SELECTIVE ANDROGEN MODULATOR (SARM) ACTION:

ANDROGEN THERAPY REVISITED

DISSERTATION

Presented in Partial Fulfillment of the Requirements for

the Degree Doctor of Philosophy in the

Graduate School of The Ohio State University

By

Christopher C Coss, B.S.

***

The Ohio State University

2008

Dissertation Committee: Approved by

Dr. James T. Dalton, Advisor

Dr. Robert W. Brueggemeier ______Dr. Thomas D. Schmittgen Advisor Dr. Mamuka Kvaratskhelia Pharmacy Graduate Program

ABSTRACT

Despite continuing advances in the clinical development of selective modulators (SARMs) for male hypogonadism, osteoporosis, muscle wasting and myriad diseases of the prostate, mechanism remains controversial. To date, mechanistic work in the selective modulator (SRM) field has been dominated by selective modulators (SERMs) where a full understanding of SERM action contributed to the development of second generation molecules with better selectivity and reduced side effects. It follows that a better understanding of SARM action could lead to improvements in rationale SARM design and even molecules tailor made for specific patient populations or disease states. The studies described herein were carried out to shed light on the molecular mechanism of aryl propionamide SARM action resulting in full efficacy in anabolic tissues (muscle and bone), while sparing androgenic tissues (prostate and skin). To this end genome wide androgen receptor (AR) promoter binding and transcriptional profiling in LNCaP prostate cancer cells was performed. In these experiments, the primary prostatic androgen 5α-dihydrostestosterone (DHT) was compared to aryl propionamide SARMs, revealing largely overlapping but distinct modes of action. These works support the existence of qualitative differences, not solely due to potency, underlying SARM mechanism.

ii A renewed therapeutic interest in androgens has created an opportunity to re- evaluate side effects that have prevented wide scale androgen therapy. Seemingly pro- athrogenic lipid profiles result from androgen treatment though links to cardiovascular disease are largely observational and conflicting. Also studies showing androgens to be hepatotoxic are confounded by existing disease states and a heavy focus on anabolic abusers. Nevertheless, the dogma surrounding dangers of androgen administration contribute to clinicians’ apprehensions in using anabolic agents to treat a cadre of ailments. The studies described herein characterize these effects for aryl propionamide SARMs arguing that elevations of serum ALT, thought to reflect liver toxicity, are actually the result of androgen mediated expression. Reductions in serum HDL-C were found to be tightly linked with anabolic efficacy in short term studies, but flexibility in the aryl propionamide pharmacophore coupled with high amenability to varied formulations offer hope toward future SARM therapies.

iii ACKNOWLEDGMENTS

I would like to first and foremost acknowledge my wife Shelley Orwick. Without her endless support, this effort would have failed long ago. I am incredibly fortunate for her caring and infinite patience.

I would also like to extend my gratitude to Drs. Wenqing Gao and Ramesh

Narayanan for their many lessons. Their teachings greatly shaped my graduate training.

I would like to thank Drs. Jeffrey Kearbey and Mitchell A. Phelps for setting me on this course and keeping me on this course, respectively. I would like to thank Dr. Victor Jin for his introduction to bioinformatics. My gratitude is also owed to my committee members Dr. Robert W. Brueggemeier, Dr. Thomas D. Schmittgen and Dr. Mamuka

Kvaratskhelia whom provided many useful suggestions. I would also like to thank GTx

Inc. for allowing me to continue my graduate research while in their employ.

Finally, I would like to convey my appreciation to my advisor Dr. James T.

Dalton for the many opportunities- past, present and future.

iv VITA

February 23, 1980...... Born – Columbus, Ohio

2002...... B.S. Molecular Genetics, The Ohio State University

B.S. Computer Science, The Ohio State University

2003-2004……………………...Graduate Teaching Assistant, The Ohio State University

2004-2006……………………...Graduate Research Associate, The Ohio State University

2006-present……………………Research Technician, GTx Inc., Memphis, TN

PUBLICATIONS

Research Publications

1. Gao W, Reiser PJ, Coss CC, Phelps MA, Kearbey JD, Miller DD, Dalton JT. “Selective androgen receptor modulator treatment improves muscle strength and body composition and prevents bone loss in orchidectomized rats.” Endocrinology, 146(11):4887-97, 2005.

1. Narayanan R, Coss CC, Yepuru M, Kearbey JD, Miller DD, Dalton JT. “Steroidal androgens and nonsteroidal, tissue-selective androgen receptor modulator, S- 22, regulate androgen receptor function through distinct genomic and nongenomic signaling pathways.” Molecular Endocrinology, 22(11): 2448-65, 2008.

FIELDS OF STUDY

Major Field: Pharmacy

v TABLE OF CONTENTS

Abstract...... ii Acknowledgments ...... iv Vita...... v Table of Contents...... vi List of Tables ...... xi List of Figures...... xiii 1. Introduction...... 1 1.1. Androgens...... 1 1.1.1 Physiological Role and Clinical Utility...... 1 1.1.2 Androgen Receptor – Structure/Function...... 3 1.2. Selective Androgen Receptor Modulators (SARMs) ...... 5 1.2.1 Discovery and Characterization of the Aryl Propionamides SARMs ...... 6 1.2.2 SARMs – Therapeutic Promise...... 7 1.2.3 SARMs – Therapeutic Concerns...... 10 1.2.4 SARM Mechanism ...... 12 1.3. Scope and Objective of Dissertation...... 14 2. SARM versus DHT: Profiling in LNCaP Prostate Cancer Cells...... 23 2.1. Introduction...... 23 2.2. Materials and Methods ...... 24 2.2.1 Materials...... 24 2.2.2 LNCaP Cell Growth Curve ...... 24 2.2.3 AR Trans-activation in COS-1 Cells...... 25 2.2.4 cDNA Microarray Study Design ...... 25

vi 2.2.5 cDNA Microarray Data Analysis ...... 26 2.2.6 Orthologous Promoter Androgen Response Element (ARE) Search...... 27 2.2.7 Gene Expression Validation...... 29 2.2.8 Electro-Mobility Shift Assay (EMSA)...... 29 2.2.9 Functional Analyses...... 31 2.3. Results...... 31 2.3.1 LNCaP Growth Curve ...... 31 2.3.2 Gene Expression Profile...... 32 2.3.3 Gene Expression Validation...... 33 2.3.4 Putative Androgen Response Elements...... 33 2.3.5 EMSA Validation of AR Binding Potential ...... 34 2.3.6 Functional Analyses ...... 35 2.4. Discussion...... 36 2.5. Acknowledgments ...... 41 3. SARM versus DHT: Genome-wide AR promoter recruitment profiling in LNCaP Prostate Cancer Cells ...... 53 3.1. Introduction...... 53 3.2. Materials and Methods ...... 54 3.2.1 Materials...... 54 3.2.2 AR Trans-activation in COS-1 Cells...... 55 3.2.3 Chromatin Immuno-precipitation (ChIP) in LNCaP...... 55 3.2.4 AR ChIP-DSL Study Design...... 57 3.2.5 ChIP-DSL Data Analysis ...... 57 3.2.6 Orthologous Promoter Androgen Response Element (ARE) Search...... 58 3.2.7 Gene Expression Analyses ...... 59 3.2.8 Functional Analyses ...... 60 3.3. Results...... 61 vii 3.3.1 AR Trans-activation ...... 61 3.3.2 PSA Enhancer ARE AR recruitment in LNCaP...... 61 3.3.3 ChIP-DSL AR Binding Profile...... 61 3.3.4 Putative Androgen Response Elements...... 63 3.3.5 Gene Expression Analyses ...... 64 3.3.6 Functional Analyses ...... 65 3.4. Discussion...... 66 3.5. Acknowledgments ...... 72 4. Androgen Regulation of the Alanine Aminotransferases ...... 85 4.1. Introduction...... 85 4.2. Materials and Methods ...... 86 4.2.1 Materials...... 86 4.2.2 Animals...... 87 4.2.3 Orthologous Promoter Androgen Response Element (ARE) Search...... 87 4.2.4 AR Trans-activation in COS-1 Cells...... 87 4.2.5 LNCaP Gene Expression...... 88 4.2.6 ALT-1 and ALT-2 Promoter Scan ChIP in LNCaP ...... 88 4.2.7 Primary Rat Hepatocyte Gene Expression (251)...... 89 4.2.8 Orchidectomized Rat Acute High Dose Study...... 90 4.3. Results...... 91 4.3.1 Putative Androgen Response Elements...... 91 4.3.2 T877A-AR Trans-activation...... 91 4.3.3 ALT Gene Expression ...... 92 4.3.4 ALT Promoter Scanning...... 92 4.3.5 ORX Rat Acute High Dose Study...... 93 4.4. Discussion...... 94 4.5. Acknowledgments ...... 101 viii 5. Androgen Regulation of Serum Lipids...... 117 5.1. Introduction...... 117 5.1.1 HDL-C Homeostasis Target ...... 118 5.1.2 Androgen Responsive Liver Control Genes...... 120 5.2. Materials and Methods ...... 121 5.2.1 Materials...... 121 5.2.2 Animals...... 121 5.2.3 Orthologous Promoter Androgen Response Element (ARE) Search...... 121 5.2.4 Primary Rat and Human Hepatocyte Experiments...... 122 5.2.5 HepG2 Transient Transfection Experiments...... 123 5.2.6 Orchidectomized Rat Time Course Study...... 123 5.2.7 Ovariectomized Rat Extended Treatment Study ...... 125 5.2.8 Intact Female Beagle Dog Study...... 126 5.3. Results...... 128 5.3.1 Putative Androgen Response Elements...... 128 5.3.2 Gene Expression Analyses in Primary Hepatocytes ...... 128 5.3.3 Transient Transfection in HepG2 Hepatocellular Carcinoma Cells...... 129 5.3.4 Orchidectomized Rat Time Course Study...... 129 5.3.5 Ovariectomized Rat Extended Treatment Study ...... 132 5.3.6 Intact Female Beagle Dog Study...... 133 5.4. Discussion...... 134 5.4.1 In Vitro Models...... 134 5.4.2 In Vivo Models ...... 135 5.4.3 Route vs. Rate – Female Beagle Dog Study...... 138 5.4.4 Conclusions and Future Directions ...... 139 5.5. Acknowledgments ...... 143 6. Summary and Conclusions ...... 165 ix Bibliography ...... 169 Appendices...... 186 Appendix A – Supplements Relevant to Chapter 2 ...... 187 Appendix B – Supplements Relevant to Chapter 3 ...... 195 Appendix C – Supplements Relevant to Chapter 4 ...... 209 Appendix D – Supplements Relevant to Chapter 5 ...... 212

x LIST OF TABLES

Table 1.1 Steroidal and Non-Steroidal AR Ligands ...... 22 Table 2.1 Characterized ARE Sequences Used in PWM ...... 49 Table 2.2 Putative AREs Examined Using EMSA...... 50 Table 2.3 Previously Reported Genes Up-Regulated ...... 51 Table 2.4 Previously Reported Genes Down or Differentially Regulated...... 52 Table 3.1 Comparative ARE Search Results by Group...... 84 Table 4.1 ORX Rat Acute High Dose Treatment Groups ...... 114 Table 4.2 ALT-1 Conserved AREs...... 115 Table 4.3 ALT-2 Conserved AREs...... 116 Table 5.1 Conserved AREs – HDL /Homeostasis and Control Genes...... 161 Table 5.2 Orchidectomized Rat Time Course Study – Treatment Groups...... 162 Table 5.3 Ovariectomized Rat Time Course Study – Treatment Groups...... 163 Table 5.4 Female Beagle Dog Study – Treatment Groups ...... 164 Table A.1 Conserved AREs in Genes 2x Up-Regulated in SARM Only...... 189 Table A.2 Conserved AREs in Genes 2x Up-Regulated in SARM and DHT...... 190 Table A.3 Conserved AREs in Genes 2x Up-Regulated in DHT Only...... 191 Table A.4 Conserved AREs in Genes 2x Down-Regulated in DHT and SARM...... 192 Table A.5 Conserved AREs in Genes 2x Down-Regulated in SARM Only...... 193 Table A.6 (Continued) Conserved AREs in Genes 2x down-regulated in SARM only 194 Table B.1 ChIP-DSL AR Binding Promoters – Group A...... 197 Table B.2 ChIP-DSL AR Binding Promoters – Group B...... 198 Table B.3 ChIP-DSL AR Binding Promoters – Group C...... 199 Table B.4 ChIP-DSL AR Binding Promoters – Group D...... 200

xi Table B.5 ChIP-DSL AR Binding Promoters – Group D (Continued) ...... 201 Table B.6 ChIP-DSL AR Binding Promoters – Group E ...... 202 Table B.7 ChIP-DSL AR Binding Promoters – Group F ...... 203 Table B.8 ChIP-DSL AR Binding Promoters – Group G...... 204 Table B.9 ChIP-DSL AR Binding Promoters – Group G (Continued) ...... 205 Table B.10 ChIP-DSL AR Binding Promoters – Group G (Continued) ...... 206 Table B.11 ChIP-DSL AR Binding Promoters – Group G (Continued) ...... 207 Table B.12 ChIP-DSL AR Binding Promoters – Group G (Continued) ...... 208 Table C.1 ALT-1 ChIP Promoter Scan Primers ...... 210 Table C.2 ALT-2 ChIP Promoter Scan Primers ...... 211

xii LIST OF FIGURES

Figure 1.1 Testosterone Metabolism...... 17 Figure 1.2 The Hypothalamic-Pituitary-Gonadal (HPG) Axis...... 18 Figure 1.3 Human Androgen Receptor – Functional Domains ...... 19 Figure 1.4 Androgen Receptor – Genomic Action ...... 20 Figure 1.5 Reverse Cholesterol Transport (RCT)...... 21 Figure 2.1 LNCaP Cell Growth in the Presence of DHT and SARM1 ...... 42 Figure 2.2 Gene Expression Profile in LNCaP...... 43 Figure 2.3 Androgen Response Element PWM WEBLOGO and Ci Vector Plot...... 44 Figure 2.4 Comparative ARE Search Dataset Flow ...... 45 Figure 2.5 EMSA Validation of Putative AREs ...... 46 Figure 2.6 GO Term Analysis of cDNA Microarray Data ...... 47 Figure 2.7 AR Transactivation in COS-1 – WT vs T877A AR...... 48 Figure 3.1 AR Transactivation in COS-1 – WT vs T877A AR...... 73 Figure 3.2 AR ChIP in LNCaP on PSA Enhancer...... 74 Figure 3.3 ChIP-DSL AR Binding Profile...... 75 Figure 3.4 Comparative ARE search – ChIP-DSL ...... 76 Figure 3.5 Putative ARE Distribution – ChIP-DSL...... 77 Figure 3.6 LNCaP Gene Expression – Controls ...... 78 Figure 3.7 LNCaP Gene Expression – Group A...... 79 Figure 3.8 LNCaP Gene Expression – Group C...... 80 Figure 3.9 LNCaP Gene Expression – Group E ...... 81 Figure 3.10 LNCaP Gene Expression – Group G...... 82 Figure 3.11 GO Term Analysis of ChIP-DSL Experiment...... 83

