The Physiological Effects of CD38 in Prostate Cancer: a Multifunctional

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The Physiological Effects of CD38 in Prostate Cancer:

A multifunctional NAD’ase capable of regulating cell metabolism, gene expression, and therapeutic response

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.

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

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

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

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 kinase

AR-A Androgen receptor antagonist

ASD Autism spectrum disorder

ASI Androgen synthesis inhibitor

Asn Asparagine

ATP Adenosine triphosphate

ATRA All-trans retinoic acid

BCa Breast cancer

BCR Biochemical recurrence

BHP Benign prostatic hyperplasia

BRCA1/2 breast cancer susceptibility gene 1/2

Ca2+ Calcium cADPR Cyclic ADP-ribose

Cam Calmodulin

CCL Chronic lymphocytic leukemia

CD38 Cluster of differentiation 38 (cyclic (ADP)-ribose synthase)

CD38-/- CD38 knockout

Cdk2 Cyclin dependent kinase 2

Cdk4/6 Cyclin dependent kinase 4/6

CDKN1A Cyclin dependent kinase 1A (p21Cip1/Waf1)

CICR Calcium induced calcium release

CO2 Carbon dioxide

CoA Coenzyme A

Complex 1 NADH : Ubiquinone oxidoreductase

CRC Colorectal cancer

CRPC Castration resistant prostate cancer

Cys Cystine

DH Dehydratase

DHT Dihydrotestosterone

DNA Deoxyribonucleic acid dsDNA Double stranded DNA

ER Endoplasmic reticulum

Er Enoyl reductase

Erk1/2 Extracellular signaling regulated kinase 1/2

ETC Electron transport chain

ETF Electron transfer flavoprotein

FAD Flavin adenine dinucleotide

FAO Fatty acid oxidation

FAs Fatty acids

FAS Fatty acid synthesis

FASN Fatty acid synthase

FDR False discovery rate

FKBP12.6 FK506 binding protein 12.6

FOXO3a Forkhead 3a

G6P Glucose-6-phosphate

Ga3P Glyceraldehyde-3-phosphate

GaCa Gastric cancer

GAPDH Glyceraldehyde-3-phosphate dehydrogenase

GDP Guanosine-5'-diphosphate

Gln Glutamine

Glu Glutamic acid

GnRH Gondatropin-releasing hormone

GnRH-A Gondatropin-releasing hormone agonists

GnRH-At Gondatropin-releasing hormone antagonists

GSIS Glucose stimulate insulin secretion

Gy Grey - unit of radiation

H+ Hydrogen

H2O Water

HAART Highly active anti-retroviral therapy

HIF-1α Hypoxia inducible factor - 1α

HIV Human immunodeficiency virus

HMG-R hydroxymethylglutaryl-CoA reductase

IDH1 Isocitrate dehydrogenase 1 (mitochondrial)

IDH2 Isocitrate dehydrogenase 2 (cytosolic)

IDH3 Isocitrate dehydrogenase 3 (mitochondrial)

IDO Indoleamine 2,3-dioxygenase

IP3 Inositol 1,4,5-triphosphate

Ka kynurenic acid

Kb Kilobase

Kd Dissociation constant kg Kilogram

xii

Km Michaelis constant : concentration of substrate at half maximum

velocity

KR Ketoreductase

KS Ketoacyl synthase

LCAT Acyl-CoA acetyltransferase

LCEH Long-chain trans-∆2-enoyl-CoA hydratase

LCHAD Long-chaing β-hydroxyacyl-CoA dehydrogenase

LCIS Live cell imaging solution

LDH Lactate dehydrogenase

LnCa Lung cancer mAB Monoclonal antibody

MAT malonyl/acetyl transferase mCRPR Metastatic castration resistant prostate cancer

MM Multiple myeloma

MMS Methyl methanesulfonate

MTP Mitochondrial trifunctional protein

NA Nicotinic acid

NAADP Nicotinic acid adenine diphosphate

xiii

NAD Nicotinamide adenine dinucleotide

NADK Nicotinamide adenine dinucleotide kinase

NADSyn Nicotinamide adenine dinucleotide Synthetase

Nam Nicotinamide

NaMN Nicotinic acid mononucleotide

NAMPT Nicotinamide phosphoribosyl transferase

NAPRT Nicotinic acid phosphoribosyltransferase

NIDDM Non-insulin-dependent diabetes mellitus

NMN Nicotinamide mononucleotide

NMNAT Nicotinamide mononucleotide adenylyltransferase

NQO1 NAD(P)H: quinone oxidoreductase 1

NR Nicotinamide riboside

NRK Nicotinamide riboside kinase

NSCLC Non-small cell lung cancer

OvCa Ovarian cancer

OT Oxytocin

PaCa Pancreatic cancer

PARP poly (ADP)-ribose polymerase

xiv

PARPi poly (ADP)-ribose polymerase inhibitor

PBEF pre-B-cell colony enhancing factor

PCa Prostate cancer

PDH Pyruvate dehydrogenase

PDK1 Pyruvate dehydrogenase kinase 1

PGC-1α Peroxisome proliferator-activated receptor gamma coactivator 1α

PI Propidium Iodide

PI3K Phosphatidylinositol 3-kinase

PIN Prostatic interepithelial neoplasia

PK Pyruvate kinase

PKA Protein Kinase A

PKB/Akt Protein kinase β / Akt

PLC Phospholipase C

PPARγ Peroxisome proliferator activated receptor γ

PPP Pentose phosphate pathway

PRPP Phosphoribosyl pyrophosphate

PSA Prostate specific antigen

PTM Post translational modification

Qa quinolinic acid

QPRT Quinolinic acid phosphoribosyl transferase

RARE Retinoic acid response element

Ribose -5P Ribose-5-Phosphase

Ribulose-5P Ribulose-5-phosphate

RNA Ribonucleic acid

ROS Reactive oxygen species

RyR Ryanodine receptor

SCLC Small cell lung cancer

Sirt1-7 Silent information regulator 1-7

SLC22A3 Solute carrie family 22 member 3

SNP Single nucleotide polymorphism

SOD Superoxide dismutase

SOD2 Superoxide dismutase 2

SSB Single strand break ssDNA Single strand DNA

TCA Tricarboxylic acid

TDO tryptophan 2,3-dioxygenase

xvi

TH Thioesterase

TNBC Triple negative breast cancer

TPC1-3 Two-pore channel 1-3

Trp Tryptophan

Vmax Maximum velocity of enzymatic reaction

xvii

ABSTRACT

Deregulation of cellular metabolism is a critical step during oncogenesis.

Tumor cells rely heavily on changes to cell metabolism to support increased macromolecule biosynthesis, sustain membrane biogenesis, and cell survival.

Nicotinamide adenine dinucleotide (NAD+) is linked all aspects of cell metabolism though its ability to act as both an oxidizing and reducing agent for hydride transfer reactions. Proper regulation of NAD+ is also vital to cells as it also serves as a substrate for three classes of NAD+ dependent enzymes: cyclic ADP-ribose synthases, sirtuins, and poly (ADP)-ribose polymerases (PARPs). The cyclic

(ADP)-ribose synthase, CD38, is the primary NAD’ase in cells and has historically been regarded as an important cell surface molecule in cells of hematopoietic lineage. The work presented herein is focused on the role of CD38 in regulating prostate cancer (PCa) cell metabolism and therapeutic response.

Prostate specific silencing of CD38 is inversely correlated with prostate cancer progression and results in increased availability of NAD+. Increased NAD+ resulting from CD38 silencing contributes to changes in the metabolic potential of

PCa cells by elevating glycolytic and mitochondrial capacity and increasing fatty acid and lipid synthesis. The presence of multiple CpG islands in the promoter of

CD38 is the proposed mechanism of CD38 silencing in PCa. Accordingly, we demonstrate treatment with the demethylating agent azacytidine is sufficient to induce CD38 mRNA levels. Our data also demonstrates treatment with all-trans retinoic acid (ATRA) is a viable strategy to rescue expression of CD38 independent of changes in epigenetic regulation. Interestingly, modulation of

xviii

NAD+ levels by CD38 results in differential expression to more than 75% of the transcriptome generating a gene expression pattern consistent with a non- proliferative phenotype. Our data establish CD38 has effects on PCa cell biology independent of its NAD’ase activity as well. As the primary consumer of NAD+, most of the activity of CD38 is devoted to production of ADP-ribose. CD38 also possess cyclase activity and is therefore able to produce cyclic ADP-ribose, a potent calcium mobilizing second messenger. Herein we demonstrate production cADPR by CD38 is active in PCa cell lines and its production is effective in eliciting changes to calcium localization. Changes in calcium localization confer protection from β-lapachone, an ortho naphthoquinone that is cytotoxic to cells when activated by NAD(P)H quinone oxidoreductase 1 (NQO1). Cumulatively, these data establish a novel connection between CD38, modulation of NAD+, and prostate tumor cell metabolism, energy homeostasis, and cell survival.

xix

CHAPTER I

GENERAL INTRODUCTION

Jeffrey P. Chmielewski and Steven J. Kridel

J. Chmielewski prepared the text and S. Kridel provided advisory and editorial assistance.

1.1 Nicotinamide Adenine Dinucleotide (NAD+) Biology

1.1.1 Historical perspective

Nicotinamide adenine dinucleotide (NAD+) is a ubiquitous molecule that functions as a cofactor or substrate for different classes of enzymes that regulate a diverse range of cellular processes. As a cofactor, NAD+ acts as both an oxidizing and reducing agent by enzymes in metabolic pathways and a multitude of other processes throughout the cell. As a substrate, three different classes of

NAD+ dependent enzymes consume NAD+: cyclic (ADP)-ribose synthetases

(CD38/CD157), sirtuins (SIRT1-7), and poly (ADP)-ribose polymerases (PARPs).

A molecule of NAD+ consists of two nucleotides, one containing an adenine base and the other nicotinamide, joined together by phosphodiester bonds (Figure 1). Sir Arthur Harden and William Young, characterized NAD+ in

1904. The pair identified a “cozymase” isolated from yeast extracts as an essential component for fermentation (1). Noble laureate Hans Von Euler-

Chelpin later identified “cozymase” as a nucleoside sugar phosphate. In 1936,

Otto Warburg attributed the ability of “cozymase” to transfer hydrogen atoms between molecules to the presence of pyridine moieties (2). Warburg is also credited with characterizing the phosphorylated form of NAD+, NADP+, which he dubbed cozymase II. Also during this period, Arthur Kornberg accurately described the synthesis of NAD+ as a series of reactions that involved the condensation of ATP with nicotinamide mononucleotide (NMN) (3-5). The work of

Drs. Friedkin and Lehninger positioned NAD+ as an essential cofactor for the tricarboxylic acid (TCA) cycle and oxidative phosphorylation in the ETC (6-8). In

2 a final series of papers published over two years, Drs. Preiss and Handler identified the complement of intermediates and enzymes that constitute the salvage pathway of NAD+ synthesis (9-12). The innovative work of these seven authors over the course of 55 years shaped the direction and understanding of cell metabolism and the many diverse roles of NAD+.

1.1.2 NAD+ biosynthesis: De novo and salvage pathways

There are two primary pathways by which NAD+ can be synthesized: the de novo and salvage pathways (1,3-5). De Novo NAD+ synthesis is a multi-step process that converts L-tryptophan into quinolinic acid (Qa) through a series of six reactions referred to as the kynurenine pathway (Figure 2A) (13). Tryptophan catabolism mainly occurs in the liver as tryptophan 2,3-dioxygenase (TDO), the rate-limiting enzyme of this pathway, is mainly expressed in this organ (14). TDO catalyzes the conversion of tryptophan into N-formylkynurenine (N-Fk) initiating de novo synthesis (15). Cells of the immune system, mainly macrophages and T cells, are also able to initiate the kynurenine pathway although this step is catalyzed by indoleamine 2,3-dioxygenase (IDO) (14,16). Following the formation of N-Fk, kynurenine formamidase and kynurenine 3-monooxygenase catalyze the formation of kynurenine and then 3-hydroxylkynurenine (3-HK), respectively

(17,18). Next, 3-HK is converted into 3-hydroxyanthranilic acid by kynureninase, which is subsequently converted into Qa by 3-hydroxyanthranilate-3,4- dioxygenase (19). In the final step of the de novo pathway, quinolinic acid phosphoribosyl transferase (QPRT) decarboxylates phosphoribosyl pyrophosphate (PRPP) transferring a ribosyl-phosphate group to Qa producing

3 nicotinic acid mononucleotide (NaMN) (15). It is important to note that production of NaMN represents a convergence of the de novo synthesis pathway and the

Preiss-Handler salvage pathway (discussed below). Similar to TDO, QPRT is expressed exclusively in the liver (14). Both enzymes are rate limiting and their colocalization to the liver confirms this organ as the principal site of de novo synthesis in mammals.

While the de novo pathway is a complex series of reactions, the salvage pathway represents a more direct means of synthesizing NAD+ from one of three precursors: nicotinic acid (Na), nicotinamide riboside (NR), or nicotinamide (Nam)

(Figure 2B). Salvage of NAD+ from Na is refered to as the Preiss-Handler pathway. In this pathway, nicotinic acid phosphoribosyltransferase (NAPRT) hydrolyzes ATP to facilitate the transfer of the phospho-ribosyl group from PRPP to Na producing NaMN (20). An additional ATP hydrolysis reaction catalyzed by nicotinamide mononucleotide adenylyltransferase (NNMAT) adds an adenosine monophosphate (AMP) moiety from ATP onto NaMN to produce nicotinic acid adenine dinucleotide (NAAD) (21-23). Lastly, nicotinamide adenine dinucleotide synthetase (NADSyn) uses a third molecule of ATP to facilitate the transamination of glutamine to NAAD forming NAD+ (10-12). NAD+ can also be salvaged from NR. In this pathway, NR is phosphorylated by nicotinamide riboside kinase (NRK) to produce nicotinamide mononucleotide (NMN) (14).

NMNAT then hydrolyzes ATP transferring an AMP onto MNM forming NAD+ (24).

The final variation of the salvage pathway consists of a similar two-step process utilizing Nam as substrate instead of NR. In the first and rate-limiting reaction,

4 nicotinamide phosphoribosyltransferase (NAMPT) converts Nam into NMN using

PRPP as a co-substrate (25,26). The second reaction uses NMNAT to convert

NMN into NAD+ as previously described. This two-step reaction represents the predominant mechanism of NAD+ synthesis in most cell types as Nam is present at much higher concentrations relative to NR and Na. Nam can be derived from diet by consuming fortified packaged foods or in the form of vitamin B3 (Na and

Nam), a common component of daily multi-vitamins. Intracellular Nam, maintained at ~10µM, is also replenished, as it is the byproduct of reactions catalyzed by NAD+ consuming enzymes: cyclic (ADP)-ribose synthase

CD38/CD157, sirtuins, and poly (ADP)-ribose polymerases (PARPs) discussed further in sections 1.3.1, 1.3.2, and 1.3.3, respectively (27-29).

NAD+ is used exclusively to support hydride transfer activity in catabolic reactions while the phosphorylated form, NADP+, when reduced to NADPH facilitates anabolic reactions. Phosphorylation of NAD+ is catalyzed by NAD+

Kinase (NADK), which requires ATP and divalent cations like magnesium, zinc, or calcium to exert its activity (30). Tissues with high-energy expenditures or metabolic activity like skeletal muscle, cardiac tissue, and liver have NAD+ concentrations of ~500 micro molar (µM). Most other tissues have slightly lower levels that hover ~300µM (15,31,32). Because NAD+ is unable to diffuse across cell membranes, it is present in distinct pools corresponding to the localization of

NAMPT and NMNAT (33,34). NAD+ concentrations are highest in the cytosol

(300µM) followed by the mitochondria (250µM) and nucleus (70µM) (35-37).

NADP is found at much lower concentrations averaging ~35µM in the cytosol

(38).

Figure 1: Structure of Nicotinamide Adenine Dinucleotide (NAD+). NAD+ consists of two nucleotides, one containing nicotinamide the other adenine, joined together by their phosphate groups.

Figure 2: De novo and salvage pathways of NAD+ synthesis. NAD+ synthesis via the de novo or salvage pathway requires multiple enzymes (red) that produce intermediates (black) that coalesce into NAD+. De novo NAD+ synthesis occurs primarily in the liver and requires a series of reactions that convert tryptophan to quinolinic acid via the kynurenine pathway. Synthesis of nicotinic acid mononucleotide (NaMN) from quinolinic acid (Qa) connects de novo synthesis to the Preiss-Handler salvage pathway. The salvage of NAD+ can proceed from nicotinic acid (Na), nicotinamide riboside (NR), or nicotinamide (Nam) and NAD

Kinase (NADK) can phosphorylate NAD+ to NADP+. IDO: indoleamine 2,3- dioxygenase, TDO: tryptophan 2,3-dioxygenase, ATP: Adenine Triphosphate,

ADP: Adenine Diphosphate, NaPRT: Nicotinic acid phosphoribosyltransferase,

NMNAT: Nicotinamide adenine mononucleotide adenyltransferase, NRK:

Nicotinamide riboside kinase, NAMPT: Nicotinamide phosphoribosyltransferase,

NADSyn: Nicotinamide adenine dinucleotide synthase, NADK: Nicotinamide adenine dinucleotide kinase, NAD+: Nicotinamide adenine dinucleotide

(oxidized), NADP+: Phosphorylated nicotinamide adenine dinucleotide (oxidized).

Mg2+: Magnesium, Ca2+: Calcium, Zn2+: Zinc.

1.1.3 Nicotinamide phosphoribosyltransferase (NAMPT) biology

NAMPT primarily functions as a type II phosphoribosyltransferase but has been reported to act as a cytokine as well (pre-B-cell colony enhancing factor:

PBEF) (39,40). NAMPT is the rate-limiting enzyme of the salvage pathway catalyzing the formation of NMN from Nam. NAMPT expression is found in almost all tissue types indicating most cells rely on salvage of Nam rather than

Na or NR for NAD+ biosynthesis (41). The NAD+ salvage pathway is indispensable for mammalian development as NAMPT knockout mice die within two weeks of gestation (42). Mice heterozygous for NAMPT are viable and develop normally despite having significantly reduced NAD+ levels in all tissues except the liver where de novo synthesis via the kynurenine pathway compensates for reduced NAD+ salvage (26,42,43). Mice heterozygous for

NAMPT also experience impaired glucose tolerance and have defects in glucose-stimulated insulin secretion (GSIS), both of which are rescued by the addition of exogenous NMN (42). Intracellular NAMPT is localized to the cytoplasm, nucleus, and mitochondria where it synthesizes distinct pools of NAD+ to support metabolic reactions and signaling activities that occur at these sites

(44,45). Most notably, by modulating cytosolic NAD+ availability, NAMPT expression is able to directly influence the activity of sirtuins and PARPs (46,47).

The multiple nodes controlled by NAMPT highlight the importance of NAD+ in maintaining cellular homeostasis and underly its importance for development and viability. It is for these reasons dysregulation of NAMPT expression is implicated in a variety of disease states.

Clinically, changes in NAMPT expression or activity are linked to the development of type II diabetes mellitus, through its ability to regulate GSIS, chronic inflammation, obesity, and multiple types of cancer (48-53). In cancer cells, many oncogenic signaling pathways regulate NAMPT expression and thus overexpression has been documented in multiple cancer types, including colorectal and ovarian carcinomas, astrocytomas, and melanoma (54-57).

Transcriptional regulation of NAMPT in cancer is controlled at multiple nodes.

Expression can be stimulated through canonical cAMP signaling, expression of nuclear receptor coactivator 6, and through pro-inflammatory cytokines such as tumor necrosis factor alpha (TNF-α), interleukin 1 beta, and interferon gamma

(48,58). Constitutive activation of c-Myc, through a positive feedback loop, is a common occurrence in colorectal cancer (CRC) and able to drive expression of

NAMPT (59). Multiple aspects of NAMPT overexpression is advantageous to cancer cells. For example, NAMPT activity is correlated with increased Notch1 signaling and activation of the tyrosine kinase C-Abl, which collectively contribute to increased cell proliferation (60,61). NAMPT also promotes tumorigenesis by blocking TNF-α induced apoptosis through regulation of the protein kinase B/Akt

(PKB/Akt) pathway and phosphorylation of extracelluar signaling-regulated kinase 1/2 (62). NAMPT overexpression mitigates the toxic effects of reactive oxygen species (ROS) that can accumulate in tumor cells because of increased metabolic activity (63-65). For instance, in breast cancer (BCa) cell lines, excess

NAD+ can be shunted into the pentose phosphate pathway (PPP) to produce

NADPH which increases the ratio of reduced to oxidized glutathione, allowing the

10 cell to neutralize more ROS (66). Specifically, resistance to oxidative stress is observed in prostate cancer (PCa) where NAMPT expression stimulates SIRT1 activity which decreases forkhead 3a (FOXO3a) mediated repression of catalase and superoxide dismutase, two critical components of the ROS defense system

(67). The reliance of cancer cells on NAD+ to sustain metabolic activity and maintain a pro-oncogenic signaling landscape highlights the importance of

NAMPT and presents an opportunity to exploit this vulnerability through the development of anti-cancer therapies.

Several small molecule inhibitors of NAMPT have been developed and evaluated for the treatment of multiple cancer types. The most well characterized and clinically relevant of these inhibitors is FK866 (Topotarget; also known as

APO866 (Apoxis)), which binds to the active site of NAMPT (68-70). Additional inhibitors of clinical significance include CHS-828 (GMX-1778), Gemin X (GMX-

1777), CB30865, and GNE-617/8 (71-74). In vitro studies of these inhibitors demonstrate short and long-term effects ranging from a rapid decline of intracellular NAD+ and reduced nucleotide metabolism in the first 12 hours to inhibition of glycolysis, oxidative phosphorylation, and ATP synthesis at 24 hours

(75-77). Longer treatments of 36-48 hours induce apoptotic cell death stemming

+ from an almost complete elimination of NAD and ATP (78-80). These effects have been validated in a variety of tumor cell lines including prostate, non-small cell lung, renal, gastric, breast, pancreatic, and hematological cancers (79,81-

86). Unfortunately, multiple phase II clinical trials have concluded NAMPT inhibitors exhibit limited efficacy when used as a single agent (72,87). The failure

11 of NAMPT inhibitors to achieve objective responses is in part related to the development of latent thrombocytopenia, identified as the dose limiting toxicity, even at relatively low doses (70). The heterogenous nature of tumors may also be responsible for the lack of clinical efficacy. For instance, serial passage of

CRC cancer cells with low concentrations of NAMPT inhibitors results in the outgrowth of cells with inherent drug resistance. Non-conservative missense mutations in the NAMPT coding region that corresponds to the active site of the protein causes amino acid changes at histidine 191 (H191R), glutamine 338

(Q338R), and glycine 217 (G217R) conferring resistance to FK866, CHS-282, and GMX1778, respectively (78,88). Given these obstacles, combination therapies are now being explored as a way to increase the effectiveness of these drugs. For example, one study is aimed at determining if tumor specific downregulation of NAPRT can be exploited to protect normal cells from the deleterious effects of NAMPT inhibition. To accomplish this, exogenous NR is supplied in conjunction with NAMPT inhibitors in the hopes that normal cells can utilize the secondary salvage pathway to maintain sufficient NAD levels (89).

Administering low dose radiation to induce DNA damage is also being explored as a way to sensitize tumor cells to NAMPT inhibitors (90).

In summary, NAMPT activity is essential for proper metabolic signaling, development, and viability in human cells. Dysregulation of NAMPT is a common occurance in multiple cancer types providing a competitive advantage to these cells by inhibiting pro-apoptotic signaling pathways and supporting the ROS defense system. The clinical relevance of NAMPT inhibitors remains to be seen

12 despite an accumulation of in vitro data that supports their effectiveness in selectively targeting tumor cells.

1.2 Utilization of NAD(P) as a cofactor for cell metabolism

Otto Warburg was the first person to understand that cancer cells reprogram their metabolic processes in favor of increased macromolecule biosynthesis. Warburg demonstrated cancer cells have a propensity to elevate glycolysis by shuttling pyruvate into lactate production, sacrificing more advantageous ATP production via oxidative phosphorylation, despite the presences of sufficient oxygen (91). Named in his honor, the “Warburg Effect” is only a small facet of the many metabolic alterations characteristic of cancer cells.

As such, deregulated cellular energetics represents one of the ten hallmarks of cancer (92,93). Remarkably, despite the vast and complex network of metabolic alterations that constitute tumor cell metabolism, these cells cannot evade their reliance for NAD+. NAD+ is an essential cofactor for enzymes in all major metabolic pathways including glycolysis, the PPP, TCA cycle, the electron transport chain (ETC), fatty acid synthesis (FAS), and fatty acid oxidation (FAO)

(94,95). The following section will detail the use of NAD+ by enzymes from each of the main metabolic pathways (Figure 3A).

