The Characterization and Role of RREB1 in Human Bladder Cancer Matthew David Nitz Sebring, Fl B.S. Wheaton College, 2003 M.S ., University of Virginia, 2009 A Dissertation presented to the Graduate Faculty of the University of Virginia in Cand idacy for the Degree of Doctor of Philosophy Department of Molecular Physiology and Biological Physics University of Virginia August, 2011 I Abstract RREB1 is a C2H2 zinc-finger protein that has previously been associated with malignancy but little is known about its expression, composition of splice variants, or role in cancer phenotypes. Total RREB1 expression decreased in human bladder cancer tumors compared to paired normal tissue. Rigorous comparison of commercial RREB1 antibodies allowed for the identification of RREB1 splice variants in the cytoplasm and nucleus on western blot. Five RREB1 splice variants were identified and designated: RREB1α, RREB1β, RREB1γ, RREB1δ, RREB1ε. Immunohistochemistry of RREB1 in a bladder cancer tissue microarray demonstrated that RREB1 expression positively correlates with the squamous cell histology. Transient depletion of RREB1 in bladder cancer cell lines (UMUC3 and KU7) decreased cell growth in vitro and similar results were seen in prostate cancer cell lines (LNCAP and PC3). siRNA duplexes that target RREB1 isoforms showed RREB1α and RREB1β, but not RREB1β alone, were necessary for in vitro UMUC3 cell growth and in vivo subcutaneous tumor growth. RREB1 depletion was found to decrease the expression of tumor associated protein CD24. Cloning of the CD24 promoter revealed a hypoxia response element, which led to the discovery of hypoxia induced CD24 RNA expression and promoter activity. RREB1 depletion decreased the expression of two additional hypoxia responsive genes (VEGFA and IGFBP3) without affecting the mRNA stability. Loss of RREB1 was found to reduce HIF1A protein levels and decrease its occupancy of the CD24 promoter. II Depletion of HIF1A caused a similar degree of cell growth inhibition as seen with RREB1 depletion. Thus, we propose the necessity of RREB1 in urologic malignancies at least partially acts by the stabilization of HIF1A protein and maintenance of downstream gene expression. III Table of Contents Abstract ............................................................................................................................... I Table of Contents ............................................................................................................. III List of Figures ................................................................................................................... V List of Tables ................................................................................................................... VII General Acknowledgments .......................................................................................... VIII Chapter 1: Introduction .....................................................................................................1 1.1 The Clinical Manifestation of Bladder Cancer .................................................................... 1 1.2 Molecular Insights into Bladder Cancer Progression ........................................................ 9 Chapter 2: The characterization of alternatively spliced RREB1 ............................... 27 2.1 Abstract .................................................................................................................................. 28 2.2 Introduction ............................................................................................................................ 29 2.3 Materials and Methods ........................................................................................................ 30 2.4 Results ................................................................................................................................... 34 2.5 Discussion ............................................................................................................................. 57 2.6 Acknowledgements .............................................................................................................. 65 Chapter 3: The Role of RREB1 in Bladder Cancer ....................................................... 66 3.1 Abstract .................................................................................................................................. 67 3.2 Introduction ............................................................................................................................ 68 3.3 Materials and Methods ........................................................................................................ 70 3.4 Results ................................................................................................................................... 74 3.5 Discussion ............................................................................................................................. 94 Chapter 4: Ral, RREB1, and Hypoxia Regulation of the CD24 Promoter ................. 96 4.1 Abstract .................................................................................................................................. 97 4.2 Introduction ............................................................................................................................ 98 4.3 Materials and Methods ...................................................................................................... 103 4.4 Results ................................................................................................................................. 106 IV 4.5 Discussion ........................................................................................................................... 131 Chapter 5: RREB1 binds and stabilizes the HIF1A transcription factor ................. 135 5.1 Abstract ................................................................................................................................ 136 5.2 Introduction .......................................................................................................................... 137 5.3 Materials and Methods ...................................................................................................... 139 5.4 Results ................................................................................................................................. 143 5.5 Discussion ........................................................................................................................... 163 Chapter 6: Discussion ................................................................................................... 165 6.1 RREB1 in bladder and prostate cancer........................................................................... 165 6.2 Defining CD24 transcriptional control .............................................................................. 178 6.3 Defining the interaction between RREB1 and HIF1A ................................................... 186 6.4 Future Directions of RREB1 .............................................................................................. 191 Chapter 7: References ................................................................................................... 195 V List of Figures Figure Page Figure 1: Low and high grade papillary carcinoma ......................................... 8 Figure 2: Bladder cancer treatment diagram ................................................. 12 Figure 3: Cycling of Ras GTPase .................................................................... 14 Figure 4: The HIF1A Pathway .......................................................................... 24 Figure 5: RREB1 alternative splicing .............................................................. 37 Figure 6: Detection of RREB1 isoforms ......................................................... 39 Figure 7: A novel RREB1 splice variant ......................................................... 42 Figure 8: RREB1 expression in cell lines ....................................................... 44 Figure 9: RREB1 expression in tumors .......................................................... 47 Figure 10: Overexpressed RREB1 protein isoforms ..................................... 49 Figure 11: Endogenous RREB1 protein isoforms ......................................... 53 Figure 12: RREB1 immunohistochemistry ..................................................... 56 Figure 13: RREB1 tissue microarray legend .................................................. 60 Figure 14: RREB1 TMA clinical associations ................................................. 62 Figure 15: RREB1 dependent growth ............................................................. 77 Figure 16: RREB1 splice variant rescue ......................................................... 79 Figure 17: RREB1 splice variant dependent growth ..................................... 84 Figure 18: Non-targeted RREB1 splice variant upregulation ........................ 87 Figure 19: RREB1 depletion xenograft tumorigenesis .................................. 91 Figure 20: RREB1delta drives xenograft tumorigenesis ............................... 93 VI Figure 21: Proposed CD24 promoter regulation .......................................... 102 Figure 22: CD24 expression is dependent on RREB1 ................................. 109 Figure 23: CD24 rescue of RREB1 depletion ............................................... 112 Figure