xiii Figure 4.1 AR Transactivation in COS-1 – T877A-AR ...... 102 Figure 4.2 LNCaP ALT-1/2 Gene Expression...... 103 Figure 4.3 Primary Rat Hepatocytes – ALT-1/2 Gene Expression ...... 104 Figure 4.4 ALT-1 9kb Promoter Region Schematic...... 105 Figure 4.5 ALT-1 Promoter Scan – DHT Results ...... 106 Figure 4.6 ALT-1 Promoter Scan – SARM3 Results ...... 107 Figure 4.7 ALT-2 9kb Promoter Region Schematic...... 108 Figure 4.8 ALT-2 Promoter Scan Results...... 109 Figure 4.9 ORX Rat Acute High Dose – Liver Gene Expression ...... 110 Figure 4.10 ORX Rat Acute High Dose Study – Levator Ani Gene Expression ...... 111 Figure 4.11 ORX Rat Acute High Dose Study – Prostate Gene Expression...... 112 Figure 4.12 ORX Rat Acute High Dose – 3 Day Tissue Weights...... 113 Figure 5.1 Primary Human Hepatocyte Time Course – Gene Expression ...... 144 Figure 5.2 Primary Rat Hepatocyte Gene Expression – Time Course ...... 145 Figure 5.3 HepG2 AR Transfection – Gene Expression...... 146 Figure 5.4 Total Plasma Cholesterol – ORX Rat Time Course...... 147 Figure 5.5 Prostate Weights – ORX Rat Time Course ...... 148 Figure 5.6 Seminal Vesicle Weights – ORX Rat Time Course...... 149 Figure 5.7 Levator Ani Weights – ORX Rat Time Course...... 150 Figure 5.8 28 Day Tissue Comparisons – ORX Rat Time Course ...... 151 Figure 5.9 ORX Rat Time Course – ORX Animal Liver Gene Expression...... 152 Figure 5.10 ORX Rat Time Course – SARM2 Treated Liver Gene Expression...... 153 Figure 5.11 ORX Rat Time Course – SARM3 Treated Liver Gene Expression...... 154 Figure 5.12 ORX Rat Time Course – SARM4 Treated Liver Gene Expression...... 155 Figure 5.13 Total Serum Cholesterol – OVX Rat Extended Treatment...... 156 Figure 5.14 Percent Bone Volume – OVX Rat Extended Treatment...... 157 Figure 5.15 OVX Rat Extended Treatment – Liver Gene Expression ...... 157 xiv Figure 5.16 Total Serum Cholesterol – Intact Female Beagle Dog...... 159 Figure 5.17 Serum HDL-C – Intact Female Beagle Dog...... 160 Figure A.1 qRT-PCR Validation of cDNA Microarray Results...... 188 Figure B.1 MA Plots of LNCaP ChIP-DSL Experiment...... 196 Figure D.1 Primary Human Hepatocyte Time Course – Control Gene Expression ...... 213 Figure D.2 Primary Rat Hepatocytes Control Gene Expression – Time Course...... 214 Figure D.3 Primary Rat Hepatocyte Control Gene Expression – Time Course (No Ligand)...... 215 Figure D.4 Primary Rat Hepatocyte Gene Expression – Time Course (No Ligand)..... 216 Figure D.5 HepG2 AR Expression Time Course...... 217 Figure D.6 HepG2 AR Transfection – Control Gene Expression ...... 218 Figure D.7 HepG2 AR Transfection Target Gene Expression – Time Course (No Ligand) ...... 219 Figure D.8 HepG2 AR Transfection Control Gene Expression – Time Course (No Ligand)...... 220 Figure D.9 Soleus Muscle Weights – ORX Rat Time Course...... 221 Figure D.10 Gastrocnemius Weights – ORX Rat Time Course ...... 221 Figure D.11 ORX Rat Time Course – ORX Animal Liver Control Gene Expression.. 223 Figure D.12 ORX Rat Time Course – SARM2 Treated Liver Control Gene Expression ...... 224 Figure D.13 ORX Rat Time Course – SARM3 Treated Liver Control Gene Expression ...... 225 Figure D.14 ORX Rat Time Course – SARM4 Treated Liver Control Gene Expression ...... 226 Figure D.15 OVX Rat Extended Treatment – Liver Control Gene Expression ...... 227

xv CHAPTER 1

1. INTRODUCTION

1.1. Androgens

1.1.1 Physiological Role and Clinical Utility

Androgens are the major circulating hormone in males and elicit their myriad effects via the intracellular androgen receptor (AR)[1]. Androgens are essential in male sexual differentiation, male secondary sexual characteristics, maintenance of anabolic tissues like skeletal muscle and bone, prostate growth, male fertility, erythropoiesis, and libido in both sexes[2]. The primary endogenous androgen is testosterone (T), which can act directly by binding the AR or indirectly by reduction to the more potent 5α- dihydrotestosterone (DHT) or aromatization to 17β-estradiol (E2) in specific tissues

(Figure 1.1).

T is reduced by 5α-reductase (5αR), which has three known functional isoforms with limited and disparate tissue distributions[3]. The importance of 5αR, and therefore

DHT, is highlighted by pathophysiologies associated with 5αR deficiencies, namely male pseudo-hermaphrodism. Conversely, prevention of T conversion to DHT is a common

1 treatment paradigm in benign prostatic hyperplasia (BPH) and prostate cancer(PCa)

prevention[4, 5]. T aromatization is catalyzed by the aromatase cytochrome p450

enzyme or CYP19. Like 5αR, this enzyme has limited tissue distribution but is found

mainly in testis, adipose, liver, brain, and hair follicles[2]. The importance of CYP19 in

men, and therefore E2, was not clear until both ERα and CYP19 deficient male patients

were described. These patients presented with a number of maladies including; early

onset osteoporosis, increased gonadotrophin levels, atherosclerosis and general

endothelial dysfunction[6]. Similar side-effects are associated with androgen deprivation

therapy (ADT), employed as a treatment paradigm for PCa, which results in both total

androgen and estrogen blockade[7]. Interestingly, estrogens have had clinical success in

the amelioration of adverse effects associated with ADT[8-10]. The metabolism of T, to

both active and inactive molecules, is critical for proper androgen signaling, though the

full complexity of steroid hormone action is not clear until regulation of T synthesis is

considered.

Steroidogenesis, testicular growth and spermatogenesis are all regulated by

gonadotrophins secreted by the pituitary[11](Figure 1.2). The primary function of the gonadotrophin luteinizing hormone (LH) is the promotion of T synthesis and secretion by

Leydig cells in the testis. Follicle stimulating hormone (FSH), another gonadotrophin,

can also drive T synthesis though its primary role is promotion of spermatogenesis in the

seminiferous tubules. Both LH and FSH production are governed by systemic levels of

gonadotrophin-releasing hormone (GnRH). GnRH is secreted by the hypothalamus and

subject to negative feedback from circulating hormones, as the lyphophillic

readily pass the blood brain barrier. Negative feedback occurs at the level of the pituitary 2 as well, where LH and FSH production directly respond to increased circulating

hormones. The importance of the so called hypothalamic-pituitary-gonadal (HPG) axis is

clear in individuals with congenital gonadotrophin receptor abnormalities having

phenotypes ranging from idiopathic infertility to pseudohermaphrodism[12, 13]. Also, the HPG axis is utilized clinically where GnRH super-agonists are used in diverse scenarios to abrogate all steroidogenesis by down-regulation of GnRH production[14]. It is not surprising given the importance and ubiquitousness of androgen signaling that the opportunities for pharmacologic intervention are many.

Traditionally, T has been used to treat hypogonadism in males. Current applications have expanded to include; anemias, either aplastic or secondary to chronic renal failure; sarcopenia or muscle wasting associated with late stage cancer, severe burns or acquired immunodeficiency syndrome (AIDS); non-pituitary growth deficiencies; breast neoplasms, acting as an anti-estrogen via the HPG axis; libido enhancement in post-menopausal women; osteoporosis and osteopenia; and hereditary angioneurotic edema to name but a few [11, 15, 16]. Recent work has even shown implications for T in metabolic disorders and Type II diabetes mellitus[17]. Continued advances in the understanding of the androgen receptor’s multitude of biological functions will only expand the possibilities for androgen therapy.

1.1.2 Androgen Receptor – Structure/Function

The androgen receptor (AR) is a ligand activated, DNA binding, transcription

factor (TF) belonging to the largest family of DNA binding TFs, the nuclear hormone

receptors[18]. AR is expressed from a single genomic on the X as a

3 919 amino-acids [1, 19]. The AR gene spans 90 kilobases with 8 total exons

encoding a number of distinct functional domains (Figure 1.3). The N-terminal domain

(NTD) contains the activation-function-1(AF-1) region which is a major co-factor interface and is requisite for full AR transactivation[20, 21]. The highly conserved DNA

binding domain (DBD) contains two cysteine motifs that recognize the

canonical androgen DNA response element (ARE), 5’-AGAACANNNTGTTCT-3’, and

also functions as the receptor dimerization interface[22, 23]. The hinge region lies

between the DBD and the C-terminal ligand binding domain (LBD). This region

contains the lysine rich nuclear localization signal (NLS), the deletion of which prevents

AR translocation to the nucleus upon ligand binding, rendering the AR transcriptionally

inert[24, 25]. The LBD contains residues capable of high affinity interactions with

specific hormones and is the least conserved region amongst the nuclear hormone

receptors[26]. The LBD also contains a ligand dependent AF-2 region that adopts a

ligand-dependent conformation dictating either co-activator or co-repressor

recruitment[27]. In the absence of ligand, apo-AR is cytosolic in an inactive

conformation bound by heat-shock (Figure 1.4). Steroids, largely bound by sex

hormone binding globulin, readily diffuse through the cell membrane activating the

receptor. Upon ligand binding, holo-AR dissociates from heat shock proteins and

homodimerizes. A sequence of poorly understood conformational changes follows,

resulting in nuclear translocation, non-covalent binding to AREs and consummate

transcriptional regulation of AR target genes[11, 28]. The ability of the same hormone

signal to affect vastly different responses in varied tissues, or even temporally distinct

effects in the same tissue, is attributed to a combination of chromatin structure, available 4 co-regulator pool, and myriad intracellular signaling cascades[27, 29, 30]. To date over

300 co-regulators are known to interact with the AR[31]. These TFs, in various combinations, supplement AR action and can themselves undergo post-translational modifications as the result of intracellular and non-genomic cues[32]. Ultimately, a nearly infinite number of cellular “states” can be defined given only current understanding of nuclear hormone receptor:co-regulator interactions resulting in at least as many biological outcomes[33].

While ubiquitous AR expression and signaling can offer vast opportunities for therapeutic intervention, steroidal AR ligands have been severely limited in their application by poor pharmacokinetics, cross-reactivity with other NRs, toxicity, and the absence of tissue selectivity[34]. Non-steroidal AR antagonists addressed several of these limitations in the 1970’s and have been successful in the treatment of prostate cancer[35, 36]. The first non-steroidal AR agonist, however, was not described until

1998[37].

1.2. Selective Androgen Receptor Modulators (SARMs)

The discovery and successful clinical development of selective estrogen receptor modulators (SERMS)[38-40] has resulted in a growing effort to discover non-steroidal ligands for other nuclear hormone receptors, namely the AR. The prototypical SARM has been defined as a compound having the following properties; antagonism or weak agonism in the prostate; full agonism in the pituitary, muscle and bone; high bio- availability; and low hepatotoxicity[41]. These attributes are preferred over steroidal non-selective androgens as the clinically useful muscle and bone could be

5 achieved in the convenience of an oral dose with reduced concern of liver toxicity, while sparing prostate from potential stimulation of either undetected or nascent neoplasia[26].

1.2.1 Discovery and Characterization of the Aryl Propionamides SARMs

The first non-steroidal AR agonists, the aryl propionamides, were serendipitously discovered by making structural modifications to bicalutamide, a non-steroidal AR antagonist (Table 1.1)[37]. These ligands showed low nanomolar affinity for the androgen receptor and most importantly full agonist activity in an AR transcriptional activation assay[37, 42]. Though the molecular pharmacology of these ligands was quite similar to DHT, the true potential of the aryl propionamides wasn’t realized until in vivo characterizations were performed. In animals, the non-steroidal backbone led to improved pharmacokinetics with high bio-availability and extended half-lifes, more similar to bicalutamide's than any previously described steroidal androgen, and surprisingly- tissue selectivity[43-45].

While rapid progress in developing aryl propionamide structure activity relationships (SARs) were being made both in vitro and in vivo, in silico molecular modeling was employed to explain the activity of current molecules and predict useful modifications to the pharmacophore[46, 47]. Successful crystallization of R- bicalutamide in the LBD of the AR showed a completely different binding mode than both steroidal molecules and what had been predicted by molecular modeling[48]. This breakthrough afforded the crystallization of many aryl propionamides with the result of highly efficient structure-based drug design for non-steroidal AR agonists[49, 50].

6 With a battery of available molecules, many proof-of-concept animal studies

showed the therapeutic promise of the aryl propionamides. SARMs showed success in rodent models of benign prostatic hyperplasia (BPH)[51], male contraception[52], muscle wasting diseases[53], and even osteoporosis[54]. This progress did not go unnoticed as many of the largest pharmaceutical companies made public their efforts in SARM development[26, 45] resulting in several SARMs entering clinical trials[34].

1.2.2 SARMs – Therapeutic Promise

Theoretically, SARMs could be used in any clinical situation calling for an AR ligand not requiring estrogenic contributions. This caveat is relevant as no SARMs to date can be aromatized to estradiol[45]. The seminal challenge of SARM development has been the separation of androgenic (secondary sex tissues) from the anabolic (muscle and bone) effects. Several SARMs have shown such separation in pre-clinical animal models as well as early clinical trials extending the possibilities of SARMs as androgen therapy[34, 54, 55].

With reduced prostate liability in men, disease states requiring long-term androgen administration, such as osteoporosis, have attracted new interest[56, 57].

SARMs offer potential synergy in treatment of osteoporosis as increased muscle mass and strength could lead to increased stimulatory mechanical bone stress and reduced falls, a major morbidity in diseases of bone frailty[58]. T effects on bone maintenance are at least partially mediated through E2, though direct anabolic and anti-resorptive effects of androgens have been characterized[59]. Current osteoporosis therapies are unsatisfactory for a number of reasons including; parenteral dosing, increased risk of osteosarcoma or

7 venous thromboembolism and singular mechanism of action[11]. An orally available,

anabolic SARM could offer a novel therapy for the treatment of both primary (age related) and secondary (i.e. xenobiotic induced) osteoporosis.

Another treatment paradigm where SARMs might offer an advantage over traditional androgen therapies is in male reversible hormonal contraception. SARMs, like T at supra-physiological doses, can abolish GnRH secretion in the hypothalamus, which in turn suppresses T production and spermatogenesis via LH and FSH,

respectively. Though considered for quite some time, T administration as the “male pill”

has shown problematic variability in spermatogenic response accompanied with

sebaceous gland induction (acne), poor lipid profiles and potential stimulation of

BPH[60, 61]. Combination therapies with progestins, to suppress GnRH, and

physiological doses of T offer a safer and more efficacious alternative. However,

impractical parenteral T formulations have prevented wide-scale use[55]. A potential

caveat in modulating the HPG-axis to this end is the reduction of libido seen in other

GnRH suppressed states. As libido is a function of free T in the central nervous system

(CNS), it is likely that any SARM with sufficient CNS potency to shutdown LH/FSH will

also maintain libido. SARMs with this very pharmacologic profile have already shown

effective, reversible, suppression of spermatogenesis in rodent models[52].

The tissue selectivity of SARMs could also afford novel therapies for BPH, a

disease that affects nearly one-half of all elderly men[62]. BPH usually presents as a

urinary obstruction comprised of both a physical blockage (enlarged prostate) and smooth

muscle dysfunction[63]. Both androgens and estrogens are believed to contribute to the

hyperplasia via stimulation of the stromal and epithelial components of the prostate 8 respectively. It follows that both AR and ER antagonism have been employed clinically using 5α-reductase and aromatase inhibitors[4, 64]. Anti-androgens have been avoided due to numerous side effects, though significant adverse effects such as increases in fat mass, gynecomastia, and prostate stromal proliferation are associated with elevated circulating estradiol following 5α-reductase inhibitor administration. Also, abrogation of all estrogen production via aromatase inhibition incurs a risk of bone liability. SARMs with very weak agonism or antagonism in the prostate and full agonist activity in bone and muscle could offer a viable therapeutic option. These molecules could compete out endogenous androgens in the prostate thus reducing prostate size, showing little effect on circulating E2 levels, and potentially providing an anabolic benefit. A SARM has already shown a favorable outcome when compared directly to finasteride, an approved

5α-reductase inhibitor, in an intact rodent model of BPH[51].