1.2.1 Glycolysis

The breakdown of glucose into pyruvate during glycolysis represents the most fundamental cell metabolism pathway. Metabolites from this pathway can serve as precursors almost every other known metabolic process. The complete

13 breakdown of glucose is a nine-step process that requires two molecules of ATP and yields the production of two net molecules of ATP and two molecules of pyruvate. The first committed step of glycolysis involves the conversion of glucose into glucose-6-phosphate (G6P) through the action of hexokinase. Three subsequent reactions produce two molecules of glyceraldehyde-3-phosphate

(G3P). The fifth reaction of glycolysis is catalyzed by glyceraldehyde-3- phosphate dehydrogenase (GAPDH) and requires two molecules of NAD+, one for each molecule of G3P. In this reaction, when bound to GAPDH, the terminal aldehyde group of G3P is oxidized by transferring its hydrogen atom onto NAD+, reducing it to NADH allowing for its release. A second molecule of NAD+ quickly binds to stabilize the carbonyl oxygen, allowing a molecule of inorganic phosphate to bind the G3P intermediate, thereby producing 1,3- bisphosphglycerate (1,3-BPG) (96). Four subsequent reactions are needed to produce pyruvate, which is then shuttled into the mitochondria to sustain the TCA cycle or, if there is insufficient oxygen, converted into lactate. Conversion of pyruvate into lactate is catalyzed by lactate dehydrogenase (LDH) and requires

NADH be oxidized to supply a hydrogen atom to reduce the carbonyl oxygen on the acetyl group (97,98).

1.2.2 Pentose Phosphate Pathway

The PPP utilizes G6P derived from the breakdown of glucose via hexokinase as substrate to synthesize NADPH and several 5-carbon sugars that support ribonucleotide biosynthesis. The PPP diverges into two distinct branches: the oxidative branch and non-oxidative branch. In the oxidative

14 branch, each molecule of G6P processed ultimately reduces two molecules of

NADP+ to NADPH and produces ribulose-5-phosphate (Ribulous-5P) through a series of three irreversible reactions (99). The production of NADPH from the

PPP is critical for cancer cells as NADPH is necessary to support fatty acid synthesis and protect against oxidative stress (99,100). The non-oxidative pathway is also necessary for cancer cells as it produces ribose 5-phosphate

(Ribose-5P); a precursor for ribonucleic acid (RNA) and deoxyribonucleic acid

(DNA) synthesis which are necessary to sustain proliferation (101,102). Subsets of cancer cells support the flow of metabolites into the PPP by controlling expression of specific isoforms of pyruvate kinase (PK), the enzyme responsible for catalyzing the final step of glycolysis. Two prominent isoforms of PK are present in mammalian cells: PKM1 and PKM2. PKM1 is principally expressed in normal cells while PKM2 expression, indirectly driven by MYC, is expressed in cancer cells (103). Expression of PKM2 is beneficial to tumor cells as it favors aerobic glycolysis and can slow glycolysis allowing glycolytic intermediates to flow into other pathways, such as G6P into the PPP (104). If the PPP becomes saturated and Ribose-5P is not needed it can re-enter glycolysis in form of G6P or fructose-6-phosphate (99).

1.2.3 TCA cycle and Electron Transport Chain

The TCA cycle and ETC are critical components of cell metabolism, generating metabolite intermediates that serve as precursors for other metabolic pathways and generating the bulk of ATP produced in the cell. The TCA cycle and ETC are connected to glycolysis through the production and use of pyruvate.

Pyruvate produced in the cytosol is transported into the mitochondria and subsequently converted to acetyl-CoA to fuel the TCA cycle. Oxidative decarboxylation of pyruvate to acetyl-CoA is catalyzed by pyruvate dehydrogenase (PDH) and requires the reduction of NAD+ to NADH (105). The mitochondrial membrane is impervious to cytosolic NAD(H) and no known transporters exist in mammalian cells. However, the electrons of NADH can be transported across the membrane through the actions of the malate and aspartate shuttles (Figure 3B) (106,107). In addition to PDH, NAD+ is required for three reactions of the TCA cycle and NADH is required in the ETC. As acetyl-

CoA enters the TCA cycle it is converted into citrate and then into isocitrate by the actions of citrate synthase and aconitase, respectively. The next step of the

TCA cycle is catalyzed by isocitrate dehydrogenase and involves the oxidative decarboxylation of isocitrate to α-ketoglutarate + CO2 (108). Three isoforms of

IDH are present in cells. IDH2 and IDH3 catalyze the oxidative decarboxylation of isocitrate in the mitochondria using NADP+ and NAD+ to oxidize isocitrate, respectively (109). IDH1 performs this reaction in the cytosol using NADP+ (110).

The α-ketoglutarate produced from IDH2/3 activity is converted into succinyl-CoA by the α-ketoglutarate dehydrogenase complex which reduces an additional molecule of NAD+ (111). The final reaction that requires NAD+ is the conversion of malate into oxaloacetate catalyzed by malate dehydrogenase (112). The

NADH derived from these reactions is oxidized by complex I (NADH: ubiquinone oxidoreductase) of the ETC and electrons passed down the chain allowing hydrogen to be pumped across the inner mitochondrial membrane generating the

16 proton gradient required by F0F1- ATP synthase to initiate production of ATP

(Figure 3C) (113).

1.2.4 Fatty acid synthesis

NADPH produced in the PPP pathway is critical for the production of fatty acids and lipids. In order to support rapid proliferation and the need for increased membrane biogenesis, many tumor cells, including prostate, develop a highly lipogenic phenotype (114). FAs serve as the building blocks for phospholipids and glycolipids that constitute cell membranes and can be covalently attached to proteins to modify their function (115,116). FAs can also be esterified with glycerol backbones, packaged into energy dense triacylglycerol, and stored in lipid droplets for later use via β-oxidation (117). Accordingly, tumor cells often overexpress fatty acid synthase (FASN) in order to increase their capacity to synthesize de novo fatty acids (FAs) and promote tumorigenesis (114,118).

FASN synthesizes palmitate, a 16-carbon saturated fatty acid, by catalyzing seven successive rounds of condensation reactions transferring 2-carbon units from malonyl-CoA onto acetyl-CoA. The functional form of FASN is a homodimer with seven distinct enzymatic domains that function sequentially to transfer carbons from malonyl-CoA into acetyl-CoA. Listed in order of their utilization, the seven domains include malonyl/acetyl transferase (MAT), acyl carrier protein

(ACP), ketoacyl synthase (KS), ketoreductase (KR), dehydratase (DH), enoyl reductase (ER), and thioesterase (TH) (119). The last two enzymatic domains,

KR and ER, each require NADPH to facilitate their enzymatic activity. These two domains work consecutively to fully reduce the double bonded oxygen attached

17 to the carbonyl carbon on the acetoacetyl-ACP intermediate, ultimately releasing water. The equation for the full reaction is summarized as follows: acetyl-CoA +

+ (7) malonyl-CoA + (14) NADPH + (14) H  Palmitate + (7) CO2 + (8) CoA + (14)

+ NADP + (6) H2O (120).

Inhibiting FASN has emerged as a potential therapeutic strategy to target tumor cells. This is because normal cells, even those with relatively high rates of proliferation, preferentially take up circulating FAs derived from diet and therefore rely very little on de novo FAS (121). Previous work in our lab has shown that modulating NAD+ levels through NAMPT blockade is sufficient to reduce fatty acid synthesis in PCa cells. An additional discussion of how modulating NAD+ levels can influence FAS can be found in Chapter II.

1.2.5 Fatty acid oxidation

During periods of limited substrate availability cells maintain the flow of acetyl-CoA into the mitochondria by shifting production from glycolysis to fatty acid oxidation (FAO). FAO, most commonly β-oxidation in mammalian cells, occurs in the mitochondrial matrix. Fatty acids are transported into the mitochondria through an orchestrated series of steps after they are converted to fatty acyl-carnitines (Figure 3D) (122). After its import into the mitochondria, acyl-

CoA undergoes successive rounds of oxidation, shortening the acyl-CoA chain by two carbons each cycle, producing acetyl-CoA to fuel the TCA cycle.

Oxidation of long chain acyl-CoA is mediated by the mitochondrial trifunctional protein (MTP) (123). The MTP contains three catalytic domains, long-chain trans-

∆2-enoyl-CoA hydratase (LCEH), long-chain β-hydroxyacyl-CoA dehydrogenase

(LCHAD), and acyl-CoA acetyltransferase (LCAT) commonly referred to as thiolase (124). MTP directly associates with phospholipids of the inner mitochondrial membrane placing it in close proximity to long chain acyl-CoA’s as they are transported into the mitochondria by carnitine palmitoyltransferase II

(125). FAO is a four-step process that begins when the acyl-CoA chain is oxidized by acetyl-CoA dehydrogenase producing trans-∆2-enoyl-CoA and reducing flavin adenin dinucleotide (FAD) to FADH2. FADH2 is then oxidized by electron transfer flavoprotein (ETF) and the hydrogens donated to coenzyme Q in the ETC (123). The LCHAD domain of the MTP, in the presence of water, then hydrolyzes trans-∆2-enoyl-CoA to β-hydroxylacyl-CoA, which is subsequently converted to ketoacyl-CoA by the LCHAD domain reducing NAD+ in the process

(123). NADH produced in this step is used by complex one of the ETC. Lastly, the thiolase domain cleaves the 2-carbon unit from acyl-CoA producing acetyl-

CoA allowing the shortened acyl-CoA to enter the cycle again. Domains of the

MTP typically only catalyze the first 2-3 rounds of oxidation, as there are homologues of each enzymatic domain that exhibit greater affinity for medium and short chain acyl-CoAs. These proteins also localize to the inner mitochondrial matrix where medium and short chain acyl-CoAs are most likely to aggregate (126).

Figure 3: Metabolic pathways and enzymes that utilize NAD+ as a cofactor.

NAD+ and NADP+ act as cofactors to facilitate hydride transfer reactions in key metabolic pathways, including glycolysis, pentose phosphate pathway (PPP), tricarboxylic acid (TCA) cycle, electron transport chain (ETC), fatty acid synthesis

(FAS), and fatty acid oxidation (FAO). A, Electrons from NADH produced in the cytosol can be transferred into the mitochondria via the malate – asparate shuttle to reduce NAD+. B, Electrons donated from NADH are first passed by complex I of the ETC across the inner mitochondrial membrane and used to pump hydrogens into the intermembrane space creating the proton gradient used to produce ATP. C, Fatty acids derived from diet or storage molecules such as triacylglycerol (TAG) can be converted into acyl-CoA and transported into the

20 mitochondria. Acyl-CoA will subsequently undergo beta-oxidation to produce acetyl-CoA to fuel the TCA cycle. ETC – electron transport chain, GAPDH –

Glyceraldehyde phosphate dehydrogenase, LDH – Lactate dehydrogenase,

Ribulous-5P: Ribulouse-5-phospate, Ribose-5P: Ribose-5-phosphate, RNA:

Ribonucleic acid, DNA: Deoxyribonucleic acid, TAG: Triacylglycerol, FAs: Fatty acids, FASN: Fatty acid synthase, PDH: Pyruvate dehydrogenase, IDH2/3:

Isocitrate dehydrogenase 2/3, α-KDH: alpha ketoglutarate dehydrogenase, MDH:

Malate dehydrogenase, MTP: Mitochondrial trifunctional protein, LCHD: long- chain β-hydroxyacyl-CoA dehydrogenase.

1.3 Utilization of NAD+ as an enzyme substrate

While NAD+ plays a critical role as a cofactor in cell metabolism, this versatile molecule has an equally important role as substrate for NAD+ dependent enzymes. As previously mentioned, the three classes of NAD+ dependent enzymes include cyclic ADP-ribose synthases, sirtuins, and PARPs.

The following sections will highlight the ways in which NAD+ is used by these enzymes, the many biological processes they regulate, and their connection to cancer biology.

1.3.1 The cyclic ADP-ribosyl synthase: CD38

1.3.1.1 CD38 gene structure

Cyclic ADP-ribose synthases are a family of type-II transmembrane glycoproteins that consist of cluster of differentiation 38 (CD38) and its homologue CD157 (BST-1). The CD38 gene is located on chromosome 4p15.32 and contains eight exons that span 76 kilobases (kb) (127,128). The 5’-region upstream of the promoter contains multiple transcriptional start points but does not contain a traditional TATA box (128). The promotor does however contain multiple areas that serve as putative binding sites for response elements and transcription factors, including an estrogen response element binding site, a binding site for nuclear factor interleukin-6, and an interferon response element binding site (Figure 4A) (129,130). Exon1 is the largest of the eight exons and contains the 5’- untranslated region, transcriptional start site, and the coding region for the transmembrane domain and a portion of the N-terminal domain

(128,131). The large expanse of the gene is due in part to a ~40kb intron that

22 separates exon 1 from exon 2 as the remainder of the exons (2-8) are contained within a 36kb region (132). In proximity to the 5’-end of intron 1 are binding sites for peroxisome proliferator activated receptor γ (PPARγ) and a binding site for the transcription factor E2A (133,134). CD38 also contains a series of CpG islands that span exon 1 and intron 1, suggesting epigenetic regulation may be one method of transcriptional regulation (128,135). In addition, present within this segment of the gene is a retinoic acid response element (RARE) (132,136). The eight exons of CD38 encode 300 amino acids that make up the 38 kilodalton (Kd) protein (Figure 4B). Crystal structure analysis demonstrates the functional protein organizes into a dimer assembling into an “L” shape, wherein the N- terminal and C-terminal domains form the two appendages linked together by a three peptide hinge (137). The N-terminal domain contains multiple α helices (1-

7), two β strands, and is encoded by amino acids 45-118 and 144-200, respectively (137). The C-terminal domain is similar in size at 123 amino acids and consists of a four-stranded parallel β sheet flanked by four α helices (137).

The two domains are stabilized along the three residue hinge by five pairs of disulphide bonds between ten cysteine residues (137). The catalytic site of the protein is centered between the two domains and requires interactions between α helices 5 and 6 of the N-terminal domain with α helix 7 and the β5 strand of the

C-terminal domain (137).

1.3.1.2 Enzymatic activity of CD38

The primary enzymatic function of CD38 is the hydrolysis of NAD+ to

ADPR, which is covalently attached to proteins to modify their function (138).

CD38 also acts as a cyclase consuming NAD+ to produce the calcium mobilizing second messenger’s cyclic ADP-ribose (cADPR) and nicotinic acid adenine diphosphate (NAADP) (Figure 4C) (139,140). In these reactions, NAD+ binds at the active site of the molecule wherein the Nam group is removed forming an activated intermediate. Cleaved Nam is recycled back into NAD+ via the salvage pathway (discussed in section 1.1.2). Whether or not the activated intermediate is hydrolyzed to ADPR or undergoes cyclization to form cADPR is dependent on the presence of water (141). The placement of a hydrophilic glutamic acid (Glu) at amino acid 146 in the binding pocket of the CD38 attracts water molecules to the activated intermediate allowing hydrolysis and production of ADPR (141,142).

Exclusion of water at this site causes intramolecular attack of the activated intermediate at the 1’ position of adenine causing rotation the activated intermediate allowing the nitrogen atom on the adenine ring to bind to the C1’- ribose forming cADPR (143). Synthesis of cADPR is extremely inefficient, requiring roughly 100 molecules of NAD+ to synthesize a single molecule of cADPR (143,144). Formation of ADPR and cADPR both occur under physiologically relevant neutral pH conditions. Under acidic conditions, the affinity for NAD+ is reduced and CD38 preferentially consumes NADP+ and Na. In this reaction, the amino group on the nicotinamide moiety of NADP+ is replaced with a hydroxyl group donated from Na producing NAADP and Nam (140). Similar to cADPR, NAADP is also a calcium mobilizing second messenger, although it targets different calcium stores. Rather than releasing calcium from the ER or sarcoplasmic reticulum, as is the case with cADPR, NAADP targets and binds to

24 calcium containing lysosomes (145,146). The final reaction catalyzed by CD38, which also occurs exclusively under acidic conditions, is the hydrolysis of cADPR to ADPR (141). CD38 is the only reported enzyme able to catalyze this reaction, suggesting an evolutionarily conserved means of reversing the formation of cADPR when calcium mobilization is not warranted.

Using site directed mutagenesis, several key residues within the CD38 binding pocket have been identified and their contribution to the catalytic activity of this enzyme resolved. Two cysteine residues, Cys119 and Cys201, located in exon 2 and 5, are critical for the production of cADPR (147). Glutamic acid 226

(Glu226), tryptophan 125 (Trp125), and tryptophan 189 (Trp189) are also important for the enzymes’ catalytic activity. Substitution of glutamine for glutamic acid at residue 226 (E226Q) completely abrogates all enzymatic activity, suggesting Glu226 is the primary catalytic residue for CD38 (141). Base substitutions at either tryptophan residue significantly lowers the kinetics of CD38 activity, signifying these residues are likely important for positioning substrates into the binding pocket (142). As previously mentioned, Glu146 is also a key amino acid. Glu146 is centrally located within the binding pocket and positioned directly opposite Glu226 at a distance of 8.1Ǻ: a distance sufficiently large enough to accommodate a water molecule. It is therefore reasonable to hypothesize that the interactions between Glu146 and Glu226 contribute to the default production of ADPR (148). As such, substituting Glu146 for a smaller uncharged alanine (Ala,E) (E147A) restricts the binding pocket excluding water and shifts the activity of the enzyme almost exclusively to the production of

25 cADPR (141). Similar reductions to the hydrolysis activity of CD38 are observed following base substitution of Glu146 with leucine (19-fold reduction) and Glu147 with asparagine (Asn,N) (7-fold reduction) (142). While these base substitutions preclude the entry of water, the default placement of the hydrophilic Glu146 residue in the binding pocket results in ~98% of the enzymatic activity of CD38 devoted to the hydrolysis of NAD+ to ADPR (149). This preference towards synthesis of ADPR has been confirmed in multiple tissues of CD38 knockout

(CD38-/-) mice which exhibit dramatic increases in NAD+ levels, ranging from 2- fold in kidney to 24-fold in cardiac tissue, despite little change in cADPR levels

(150). Additionally, extracts from the plasma membrane, mitochondria, sarcoplasmic reticulum, and nuclei of these tissues have virtually no NAD’ase activity, suggesting CD38 is the main NAD’ase in cells (151).

In addition to the amino acid substitutions discussed above, the presence of zinc ions, serine phosphorylation, ATP, and glycosylation have been found to influence the enzymatic activity of CD38. From a panel of metal ions, zinc was shown to shift the enzymatic activity of CD38 away from hydrolysis to cyclization, presumably by causing a conformational change in CD38 the prevents water from entering the catalytic site (152). Preferential production of cADPR is also observed following activation of protein kinases A (PKA) that results in elevated serine phosphorylation (153). In pancreatic beta cells stimulated with glucose, accumulation of ATP inhibits the hydrolyzing activity of CD38 allowing accumulation of cADPR (154). This may be an evolutionarily conserved means of sustaining calcium mobilization in these cells, as sustained calcium release is

26 required for insulin production in response to glucose stimulation (155,156).

Multiple reports also suggest glycosylation of CD38 is an important regulator of its enzymatic activity. Time dependent deglycosylation of CD38 demonstrated multiple bands of the CD38 that ultimately resolve to a minimum molecular weight of 33kda (157). The specific banding pattern observed during deglycosylation is consistent with the four N-linked glycosylation sites present in the protein (158,159). N-linked glycosylation is a common post translational modification (PTM) well characterized to regulate protein function (160). In line with this, deglycosylation of CD38 reduced its catalytic activity (Vmax) by 42% and

+ its Km for nicotinamide guanine dinucleotide, a fluorescent analog of NAD , by

48% (157). Site directed mutagenesis of the four asparagine residues (N100,

N164, N209, and N219) yielded similar results providing additional lines of evidence as to the importance of glycosylation in regulating CD38 activity (161).

CD38 is expressed in numerous cell types of the hematopoietic system, including lymphocytes, myeloid cells, natural killer cells, platelets, and erythrocytes (162). In lymphocytes, CD38 expression oscillates and is highest during activation and maturation as B cells and T cells (163-165). Increased expresion of CD38 is also a marker of maturation in cells of myeloid lineage. For example, CD38 is low in monocytes and is quickly amplified when these cells develop into mature dendritic cells (166). Other differentiated members of myeloid lineage, including granulocytes, osteoclasts, and osteoblasts express

CD38 (167-169). CD38 is also found in almost every solid tissue type, with the highest levels of expression in brain, spleen, urinary bladder, prostate, lung,

27 intestine, pancreas, and kidney (170-174). Intracellular CD38 is localized to the cell membrane, endoplasmic reticulum (ER), Golgi, mitochondria, and nucleus

(175-177). Initially characterized as an ectoenzyme with a type-II transmembrane domain, the presence of CD38 on the cell surface presented a topological paradox wherein; CD38 was not in proximity to cytosolic sources of NAD+ and the formation of cyclic ADP-ribose (cADPR) at these locations would not permit interaction with intracellular ryanodine receptors (178). This paradox was resolved by investigations into the function of connexin-43 hemichannels.

Connexin-43 hemichannels allow transport of NAD+ down its concentration gradient to the extracellular active site of CD38. This allows for the synthesis of cADPR and ADPR, which may then pass back through selective pores in the cell membrane (150,179). This paradox was also solved by the understanding that the CD38 transmembrane domain can also adopt a type-III orientation, placing the catalytic domain inside the cell (180). The coexistence of both forms is supported by the fact that an equal number of positive residues flank the transmembrane domain and an understanding that the disulfide bonds linking the cysteine residues withstand the reductive environment of the cytosol (181).

1.3.1.3 Physiological processes regulated by CD38

CD38 governs a variety of physiological processes through its production of ADPR and cADPR. Post-translational modification of proteins through the addition of ADP-ribose polymers governs many important activities throughout the cell, including transcriptional regulation, calcium mobilization, DNA repair, cell cycle regulation, and cell death (182-184). ADP-ribosylation comes in three

28 distinct forms: (i) mono (ADP)-ribosylation, (ii) poly (ADP)-ribosylation, and (iii) cyclic (ADP)-ribosylation. The physiological consequence of poly (ADP)- ribosylation is discussed below in section 1.4.3.1. The other products of CD38’s enzymatic activity, cADPR and NAADP, are potent calcium mobilizing second messengers. The ER, sarcoplasmic reticulum (muscle cells), and to a lesser extent lysosomes, represent the main calcium stores in cells (185). In the canonical calcium mobilization pathway an external stimuli activates phospholipase C (PLC), thereby activating inositol 1,4,5-triphosphate (IP3).

Activated IP3 initiates a signaling cascade that ultimately releases calcium from the ER (186). Alternatively, cyclic ADP-ribose and NAADP represent efficient non-canonical means of calcium release in cells. Cyclic ADP-ribose specifically targets ER stores of calcium by binding to ryanodine receptors (RyRs), while

NAADP interacts with lysosomes to target endocytic sources of calcium

(187,188). Despite only requiring sub-nanomolar concentrations of cADPR to activate RyRs, this cascade requires two effector proteins: calmodulin (CaM) and

FK506 binding protein 12.6 (FKBP12.6) (189,190). The exact mechanisms of how CaM activates RYRs is the subject of debate in the literature with two predominant models: (i) the messenger model and (ii) the modulator model

(191). In the messenger model, cytosolic cADPR levels are elevated in response to an external stimulus and synergizes with CaM to facilitate release of calcium from the ER via the RyR (189). Alternatively, the modulator model posits that persistant low concentrations of cADPR and CaM exist in the cytosol. In response to receptor mediated release of calcium or calcium release by the IP3

29 pathway, a second more robust wave of calcium is released from the ER (191).

Irrespective of which model reflects the true mechanism, it is understood that complete activation of RyR’s requires the binding of cADPR to FKBP12.6 localized at the RyR (190,192).

The production of NAADP by CD38 strictly under acidic conditions aligns with the molecules’ ability to mobilize calcium from lysosomes that are acidic in nature. There are two lines of evidence as to how NAADP stimulates calcium efflux from lysosomes. First, NAADP can act through H+-ATPase or H+/Ca2+ channels present on lysosomes as NAADP mediated stimulation is sensitive to proton pump inhibitors and ionophores which depolarize proton gradients (145).

Second, NAADP complexes with and stimulate two-pore channels (TPC1-3); a class of voltage-gated ion channel localized to lysosomal membranes (193). The physiological relevance of calcium efflux under acidic conditions is not settled in the literature. For example, similar to cADPR, NAADP can regulate calcium efflux in response to insulin and glucose stimulation in pancreatic β-cells (146). NAADP also plays role in potentiating the calcium induced calcium release (CICR) mechanism facilitated by the RyR (194). In arterial smooth muscle cells, contraction is dependent on activation of the CICR triggered by NAADP mediated release of calcium from lysosomes colocalized to the SR (195). NAADP dependent activation of TPC2 and lysosomal calcium release is also linked to regulation of neuronal differentation in mouse embryonic stem cells (196,197).

Despite the relevance of NAADP to these processes, a broader understanding of

30 the signaling cascade that stimulates the synthesis of NAADP by CD38 and its relevance to cell physiology is unknown.

1.3.1.4 CD38 in Cancer

Dysregulation of CD38 is linked to the development and progression of both hematopoeitic malignancies and solid tumors. In chronic lymphocytic leukemia (CLL), expression of CD38 serves as a negative prognostic marker as it is highly correlated with more aggressive disease and lower overall survival

(198,199). CD38 is also important in the diagnosis and treatment of multiple myeloma (MM) as 80-100% of MM cells have increased expression of the protein

(200). The association between CD38 and CLL is tightly linked to the chronic nature of this disease, indicative of a gradual accumulation of genetic alterations and favorable environment that supports neoplastic transformation. Accordingly, the oscillation of CD38 tied to B cell maturation and the ability of CD38 to activate

B cell antigen receptor signaling confers a survival advantage to a single clone that eventually progresses into full-fledged CLL (201). This is supported by multiple lines of evidence demonstrating that its catalytic activity is distinct from its receptor functions in B and T cells (201). Given its elevated expression and links to cell maturation in lymphoid cells, targeting CD38 has emerged as a possible therapeutic intervention. As such, multiple monoclonal antibodies (mAB) have entered into clinical trials for the treatment of MM, either as frontline therapy or in combination with existing standard of care chemotherapy (200). The most clinically advanced mAB in development is daratumumab; labeled under the tradename Darzalex (202). Daratumumab was selected from a panel of mAB’s

31 based on its strong binding affinity for CD38 (Kd = 4.36nM) and its ability to induce multiple mechanisms of cell death (203). Antibody mediated cell death pathways include induction of complement-dependent cytotoxicity, antibody- mediated cell-mediated cytotoxicity, antibody-mediated cellular phagocytosis, and apoptosis associated with crosslinking FcγR proteins (203). The potential of treating patients with all-trans retinoic acid (ATRA) (Vitamin A) to induce CD38 expression has also been an area of interest given the presence of the RARE within intron 1 (previously discussed) (204). In myeloid leukemia cell lines, ATRA directly binds to the RARE stimulating expression of CD38 (204). ATRA also inhibits expression of DNA methyltransferases in these cell lines that may contribute to its ability to induce expression of CD38 (205).

