer cells. Future Oncol 5, 153-6 (2009). 265. Chabner, B.A. & Roberts, T.G., Jr. Timeline: Chemotherapy and the war on cancer. Nat Rev Cancer 5, 65-72 (2005). 266. Pantel, K., Schlimok, G., Braun, S., Kutter, D., Lindemann, F., Schaller, G., Funke, I., Izbicki, J.R. & Riethmuller, G. Differential expression of proliferation-associated molecules in individual micrometastatic carcinoma cells. J Natl Cancer Inst 85, 1419-24 (1993). 267. Aguirre-Ghiso, J.A. Models, mechanisms and clinical evidence for cancer dormancy. Nat Rev Cancer 7, 834-46 (2007). 268. To, K., Fotovati, A., Reipas, K.M., Law, J.H., Hu, K., Wang, J., Astanehe, A., Davies, A.H., Lee, L., Stratford, A.L., Raouf, A., Johnson, P., Berquin, I.M., Royer, H.D., Eaves, C.J. & Dunn, S.E. Y-box binding protein-1 induces the expression of CD44 and CD49f leading to enhanced self-renewal, mammosphere growth, and drug resistance. Cancer Res 70, 2840-51 (2010). 269. Kashihara, M., Azuma, K., Kawahara, A., Basaki, Y., Hattori, S., Yanagawa, T., Terazaki, Y., Takamori, S., Shirouzu, K., Aizawa, H., Nakano, K., Kage, M., Kuwano, 190  M. & Ono, M. Nuclear Y-box binding protein-1, a predictive marker of prognosis, is correlated with expression of HER2/ErbB2 and HER3/ErbB3 in non-small cell lung cancer. J Thorac Oncol 4, 1066-74 (2009). 270. Ivanov, P., Emara, M.M., Villen, J., Gygi, S.P. & Anderson, P. Angiogenin-induced tRNA fragments inhibit translation initiation. Mol Cell 43, 613-23 (2011). 271. van Zalen, S., Jeschke, G.R., Hexner, E.O. & Russell, J.E. AUF-1 and YB-1 are critical determinants of beta-globin mRNA expression in erythroid cells. Blood 119, 1045-53 (2012). 272. Shapiro, I.M., Cheng, A.W., Flytzanis, N.C., Balsamo, M., Condeelis, J.S., Oktay, M.H., Burge, C.B. & Gertler, F.B. An EMT-driven alternative splicing program occurs in human breast cancer and modulates cellular phenotype. PLoS Genet 7, e1002218 (2011). 273. Yilmaz, M. & Christofori, G. EMT, the cytoskeleton, and cancer cell invasion. Cancer Metastasis Rev 28, 15-33 (2009). 274. Liu, M., Faury, D., Sollier, C., Meehan, B., Gerges, N., Dong, Z., Siegel, P., Korshunov, A., Pfister, S., Rak, J. & Jabado, N. Sub-cellular localization of Y-Box Protein 1 regulates proliferation, invasion, and increased mesenchymal phenotype in astrocytomas. American Society of Human Genetics/ICHG 2011 Meeting (2011). 275. Bargou, R.C., Jurchott, K., Wagener, C., Bergmann, S., Metzner, S., Bommert, K., Mapara, M.Y., Winzer, K.J., Dietel, M., Dorken, B. & Royer, H.D. Nuclear localization and increased levels of transcription factor YB-1 in primary human breast cancers are associated with intrinsic MDR1 gene expression. Nat Med 3, 447-50 (1997). 276. Wu, J., Lee, C., Yokom, D., Jiang, H., Cheang, M.C., Yorida, E., Turbin, D., Berquin, I.M., Mertens, P.R., Iftner, T., Gilks, C.B. & Dunn, S.E. Disruption of the Y-box binding 191  protein-1 results in suppression of the epidermal growth factor receptor and HER-2. Cancer Res 66, 4872-9 (2006). 277. Silveira, C.G., Krampe, J., Ruhland, B., Diedrich, K., Hornung, D. & Agic, A. Cold-shock domain family member YB-1 expression in endometrium and endometriosis. Hum Reprod 27, 173-82 (2012). 278. Wang, X., Hao, N., Dohlman, H.G. & Elston, T.C. Bistability, stochasticity, and oscillations in the mitogen-activated protein kinase cascade. Biophys J 90, 1961-78 (2006). 