Perhaps the greatest therapeutic opportunity for SARMs lies in androgen therapy for women. Androgen administration in women has been largely forsaken due to unacceptable side-effects of virilization. Virilization encompasses a number of female- to-male transformations including; clitoral enlargement, increased muscle strength, acne, hirsutism, frontal hair thinning, deepening of the voice, and menstrual disruption.

SARMs spare androgenic tissues and therefore have reduced virilization liability in women. SARMs have been suggested as hormone replacement therapy (HRT), libido enhancement, and osteoporosis treatment in post-menopausal women[34]. SARMs also offer hope of androgen therapy for women in a number of non sex-specific disease states already treatable by androgens in men.

9 1.2.3 SARMs – Therapeutic Concerns

The dissociation from a steroidal back bone and the development of tissue selectivity enabled SARMs to overcome many of the previous limitations of androgen therapies. However, some therapeutic concerns remain even in light of dynamic SARM pharmacology. Novel liabilities unique to SARMs have also arisen as androgen administration is considered in broader patient populations. Of particular consideration

are AR polymorphisms known to dictate AR response.

The androgen receptor contains a polymorphic stretch of CAG, or glutamine, repeats in the NTD varying in length from 12 to 25 amino acids in healthy normal people[65]. In some people, longer stretches up to 70 repeats have been characterized.

AR activity is inversely proportional to repeat length. In extreme cases, longer polyglutamine stretches manifest themselves as Kennedy’s disease or Spinal Bulbar

Muscular Atrophy (SAMBA)[66]. The concern for SARM therapy lies in shorter repeats, where the receptor is comparably hyperactive. There is potential for established safe and efficacious SARM dosing regimens to “lose” tissue selectivity in certain people as their response to ligand is more severe. This concern can be addressed clinically by correlating CAG repeat length to SARM efficacy and tissue selectivity.

A major therapeutic limitation of traditional androgen therapies, namely synthetic

steroidal analogs, is liver toxicity. Chemical modifications made to the 17 carbon in T

protect the parent molecule from metabolism, affording oral dosing, but can result in

severe liver toxicity[2]. Studies have suggested this hepatotoxicity is common to the 17α

alkyl group found in a number of steroid analogs and not due to increased liver exposure

10 from oral dosing [67]. One such T derivative, 17α-methyltestosterone, was in widespread use in the 1950’s but was removed from the market due to adverse liver effects[2]. Non- steroidal AR antagonists are also plagued by a host of liver pathologies including; cirrhosis, hepatitis, and even heptocellular carcinoma[68]. When considering treatment of otherwise healthy individuals for age-related maladies, the aforementioned liver liabilities are unacceptable. The clinical evaluation of numerous steroidal analogs suggests that varied structure and/or dosage formulations could circumvent this problem[2, 11], though some concerns of liver toxicity might be misdiagnosed and the result of apprehension derived largely of convention[69-71]. The liver toxicity of

SARMs, whose appeal is due in part to orally availability, remains largely unknown, though flexibility in the pharmacophore is promising in terms of evaluating various efficacious molecules for adverse liver effects.

Another therapeutic concern for SARMs are the well characterized effects of androgen therapy on serum lipid profiles[72, 73]. Androgens reliably reduce high density lipoprotein cholesterol (HDL-C) in an effect that appears worse at supra-physiological doses and with oral dosage forms[74-76]. HDL-C is thought to be cardio-protective and shows an inverse relationship with risk of cardiovascular disease (CVD)[77, 78].

Traditionally, the ratio of HDL-C, good cholesterol, to low density lipoprotein (LDL-C), bad cholesterol, is a highly prognostic CVD marker[79], though low levels of HDL-C have been established as an independent risk factor[80]. HDL-C is thought to elicit its cardioprotective effects via reverse cholesterol transport, though the relationship is decidedly complex as drugs specifically designed to raise HDL-C showed increased cardiovascular mortality and morbidity[81]. 11 Reverse cholesterol transport is the means by which excess cholesterol is

transported out of diverse tissues in the body, carried by circulating HDL-C to the liver or intestine where they are absorbed, bile acid conjugated and excreted (Figure 1.5)[82].

This process is heavily researched, as CVD is the leading cause of death in the developed

world, and amazingly complex[80]. An added complication is species diversity in lipid

homeostasis mechanisms that result in conflicting in vitro and in vivo evidence depending

on the model system used[83]. It is unclear whether androgens are affecting HDL-C

synthesis or metabolism, though selective removal of large buoyant HDL-C particles

would suggest metabolism is a more likely culprit[74, 84, 85]. Adding even further

confusion are myriad studies linking low serum T to CVD[86, 87] and suggesting the

cardio-protective effects of exogenous T administration [2, 88]. Like liver toxicity,

cardio-vascular risk assessment can only be performed in the clinic. SARMs have the

benefit of numerous efficacious chemophores with likely varied lipid effects.

1.2.4 SARM Mechanism

The molecular mechanism by which SARMs are capable of achieving tissue

selectivity is unknown. Current mechanistic insight is derived almost completely from

the more established field of selective estrogen receptor modulators (SERMs)[39, 89].

Estrogen action is mediated through two nuclear hormone receptors, ERα and ERβ,

which exhibit different biological activities. Tissue specific expression of these isoforms coupled with ligand specific receptor conformations and co-regulator recruitment have successfully explained SERM pharmacology. However, a singular AR is responsible for

12 androgen action, ruling out part of the SERM hypothesis, though AR receptor conformation and co-regulator recruitment have been active areas of research.

Like the ERs, AR interacts with a number of TFs that modulate its action. These interactions are dictated by both the cellular “state” and the conformation assumed by the receptor upon ligand binding. Kang et al showed different co-regulators are recruited, with distinct temporal relationships as well, by agonist-bound AR versus antagonist- bound AR to the prostate specific antigen promoter[90]. In a higher resolution assay,

McDonnell et al used peptide phage display to show distinct co-regulator interactions not only between SARM and DHT-bound AR, but between SARMs with varied pharmacology[91-93]. These studies suggest that SARMs could recruit different co- activator complexes in androgenic tissues than endogenous androgens and similar ones in anabolic tissues, thus explaining the tissue selectivity.

Another potential explanation is the role of 5α-reductase[94]. 5αR is almost exclusively expressed in androgenic tissues where T’s androgenic signal is amplified by reduction to DHT[3]. When co-administered with a 5αR inhibitor in castrated rats, T displays tissue-selective pharmacology quite similar to SARMs[95]. No SARMs to date directly interact with 5αR making the reduced potency of SARMs versus DHT in androgenic tissues a plausible explanation for SARMs’ tissue selective pharmacology[94,

96].

Whatever the mechanism, a full characterization SARM action would likely improve the rationale design of future SARMs. Establishing just what makes a SARM selective could revolutionize non-steroidal AR ligand development resulting in molecules tailor made for specific patient populations or disease states. 13 1.3. Scope and Objective of Dissertation

Previous work in our laboratory was devoted to pre-clinical development of the aryl-propionamide SARMs with a heavy emphasis on mechanism of action. Previous

work excluded potential explanations including active metabolite action and tissue

distribution of parent compound[96]. This research project utilized several aryl

propionamide SARMs in an extension of previous pre-clinical development and

mechanistic work. Specifically, its objectives were the following;

I. Determine if differences in potency between SARMs and DHT can explain

SARM pharmacology in prostate. One plausible mechanistic explanation for tissue-

selectivity is the reduced potency of SARMs compared to DHT in androgenic tissues.

Genome-wide expression profiling in LNCaP PCa cells revealed largely over-lapping but

distinct mRNA signatures between equi-efficacious concentrations of ligand. A follow-

up study evaluating genome-wide AR promoter occupancy following stimulation with

saturating concentrations of either SARM or DHT displayed similar but distinct AR

binding profiles.

II. Determine direct AR transcriptional targets as well as potentially ligand-specific

AR regulation. Establishing ligand-specific AR regulation could elucidate gene products

causal in SARM tissue-selectivity. More than 2000 AR regulated transcripts were

determined from gene expression profiles of SARM and DHT. Most affected genes were

regulated in similar directions to varying degrees by each ligand. A comparative

bioinformatics method was employed to establish putative direct AR targets from gene

expression profiles. More than 300 bone fide direct targets of AR regulation were

14 determined using Chromatin Immuno-Precipitation (ChIP) on Chip technology.

However, AR promoter occupancy was found to be a poor predictor of mRNA

regulation. Several direct targets of AR action were characterized by dose response and

time course analyses yielding differential regulation not apparent at a single

concentration and time.

III. Determine if the alanine amino-transferases, ALT-1 and ALT-2, are AR

regulated. Elevation of serum ALT is traditionally associated with liver toxicity.

Demonstrating ALT induction by drug action could explain asymptomatic ALT elevation

common in androgen administration. ALT-2, a bone fide direct target, showed androgen

mediated mRNA regulation in LNCaP cells with several AR ligands. In male rats, ALT-

2 but not ALT-1, was repressed following castration in both muscle and prostate tissues.

ALT-2 mRNA levels were maintained or induced following DHT or SARM

administration whereas ALT-1 showed no change. ChIP reactions were used to scan the

proximal promoter regions of each gene, but no AR loading was found.

IV. Determine the mechanism by which androgens reduce serum high density

lipoprotein cholesterol (HDL-C). Elucidating a molecular target causal in androgens’

lipid effects could permit directed screening and rationale development of SARMs with

reduced lipid effects. Androgen-mediated mRNA regulation of several genes involved in

reverse cholesterol transport were studied in a plethora of cellular and animal models.

No molecular culprit emerged, as most regulation was detected concurrently with

reductions in HDL-C but not before. The timing of these changes pointed toward genes

responding to serum lipid flux, not causing it. A multiple route of administration and

divided dose study were performed to evaluate the impact of hepatic exposure and peak 15 liver concentrations on androgen mediated reductions in serum HDL-C. No differences were seen between treatment groups suggesting the effect is not sourced in the liver.

16 5α-dihydrotestosterone 17β-estradiol Function spermatogenesis, muscle, bone turn-over, negative feedback (CNS)

p45 0ar om 5αR Tissue Tissue ACTIVE testis, adipose, liver, brain prostate, hair follicles, skin Function Function behavior, bone growth, Testosterone external virilization, sebum anti-atherogenic production

conjugation or oxidation hydroxylation

conjugation

INACTIVE hydroxylation reduction

Figure 1.1 Testosterone Metabolism A schematic of tissue-specific testosterone metabolism into both active and inactive molecules. Figure adapted from Neilshlag et al and Gao et al[2, 45].

17 Hypothalamus GnRH

Anterior LH Pituitary FSH

Target Tissue Gonads

Target Tissue Androgens Estrogens Product

Figure 1.2 The Hypothalamic-Pituitary-Gonadal (HPG) Axis A single hypothalamic releasing factor, gonadotrophin releasing hormone (GnRH), controls the synthesis and release of both gonadotrophins (LH and FSH) in males and females. Gonadal steroid hormones cause feedback inhibition at the level of the pituitary and the hypothalamus. Figure adapted from Goodman and Gilman’s Pharmacological Basis of Therapeutics[11]

18

Figure 1.3 Human Androgen Receptor – Functional Domains A schematic representation of the 110 kD hAR protein spanning 8 exons. Amino acids are numbered and allosteric interaction motifs of importance are highlighted. Figure adapted from Gao et al.[26]

19 SHBG

DHT Extra-Cellular Cytoplasm Nucleus

Figure 1.4 Androgen Receptor – Genomic Action A schematic representation of AR genomic action with DHT as the ligand. Both co- activators and repressors are recruited, though only co-activators are shown here. SHBG – sex hormone binding globulin, HSP – heat shock proteins, GTA – general transcription apparatus, DHT – dihydrotestosterone, ARA70 – coactivator 4. Adapted from Feldman et al[28].

20 Bile Salts Excretion

Liver GI-tract SR-B1 LDLR

apoA-1 LCAT PLTP CETP HL

HDL-n HDL-s HDL-l CE

Effluxed cholesterol Extra-cellular/Vascular

SR-B1 Passive ABCA1 diffusion Caveolin Cyp27A1

Intracellular cholesterol pool

Cellular

Figure 1.5 Reverse Cholesterol Transport (RCT) RCT delivers free cholesterol from cells to the liver or intestine for excretion. Cholesterol is effluxed by transporters like ATP-binding membrane cassette transporter A1 (ABCA1) and scavenger receptor B1 (SR-B1), caveolin, and Sterol 27-hydroxylase (CYP27A1). Effluxed cholesterol is adsorbed by apolipoprotein-A1 (APOA1) containing nascent HDL (HDL-n) to form small mature HDL (HDL-s) via lecithin:cholesterol acyltransferase (LCAT). HDL-s can gain cholesterol esters via phospholipid transfer protein (PLTP) to become large HDL (HDL-l). HDL-l can be metabolized to HDL-s by hepatic lipase (HL) or cholesterol ester transfer protein (CETP) giving off cholesterol esters (CE). Cholesterol is eventually delivered by HDL-l or as CE to the liver where it’s absorbed via SR-B1 or low-density lipoprotein receptors (LDLR) respectively and finally excreted as bile salts. Figure adapted from Ohashi et al[82]

21

RBA Name Compound Structure Activity Ref. (%)

5α-dihydrotestosterone 100 Full Agonist [97]

Testosterone 28 Agonist [97]

R-Bicalutamide .4 Antagonist [97]

Partial to Aryl Proprionamide 1-11 Full [43, 52, 98] SARM Agonists

Table 1.1 Steroidal and Non-Steroidal AR Ligands Relative binding affinity (RBA) is represented as a percentage of DHT.

22 CHAPTER 2

2. SARM VERSUS DHT: GENE EXPRESSION PROFILING IN LNCAP

PROSTATE CANCER CELLS

2.1. Introduction

SARMs are named for being fully efficacious in muscle and bone while having

only weak agonism in prostate. Previous work on the aryl propionamide SARMs

excluded active metabolite action and tissue distribution of parent compound as potential

drivers of tissue selectivity for this class of SARMs[96]. Prostate tissue has been the

primary focus of continued work on the molecular mechanism of SARM action following

the rationale that a tissue displaying disparate pharmacological response might yield

more readily attainable mechanistic answers. One explanation for the reduced efficacy of

SARMs in prostate is the lack of “amplification” of the androgenic signal by 5α-

reductase, essentially arguing SARMs are equipotent to endogenous androgens in

anabolic tissues, but less potent than endogenous androgens in androgenic tissues that

commonly convert T to DHT[94]. This hypothesis was tested by performing gene

expression profiling of the endogenous prostatic androgen DHT and an aryl-

propionamide SARM in prostate cancer cells. If in vivo pharmacological differences 23 between the molecules are due to potency alone, gene expression profiles at equi- efficacious concentrations of ligand should overlap.

2.2. Materials and Methods

2.2.1 Materials

COS-1 monkey kidney cells and LNCaP human prostate cancer cells were purchased from ATCC (Manasas, Virginia). RPMI-1640 medium and Trizol® reagent were purchased from Invitrogen Corp. (Carlsbad,California). Trypsin-EDTA.(0.25%) and DMEM were purchased from MediaTech (Manasas, Virginia). Charcoal-treated fetal bovine serum (cFBS) was purchased from HyClone (Logan, UT).RNeasy Mini Kit was purchased from Qiagen Inc. (Valencia, CA). Affymetrix® U133A Array was purchased from Affymetrix Inc. (Santa Clara, CA). DHT was purchased from

Sigma-Aldrich (St. Louis, Missouri). SARM1 was synthesized using previously reported methods[44].

2.2.2 LNCaP Cell Growth Curve

LNCaP cells were cultured according to ATCC’s recommendation. Cells were plated in 96 well plates at a seeding density of 5000 cells/well. 48 hours before treatment, cells were switched to phenol red-free RPMI-1640 medium supplemented with

10% charcoal-stripped FBS. Phenol red-free medium was used to avoid the stimulatory effects of phenol red on LNCaP cell growth[100]. After 48 hours of androgen deprivation, cells were treated with various concentrations of DHT (10E-4 nM to 10E+2 nM) or SARM1 (10E-3 nM to 10E+3 nM) for 24, 48, or 96 hours (n=5). The cell number 24 after different treatment periods was determined using the colorimetric sulforhodamine

(SRB) assay.

