The Physiological Effects of CD38 in Prostate Cancer: A multifunctional NAD’ase capable of regulating cell metabolism, gene expression, and therapeutic response BY Jeffrey P. Chmielewski A Dissertation Submitted to the Graduate Faculty of WAKE FOREST UNIVERSITY GRADUATE SCHOOL OF ARTS AND SCIENCES In Partial Fulfillment of the Requirements For the Degree of DOCTOR OF PHILOSOPHY Cancer Biology May, 2018 Winston-Salem, North Carolina Approved By: Steven J. Kridel, Ph.D., Advisor W. Todd Lowther, Ph.D., Chair Anthony Molina, Ph.D. Lance Miller, Ph.D. Ravi Singh, Ph.D. DEDICATION This work is dedicated to my wife: an enduring pillar of support throughout this process; to my children: realize your potential, and pursue your dreams – regardless of the obstacle; and to my family: thank you for the encouragement and assistance during this period. ii TABLE OF CONTENTS List of Figures and Tables ......................................................................... vi List of Abbreviations ................................................................................ viii Abstract .................................................................................................. xviii CHAPTER I GENERAL INTRODUCTION 1.1 Nicotinamide Adenine Dinucleotide (NAD+) Biology 1.1.1 Historical Perspective .............................................................2 1.1.2 NAD+ Biosynthesis: De novo and salvage pathways…...…….3 1.1.3 Nicotinamide phosphoribosyltransferase (NAMPT) biology ....9 1.2 Utilization of NAD(P) as a cofactor for cell metabolism .................. 13 1.2.1 Glycolysis .............................................................................. 13 1.2.2 Pentose Phosphate Pathway ................................................ 14 1.2.3 TCA cycle and Electron Transport Chain .............................. 15 1.2.4 Fatty Acid Synthesis ............................................................. 17 1.2.5 Fatty Acid Oxidation .............................................................. 18 1.3 Utilization of NAD+ as an enzyme substrate .................................. 22 1.3.1 The Cyclic ADP-Ribose Synthase: CD38 ............................. 22 1.3.1.1 CD38 gene structure ............................................... 22 1.3.1.2 Enzymatic activity of CD38 ...................................... 23 1.3.1.3 Physiological processes regulated by CD38 ........... 28 1.3.1.4 CD38 in Cancer ....................................................... 31 1.3.1.5 CD38 in cell physiology and other diseases ............ 34 iii 1.3.2 Sirtuins .................................................................................. 40 1.3.2.1 Intracellular localization and function ...................... 40 1.3.2.2 Sirtuin/NAD+ Axis in cell metabolism ....................... 41 1.3.2.3 Sirtuins in Cancer .................................................... 43 1.3.3 Poly (ADP)-Ribose Polymerases .......................................... 47 1.3.3.1 PARPs localization and Function ............................ 47 1.3.3.2 PARPs in Cancer .................................................... 48 1.4 Prostate Cancer ............................................................................. 51 1.4.1 Current epidemiology ............................................................ 51 1.4.2 Development and progression of prostate cancer................. 52 1.4.3 Treatment options for localized and metastatic disease ....... 54 1.5 Overview ........................................................................................ 59 1.6 References .................................................................................... 60 CHAPTER II CD38 INHIBITS PROSTATE CANCER CELL METABOLISM AND PROLIFERATION BY REDUCING CELLULAR NAD+ POOLS 2.1 Abstract ........................................................................................ 114 2.2 Introduction .................................................................................. 115 2.3 Materials and Methods................................................................. 117 2.4 Results ......................................................................................... 128 2.5 Discussion ................................................................................... 138 2.6 Figures ......................................................................................... 143 2.7 References .................................................................................. 155 iv CHAPTER III CD38 MEDIATED CALCIUM MOBILIZATION PROTECTS PROSTATE CANCER CELLS FORM THE CYTOTOXIC EFFECTS OF β-LAPACHONE 3.1 Abstract ........................................................................................ 164 3.2 Introduction .................................................................................. 