279. U.S. Cancer Statistics Working Group. United States Cancer Statistics:1999 Incidence. Atlanta (GA): Department of Health and Human Services, Centers for Disease Control and Prevention and National Cancer Institute.  (2002). 280. May, C.D., Sphyris, N., Evans, K.W., Werden, S.J., Guo, W. & Mani, S.A. Epithelial-mesenchymal transition and cancer stem cells: a dangerously dynamic duo in breast cancer progression. Breast Cancer Res 13, 202 (2011). 281. Lasham, A., Samuel, W., Cao, H., Patel, R., Mehta, R., Stern, J.L., Reid, G., Woolley, A.G., Miller, L.D., Black, M.A., Shelling, A.N., Print, C.G. & Braithwaite, A.W. YB-1, the E2F pathway, and regulation of tumor cell growth. J Natl Cancer Inst 104, 133-46 (2012). 282. Lee, C.H., Xue, H., Sutcliffe, M., Gout, P.W., Huntsman, D.G., Miller, D.M., Gilks, C.B. & Wang, Y.Z. Establishment of subrenal capsule xenografts of primary human ovarian tumors in SCID mice: potential models. Gynecol Oncol 96, 48-55 (2005). 283. Cutz, J.C., Guan, J., Bayani, J., Yoshimoto, M., Xue, H., Sutcliffe, M., English, J., Flint, J., LeRiche, J., Yee, J., Squire, J.A., Gout, P.W., Lam, S. & Wang, Y.Z. Establishment in 192  severe combined immunodeficiency mice of subrenal capsule xenografts and transplantable tumor lines from a variety of primary human lung cancers: potential models for studying tumor progression-related changes. Clin Cancer Res 12, 4043-54 (2006). 284. Watahiki, A., Wang, Y., Morris, J., Dennis, K., O'Dwyer, H.M., Gleave, M., Gout, P.W. & Wang, Y. MicroRNAs associated with metastatic prostate cancer. PLoS One 6, e24950 (2011). 285. Al-Romaih, K., Somers, G.R., Bayani, J., Hughes, S., Prasad, M., Cutz, J.C., Xue, H., Zielenska, M., Wang, Y. & Squire, J.A. Modulation by decitabine of gene expression and growth of osteosarcoma U2OS cells in vitro and in xenografts: identification of apoptotic genes as targets for demethylation. Cancer Cell Int 7, 14 (2007). 286. Westerfield, M. The Zebrafish Book. A Guide for the Laboratory Use of Zebrafish (Danio rerio), 3rd Edition. Eugene, OR, University of Oregon Press, 385.   .  (1995). 287. Corkery, D.P., Dellaire, G. & Berman, J.N. Leukaemia xenotransplantation in zebrafish--chemotherapy response assay in vivo. Br J Haematol 153, 786-9 (2011). 288. Vieira, S.C., Zeferino, L.C., Da Silva, B.B., Aparecida Pinto, G., Vassallo, J., Carasan, G.A. & De Moraes, N.G. Quantification of angiogenesis in cervical cancer: a comparison among three endothelial cell markers. Gynecol Oncol 93, 121-4 (2004). 289. Debnath, J., Muthuswamy, S.K. & Brugge, J.S. Morphogenesis and oncogenesis of MCF-10A mammary epithelial acini grown in three-dimensional basement membrane cultures. Methods 30, 256-68 (2003). 290. Liu, S. & Leach, S.D. Zebrafish models for cancer. Annu Rev Pathol 6, 71-93 (2011). 193  291. Talmadge, J.E. & Fidler, I.J. AACR centennial series: the biology of cancer metastasis: historical perspective. Cancer Res 70, 5649-69 (2010). 292. Singh, B., Tai, K., Madan, S., Raythatha, M.R., Cady, A.M., Braunlin, M., Irving, L.R., Bajaj, A. & Lucci, A. Selection of metastatic breast cancer cells based on adaptability of their metabolic state. PLoS One 7, e36510 (2012). 293. Takahashi, M., Shimajiri, S., Izumi, H., Hirano, G., Kashiwagi, E., Yasuniwa, Y., Wu, Y., Han, B., Akiyama, M., Nishizawa, S., Sasaguri, Y. & Kohno, K. Y-box binding protein-1 is a novel molecular target for tumor vessels. Cancer Sci 101, 1367-73 (2010). 