2.2.3 AR Trans-activation in COS-1 Cells

COS-1 cells were cultured according to ATCC guidelines. Cells were trypsinized

and plated in 24-well plates (Corning) at a density of 125,000 cells per well in phenol

red-free DMEM media supplemented with 5% charcoal stripped FBS. 24 hours later

cells were incubated in Opti-MEM (Invitrogen) for 30 minutes before transient

transfection using FugeneHD (Roche) in accordance with manufacturer’s instructions.

Aliquots of GRE-Firefly Luciferase (0.25 µg)[101] and CMV-Renilla Luciferase (5 ng)

plasmids were added along with either 12.5 ng of empty pCR3.1 (Invitrogen), wtAR or

pCR3.1 T877A AR[102] plasmids. Cells recovered for 12 hours in phenol red-free

DMEM media supplemented with 5% charcoal stripped FBS. Cells were treated for 24

hours in phenol red free DMEM media supplemented with 5% charcoal stripped FBS

containing increasing concentrations of DHT or SARM1 (.01 nM – 1000 nM) or vehicle

(0.1% ethanol in media)(n=3). Following treatment, cells were assayed for transcriptional activity using Dual-Glo (Promega) using manufacture’s guidelines and a

Victor 3V Multi-label Counter (Perkin-Elmer).

2.2.4 cDNA Microarray Study Design

LNCaP cells were sub-cultured in accordance with ATCC’s recommendations in

15 cm plates. 48 hours prior to treatment, cells were switched to phenol red-free RPMI-

1640 medium supplemented with 10% charcoal stripped FBS. Cells were treated with

25 DHT (1 nM), SARM1 (1 nM), or vehicle (0.01% ethanol in culture medium) for 24

hours. The experiment was performed in biological triplicate at one time. Total RNA was isolated using Trizol® reagent and cleaned up using RNeasy Mini Kit in accordance

with the manufacturers’ instructions. Prior to microarray analysis, the integrity of the

RNA sample was confirmed by capillary electrophoresis. Microarray analysis using

Affymetrix® human genome U133A gene chip was performed by Dr. Herbert Auer in

the OSU Comprehensive Cancer Center Microarray Unit. The protocols for all the

procedures are available on line: www.dnaarrays.org.

2.2.5 cDNA Microarray Data Analysis

Gene expression levels were estimated from GeneChip® PM probe intensities by

means of an enhanced version of the Li-Wong PM (perfect match)-only algorithm [103].

The enhanced algorithm: 1) scales all PM and MM (mismatch) probe intensities so as to

minimize between-array differences in the scaled MM probe intensity distributions; 2)

applies between-array regression analysis to the scaled PM probe intensities in order to

estimate PM-specific sensitivities, excluding any PM probes that fail to show

significantly positive sensitivities; 3) estimates gene expression levels by regressing

scaled PM probe intensities on estimated PM probe sensitivities within each probe set,

excluding any PM probes that show significant non-monospecificity; 4) tests a probe-

level GLM (General Linear Model) within each probe set in order to estimate the p-

values for between-array differential gene expression. The estimated p-values can be

several orders of magnitude lower than 0.05, as required by the Bonferroni correction

26 which applies when simultaneously testing thousands of genes for significant differential expression[104].

All groups were competitively hybridized to untreated androgen-deprived cells.

Normalized probe intensities for each probe set in each treatment were used in pair-wise

comparisons between DHT-treated cells and vehicle-treated cells, SARM1-treated cells

and vehicle-treated cells, and between DHT-treated cells and SARM1-treated cells. The

difference in gene expression was considered significant only when the negative log of

the estimated p value was no less than 6 (NLP>=6).

2.2.6 Orthologous Promoter Androgen Response Element (ARE) Search

Matrix Construction 28 well-characterized AREs (Table 2.1) were compiled from

Natermet et. al. [105]. The position weight matrix (PWM) was using the MatInd

method[106]. All 15 sequences were aligned and the relative frequency of each

base at each position was calculated. Consensus index vector (Ci ) values were calculated

for each position i in the matrix:

⎡ ⎤ Ci (i) = (100 / ln 5)× ⎢ ∑ P(i, b)× ln P(i,b) + ln 5⎥ ⎣b∈A,G,T ,C,gap ⎦

0 ≤ Ci ≤ 100

This resulted in a Ci vector for the AR matrix (Figure 2.3).

Data Retrieval For all 107 genes showing 2x or greater regulation, the NetAffx

Analysis Center (http://www.affymetrix.com/analysis/index.affx) was used to retrieve

Unigene IDs. These Unigene IDs were used in human and mouse promoter sequence

27 retrieval from Orthologous Mammalian Gene Promoters Database

(http://bioinformatics.med.ohiostate.edu/OMGProm/) or via UCSC’s Genome Browser

(http://genome.cse.ucsc.edu/). All assignments of orthology were based on Homologene

(http://www.ncbi.nlm.nih.gov/sites/entrez?db=homologene) declarations. Due to often

conflicting annotations of the transcriptional start site (TSS), 7kb surrounding the TSS

(5kb upstream and 2 kb downstream) were retrieved. In the case where multiple

transcriptional variants were defined, all transcripts were retrieved and examined. Only

92 of 107 genes had an annotated human TSS at the time of query. Those sequences

have a TSS were then queried for orthologous mouse promoters with annotated TSSs.

Ortholgous promoters were then aligned using ClustalW. A minimum identity of 70%

was required to be carried further. Only 82 of the remaining 92 genes met these criteria.

Figure 2.4 contains a summary of the data set flow.

Matrix Searching Using a modified version of the Perl script MatScan, human

sequences were searched, 15 base pairs at a time, using the MatInspector[106]

methodology. First identity to the core sequence, defined as the 5 consecutive bases with

the high possible sum of Ci values, was required. In the AR matrix, bases 5-10 made up

the core. If core identity was achieved, then the matrix similarity score was calculated as

follows:

⎡ n ⎤ ⎡ n ⎤ mat _ sim = ⎢∑ Ci ( j)× score(b, j)⎥ /⎢∑Ci ( j)× max_ score(b, j)⎥ ⎣ j=1 ⎦ ⎣ j=1 ⎦ 0 ≤ mat _ sim ≤1

Where Ci(j) is the consensus index value at postion j, n is the length of the consensus matrix, score(b, j) is the matrix value for base b at postion j and max_score(j) is the

28 product of the frequency of the most conserved based at postion b and the Ci value. If a mat_sim score of .9 or greater was detected then 400 bases, in both 5’ and 3’ directions, from the first position in the human sequence were searched in the orthologous mouse sequence. If a mouse mat_sim score of .9 or greater was detected a putative ARE was reported.

2.2.7 Gene Expression Validation

qRT-PCR was performed on gene target representing several types of regulation detected in the cDNA microarray experiment on and ABI 7900HT thermo-cycler. Primer pairs were designed using PrimerExpress© (Applied Biosystems) and qRT-PCR performed with SYBR Green© 2X master mix. Melting curves were created to check for single amplicons of the correct size. Fold changes were calculated using the 2-∆∆Ct method[107] with 18s used as an internal control.

2.2.8 Electro-Mobility Shift Assay (EMSA)

AR DBD Expression and Purification. AR-DBD (AA549-650) was cloned as a glutathione-S-transferase (GST) fusion protein into the pGEX6P-1 vector (Amersham,

Piscataway, NJ) and expressed in E. coli BL21 DE3. Cells were grown in 2X YT media at 37 ºC and induced at 37 ºC for 3 hours with 30 µM IPTG. Cells were lysed in a buffer containing 150 mM NaCl, 50mM Tris pH 8.0, 5 mM EDTA, 10% glycerol, 1 mg/mL lysozyme, 10 U/ml DNase I, 10 mM MgCl2, 10 mM DTT, 0.5% CHAPS, and 100µM

PMSF by 3 cycles of freeze-thaw. Lysates were centrifuged at 20,000g for 1hr. Lysate supernatants were then incubated for 2 hours at 4 °C with glutathione Sepharose resin

29 (Amersham), and washed with 150 mM NaCl, 50 mM Tris pH 8.0, 5 mM EDTA, and

0.5% CHAPS five times. The GST tag was cleaved on-column in a buffer containing

150 mM NaCl, 50 mM Tris pH 8.0, 1 mM EDTA, and 1 mM DTT for 12 hours at 4 ºC using PreScission Protease (Amersham, Piscataway, NJ). The supernatant containing the cleaved DBD was then diluted 3-fold in a buffer containing 10mM Hepes pH 7.2, 10% glycerol, 500mM NaCl, 1 mM DTT, and 0.03% n-octyl-β-glucoside, loaded onto an HP

SP cation exchange-column (Amersham), and eluted with a gradient of 50 mM to 500 mM NaCl in the same dilution buffer. Fractions were examined by SDS-page gel electrophoresis. The most concentrated fractions were combined and protein content determined by Bradford Assay.

EMSA EMSAs were performed according to Hope and Struhl[108] with modifications.

35mers were synthesized in perfect complement pairs (IDT, Coralville, IA) with 10 base pairs of genomic DNA flanking the putative ARE (Table 2.2). The oligo pairs were mixed and heated to 90 ºC then allowed to anneal at room temperature. The annealed pairs were then 5’ labeled using T4 Polynucleotide Kinase (NEB, Ipswitch, MA) and [γ-

32P]-ATP (PerkinElmer, Waltham, Mass.) according to the manufacturer’s instructions.

Binding reactions including labeled probes, 2 µg polydI:dC, and purified AR-DBD were then incubated in a buffer containing 12 mM HEPES pH 7.9, 2 mM DTT, 3 mM MgCl2,

.5 mM EDTA, 50 mM KCl, and 15% glycerol at room temperature for 30 minutes. The labeled oligos were added at 200-fold molar excess to the AR-DBD. This reaction was then loaded onto a 5% polyacrylamide gel and electrophoresed in .25x TBE. Gels were then dried and exposed to either Biomax X-ray film (Kodak, Rochester, NY) or a storage phosphor screen for visualization. 30 2.2.9 Gene Ontology Functional Analyses

Functional annotations were reported in a similar fashion to Kazmin et al. [92].

GO declarations for all genes showing significant regulation (2130) were retrieved using

NetAffix (http://www.affymetrix.com/analysis/index.affx) as a batch. Gene Ontology annotations were retrieved for 1981 or 93% of the search terms. A combination of Perl scripts and Microsoft Excel were used to bin terms by treatment and determine GO terms differing in distribution from the “Total Unique” group.

2.3. Results

2.3.1 LNCaP Growth Curve

DHT and SARM1 stimulated cell growth was determined using LNCaP cells

(passage # 38) after 48 hours of androgen deprivation. Only very small changes in cell number were observed after the first 24 hours of treatment, in accordance with the near

48 hour doubling time of LNCaP cells[100] (Figure 2.1). The 48 hour treatment groups showed greater than 50% increases in cell number in both DHT and SARM1 treated cells at concentrations 10 nM and higher. When treatments were carried out to 96 hours,

100% increases in cell number were detected in both the SARM1 and DHT groups at 1 nM. SARM1 maximal cell growth induction was nearly 300% at 10 nM, whereas DHT peaked at 200% at 1 nM, decreasing at both 10 and 100 nM. This biphasic behavior of the endogenous androgen DHT on LNCaP cell growth is a well-characterized phenomenon [109]. The 1 nM treatment showed the least difference in cell number

31 across all examined times and at 24 hours showed very little cell growth. These conditions were chosen for gene expression profiling for the following reasons; 1) they resulted in equally efficacious cell growth between ligands, 2) they maximize treatment time but minimize potentially confounding increases in cell number and 3) minimize the likelihood of detecting indirect AR transcriptional regulation.

2.3.2 Gene Expression Profile

The Affymetrix HU133A microarray contains 18,400 transcripts and variants, representing 14,500 well-characterized human genes. When treated for 24 hours with 1 nM of ligand, 2077 transcripts showed significant expression changes in ligand-treated cells compared to the vehicle control (Figure 2.2). 843 of these genes were regulated by both SARM1 and DHT in a similar fashion (black dots), split nearly equally in the up and down directions, 469 and 374, respectively. 1015 genes were regulated by SARM1 but not DHT (green dots). These transcripts were primarily down regulated, with 911 showing repression versus 104 showing induction. Conversely, 214 genes were regulated by DHT alone (red dots). These genes were overwhelmingly up-regulated with 209 genes showing induction versus only 10 showing repression. Another 26 transcripts showed significant regulation between the ligands, but not compared to vehicle-treated cells (blue dots).

Most regulated transcripts were regulated in a similar fashion by SARM1 and

DHT as evidenced by a near 1:1 correspondence in fold change with an r2 value of .89

(Figure 2.2). Genes regulated only by SARM were 90% down regulated as opposed to genes regulated only by DHT which were 98% up regulated. No genes regulated in

32 opposite directions achieved 2-fold or higher regulation, a commonly accepted cut-off for regulation of likely biological consequence.

When compared to existing gene expression profiling studies in prostate, a large number of previously reported AR regulated genes were detected[105, 110, 111]. In considering just the 107 genes showing at least 2-fold or greater regulation in SARM1 or

DHT, 36 were reported elsewhere as AR regulated (Table 2.3 - Table 2.4).

2.3.3 Gene Expression Validation

In an effort to validate the competitive hybridization experiments, 7 targets were chosen for standard qRT-PCR analysis using the same treatment conditions;

1) KLK2, KLK3, MAF; SARM1 and DHT versus Control up regulated.

2) PAK2, MME; SARM1 versus Control down regulated.

3) BTG3; DHT versus Control down regulated.

4) IRS1; SARM1 versus DHT

These targets were reproducibly regulated when assayed by qRT-PCR with the magnitudes of regulation sharing a near perfect 2:1 correspondence (Figure A.1).

2.3.4 Putative Androgen Response Elements

Of the 107 genes regulated by 2-fold or more, only 82 met the criteria needed for the comparative PWM search. 48 conserved putative AREs representing 30 different genes were found. The search method found the characterized KLK2 ARE [110] (Table

A.2) that was used in matrix construction in the data set as well as AREs in the

TMPRSS2 gene enhancer, a gene with well characterized direct AR regulation[112].

33 These two genes are considered sound positive controls. Also two negative controls, 18S ribosomal RNA and RNA Pol IIB, were searched and did not return conserved AREs. 13

AREs from 6 genes of 19 searched (31.5%) were found in those transcripts up-regulated

2-fold or more by SARM1 only (Table A.1). 12 AREs from 7 genes of 12 searched

(58%) were found in those transcripts up-regulated 2-fold or more by SARM1 and DHT, including one on an intron-exon boundary in the GNMT gene (Table A.2). Only 1 ARE from 1 gene of 3 searched (33%) was found in those transcripts up-regulated 2-fold or more by DHT alone (Table A.3). A single ARE from 1 gene of 8 searched (12.5%) was found in those transcripts down-regulated by 2-fold or more by both ligands (Table A.4).

Finally, 22 AREs from 14 genes of 40 searched (35%) were found in those transcripts down-regulated 2-fold or more by SARM1 only (Table A.5-Table A.6). Only 3 or 6% of the AREs were down stream of the TSS in exons. 9 or 19% of the AREs were found within introns downstream of the TSS with another 9 landing in the promoter region (0 to

-2000 base pairs). The greatest number of conserved AREs, 27 or 56%, was found in the enhancer region (-2001 to -5000 base pairs).