While increased expression of CD38 is observed in leukemic and myeloid cells, the opposite is true in solid tumors. Decreased expression of CD38 and the subsequent loss of its NAD’ase activity are contributing factors to the development and progression of PaCa and PCa. Loss of CD38 expression and is corresponding NAD’ase activity is observed in multiple PaCa cell lines. Restoring expression of CD38 in these cell lines sensitizes cells to Nampt blockade, reduces clonogenic potential, and induces cellular senescence via activation of the cell cycle inhibitor p21cip1 (206). In PCa, decreased expression of CD38 is observed in a subset of luminal progenitor cells around sites of inflammation.

These CD38low cells have increased NF-κB signaling and are able to initiate PCa, suggesting they may be a driving force for the development of PCa. In line with this, expression of CD38 is found to decrease proportionately during the

32 progression from benign prostatic hyperplasia (BPH) to prostatic intraepithelial neoplasia, PCa, and metastatic disease (207). These results were further confirmed by characterization of the differences in the CD profile and analysis of changes that occur in the transcriptome of primary and metastatic prostate tissue

(208). Low levels of CD38 are also prognostic of biochemical recurrence and increased likelihood of developing metastasis (209).

The connection between loss of CD38 and the development of PaCa and

PCa suggests restoring its expression may be of clinical value. Interestingly, treatment of ATRA in mouse models of spontaneous prostate cancer induces hypomethylation of histones and contributes to reduced cell proliferation and induced apoptosis (210). These effects are mediated by induction of the cell cycle inhibitors p21Cip1 and p16Ink4 and down-regulation of pro-proliferative proteins such as cyclin dependent kinase 2 (Cdk2), Cdk4/6, and Cyclin D1 (211).

Treatment with ATRA is also sufficient to inhibit the proliferation and clonogenicity of androgen refractory PC3 cells and androgen sensitive LNCaP cells, although these studies provided no concrete evidence as to how the effects of ATRA were mediated (212,213). Additionally, there is some anecdotal evidence from a large-scale epidemiological study that investigated the connection between serum levels of retinoids with the incidence of PCa. From this study, men in the lowest quartile of the cohort had a 2.4x greater risk of developing PCa compared to those in the highest quartile (214). Our own investigation into the effects of treatment with ATRA in PCa is presented in section chapter II.

Despite the established links between CD38 and PCa, there has yet to be an investigation into the relationship between CD38 and cell metabolism.

Therefore, the focus of chapter II is to define how CD38 and its regulation of intracellular NAD+ levels influences cell metabolism in PCa.

1.3.1.5 CD38 in cell physiology other disease states

In addition to its functions in cancer, CD38 plays a pivotal role in general physiology and pathology. Studying CD38-/- mice has significantly contributed to the understanding of CD38 and its ability to regulate physiological processes throughout the body. CD38-/- mice are viable and appear to have no major physiological problems despite an almost complete loss of NAD’ase activity in all tissues and minimal cADPR production, indicating CD38 is dispensible for viability (150,151). However, through careful investigation, these mice have revealed CD38 is able to protect against diet-induced obesity and is involved in the pathology of neurodevelopmental disorders (215-217). Separate from the work in CD38-/- mice, studying dysregulation of CD38 in relation to various diseases has demonstrated the importance of CD38 to human immunodeficiency virus (HIV) infections, diabetes, and aging (218).

CD38-/- mice were generated out of the desire to develop a better understand the role of NAD+ in pancreatic β-cell dysfunction and the onset of diabetes. The CD38/NAD+ axis connects to diabetes through the ability of CD38 to mediate production of cADPR. High concentrations of cADPR are necessary for pancreatic β-islet cells following glucose stimulation. When stimulated with glucose, these cells increase production of cADPR thereby mobilizing calcium

34 and initiating the release of insulin (219). Accordingly, the absence of CD38 in these mice leads to an accumulation of plasma glucose and insulin insensitivity consistent with type-2 diabetes mellitus. The same group that generated the

CD38-/- mouse later established a connection between non-insulin-dependent diabetes mellitus (NIDDM) and CD38 in humans. This group demonstrated 14% of men with NIDDM had autoantibodies against CD38 and that these antibodies were capable of preventing CD38 mediated production of cADPR (220). The development of type 2 diabetes is often associated with obesity providing a potential link between CD38 and obesity. Interestingly, while not linked to pancreatic β-islet function or insulin secretion, CD38-/- mice are also protected from diet-induce obesity conferred from increased energy expenditure (221).

These effects are mediated by Silent information regulator 1 (SIRT1) dependent activation of peroxisome proliferator-activated receptor gamma coactivator 1α

(PGC-1α), which regulates energy metabolism and mitochondrial membrane biogenesis (222). Resveratrol, a SIRT1 activator, has a very similar effect on activation of PGC-1α, energy metabolism, and has been established to increase lifespan in C. elegans and the short-lived fish N. furzeri (223). Age-associated pathologies resulting from nuclear and mitochondrial dysfunction are tightly linked to decreased NAD+ levels (224). Accordingly, using CD38-/- mice and littermate controls, increased expression of CD38 and decreased levels of NAD+ have been determined to correlate with age and is associated with diminished

SIRT3 activity leading to mitochondrial dysfunction (225). This provides further evidence of the association between expression of CD38 and aging.

Anecdotal evidence from observing CD38-/- mice suggested both female and male mice had marked changes to maternal nurturing and social behavior, respectfully (217). Upon further investigation, it was revealed that elevated calcium in the oxytocinergic neurohypophysial axon terminals of these mice contributed to diminished oxytocin (OT) release; reestablishing OT levels rescued social behavior and maternal care (217). In humans, alterations in the

OT release system are thought to play are role in the etiology social deficits associated with autism spectrum disorder (ASD) and other neuropsychiatric disorders (226). Interestingly, SNPs in CD38 (rs6449197 and rs3796863) are highly correlated with patients diagnosed with high-funciton autism and evidence suggests these SNPs may be heredity (227,228).

Until the introduction of highly active antiretroviral therapy (HAART), death closely followed the progression from HIV infection to acquired immunodeficiency syndrome (AIDS). A hallmark to the progression from HIV-infection to AIDS is the elimination of antigen presenting CD4+ T-cells followed by a rapid and persistant increase in cytotoxic CD8+ T-cells (229). Because expression of CD38 is increased during lymphocyte maturation, an increase in the population of

CD8+/CD38+ T lymphocytes following HIV infection has become an important prognostic factor for the likelihood of developing AIDS (230). Accordingly, an increase in the number of CD8+/CD38+ T-cells is predictive of CD4+ T-cell decline before it is clinically detectable. The presence of CD8+/CD38+ cells also signals underlying chronic activation of the immune system and enrichment of these cells is indicative of poor clinical response to HAART (231-233).

Collectively, these data demonstrate the complexity and importance of

CD38 to many biological processes. It also serves to highlight the general interest in understanding the physiology and pathology of CD38. By advancing the knowledge of the many roles for CD38 in normal biology, we will be able to develop a deeper understanding of its role in different types of cancer.

Figure 4: The cyclic ADP-ribose synthase CD38 is the primary NAD’ase in cells. CD38 is a type-II/III transmembrane glycoprotein. A, The CD38 gene is located on chromosome 4p15.32 and consists of 7 introns and 8 exons that span

76kb. Key transcription factor binding sites and regulatory response elements are located in the 5’-UTR and intron 1 in close proximity to the first exon. B, Exon 1 contains the coding region for the intercellular (IC) and transmembrane (TM) domains. A portion of the 3’ end of exon 1 and exons 2-8 contain the coding region for the catalytic subunits that extend into the extracelluar space, cytoplasm, or into the mitochondrial matrix depending on CD38 localization. The eight exons encode 300 amino acids (AA) that make up the protein. C, The

38 principle function of CD38 is the hydrolysis of NAD+ to ADP-ribose (ADPR). The presence of zinc (Zn2+) at the NAD+ binding site causes a small confirmation change that cause the exclusion of water shifting the catalytic activity to production of cyclic ADP-ribose. Nicotinamide (Nam) in a biproduct of both reactions and can be recycled to NAD+ via the NAMPT catalyzed salvage pathway. Portions of this figure were adapted from (129,234).

1.3.2 Sirtuins

1.3.2.1 Intracellular localization and function

Sirtuins, silent information regulator 1-7 (SIRT1-7), are a family of class III

NAD+ dependent protein deacetylases. Sirtuins are distributed throughout the cell with localization to the nucleus (SIRT1,-6,-7), mitochondria (SIRT3, -4, -5), and cytoplasm (SIRT1, -2) (235). Post-translational modification of proteins by the addition of acetyl groups to the ε-amino group of lysine residues is a common form of protein regulation in eukaryotes. Adding an acetyl group neutralizes positive charges on lysine residues thereby effecting the electrostatic properties of the protein (236). SIRTs1-3 are the best-characterized members of the sirtuin family and function exclusively as protein deacetylase. Sirtuins 4, 6, and 7 also function exclusively as protein deacetylases but with decidedly lower activity and relatively fewer substrates compared to SIRT1-3 (235,237). Despite being a member of this family, SIRT5 does not possess any deacetylase activity. Rather,

SIRT5 functions as an NAD+ dependent lysine desuccinylase and demalonylase, two types of post-translational modification that can dramatically alter protein function (238). Each family member contains a conserved catalytic core comprised of a Rossmann fold domain that forms a binding pocket for the Nam and ribose groups of NAD+ (239). The requirement for NAD+ distinguishes sirtuins from other classes of protein deacetylases, such as class I and II histone deacetylases. Aside from SIRT6, each sirtuin must be bound to an acetylated substrate in order to bind NAD+, suggesting SIRT6 may have a complementary role as an NAD+ sensor (237). When bound to acetylated lysine residues and in

40 association with NAD+, sirtuins cleave and release the Nam group from NAD+ allowing the acetyl group on lysine to be transferred to the 2’-hydroxyl group of the ADP-ribosyl moiety forming 2-O-ADP-ribose (Figure 5A) (240). Nam is a product inhibitor that if allowed to accumulate can block the deacetylase reaction by binding the active site and reforming NAD+ (239). However, this rarely happens under normal physiological conditions as Nam produced during these reactions is recycled back into NAD+ via the salvage pathway.

1.3.2.2 Sirtuin/NAD+ Axis in cell metabolism

The association between NAD+ availability and sirtuin activity is tightly linked to the energy status of the cell and cell metabolism given the interdependence on NAD+ availability. Colocalization sirtuins and different isoforms of NMNAT to the nucleus, mitochondria, and cytosol, key areas of metabolic activity, suggests compartmentalized control of NAD+ synthesis and sirtuin activity (241,242). Accordingly, changes in NAD+ synthesis either through overexpression or through blockade of NAMPT have been well documented to influence sirtuin activity (45,46). Interestingly, calorie restriction induces expression of NAMPT and increases NAD+ levels through activation of AMP- activated protein kinase (AMPK) (243,244). AMPK is regarded as the master regulator of cell metabolism and is activated in response to increases in the

AMP/ATP ratio (245). Caloric restriction also enhances transcription of SIRT1 and SIRT3, providing additional evidence for the link between NAD+, sirtuins, and cell metabolism (246,247). SIRT1 is regarded as the master regulator of cell metabolism as it activates multiple transcription factors and enzymes in response

41 to the energy status of the cell (Figure 5B) (248). To maintain ATP production during extended periods of fasting, hepatic SIRT1 activates the transcription factor peroxisome proliferator-activated receptor gamma coactivator 1α (PGC-

1α) which activates transcription of genes that support fatty acid oxidation

(222,249). In response to hypoxia, SIRT1 deacetylates the transcription factors hypoxia inducible factor 1α and 1β (HIF-1α/1β) activating transcription of multiple genes ATP production from oxidative phosphorylation to glycolysis (250,251).

SIRT1 also directly supports fatty acid and lipid synthesis through activation of acetyl-CoA synthetase 1 (AceCoA1) in the cytosol (252). SIRT3 performs a similar function in the mitochondria by activating acetyl-CoA synthetase 2

(AceCoA2) which synthesizes acetyl-CoA to support the TCA cycle and ATP production (253). SIRT3 also supports ATP production by regulating the acetylation status of multiple components of complex 1 in the ETC (Figure 5C)

(254). In line with this, basal levels of ATP are dramatically reduced in multiple tissues of SIRT3 knockout mice (254). SIRT3 knockout mice also exhibit increased oxidative stress resulting from reduced activation of superoxide dismutase 2 (SOD2), a major component of the anti-oxidant stress response

(255). The multiple nodes that connect NAD+ levels to sirtuin activity and their influence on cell metabolism highlights the diverse array of networks that are interconnected in an effort to maintain a stable flow of energy for the cell.

1.3.2.3 Sirtuins and Cancer

Sirtuin activity is tied to cancer through regulation of many different proteins and pathways, including many of the metabolic pathways described above. The classification of sirtuins as both tumor suppressors and promoters is highly context dependent, as a single sirtuin may exert opposing effects in cancers of different origin. In many cases, SIRT1 is regarded as a tumor promoter through its regulation of proteins involved in cell cycle regulation, DNA damage repair, proliferation, and survival under different types of metabolic stress (256). Overexpression of SIRT1 has been documented in multiple types of solid tumors, including prostate, breast, ovarian, colorectal, pancreatic, and gastric cancers, wherein its inhibition induces cell death (257-262). In PCa,

SIRT1 acts as a tumor promoter by deacetylating and inactivating the tumor suppressor p53, increasing the activity of matrix metalloproteinase-2 (MMP2), and conferring chemoresistance (263-265). SIRT1 dependent chemoresistance is mediated by the multidrug resistance protein P-glycoprotein which protects cells from DNA damaging agents like doxorubicin (266). Treatment with sirtinol, a potent SIRT1 inhibitor, can sensitize PCa cells to chemotherapeutic agents like doxorubicin, camptothecin, and cisplatin (263). SIRT2 mediated deacetylation of cortactin promotes cell migration and invasion in bladder cancer and is associated with poor prognosis in PCa (267,268). Paradoxically, SIRT2 acts as a tumor suppressor in non-small cell lung cancer (NSCLC), BCa, and metastatic

CRC (269-271). SIRT3 is also an important mediator of tumor cell viability by mitigating the effects of ROS through direct activation of IDH2, superoxide

43 dismutase (SOD), and glutathione, all of which are pillars of the anti-oxidant response machinery (272,273). SIRT6 can act as both a tumor suppressor and promoter as inhibition induces cell death in PaCa and PCa, but overexpression confers chemoresistance and survival in BCa (274-277). SIRT7 is generally regarded as a tumor promoter as its deacetylase activity is linked to increased proliferation and survival in colorectal, gastric, and breast cancer (278-280).

Collectively, sirtuin activity governs a significant portion of the regulatory networks and key proteins that underly tumor cell biology. Accordingly, the connection between NAD+ levels and sirtuin activity emphasizes the importance of understanding how cancer cells regulate NAD+ homeostasis and presents an opportunity to develop new therapeutic strategies to target tumor cells.

Figure 5: Sirtuins consume NAD+ to facilitate lysine deacetylation. Sirtuins

1-4,6, and 7 are NAD+ dependent protein deacetylases that consume NAD+ in order to remove acetyl groups from lysine residues. A, Sirtuins cleave the nicotinamide moiety of NAD+ and transfer the acetyl group from the target protein onto the 2’ hydroxyl group of the ribose ring distal to adenine producing 2-O-

ADP-ribose. B, SIRT1, localized to the cytosol and nucleus, is regarded as the master regulator of cell metabolism through its ability to regulate several metabolic processes, including fatty acid synthesis (FAS) and fatty acid oxidation

(FAO) in response to the energy status of the cell. C, SIRT3, localized to the mitochondria, performs equally important functions by deacetylating acetyl-CoA synthetase 2 (AceCS2), subunits of complex I of the ETC, and regulating the

45 activity of isocitrate dehydrogenase 2 (IDH2) and superoxide dismutase 2

(SOD2) to mitigate the accumulation of reactive oxygen species (ROS). P-GP: P- glycoprotein, AceCS1: Acetyl-CoA synthetase 1, PGC-1α: peroxisome proliferator-activated receptor gamma coactivator 1α.

1.3.3 Poly (ADP)-Ribose Polymerases

1.3.3.1 PARPs localization and Function

The PARP super family of proteins consists of roughly ten proteins classified according to their function: DNA-dependent PARPs (PARP-1 and

PARP-2), tankyrases, CCCH-type PARPS, and macroPARPs (184). PARP-1 is the most extensively studied member of the PARP family with activity that extends into multiple cell processes, including the DNA damage response, epigenetic regulation through histone modification, cell differentiation, and necrosis (28,281,282). Localized to the nucleus, PARP-1 consists of three functional domains: an N-terminal domain that houses two zinc fingers to facilitate DNA binding, a central autoregulatory domain, and a C-terminal domain that confers the catalytic activity of the enzyme (283). The most notable activity of PARP-1 is its ability to initiate the DNA damage response by binding at the site of single strand breaks (SSB). PARP-1 recognizes and binds to single strand

DNA (ssDNA) where it hydrolyzes NAD+ cleaving the Nam group and attaching the negatively charged ADP-ribosyl moiety onto positively charged amino acids

(Figure 6A). Reducing the positive charge of amino acids on histones causes the

DNA wrapped around histones to relax, allowing for the recruitment of DNA repair machinery (28,284). ADP-ribosylation at these sites can range from a few to several hundred polymers that form large branched structures. PARP-1 activity is subject to negative regulation by auto-poly (ADP)-ribosylation or through a negative feedback loop with nicotinamide (28). The array of PARP activity in normal cells suggests it is a steady consumer of NAD+ and in effect may

47 compete with other NAD+ consuming enzymes such as SIRT1 for substrate. In line with this, there have been several reports that show SIRT1 knockdown leads to increased PARP-1 activity; the reverse situation also holds true (285,286). The antagonistic relationship between the two proteins may be beneficial to cells as it would not be advantageous to promote SIRT1 activity and effect metabolism under conditions of extensive DNA damage. Consequently, when DNA damage is low, metabolism should be more productive.

1.3.3.2 PARPs in Cancer

Increased expression of PARP-1 is observed in a variety of tumors, including triple-negative breast cancer (TNBC), CRC, PaCa, and PCa (287-290).

Specifically in PCa, PARP-1 expression positively correlates with Gleason score

(291). The effect of PARP-1 activity in response to alkylating agents or ionizing radiation has been well-documented (292,293). Cancer cells exposed to these agents typically follow one of three fates (Figure 6B). In response to mild genotoxic stress, PARP-1 activation initiates cell-cycle arrest, allowing for recruitment of DNA repair machinery (294). In the event DNA damage is more severe, cells will initiate the apoptotic cascade, wherein activated caspases cleave PARP-1, removing catalytic activity (281). In the event of catastrophic

DNA damage, cells have a propensity to over activate PARP-1, leading to rapid consumption of NAD+ and causing a subsequent decline in ATP and initiation of necrosis (295). This process is strictly NAD+ dependent as repletion of NAD+ prevents necrosis (47). Because of its central role in regulating the DNA damage

48 response, several PARP inhibitors (PARPi) have been developed as adjuvant chemotherapy.

The first PARPi study was published in 1995 (296). Since then there have been numerous iterations of these drugs that have been tested in more than 200 phase I-IV clinical trials (297). In 2014, olaparib, under the trade name

Lynparza®, gained FDA approval for the treatment of BCa patients with deficiencies in breast cancer susceptibility genes (BRCA1/2) (298). The efficacy of olaparib in patients with PCa that harbor similar loss of function mutations in

DNA damage repair proteins is currently under investigation (299). Newer generation PARP inhibitors, rucaparib and talazoparib, are also being evaluated as treatments for advanced BCa, ovarian cancer (OvCa) and small cell lung cancer (SCLC) (300-302). In clincial trials, Rucaparib has shown promise in treating hematological malignancies such as acute leukemias when used in combination with the antimetabolite pyrimidine analog, fluorouracil (303). Similar to most chemotherapy drugs, PARP inhibitors have off target effects and dose- limiting toxicities. Orlaparib is particulary toxic to normal tissues when used in combination with commonly used chemotherapy agents like dacarbazine, cyclophosphamide, and paclitaxel (304-306).

Figure 6: Poly (ADP)-ribose polymerases require NAD+ to ADP-ribosylate target proteins. PARPs require NAD+ to covalently attach molecules of ADP- ribose onto proteins to modify their funciton. A, PARPs cleave nicotinamide

(Nam) from NAD+ and transfer ADP-ribose at the 1’ hydroxyl group distant to the adenine moiety or at the 2’ hydroxyl group on the ribose proximal to the adenine moiety. Up to 50 molecules of ADP-ribose can be attached to proteins forming poly (ADP)-ribose polymers. Cleaved Nam is recycled back into NAD+ via the

NAMPT catalyzed salvage pathway. B, Varying levels of PARP activity can be achieved in response to ionizing radiation or alkylating agents.

1.4 Prostate Cancer

1.4.1 Current epidemiology

Last year there were 1.6 million new cancer diagnoses and over 600,000 deaths attributed to cancer making cancer the second leading cause of death in the United States (307). Although lung cancer has the highest mortality rate among all sub-types, PCa is the most frequently diagnosed and has the third highest mortality rate with an estimated 161,000 new cases and 26,000 deaths attributed to the disease in 2017 (307). The primary risk factors for the development of PCa are age, race, and family history. PCa is primarily a disease of the aged, as two thirds of new diagnoses are in men above age 65 and men

70+ have a 30x greater risk of developing the disease relative to men under 50

(308). Race also plays a significant role, as African-American males are 73% more likely to develop PCa during their lifetime and 2.3x more likely to die from the disease, relative to caucasians (307). There is no conclusive data to account for the discrepancy of cancer incidence between races. Family history and genetic predisposition are also linked to incidents of PCa, accounting for roughly

5-15% of all cases (309). Inherited mutations (germline) in the DNA repair proteins BRCA1/2 (breast cancer susceptibility genes 1/2) and the transcription factor Hox13B are most closely associated with increased risk of developing

PCa, as ~2% of early onset PCa harbor these mutations (310-313). The aforementioned risk factors (age, race, family history, and genetic predisposition) represent non-modifiable risk factors. Modifiable risk factors such as smoking, body mass index, exercise, and diet are also linked to PCa. Cigarette smoking is

51 not associated with an increased risk of developing PCa; however, it has been linked to the development of aggressive disease, higher rates of metastatic disease, and higher mortality (314,315). Early epidemiological studies of the association between BMI and PCa risk presented conflicting results. These disagreements were resolved by a meta-analysis that included dozens of cohorts and conclude obesity is weakly associated with the risk of developing PCa, but more significantly correlated with disease progression (316,317). The development of obesity coincides with the adoption of a Western style diet characterized as high in saturated fats, protein from red meat, and “empty” carbohydrates, but low in fruits, vegetables, whole grains, and lean protein.

Some cohort studies have identified components of a Western diet, such as intake of animal fats, with increased risk of PCa and higher rates of advanced disease and mortality, although studies that are more rigorous are required to establish a definitive association between diet and PCa (318-320).

1.4.2 Development and progression of prostate cancer

Prostate cancer is a progressive disease with well-defined parameters that describe the advancement of localized and metastatic disease. The severity of

PCa burden is graded using the TNM system which utilizes a combination of factors, including Gleason score (T) (a measurement of the primary tumors size and level of differentiation; graded T1-5), whether or not there is lymph node involvement (N) (graded 1 = regional lymph nodes, 2 = involvement of distant lymph node), and metastatic burden (M) (graded 0 = no metastatic burden, 1a = metastasis to lymph nodes, 1b = metastasis to bone, 1c = metastasis to other

52 organs) (321,322). Four distinct stages encompass the progression of PCa.

Stage I, characterized by a nondistinct enlargement of the prostate gland, is referred to as benign prostatic hyperplasia (BPH). Progression to stage II, prostatic intraepithelial neoplasia (PIN), requires cells lining the ducts and acini of the prostate to begin proliferating changing the morphology of these structures

(323,324). Stage III, adenocarcinomas, is distinguished by an outgrowth of cells into the surrounding tissue, specifically involving penetration of the capsular, an outer lining of the prostate gland, and invasion into the peri-seminal and seminal vesicles with no detectable disease in the pelvic lymphnodes (325,326). Stage IV cancer requires disease to have spread to the surrounding lymphnodes or evidence that the primary tumor has metastasized. PCa predominantly metastasizes to bone, lungs, liver, pleura (a set of membranes lining the lungs), and adrenal glands. Bone is the most common site of metastasis, as roughly

90% of patients that develop metastatic PCa have involvement of the bone (327).