294. DeLisser, H.M., Christofidou-Solomidou, M., Strieter, R.M., Burdick, M.D., Robinson, C.S., Wexler, R.S., Kerr, J.S., Garlanda, C., Merwin, J.R., Madri, J.A. & Albelda, S.M. Involvement of endothelial PECAM-1/CD31 in angiogenesis. Am J Pathol 151, 671-7 (1997). 295. Harris, M.B., Gieser, P., Goorin, A.M., Ayala, A., Shochat, S.J., Ferguson, W.S., Holbrook, T. & Link, M.P. Treatment of metastatic osteosarcoma at diagnosis: a Pediatric Oncology Group Study. J Clin Oncol 16, 3641-8 (1998). 296. Goorin, A.M., Harris, M.B., Bernstein, M., Ferguson, W., Devidas, M., Siegal, G.P., Gebhardt, M.C., Schwartz, C.L., Link, M. & Grier, H.E. Phase II/III trial of etoposide and high-dose ifosfamide in newly diagnosed metastatic osteosarcoma: a pediatric oncology group trial. J Clin Oncol 20, 426-33 (2002). 297. Delattre, O., Zucman, J., Plougastel, B., Desmaze, C., Melot, T., Peter, M., Kovar, H., Joubert, I., de Jong, P., Rouleau, G. & et al. Gene fusion with an ETS DNA-binding domain caused by chromosome translocation in human tumours. Nature 359, 162-5 (1992). 194  298. Shepard, J.L., Amatruda, J.F., Stern, H.M., Subramanian, A., Finkelstein, D., Ziai, J., Finley, K.R., Pfaff, K.L., Hersey, C., Zhou, Y., Barut, B., Freedman, M., Lee, C., Spitsbergen, J., Neuberg, D., Weber, G., Golub, T.R., Glickman, J.N., Kutok, J.L., Aster, J.C. & Zon, L.I. A zebrafish bmyb mutation causes genome instability and increased cancer susceptibility. Proc Natl Acad Sci U S A 102, 13194-9 (2005). 299. Shepard, J.L., Stern, H.M., Pfaff, K.L. & Amatruda, J.F. Analysis of the cell cycle in zebrafish embryos. Methods Cell Biol 76, 109-25 (2004). 300. Feitsma, H. & Cuppen, E. Zebrafish as a cancer model. Mol Cancer Res 6, 685-94 (2008). 301. Payne, E. & Look, T. Zebrafish modelling of leukaemias. Br J Haematol 146, 247-56 (2009). 302. Stoletov, K. & Klemke, R. Catch of the day: zebrafish as a human cancer model. Oncogene 27, 4509-20 (2008). 303. Berman, J., Hsu, K. & Look, A.T. Zebrafish as a model organism for blood diseases. Br J Haematol 123, 568-76 (2003). 304. Detrich, H.W., 3rd, Westerfield, M. & Zon, L.I. Overview of the Zebrafish system. Methods Cell Biol 59, 3-10 (1999). 305. Cheng, H., Clarkson, P.W., Gao, D., Pacheco, M., Wang, Y. & Nielsen, T.O. Therapeutic Antibodies Targeting CSF1 Impede Macrophage Recruitment in a Xenograft Model of Tenosynovial Giant Cell Tumor. Sarcoma 2010, 174528 (2010). 306. Kortmann, U., McAlpine, J.N., Xue, H., Guan, J., Ha, G., Tully, S., Shafait, S., Lau, A., Cranston, A.N., O'Connor, M.J., Huntsman, D.G., Wang, Y. & Gilks, C.B. Tumor growth 195  inhibition by olaparib in BRCA2 germline-mutated patient-derived ovarian cancer tissue xenografts. Clin Cancer Res 17, 783-91 (2011). 307. Zagzag, D., Zhong, H., Scalzitti, J.M., Laughner, E., Simons, J.W. & Semenza, G.L. Expression of hypoxia-inducible factor 1alpha in brain tumors: association with angiogenesis, invasion, and progression. Cancer 88, 2606-18 (2000). 308. Wu, H.C. & Chang, D.K. Peptide-mediated liposomal drug delivery system targeting tumor blood vessels in anticancer therapy. J Oncol 2010, 723798 (2010). 309. Motzer, R.J., Hutson, T.E., Tomczak, P., Michaelson, M.D., Bukowski, R.M., Rixe, O., Oudard, S., Negrier, S., Szczylik, C., Kim, S.T., Chen, I., Bycott, P.W., Baum, C.M. & Figlin, R.A. Sunitinib versus interferon alfa in metastatic renal-cell carcinoma. N Engl J Med 356, 115-24 (2007). 