2.3.5 EMSA Validation of AR Binding Potential

12 putative AREs detected in the bioinformatics search were evaluated for in vitro

AR-DBD binding along with a characterized high affinity sequence, SRE_1[113], and a random sequence from the PSA promoter region (PSA_NC) (Table 2.2). These ARE’s represent induced and repressed genes as well as those regulated by both ligands. Also the AREs chosen spanned the range of matrix match scores from .90 (CITED2_2) to .98

(KLK2_1). SRE_1 bound the AR-DBD and was reversed by including a 100x excess of

34 un-labeled probe (Figure 2.5). Expected Protein:DNA interactions were also detected between the AR and the KLK_2 ARE and the two AREs examined from the TMPRSS2 regulatory region. Taken with the lack of binding detected in the PSA_NC experiment, the assays’ results are in accordance with known AR:DNA interactions. On the whole,

58% percent of the AREs tested bound the AR-DBD with no discernable relationship between matrix score and binding as both the highest and lowest scoring oligos bound.

57% of the AREs tested belonging to genes up-regulated by SARM1 and DHT bound the

AR, whereas only 25% of theose belonging to repressed genes bound the AR DBD. The difference in signal strength seen between probes is likely the result of T4 polynucleotide’s varied 5’base labeling efficiency.

2.3.6 Functional Analyses

1981 of the 2130 significantly regulated transcripts were functionally annotated with one or more Gene Ontology terms (Figure 2.6). Two GO classifications were examined as positive controls; 1) 209 regulated transcripts were annotated with the biological process “Gene Expression”. The large number of transcripts with this annotation is expected as androgens are known to trigger genomic signaling cascades.

The large overlap of the two treatments in this functional classification coincides with the similar gene expression profiles (Figure 2.2). However, DHT maintained singular regulation over 44 of these transcripts while SARM only regulated 2 singularly. 2) 19 transcripts were annotated specifically with the biological process “Response to Hormone

Stimulus” and again their distribution was enriched for regulation by both ligands. This annotation is reflective of the ligands acting through a nuclear hormone receptor with

35 largely overlapping results. “Positive Regulation of Cell Proliferation” was enriched for

BOTH as well as DHT only. This functional classification indicates that DHT uniquely regulates 11 genes known to positively affect cell proliferation, whereas SARM1 regulates only 1. The terms “Cholesterol Metabolic Process” and “Cholesterol

Biosynthetic Process” are enriched for overlap and imply similar regulation of the processes by both ligands. The biological process “Muscle Development” was very reflective of the “Total Unique” distribution which can be interpreted as both ligands regulating transcripts involved in “Muscle Development” but greater than 50% of those transcripts are unique to either SARM1 or DHT.

2.4. Discussion

With the goal of gaining insight into SARMs’ molecular mechanism of action, cDNA microarray analysis was performed in prostate cancer cells comparing equally efficacious concentrations of SARM1 with the primary prostatic endogenous androgen

DHT. However, profiling of SARM1 and DHT were performed in a prostate cancer cell line with a known LBD mutation (T877A) that has been shown to concomitantly reduce the AR binding affinity of DHT and increase the SARM’s affinity via trading a hydrogen bond[102]. These changes in affinity also resulted in an increase in the transcriptional activity of the SARM in T877A AR over WTAR with an apparent reduction in both the potency and efficacy of DHT in the mutant receptor (Figure 2.7). Interestingly, identical transcriptional activities in a transient transfection system, properly reflecting the

LNCaP’s mutant receptor, do not agree with the disparate growth properties in LNCaP with extended treatment (Figure 2.1). This alone suggests the existence of potential

36 qualitative differences, not due to varied potency alone, in these androgens’ mechanism of action. However, the stability of DHT in culture at 96 hours is in doubt as reports of its in vitro inactivation by 3-hydroxysteroid dehydrogenase (3-HSD) or by 3ß-HSD in prostate cancer cells exist[114, 115]. Nevertheless, differences between the LNCaP model and the in vivo situation could explain in part the largely overlapping, but yet distinct, gene expression profiles seemingly at odds with the disparate in vivo pharmacology[53].

An early time point was chosen for expression profiling, which in theory limited the detection of secondary transcriptional events. Direct targets of AR action are more likely to yield mechanistic information as they are considered “closer” to the initial stimulus of ligand addition[112]. To further discern direct AR targets, a bioinformatics search for Androgen Response Elements was performed. Simple sequence matching to the canonical ARE is too rigid to incorporate what is known to be rather diverse ARE sequence content (Table 2.1). Also, only slightly more complicated mis-match allowance techniques do not account for known invariant bases required for AR binding[113]. The MatInd methodology[106] integrates known sequence variety by using a PWM and allows an assignment of a core sequence with separate matching thresholds than the matrix as a whole. The combination of these features result in a flexible tool capable of predicting binding sites with high fidelity.

MatScan, an implementation of the MatInd methodology, was used to search the promoter regions of all genes regulated by 2-fold or more. AREs returned from the search largely bound the AR (Figure 2.5) displaying the potential for direct regulation in

37 vivo. 30 genes with AREs conserved between human and mouse were considered potential direct targets of AR action.

Two novel potential direct targets of AR are involved in normal and aberrant proliferation, including the transcription repressor Snai2 (SLUG) which was up-regulated by both SARM1 (8.96 fold) and DHT (2.34 fold) and bound the AR in EMSA analysis.

This gene is known to regulate the epithelial-mesenchymal transition in metazoan development and provide resistance to pro-apoptotic signals in breast cancer cells [116].

Though SLUG’s role in prostate is poorly understood, SARM1’s greater SLUG induction could at least partially differences in the prostatic effects of DHT and SARMs. In vivo studies of the effects of SARM and DHT on Snai2 in the prostate are needed to better understand its role, if any. Similarly, Catenin (CTNNAL1) upregulated by SARM1 (3.97 fold) and DHT (2.76 fold), is a scaffold protein involved in Rho signaling during actin cytoskeleton mobilization[117]. Over expression of CTNNAL1 resulted in increased growth in several cellular systems and could potentially contribute to ligand differences in LNCaP growth seen at later time points.

Glycine-N-methyltransferase (GNMT) (SARM1 up 2.12 fold and DHT up 2.12 fold, EMSA confirmed) has no direct role in cell growth. However, recent work has classified GNMT as a tumor susceptibility gene in both liver and prostate cancers.

GNMT affects genetic stability by regulating the ratio of S-adenosylmethionine to S- adenosylhomocysteine, binding to folate, and interacting with environmental carcinogens, such as benzo(a)pyrene. Loss of heterozygosity at the GNMT locus was associated with prostate cancer and GNMT was down-regulated in tumor tissue from patients[118].

Establishing GNMT as a direct transcriptional target of the AR in prostate could offer a 38 potential mechanism for the development of androgen-independent cancer following androgen deprivation therapy. Anti-androgens, while blocking endogenous growth signals, would likely repress GNMT expression creating genomic instability and such epigenetic events are characterized as requisite in prostate cancer progression[119].

Gene Ontology functional analyses of all regulated genes, including transcripts regulated less than two fold and likely indirect AR targets, both confirmed similarity between the ligands’ pharmacology and provided insight into potential differences into mechanisms of action (Figure 2.6). Interestingly, “Positive Regulation of Cell

Proliferation” was enriched for overlap and decreased for SARM1 which was more reflective the increased growth potential of DHT over SARM1 in actual prostate tissue than in the model system used[53]. Also, 34 genes involved in Muscle development were regulated in LNCaP cells. Though NR transcriptional networks are distinct between tissues[27], if extrapolated to muscle tissue, the distribution of “Muscle Development” transcripts would suggest that SARM1 and DHT regulate muscle development in distinct ways.

Combining gene expression profiling with bioinformatic determination of potential direct targets of AR action permitted both a genome wide evaluation of each ligands’ transcriptional signature and an in silico identification of initiators of AR signaling cascades in LNCaP cells. Though AR gene expression profiles are known to be both time and dose dependent[92], resource constraints dictated evaluation of a single time point and a single concentration. This paradigm resulted in distinct but largely overlapping expression profiles and a small number of putative direct AR targets. It is likely given the subtle differences detected, in spite of inherent model corrections for 39 disparate potency and efficacy combined with conditions chosen to be equally efficacious in cell growth, that expanded dose response and time course analyses would elicit greater wholesale differences in gene expression profiles. Also as transcription factor binding is known to occur at great distances from transcribed regions of the genome [112, 120], an expanded ARE search would have likely produced more putative direct targets. As an alternative methodology, chromatin immuno-precipitation (ChIP) assays can be used to identify protein:DNA interactions, and more specifically bone fide direct targets of transcription factors. Using ChIP, AR:DNA interactions can be captured immediately following ligand addition that result in transcriptional regulation at varied time points thereafter. This potentially increases the information content at a single time point when compared to gene expression profiling. If applied on a genome-wide scale, ChIP-on-

Chip is thought to afford the most direct evaluation of both transcription factor binding and potential elucidation of ligand specific genomic signaling cascades.

Despite efforts to control for disparate potencies between SARM1 and DHT, their gene expression profiles in LNCaP were distinct. However, truly differential regulation was not apparent, as most transcripts were regulated in similar directions to varying degrees by each ligand. These differences, though subtle, suggest reduced potency alone cannot explain SARM pharmacology in the prostate.

40 2.5. Acknowledgments

LNCaP cell growth experiments, sample RNA isolation, and expression validation were performed by Dr. Wenqing Gao. AR-DBD cloning and purification were completed in large part by the combined efforts of Drs. Wenqing Gao and Casey Bohl. cDNA microarray data analysis was performed by Dr. Karl Kornacker. WT-AR and

T877A-AR expression plasmids were provided by Dr. Casey Bohl. GRE-LUC plasmid was provided by the laboratory of Dr. Nancy Weigel. Access to the OMGprom database and MatScan were graciously provided by Drs. Victor Jin and Ramana Davaluri.

41 LNCaP Cell Growth Curve 350 DHT-24 hours 300 DHT-48 hours DHT-96 hours SARM1 -24 hours 250 SARM1 -48 hours SARM1 -96 hours

200

150

100

50 % of Androgen-deprived LNCaP Cells Androgen-deprived of %

0 10E-4 10E-3 10E-2 10E-1 10E0 10E+1 10E+2 10E+3

Concentration (nM)

Figure 2.1 LNCaP Cell Growth in the Presence of DHT and SARM1 Following 48 hours of hormone deprivation in charcoal-stripped media cells were treated with the indicated concentrations of ligand for 24, 48 and 96 hours (n=4-5). Cell number was determined using the SRB assay and values are reported as a percentage of untreated cells.

42 Gene Expression Profile in LNCaP 10

8

6

4

2

R2 = .896 0 y = 1.057320x - .026635

-2

Fold Change 1 nM SARM 24 hours SARM 1 nM Fold Change -4

-6 -4 -2 0 2 4 6

Fold Change 1 nM DHT 24 hours

Figure 2.2 Gene Expression Profile in LNCaP Fold change in expression level compared to the vehicle control was plotted. Transcripts were grouped based on significant changes observed during pair-wise comparison. (●) DHT vs Ctrl (NLP>=6) and SARM1 vs Ctrl (NLP>=6), 843 transcripts. (●) DHT vs Ctrl (NLP>=6), but not SARM1 vs Ctrl, 219 transcripts. (●) SARM1 vs Ctrl (NLP>=6), but not DHT vs Ctrl, 1015 transcripts. (●)DHT vs SARM1 (NLP>=6), but not DHT vs Ctrl or SARM1 vs Ctrl, 53 transcripts. A total of 2130 transcripts showed significant differences in pair wise comparison.

43

100

90

80

70

60 Ci 50

40

30

20

10 123456789101112131415 Position

Figure 2.3 Androgen Response Element PWM WEBLOGO and Ci Vector Plot WEBLOGO representation of the information content at each position in the 15 base pair ARE alignment (Top). Ci vector plot of ARE alignment created by using the MatInd methodology (Bottom).

44 Microarray Analysis 107 genes - 100% 2 fold change

Retrieve 7kb Human Promoters from MPromDb No annotated TSS

Homolgene assignment

Retrieve 7kb Mouse Promoters 92 genes - 86% from MPromDb

ClustalW global alignment 70% identity threshold Inadequate Homology

Search Human Promoters w/ ARE PWM 82 genes - 77% 100% Core, 90% overall threshold

Comparative Search Mouse homolog in corresponding 800bp window PMW Search

100% Core, 90% overall threshold

Putative ARE Data Set 30 genes

Figure 2.4 Comparative ARE Search Dataset Flow A graphical representation of how the cDNA microarray data set changed at each step in the comparative ARE search. 77% of the genes regulated by 2x or more were subjected to the search resulting in 30 genes with 1 or more conserved AREs.

45 ABCA1_1 ABCA1_2 CITED2_1 CITED2_2 FACL3_1 GNMT_1 KLK2_1* AR-DBD -+ + -+ + -+ + -+ + -+ + -+ + -+ XS-COLD SRE --+ --+ --+ --+ --+ --+ --

GNMT_2 GNMT_3 SRE_1 SNAI2_1 TMPRSS2_1 TMPRSS2_2 PSA_NC* AR-DBD -+ + -+ + -+ + -+ + -+ + -+ + -+ + XS-COLD SRE --+ --+ --+ --+ --+ --+ --+

Figure 2.5 EMSA Validation of Putative AREs GST-tagged purified AR-DBD was incubated with radio-labeled synthesized oligonucleotides represented in Table 2.1 and examined on a native protein gel. Reactions producing band retardation indicate Protein/DNA binding. The addition of an excess of unlabeled SRE control probe reversed the shift indicating specific binding. (*analyzed in a different experiment visualized by phosphor imaging).

46

Figure 2.6 GO Term Analysis of cDNA Microarray Data Genes significantly regulated up or down in the cDNA microarray experiment, also having functional GO annotations, are represented in the “Total Unique” chart by treatment. Specific GO terms, with the number of regulated genes sharing that GO term in parentheses, are represented as well.

47 AR Transactivation - COS-1 cells 0.6

DHT (WT) 0.5 SARM1 (WT) DHT (T877A) SARM1 (T877A) 0.4

0.3 RLU 0.2

0.1

0.0

e icl nM nM nM nM nM nM eh 1 .1 1 0 0 0 V .0 1 10 00 1

Figure 2.7 AR Transactivation in COS-1 – WT vs T877A AR Dose response curves of both DHT and SARM1 in a transient transfection system. When comparing the dose response in WT versus T877A, reduced potency and efficacy of SARM1 versus DHT in WT are lost in the T877A mutant.

48

Gene Sequence Score PSCA hi-affinity GGAACTttcCGTCCT 0.91 AR 1 CTTTCTgaaTGTCCT 0.84 AR 2 AGTACTcctGGATGG 0.77 CDKN1A/p21 AGCACGcgaGGTTCC 0.89 F9 AGCTCAgctTGTACT 0.94 KLK2 GGAACAgcaAGTGCT 0.98 KLK3/PSA 3 GGAACAtatTGTATC 0.92 MME CTCACAaagAGTTCT 0.86 PIGR GGCTCTttcAGTTCT 0.90 FGF8 F1R1 GGGCCTggcTGTGCT 0.89 PEM ARE-2* AGCACAtcgTGCTCA 0.92 PEM ARE-1* ATCTCAttcTGTTCC 0.87 NEP GTCACAaagAGTTCT 0.90 Probasin G-1 GGGACAtaaAGCCCA 0.89 Probasin G-2 ATGACAcaaTGTCAA 0.83 PSA Enhancer V GGGACAactTGCAAA 0.85 PSA Enhancer III AGGACAgtaAGCAAG 0.84 PSA Enhancer IV AGATCAtgaAGATAA 0.82 SLP 2 AGAACTggcTGACCA 0.89 Probasin ARE1 ATAGCAtctTGTTCT 0.89 Probasin ARE2 AGTACTccaAGAACC 0.89 PSA Enhancer III GGAACAtatTGTATT 0.93 PSA ARR GGATCAgggAGTCTC 0.87 PSA ARE AGAACAgcaAGTGCT 0.99 SLP 3 AGAACAggcTGTTTC 0.94 SC GGCTCTttcAGTTCT 0.90 KLK-2 GGAACAgcaAGTGCT 0.98 C3 AGTACGtcaTGTTCT 0.96 Consensus(Transfac) GGTACAnnnTGTTCT 0.98

Table 2.1 Characterized ARE Sequences Used in PWM Known androgen receptor binding sites used to construct the position weight matrix were used in the sequence search and the matrix score assigned when searched with the assembled matrix. Colored shading indicates identical C and G bases at positions 5 and 11. * indicates murine sequences.