The mechanisms underlying the preferential homing of PCa cells to bone are not well understood and remain an area of intense investigation. The progression of

PCa is not linear with respect to time. Instead, PCa progresses through a series of waves with peaks corresponding to increased burden requiring intervention followed by periods of dormancy. Depending on the aggressiveness of the tumor, dormancy can last as long as a decade after treatment of localized disease or as short as a few months in the case of advanced stage PCa (Figure 7) (328,329).

1.4.3 Treatment options for localized and metastatic disease

Patients who present with localized or regional disease at the time of diagnosis have a 99% 5-year overall survival (OS) rate, due in part to the long dormancy period associated with frontline therapy (307). Unfortunately, this falls to 29% for patients who present with metastatic disease, emphasizing the need for early detection (307). Men with elevated PSA levels are typically subjected to biopsies of the prostate gland in an attempt to discern indolent from aggressive disease. Because PCa is a disease of the aged, overdiagnosis of indolent PCa is a common occurrence, causing patients who may otherwise outlive the disease to undergo unnecessary treatment. Overdiagnosis has been addressed by the adoption of active surveillance that consists of more frequent PSA tests, digital rectal exams, and transrectal ultrasound for men with low Gleason grade biopsies (330). Men who present with aggressive disease at the time of diagnosis often elect to receive external-beam radiation therapy, brachytherapy (placement of radioactive beads), radical prostatectomy (RP), or in select cases androgen depravation therapy (ADT) (331). Despite these treatment options, 20-40% of men who undergo RP and 30-40% of men who receive radiation therapy will experience biochemical recurrence (BCR) within 7-9 years. BCR is defined as a recurrent and persistant rise in PSA level. Of the men who experience BRC roughly 25% will develop metastatic disease within five additional years (332).

ADT, either in isolation or in combination with radiotherapy, is the main salvage therapy in men who experience BCR following external-beam radiation or brachytherapy (332). Androgen deprivation can be achieved surgically through

54 orchiectomy or medically through the use of gondatropin-releasing hormone agonists (GnRH-A), GnRH antagonists (GnRH-At), androgen synthesis inhibitors

(ASIs), androgen receptor antagonists (AR-A), or 5α-reductase inhibitors (5-

ARI’s) (333). Leuprolide and Goserelin are the tradenames for the leading

GnRH-A’s and Abarelix is the tradename for the most commonly used GnRH-At

(333). Both drugs target the production of testosterone though different means of blocking the signaling cascade in the anterior pituitary that produces hormones to stimulate testosterone synthesis. GnRH-Ats directly binding to GnRH receptors inhibiting their activity causing an immediate decline in testosterone levels without an accompanying surge of testosterone that is indicative of GnRH-As

(334). ASI’s are an alternative way to block downstream testosterone biosynthesis by inhibiting the activity of cytochrome p450 (CYP) enzymes (335).

Ketoconazole and Liarozole are the tradenames of the most commonly used

ASI’s. AR-As represent the fourth way to achieve androgen ablation. AR-As were prescribed under the tradenames Flutamide, Bicalutamide, and Nilutamide; however, a more potent drug, Enzalutamide, is now commonly used in the clinic

(336). These drugs work by binding the ligand-binding domain on the androgen receptor preventing the association of testosterone and the subsequent activation of the AR. Lastly, 5-ARIs block the activity of 5-alpha reductase preventing the conversion of testosterone to dihydrotestosterone (DHT), a potent growth activator for stromal and epithelial prostate cells (337).

Despite the efficacy of ADT for recurrent or metastatic PCa, a subset of patients fail to enter long term remission and develop recurrent castration

55 resistant prostate cancer (CRPR), typically within 14-20 months of being placed on ADT (328,338). Androgen independent disease is the most progressive form of PCa and to date has no curable therapy. Androgen independence can be achieved through four prominent pathways; hypersensitivity / AR gene amplification pathway, promiscuous pathway, “outlaw” pathway, and the bypass pathway (339,340).

Treatment of mCRPC has had relatively little advancement over the last two decades. Mitoxantrone, a Type II topoisomerase inhibitor, became standard of care for the treatment of mCRPC but has sense been replaced by docetaxel.

Taxanes like docetaxel inhibit cell division by stabilizing guanosine-5’- diphosphate molecules bound to tubulin in the microtubule preventing their disassembly which is required for cytokinesis (341). These drugs are effective chemotherapies given the rapid cell division inherent to cancer cells. Docetaxel is particularly effective in PCa as it also induces phosphorylation of the anti- apoptotic protein BCL-2 resulting in caspase activation and apoptosis (342,343).

Despite this dual effect, approximately half of all PCa patients treated with docetaxel do not achieve clinical remission and most will eventually develop taxane resistance (344,345). Patients who experience BCR following treatment with docetaxel are typically placed on cabazitaxel as salvage therapy (346-348).

The mortality associated with mCRPC combined with the limited efficacy of taxanes highlights the need for new treatment options.

To meet this need, new types of drugs including Sipuleucel-T (Provenge),

Abiraterone, Enzalutamide, and Radium-223 are being used as last line salvage

56 therapy for patients who do not respond to cabazitaxel (349-352). Sipuleucel-T is a first of its kind autologous active cell immunotherapy that harnesses an individual’s immune system to targe PCa cells (353). Sipuleucel-T therapy requires leukapheresis and the ex vivo activation of isolated antigen presenting cells including T-cells, monocytes, B-cells, and natural killer cells (354).

Abiraterone acetate is an ASI that blocks production of testosterone by inhibiting cytochrome p450 C17 (355). Enzalutamide, an AR-A originally approved as frontline ADT (previously described), is now under investigation as salvage therapy for mCRPC. Radium-223 dicholride is a systemic radiopharmaceutical with a relatively long half-life of 11.4 days that emits four high-energy alpha particles during its decay (356). Radium-223 provides additional therapeutic benefits relative to external beam radiation as Radium-223 is a calcium mimetic localizing it to sites of active mineralization, high osteoblast activity, in bone

(357). This is particularly advantageous for the treatment of PCa as 90% of patients with metastatic disease have bone involvement (327). Despite preclinical evidence exhibiting the efficacy of these drugs, they target many of the vulnerabilities PCa ultimately adapts to and overcomes. This highlights the need to develop new classes of drugs that target other cellular processes or pathways

PCa cells depend on; cell metabolism being one of them.

Figure 7: Development, progression, and treatment of PCa. PCa progresses along a well-defined series of stages that describes the advancement of localized and metastatic disease. Treatment of localized disease confers the longest period of dormancy with increasingly shorter treatment-dormancy-relapse cycles as PCa progresses. Ultimately, a significant portion of individuals that develop metastatic disease will fail all available treatment options highlighting the need to develop novel treatment strategies for mCRPC.

1.5 Overview

Energy production is the most basic need for tumor cells. Cell metabolism is an orchestrated process involving numerous interconnected pathways all of which require the presence of NAD+ produce the metabolites necessary to sustain proliferation and cell survival. The critical importance of NAD+ for cell metabolism combined with the diverse processes regulated by NAD+ dependent enzymes emphasizes the need to develop a better understand of how tumor cells regulate NAD+ inorder to gain a competitive advantage. The purpose of this work is to provide an understanding of how dysregulation of CD38 and modulation of

NAD+ contribute to PCa cell metabolism. This work also establishes a novel role for CD38 in regulating calcium mobilization and its ability to confer protection from cytotoxic stress.

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112

CHAPTER II

CD38 Inhibits Prostate Cancer Cell Metabolism and Proliferation by

Reducing Cellular NAD+ Pools

Jeffrey P. Chmielewski 1, Sarah C. Bowlby 1, Frances B. Wheeler 1, Lihong Shi 1,

Guangchao Sui 1, Amanda L. Davis 1, Ralph B. D’Agostino Jr. 2, 3, Lance D.

Miller1, 2, S. Joseph Sirintrapun 5, Scott D. Cramer 4, Steven J. Kridel 1, 2.

1Department of Cancer Biology, Wake Forest School of Medicine, Winston-

Salem, North Carolina; 2Comprehensive Cancer Center at Wake Forest Baptist

Medical Center, Winston-Salem, North Carolina; 3Public Health Sciences-

Department of Biostatistical Sciences, Wake Forest School of Medicine, Winston-

Salem, North Carolina; 4Department of Pharmacology, University of Colorado

Denver, Aurora, Colorado; 5Department of Pathology, Memorial Sloan Kettering

Cancer Center, New York, New York.

The following chapter is formatted for submission to Molecular Cancer Research.

Stylistic variations are due to journal requirements.

113

2.1 Abstract

Tumor cells require increased rates of cell metabolism to generate the macromolecules necessary to sustain proliferation. They rely heavily on nicotinamide adenine dinucleotide (NAD+) as a cofactor for multiple metabolic enzymes in anabolic and catabolic reactions. NAD+ also serves as a substrate for poly ADP-ribose polymerases (PARPs), sirtuins, and cyclic ADP-ribose synthases. Dysregulation of the cyclic ADP-ribose synthase CD38, the main

NAD’ase in cells, is reported in multiple cancer types. This study demonstrates a novel connection between CD38, modulation of NAD+, and tumor cell metabolism. Expression of CD38 is inversely correlated with prostate cancer progression and treatment with all-trans retinoic acid (ATRA) represents a viable strategy to restore its expression. Induced expression of CD38 lowers intracellular NAD+ levels resulting in cell cycle arrest through activation of p21Cip1.

The metabolic consequence of CD38 expression extends to diminished glycolytic and mitochondrial potential, activation of AMP-activated protein kinase (AMPK), and inhibition of fatty acid and lipid synthesis. Pharmacological inhibition of nicotinamide phosphoribosyltransferase (NAMPT) yields similar results to CD38, demonstrating that CD38 expression mimics NAMPT inhibition. Modulation of

NAD+ by CD38 also induces differential expression to more than 75% of the transcriptome producing a unique gene expression signature indicative of a non- proliferative phenotype. Altogether, these data establish a novel role for the

CD38-NAD+ axis in the regulation of cell metabolism and development of prostate cancer.

114

2.2 Introduction

Nicotinamide adenine dinucleotide (NAD+) metabolism is associated with everything from aging and healthy lifespan, to diabetes, neurocognitive function, and cancer, among others (1,2). NAD+ connects to cellular metabolism in various ways. First, it is a necessary cofactor for multiple anabolic and catabolic reactions, in doing so providing a crucial link to energy production and macromolecular synthesis (3). For example, NAD+ is reduced to NADH during glycolysis and the TCA cycle, producing an electron carrier for subsequent adenosine triphosphate (ATP) generation. Conversely, the phosphorylated form,

NADPH, is a cofactor for fatty acid synthesis. Because deregulated cellular metabolism is an established hallmark of oncogenic transformation (4), it is reasonable to assume NAD+ metabolism must be regulated accordingly.

There are multiple pathways by which NAD+ can be synthesized. Principle among them are the de novo and salvage pathways. Most tissues and oncogenic cells synthesize NAD+ via the salvage pathway (5). The salvage pathway is a two-step reaction that recycles nicotinamide into NAD+ and is catalyzed by nicotinamide phosphoribosyltransferase (NAMPT). NAD+ turnover in cancer cells is dramatically increased relative to non-proliferating healthy cells (6), making

NAD+ metabolism an attractive therapeutic target. NAD+ levels can also be modulated by enzymatic consumption. There are three classes of enzymes that consume NAD+: the poly-ADP ribose polymerases (PARPs), sirtuins, and cyclic

ADP-ribose synthases (7-9). While PARPs and sirtuins have multiple roles in cancer biology, including response to DNA damaging reagents and modulation of

115 protein modification, among others, CD38 is the primary NAD’ase in mammalian cells (10). CD38 is a cyclic ADP-ribose synthase that localizes to the cell membrane and elsewhere throughout the cell including the endoplasmic reticulum (ER), golgi, and mitochondria (11,12). CD38 hydrolyzes NAD+ to produce ADP-ribose (ADPR), which is covalently attached to proteins to modify their function (9). Alterations in CD38 expression occur in a variety of cancers including hematological malignancies, glioma, pancreatic cancer, and prostate cancer (PCa) (13-15). Decreased expression of CD38 in prostate luminal cells is sufficient to drive disease progression and is linked to lower overall survival (16).

Expression of CD38 is also inversely correlated with growth inhibition, disease progression, and overall survival in pancreatic cancer (17). Although expression of CD38 is linked to disease progression, the metabolic consequences of CD38 expression in PCa have not been defined.

We previously demonstrated that NAMPT activity is required to support fatty acid and lipid synthesis in PCa (18). Therefore, we hypothesized that CD38, through its NAD’ase activity, also regulates energy production and lipid synthesis in PCa. The data presented herein demonstrate CD38 has NAD’ase activity in

PCa cells and that expression correlates with disease progression, affects multiple metabolic processes, and ultimately reprograms cells in favor of a non- proliferative state. In addition, we provide evidence that expression of CD38 in

PCa is epigenetically regulated by methylation. Collectively, our data suggests loss of CD38 is a key event in oncogenic transformation in prostate that confers a

116 metabolic advantage to tumor cells, thus providing new insight into the mechanisms underlying tumor cell metabolism.

2.3 Materials and Methods

Materials

Antibodies for acetyl-CoA carboxylase 1 (ACC1), phospho-ACC1 (S79),

AMPK, phospho-AMPK (T172), Sirt1, and Sirt3 (C73E3) were obtained from Cell

Signaling Technologies (Beverly, MA, USA), antibodies against CD38 and

FOXO1 were from Abcam (Cambridge, MA, USA), antibodies for SLC22A3 and

β-Actin were purchased from Sigma-Aldrich (St. Louis, MO, USA) and antibody for Ac-FOXO1 was purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX,

USA). Secondary antibodies for goat-anti-mouse and goat-anti-rabbit were purchased from BioRad (Hercules, CA, USA). The NAMPT inhibitor FK866 was obtained from Cayman Chemical Company (Ann Arbor, MI, USA). Nicotinamide adenine dinucleotide (NAD+), nicotinamide mononucleotide (NMN), doxycycline hyclate, and puromycin dihydrochloride were purchased from Sigma-Aldrich (St.

Louis, MO, USA). 14C-Acetate was purchased from PerkinElmer (Boston, MA,

USA) and 14C – Choline from American Radiolabeled Chemicals (St. Louis, MO,

USA). Retinoic acid (all-trans-Retinoic acid; ATRA) and 5-Azacytidine (Aza) were obtained from Sigma-Aldrich (St. Louis, MO, USA). All primers for qPCR were purchased from Integrated DNA Technologies (San Jose, CA, USA).

117

Cell Culture, drug treatments, and transfection

LNCaP, PC-3, and DU145 cell lines were obtained from the American

Type Culture Collection (ATCC, Manassas, VA, USA). Normal prostate epithelial cells (PRECs) were obtained from Lonza (Switzerland). Cells were routinely subjected to mycoplasma testing. PCa cell lines were cultured in RPMI 1640

(Gibco) supplemented with 10% fetal bovine serum (FBS), Penicillin (100 units/ml), and Streptomycin (100 µg/mL) at 37°C and 5% CO2. PRECs were cultured in CloneticsTM prostate cell growth medium as directed by the manufacturer. Cells were monitored for mycoplasma on a quarterly basis. Cell lines and clones were used over 10-20 passages following thawing. FK866 was dissolved in DMSO and used at the concentrations and times as indicated.

Vehicle control cells were treated with DMSO (0.01%). NMN (500µM) and NAD+

(100µM) were dissolved in sterile water and cotreated with FK866 at the time of administration.

For transfection, cells were cultured in complete RPMI 1640 and 10% FBS without antibiotics for 48Hrs prior to seeding. After seeding, cells were transfected with varying amounts (500ng-2µg) of plasmid DNA containing either the CD38 gene under the direction of a doxycycline inducible promoter or the same vector without CD38. Cells were transfected using Lipofectamine LTX with

Plus Reagent per manufactures instructions (Thermo Fisher, Waltham, MA

USA). Transfected cells were selected with puromycin (4µg/ml) (Sigma-Aldrich,

St. Louis, MO, USA) until individual colonies were formed. Clonal populations were established from single colonies and maintained on puromycin (1µg/ml) to

118 retain selection and CD38 was induced with doxycycline (1µg/ml) for the indicated times.

Immunohistochemistry of CD38 expression

CD38 staining intensity was determined from benign, prostatic intraepithelial neoplasia (PIN), and prostate adenocarcinoma cores from a tissue microarray obtained from BioMax Inc. (Rockville, MD, USA). Slides were deparaffinized and rehydrated by xylene/ethanol wash. Slides were then incubated with antigen unmasking solution in a pressure cooker at 97°C for 30 minutes. Cooled slides were blocked in 1% bovine serum albumin (BSA) in 1x phosphate-buffered saline (PBS) for 10 minutes at room temperature. CD38 antibody was incubated at a 1:100 dilution in 1% BSA overnight at 4°C. Slides were washed three times with PBS and incubated with horseradish peroxidase

(HRP) secondary antibodies, incubated for 10 minutes in 3, 3-diaminobenzidine

(DAB) solution, washed three times in 1x PBS, and counterstained with hematoxylin. TMA cores were independently scored by staining intensity ranging from 0 to 3 with 0 representing the lowest intensity.

Western blotting

Cells were treated as indicated for individual experiments, harvested, washed with 1x cold PBS, and lysed in buffer containing 1% Triton X-100, 20mM

Tris-HCl (pH 8.3), 5mM EDTA, and protease inhibitors (1mM sodium orthovanadate, 1mM sodium fluoride, 200nM okadaic acid, 5µg/ml aprotinin, pepstatin, and leupeptin, and 200µM PMSF). Protein concentrations for individual samples were assayed using BioRad DC reagents, using BSA as a standard

119 curve. Samples were resolved via SDS-PAGE, and transferred to nitrocellulose blotting membrane. Membranes were blocked and incubated with primary and secondary antibodies as previously described (18). Enhanced chemiluminescence (Perkin Elmer Life science, Boston, MA, USA) was used to detect immuno reactive bands.

Total NAD+ quantification

Quantification of total cellular NAD+ was performed using an EnzyChrom

NAD+/NADH Assay Kit (Hayward, CA, USA) as described previously (18). Briefly, cells were seeded (1.5 x 105 cells/dish) in triplicate in 10cm dishes and treated as indicated. After treatement, cells were harvested by trypsinization, counted, and

NAD+ determined according to manufacturer’s instructions. Total NAD+ levels were normalized to protein from an equivalent number of cells and made relative to vehicle treated cells.

Cell count and viability assays

PCa cells were plated in 6-well plates in triplicate and allowed to adhere for 24Hrs prior to treatment with doxycycline (1µg/ml) or ATRA (Sigma-Aldrich,

St. Louis, MO, USA). At the indicated time points, cells were trypsinized, pelleted, and resuspended in a known volume. Cell counts were established using a

Countess automated cell counter. Live versus dead cells were quantified by trypan blue exclusion using a Countess automated cell counter. Cell growth was determined by normalizing cell numbers at the indicated time points to the number of cells plated. Doubling times were determined using relative growth values from T24 and T96 time points.

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The viability of cells expressing CD38 treated with FK866 was determined using Cell Titer-Glo 2.0 (Promega, Madison, WI, USA). Briefly, cells treated with vehicle or doxycycline (1µg/mL) were plated in 96-well plates (N=7) and allowed to adhere for 8Hrs prior to the addition of FK866. Luminescence was detected using a FLUOstar Optima spectrometer (BMG Labtech, Cary, NC, USA) and viability was determined relative to control.

Cell Cycle Analysis

PCa cells were treated with doxycycline (1µg/ml) or vehicle for 96Hrs.

They were then trypsinized, washed twice in cold PBS, and fixed in cold 70% methanol for 30 minutes at 4°C. Following fixation, cells were washed twice with

PBS and treated with RNase A (100µg/ml) for 15 minutes (Thermo Fisher,

Waltham, MA, USA) followed by staining with propidium iodide (50µg/ml)

(Invitrogen, Carlsbad, CA, USA). Flow cytometry was performed on a Becton

Dickinson Accuri C6 analyzer (Franklin Lakes, NJ, USA) and analyzed using

FlowJo V10 software (FlowJo, LLC., Ashland, Oregon, USA). The percent of cells in each phase of the cell cycle was determined using the Watson

Paragmatic algorithm available in the FlowJo software.

Determination of CD38 gene methylation status

PCa cells were seeded into 10cm dishes and allowed to adhere overnight before treating with ATRA (1.2µM) or azacytidine (5µM) for 96Hrs (Sigma-

Aldrich, St. Louis, MO, USA). They were then trypsinized, washed twice with

PBS, and lysed in RLT lysis buffer supplemented with 2-Mercaptoethanol

(140µM) (Sigma-Aldrich, St. Louis, MO, USA). DNA/RNA purification was

121 performed using an AllPrep DNA/RNA Mini Kit (Qiagen, Germantown, MD, USA).

Pyrosequencing assays were designed using PyroMark Assay Design 2.0

(version 2.0.1.15) to sequence two target regions. Region 1 sequenced the genomic region chr4:15,780,321-15,780,394 (Human genome build

GRCh37/hg19) and contained 13 CpG sites. Primers for region 1 were as follows: forward, 5’ –TGGGTATTGAGGGGATA GTAG-3’; reverse (biotinylated),

5’-AACCCCAAACTCCCTACTCAACA-3’; sequencing, 5’-

TTGAGGGGATAGTAGGGT-3’. Region 2 sequenced genomic region chr4:15,780,514-15,780,586 and contained 6 CpG sites. Primers for region 2 were as follows: forward (biotinylated), 5’-TGGTGTTGAGTAGGGAGT-3’; reverse, 5’-CCAC AAACTTTACAAACCACTTAAATATCC-3’; sequencing, 5’-

AAAATAAACTACAAC-3’. Genomic DNA (1μg) from each sample (N=2) was treated with sodium bisulfite using the EZ 96-DNA methylation kit (Zymo

Research, CA, USA), following the manufacturer's standard protocol.

Pyrosequencing was performed according to the PyroMarkMD protocol and sequenced with a PyroMark Q96 MD instrument. Methylation analysis was performed with Pyro Q-CpG (version 1.0.9).

Quantitative real time PCR gene expression analysis

PCa cells were seeded into 10cm dishes and treated with either ATRA

(1.2µM) or azacytidine (5µM) for 96Hrs (Sigma-Aldrich, St. Louis, MO, USA).

LNCaP cells treated with doxycycline (1µg/ml) for 96Hrs served as positive control. Cells were then harvested, washed twice with PBS, and lysed in RLT lysis buffer supplemented with 2-Mercaptoethanol (140µM) (Sigma-Aldrich, St.

122

Louis, MO, USA). DNA/RNA purification was performed using an AllPrep

DNA/RNA Mini Kit (Qiagen, Germantown, MD, USA). qPCR was performed using a Roche LightCycler II and 1-Step Brilliant SYBR Green qRT-PCR master mix (Agilent Technologies, Santa Clara, CA, USA). Forward and reverse primer sequences for CD38 primer sets 1 and 2 and β-ACTIN were as follows: CD38

Forward 1: 5’-CGCGATGCGTCAAGTACACTGAA-3’, CD38 Reverse 1: 5’-

CGGTCTGAGTTCCCAACTTCATTAG-3’; CD38 Forward 2 : 5’-

GGAGAAAGGACT: GCAGCAACAACC-3’, CD38 Reverse 2: 5’-

CTGCGGGATCCATTGAGCATCACAT-3’; β-ACTIN Forward : 5’- CGG AGT

ACT TGC GCT CAG GA – 3’, β-ACTIN Reverse: 5’ – CCA CGA AAC TAC CTT

CAA CTC CAT CAT G-3’. Roche LightCycler 489 instrument II protocol was as follows: 1x cycle at 50°C for 30Min, 1x cycle at 95°C for 10Min, 45x cycles using three-step amplification (30Sec at 95°C, 30Sec at 55°C, 1Min at 68°C), rest at

40°C. Fold changes in gene expression were determined using the ∆∆Ct method as previously described (19).

Measurement of extracellular acidification rates (ECAR) and oxygen consumption rates (OCR)

PCa cells were either pretreated with vehicle, FK866 (100nM), FK866 +

NMN (500µM), or FK866 + NAD+ (100µM) for 24Hrs or doxycycline 72Hrs prior to plating in 24-well plates and incubated overnight at 37°C (6 x 105 cells/well; XF24 plates, Agilent, Santa Clara, CA, USA). To measure ECAR and OCR, RPMI culture medium was changed to minimal DMEM assay media supplemented with glutamine (2mM) (ECAR) or XF Assay Medium supplemented with glucose

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(25mM) and sodium pyruvate (1mM) (OCR) followed by a 1Hr incubation at 37°C in the absence of CO2. Baseline ECAR and OCR were measured on a Seahorse

XF24 extracellular flux analyzer (Agilent; Santa Clara, CA, USA). Basal glycolytic and mitochondrial functions were established following injection of glucose

(80mM) or oligomycin (8µM), respectively. Maximum glycolytic and mitochondrial potential were achieved by injecting oligomycin (90µM) or carbonyl cyanide 4-

(trifluoromethoxy) phenylhydrazone (FCCP) (4.5µM), respectively. Glycolytic and mitochondrial inhibition were achieved following injection of 2-Deoxy-D-glucose

(1M) or a combination of rotenone and antimycin A (10µM each), respectively. All measurements were normalized to cell counts determined at the conclusion of each assay.

Quantification of glucose and lactate levels

PCa cells were plated in 6-well plates in triplicate and allowed to adhere overnight prior to treatment with doxycycline (1µg/ml) for 96Hrs. To determine the amount of glucose consumed or lactate produced the existing culture media was removed, cells were rinsed with PBS, and 1.5mL of fresh RPMI was added.