310. Ratain, M.J., Eisen, T., Stadler, W.M., Flaherty, K.T., Kaye, S.B., Rosner, G.L., Gore, M., Desai, A.A., Patnaik, A., Xiong, H.Q., Rowinsky, E., Abbruzzese, J.L., Xia, C., Simantov, R., Schwartz, B. & O'Dwyer, P.J. Phase II placebo-controlled randomized discontinuation trial of sorafenib in patients with metastatic renal cell carcinoma. J Clin Oncol 24, 2505-12 (2006). 311. Hurwitz, H., Fehrenbacher, L., Novotny, W., Cartwright, T., Hainsworth, J., Heim, W., Berlin, J., Baron, A., Griffing, S., Holmgren, E., Ferrara, N., Fyfe, G., Rogers, B., Ross, R. & Kabbinavar, F. Bevacizumab plus irinotecan, fluorouracil, and leucovorin for metastatic colorectal cancer. N Engl J Med 350, 2335-42 (2004). 312. Postel-Vinay, S., Veron, A.S., Tirode, F., Pierron, G., Reynaud, S., Kovar, H., Oberlin, O., Lapouble, E., Ballet, S., Lucchesi, C., Kontny, U., Gonzalez-Neira, A., Picci, P., Alonso, J., Patino-Garcia, A., de Paillerets, B.B., Laud, K., Dina, C., Froguel, P., Clavel-196  Chapelon, F., Doz, F., Michon, J., Chanock, S.J., Thomas, G., Cox, D.G. & Delattre, O. Common variants near TARDBP and EGR2 are associated with susceptibility to Ewing sarcoma. Nat Genet 44, 323-7 (2012). 313. Weber, G.F. & Ashkar, S. Stress response genes: the genes that make cancer metastasize. J Mol Med (Berl) 78, 404-8 (2000). 314. de Herreros, A.G., Peiro, S., Nassour, M. & Savagner, P. Snail family regulation and epithelial mesenchymal transitions in breast cancer progression. J Mammary Gland Biol Neoplasia 15, 135-47 (2010). 315. Ashizuka, M., Fukuda, T., Nakamura, T., Shirasuna, K., Iwai, K., Izumi, H., Kohno, K., Kuwano, M. & Uchiumi, T. Novel translational control through an iron-responsive element by interaction of multifunctional protein YB-1 and IRP2. Mol Cell Biol 22, 6375-83 (2002). 316. Bert, A.G., Grepin, R., Vadas, M.A. & Goodall, G.J. Assessing IRES activity in the HIF-1alpha and other cellular 5' UTRs. RNA 12, 1074-83 (2006). 317. Dhawan, L., Liu, B., Pytlak, A., Kulshrestha, S., Blaxall, B.C. & Taubman, M.B. Y-box binding protein 1 and RNase UK114 mediate monocyte chemoattractant protein 1 mRNA stability in vascular smooth muscle cells. Mol Cell Biol 32, 3768-75 (2012). 318. Svitkin, Y.V., Evdokimova, V.M., Brasey, A., Pestova, T.V., Fantus, D., Yanagiya, A., Imataka, H., Skabkin, M.A., Ovchinnikov, L.P., Merrick, W.C. & Sonenberg, N. General RNA-binding proteins have a function in poly(A)-binding protein-dependent translation. EMBO J 28, 58-68 (2009). 319. Vaiman, A.V., Stromskaya, T.P., Rybalkina, E.Y., Sorokin, A.V., Guryanov, S.G., Zabotina, T.N., Mechetner, E.B., Ovchinnikov, L.P. & Stavrovskaya, A.A. Intracellular 197  localization and content of YB-1 protein in multidrug resistant tumor cells. Biochemistry (Mosc) 71, 146-54 (2006). 320. Homer, C., Knight, D.A., Hananeia, L., Sheard, P., Risk, J., Lasham, A., Royds, J.A. & Braithwaite, A.W. Y-box factor YB1 controls p53 apoptotic function. Oncogene 24, 8314-25 (2005). 321. Sun, S., Ning, X., Zhang, Y., Lu, Y., Nie, Y., Han, S., Liu, L., Du, R., Xia, L., He, L. & Fan, D. Hypoxia-inducible factor-1alpha induces Twist expression in tubular epithelial cells subjected to hypoxia, leading to epithelial-to-mesenchymal transition. Kidney Int 75, 1278-87 (2009). 