49 ARE EMSA ARE Forward(5'-3')/Reverse(3'-5') Primer Matrix Score ABCA1_1 (-) tggtgagcacTGGACTCTATGTTCAggtgctgagg 0.92 accactcgtgACCTGAGATACAAGTccacgactcc

ABCA1_2 (-) ctcccagtatTGATCATTGTGTTCTgagttggggg 0.92 gagggtcataACTAGTAACACAAGActcaaccccc

CITED2_1 (-) ctaaaatactGCCTGAAGCACACGTtcgtcgtctt 0.91 gcttttatgaCGGACTTCGTGTGCAagcagcagcc

CITED2_2 (+) tggcacttggAGGACATTCAGTGGAcggatgacaa 0.90 accgtgaaccTCCTGTAAGTCACCTgcctactctt

FACL3_1 (-) tgcttccaagCGTACACACAGTGCTttggtgtttg 0.93 acgaaggttcGCATGTGTGTCACGAaaccacaaac

GNMT_1 (+) cgtagcctgtGGCACTGGGTGAGCCcaggccgggg 0.91 gcatcggacaCCGTGACCCACTCGGgtccggcccc

GNMT_2 (-) cgcaggatggTGGACAGCGTGTACCggacccgctc 0.96 gcgtcctaccACCTGTCGCACATGGcctgggcgag

GNMT_3 (-) agggacccccACGTGTCGGACATGActcctacacc 0.96 tccctgggggTGCACAGCCTGTACTgaggatgtgg

SNAI2_1 (+) aaaagttgttAGGGCACAAAGTGCTccatttcttg 0.91 ttttcaacaaTCCCGTGTTTCACGAggtaaagaac

TMPRSS2_1 (+) ttaggaggatAGAACATCTCGTTGTataatgtgat 0.91 aatcctcctaTCTTGTAGAGCAACAtattacacta

TMPRSS2_2 (+) taatctctttGGGACAATGAGTCATtcacaggaaa 0.92 attagagaaaCCCTGTTACTCAGTAagtctccttt

KLK2_1 (-) tagtatgtgtGGAACAGCAAGTGCTggctctccct 0.98 atcatacacaCCTTGTCGTTCACGAccgagaggga

SRE_1 (+) atgcattgGGTACATCTTGTTCAcatagaca 0.97 tacgtaacCCATGTAGAACAAGTgtatctgt

PSA_NC (-) ggtgtgggagGGGGTTGTCCAGCCTccagcagcat NA* ccacaccctcCCCCAACAGGTCGGAggtcgtcgta

Table 2.2 Putative AREs Examined Using EMSA 11 putative AREs with varied matrix scores from 6 genes were tested for AR DBD binding using EMSA. The ARE is in BOLD flanked by 10 base pairs of genomic sequence in lower case. The SRE_1 sequence was used as the positive control and the excess cold probe. The PSA_NC sequence was randomly chosen from the PSA promoter and used as a negative control. (* insufficient core match to provide matrix score)

50 SARM1 and DHT 2x up-regulated Fold Change Accession Gene Description DHT vs Veh. SARM1 vs. Veh. NM_000860 HPGD hydroxyprostaglandin dehydrogenase 3.07 5.29 NM_000693 ALDH1A3 aldehyde dehydrogenase 2.38 2.87 NM_014762 DHCR24 24-dehydrocholesterol reductase 2.27 2.44 M16768 TRGV9 T cell receptor gamma variable 9 4.7 6.4 NG_001336 TRG@ T cell receptor gamma locus 4.41 6.09 NM_005551 KLK2 kallikrein 2, prostatic 3.31 4.78 NM_001648 KLK3 kallikrein 3, (prostate specific antigen) 2.1 2.4 NM_005656 TMPRSS2 transmembrane protease, serine 2 2.03 2.71

SARM1 only 2x up-regulated NM_004457 FACL3 fatty-acid-Coenzyme A ligase 3 1.66 2.06 NM_014350 GG2-1 TNF-induced protein 1.84 3.23 v- musculoaponeurotic fibrosarcoma oncogene NM_005360 MAF 1.71 4.04 homolog NM_012081 ELL2 elongation factor, RNA polymerase II 1.51 2.38 serine threonine kinase 39 (STE20/SPS1 homolog, NM_013233 STK39 1.77 2.19 yeast) NM_000875 IGF1R insulin-like growth factor 1 receptor 1.51 2.15 NM_004117 FKBP5 FK506 binding protein 5 1.96 3.18 NM_021082 SLC15A2 solute carrier family 15 (H+/peptidetransporter) 1.49 2.01 NM_023929 RINZF zinc finger protein RINZF 1.67 2.39 NK3 transcription factor related, locus 1 NM_006167 NKX3-1 1.72 2.02 (Drosophila)

Table 2.3 Previously Reported Genes Up-Regulated List of genes up-regulated in cDNA microarray experiment that have been previously reported as AR regulated [105, 110, 111].

51 SARM1 and DHT 2x down-regulated Accession Gene Description DHT vs Veh. SARM1 vs. Veh. NM_001077 UGT2B17 UDP glycosyltransferase 2 family polypeptide B17 -2.07 -3.53

NM_001076 UGT2B15 UDP glycosyltransferase 2 family polypeptide B15 -2.2 -3.35

NM_000598 IGFBP3 insulin-like growth factor binding protein3 -2.61 -3.22 SARM1 only 2x down-regulated NM_053039 UGT2B28 UDP glycosyltransferase 2 family polypeptide B28 -1.71 -2.27 dopa decarboxylase (aromatic L- NM_000790 DDC -1.89 -2.48 decarboxylase) NM_001260 CDK8 cyclin-dependent kinase 8 -1.53 -2.07 NM_004938 DAPK1 death-associated protein kinase 1 -1.88 -2.92 NM_002538 OCLN occludin -1.53 -2.24

NM_005941 MMP16 matrix metalloproteinase 16 (membrane-inserted) -1.73 -3.13

NM_000055 BCHE butyrylcholinesterase -1.65 -2.43 NM_020245 TULP4 tubby like protein 4 -1.72 -2.42 NM_005025 SERPINI1 serine (or cysteine) proteinase inhibitor -1.68 -2.53 ATP-binding cassette, sub-family A (ABC1), NM_005502 ABCA1 -1.55 -2.01 member ATP-binding cassette, sub-family G (WHITE), NM_004915 ABCG1 -1.99 -2.78 member 1 SARM1 and DHT different NM_002737 PRKCA protein kinase C -1.2 1.27 NM_005544 IRS1 insulin receptor substrate 1 -1.26 1.33 v-maf musculoaponeurotic fibrosarcoma oncogene NM_005461 MAFB 1.17 -1.05 homolog B (avian)

Table 2.4 Previously Reported Genes Down or Differentially Regulated List of genes down or differentially regulated in cDNA microarray experiment that have been previously reported as AR regulated [105, 110, 111]

52 CHAPTER 3

3. SARM VERSUS DHT: GENOME-WIDE AR PROMOTER RECRUITMENT

PROFILING IN LNCAP PROSTATE CANCER CELLS

3.1. Introduction

Chromatin immuno-precipitation (ChIP) experiments often elucidate initiators of genomic signaling cascades when performed on acute time-scales[121]. If performed on a genome-wide scale (ChIP-on-Chip), profiles emerge, and transcription factor target genes can be captured that may not reveal transcriptional regulation until long and varied time points thereafter[122]. ChIP-on-Chip utilizes two major technologies; tiled arrays, comprised of array elements covering both coding and non-coding portions of the genome, and promoter arrays made of elements corresponding only to proximal regulatory regions of defined expressed elements[123]. The former offers greater resolution in transcription factor binding but requires several dozen chips to cover even a single human chromosome[124]. The latter is more cost effective, covering an entire eukaryotic genome on a single chip, but assumes transcription factor binding is largely proximal to the transcriptional start site (TSS), a currently waning notion[120]. In the following study, the ChIP-DSL (DNA Selection and Ligation) strategy was coupled with 53 the H20K human promoter array to examine genome-wide AR binding profiles in LNCaP cells treated with SARM or DHT.

To test the hypothesis that the disparate in vivo pharmacology of DHT and

SARM1 in the prostate was due to potency differences alone, gene expression profiling at equi-efficacious concentrations of ligand in LNCaP cell growth was evaluated. As an alternate test to the same hypothesis, genome-wide AR promoter binding was performed using DHT and SARM2 concentrations resulting in 100% ligand-receptor occupancy and maximal trans-activation in the LNCaP mutant AR. If varied potency underlies the mechanism of SARM2 tissue selectivity, AR binding profiles at saturating ligand concentrations should overlap.

3.2. Materials and Methods

3.2.1 Materials

COS-1 monkey kidney cells and LNCaP human prostate cancer cells were purchased from American Type Culture Collection (Manasas, Virginia). Trypsin-

EDTA(0.25%) DMEM and RPMI-1640 medium were purchased from MediaTech

(Manasas, Virginia). Trizol® reagent were purchased from Invitrogen Corp.

(Carlsbad,California). Charcoal-treated fetal bovine serum (cFBS) was purchased from

Atlanta Bilogicals (Atlanta, Georgia). DHT was purchased from Sigma-Aldrich (St.

Louis, Missouri). ChIP-DSL services were purchased from Aviva Systems Biology (San

Diego, CA). SARM2 was synthesized using previously reported methods[98]. SARM2 was chosen for its improved pharmacokinetic and pharmacologic properties[98]

54 3.2.2 AR Trans-activation in COS-1 Cells

Transient transfections were carried out as previously described (2.2.3) except that cells were treated for 24 hours in phenol red-free DMEM media supplemented with

5% charcoal-stripped FBS containing .1% either ethanol vehicle or increasing concentrations of DHT or SARM2 (.01 nM – 1000 nM) dissolved in vehicle (n=3).

3.2.3 Chromatin Immuno-precipitation (ChIP) in LNCaP

Cell Culture and ChIP Low passage LNCaP cells were cultured according to ATCC’s guidelines. Cells were plated in 15 cm dishes at 15 x106 cells per dish in phenol red-free

RPMI supplemented with 1% charcoal-stripped FBS. Media was replaced on days 2 and

4. On the fifth or sixth day, cells were treated with fresh phenol red-free RPMI supplemented with 1% charcoal-stripped FBS containing .1% ethanol alone, 10 nM DHT or 100 nM SARM2 dissolved in ethanol for 2 hours. ChIP assays were performed as described by Narayanan et al.[125]. Cross-linking was performed by incubation with 1% formaldehyde (final concentration) at 37°C for 10 min. The cells were washed with 1×

PBS twice, scraped in 1 ml of PBS containing protease inhibitors ([1 mg each of aprotinin, leupeptin, antipain, benzamidine HCl, and pepstatin/ml], 0.2 mM phenylmethylsulfonyl fluoride, and 1 mM sodium vanadate), pelleted, and resuspended in

SDS lysis buffer (1% SDS, 10 mM EDTA, 50 mM Tris-HCl [pH 8.1]). After lysis on ice for 20 min, the cell extract was sonicated (Branson SONIFER 250) at 4°C 10 times for 10 s each at constant duty cycle, with an output of 30% and a 1 minute incubation on ice after every sonication, yielding 300-800 bp fragments. Cellular debris were pelleted at

55 13,000 rpm for 10 min at 4°C, and the supernatant diluted 5-fold with ChIP dilution buffer (0.01% SDS, 1.1% Triton X-100, 1.2 mM EDTA, 16.7 mM Tris HCl [pH 8.1], 167 mM NaCl). A portion of the diluted supernant was removed as an input DNA fraction then the remaining Protein:DNA complexes were pre-cleared with 50 µl of 1:1 protein A-

Sepharose beads(GE, United Kingdom) and 2 µg sheared salmon sperm DNA

(Invitrogen, Carlsbad, CA) in TE for 3 hours at 4°C. Following pre-clearing the remaining samples were divided and incubated with 4 µg of AR441 (Santa Cruz

Biotechnology, Santa Cruz, CA) antibody and 2 µg of sheared salmon sperm DNA rotating overnight at 4°C. The protein:DNA:antibody complexes were precipitated by incubating with 100 µl of 1:1 protein A-Sepharose beads and 2 µg of salmon sperm DNA at 4°C for 3 h. The beads were pelleted and washed three times with low-salt wash buffer

(0.1% sodium dodecyl sulfate [SDS], 1% Triton X-100, 2 mM EDTA, 20 mM Tris HCl

[pH 8.1], 0.15 M NaCl), and three times with 1× TE (10 mM Tris HCl, 1 mM EDTA; pH

8.0). The DNA-protein complex was eluted by extracting the beads with 50 µl of freshly prepared extraction buffer (1% SDS, 0.1 M NaHCO3) three times. Protein:DNA complexes were reverse cross-linked by incubating at 65°C for 6 h. The remaining DNA was extracted with a QIAquick PCR purification kit (QIAGEN, Valencia, Calif.). qRT-PCR Analysis The success of ChIP reactions was assessed by monitoring transcription factor loading on the PSA enhancer region using previously reported primer-probes[90]. IP and input DNA were quantitated by the relative standard curve method using serial dilutions of LNCaP genomic DNA and 2x Universal Master Mix qRT-PCR reagents on a 7300 Real Time PCR System (Applied Biosystems). Fold

56 recruitment was reported as the ratio (Treated IP DNA/Treated Input DNA) to (Vehicle

IP DNA/Vehicle Input DNA).

3.2.4 AR ChIP-DSL Study Design

Low passage LNCaP cells were cultured according to ATCC’s guidelines. Cells were plated in 15 cm dishes at 15 x106 cells per dish in phenol red-free RPMI supplemented with 1% charcoal-stripped FBS. Media was replaced on days 2 and 4. On the sixth day, cells were treated with fresh phenol red-free RPMI supplemented with 1% charcoal-stripped FBS containing .1% ethanol alone, 10 nM DHT or 100 nM SARM2 dissolved in ethanol for 2 hours. Cells were fixed by adding 37% formaldehyde solution to a final concentration of 1% at 37 ºC for 15 minutes. Following fixation, cells were washed with cold PBS containing a protease inhibitor cocktail (Roche) and collected by scraping. Cells were pelleted by centrifugation and flash frozen in a dry ice/ethanol bath.

Treatment-blinded, frozen cell pellets were shipped on dry-ice to Aviva Biosystems for

ChIP-DSL analysis using 1µg AR-441 human androgen receptor antibody (Santa Cruz).

The merits and details of ChIP-DSL as a ChIP-on-Chip technology are discussed at length by Kwon et al. [126]. Each treatment was repeated 4 times across a 2 month span using LNCaP cells of passage 4 to 11.

3.2.5 ChIP-DSL Data Analysis

Following competitive hybridization of labeled AR-ChIP DNA(red) and Input

DNA (green) samples, H20K promoter microarray chips (Aviva Biosystems) were scanned using a Genepix scanner. The resulting image files were background subtracted

57 global lowess normalized[127] using GenePix Acuity 4.0 (Molecular Devices, Toronto,

Canada). M and A values were calculated in the following way:

M = log2 (R) − log2 (G)

1 A = × ()log (R) + log (G) 2 2 2

R and G are defined as normalized AR ChiP dye and Input DNA dye intensities, respectively. M versus A plots were generated with normalized values and visually inspected for data spread between replicates (Figure B.1) A one-sample T-test was performed on M values across replicates with a null hypothesis of M = 0 (No enrichment in ChIP versus Input) . Promoter elements having p <.02 were further considered for the magnitude of their binding. From this group, only promoters with M > 1.4 (i.e. 2.5 fold or greater enrichment of ChIP over input DNA) across all 4 replicates were considered positive for AR binding.