Samples were taken immediately following media change (T0) and after 12Hrs.

Glucose and lactate levels were determined using Biovision Glucose and Lactate

Assay Kits (Milpitas, CA, USA). Glucose and lactate concentrations were normalized to cellular protein.

Fatty acid and lipid synthesis

Fatty acid and lipid synthesis were determined by incorporation of 14C- acetate or 14C-choline into lipid as previously described (18). Briefly, 5x105 cells

124 treated with vehicle or doxycycline (1µg/ml) for 72Hrs were seeded in 24-well plates for 24Hrs (N = 4 wells per treatment). Cells were incubated with 14C- acetate (1µCi) or 14C-choline (1µCi) for 2Hrs. Cells were then harvested, washed with PBS, and lysed in hypotonic buffer (20mM Tris-HCL (pH 7.5), 1mM EDTA, and 1mM DDT). The lipid fraction was extracted using chloroform/methanol (2:1), and incorporation of labeled acetate or choline into newly synthesized lipid was determined by scintillation counting and normalized to cellular protein.

Metabolite Profiling

Metabolomic profiling was conducted on PC3 cells treated with vehicle or

FK866 (100nM) for 24Hrs (N = 4). Samples were collected and submitted for analysis as directed by Metabolon, Inc. Onsite sample preparation was carried out using an automated MicroLab STAR© system utilizing a series of proprietary organic and aqueous extractions to remove the protein fraction. Samples were analyzed on gas chromatography/ mass spectrometry (GC/MS) and liquid chromatography/ tandem mass spectrometry (LC/MS2) platforms and analyzed against known libraries of purified compounds as previously described (20).

Statistical significance for pair-wise comparisons was determined using Welch’s t-tests and/or Wilcoxon’s rank sum tests. Compound clustering and classification was performed using random forest analyses (21). All statistical modeling was performed using R.

Genomic Analysis

RNASeq analysis was conducted in the Cancer Genomics Shared

Resource of Wake Forest Baptist Comprehensive Cancer Center. LNCaP cells

125 transfected with either an empty vector or CD38 expression construct were plated (5 x 105 cells/dish) in 10cm dishes and treated with doxycycline (1µg/ml) for 96Hrs (N = 5 per group). Total RNA was purified from cell lines using the

RNeasy Plus Micro Kit (Qiagen, Germantown, MD, USA) with genomic DNA removal. RNA integrity (RIN) was assessed via electrophoretic tracing on an

Agilent Bioanalyzer. RNASeq libraries were constructed from samples (RIN>7.0) using an Illumina TruSeq Stranded Total RNA kit with Ribo-Zero Gold rRNA depletion. Libraries were sequenced on an Illumina NextSeq 500 DNA sequencer using 75-nt single end reads generating 45 to 55 million uniquely mapped reads/sample. The quality of raw sequencing reads was evaluated by FASTQC analysis (Babraham Bioinformatics). Read alignment was conducted using the

STAR sequence aligner and gene counts established using Feature Counts software (22,23). Differentially expressed genes (DEGs) were verified using the

DESeq2 algorithm and statistically significant results defined as p ≤ 0.05 after adjusting for false discovery (Benjamini-Hochberg) (24).

Gene set enrichment analysis was performed as described by

Subramanian et al. (25). Briefly, genes with base mean values below five reads were eliminated from analysis and log2 fold changes were arranged in descending order. Gene symbols were loaded into GSEA software and run against hallmark gene symbol v6.0 with a false collapsable data set run at 1000 permutations.

CD38 gene expression and methylation status were investigated using the

TCGA prostate adenocarcinoma (PRAD) provisional dataset (N = 491) as

126 accessed through the cBioPortal Web Resource (26). Methylation (HM450) beta- values for CD38 were plotted against CD38 RNASeq (V2 RSEM) expression values using the cBioPortal query interface (http://www.cbioportal.org/). Pearson and Spearman correlations were used to evaluate the relationship between the two measures.

Survival analysis of SLC22A3 expression as a predictor of biochemical recurrence was performed using GraphPad Prism version 5.00 for Windows (San

Diego, CA, USA). Survival and expression data was derived from patient samples from two independent cohorts (27,28). SLC22A3-low expression was defined as values one standard deviation below the mean relative to the remainder (SLC22A3-High). Statistical significance was derived using log rank

(Mantel Cox) analysis.

Statistical Analysis

Data were graphically displayed using bar-charts or box-plots to allow groups to be compared visually. Pairwise comparisons were made using unpaired two-tailed t-tests. Groups were compared using one-way analysis of variance (ANOVA) models. If there was evidence of an overall difference among groups (i.e., significant F-test in ANOVA) subsequent pairwise comparisons were made using multiple comparison adjustments Tukey’s HSD (honest significant difference) as appropriate to determine which groups differed from each other.

For gene expression analysis, a linear contrast was fit in the ANOVA model to examine whether there was a linear relationship in the outcome going across the groups ordered Benign, PIN, PCa, Met-HR. Levels of significance were denoted

127 by three groupings, *p ≤ 0.05, **p ≤ 0.01, and ***p ≤ 0.001, comparisons with p- values between 0.01 and 0.05 considered as marginally significant to account for the multiple comparisons being made, whereas p-values less than 0.01 were considered as significant accounting for multiple comparisons.

2.4 Results

CD38 expression is inversely correlated with prostate cancer progression.

In order to understand the role of CD38 in PCa, protein expression was first determined in normal prostate epithelial cells (PREC) and the tumorigenic

C4-2, PC3, LNCaP, and DU145 cell lines. PREC’s expressed CD38 whereas expression was nearly absent in the tumorigenic lines (Figure 1A). Consistent with CD38 expression and its reported NAD’ase activity, PREC’s had lower total

NAD+ levels compared to tumorigenic lines with 2.5 nmole/µg protein, whereas the tumorigenic lines ranged from 5.23-11.8 nmoles/µg protein (p ≤ 0.05, Figure

1A). It has been demonstrated that low CD38 expression is associated with both biochemical recurrence and metastasis (16). The GEO dataset GSE6099 was used to delve deeper and determine if CD38 expression is decreased with PCa progression (29). ANOVA suggested a highly significant group effect between benign, prostatic interepithelial neoplasia (PIN), PCa, and metastatic hormone refractory (Met-HR) disease states (F = 23.5, p ≤ 0.001) (Figure 1B). Pairwise comparisons revealed benign samples had significantly higher CD38 expression relative to each of the other stages (p ≤ 0.001). Furthermore, expression of CD38 in PIN margins was greater relative to both PCa (p ≤ 0.05) and Met-HR (p ≤

128

0.019). There was no difference between PCa and Met-HR. Linear contrast analysis (1 degree of freedom) of the trend in expression across all four groups was found to be highly significant (F = 54.33, p ≤ 0.001) (Figure 1B). CD38 protein expression was also determined by immunohistochemistry (IHC). ANOVA followed by post-hoc Student-Newman-Keuls analysis demonstrated a 24% reduction of CD38 staining intensity in benign vs. PIN, 36% decrease in PIN vs

PCa, and a 50% decrease in benign vs PCa (p ≤ 0.001, Figure 1C). Overall these results indicate loss of CD38 expression during PCa progression occurs in a stepwise manner and is correlated with elevated NAD+ levels.

Expression of CD38 reduces total NAD+ levels and inhibits cell proliferation.

To determine if CD38 acts as a NAD’ase in PCa, total NAD+ levels were determined following CD38 induction. Doxycycline induction of CD38 in DU145,

LNCaP, and PC3 cells was confirmed by western blot and was sufficient to reduce total NAD+ levels in DU145 cells by 31%, in LNCaP cells by 29%, and in

PC3 cells by 91% relative to vehicle (p ≤ 0.05, Figure 2A). DU145 empty vector cells treated with doxycycline showed no significant change in total NAD+ (Figure

2A). Sirtuins require NAD+ for their catalytic activity and FOXO1 is one SIRT1 substrate whose activity is regulated by acetylation and affected by changes in

NAD+ levels (30). Therefore, we next determined the impact of CD38 on FOXO1 acetylation. Expression of CD38 increased FOXO1 acetylation 3.4-fold, similar to what was observed following treatment with Sirtinol (Figure 2B) or the NAMPT inhibitor FK866 (Figure 2B). The effect on acetylation was independent of CD38-

129 induced change in Sirt1 and 3 expression (Figure 2C). The impact of CD38 expression on cell proliferation was determined by trypan blue exclusion in all three cell lines. CD38 induced a significant proliferation blockade at multiple time points with the most significant effects occurring in DU145 (34%, p ≤ 0.01),

LNCaP (32%, p ≤ 0.001), and PC3 (21%, p ≤ 0.001) cells at 96Hrs post induction, without increasing dead cells. This demonstrates that CD38 is sufficient to inhibit proliferation by limiting NAD+ (Figures 2D, G, and J).

Decreased cell numbers were accompanied by a small but significant G0/G1 blockade and subsequent reduction of cells in S and G2/M (Figures 2E, H, and

K). Moreover, CD38 expression increased the doubling time of DU145 cells from

44 hrs to 55.6 hrs, of LNCaP cells from 36 hrs to 44.5 hrs, and of PC3 cells from

44 hrs to 59.8 hrs (p < 0.01) (Figures 2F, I, and L). Although CD38 reduced proliferation, cells expressing CD38 remained sensitive to NAMPT inhibition by

FK866, as has been previously demonstrated in pancreatic cancer (17).

Exposure of CD38 positive cells to low dose FK866 (2.5nM) was sufficient to reduce viability by 17% relative to vehicle controls (p ≤ 0.01). Higher doses of 5 and 10nM had more robust effect leading to a 36% and 39% reduction in viability, respectively (p ≤ 0.001, Figure 2M). These data reveal the critical requirement for

NAD+ by PCa cells.

ATRA induces expression of CD38 independent of changes to epigenetic regulation.

There is a retinoic acid response element (RARE) in the CD38 promoter and RA can induce CD38 expression in some cells (31,32). PCa cells were

130 treated with all-trans retinoic acid (ATRA) to see if CD38 expression can be induced and overcome silencing. Treatment with ATRA was sufficient to induce expression of CD38 in three PCa cell lines, although there was no dose effect over the range tested. ATRA also reduced total NAD+ in LNCaP and DU145 cells (p ≤ 0.01), which correlated with a significant decrease in cell number (p ≤

0.01, Figure 3B-C). These data provide another demonstration of the connection between CD38 and NAD+ in PCa. The presences of CpG islands spanning exon

1 and intron 1 of the CD38 gene can be methylated (33,34). The Cancer

Genome Atlas (TCGA) prostate adenocarcinoma (PRAD) provisional data set demonstrated that there is a significant inverse correlation between CD38 expression and CD38 methylation (Pearson R = -0.667, Spearman = -0.826) suggesting that CD38 may be subject to negative regulation by methylation in

PCa (Figure 3D). LNCaP and PC3 cells were treated with ATRA and azacytidine to determine their impact on the methylation status of 19 potential methylation sites (Figure 3E and G). While there is some difference in the overall methylation status of CD38 amongst these sites, ATRA did not impact the methylation status of either site, despite significant CD38 expression in LNCaP and PC-3 cells (p ≤

0.001, Figure 3F and H). On the other hand, azacytidine reduced the overall methylation of CD38 in PC3 (p ≤ 0.05) but not LNCaP cells while increasing

CD38 expression in both cell lines (p ≤ 0.001, Figure 3F and H). These data suggest that ATRA can induce CD38 expression, but not through changes in the

CpG sites analyzed here.

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CD38 reduces glycolytic and mitochondrial capacity.

The role of CD38 in regulating cellular metabolism was determined at several levels. The effect of CD38 on glycolysis was determined by seahorse analysis. Figure 4A illustrates the influence of CD38 on the extracellular acidification rate (ECAR) of DU145 cells. Quantification of the ECAR demonstrates that CD38 reduced basal glycolysis and glycolytic capacity by 32% and 44%, respectively (p ≤ 0.05, Figure 4B). Similarly, CD38 in PC3 cells reduced basal glycolysis by 27% and glycolytic capacity by 30% (p ≤ 0.05, Figure

4C). CD38 did not affect either basal glycolysis or glycolytic capacity in LNCaP cells (Figure 3D). Mitochondrial stress assays were performed to determine the effect of CD38 expression on cellular oxygen consumption rates (OCR). Figure

4E illustrates the influence of CD38 on the OCR of LNCaP cells. When quantified, basal respiration was reduced by 24% (p ≤ 0.05), maximal respiration by 45% (p ≤ 0.01), and spare respiratory capacity by 25% (p ≤ 0.05) (Figure 4F).

In PC3 cells, CD38 reduced basal respiration by 43% (p ≤ 0.001) maximal respiration by 39% (p ≤ 0.01), and spare respiratory capacity by 27% (p ≤ 0.05)

(Figure 4G). In DU145 cells, CD38 reduced basal OCR by 36% (p ≤ 0.05), maximal respiration by 39% (p ≤ 0.05), and spare respiratory capacity by 35% (p

≤ 0.05) (Figure 4H). In accordance with decreased ECAR, CD38 reduced glucose consumption from 14.3 to 7.1 µM/µg protein and 15.2 to 8.1 µM/µg protein in DU145 and PC3 cells, respectively (p ≤ 0.001, Figure 4I and J). A concomitant decrease in lactate production from 23.2 to 18.4 µM/µg protein in

DU145 cells (p ≤ 0.001) and 25.3 to 16 µM/µg protein in PC3 cells (p < 0.001)

132 accompanied decreased glucose consumption (Figure 4I and J). These data provide novel insight into metabolic regulation by CD38 and suggest CD38 silencing is required for full metabolic capacity in PCa cells.

Pharmacological inhibition of NAMPT reduces glycolytic and mitochondrial capacity.

Because CD38 expression may mimic inhibition of NAD+ synthesis, the metabolic consequences of NAMPT inhibition were determined. Figure 5A illustrates the impact of NAMPT inhibition by FK866 on the ECAR of PC3 cells.

NAMPT inhibition reduced basal glycolysis and glycolytic capacity by 30% and

49%, respectively (p ≤ 0.001, Figure 5B), both of which were fully restored with exogenous NMN or NAD+. Similar results were observed in DU145 cells where basal glycolysis was reduced by 29% (p ≤ 0.05) and glycolytic capacity by 40%

(p ≤ 0.05) (Figure 5C). Similar to the effects of CD38 on ECAR, there were no observed differences in LNCaP cells following NAMPT inhibition (Figure 5D). We next determined the consequence of NAMPT inhibition on OCR in PC3 cells

(Figure 5E). Maximal respiration and spare respiratory capacity were reduced by

37% (p ≤ 0.01) and 35% (p ≤ 0.01), respectively (Figure 5F). Supplementation of

NMN and NAD+ rescued the effect of FK866. DU145 cells had a more pronounced reduction in maximal respiration and spare respiratory capacity, being reduced by 54% (p ≤ 0.01) and 56% (p ≤ 0.01), respectively (Figure 5G).

No changes in either measurement were detected in LNCaP cells (Figure 5H).

To compliment glycolytic and mitochondrial flux assays, metabolite levels were measured in PC3 cells following NAMPT inhibition (Figure 5I-K). As

133 expected, treatment with FK866 reduced the NAD+ metabolites nicotinamide,

NAD+, NADH, and NADP+ by 87%, 86%, 71%, and 95% relative to vehicle, respectively (p ≤ 0.001, Figure 5I). Metabolites involved in glycolysis upstream of glyceraldehyde phosphate dehydrogenase (GAPDH), the reaction in glycolysis that utilizes NAD+ as a cofactor, were increased. Specifically, glucose, glucose-6- phosphate, and sorbitol were increased by 56%, 91%, and 35% relative to vehicle, respectively (p ≤ 0.05, Figure 5J). Lactate, the byproduct of glycolysis, was decreased by 14% (p ≤ 0.05, Figure 5J). Metabolites in the tricarboxylic acid

(TCA) cycle, which contains multiple enzymes that utilize NAD+ as a cofactor, were also affected. Citrate and succinate were reduced to 77% (p ≤ 0.05) and

36% (p ≤ 0.001) of vehicle (Figure 5K). Fumarate and malate, metabolites upstream of malate dehydrogenase, an NAD+ dependent enzyme, were increased 207% (p ≤ 0.001) and 218% (p ≤ 0.001) relative to vehicle (Figure 5K).

These data directly align with the effects of CD38 on glycolytic and mitochondrial metabolism, providing additional evidence to the importance of NAD+ balance, and that CD38 expression closely mimics NAMPT inhibition.

Expression of CD38 Activates AMPK and Inhibits Fatty Acid and Lipid

Synthesis.

We previously reported that NAMPT inhibition activates AMP-activated protein kinase (AMPK) and subsequently inactivates acetyl-CoA carboxylase 1

(ACC1), resulting in decreased fatty acid and lipid synthesis (18). Similarly, CD38 increased the pAMPK/AMPK ratio in PC3, DU145 and LNCaP cells by 1.86-fold,

3.95-fold, and 1.79-fold, respectively (Figure 6A). Consistent with AMPK

134 activation, the ratio of pACC to total ACC in PC3, DU145 and LNCaP cells increased by 2.39-fold, 10.85-fold, and 1.94-fold, respectively. There was little change in FASN levels (Figure 6A). In line with inactivation of ACC1, incorporation of 14C-acetate into fatty acid was reduced by 34% in PC3 cells,

20% in DU145 cells, and by 48% in LNCaP cells (p ≤ 0.01, Figure 6B).

Expression of CD38 had similar effects on the incorporation of 14C-choline into the lipid fraction. Lipid synthesis was reduced by 35%, 32%, and 26% in PC3,

DU145 and LNCaP cells, respectively (p ≤ 0.01, Figure 6C). Taken together, these results indicate that CD38 impedes energy production and substrate flow, resulting in activation of AMPK and the subsequent inhibition of fatty acid and lipid synthesis.

CD38 reprograms the transcriptome of prostate cancer cells.

Because CD38 affected multiple metabolic processes, RNASeq was used to determine the extent to which CD38 was altered gene expression and biological processes on a global scale. Of the 34,000 genes analyzed, 14,409

(41%) genes had significant differential expression following induction of CD38 (p

≤ 0.05, FDR-adjusted). The data were further gated to include only transcripts with a log2 fold-change of 1 ≤ x ≤ -1, revealing 1082 (3.1%) up-regulated and 828

(2.4%) down-regulated genes (data not shown). Gene set enrichment analysis was performed to define molecular signatures consistent with the RNASeq expression profile (Figure 7A- C). Consistent with the effects of CD38 on proliferation, multiple pathways associated with reduced proliferation were affected by CD38 expression. Among them, the E2F targets (q-value ≤ 0.0001)

135 were the most significant (Figure 7A). As an example, CDKN1A expression was increased 2.85-fold by CD38 expression, consistent with a non-proliferative phenotype. Similar to what has been reported in pancreatic cancer, CD38 expression caused an increase in p21Cip1 protein expression of 1.53-fold, 6.0-fold, and 1.96-fold in PC3, DU145, and LNCaP cells, respectively, following CD38 expression. Other pathways, including Notch signaling (q-value = 0.005), and

G2M checkpoint (q-value ≤ 0.0001), were also highly correlated as signatures that exhibited decreased expression following induction of CD38 (Figures 7B-C).

As each pathway carries an important role in cell proliferation, these data establish a connection between expression of CD38 and a reprogramming of the transcriptome in favor of a non-proliferative phenotype. Although the metabolic consequences of CD38 expression in PCa were broad, it appears that the effects are primarily attributed to changes in cellular NAD+ and not decreases in metabolic gene expression. In general, the relative gene expression levels of enzymes involved in glycolysis, the TCA cycle, fatty acid synthesis, and the electron transport chain (ETC) were below the 2-fold cut off despite multiple transcripts achieving statistical significance (Table 1). It is important to note that of the glycolytic enzymes, there was no change in glyceraldehyde phosphate dehydrogenase (GAPDH), the only enzyme in glycolysis that requires NAD+. The expression levels of genes encoding TCA cycle enzymes were similarly unaltered, with the greatest changes occuring in pyruvate dehydrogenase kinase

(PDK1) (1.49-fold), aconitase 1 (ACO2) (1.5-fold), and isocitrate dehydrogenase

1 (IDH1) (1.5-fold). These data provide further evidence that decreased

136 mitochondrial output is not the result of decreased gene expression but rather limited availability of NAD+ to facilitate enzymatic activity. The single greatest change of any metabolic gene occurred in FASN, which was reduced 1.92-fold (p

≤ 0.0001, FDR-adjusted), although there was no detectable change in protein levels. In addition to gene set enrichment analysis, the most differentially expressed gene in the data set was selected for analysis. SLC22A3 expression increased more than 300-fold (Figure 7D). SLC22A3 protein expression was also increased in LNCaP cells following induction of CD38 and NAMPT inhibition

(Figure 7D). Interestingly, the single nucleotide polymorphism (SNP) rs936455 in

SLC22A3 is the single most highly correlated SNP with PCa (35). SLC22A3 is also one of the most significantly down-regulated genes in high-Gleason grade tumors (29). Therefore, the correlation between biochemical recurrence and

SLC22A3 expression was determined in the Taylor and Yu cohorts which contain

181 primary and 37 metastases or 66 primary and 83 normal (adjacent or donor) samples, respectively (27,28). Consistent with the inverse association between

SLC22A3 and high-Gleason grade tumors, high SLC22A3 was associated with increased time to biochemical recurrence in the Taylor and Yu cohorts (Figure

7E). These data establish a novel connection between CD38 and cellular processes that regulate proliferation and are associated with PCa progression.

137

2.5 Discussion

Tumor cells require an available pool of NAD+ for appropriate metabolic control and post-translational protein modification, among other functions, to support survival and proliferation. Intracellular NAD+ levels can be modulated by increased synthesis, in particular through the salvage pathway, where NAMPT is the rate-limiting enzyme. In fact, NAMPT expression is increased in several cancers, partially illustrating the importance of NAD+ in tumor biology (36). One of the principle roles of NAD+ is as a cofactor in metabolic reactions, serving to support hydride transfer reactions. While these reactions affect whether NAD+ is oxidized or reduced, they do not consume or alter NAD+ levels. CD38 is the primary NAD’ase in mammalian cells (10). Here we demonstrate that CD38 expression is decreased in PCa and regulates the metabolic potential of PCa cells. This provides a mechanistic explanation for why loss of CD38 in prostate luminal progenitor cells results in PCa, particularly in response to inflammation

(16).

Although we and others have demonstrated that CD38 expression is reduced in PCa, the mechanism by which CD38 expression is decreased is unknown. The CD38 gene is located on chromosome 4p15.32; a region not frequently lost in PCa (37). Using the TCGA prostate adenocarcinoma (PRAD) provisional data set, we establish a strong negative correlation between CD38 methylation and expression. This suggests that epigenetics could be a determinant of CD38 expression in PCa. Interestingly, CD38 has CpG islands that span exon 1 and intron 1 that may be potential epigenetic regulatory sites

138

(34). Moreover, CD38 also has a retinoic acid response element (RARE) in its promoter region (33). The data demonstrate that ATRA is sufficient to induce

CD38 expression, but not through the CpG sites tested. Similarly, azacytidine can induce CD38 expression, although likely through other CpG sites as well since expression in LNCaP was independent of changes in the examined sites.

Overall, the data suggest that strategies to induce CD38 expression could be one mechanism to decrease NAD+ in PCa and perhaps affect therapeutic response and hint that there are epigenetic or other elements in CD38 that regulate its expression.

The data also demonstrate a direct link between CD38 and metabolic capacity in PCa. Expression of CD38 reduced the glycolytic and mitochondrial potential of PCa cell lines reflected by decreases in ECAR and OCR. This is in line with decreased NAD+ levels and reduced proliferation. Interestingly, decreased CD38 expression in PCa is opposite of that in aging where CD38 expression increases with age (38). On the other hand, in both cases CD38 regulates mitochondrial metabolism. It is worth noting that, while CD38 reduced the OCR in LNCaP cells, there was no observed effect on ECAR. This is in line with previous reports, including ours, that some cells are resistant to NAMPT inhibition, perhaps indicating that NAD+ can exist in distinct pools in some cells or tissues (18,39,40). It appears that most of the metabolic consequences of CD38 expression are due to diminished total NAD+ as the majority of metabolic enzymes showed no change at the mRNA level following CD38 expression.

However, it does not rule out the enzymes being affected by acetylation as CD38

139 induces acetylation of the Sirt1 substrate FOXO1 and also affects the acetylation status of P53 (41). Because sirtuins can regulate metabolic processes including

ATP production, anti-oxidative stress response, lipid synthesis, and the metabolic switch to anaerobic glycolysis (42,43) we posit that CD38 impacts cellular metabolism through acetylation and depleted metabolite flow by limiting available

NAD+.

PCa has a lipogenic phenotype indicated by high expression of lipogenic enzymes and increased uptake of metabolic tracers that indicate lipogenesis

(44). We previously reported the link between NAD+ synthesis and lipogenesis in

PCa (18). In line with this, CD38 expression induced AMPK activation and subsequent phosphorylation of the canonical AMPK substrate ACC1.

Accordingly, CD38 reduced fatty acid and lipid synthesis, suggesting that reduced CD38 expression is required in some part to support the lipogenic phenotype associated with PCa. Interestingly, CD38 does not have a significant impact on ACC1 or FASN expression, suggesting that CD38 regulates bioenergetics and substrate flow through the pathway, but that oncogenic drivers control enzyme expression. It is noteworthy that CD38 expression in PCa mimics the metabolic consequences of NAMPT inhibition, as assessed by OCR, ECAR, and lipid synthesis, suggesting that alterations in NAD+ levels, regardless of the cause, yield similar results.