165 3.3 Materials and Methods................................................................. 167 3.4 Results ......................................................................................... 175 3.5 Discussion ................................................................................... 184 3.6 Figures ......................................................................................... 189 3.7 References .................................................................................. 203 CHAPTER IV GENERAL DISCUSSION 4.1 Targeting NAD+ in cancer ............................................................ 209 4.2 Future directions .......................................................................... 213 4.3 Overall impact and significance ................................................... 215 4.3 References .................................................................................. 217 Curriculum Vitae ..................................................................................... 220 v LIST OF FIGURES AND TABLES CHAPTER I Figure 1: Structure of Nicotinamide Adenine Dinucleotide (NAD+) ............6 Figure 2: De novo and salvage pathways of NAD+ synthesis ....................7 Figure 3: Metabolic pathways and enzymes that utilize NAD+ as a cofactor .................................................................................................................. 20 Figure 4: The cyclic ADP-ribose synthase CD38 is the primary NAD’ase in cells ………………………………………………………………………………….....38 Figure 5: Sirtuins consume NAD+ to facilitate lysine deacetylation……....45 Figure 6: Poly (ADP)-ribose polymerase’s require NAD+ to ADP-ribosylate target proteins…………………………………………………………………………..50 Figure 7: Development, progression, and treatment of PCa ................... ..58 CHAPTER II Figure 1: CD38 expression is inversely correlated with prostate cancer progression. ............................................................................................ 143 Figure 2: CD38 reduces total NAD+ and inhibits cell proliferation. ......... 144 Figure 3: ATRA induces expression of CD38 independent of changes to epigenetic regulation. ............................................................................. 146 Figure 4: CD38 reduces glycolytic and mitochondrial capacity. ............. 147 Figure 5: Pharmacological inhibition of NAMPT reduces glycolytic and mitochondrial capacity ............................................................................ 149 vi Figure 6: Expresion of CD38 activates AMPK and inhibits fatty acid and lipid synthesis ................................................................................................ 151 Figure 7: CD38 reprograms the transcriptome of PCa cells .................. 152 Table 1: CD38 does not alter metabolic pathway gene expression. ...... 154 CHAPTER III Figure 1: CD38 expression protects cells from β-Lapachone induced cell death ............................................................................................................... 189 Figure 2: Pharmacological inhibition of NAMPT sensitizes cells to the effects of β-Lapachone .......................................................................................... 191 Figure 3: CD38 mediated protection from β-Lapachone is independent of its NAD’ase activity ..................................................................................... 193 Figure 4: Expression of CD38 protects cells form β-Lapachone mediated loss of mitochondrial membrane potential .......................................................... 195 Figure 5: Pharmacological inhibition of NAMPT sensitizes cells to β-Lapachone mediated loss of mitochondrial membrane potential ............................... 197 Figure 6: CD38 mediated calcium mobilization confers protection from β- Lapachone ............................................................................................. 199 Figure 7: Punctate calcium staining associated with expression of CD38 localizes to mitochondria ........................................................................ 201 CHAPTER IV Figure 1: Correlation between expression of CD38 and BCR ............... 212 Figure 2: Key components controling NAD+ homeostasis ..................... 216 vii LIST OF ABBREVIATIONS α Alpha β Beta β-Lap β-Lapachone µM Micromole γ Gamma 1,3-BPG 1,3-biphosphoglycerate 5-ARI 5α-reductase inhibitor Å Angstrom ACC Acetyl-CoA carboxylase AceCoA1 Acetyl-CoA synthase 1 (cytosolic) AceCoA2 Acetyl-CoA synthase 2 (mitochondrial) ACO2 Aconitase 1 ACYL ATP citrate lyase ADPR ADP-ribose ADT Androgen deprivation therapy AIDS Acquired immunodeficiency syndrome Ala Alanine viii AMP Adenosine monophosphate AMPK AMP-activated protein