3.2.6 Orthologous Promoter Androgen Response Element (ARE) Search

Promoter searching was carried out as previously described (2.2.6) with the following changes;

Data Retrieval mRNA reference sequence numbers provided by AVIVA for each array element were used to retrieve Unigene IDs from NCBI’s Gene database

(http://www.ncbi.nlm.nih.gov/sites/entrez?db=gene) using NCBI’s E-utilities. Unigene

ID’s were then used to query the Homolgene database

(http://www.ncbi.nlm.nih.gov/sites/entrez/query.fcgi?db=homologene) and retrieve both corresponding mouse and rat homolgous Unigene IDs if they existed. All sequence data

58 was then retrieved from NCBI’s nucleotide database according to the genomic addresses of Exon 1 (TSS) of the 3’ most isoform of each gene. No minimum identity was required following ClustalW alignment.

When searching all 20,831 elements on the array,16,039 (77%) contained human reference sequence data. Of those, 10,701 (67%) had human, mouse and rat data. 2664

(16%) had only human sequence data. 1,599 (10%) had only human and mouse data with the remaining 1075 (7%) having only human and rat data.

When searching the 337 elements that bound the AR, 268 (80%) contained human reference sequence data. Of those, 182(68%) had human, mouse and rat data. 43 (16%) had only human sequence data. 27(10%) had only human and mouse data with the remaining 16 (6%) having only human and rat data.

Matrix Searching Putative ARE’s were declared if an AR matrix score of greater than or equal to .9 with a threshold score of 1 were detected across all three species or just human and one of the rodent species. All hits were required to be with in an 800 bp

(400bp in either direction) window centered on the human ARE.

3.2.7 Gene Expression Analyses

Low passage LNCaP cells were cultured according to ATCC’s guidelines. Cells were plated in 10 cm dishes at 4 x106 cells per dish in phenol red-free RPMI supplemented with 1% charcoal-stripped FBS. Media was replaced on day 2. On the fifth day, cells were treated with fresh phenol red-free RPMI supplemented with 1% charcoal-stripped FBS containing .1% ethanol alone, 10 nM DHT, 100 nM DHT, 10 nM

SARM2 or 100 nM SARM2 dissolved in ethanol for 12, 24,48 or 72 hours (n=3). Cells

59 were harvested using Trizol and total RNA extractions performed. RNA was reconstituted in DEPC treated molecular biology grade water and diluted to 200 ng per ul. cDNA was generated using the High Capacity cDNA Kit with RNase inhibitor

(Applied Biosystems) and 2 ug of total RNA from each sample. Control cDNA was diluted 1:100 for 18S analysis. All genes were assayed using Taqman Assays (Applied

Biosystems) and 2x Universal Master Mix qRT-PCR reagents on a 7300 Real Time PCR

System (Applied Biosystems). Data was analyzed using the 2-∆∆Ct method[107], normalized to 18S, comparing each treated sample back to the vehicle samples from the same time point. Positive fold change values are represented as 2-(∆∆Ct) (Low Error Bar:

2-(∆∆Ct + SD∆∆Ct), High Error Bar: 2-(∆∆Ct - SD∆∆Ct)) as suggested by Yuan et al.[128].

Negative fold change values are represented as -2 ∆∆Ct (Low Error Bar: -2(∆∆Ct - SD∆∆Ct),

High Error Bar: -2(∆∆Ct + SD∆∆Ct)) for easier interpretation of down regulated transcripts.

One-way ANOVA followed by pair wise two tailed Student’s t-tests (Fisher’s LSD) were performed on ∆∆Ct values with a threshold set at p<.05 when determining significant differences.

3.2.8 Functional Analyses

Functional annotations were reported in a similar fashion to Kazmin et al. [92].

GenBank accessions from all genes showing AR recruitment (337) were submitted to

DAVID (http://david.abcc.ncifcrf.gov/) as a batch. Gene Ontology annotations were retrieved for 276 or 82% of the search terms. A combination of Perl scripts and

Microsoft Excel were used to bin terms by treatment and determine GO terms differing in distribution from the “Total Unique” group. From Figure 3.3, ALL ON includes area G,

60 BOTH OFF includes area C, DHT ON includes areas A and D and SARM ON includes areas F, B and E. Those promoters recruiting AR in response to SARM and DHT were included in the SARM ON but not DHT ON.

3.3. Results

3.3.1 AR Trans-activation

The capacity for trans-activation in a transient transfection system with both WT

AR and T877A-AR were measured for each ligand. The control transfecton, pCR3.1 with GRE-LUC showed no activity (data not shown). Much like SARM1 discussed in

Chapter 2 (Figure 2.7), SARM2 showed increased potency and efficacy in a system using

LNCaP’s mutant AR (Figure 3.1). Dose response curves for SARM2 and DHT were highly similar in the T877A mutant at a treatment time of 24 hours.

3.3.2 PSA Enhancer ARE AR recruitment in LNCaP

AR loading on the PSA enhancer was assayed using conditions identical to the

ChIP-DSL experiment. Both 10 nM DHT and 100 nM SARM2 showed similar and significant AR loading to this control genomic region (Figure 3.2). AR loading following

DHT treatment coincides with previous work in LNCaP[112].

3.3.3 ChIP-DSL AR Binding Profile

ChIP-DSL analyses were performed on LNCaP cells treated with both 100 nM

SARM2 and 10 nM DHT for 2 hours. These concentrations were choosen for the following reasons; 1) A 10 fold increase in SARM2 over DHT corrects for reduced 61 SARM2 AR binding affinity, ensuring 100% receptor occupancy for both ligands; 2)

These concentrations showed similar AR loading on the PSA enhancer region; 3) These concentrations maximally stimulated transcriptional activation in a T877A-AR system.

-1.5kb to +0.5kb (Relative to the TSS) promoter elements showing statistically significant (p <.02) 2.5 fold or greater increases in normalized AR-ChIP signal over

INPUT across all 4 biological replicates were considered AR positive binders. These selection criteria resulted in a total of 337 (1.69%) of the more the 20,000 assayed promoters occupying the AR (Figure 3.3). Of the 337, 32 (9.5%, Group A, Table B.1) recruited the AR only in response to DHT treatment. Only 6 (1.7%, Group B, Table B.2) promoters uniquely recruited the AR in response to SARM2. 45 (13%, Group C, Table

B.3) promoters were occupied by AR only the absence of ligand. 62 (18%, Group D,

Table B.4 and Table B.5) promoters were occupied by AR only in the absence of

SARM2. Only 4 (1.1%, Group E, Table B.6) promoters shared AR occupancy following ligand addition with 6 (1.7%, Group F, Table B.7) promoters occupied only in the absence of DHT. In the largest group, 182 (54%, Group G, Table B.8 through Table

B.12), AR recruitment was found in common between all treatments at the 2 hour time point. In this AR binding profile, the ‘ON’ signal (Groups A, E and B) includes 42 promoters which was much smaller than the ‘OFF’ signal (Groups C, D, and F) of 113 promoters. The majority of promoters maintained AR loading irrespective of ligand or vehicle (Group G).

62 3.3.4 Putative Androgen Response Elements

The comparative ARE search previously described was employed with minor modifications to evaluate the number of conserved AREs found in a group of promoter regions experimentally determined to bind the AR. Though the ChIP-DSL technology captures genomic DNA within only approximately -1.5kb to +0.5kb (relative to the TSS) of the gene, the full -5kb to +2.0kb search was performed. The percentage of genes with sufficient information to be searched was similar between the entire H20K array and the subset of 337 AR positive binders at 65% (Figure 3.4). Of the genes sufficiently annotated, slightly more than 50% were ARE positive for one or two rodent species conservation in both the full array and the AR binders. Not surprisingly, the majority of detected AREs were only conserved between human and one rodent species (80%) in both the search of all array elements and the subset of AR binders.

7029 genes from the entire H20K array had sufficient information to be searchable and contained one or more conserved AREs. Only 123(1.7%) of these putative AR binders were members of the ChIP-DSL AR positive data set. When those

AREs were examined based on ligand dependencies of AR recruitment (Figure 3.3), no clear relationship between ligand and the presence of conserved ARE’s was evident

(Table 3.1). Most groups showed a near 50/50 split reflective of the trend in the search results as a whole. The only group with a large number of genes apparently enriched for conserved AREs was Group C.

63 The spatial distribution, relative to the TSS, of the detected AREs was nearly uniform for the entire H20K array, but showed strong skew away from the TSS toward -

5kb in the AR binding data set (Figure 3.5).

3.3.5 Gene Expression Analyses

All well-annotated (i.e., containing a Refseq number assigned by NCBI) ‘ON’ signal genes (Groups A, B and E) were assayed for ligand-mediated transcriptional regulation via dose response and time course analyses in LNCaP cells. Figure 3.6 shows the dose and time dependencies of three well characterized AR regulated genes following treatment. In all three cases, DHT induction was more rapid, reaching maximal response at 12 or 24 hours. SARM induction was slower but sustained throughout 72 hours. Also

SARM2 was more efficacious in all three control genes’ expression. 21 of the 32 genes in Group A (AR ON, DHT only) were assayed. Only GPLD1 displayed significant regulation, which was repressed in a time- and dose-dependent manner by both ligands

(Table B.1, Figure 3.7). 5 of 6 genes in Group B (AR ON, SARM2 only) were assayed with no regulation detected (Table B.2), while 3 of 4 genes in Group E (AR ON, DHT and SARM2) were assayed. Only ALK in Group E showed regulation, comprised of induction by both DHT and SARM2 at 24 hours that decreased with time (Table B.6,

Figure 3.9). Nearly 70% of the AR ‘ON’ signal was assayed for transcriptional effects with less than 5% actually exhibiting regulation. 3 genes each from the ligand specific

‘OFF’ signal groups (Group D, AR OFF- SARM2 only, Table B.4 and Table B.5; Group

F, AR OFF-DHT only, Table B.7) were chosen for evaluation based on potentially interesting growth properties (FGF6[129], IRLB[130]) or relatedness to known AR

64 regulated genes (KLK6[131]). No regulation was detected in any of the gene targets examined. 3 genes were selected using similar criteria from the combined ‘OFF’ signal

Group C (Table B.3). In this group TMPRSS3[132] showed rapid, dose-dependent repression by both DHT and SARM2 (Figure 3.8).

Finally, three genes were chosen from the largest group, to which AR was recruited irrespective of ligand, Group G (Table B.8 through Table B.12). These targets were selected for known AR recruitment (AR, [110]), potentially interesting growth properties (CSRP2[133]), and interest derived from concurrent studies (GPT-2, Chapter

4). Interestingly, AR was selectively down-regulated by SARM2. GPT-2 was both repressed by DHT at later time points and induced by SARM2 (Figure 3.10).

3.3.6 Functional Analyses

Functional analyses using the Gene Ontology were rendered more difficult by the reduced size of the AR binding data set compared to the cDNA microarray experiment.

However, useful relationships were still evident (Figure 3.11). “Gene Expression” was presented as a positive control as a large number of the genes recruiting AR are expected to be involved in transcriptional regulation. Also the distribution of genes with this annotation was highly reflective of the “Total Unique” groups supporting this GO term as a control. The “Cell Proliferation” annotation was enriched for ALL ON indicative of an overlap in the ligands’ ability to regulate cellular proliferation in LNCaP cells. “Cell

Differentiation” was enriched for DHT ON, suggesting an increased capacity for DHT in mediating cellular differentiation in LNCaP. “Phosphorylation” was highly enriched for

SARM ON, suggesting an increased kinase signaling capacity for SARM2 versus DHT.

65 “Anatomical Structure Development” was slightly enriched for Both OFF, suggesting an overlap in the developmental capacities of the ligands. Finally, “Macromolecule

Biosynthetic Process” was marginally enriched for both SARM ON and DHT ON indicating a common ability to, but distinct mode of, regulating biosynthetic processes.

3.4. Discussion

Genome-wide AR promoter recruitment was determined for both 10 nM DHT and

100 nM SARM2 at 2 hours in LNCaP cells. A ten-fold excess of SARM2 over DHT was chosen to correct for the reduced AR binding affinity for SARM2 when compared to

DHT[45]. These concentrations assured near 100% ligand-receptor occupancy and resulted in maximal trans-activation for both ligands (Figure 3.1). These concentrations also demonstrated similar AR loading capacity on the PSA enhancer ARE (Figure 3.2) with DHT’s AR loading in agreement with previous work showing 10 nM to be near maximal[112]. The two hour time point is slightly longer than ‘acute’ time points from previous work[112, 134], but has been shown to catch maximal AR loading on the PSA enhancer[90]. The profiles for SARM2 and DHT resulting from the aforementioned conditions were again largely over-lapping but distinct (Figure 3.3). Surprisingly, greater than half of the AR:DNA interactions detected were maintained irrespective of ligand

(Group G) and only 4 promoters recruited the AR in response to both ligands (Group E).

DHT uniquely recruited AR to almost 6 times as many promoters as SARM2, though the greatest differences lay in the ‘OFF’ signal, or ligand-specific abandonment of AR from a promoter.

66 Not normally monitored in a singleton ChIP assay, the OFF signal has been studied with increasing interest in ChIP-on-Chip experiments[126, 135]. In this experiment, 45 promoters lost AR in response both ligands (Group C), compared to 4 that recruited AR (Group E). This suggests the commonalties in SARM2 and DHT function in LNCaP cells are the result of increased AR motility that removes AR from promoter as opposed to delivering it. This notion is supported by the thorough examination of all

‘ON’ signal promoters that resulted in less than 5% showing regulation. Also, Group D suggests 62 promoters lose the AR in response to SARM2 but not DHT, ten times the number selectively recruiting AR following SARM treatment. ‘ON’ or ‘OFF’, AR motility under these conditions proved a poor predictor of transcriptional regulation.

Very few of the gene targets showing varied AR occupancy following treatment revealed transcriptional regulation when evaluated by both time course and dose response. Given this fact, it is not surprising that a very poor overlap between the DHT gene expression profile and AR binding profile was realized. Of the 1055 genes significantly regulated by 1 nM DHT at 24 hours(Chapter 2), only 11 were also found in the ChIP-DSL DHT AR binding profile. 8 fell into the largest group G, 2 into Group D with only one gene, CAD, showing both ligand-mediated transcriptional regulation and

AR recruitment in Group A.

Though few AR binders proved to be regulated, those that did showed both time and dose dependent effects. These time and concentration dependencies highlight the limitations of the ‘one dose, one time’ snap shot approach to high density experiments often dictated by resource constraints. For example, when looking at control genes, PSA expression (100 nM of each ligand) at 24 hours is almost identical between ligands, but at 67 72 hours SARM2 supersedes DHT by nearly 3-fold (Figure 3.6). 10 nM ALK induction at 24 hours is greater in DHT, but the reverse is true at 48 hours (Figure 3.9). Perhaps most interesting was the ligand-specific regulation of AR gene expression (Figure 3.10), where SARM2 repressed AR expression as early as 24 hours and DHT showed no effect.

Though not thoroughly examined here, the down regulation of AR in prostate by

SARM2, but not DHT, offers a particularly elegant explanation of tissue selectivity worthy of further inquiry.

Transcriptional regulation of both GPT-2 (Figure 3.10) and GPLD1 (Figure 3.7) were of particular interest. GPT-2 is an alanine-amino transferase, indistinguishable from

GPT-1 (ALT-1) by enzymatic assay[136]. Increases in serum ALT activity are seen as a marker of liver toxicity [137]. AR-mediated transcriptional regulation, albeit in prostate, argues that elevated serum ALT levels following androgen therapy could be due in part to

AR action, not toxicity. GPLD1 is associated with high density lipoproteins in the vascular space [138] and functions by hydrolyzing the inositol phosphate linkage in proteins anchored by phosphatidylinositol glycans (GPI-anchor) to release these proteins from the membrane [139]. Though its role in HDL-C homeostasis is unknown, regulation of GPLD1 by androgens could contribute to the well-characterized androgen- mediated serum lipid effects and warrants further study.

Determination of genome-wide AR promoter occupancy offered a direct biological evaluation of the in silico comparative androgen response element search.