322. Favaro, E., Nardo, G., Persano, L., Masiero, M., Moserle, L., Zamarchi, R., Rossi, E., Esposito, G., Plebani, M., Sattler, U., Mann, T., Mueller-Klieser, W., Ciminale, V., Amadori, A. & Indraccolo, S. Hypoxia inducible factor-1alpha inactivation unveils a link between tumor cell metabolism and hypoxia-induced cell death. Am J Pathol 173, 1186-201 (2008). 323. Moulder, J.E. & Rockwell, S. Tumor hypoxia: its impact on cancer therapy. Cancer Metastasis Rev 5, 313-41 (1987). 324. Ryan, H.E., Poloni, M., McNulty, W., Elson, D., Gassmann, M., Arbeit, J.M. & Johnson, R.S. Hypoxia-inducible factor-1alpha is a positive factor in solid tumor growth. Cancer Res 60, 4010-5 (2000). 325. Batra, S., Reynolds, C.P. & Maurer, B.J. Fenretinide cytotoxicity for Ewing's sarcoma and primitive neuroectodermal tumor cell lines is decreased by hypoxia and synergistically enhanced by ceramide modulators. Cancer Res 64, 5415-24 (2004). 198  326. Wang, G.L., Jiang, B.H., Rue, E.A. & Semenza, G.L. Hypoxia-inducible factor 1 is a basic-helix-loop-helix-PAS heterodimer regulated by cellular O2 tension. Proc Natl Acad Sci U S A 92, 5510-4 (1995). 327. Minchinton, A.I., Wendt, K.R., Clow, K.A. & Fryer, K.H. Multilayers of cells growing on a permeable support. An in vitro tumour model. Acta Oncol 36, 13-6 (1997). 328. Michel, G., Minet, E., Mottet, D., Remacle, J. & Michiels, C. Site-directed mutagenesis studies of the hypoxia-inducible factor-1alpha DNA-binding domain. Biochim Biophys Acta 1578, 73-83 (2002). 329. Fukuda, T., Ashizuka, M., Nakamura, T., Shibahara, K., Maeda, K., Izumi, H., Kohno, K., Kuwano, M. & Uchiumi, T. Characterization of the 5'-untranslated region of YB-1 mRNA and autoregulation of translation by YB-1 protein. Nucleic Acids Res 32, 611-22 (2004). 330. Spriggs, K.A., Stoneley, M., Bushell, M. & Willis, A.E. Re-programming of translation following cell stress allows IRES-mediated translation to predominate. Biol Cell 100, 27-38 (2008). 331. Hui, A.S., Bauer, A.L., Striet, J.B., Schnell, P.O. & Czyzyk-Krzeska, M.F. Calcium signaling stimulates translation of HIF-alpha during hypoxia. FASEB J 20, 466-75 (2006). 332. Kim, W.Y. & Kaelin, W.G. Role of VHL gene mutation in human cancer. J Clin Oncol 22, 4991-5004 (2004). 333. Zhou, J. & Brune, B. Cytokines and hormones in the regulation of hypoxia inducible factor-1alpha (HIF-1alpha). Cardiovasc Hematol Agents Med Chem 4, 189-97 (2006). 199  334. Bommert, K.S., Effenberger, M., Leich, E., Kuspert, M., Murphy, D., Langer, C., Moll, R., Janz, S., Mottok, A., Weissbach, S., Rosenwald, A., Bargou, R. & Bommert, K. The feed-forward loop between YB-1 and MYC is essential for multiple myeloma cell survival. Leukemia 27, 441-50 (2013). 335. Woolley, A.G., Algie, M., Samuel, W., Harfoot, R., Wiles, A., Hung, N.A., Tan, P.H., Hains, P., Valova, V.A., Huschtscha, L., Royds, J.A., Perez, D., Yoon, H.S., Cohen, S.B., Robinson, P.J., Bay, B.H., Lasham, A. & Braithwaite, A.W. Prognostic association of YB-1 expression in breast cancers: a matter of antibody. PLoS One 6, e20603 (2011). 336. Darb-Esfahani, S., Loibl, S., Muller, B.M., Roller, M., Denkert, C., Komor, M., Schluns, K., Blohmer, J.U., Budczies, J., Gerber, B., Noske, A., du Bois, A., Weichert, W., Jackisch, C., Dietel, M., Richter, K., Kaufmann, M. & von Minckwitz, G. Identification of biology-based breast cancer types with distinct predictive and prognostic features: role of steroid hormone and HER2 receptor expression in patients treated with neoadjuvant anthracycline/taxane-based chemotherapy. Breast Cancer Res 11, R69 (2009). 