Although CD38 clearly affects cellular metabolism, it also impacts pathways that control proliferation, including Notch signaling, E2F targets, and

G2M checkpoint. Notch signaling has both pro and anti-proliferative potential in

140

PCa and is linked to metastatic disease (45,46). Interestingly, Notch signaling can be triggered by Sirt1 activation and modulated in response to variations in

NAMPT expression (47). Additionally, activated NOTCH is a known repressor of p21Cip1 expression in PCa (48). Therefore, it is reasonable that CD38 expression suppressed NOTCH signaling and increased p21Cip1 expression in the E2F signaling hallmark. In addition to indicators of reduced proliferation, CD38 also affects SLC22A3 expression. Decreased SLC22A3 expression is associated with tumor progression and poor patient survival in a variety of solid tumors including ovarian, pancreatic, and PCa (49,50). Moreover, SLC22A3 is among the most down-regulated genes in high-Gleason grade PCa (29,51). Our data demonstrate an inverse correlation between SLC22A3 and recurrence free survival, and the same is also true for CD38 (16), suggesting a correlation between the two genes in PCa. Silencing of SLC22A3 in prostate is regulated by genetic polymorphisms in the proximal promoter and aberrant methylation of the promoter region (52). In

PCa cell lines, the SLC22A3 promoter is variably methylated, with LNCaP cells being less methylated than in DU145 and PC3 cells (52). This may explain why

CD38 induces SLC22A3 in LNCaP cells but not PC3 and DU145 cells. While the role for SLC22A3 in tumor biology remains to be determined, the loss of expression during progression is striking and suggests that it may inhibit tumor growth and metastasis. These data provide the first demonstration that metabolism can regulate SLC22A3 expression.

Collectively, these data suggest a novel connection between CD38, modulation of NAD+, and tumor cell metabolism. We propose a model in which

141 silencing of CD38 elevates NAD+ bioavailability and reprograms prostate cells in favor of macromolecule biosynthesis and a pro-proliferative phenotype. The finding that low CD38 expression in luminal progenitor cells is sufficient to initiate

PCa (16), combined with our metabolic and gene expression data, provides a critical connection between CD38, NAD+ metabolism, and PCa progression.

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2.6 Figures

Figure 1: CD38 expression is inversely correlated with prostate cancer progression. A, CD38 protein expression was determined by western blot in

PREC and PCa cells. Colorimetric determination of total NAD+ levels in PREC and PCa cell lines (N = 3). B, Quantification of relative CD38 expression from patient samples contained in GEO database GSE6099 containing benign (N =

22), PIN (N = 13), PCa (N = 29), and Met-HR (N = 17) samples (29). C, CD38 expression was determined by immunohistochemistry (IHC) on a tissue microarray containing benign (N = 37), PIN (N = 17), and PCa (N = 37). Staining intensity was independently scored on scale of 0-3. (*p ≤ 0.05, **p ≤ 0.01, ***p ≤

0.001)

143

Figure 2: CD38 reduces total NAD+ and inhibits cell proliferation. A, Total

NAD+ levels were measured in PCa cells following treatement with vehicle

(dH2O) or doxycycline (1µg/ml) for 96Hrs (N = 3). Expression of CD38 and β-

144

Actin in PCa cells determined by western blot following 96Hr treatment with doxycycline (1µg/ml). B, Expression of acetylated-FOXO1, total FOXO1, and β-

Actin in PC3 cells determined by western blot in response to CD38 expression or treatment with FK866 (100nM). Treatment with Sirtinol (100µM) and Trichostatin

A (500µM) for 18Hrs served as positive control. The ratio of acetylated/total

FOXO1 was determined by densitometry of single bands using β-Actin as a loading control. C, Expression of Sirt1, Sirt 3, and β-Actin in PCa cells was determined by western blot following 96Hr treatment with doxycycline (1µg/ml).

Changes in Sirt1 or Sirt3 expression were determined by densitometry of single bands using β-Actin as a loading control. D, G, and J, Relative cell counts of live and dead PCa cells determined by trypan blue exclusion of cells treated with doxycycline (1µg/ml) for the indicated times (N = 3). E, H, and K, Cell cycle analysis of PCa cells treated with doxycycline (1µg/ml) for 96Hrs. Signal intensity of incorporated propidium iodide corresponding to DNA content was used to gate cells into G0/G1, S, and G2/M phases (N=3). F, I, and L, Doubling time of PCa cells from following 96Hr treatment with doxycycline (1µg/ml) (N=3). M, Viability of DU145 cells treated with doxycycline (1µg/ml) for 72Hrs followed by 24Hr treatment with FK866 at the indicated concentration (N=3). (*p ≤ 0.05, **p ≤ 0.01,

***p ≤ 0.001)

145

Figure 3: ATRA induces expression of CD38 independent of changes to epigenetic regulation. A, Expression of CD38 following 96Hr treatment with

ATRA at the indicated concentration. B-C, NAD+ levels and cell counts in LNCaP and DU145 cells treated for with ATRA for 96Hrs (N=3). D, CD38 expression as a function of methylation status determined using data from the TCGA prostate adenocarcinoma (PRAD) provisional data set (N = 491). E & G, Pyrosequencing data from PC3 and LNCaP cells representing the percent of methylated CG sites in two segments of intron 1 of the CD38 gene following 96Hr treatment with

ATRA (1.2µM) or azacytidine (5µM) (N=2). F & H, Fold change in CD38 mRNA in response to treatment with ATRA (1.2µM) or azacytidine (5µM) for 96Hrs. (*p ≤

0.05, **p ≤ 0.01, ***p ≤ 0.001)

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Figure 4: CD38 reduces glycolytic and mitochondrial capacity. A,

Representative trace of a glycolytic stress test in DU145 cells following treatment with doxycycline (1µg/ml) for 96Hrs to induce expression of CD38 (N = 3). B-D,

Quantification of basal glycolysis and glycolytic capacity in DU145, PC3, and

LNCaP cells treated with doxycycline (1µg/ml) for 96Hrs (N = 3). E,

Representative trace of a mitochondrial stress test in DU145 cells following treatment with doxycycline (1µg/ml) for 96Hrs (N = 3). F-H, Quantification of

147 basal respiration, maximal respiration, and spare respiratory capacity in DU145,

PC3, and LNCaP cells treated with vehicle (dH2O) or doxycycline (1µg/ml) for

96Hrs (N = 3). I-J, Quantification of glucose consumed or lactate produced in

PC3 and DU145 cells over a period of 12Hrs following 96Hr treatment with doxycycline (1µg/ml) (N=6). (*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001)

148

Figure 5: Pharmacological inhibition of NAMPT reduces glycolytic and mitochondrial capacity. A, Representative trace of a glycolytic stress test in

PC3 cells following treatment with FK866 (100nM) for 24Hrs in the absence or presence of exogenous NMN (500µM) or NAD+ (100µM) (N = 3). B-D,

Quantification of basal glycolysis and glycolytic capacity in DU145, PC3, and

LNCaP cells (N = 3). E, Representative trace mitochondrial stress test in PC3 cells following treatment with FK866 (100nM) for 24Hrs in the absence or

+ presence of exogenous NMN (500µM) or NAD (100µM) (N = 3). F-H,

Quantification of basal respiration, maximal respiration, and spare respiratory capacity in PC3, DU145, and LNCaP cells (N = 3). I–K, Scaled intensity of nicotinamide containing metabolites (I), glycolytic metabolites (J), and TCA cycle

149 metabolites (K) in PC3 cells following treatment with FK866 (100nM) for 24Hrs.

(*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001)

150

Figure 6: Expression of CD38 activates AMPK and inhibits fatty acid and lipid synthesis. A, Expression of FASN, phospho-ACC, ACC, phospho-AMPK,

AMPK, and β-Actin determined by western blot in PC3, DU145, and LNCaP cells following induction of CD38 by doxycycline (1µg/ml). Phospho-to-total ratios were determined by densitometry of single bands using β-Actin as a loading control. B,

Fatty acid synthesis in PC3, DU145, and LNCaP cells as determined by incorporation of 14C-acetate into lipid following induction of CD38 for 96Hrs (N =

3). C, Lipid synthesis in PC3, DU145, and LNCaP cells as determined by incorporation of 14C-choline into lipid following induction of CD38 for 96Hrs (N =

3). (*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001)

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Figure 7: CD38 reprograms the transcriptome of PCa cells. A, Gene set enrichment plot of genes included within RNASeq analysis with homology to an established E2F target hallmark signature (M5925) containing genes encoding cell cycle related targets of E2F transcription factors. Bar graph represents gene expression values from RNASeq data for a subset of genes contained in the E2F hallmark gene list. Expression of the cell cycle inhibitor p21Cip1 was determined by western blotting following treatment with Doxycycline (1µg/ml) for 96Hrs with

β-Actin as a loading control. B, Gene set enrichment plot of the genes in our

RNASeq analysis that are homologous to the hallmark NOTCH signaling gene set (M5903) consisting of genes up regulated by activation of NOTCH signaling.

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Bar graph represents relative expression levels of a subset of genes within the

NOTCH signaling hallmark indicative of a non-proliferative phenotype. C, Gene set enrichment plot of genes that exhibit a similar expression pattern to genes contained within the hallmark G2M checkpoint (M6901), which consists of genes involved in progression through the G2/M checkpoint. Bar graph represents relative expression of genes from RNASeq analysis contained within the G2M checkpoint hallmark consistent with a non-proliferative phenotype. D, SLC22A3 expression in LNCaP cells following induction of CD38. Expression of SLC22A3 in LNCaP cells determined by western blot following treatment with FK866

(100nM) for 24Hrs or doxycycline (1µg/ml) for 96Hrs, respectively. E, Recurrence free survival in two patient cohorts as a function of SLC22A3 expression (27,28).

(*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001)

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Table 1: CD38 does not alter metabolic pathway gene expression.

Metabolic pathway gene expression as a function of CD38. Fold changes are expressed relative to vehicle control ± standard deviation (N=5).

154

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162

CHAPTER III

CD38 Mediated Calcium Mobilization Protects Prostate Cancer Cells from

the Cytotoxic Effects of β-Lapachone

Jeffrey P. Chmielewski and Steven J Kridel

This chapter will be submitted for publication upon completion of further studies.

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3.1 Abstract

Calcium plays an integral role in regulating many processes throughout the cell including cell-to-cell signaling, mitochondrial function, and regulation of apoptosis. The cyclic (ADP)-ribose synthase CD38, the main NAD’ase in cells, is a multi-function protein capable of producing both ADP-ribose and cyclic ADP- ribose. Cyclic ADP-ribose is a potent calcium mobilizer able to release calcium by binding and activating ryanodine receptors on the endoplasmic reticulum.

Dysregulation of CD38 is reported in multiple cancer types however; to date there has not been an established role for cADPR in prostate cancer. This study demonstrates a novel connection between CD38, production of cADPR, and protection from the ortho naphthoquinone, β-Lapachone. The cytotoxic effects of

β-Lapachone are conferred by a rapid depletion of NAD+ resulting from PARP-1 hyperactivation and an accumulation of reactive oxygen stemming from a futile cycle initiated by NAD(P)H quinone oxidoreductase 1 (NQO1). Expression of

CD38 is lost in prostate cancer and consequently restoring its expression protects cells from the detrimental effects of β-Lapachone through its ability to produce cADPR and mobilize calcium rather than its NAD’ase activity. In line with

NQO1 mediated NAD+ depletion, pharmacological inhibition of nicotinamide phosphoribosyltransferase (NAMPT) dramatically sensitizes cells to β-

Lapachone. Collectively, these data demonstrate β-Lapachone may be a novel chemotherapy in the treatment of PCa given the changes in CD38 expression inherent to this disease. Furthermore, NAMPT inhibition may represent an effective strategy to sensitize cells to β-Lapachone.

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3.2 Introduction

Because PCa is the most frequently diagnosed cancer and has the third highest mortality rate in the US, there is a need to develop novel drugs to treat this disease. NAD(P)H: quinone oxidoreductase 1 (NQO1), a protease present in most eukaryotic cells, has recently received attention as it appears to play an important role in cancer chemoprevention (1). β-Lapachone is activated through its interaction with NQO1, initiating a futile cycle where it is reduced to hydroquinone oxidized to semiquinone and finally oxidized a second time back into β-Lapachone. This futile cycle generates large amounts of cytotoxic reactive oxygen species (ROS) and dramatically reduces the NAD+/NADH ratio (2,3).

Treatment of PCa cells with β-Lapachone also hyper-activates PARP-1, leading to a reduction in total NAD+ availability (4). NAD+ is required by tumor cells to maintain appropriate metabolic activity and post-translational modification. NAD+ is unique in that it acts as both a cofactor for hydride transfer reactions and as a substrate for NAD+-consuming enzymes such as the cyclic (ADP)-Ribose synthase CD38, poly(ADP)-ribose polymerases (PARPs), and sirtuins (5-7).

Intracellular NAD+ levels can either be modulated by increased synthesis, particularly through the salvage pathway where NAMPT is the rate-limiting enzyme, or through decreased catabolism from diminished activity of NAD+ consuming enzymes (7). We have previously demonstrated a link between

CD38, modulation of NAD+, and tumor cell metabolism (Chapter II). CD38 is a type-II/III transmembrane glycoprotein that consumes NAD+ as part of its catalytic activity. The primary enzymatic function of CD38 is the hydrolysis of

165

NAD+ to ADPR (8). However, CD38 can also act as a cyclase consuming NAD+ to produce the calcium mobilizing second messenger, cyclic ADP-ribose (cADPR)

(9). Alterations in CD38 expression occur in a variety of cancers, including hematological malignancies, glioma, pancreatic cancer, and prostate cancer

(PCa) (10-12). Decreased expression of CD38 in prostate luminal cells is sufficient to drive disease progression and is linked to lower overall survival (13).

Cells treated with β-Lapachone tend to die independent of the normal apoptotic pathway. Accordingly, β-Lapachone initiates cell death independent of

Bax or Bak activation as mitochondrial membrane depolarization can be achieved by PARP-1 dependent NAD+ depletion (14). β-Lapachone induced cell death also requires calcium release from the endoplasmic reticulum (ER) to activate the calcium-dependent cystein protease, calpain (15). Calcium mobilization directly regulated by CD38 is critically important for the proper contraction of smooth muscle cells, glucose stimulated insulin release in pancreatic β-islets cells, and noncanonical means of calcium mobilization, such as that in the calcium induced calcium release (CICR) response (16-20).

Therefore, we hypothesized that loss of CD38, through its diminished production of cADPR, would sensitize cells to the effects of β-Lapachone. The data presented herein demonstrate restoring expression of CD38 in PCa increases viability and clonogenic survival, prevents mitochondrial membrane depolarization, and inhibits calcium flux in response to treatment with β-

Lapachone. In addition, we provide evidence that expression of CD38 and its associated production of cADPR is sufficient to mobilize calcium PCa cell lines.

166

These effects are independent of the NAD’ase activity of CD38 as NAMPT blockade yielded strikingly different results. Collectively, our data suggest decreased expression of CD38 as well as NAMPT inhibition may uniquely sensitize PCa cells to the effects of β-Lapachone. These data also demonstrate the potential use of β-Lapachone in the treatment of PCa.

3.3 Materials and Methods

Materials

Antibodies for NAD(P)H Quinone Dehydrogenase 1 (NQO1), Poly(ADP)- ribose polymerase (PARP), and cleaved poly(ADP)-ribose polymerase (C-PARP) were obtained from Cell Signaling Technologies (Beverly, MA, USA), the antibody for β-Actin was purchased from Sigma-Aldrich (St. Louis, MO, USA), and the antibody for poly(ADP)-ribose was purchased from Trevigen

(Gaithersburg, MD, USA). Secondary antibodies for goat-anti-mouse and goat- anti-rabbit were purchased from BioRad (Hercules, CA, USA). The NAMPT inhibitor FK866 was obtained from Cayman Chemical Company (Ann Arbor, MI,

USA). Nicotinamide mononucleotide (NMN), doxycycline hyclate, and puromycin dihydrochloride were purchased from Sigma-Aldrich (St. Louis, MO, USA). 14C-

Acetate was purchased from PerkinElmer (Boston, MA, USA). The JC-1 dye

(5,5’6,6’–tetrachloro-1,1’,3,3’–tetraethyl-benzimidazolylcarbocyanine idodide),

Fluo-4 calcium imaging kit, MitoTracker Orange, ER-Tracker Red (BODIPYTM TR

Glibenclamide), LysoTracker Red DND-99, and Hoechst 33342 were purchased from Molecular Probes, Inc. (Eugene, OR, USA). EnzyChrom NAD+/NADH assay

167 kits were purchased from BioAssay Systems (Hayward, CA, USA). The CellTiter-

Glo 2.0 assay kit was obtained from Promega (Madison, WI, USA). Dicoumarol

(3,3-Methylenebis(4-hydroxycoumarin) and staurosporine were purchased from

Sigma-Aldrich (St. Louis, MO, USA). β-Lapachone (3,4-dihydro-2,2-dimethyl-2H- naph-tho[1,2b]pyran-5,6-dione) was generously provided by Dr. David A.

Boothman (Indiana University, Indianapolis, IN, USA).

Cell Culture and drug treatments

PC-3 and DU145 PCa cell lines were obtained from the American Type

Culture Collection (ATCC, Manassas, VA, USA). PCa cell lines were cultured in

RPMI 1640 (Gibco) supplemented with 10% fetal bovine serum (FBS), Penicillin

(100 units/ml), and Streptomycin (100 µg/mL) at 37°C and 5% CO2. For transfection, cells were cultured in complete RPMI 1640 and 10% FBS without antibiotics for 48Hrs prior to seeding. After seeding, cells were transfected with varying amounts (500ng-2µg) of plasmid DNA containing either the CD38 gene under the direction of a doxycycline inducible promoter or the same vector without CD38. Cells were transfected using Lipofectamine LTX with Plus

Reagent per manufacturer’s instructions (Thermo Fisher, Waltham, MA USA).

Transfected cells were selected with puromycin (4µg/ml) (Sigma-Aldrich, St.

Louis, MO, USA) until individual colonies were formed. Clonal populations were established from single colonies and maintained on puromycin (1µg/ml) to retain selection and CD38 was induced with doxycycline (1µg/ml) for the indicated times. Cell lines and clones were used for 2-20 passages following thawing and monitored for mycoplasma on a quarterly basis. FK866 was dissolved in DMSO

168 and used at the concentrations and times as indicated. Vehicle control cells were treated with DMSO (0.01%). Nicotinamide mononucleotide (NMN) was dissolved in sterile water and delivered at the same time as FK866 or β-Lapachone. β-

Lapachone was diluted to a stock concentration of 50mM in DMSO, aliquoted for one time use, and used at the indicated concentrations. Dicoumarol was resuspended in sterile water and used at 50mM when cotreated with β-

Lapachone.

Western blotting

Cells were treated as indicated for individual experiments, harvested, washed once with PBS, then lysed in buffer containing 1% Triton X-100, 20mM

Tris-HCl (pH 8.3), 5mM EDTA, and protease inhibitors (1mM sodium orthovanadate, 1mM sodium fluoride, 200nM okadaic acid, 5µg/ml aprotinin, pepstatin, leupeptin and 200µM PMSF). Protein concentrations for individual samples were assayed using BioRad DC reagents (Hercules, CA, USA), using bovine serum albumin (BSA) as a standard curve. Samples were resolved via

SDS-PAGE and transferred to nitrocellulose blotting membrane. Membranes were blocked and incubated with primary and secondary antibodies as previously described (21). Enhanced chemiluminescence (Perkin Elmer Life Science,

Boston, MA, USA) was used to detect immuno reactive bands.

Cell viability assays

The viability of cells treated with β – Lapachone was determined using

Cell Titer-Glo 2.0. In experiments involving expression of CD38, cells were treated with vehicle or doxycycline (1µg/mL) for 72Hrs, 5x103 cells were then

169 seeded into 96-well plates (N=7) and allowed to adhere for 8Hrs prior to 2Hr treatment with β-Lapachone in the presence or absence of dicoumarol (50µM) as indicated. In experiments involving NAMPT inhibition, 5x103 cells were seeded into 96-well plates (N=7) and allowed to adhere overnight. Cells were then treated with FK866 in the presence or absence of NMN (500µM) for 24Hrs prior to 2Hr treatment with β-Lapachone (5µM) in the presence or absence of dicoumarol (50µM) or NMN (500µM). After treatement with β-Lapachone all cells were rinsed with RPMI and allowed to recover overnight before assessing cell viability. Cell TiterGlo-2.0 was added to cells according to manufacturer’s instructions, luminescence detected using a FLUOstar Optima spectrometer

(BMG Labtech, Cary, NC, USA) and viability determined relative to vehicle control.

The viability of cells treated with β-Lapachone was also determined using propidium iodide (PI). In experiments involving expression of CD38, 3x104 cells were seeded in 6-well plates in triplicate and treated with vehicle or doxycycline

(1µg/ml) for 72Hrs prior to treatment with β-Lapachone. For experiments involving NAMPT inhibition, 2x105 cells were seeded in 6-well plates and allowed to adhere before being treated with FK866 for 24Hrs. Following initial treatments, all cells were treated with vehicle or β-Lapachone in the presences or absence of dicoumarol for 2Hrs, washed with RPMI, and allowed to recover overnight. Cells treated with staurosporine (1µM) for 4Hrs served as a positive control. Adherent cells were trypsinized, pelleted with unattached cells from culture media, rinsed with PBS, and resuspended in live cell imaging solution (LCIS) (130mM NaCl,

170

5mM KCl, 1.5mM CaCl2, 1mM MgCl2, 25mM HEPES, 5mM glucose, 1mg/ml

BSA, pH 7.5) containing propidium iodide (3µg/ml) for 15 minutes. Flow cytometry was performed on a Becton Dickinson Accuri C6 analyzer (Franklin

Lakes, NJ, USA) and analyzed using FlowJo V10 software (FlowJo, LLC.,

Ashland, Oregon, USA). Viability was determined relative to vehicle control.

Clonogenic survival assays

In experiments involving expression of CD38, cells were seeded in 10cm dishes and treated with vehicle or doxycycline (1µg/ml) for 48Hrs, then trypsinized and plated at low density (600 cells/well) in 6-well plates in triplicate, and allowed to adhere overnight. Cells were then treated with β-Lapachone for

2Hrs, rinsed with RPMI, and maintained until visible colonies (>40 cells/colony) were formed. Cells induced for expression of CD38 were maintained in doxycycline-supplemented media for the duration of the post-treatment period.

For experiments involving NAMPT inhibition, PCa cells were plated at low density (600 cells/well) in 6-well plates, in triplicate, and allowed to adhere overnight. Cells were then treated with FK866 ± NMN (500µm) for 24Hrs followed by a 2Hr treatment with β-Lapachone in the presence or absence of dicoumarol

(50µM) or NMN (500µM), rinsed with RPMI, and maintained in complete RPMI until visible colonies (>40 cells/colony) were formed. In all experiments, colonies were rinsed twice with PBS, fixed (10% methanol, 10% glacial acetic acid, in dH2O), stained with crystal violet staining solution (0.4% crystal violet, 20% ethanol, in dH2O), and rinsed to remove unbound crystal violet. Crystal violet bound to colonies was solubilized with 33% glacial acetic acid, optical density

171

(OD) determined using a spectrophotometer (540n), and OD values made relative to vehicle control.

Total NAD+ quantification

Quantification of total cellular NAD+ was performed using an EnzyChrom

NAD+/NADH Assay Kit (Hayward, CA, USA) as described previously (21). Briefly, for experiments involving expression of CD38, 3x105 cells were seeded into

10cm dishes in triplicate and treated with vehicle or doxycycline (1µg/ml) for

96Hrs. For experiments involving NAMPT inhibition, 5x105 cells were seeded into

10cm dishes in triplicate and allowed to adhere overnight prior to 24Hr treatment with FK866 ± NMN (500µm). All cells were treated for 2Hrs with β-Lapachone in the presence or absence of dicoumarol (50mM) or NMN (500µM) as indicated.

Cells were then harvested, counted, and total NAD+ determined using

EnzyChrom NAD+/NADH assay kits (Hayward, CA, USA). Total NAD+ levels were normalized to protein from an equivalent number of cells.

Fatty acid and lipid synthesis

Fatty acid synthesis was determined by incorporation of 14C-acetate into lipid as previously described (21). Briefly, in experiments involving expression of

CD38, 6x105 cells treated with vehicle or doxycycline (1µg/ml) for 72Hrs were seeded in 24-well plates (N=4) and allowed to adhere overnight. For experiments involving NAMPT inhibition, 6x105 cells were seeded into 24-well plates (N=4), allowed to adhere overnight, and treated with FK866 (5nM) for 24Hrs. Cells were treated with β-Lapachone in the presence or absence of dicoumarol (50µM) and

NMN (500µM) for 2Hrs as indicated followed by 2Hr incubation with 14C-acetate

172

(1µCi). Cells were then harvested, washed with PBS, and lysed in hypotonic buffer (20mM Tris-HCL (pH 7.5), 1mM EDTA, and 1mM DDT). The lipid fraction was extracted using chloroform/methanol (2:1), and incorporation of labeled acetate into newly synthesized lipid was determined by scintillation counting, normalized to cellular protein, and made relative to vehicle control.

