ROLE OF THE VITAMIN D RECEPTOR IN 1,25-DIHYDROXYVITAMIN D3- MEDIATED GROWTH ARREST AND APOPTOSIS A Dissertation Submitted to the Graduate School of the University of Notre Dame In Partial Fulfillment of the Requirements For the Degree of Doctor of Philosophy by Meggan E. Valrance, B.S. JoEllen Welsh, Director Graduate Program in Biological Sciences Notre Dame, Indiana July 2007 © Copyright by Meggan E. Valrance 2007 All Rights Reserved ROLE OF THE VITAMIN D RECEPTOR IN 1,25-DIHYDROXYVITAMIN D3- MEDIATED GROWTH ARREST AND APOPTOSIS Abstract by Meggan E. Valrance 1,25-dihydroxyvitamin D3 (1,25D) and its cognate nuclear receptor, the vitamin D receptor (VDR), regulate calcium homeostasis via transcriptional activation of target genes. 1,25D is also known to mediate growth arrest and apoptosis in a variety of cancer cell lines and tumors, however, the role of the VDR and the role of transcriptional activation in this process remain unclear. To investigate the role of the VDR in mediating the anti-cancer properties of 1,25D, two murine mammary tumor cell lines with differential VDR expression were characterized. WT145 cells, which express transcriptionally functional VDR protein, were growth inhibited and rendered apoptotic by 1,25D and synthetic vitamin D analogs. In contrast, KO240 cells, which express no detectable VDR protein or mRNA, were neither growth inhibited nor rendered apoptotic by 1,25D, at doses as high as 1μM. Collectively, these data indicate that functional VDR protein is Meggan E. Valrance required for the anti-cancer effects of 1,25D and structurally related vitamin D based therapeutics in vitro. To determine whether the anti-cancer actions of vitamin D are mediated via the VDR in vivo, nude mice bearing tumors derived from WT145 or KO240 cells were treated with EB1089, a synthetic analog of 1,25D, or placebo for six weeks. An untreated subset of tumor-bearing mice was exposed to ultraviolet (UV) light, to activate endogenous 1,25D production. Both EB1089 and UV light exposure decreased volume of WT145 tumors through decreased tumor cell proliferation and increased tumor cell apoptosis. No effects of either EB1089 or UV treatments were observed in KO240 tumors, indicating that the vitamin D pathway mediates its anti-tumor effects in vivo via tumor-cell VDR. KO240 cells stably expressing wild-type VDR and VDR point mutants from hereditary rickets patients were created and characterized, to further examine the mechanism of VDR in growth regulation. KO cells stably expressing VDR were growth inhibited by 1,25D and its structural analogs, indicating that the vitamin D growth regulatory pathway could be reconstituted in VDR null cells. Cells expressing mutant VDRs were differentially affected by 1,25D and analogs; VDR that lack DNA binding ability were growth inhibited by physiological doses of 1,25D while ligand binding domain mutants were not. This suggests that the anti-cancer effects of 1,25D, while VDR mediated, are in part mediated via novel, DNA-independent mechanisms. To Jamie, my source of sanity in an insane world. I could never have done this without your love, your help, your sense of humor, or your understanding. Thank you for being you, for loving and understanding me, and for putting up with my many mental breakdowns while writing. And, to my grandmother Maxine, my mother Kathy, and my aunt Charlie - this is for you. I couldn’t save you, but I hope beyond hope that my work can save others, so that no other woman has to be as strong and as brave as the three of you were. You are in my thoughts always. ii CONTENTS LIST OF TABLES .................................................................................................vi LIST OF FIGURES ..............................................................................................vii LIST OF ABBREVIATIONS ..................................................................................xi ACKNOWLEDGMENTS .....................................................................................xiii Chapter 1: LITERATURE REVIEW ...................................................................... 1 1.1 Introduction .......................................................................................... 1 1.2 1,25D Biosynthesis .............................................................................. 2 1.3 Classical 1,25D/VDR Signaling............................................................ 4 1.4 Non-Classical VDR Signaling Pathways............................................ 11 1.5 Vitamin D3 and Cancer ...................................................................... 14 1.6 Dissertation Objectives ...................................................................... 18 Chapter 2: CHARACTERIZATION OF WT145 AND KO240 MURINE MAMMARY TUMOR CELLS IN STEROID-FREE MEDIA ....................... 20 2.1 Introduction ........................................................................................ 20 2.2 Materials and Methods....................................................................... 23 2.3 Results............................................................................................... 27 2.4 Discussion ......................................................................................... 36 iii Chapter 3: INDUCTION OF APOPTOSIS BY 1,25D IN MURINE MAMMARY TUMOR CELLS REQUIRES VDR PROTEIN EXPRESSION .................. 39 3.1 Introduction ........................................................................................ 39 3.2 Materials and Methods....................................................................... 43 3.3 Results............................................................................................... 46 3.4 Discussion ......................................................................................... 54 Chapter 4: REQUIREMENT OF VDR FOR THE ANTI-CANCER ACTION OF SYNTHETIC STRUCTURAL ANALOGS OF 1,25D AND OTHER VITAMIN D METABOLITES..................................................................... 57 4.1 Introduction ........................................................................................ 57 4.2 Materials and Methods....................................................................... 60 4.3 Results............................................................................................... 63 4.4 Discussion ......................................................................................... 71 Chapter 5: VDR-DEPENDENT INHIBITION OF MAMMARY TUMOR GROWTH IN VIVO BY EB1089 AND ULTRAVIOLET RADIATION ........ 75 5.1 Introduction ........................................................................................ 75 5.2 Materials and Methods....................................................................... 78 5.3 Results............................................................................................... 82 5.4 Discussion ......................................................................................... 94 Chapter 6: CREATION OF VECTORS SUITABLE FOR STABLE EXPRESSION OF WILD-TYPE AND MUTANT VDRS IN MURINE MAMMARY CELLS.................................................................................. 99 6.1 Introduction ........................................................................................ 99 6.2 Materials and Methods..................................................................... 103 iv 6.3 Results............................................................................................. 110 6.4 Discussion ....................................................................................... 120 Chapter 7: CREATION AND CHARACTERIZATION OF KO240 CELLS STABLY EXPRESSING WILD-TYPE HUMAN VDR.............................. 124 7.1 Introduction ...................................................................................... 124 7.2 Materials and Methods..................................................................... 127 7.3 Results............................................................................................. 131 7.4 Discussion ....................................................................................... 143 Chapter 8: CREATION AND CHARACTERIZATION OF KO240 CELLS STABLY EXPRESSING HVDRR-2 POINT-MUTANT VDRS ................. 148 8.1 Introduction ...................................................................................... 148 8.2 Materials and Methods..................................................................... 151 8.3 Results............................................................................................. 155 8.4 Discussion ....................................................................................... 163 GENERAL DISCUSSION ................................................................................. 169 REFERENCES: ................................................................................................ 177 v LIST OF TABLES Table 6.1. Nucleotide positions and base changes for VDR mutant constructs................................................................................................107 Table 6.2. A comparison of the wild-type VDR sequence with the point mutations selectively created................................................................. 115 Table 7.1. 1,25D analogs induce growth arrest in KOhVDR cells........................ 142 Table 8.1. Transactivation activity of the VDR in KOmutant stable cell lines........ 157 Table 8.2. 1,25D analogs have differential growth effects in KOmutant cells. .... 161 vi LIST OF