337. Dong, J., Akcakanat, A., Stivers, D.N., Zhang, J., Kim, D. & Meric-Bernstam, F. RNA-binding specificity of Y-box protein 1. RNA Biol 6, 59-64 (2009). 338. Ohga, T., Uchiumi, T., Makino, Y., Koike, K., Wada, M., Kuwano, M. & Kohno, K. Direct involvement of the Y-box binding protein YB-1 in genotoxic stress-induced activation of the human multidrug resistance 1 gene. J Biol Chem 273, 5997-6000 (1998). 339. Comerford, K.M., Wallace, T.J., Karhausen, J., Louis, N.A., Montalto, M.C. & Colgan, S.P. Hypoxia-inducible factor-1-dependent regulation of the multidrug resistance (MDR1) gene. Cancer Res 62, 3387-94 (2002). 200  340. Galban, S., Kuwano, Y., Pullmann, R., Jr., Martindale, J.L., Kim, H.H., Lal, A., Abdelmohsen, K., Yang, X., Dang, Y., Liu, J.O., Lewis, S.M., Holcik, M. & Gorospe, M. RNA-binding proteins HuR and PTB promote the translation of hypoxia-inducible factor 1alpha. Mol Cell Biol 28, 93-107 (2008). 341. Schepens, B., Tinton, S.A., Bruynooghe, Y., Beyaert, R. & Cornelis, S. The polypyrimidine tract-binding protein stimulates HIF-1alpha IRES-mediated translation during hypoxia. Nucleic Acids Res 33, 6884-94 (2005). 342. Cobbold, L.C., Wilson, L.A., Sawicka, K., King, H.A., Kondrashov, A.V., Spriggs, K.A., Bushell, M. & Willis, A.E. Upregulated c-myc expression in multiple myeloma by internal ribosome entry results from increased interactions with and expression of PTB-1 and YB-1. Oncogene 29, 2884-91 (2010). 343. Coles, L.S., Bartley, M.A., Bert, A., Hunter, J., Polyak, S., Diamond, P., Vadas, M.A. & Goodall, G.J. A multi-protein complex containing cold shock domain (Y-box) and polypyrimidine tract binding proteins forms on the vascular endothelial growth factor mRNA. Potential role in mRNA stabilization. Eur J Biochem 271, 648-60 (2004). 344. Ohga, T., Koike, K., Ono, M., Makino, Y., Itagaki, Y., Tanimoto, M., Kuwano, M. & Kohno, K. Role of the human Y box-binding protein YB-1 in cellular sensitivity to the DNA-damaging agents cisplatin, mitomycin C, and ultraviolet light. Cancer Res 56, 4224-8 (1996). 345. Ise, T., Nagatani, G., Imamura, T., Kato, K., Takano, H., Nomoto, M., Izumi, H., Ohmori, H., Okamoto, T., Ohga, T., Uchiumi, T., Kuwano, M. & Kohno, K. Transcription factor Y-box binding protein 1 binds preferentially to cisplatin-modified DNA and interacts with proliferating cell nuclear antigen. Cancer Res 59, 342-6 (1999). 201  346. Laughner, E., Taghavi, P., Chiles, K., Mahon, P.C. & Semenza, G.L. HER2 (neu) signaling increases the rate of hypoxia-inducible factor 1alpha (HIF-1alpha) synthesis: novel mechanism for HIF-1-mediated vascular endothelial growth factor expression. Mol Cell Biol 21, 3995-4004 (2001). 347. Zhou, J., Callapina, M., Goodall, G.J. & Brune, B. Functional integrity of nuclear factor kappaB, phosphatidylinositol 3'-kinase, and mitogen-activated protein kinase signaling allows tumor necrosis factor alpha-evoked Bcl-2 expression to provoke internal ribosome entry site-dependent translation of hypoxia-inducible factor 1alpha. Cancer Res 64, 9041-8 (2004). 348. Young, R.M., Wang, S.J., Gordan, J.D., Ji, X., Liebhaber, S.A. & Simon, M.C. Hypoxia-mediated selective mRNA translation by an internal ribosome entry site-independent mechanism. J Biol Chem 283, 16309-19 (2008). 