Microscopy, calcium mobilization, colocalization

All images were captured using an Olympus IX83 inverted laser scanning confocal microscope equipped with a 100x oil immersion objective. Cells were maintained at 37°C with 5% CO2 during image acquisition. Confocal images of

Fluo-4 fluorescence were collected using a 488nm excitation light from an argon/krypton laser, a 560nm dichroic mirror, and a 500-550nm band-pass barrier filter.

For calcium mobilization assays cells were cultured in µ-Dish 35mm glass bottom culture dishes (Ibidi, Fitchburg, WI, USA). In experiments involving CD38 expression, 3x104 cells were seeded in imaging dishes and treated with vehicle or doxycycline (1µg/ml) for 96Hrs prior to analysis. Experiments involving

NAMPT inhibition, 2x105 were seeded into imaging dishes, allowed to adhere overnight, then treated with FK866 (10nM) 24Hrs. Cells were rinsed twice with live cell imaging solution (LCIS) (130mM NaCl, 5mM KCl, 1.5mM CaCl2, 1mM

MgCl2, 25mM HEPES, 5mM glucose, 1mg/ml BSA, pH 7.5), and loaded with the calcium sensitive fluorescent indicator Fluo-4AM according to manufacturer’s suggestion. β-Lapachone (8µM) was added to cells after two sets of basal images were taken from each field of view. After the addition of β-Lapacone,

173 images from each field of view were acquired approximately every 90Sec. over the course of 30 minutes. The mean pixel intensity from cells was determined using Olympus Fluoview FV10-ASW (Version 4.2) software. Basal fluorescence intensity of individual cells was compared to peak intensity approximately 25 minutes after the addition of β-Lapachone and the difference reported as the change in mean fluorescence intensity (∆MFI). The percent of cells with punctate calcium staining was scored by counting the number of cells with punctate or ubiquitous calcium staining from approximately five fields of view for three independent experiments.

For colocalization experiments, 3x104 PCa cells were seeded in imaging dishes and treated with vehicle or doxycycline (1µg/ml) for 96Hrs. Cells were then washed twice with LCIS and loaded with Fluo-4 as previously described.

Cells were colabeled with Mitotracker (5µM), LysoTracker (5µM), ERTracker

(2µM), and Hoechst 33342 (2.5µM) during the required 30Min. room temperature

Fluo-4AM activation period, washed twice with LCIS, and imaged in fresh LCIS at

37°C.

Determination of mitochondrial membrane potential

In experiments involving expression of CD38, 3x104 cells were seeded in

6-well plates in triplicate and cultured with vehicle or doxycycline (1µg/ml) for

96Hrs. In experiments involving NAMPT inhibition, 2x105 cells were seeded in 6- well plates, allowed to adhere overnight, and then treated with FK866 for 24Hrs.

Following initial treatment, all cells were trypsinized, washed with PBS, resuspended in 1mL of PBS, and then loaded with JC-1 dye (1µg/ml) at 37°C

174 and 5% CO2 for 30Mins. Cells were then pelleted, washed with PBS, resuspended at ~1x106 cells/mL and analyzed on a Becton Dickinson Accuri C6 flow cytometer (Franklin Lakes, NJ, USA). JC-1 monomer and aggregate emissions were excited at 488nM and data analyzed using FlowJo V10 software

(FlowJo, LLC., Ashland, Oregon, USA). Shifts in emission spectra were plotted on bivariant dot plots, on a cell-by-cell basis, to determine the mitochondrial membrane potential of treated and control cells.

Statistical Analysis

Data were graphically displayed using bar-charts or box-plots to allow groups to be compared visually. Pairwise comparisons were made using unpaired two-tailed t-tests and significance denoted as: *p ≤ 0.05, **p ≤ 0.01, and

***p ≤ 0.001.

3.4 Results

CD38 Expression protects cells from β-Lapachone induced cell death.

In order to understand if β-Lapachone could be a useful treatment in PCa, we first determined if NQO1 is expressed in PCa cell lines. Expression of NQO1 was present in DU145’s but absent in PC3 and LNCaP’s. Treatment with doxycycline for 96Hrs to induce expression of CD38 did not change the relative expression of NQO1 (Figure 1A). Consistent with its reported effects, DU145 cells treated with β-Lapachone had a 75% reduction in cell viability (p ≤ 0.0001)

(Figure 1B). Induced expression of CD38 conferred a 40% increase in cell viability (p ≤ 0.01) despite retained sensitivity to β-Lapachone (p ≤ 0.05). The

175 addition of dicoumarol was able to rescue viability in all experimental groups. The effect on cell viability was confirmed using the membrane impermeant dye propidium iodide (PI). When incorporated into dead cells, PI intercalates DNA increasing its fluorescence intensity 20-30 fold and shifting its excitation wavelength from red to green. Using this method of detection, treatment with β-

Lapachone reduced viability by 58% in vehicle treated cells (p ≤ 0.001) with no detectable change in cells induced for expression of CD38 (Figure 1C).

Reducing the concentration of β-Lapachone from high dose (6µM) to low dose

(4µM) failed to decrease cell viability in vehicle treated or cells induced for expression of CD38, suggesting the damaging effects of β-Lapachone are tightly controlled. Cells treated with staurosporine (1µM) served as a positive control and exhibited similar levels of cell death as CD38 nonexpressing cells treated with high dose β-Lapachone (6µM) (p ≤ 0.001). Treatment with β-Lapachone also reduced clonogenic survival by 50% in vehicle treated cells (p ≤ 0.001) with no detectable change in cells expressing CD38 (Figure 1D). β-Lapachone mediated cell death occurs independent of the normal apoptotic cascade although it does involve cleavage of poly (ADP)-Ribose polymerase 1 (PARP1) (22). Accordingly, we observed a concentration dependent increase of cPARP in cells treated with

β-Lapachone. There was no detectable accumulation of cPARP following treatment with 4µM β-Lapachone. However, vehicle treated cells had a 37%,

95%, and 130% increase in cPARP when treated with 6-10µM β-Lapachone relative to a 4%, 25% and 52% increase in cells expressing CD38, respectively

(Figure 1E). Collectively, these results indicate the toxic effects of β-Lapachone

176 are highly concentration dependent and suggest CD38 confers some form of protection.

Pharmacological inhibition of NAMPT sensitizes cells to the effects of β-

Lapachone.

To determine if NAMPT inhibition can sensitize cells to β-Lapachone,

DU145 cells were treated with varying concentrations of the NAMPT inhibitor

FK866. In isolation, β-Lapachone was sufficient to reduce cell viability by 13% (p

≤ 0.01) with no significant change in cells treated with 5nM or 10nM FK866

(Figure 2A). When cells were treated with 5nM and 10nM FK866 prior to treatment with β-Lapachone, viability was reduced by 43% and 75%, respectively

(p ≤ 0.001). Reductions in viability were rescued by the addition of both dicoumarol and nicotinamide mononucleotide (NMN); the product of the NAMPT catalyzed reaction in the salvage pathway. The effect of β-Lapachone on cell viability was confirmed cells stained with PI. In isolation, treatment with β-

Lapachone increased the percent of dead cells from 3% to 28%, whereas treatment with FK866 at either concentration had increased viability (Figure 2B).

However, treatment with 5nM and 10nM FK866 prior to low dose β-Lapachone

(4µM) increased the percent of dead cells from 5% and 7% to 28% and 30%, respectively. NAMPT inhibition at the same concentrations of FK866 and high dose β-Lapachone (6µM) increased the percent of dead cells even further to

44% and 83%, respectively. Similar to these effects, when treated together,

FK866 and β-Lapachone reduced the clonogenic survival of DU145 cells beyond that achieved in isolation indicating synergy. In isolation, high dose β-Lapachone

177 reduced clonogenic survival by only 14% (p ≤ 0.05). However, a 64% and 83% decline in clonogenic survival was observed in cells treated with 5nM and 10nM

FK866 followed by β-Lapachone, respectively (p ≤ 0.001, Figure 2C). These effects were fully rescued by treating with dicoumarol or NMN. When taken together, these results suggest pharmacological inhibition of NAMPT could represent a useful strategy to enable the use of lower therapeutic doses of β-

Lapachone in the clinic.

Expression of CD38 prevents immediate and sustained β-Lapachone induced depletion of NAD+

Because CD38 is the main NAD’ase in cells, we expected expression of

CD38 and NAMPT inhibition to yield similar results on total NAD+ levels in response to β-Lapachone. Treatment with FK866 almost completely abolished

NAD+ reducing levels from 4.13 pMole/µg protein to 0.31 pMole/µg protein (p ≤

0.001, Figure 3A). Treatment with β-Lapachone was able to reduce total NAD+ levels 50% further to 0.18 pMole/µg protein (p ≤ 0.05). While not to the extent of

NAMPT inhibition, induction of CD38 and β-Lapachone independently reduced total NAD+ levels from 6.27 (pMole/µg protein) to 3.66 and 3.92 pMole/µg protein, respectively (p ≤ 0.001, Figure 3D). However, unlike NAMPT inhibition, β-

Lapachone was insufficient to reduce total NAD+ levels beyond that achieved by induction of CD38 alone. This suggests loss of NAD+ may occur soon after the addition of β-Lapachone and be maintained for at least two hours, coinciding with our collection time point. One method of detecting the immediate effects of β-

Lapachone is the accumulation of (ADP)-ribose (PAR) polymers. PARP1 is

178 hyper-activated in response to β-Lapachone resulting in an immediate depletion of NAD+ and accumulation of PAR (4). Therefore, we assessed the time dependent accumulation of PAR in cells treated with β-Lapachone following

NAMPT blockade or induction of CD38. Syntheis of PAR was detected as soon as 5 minutes and sustained for at least 30 minutes following the addition of β-

Lapachone (Figure 3B, E). Because NAMPT inhibition essentially abolished

NAD+ levels, there was no detectable accumulation of PAR in these cells in response to β-Lapachone. On the other hand, while expression of CD38 induced a slight increase in the basal levels of PAR, peak PAR accumulation occuring 10 minutes after the addition β-Lapachone was reduced by 50% relative to vehicle

(p ≤ 0.001, Figure 3E). At 15 minutes dicoumarol was able to reduce PAR to background levels, providing additional evidence that NAD+ depletion occurs almost immediately after the addition of β-Lapachone. We next determined if long-term depletion of NAD+ by β-Lapachone could influence the rates of fatty acid synthesis (FAS), a metabolic pathway that relies heavily on the availability of the NAD+ derivative NADPH. Consistent with the effects modulating NAD+ by expression of CD38 (section 2.4) or NAMPT inhibition, incorporation of 14C- acetate into fatty acids was reduced by 23% and 21%, respectively (p ≤ 0.01,

Figure 3C , F) (23). In isolation, β-Lapachone reduced the FAS by 14-24% (p ≤

0.05) with a more dramatic 72% reduction following NAMPT blockade (p ≤

0.001). These effects were fully restored by dicoumarol or exogenous NMN.

Similar to its effects on NAD+ levels, β-Lapachone was unable to lower the rate of

FAS beyond that achieved by expression of CD38 alone. Collectively, these data

179 demonstrate the protection effects of CD38 expression extended to NAD+ levels and metabolic activity. However, these effects are independent of its NAD’ase activity as NAMPT inhibition sensitizes cells to β-Lapachone.

CD38 protects cells from β-Lapachone mediated decreases in mitochondrial membrane potential.

PAR accumulation resulting from β-Lapachone and PARP1 hyperactivation can be cytotoxic to cells and induce translocation of apoptosis- inducible factor (AIF) causing mitochondrial membrane depolarization (24).

Accordingly, because expression of CD38 blunted PAR accumulation, we determined if CD38 was sufficient to retain mitochondrial membrane potential.

Figure 4A illustrates the influence of CD38 on mitochondrial membrane potential in DU145 cells using the membrane-permeant dye JC1. In isolation, expression of CD38 and low dose β-Lapachone had no effect on membrane potential, indicated by no change in the percent of cells with polarized membranes (upper- right quadrant) (Figure 4A). Cells treated with high dose β-Lapachone (6µM) reduced the percent of cells with polarized membranes from 80% to 52% (p ≤

0.001) with 70% of these cells shifting to the depolarized (lower-right quadrant) (p

≤ 0.01) (Figure 4B). Cells induced for expression of CD38 and treated with high dose β-Lapachone (6µM) had no change in the distribution of cells to any quadrant. These results suggest CD38 mediated protection from β-Lapachone is in part confered by its ability to retain mitochondrial membrane potential.

180

Pharmacological inhibition of NAMPT sensitizes cells to β-Lapachone mediated loss of mitochondrial membrane potential.

Because NAMPT blockade sensitized cells to NAD+ depletion and induced cell death in response to treatment with β-Lapachone, the consequence of

NAMPT inhibition on mitochondrial membrane potential were also determined.

Figure 5A illustrates the impact of NAMPT inhibition by FK866 on the mitochondrial membrane potential of DU145 cells. In isolation, treatment with

5nM and 10nM FK866 had no significant effects on membrane potential.

However, NAMPT blockade 24Hrs prior to treatment with low dose β-Lapachone

(4µM) was sufficient to reduce the number of cells with polarized mitochondrial membranes by 16% and 20% in cells treated with 5nM and 10nM FK866, respectively (p ≤ 0.05). Cells treated with high dose β-Lapachone (6µM) had a more pronounced effect, reducing the number of cells with polarized membranes by 49% and 52% when treated with FK866 5-10nM FK866, respectively (p ≤

0.001). Unlike the effects of β-Lapachone on its own, cells cotreated with FK866 were disproportionately represented in the quadrant associated with cell death.

Accordingly, treatment with FK866 prior to low dose β-Lapachone increased the number of depolarized cells by 4% and 5% (5nM FK866, p ≤ 0.05) and the number of dead cells by 11% and 14% (10nM FK866), respectively (p ≤ 0.01,

Figure 5B). NAMPT inhibition in combination with high dose β-Lapachone (6µM) increased the number of depolarized cells by 12% and 21% (p ≤ 0.01) while the number of dead cells increased to 52% and 55% in cells treated with 5nM and

10nM FK866, respectively (p ≤ 0.001). These data directly align with the

181 importance of NAD+ in maintaining cell viability, providing additional evidence to the synergistic effects achieved by treating with β-Lapachone and blocking cells main mechanism of NAD+ synthesis.

CD38 mediated calcium mobilization confers protection from β-Lapachone

To determine if changes in calcium directly contribute to the ability of

CD38 to protect cells form β-Lapachone, DU145 cells induced for expression of

CD38 were loaded with the calcium sensitive fluorescent indicator Fluo-4AM

(Figure 6A). Expression of CD38 reduced basal fluorescence intensity by 63% (p

≤ 0.05) and peak flourescence intensity by 234% (p ≤ 0.001) relative to vehicle treated cells (Figure 6B). Vehicle treated cells exhibited a 36% increase (p ≤

0.01) fluorescence intensity after treating with β-Lapachone, whereas cells expressing CD38 had no change, indicating a lack of calcium release.

Interestingly, 60% of cells induced for expression of CD38 had a unique punctate phenotype whereas only 12% of vehicle treated cells appeared punctate (p ≤

0.001, Figure 6C). To determine if these effects were generally associated with expression of CD38 and not inherent to expression in DU145 cells, PC3 cells were also induced for expression of CD38 and loaded with Fluo-4AM (Figure

6D). Similar to DU145’s, 89% of PC3 cells appeared with punctate calcium staining whereas only 5% of vehicle treated cells had an indication of punctate signaling (p ≤ 0.001, Figure 6E). To verify reduced NAD+ levels were not a contributing factor to the punctate appearance of CD38 expressing cells, we next stained NAMPT inhibited cells with the calcium sensitive dye Fluo-4AM (Figure

6F). Basal levels of calcium staining and peak flourescence intensity following

182 treament with β-Lapachone were increased 43% and 41% in NAMPT inhibited cells, respectively (p ≤ 0.05) (Figure 6G). In response to β-Lapachone, NAMPT inhibition did not influence calcium flux as there was no signficant difference in the change in mean fluoresence intensity in NAMPT inhibited cells relative to vehicle controls (Figure 6G, right panel). Additionally, reduced NAD+ levels were not a contributing factor to the punctate calcium staining observed in CD38 expressing cells, as NAMPT inhibition did not induce a punctate staining pattern

(Figure 6H). Collectively, these data suggest calcium mobilization and not the

NAD’ase activity of CD38 is responsible for conferring protection from β-

Lapachone.

Punctate calcium staining associated with expression localizes to mitochondria.

Because cADPR produced by CD38 is a nonspecific calcium mobilizer, we stained multiple organelles whose function is known to be influenced by changes in cADPR. Figure 7 illustrates the costaining of calcium with fluorescent dye useful in visualizing mitochondria, the ER, and lysosomes. In DU145 and PC3 cells, mitochondria appeared compacted around a single pole at the periphery of the nucleus in vehicle treated cells. In cells expressing CD38, the morphology of mitochondria shifted to a much more diffuse appearance with localization around the entire periphery of the nucleus (Figure 7A, B). Staining of the ER resulted in a very similar pattern of polarized compact staining in vehicle treated cells and a diffuse nonpolarized appearance in cells expressing CD38 (Figure 7 C, D). In

DU145 cells, expression of CD38 had no apparent effect on the appearance,

183 size, or quantity of lysosomes (Figure 7E). Colocalization analysis demonstrated a small but significant increase in calcium localized to the Mitochondria, ER, and

Lysosomes DU145 cells, 3.71%, 3.87%, and 2.23%, respectively (Figure 7G).

PC3 cells demonstrated a similar 7.25% increase in calcium colocalized to mitochondria with no detectable increase in ER colocalization (Figure 7F).

3.5 Discussion

We have previously demonstrated that loss of CD38 expression and its corresponding NAD’ase activity plays an important role in regulating cell metabolism in PCa (Chapter II). However, a biological role for CD38 and production of the calcium-mobilizing second messenger, cADPR, in PCa is unknown. Here we demonstrate that the (ADP)-ribose cyclase activity of CD38 is not only active in PCa cell lines, but confers protection from the cytotoxic effect of the ortho naphthoquinone, β-Lapachone. This work provides useful insight into understanding the utility of β-Lapachone as a novel chemotherapy for the treatment of pancreatic and prostate cancer, two tumor types where expression of CD38 is decreased (13,25).

Activation of β-Lapachone by NQO1 results in a rapid reduction in the

NAD+/NADH ratio and consumption of NAD+ stemming from PARP-1 hyperactivation. Changes to intercellular NAD+ levels can also be achieved by blocking synthesis, in particular through NAMPT inhibition, or by increased catabolism through expression of CD38. Therefore, we reasoned that treating cells with the NAMPT inhibitor FK866 or restoring expression of CD38 would be

184 an effective strategy to sensitize cells to the effects of β-Lapachone. Consistent with what others have reported, we demonstrate expression of NQO1 is variable between PCa cell lines and is highest in DU145 cells (26). Reducing total NAD+ levels by treating cells FK866 sensitized cells to β-Lapachone resulting in synergistic decreases in cell viability and clonogenic survival. While expression of

CD38 did not alter the levels of NQO1, it did provide protection from β-

Lapachone. DU145 cells expressing CD38 and treated with β-Lapachone had markedly improved viability and clonogenic survival relative to empty vector controls. In line with this, cells expressing CD38 also had blunted accumulation of cleaved PARP. Protection from β-Lapachone was not due to reduced NAD’ase activity as cells expressing CD38 had significantly lower NAD+ levels.

Interestingly, in cells treated with FK866, β-Lapachone was sufficient to lower

NAD+ beyond that achieved by NAMPT inhibition alone. Conversely, β-

Lapachone was unable to lower total NAD+ levels beyond that achieved by expression of CD38. This suggests β-Lapachone may be subject to different types of processing in cells induced to express CD38.

These data also demonstrate that the down-stream effects of β-

Lapachone are mediated differently in cells expressing CD38. A hallmark of β-

Lapachone activity is hyperactivation of PARP-1 leading to an accumulation of cytotoxic (ADP)-ribose polymers. In NAMPT inhibited cells, there was a complete absence of PAR accumulation. This suggests the decrease in NAD+ levels achieved by FK866 effectively prohibited PARP-1 activity. In this case, it is reasonable to believe NAMPT inhibited cells die via necrosis rather than an

185 accumulation of PAR and ROS (4,24). Similarly, despite CD38 expressing cells having relatively more NAD+, β-Lapachone mediated PAR accumulation was reduced relative to vehicle controls. The exact mechanisms contributing to this remain unclear, although it is reasonable to posit an accumulation of

Nicotinamide (Nam) may contribute to these effects (27). Nam is a bi-product of reactions catalyzed by all three classes of NAD+ consuming enzymes and is a known inhibitor of PARP-1 activity. Accordingly, localized increases in Nam, resulting from CD38 activity, may prevent full activation of PARP-1 and contribute to the blunted accumulation of PAR. β-Lapachone also appears to have varying effects on cell metabolism in cells treated with FK866 and those expressing

CD38. We have previously established that NAMPT inhibition as well as expression of CD38 are sufficient to lower the rates of FAS in PCa cells (23).

Similar to its effects on NAD+, treatment with β-Lapachone lowered the rates of

FAS below those achieved by NAMPT inhibition alone, but had no additional effects on cells expressing CD38. Collectively, these data demonstrate that

CD38, rather than sensitizing cells to β-Lapachone, confers protection and that this protection is independent of its NAD’ase activity.

Rises in intracellular calcium are dependent on bioactivation of β-

Lapachone by NQO1, suggesting it plays an integral role in down-stream events.

In fact, cell death resulting from treatment with β-Lapachone is unique in that it utilizes calcium and the subsequent activation of calpains, rather than canonical means of apoptosis (22). Accordingly, extensive mitochondrial calcium accumulation and subsequent depolarization of mitochondrial membrane

186 potential is associated with activation of β-Lapachone, but on its own is not sufficient to initiate cell death (22). These data demonstrate expression of CD38 confers protection from β-Lapachone induced mitochondrial membrane depolarization. Conversely, NAMPT inhibition synergistically enhanced the effects of β-Lapachone, bypassing a depolarized state and shunting mitochondria into a staining pattern characteristic of cell death. In an attempt to visualize changes in calcium mobilization, cells were stained with the calcium sensitive fluorescent dye, Fluo-4AM. NAMPT inhibited cells had basal levels of calcium staining commensurate with vehicle treated cells following the addition of β-

Lapachone, suggesting these cells are primed with additional calcium.

Accordingly, when treated with β-Lapachone, the massive change in cytosolic calcium in these cells is likely a contributing factor to the observed changes in membrane potential and viability, in addition to decreased NAD+ levels. In stark contrast to NAMTP inhibition, cells expressing CD38 not only had lower basal levels of calcium, but also failed to flux calcium in response to β-Lapachone.

Consequently, the protection effects of CD38 appear to be mediated by calcium mobilization. In line with this, the vast majority of cells expressing CD38 had a unique punctate appearance. The punctate calcium pattern was not inherent to

DU145 cells as PC3’s expressing CD38 also had this appearance. Costaining calcium with fluorescent dyes targeting the ER, mitochondria, and lysosomes indicated that calcium is increased across all organelles in DU45 cells and upregulated slightly in the mitochondria of PC3 cells. The slight increase in calcium colocalized to these organelles likely does not account for the total

187 amount of calcium chase in response to expression of CD38. Understanding the localization of calcium will be imperative to understanding both mechanisms of calcium sequestration in PCa and protection from β-Lapachone. One possibility is that cADPR targets and activates ER stores of calcium by binding to ryanodine receptors (RyRs), releasing calcium or allowing calcium to slowly leak into the cytosol (28,29). Subsequently, in an effort to avoid toxicity, a portion of the leaked calcium may be sequestered by mitochondria or shuttled out of the cell by membrane bound calcium ATPase transporters (30,31). This scenario is plausible as only sub-nanomolar concentrations of cADPR are required to activate RyRs (32,33).

Collectively, these data demonstrate a novel connection between CD38, production of cADPR, and protection from β-Lapachone. We propose a model in which CD38 and its ability to produce cADPR mobilizes calcium in such a way that prevents its utilization in calpain mediated cell death. These effects are independent of the NAD’ase activity of CD38 as NAMPT inhibition yields dramatically different results. Therefore, because expression of CD38 is decreased in PCa, treatment with β-Lapachone may prove to be an effective chemotherapeutic in the treatment of this disease.

