THE ROLE OF HYPOXIA IN MODULATING GLIOMA CELL TUMORIGENIC POTENTIAL BY JOHN MICHAEL HEDDLESTON Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy Dissertation Advisor: Jeremy N. Rich, M.D., M.H.Sc. Program in Cell Biology CASE WESTERN RESERVE UNIVERSITY August, 2011 CASE WESTERN RESERVE UNIVERSITY SCHOOL OF GRADUATE STUDIES We hereby approve the thesis/dissertation of John Michael Heddleston ______________________________________________________ Doctor of Philosophy candidate for the ________________________________degree *. Danny Manor (signed)_______________________________________________ (chair of the committee) Jeremy N. Rich ________________________________________________ George R. Stark ________________________________________________ Erik Andrulis ________________________________________________ ________________________________________________ ________________________________________________ June 17, 2011 (date) _______________________ *We also certify that written approval has been obtained for any proprietary material contained therein. Dedications I would like to dedicate this dissertation to wife and family. Katie, without your constant love and support, I would have not been able to accomplish all that I have. Your gentle words of encouragement and sympathetic ear has kept me sane and able to push on, even when I didn’t think I could. To my parents, Mike and Gi, and my sister, Sara, you have helped shaped the person I am today. I owe everything to your love and guidance. As I move forward in my career, I hope I can achieve great things that make you proud. All my love, John Table of Contents Chapter 1.1: Introduction ..................................................................................... 1 1.2: Identification of Glioma Stem Cells .................................................... 4 1.3: Functional Characteristics of GSCs ................................................... 5 1.4: Other Characteristics of GSCs .......................................................... 8 1.5: Immunophenotypic Characteristics of GSCs ................................... 12 1.6: CD133 as a Marker for GSCs .......................................................... 14 1.7: Alternative Cell Surface Markers for GSCs ..................................... 17 1.8: Identification of GSCs Independent of Surface Markers ................. 19 1.9: GSC Regulators on the Cell Surface ............................................... 21 1.10: Intracellular GSC Regulators ......................................................... 26 1.11: Perivascular Microenvironmental Regulation of GSCs .................. 30 1.12: Hypoxia in Glioma ......................................................................... 36 1.13: GSCs, Hypoxia, and HIF2α ........................................................... 42 1.14: Epigenetics .................................................................................... 45 1.15: Induced Pluripotency ..................................................................... 48 1.16: Epigenetic Modifiers in Glioma ...................................................... 49 1.17: Targeting GSCs via the Microenvironment .................................... 53 Chapter 2.1: Hypoxia Promotes the GSC Phenotype Through HIF2α .............. 56 i 2.2: The Role of HIFs in GSC Self-Renewal and Tumor Growth ........... 60 2.3: Hypoxia Reprograms Towards a Stem Phenotype ......................... 62 2.4: HIF2α Promotes a Glioma Stem Cell State ..................................... 68 2.5: Discussion ....................................................................................... 72 2.6: Materials and Methods .................................................................... 78 Chapter 3.1: The Hypoxia Response in Glioma Cells Requires Epigenetic Modifying Factor, MLL1 ..................................................................................... 83 3.2: Hypoxia Increases Expression of MLL1 .......................................... 87 3.3: HIF1α and HIF2α are Necessary for MLL Induction ........................ 89 3.4: MLL1 can be Efficiently Targeted with shRNA ................................ 93 3.5: MLL1 is Required for HIF2α Expression .......................................... 95 3.6: MLL1 is Preferentially Expressed in GSCs .................................... 101 3.7: Hypoxia Regulates MLL1 Expression in GSCs ............................. 105 3.8: Targeting MLL1 Reduces VEGF in GSCs ..................................... 108 3.9: Loss of MLL1 Inhibits GSC Growth and Self-Renewal .................. 108 3.10: Inhibition of MLL1 Reduces GSC Tumorigenicity ........................ 112 3.11: Discussion ................................................................................... 113 3.12: Materials and Methods ................................................................ 118 Chapter 4.1: Discussion .................................................................................. 126 4.2: Glioma Cells and Phenotypic Plasticity ......................................... 129 ii 4.3: Epigenetics of Glioma Cells ........................................................... 133 4.4: The Importance of MLL1 in HIF2α Signaling ................................. 135 4.5: Future Directions ........................................................................... 139 Bibliography ..................................................................................................... 143 iii List of Figures 1: Microenvironmental factors modulate intrinsic pathways ................... 31 2: Hypoxia enchances neurosphere formation and proliferation ............ 64 3: Exposure to hypoxia increases stem gene transcripts ....................... 67 4: Ectopic expression of nondegradeable HIF2α causes morphological and phenotype changes in nonstem glioma cells ................................... 69 5: Overexpression of HIF2α in nonstem glioma cells increases tumorigenic capacity ............................................................................... 71 6: The role of hypoxia in the glioma stem cell hypothesis ...................... 77 7: MLL1 is a hypoxia responsive gene in nonstem glioma cells ............. 88 8: HIF1α and HIF2α are required for the MLL1 hypoxia response ......... 91 9: MLL1 is hypoxia responsive but not a direct binding target of HIF2α ...................................................................................................... 92 10: MLL1 can be successfully targeted by shRNA ................................. 94 11: Targeting MLL1 via shRNA inhibits expression of HIF2α, but not HIF1α .......................................................................................... 96 12: Targeting MLL1 by shRNA inhibits downstream hypoxic response ................................................................................................. 98 13: MLL1 knockdown inhibits HIF2α expression and the downstream hypoxic response .................................................................................... 99 14: MLL1 modulates HIF2A transcription by chromatin regulation ....... 100 15: MLL1 expression is elevated in GSCs compared to nonstem glioma cells ....................................................................................................... 102 16: GSCs form neurospheres expressing stem cell markers ............... 103 17: MLL1 co-localizes with GSC enrichment marker, CD133 .............. 104 18: MLL1 is regulated by hypoxia and HIF2α in GSCs ......................... 106 iv 19: Pharmacological inhibition of HIF2α reduces MLL1 expression ..... 107 20: Targeting MLL1 reduces GSC-mediated VEGF production and endothelial cell proliferation .................................................................. 109 21: MLL1 knockdown decreases the growth of GSCs .......................... 110 22: Targeting MLL1 via shRNA reduces neurosphere formation in GSCs ................................................................................................ 111 23: MLL1 reduces tumor propagation in mice ...................................... 114 24: Treatment with doxycycline activates inducible construct .............. 141 v Acknowledgements I would like to thank all current and previous members of the Rich Lab. Without your guidance this dissertation and my growth as a scientist would not have been possible. I have made life-long friends in this lab and I look forward to staying in contact with all of you throughout life. Although there are too many fun times to describe in this dissertation, I hope all of you know how important you are in my life and how much fun I had while in the Rich Lab. In particular, I want to thank Justin Lathia, Monica Venere, and Anita Hjelmeland. Justin, as one of the only remaining Duke Transfers I fondly remember the fun we had playing sand volleyball and not having to deal with snow for the better part of the year while in North Carolina. If I have learned anything from you, I will at least be moving forward with a honed skill in making nicknames and using cufflinks even though they serve no real purpose. I wish you all the best in your research endeavors and I know you’ll be amazingly successful. I’m sure that I’ll be back to the area to check in and see how large your lab has grown and how many Nature publications you have. Monica, even though you have only been in the lab a short year, you have spent a good part of that year dealing with this annoying graduate student and resisting the urge to unleash your