349. Braunstein, S., Karpisheva, K., Pola, C., Goldberg, J., Hochman, T., Yee, H., Cangiarella, J., Arju, R., Formenti, S.C. & Schneider, R.J. A hypoxia-controlled cap-dependent to cap-independent translation switch in breast cancer. Mol Cell 28, 501-12 (2007). 350. Mani, S.A., Guo, W., Liao, M.J., Eaton, E.N., Ayyanan, A., Zhou, A.Y., Brooks, M., Reinhard, F., Zhang, C.C., Shipitsin, M., Campbell, L.L., Polyak, K., Brisken, C., Yang, J. & Weinberg, R.A. The epithelial-mesenchymal transition generates cells with properties of stem cells. Cell 133, 704-15 (2008). 351. Rauen, T., Raffetseder, U., Frye, B.C., Djudjaj, S., Muhlenberg, P.J., Eitner, F., Lendahl, U., Bernhagen, J., Dooley, S. & Mertens, P.R. YB-1 acts as a ligand for Notch-3 receptors and modulates receptor activation. J Biol Chem 284, 26928-40 (2009). 202  352. Notredame, C., Higgins, D.G. & Heringa, J. T-Coffee: A novel method for fast and accurate multiple sequence alignment. J Mol Biol 302, 205-17 (2000). 353. Di Tommaso, P., Moretti, S., Xenarios, I., Orobitg, M., Montanyola, A., Chang, J.M., Taly, J.F. & Notredame, C. T-Coffee: a web server for the multiple sequence alignment of protein and RNA sequences using structural information and homology extension. Nucleic Acids Res 39, W13-7 (2011). 354. Lorenz, R., Bernhart, S.H., Honer Zu Siederdissen, C., Tafer, H., Flamm, C., Stadler, P.F. & Hofacker, I.L. ViennaRNA Package 2.0. Algorithms Mol Biol 6, 26 (2011). 355. Gruber, A.R., Lorenz, R., Bernhart, S.H., Neubock, R. & Hofacker, I.L. The Vienna RNA websuite. Nucleic Acids Res 36, W70-4 (2008). 356. El Naggar, A., Clarkson, P., Zhang, F., Mathers, J., Tognon, C. & Sorensen, P.H. Expression and stability of hypoxia inducible factor 1alpha in osteosarcoma. Pediatr Blood Cancer 59, 1215-22 (2012). 357. Vemulapalli, S., Mita, A., Alvarado, Y., Sankhala, K. & Mita, M. The emerging role of mammalian target of rapamycin inhibitors in the treatment of sarcomas. Target Oncol 6, 29-39 (2011). 358. Wachtel, M. & Schafer, B.W. Targets for cancer therapy in childhood sarcomas. Cancer Treat Rev 36, 318-27 (2010). 359. Wang, C. Childhood rhabdomyosarcoma: recent advances and prospective views. J Dent Res 91, 341-50 (2012). 360. Koh, M.Y., Spivak-Kroizman, T.R. & Powis, G. HIF-1alpha and cancer therapy. Recent Results Cancer Res 180, 15-34 (2010).  

Cite

Citation Scheme:

        

Citations by CSL (citeproc-js)

Usage Statistics

Share

Embed

Customize your widget with the following options, then copy and paste the code below into the HTML of your page to embed this item in your website.
                        
                            <div id="ubcOpenCollectionsWidgetDisplay">
                            <script id="ubcOpenCollectionsWidget"
                            src="{[{embed.src}]}"
                            data-item="{[{embed.item}]}"
                            data-collection="{[{embed.collection}]}"
                            data-metadata="{[{embed.showMetadata}]}"
                            data-width="{[{embed.width}]}"
                            async >
                            </script>
                            </div>
                        
                    
IIIF logo Our image viewer uses the IIIF 2.0 standard. To load this item in other compatible viewers, use this url:
http://iiif.library.ubc.ca/presentation/dsp.24.1-0165716/manifest

Comment

Related Items