188

3.6 Figures

Figure 1: CD38 expression protects cells from β-Lapachone induced cell death. A, Expression of NQO1 and β-Actin in PCa cells determined by western blot. PC3, LNCaP, and DU145 cells in the first three lanes represent cell lines not transfected with CD38. The remaining lanes represent established cell lines stably transfected with CD38 or empty vector and treated with vehicle or doxycycline (1µM) for 96Hrs. B, Viability of DU145 cells treated with vehicle or doxycycline (1µM) for 72Hrs followed by 2Hr treatment with β-Lapachone (6µM)

189 in the presence or absence of dicoumarol (50µM) and 24Hr recovery in fresh media (N = 3). C, Percent of live and dead cells treated with vehicle or doxycycline (1µM) for 96Hrs followed by 2Hr treatment with β-Lapachone at the indicated concentration, and 24Hr recovery in fresh media. Cells were stained with propidium iodide (PI) (3µM) and signal intensity of incorporated propidium iodide corresponding to live/dead cells determined by flow cytometry. Cells treated with staurosporine (1µM) for 4Hrs served as a positive control (N=3). D,

Clonogenic survival of DU145 cells treated with vehicle or doxycycline (1µM) for

96Hrs followed by treatment with β-Lapachone (6µM) ± dicoumarol (50µM) for

2Hrs (N=3). E, Expression of PARP, cleaved-PARP (cPARP), and β-Actin detected by western blot. Cells were treated with vehicle or doxycycline (1µM) to induce expression of CD38 for 72Hrs prior to 2Hr treatment with β-Lapachone at the indicated concentration followed by 24Hr recovery in fresh media. Cells treated with staurosporine (1µM) for 4Hrs followed by 24Hr recovery served as a positive control (N=3). (*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001)

190

Figure 2: Pharmacological inhibition of NAMPT sensitizes cells to the effects of β-Lapachone. A, Viability of DU145 cells treated with FK866 ± NMN

(500µM) followed by 2Hr treatment with β-Lapachone (5µM) in the presence or

191 absence of dicoumarol (50µM) or NMN (500µM) and 24Hr recovery in fresh media (N=3) B, Percent of live and dead DU145 cells determined by propidium iodide staining. Cells were treated as described in “A”, at the indicated concentration of β-Lapachone, followed by staining with propidium iodide (PI)

(3µM). Signal intensity of incorporated PI corresponding to live/dead cells determined by flow cytometry (N=1). C, Clonogenic survival of DU145 cells treated with FK866 ± NMN (500µM) for 24Hrs followed by 2Hr treatment with β-

Lapachone (6µM) in the presence or absence of dicoumarol (50µM) or NMN

(500µM). Following all treatments cells were cultured in fresh media until well- defined colonies were established (N=3). (*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001)

192

Figure 3: CD38 mediated protection from β-Lapachone is independent of its

NAD’ase activity. A, Total NAD+ levels in DU145 cells treated with FK866 ±

NMN (500µM) for 24Hrs followed by 2Hr treatment with β-Lapachone (5µM) in

193 the presence or absence of dicoumarol (50µM) and NMN (500µM) (N=3). B,

Accumulation of poly(ADP)-ribose polymers (PAR) detected by western blot in

DU145 cells treated with FK866 (5nM) for 24Hrs followed by treatment with β-

Lapachone (10µM) in the presence or absence of dicoumarol (50µM) for the indicated time. C, Fatty acid synthesis in cells as determined by incorporation of

14C-acetate into lipid. Cells were treated with FK866 ± NMN (500µM) for 24Hrs followed by 2Hr treatment with β-Lapachone in the presence or absence of NMN

(500µM) or dicoumarol (50µM), and then treated for 2Hrs with 14C-acetate (1µCi),

(N=4). D, Total NAD+ levels in DU145 cells treated with vehicle or doxycycline

(1µM) for 96Hrs followed by 2Hr treatment with β-Lapachone (6µM) in the presence of absence of dicoumarol (50µM). E, Accumulation of PAR in DU145 cells determined by western blot. Cells were treated with vehicle or doxycycline

(1µM) for 96Hrs followed by treatment with β-Lapachone (10µM) for the indicated amount of time. Cells treated with β-Lap + dicoumarol (50µM) were treated for

15Min. Changes in PAR accumulation were determined by densitometry of single bands using β-Actin as a loading control and plotted relative to vehicle. F, Fatty acid synthesis in DU145 cells as determined by incorporation of 14C-acetate into lipid. Cells were treated with vehicle or doxycycline (1µM) for 96Hrs followed by

2Hr treatment with β-Lapachone (6µm) in the presence or absence of dicoumarol

(50µM) prior to 2Hr prior to treatment with 14C-acetate (1µCi), (N=2). (*p ≤ 0.05,

**p ≤ 0.01, ***p ≤ 0.001)

194

Figure 4: Expression of CD38 protects cells from β-Lapachone mediated loss of mitochondrial membrane potential. Changes in mitochondrial membrane potential in response to β-Lapachone were measured with JC-1 dye in control or CD38 expressing DU145 cells. A, Cells were treated with vehicle or doxycycline (1µM) for 96Hrs followed by 2Hr treatment with β-Lapachone before being assayed for changes in mitochondrial membrane potential (N=3). Cells

195 stained with JC1 in the upper right-hand quadrant are live cells exhibiting high mitochondrial membrane potential, cells in the lower right-hand quadrant exhibit lower mitochondrial membrane potential, and cells in the lower left-hand quadrant had low membrane potential and JC-1 incorporation, indicative of dead cells. B, Data represented in “A” displayed graphically to show the distribution of mitochondrial membrane potential in cells treated with doxycycline and β-

Lapachone as described (N=3). (*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001)

196

Figure 5: Pharmacological inhibition of NAMPT sensitizes cells to β-

Lapachone mediated loss of mitochondrial membrane potential. Changes in mitochondrial membrane potential in response to β-Lapachone were measured with JC-1 dye in control or FK866 treated DU145. A, Cells were treated for 24Hrs

197 with FK866 followed by 2Hr treatment with β-Lapachone at the indicated concentrations and assayed for changes in mitochondrial membrane potential

(N=3). Cells stained with JC1 in the upper right-hand quadrant are live cells exhibiting high mitochondrial membrane potential, cells in the lower right-hand quadrant exhibit lower mitochondrial membrane potential, and cells in the lower left-hand quadrant had low membrane potential and JC-1 incorporation, indicative of dead cells. B, Data represented in “A” displayed graphically to show the distribution of mitochondrial membrane potential in cells treated with FK866 and β-Lapachone as described (N=3). (*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001)

198

Figure 6: CD38 mediated calcium mobilization confers protection from β-

Lapachone. Intracellular calcium levels were measured in live DU145 and PC3 cells via confocal microscopy using the calcium indicator dye, Fluo-4AM. A,

199

Representative images of DU145 cells treated for 96Hrs with vehicle or doxycycline (1µM), loaded with Fluo-4AM, and then treated with β-Lapachone

(8µM). β-Lapachone was added to cells after basal images were recorded.

Images were collected approximately every 90Sec. for 30Min. from multiple fields of view within each treatment condition. Presented images are representative of changes in Fluo-4AM fluorescence intensity before and after β-Lapachone treatment. B, Quantification and distribution of mean fluorescence intensity (MFI) and the change in MFI from individual cells treated as described in “A” (N=3). C,

Quantification of the number of cells exhibiting ubiquitous or punctate calcium staining following induction of CD38 for 96Hrs. D-E, Representative images of

PC3 cells loaded with Fluo-4AM following induction of CD38 for 96Hrs. The number of cells exhibiting ubiquitous or punctate calcium staining was then determined and is represent as a percent of total cells (N=3). F, DU145 cells treated with vehicle or FK866 (10µM) for 24Hrs, loaded with Fluo-4AM, and treated with β-Lapachone (8µM). β-Lapachone was added to cells and images collected as described in “A.” G-H, MFI, change in MFI, and calcium staining pattern in DU145 cells treated with FK866 and β-Lapachone as described in “F”

(N=3). (*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001

200

Figure 7: Punctate calcium staining associated with expression of CD38 localizes to mitochondria. Live DU145 and PC3 cells induced to express CD38 for 96Hrs were triple labeled to visually determine calcium localization to the mitochondria, endoplasmic reticulum, or lysosomes. A-B, Cells treated with

201 vehicle or doxycycline (1µM) for 96Hrs then stained with Fluo-4AM and

MitoTracker Orange to visualize calcium and mitochondria localization. C-D,

Cells treated with vehicle or doxycycline (1µM) for 96Hrs then stained with Fluo-

4AM and ERTracker-Red (5µM) to visualize calcium and ER localization. E,

DU145 cells treated with vehicle or doxycycline (1µM) for 96Hrs then stained with

Fluo-4AM and LysoTracker-Red DND-99 (5µM) to visualize calcium and

Lysososme localization. All cells were stained with Hoechst 33343 (2.5µM) to visualize the nucleus.

202

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2. Pink JJ, Planchon SM, Tagliarino C, Varnes ME, Siegel D, Boothman DA.

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3. Huang X, Motea EA, Moore ZR, Yao J, Dong Y, Chakrabarti G, et al.

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11. Liu AY, Roudier MP, True LD. Heterogeneity in Primary and Metastatic

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207

CHAPTER IV

GENERAL DISCUSSION

Jeffrey P. Chmielewski and Steven J. Kridel

J. Chmielewski prepared the text and S. Kridel provided advisory and editorial assistance.

208

4.1 Targeting NAD+ in cancer

Cell metabolism is the collective effort of several interconnected pathways working in concert to provide cells with the molecules necessary to remain viable.

Accordingly, all tumor cells deregulate cellular energetics in favor of macromolecule synthesis to some degree in order to support increased proliferation and survival. The pathways of cell metabolism are interconnected through their requirement for NAD+ as a cofactor. The data presented herein demonstrate the importance of CD38 as an NAD’ase as well as how its dysregulation contributes to cell metabolism in PCa.

In Chapter II, we demonstrate a strong correlation between decreased expression of CD38 and PCa progression (Chapter II, Figure 1B) (1). Data derived from two independent cohorts containing 181 primary and 37 metastatic samples or 66 primary and 83 normal (adjacent or donor) samples, respectively, also established expression of CD38 correlates with recurrence free survival

(Figure 1) (2,3). Several lines of evidence indicate CD38, through its NAD’ase activity, has the capacity to impede metabolic capacity at multiple nodes leading to a nonproliferative phenotype both metabolically as well as transcriptionally.

Collectively, these data demonstrate expression of CD38 may serve as a prognostic marker for PCa progression or aggressiveness. Determining relative expression of CD38 from needle biopsies could have clinical benefit in guiding the course of therapy at the time of diagnosis. In line with this, we demonstrate that treatment with ATRA is sufficient to drive expression of CD38 in PCa cell lines. Hypermethylation of the promoter region of CD38 is thought to be a

209 contributing factor that drives gene silencing in PCa. While we did not detect significant changes in the methylation status of CpG sites tested, this does not rule out ATRA is able to alter the methylation status of other CpG sites. It is also conceivable ATRA may induce CD38 gene expression through direct activation of the RARE in addition to its influence on the methylation status of CpG sites not tested.

Targeting NAD+ synthesis via pharmacological inhibition of NAMPT remains a viable strategy for targeting tumor cell metabolism (4-6). However, if

NAMPT inhibitors are to progress clinically there will be a need to address the sensitivity of normal cells, which underlies its dose-limiting toxicities (7). The data presented in Chapter III establishes a compelling argument for the utility of co- treatment of FK866 and β-Lapachone in PCa. Specifically, β-Lapachone sensitize cells to low dose FK866, which is clinically significant as tumor cells specifically upregulate NQO1 and rely heavily on NAD+ salvage via the NAMPT pathway (8). The data presented herein establish NAMPT blockade is effective in inhibiting glycolysis and mitochondrial function. This coincides with our previous report demonstrating NAMPT inhibition is sufficient inhibit de nono lipogenesis and induce apoptosis in PCa (Chapters II and III) (9). Interestingly, our data demonstrates when treated in combination with β-Lapachone, the effective dose of FK866 can be dramatically lowered (Chapter III, Figure 2A-C). On its own treatment with FK866 sensitizes PCa cells to oxidative stress through reduced

NAD+ levels and SIRT1 activity – both of which are required for the antioxidant proteins catalase and SOD (10-12). When activated by NQO1, the effects of β-

210

Lapachone compound the effects of NAMPT inhibition. This is primarily through blockade of NAD+ synthesis and the rapid consumption of available NAD+ (13).

β-Lapachone has been available since the mid-1990’s, but its utility as a chemotherapeutic agent has only garnered attention over the last five years.

Accordingly, only a few groups have investigated the potential of NAMPT inhibition in combination with β-Lapachone. While the combination therapy has been investigated in NSCLC and PaCa, we are the first to demonstrate the effects in PCa (14-16). Consistent with what these groups have reported,

NAMPT inhibition sensitizes cells to β-Lapachone suggesting lower therapeutic doses of both may be achieved in the clinical. Ultimately, the utility of both drugs circles back to their ability to reduce total NAD+ levels as well as the NAD+/NADH ratio, again highlighting the importance of NAD+ to tumor cells and metabolism.

The protection effects of CD38 expression also present compelling evidence for the utility of the combined treatment in PCa. Because expression of CD38 is high in normal prostate epithelial cells these cells would likely be protected from the cytotoxicity associated with β-Lapachone.

211

Figure 1: Correlation between expression of CD38 BCR. Recurrence free survival in two patient cohorts as a function of CD38 expression (2,3). (*p ≤ 0.05,

**p ≤ 0.01, ***p ≤ 0.001)

212

4.2 Future directions

The experiments performed in Chapter II establish a role for CD38 in the direct modulation of NAD+ and its impact on tumor cell metabolism. The ability of

CD38 to illicit changes in acetylation of FOXO1 suggests the function of sirtuins can be regulated by CD38 (Chapter II, Figure 2B). Interestingly, primary PCa tissue microarrays reveal elevated levels of SIRT1 relative to areas of benign growth and PIN (17,18). Sirt1 levels are also increased in androgen-refractory

PCa cell lines compared to androgen-sensitive cells (19). Given expression of

SIRT1 is in direct contrast to that of CD38, it suggests advanced disease has a greater reliance on NAD+ and sirtuin activity. Therefore, understanding the relationship between CD38 and sirtuin activity would represent a viable next step.

Detecting how expression of CD38 alters the function of SIRT1 or SIRT3 could easily be determined using the Fluor de Lys Fluorometric assay kit (abcam).

Using this system, Sirt1 and Sirt3 enzymatic activity could be directly determined from nuclear (Sirt1) or mitochondrial (Sirt3) isolates as well as whole cells lysates. If sirtuin activity was inhibited, this would provide direct evidence of the ability of CD38 to alter sirtuin activity. These results could be compared to the effects of SIRT1 and SIRT3 inhibitors EX525 and AGK7, respectively, to provide an understanding of the relative level of inhibition.

The data presented in Chapter III, suggests expression of CD38 is sufficient to induce a small increase in (ADP)-ribose – an indication of PARP-1 activity. However, when stressed it appears the activity of PARP-1 is blunted

(Chapter III, Figure 3E). To determine the extent to which expression of CD38

213 modulates PARP-1 activity, cells could be treated with an alkylating agent such as methyl methanesulfonate (MMS) and the accumulation of dsDNA breaks measured. In response to alkylating agents (such as MMS), dsDNA breaks increases the presence of γH2Ax (a histone H2a variant), providing a functional read out of downstream PARP-1 activity and the DNA repair response. This would provide an additional line of evidence as to the impact of CD38 on PARP-1 activity and the DNA repair process.

Lastly, Chapter III establishes a role for CD38, production of cADPR, and calcium mobilization in PCa. The data gathered during these experiments suggests calcium is chased from its normal sites of storage and at least in part taken up by the ER, Mitochondria, and to a certain extent lysosomes. However, the percent of colocalized calcium to these organelles does not account for the total amount of calcium released upon expression of CD38. Therefore, it would be worthwhile to determine if calcium (released because of CD38) is pumped out of the cell to maintain an appropriate level in the cytosol. Our data demonstrate expression of CD38 results in changes to more than 75% of the transcriptome.

Accordingly, it would be interesting to determine if changes in calcium mobilization are the coordinated effort of calcium transporters whose expression change in response to CD38 expression or other means. Blocking classes of calcium transporters and determining if calcium localization is restored to its normal ubiquitous phenotype is one strategy to assess their involvement.

214

4.3 Overall significance and impact

Prostate cancer is the most diagnosed cancer of all subtypes, and the third highest in associated mortality. A large percentage of the patients that develop distant metastases will sucrum to the disease. These realities highlight the need to develop new treatments and diagnostics for patients with aggressive

PCa. The data presented herein is the first to establish the connection between

CD38, NAD+, and cell metabolism in PCa. Furthermore, this work establishes that in addition to its function as and NAD’ase, CD38 may regulate more processes and pathways then originally conceived. More than 75% of the transcriptome is affected by changes expression of CD38. We also demonstrate

CD38 and its ability to influence calcium mobilization is important in protecting cells from cytotoxic stress. The fact that expression of CD38 confers protection from β-Lapachone but NAMPT inhibition sensitizes cells to this drug is promising.

Determinating at what node expression of CD38 and NAMPT inhibition elicit their effects will highlight vulnerabilities inherent to PCa and may advance β-

Lapachone as a potential new drug for the treatment of this disease. When considered as a whole, our work establishes an essential role for NAD+ in cell metabolism and survival (Figure 2).

215

Figure 2: Key components controling NAD+ homeostasis. Our data suggest

PCa cell metabolism has an undeniable requirement for NAD+. Changes in the activity of CD38, sirtuins, or PARPs can modulate NAD+ levels thereby effecting cell metabolism. Silencing of CD38 represents one mechanism, of which PCa subverts the limitations of NAD+ to increase metabolic potential and survival.

Decreasing NAD+ through NAMPT inhibition, and treating cells with β-

Lapachone, has promising therapeutic potential in the treatment of this disease.

216

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JEFFREY P. CHMIELEWSKI, PhD,MBA [email protected]

Accomplished and motivated scientific researcher with a history of academic achievement in both business and science. Demonstrated success in mentoring, public speaking, organizational leadership roles, and adept at facilitating communication between stakeholders at all levels. Six years of experience in trending therapeutic strategies with the ability to interpret complex data and effectively communicate scientific and medical information to a variety of audiences. Comprehensive understanding of business principles with a focus on client management, resource allocation, and team development. Able to prioritize competing demands under pressure in results driven environment.

Areas of expertise include:

 Cancer Biology  Detailed Documentation  Molecular Biology & Biotechnology  Experimental Design  Statistical Analysis  Presentations & Public Speaking  Pre-clinical Research  Stakeholder Relationship Management

EDUCATIONAL BACKGROUND

WAKE FOREST UNIVERSITY Winston-Salem, NC Ph.D. Candidate: Cancer Biology – May 2018 MBA – August 2016

EAST CAROLINA UNIVERSITY Greenville, NC M.S., Molecular Biology & Biotechnology – May 2012

UNIVERSITY OF NORTH CAROLINA AT GREENSBORO Greensboro, NC B.S., Biotechnology – May 2010

ACADEMIC PROJECTS

WAKE FOREST UNIVERSITY Tumor Cell Metabolism Project  Pre-clinical research: Investigate mechanisms underlying tumor cell metabolism and the therapeutic potential of novel combination therapies in the treatment of prostate cancer  Built scientific and technical expertise in the molecular and genetic alterations that contribute to oncogenesis in multiple solid tumor types  Presented project findings to a variety of audiences at local and international conferences

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[email protected]

EAST CAROLINA UNIVERSITY Chromosome Condensation Project  Identified and characterized a novel gene responsible for maintaining chromosome condensation during cell division and created a unique method for determining chromosome compaction  Led project team members in compiling, analyzing, and publishing project results

PROFESSIONAL & LEADERSHIP EXPERIENCE

WAKE FOREST UNIVERSITY Winston-Salem, NC External Consultant, Organizational Leadership & Behavior – 2015 – 2016  Advised prominent law group on best practices in organizational leadership and behavior. Provided evidenced based evaluation of work place practices and delivered a detailed action plan aimed at improving management staff relations  Developed reports, presentations, and charts for review by University leadership External Consultant, Marketing – 2015 – 2016  Initiated integrated marketing strategy and identified target market for a medical device start-up company bringing a first of its kind product to market  Conducted interviews with Key Opinion Leaders (KOLs) in gerontology and medical adherence to understand the needs of intended market and current treatment landscape Undergraduate Mentor – 2015 – 2016  Conceptualized, planned, and interpreted independent research projects for students  Set timelines and project milestones, ensured students maintained accountability for project goals  Coordinated with student throughout project duration to assist with research and strategy  Review student progress and advised students regarding publication options

EAST CAROLINA UNIVERSITY Greenville, NC Graduate Teaching Assistant, General biology and Cell Physiology Labs – August 2010 – July 2012  Created and implemented lesson plans designed to complement corresponding lecture based course  Crafted mid-term and final exams to assess comprehension of learning objectives  Trained and mentored new teaching assistants in effective delivery and structure of lectures

UNIVERSITY OF NORTH CAROLINA AT GREENSBORO Greensboro, NC Experiential Education Facilitator - May 2008 – August 2010  Created individualized programming tailored to the specific needs of clients in the areas of leadership, communication, effective decision making, and enacting support from team members.  Use of initiatives and low/high course elements to facilitate team development

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[email protected]

HONORS & AWARDS

 Selected for Appointment to Lipid Sciences T32 Training Grant, Wake Forest University – 2017  Selected for Appointment to Cancer Biology T32 Training Grant, Wake Forest University – 2013  Appointed to the Molecular and Cellular Biosciences Honor Council, Wake Forest University – 2012  Merit Based Travel Grant, Graduate Professional Student Senate (GPSS), East Carolina University – 2012  McDaniel’s Scholarship; Department of Biology, East Carolina University – 2011  Next Step Scholarship; Department of Biology, East Carolina University – 2011  Merit Based Travel Grant, Graduate Professional Student Senate (GPSS), East Carolina University – 2011  Merit Based Travel Grant, Department of Biology, East Carolina University – 2011  Oral Presentation Award, RENCI Visualization Challenge, East Carolina University – 2010

PUBLICATIONS

 Chmielewski, Jeffery P., Kridel, Steven J. (2018) Mobilization of Calcium by CD38 Protects Prostate Cancer Cells from β-Lapachone Mediated Cell Death. – Manuscript in progress  Chmielewski, Jeffrey P., Bowlby, Sarah., Wheeler, Frances., Shi, Lihong., Sui, Guangchao., Davis, Amanda., D’Agostino Jr., Ralph., Miller, Lance., Sirintrapun, Joseph S., Cramer, Scott., Kridel, Steven. (2017) CD38 Inhibits Prostate Cancer Cell Metabolism and Proliferation by Reducing Cellular NAD+ Pools. Molecular Cancer Research.  Binder, A., Wakimoto, B., Davis, C., Chmielewski, J., Tomkiel, J. (2017) Versager is expressed at the histone-to-protamine transition during spermiogenesis and is required for embryonic chromosome transmission in drosophila melanogaster. Research Journal of Developmental Biology. Accepted for publication (online early access only).  Chmielewski, JP., Henderson, L., Smith CM., Christensen, TW. (2012) Drosophila Psf2 has a role in chromosome condensation. Chromosoma; Vol.121. 6:585-596.  Chmielewski, Jeffrey P., Chistensen, Tim W. (2011) Novel method for determining chromosome compaction and DNA content of salivary gland nuclei in Drosophila. Drosophila information services; 94:146-55.

POSTER PRESENTATIONS

 American Association for Cancer Research Special Conference on Metabolism and Cancer: Bellevue, Washington, CD38 and Nampt Regulate Tumor Cell Metabolism through Modulation of NAD+. 2015  East Carolina University Research and Creative Achievement Week, Greenville, NC, "Psf2: A Role in Chromosome Condensation" – 2012  53rd Annual Genetics Society of America International Research Conference, Chicago, IL, "Psf2: A Role in Chromosome Condensation" – 2012  Sigma Xi International Research Conference, Raleigh, NC, "Psf2: A role in chromosome condensation" – 2011  East Carolina University Research and Creative Achievement Week, Greenville, NC, "Investigating the Role of Psf2 in Maintaining Genomic Integrity" – 2010

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[email protected]

 52nd Annual Genetics Society of America International Research Conference, San Diego, CA, "Investigating the Role of Psf2 in Maintaining Genomic Integrity" – 2010  North Carolina Academy of Sciences 107th Annual Meeting, Greensboro, NC. "Characterization of a Male Chromosome Loss Mutation in Drosophila Melanogaster" – 2010

ORAL PRESENTATIONS

 Wake Forest University – School of Medicine: Department of Lipid Sciences Seminar Series. Wake Forest University, Winston-Salem, NC. Title: Store-Operated Ca2+ Entry Controls Induction of Lipolysis and the Transcriptional Reprogramming to Lipid Metabolism. 2017  Wake Forest University -School of Medicine: Department of Cancer Biology Seminar Series: Wake Forest University, Winston-Salem, NC, Title: Loss of CD38 Promotes Prostate Cancer via Modulation of NAD+. – 2016  Research Progress Report Seminar Series: Wake Forest University, Winston-Salem, NC, "CD38 and Nampt Regulate Prostate Cancer Metabolism and Survival through modulation of NAD+." – 2015  Wake Forest University - School of Medicine: Department of Cancer Biology Seminar Series: Wake Forest University, Winston-Salem, NC, "Regulation of Prostate Cancer Survival and Metabolism via the Interplay of Nampt, CD38, and Sirtuins" – 2014  Research in Progress Seminar Series, East Carolina University, Greenville, NC, "DrosophilaPsf2 has a role in Chromosome Condensation" – 2012  Research in Progress Seminar Series, East Carolina University, Greenville, NC, "Characterizing the role of Psf2 in maintaining genomic integrity" – 2011  RENCI Visualization Challenge, East Carolina University, Greenville, NC, "Cytological Analysis of Polytene Chromosomes in the Salivary Glands of D. melanogaster" – 2010  Undergraduate research expo, Greensboro, NC" Characterization of a Male Chromosome Loss Mutation in Drosophila Melanogaster" – 2010

PROFESSIONAL AFFILIATIONS

 Founding father of Pi Kappa Alpha International Fraternity – Lambda Rho Alumni Association: Served on executive leadership team – 2016 – Present  Healthcare Strategy Conference & Case Competition Leadership Team, Wake Forest University – 2016 – 2017  Golden Key International Honour Society – 2011 – Present  Sigma Xi Research Society – 2011 – Present  Genetics Society of America – 2010 – Present  Founding father of Pi Kappa Alpha International Fraternity – Lambda Rho Chapter: Served on executive leadership team – 2008 – 2010

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