Though only 65%of the 337 genes with varied AR occupancy had sufficient sequence information to be searched, a measly 121 or 35%percent had a conserved ARE (Figure

3.4). This low percentage of direct AR targets containing AREs is in agreement with 68 similar analyses where only 27% of promoters binding AR following R1881 administration contained something similar to the 15bp canonical ARE[134]. As a percentage of searchable targets containing an ARE, the AR binding profile surpassed the cDNA microrray, with 55%and 36% ARE (+) genes, respectively. Ideally, this increase is the result of the ChIP-DSL experiment only identifying direct AR targets, but the inclusion of rat homology criteria in the search of AR binders likely contributed to the increased number of conserved AREs detected in the promoter array study. It is also interesting to note that the only group showing a significant increase in ARE(+) versus

ARE(-) searched promoters was Group C(Table 3.1), the both ‘OFF’ signal, whose likely importance has been previously discussed.

It is reasonable to assume that if AR:DNA promoter interactions are dictated by the presence of AREs, that a subset of promoters biologically determined to bind the AR would be enriched for the presence of AREs when compared to all promoters on the whole. To test this hypothesis, the comparative ARE search was run on all genes on the

H20k promoter array along with the subset of AR binders (Figure 3.4). The percentage of searchable targets and percentage of ARE(+) targets were in reality almost identical between the two data sets. Three experimental possibilities could explain this result;

First, a large portion of AR:DNA interactions are mediated by a non-canonical ARE, like the 6 base pair half site found enriched in AR binders by Massie et al[134]. Second, indirect AR:DNA interactions comprised of AR protein:protein interactions with another

TF bound to DNA largely contributed to the AR binding profile. Third, a large portion of the AR:DNA interactions are distal to the TSS and only result in a promoter array hit based on chromosomal looping in which the AR binds AREs located several kilo-bases 69 from the TSS [140]. Any of these alone or in combination could explain the lack of ARE enrichment in the bona fide AR direct target data set. Conversely, the presence of a conserved ARE was not predictive of AR binding. More than 7000 of the total elements on the promoter array had conserved AREs, but less than 2%of these showed AR occupancy in the ChIP-DSL experiment. When considering the spatial distribution of the detected AREs, the location of an ARE in the promoter array as a whole was nearly equally likely anywhere within the 7kb search window (Figure 3.5). The subset of AR binders however, showed a skew away from the TSS. This suggests that the majority of the AR:DNA interactions, if dictated by 15 base-pair AREs, are occurring distal to the promoter in the enhancer or even further upstream.

This experiment largely failed in discovering novel initiators of AR-mediated genomic signaling in LNCaP. A more through analyses of the ‘OFF’ signal genes or earlier time points in the gene expression analyses could potentially reveal more AR mediated regulation that was expected, but mostly missing, in the studies performed on the ‘ON’ signal elements. Another approach taken by others is combined analyses with

RNA Pol II Chip-on-ChIP as the presence of general transcriptional machinery is requisite in mRNA regulation[126]. With the combined information of both AR and Pol

II promoter binding, a clearer picture of AR-mediated transcriptional regulation, both

‘ON’ and ‘OFF’, would form. This would likely permit more successful gene expression analyses. Tiled arrays could be employed as even another possiblilty. Work with such arrays has shown robust nuclear hormone receptor binding at sites located mega-bases from any known coding region[124]. Several lines of evidence, including this study,

70 point toward the importance of distal TF binding in understanding the full complexities of eukaryotic transcriptional regulation[112, 120].

Much like the cDNA microarray experiment, the genome-wide AR binding experiment performed with SARM2 and DHT, with corrective efforts made for discrepancies in potency, resulted in profiles more similar than different. At first look this experiment supports potency as the basis for SARM tissue selectivity. However, when individual genes were examined via expanded dose response and time course, clear differences emerged pointing toward a qualitative difference is these ligands’ activities in

LNCaP cells.

71 3.5. Acknowledgments

ChIP-DSL services were purchased AVIVA Systems Biology in a collaboration managed by Drs. Jeff Falk and Lingxun Duan. WT-AR and T877A-AR expression plasmids were provided by Dr. Casey Bohl. GRE-LUC plasmid was provided by the laboratory of Dr. Nancy Weigel. ChIP protocol and training was provided by Dr.

Ramesh Narayanan.

72 AR Transactivation - COS-1 cells 0.7

DHT (WT) 0.6 SARM2 (WT) DHT (T877A) 0.5 SARM2 (T877A)

0.4

0.3 RLU

0.2

0.1

0.0

-0.1 le ic nM nM nM nM nM nM eh 1 .1 1 0 0 0 V .0 1 10 00 1

Figure 3.1 AR Transactivation in COS-1 – WT vs T877A AR Dose response curves of both DHT and SARM2 in a transient transfection system. When comparing the dose response in WT versus T877A AR, reduced potency and efficacy of SARM2 in WT are lost in the T877A mutant.

73 ChIP in LNCaP - 2 Hour Treatment 6

AR * 5

4 *

3

FC vs. Vehicle vs. FC 2

1

0 Veh 10 nM DHT Veh 100 nM SARM2 PSA Enhancer ARE

* p<.006 Treatment versus Vehicle (One-Tailed Student's T-test)

Figure 3.2 AR ChIP in LNCaP on PSA Enhancer Control ChIP experiments showing similar responses in AR recruitment to the PSA- Enhancer ARE in both 10 nM DHT and 100 nM SARM2 treatments.

74 Vehicle 10 nM DHT C D A 62 45 32 182 6 G 4 F E

6

B

100 nM SARM2

Figure 3.3 ChIP-DSL AR Binding Profile A Venn diagram representing promoters binding the AR in the presence of Vehicle, SARM2, or DHT. (A) – AR ON, DHT only; (B) – AR ON, SARM2 only; (C) – AR OFF, DHT & SARM2; (D) – AR OFF, SARM2 only; (E) – AR ON, DHT & SARM2; (F) – AR OFF, DHT only; (G) – AR ON, ALL.

75 Comparative ARE Search - ChIP-DSL

100

AR Binding Array Elements Total Array Elements % ARE(+) 80

% Total

60 % Searchable

40 % ARE(+)

20

0 rs s d d te ter rve rve mo se se ro omo n n P Pr Co Co le ) R R ab (+ M H ch RE H or ar A M Se H

Figure 3.4 Comparative ARE search – ChIP-DSL Human H20k array data set breakdown showing from left to right; the percentage of the total elements having sufficient information to search, the percentage of searchable elements having conserved AREs, the percentage of detected AREs conserved across all three species, and the percentage of detected AREs conserved across human and only one rodent species. Both the AR positive and the total H20k array are represented.

76 Putative ARE Distibution - ChIP-DSL

120 AR Binding Elements All Array Elements

100

80

60

40

% of Max number of AREs per kb number of AREs per % of Max 20

0 kb kb kb kb kb kb kb -4 -3 -2 -1 o 0 +1 +2 to to to to 1 t to to -5 -4 -3 -2 - 0 +1

BP Relative to TSS

Figure 3.5 Putative ARE Distribution – ChIP-DSL Detected conserved AREs (human and one rodent species) are presented as a distribution within the 7kb region searched. Both the AR positive and the total H20k array are represented.

77 * 10 * * 8 * 6

NKx3.1 4

2

400 12 Hrs 24 Hrs 48 Hrs* 72 Hrs * 30 *

* 20 * TMPRSS2 TMPRSS2 10 *

0 12 Hrs 24 Hrs 48 Hrs 72 Hrs* 200 Veh * 10 nM DHT * 150 100 nM DHT 10 nM SARM2 100 nM SARM2

PSA 100

50 * 0 12 Hrs 24 Hrs 48 Hrs 72 Hrs *p<.05 Fisher's LSD vs. Vehicle

Figure 3.6 LNCaP Gene Expression – Controls Values are represented as mean + SD (n=3), as described in the methods section, fold change versus vehicle group from same time point. Control line represents no change.

78 0

-1

* * * -2 * * * -3 * * GPLD1

-4 Veh * 10 nM DHT 100 nM DHT 10 nM SARM2 -5 * 100 nM SARM2

-6 12 Hrs 24 Hrs 48 Hrs 72 Hrs *p<.05 Fisher's LSD vs. Vehicle

Figure 3.7 LNCaP Gene Expression – Group A Values are represented as mean + SD (n=3), as described in the methods section, fold change versus vehicle group from same time point. Control line represents no change.

79 0

-2

* * -4

-6 * * TMPRSS3 -8 Veh * 10 nM DHT -10 100 nM DHT 10 nM SARM2 * 100 nM SARM2 -12 12 Hrs 24 Hrs 48 Hrs 72 Hrs *p<.05 Fisher's LSD vs. Vehicle

Figure 3.8 LNCaP Gene Expression – Group C Values are represented as mean + SD (n=3), as described in the methods section, fold change versus vehicle group from same time point. Control line represents no change.

80 6 * Veh 10 nM DHT 100 nM DHT 4 10 nM SARM2 * 100 nM SARM2 * *

* * * 2 ALK

0

-2 *

12 Hrs 24 Hrs 48 Hrs 72 Hrs *p<.05 Fisher's LSD vs. Vehicle

Figure 3.9 LNCaP Gene Expression – Group E Values are represented as mean + SD (n=3), as described in the methods section, fold change versus vehicle group from same time point. Control line represents no change.

81 3

2

1

0 AR

-1

-2 * * * * * * -3 4 12 Hrs 24 Hrs 48 Hrs* 72 Hrs

*

2 * *

0 GPT-2

-2 Veh 10 nM DHT * 100 nM DHT 10 nM SARM2 100 nM SARM2 * -4 *

12 Hrs 24 Hrs 48 Hrs 72 Hrs *p<.05 Fisher's LSD vs. Vehicle

Figure 3.10 LNCaP Gene Expression – Group G Values are represented as mean + SD (n=3), as described in the methods section, fold change versus vehicle group from same time point. Control line represents no change.

82

Figure 3.11 GO Term Analysis of ChIP-DSL Experiment Genes recruiting the AR to their proximal promoter, also having functional GO annotations, are represented in the “Total Unique” chart by AR loading pattern. Specific GO terms, with the number of promoters recruiting AR common to that GO term in parentheses, are represented as well.

83 Group Total Genes Total Searchable ARE(+) ARE(-) A 32 20 45% 55% B 6 5 40% 60% C 45 34 65% 35% D 62 42 52% 48% E 4 3 66% 34% F 6 4 75% 25% G 182 117 53% 47%

Table 3.1 Comparative ARE Search Results by Group

84 CHAPTER 4

4. ANDROGEN REGULATION OF THE ALANINE AMINOTRANSFERASES

4.1. Introduction

Glutamate pyruvate transaminases (GPTs, also known as alanine amino- transferases or ALTs) catalyze the reversible transamination between alanine and 2- oxoglutarate to form pyruvate and glutamate playing an essential role in the intermediary metabolism of glucose and amino acids[136]. In tissues, such as muscle, where amino acids are a primary fuel source, amino groups are collected from glutamate via trans- amination. ALTs transfer the α-amino group from glutamate to pyruvate to form 2- oxoglutarate and alanine, the major amino acid present in fasting blood. Circulating alanine is then absorbed by the liver for glucose generation from pyruvate, reversing the

ALT reaction and completing the alanine-glucose cycle[141]. Two ALT isoforms with

80%homology and undistinguishable transaminase activities have been characterized to date[136]. Interestingly, these enzymes have highly disparate tissue expression, suggesting possibly varied biological roles. ALT1, the first characterized, is primarily expressed in liver, kidney and adipose, while ALT2 shows more abundant expression overall with the majority of transcripts detected in prostate, muscle, and brain[142]. 85 ALT is best known as a surrogate marker of liver injury where several criteria involving serum ALT elevation are used to diagnose both hepatocellular disease and drug-induced damage[137]. Toxicity-induced ALT elevations result from porous necrotic liver tissue leaking enzymes into circulation as opposed to a coordinated physiological response such as increased gene expression. Serum ALT elevations following androgen administration have been reported with several compounds in both human and animal models [143-146]. Though liver function tests (LFTs) used to determine ALT activity offer fast and convenient information to clinicians, their interpretation is often debated[147] and several factors have been shown to contribute to serum ALT levels and confound standard LFT diagnoses including; myriad existing disease states, alcohol abuse, and intense muscle damage [69, 148-150].

The presence of several conserved AREs in the promoters of both ALTs combined with AR promoter occupancy data indicating ALT2 is a bona fide direct target of the AR led to the hypothesis that multi-tissue androgen regulation of ALT transcription might contribute to elevated serum ALT, as opposed to or instead of liver toxicity alone. With this hypothesis in mind, androgen-mediated gene expression of the

ALTs was studied in both in vitro and in vivo models.

4.2. Materials and Methods

4.2.1 Materials

HepG2 hepatocellular carcinoma cells, COS-1 monkey kidney cells and LNCaP human prostate cancer cells were purchased from ATCC (Manassas, Virginia). Trypsin

86 (0.25%)-EDTA, DMEM, PBS and RPMI-1640 medium were purchased from MediaTech

(Manasas, Virginia). Trizol® reagent and restriction enzymes were purchased from

Invitrogen Corp. (Carlsbad, California). Charcoal-stripped fetal bovine serum (cFBS) was purchased from Atlanta Bilogicals (Atlanta, Georgia). DHT was purchased from Sigma-

Aldrich (St. Louis, Missouri). Protease inhibitor compounds were purchased from both

Fisher Scientific (Pittsburg, PA) and Sigma Aldrich.

4.2.2 Animals

Sprague-Dawley rats were purchased from Harlan Biosciences (Indianapolis, IN).

Animals were maintained on a 12-hour light-dark cycle with food and water available ad libitum. The animal protocol was reviewed and approved by the Institutional Laboratory

Animal Care and Use Committee of The University of Tennessee.

4.2.3 Orthologous Promoter Androgen Response Element (ARE) Search

ARE searches were carried out as previously described (3.2.6) except that the search was extended from a 7kb region to a 8kb region from(-6kb to +2kb) and the window surrounding the human ARE was expanded to 800 bp in each direction when considering ARE’s conserved between human and rodent species.

4.2.4 AR Trans-activation in COS-1 Cells

Transient transfections were carried out as previously described (2.2.3) except only T877A-AR was compared to pCR3.1, no WT transfections were performed. Also cells were treated for 24 hours in phenol red-free DMEM media supplemented with 5%

87 vFBS containing .1% either ethanol vehicle or increasing concentrations of DHT or

SARM3 (.01 nM – 1000 nM) dissolved in vehicle (n=3). SARM3 is a structural analog to SARM1 and SARM2 with greater in vitro potency and efficacy.

4.2.5 LNCaP Gene Expression

Low passage LNCaP cells were cultured according to ATCC’s guidelines. Cells were plated in 6 well plates at 3 x106 cells per well in phenol red-free RPMI supplemented with 1% cFBS. Media was replaced on day 2. On the fifth day, cells were treated with fresh phenol red-free RPMI supplemented with 1% cFBS containing .1% ethanol alone, 100 nM DHT, or 100 nM SARM3 dissolved in ethanol for 24,48 or 72 hours (n=3). Cells were harvested using Trizol and total RNA extractions performed.

RNA was reconstituted in DEPC-treated molecular biology grade water and diluted to

200 ng per ul. cDNA was generated using the High Capacity cDNA Kit with RNase inhibitor (Applied Biosystems) and 2 ug of total RNA from each sample. All genes were assayed using Taqman Assays (Applied Biosystems) and 2x Universal Master Mix qRT-

PCR reagents on a 7300 Real Time PCR System (Applied Biosystems). Data was analyzed using the 2-∆∆Ct method[107], normalized to 18S, with previously described statistical analysis and representation (3.2.7).

4.2.6 ALT-1 and ALT-2 Promoter Scan ChIP in LNCaP

ChIP reactions were performed as previously described (3.2.3) with the following exceptions; 2x10cM CellBind(Corning) dishes plated at 1x107 cells per dish were used for each replicate of each treatment, RNA PolII (Upstate, Temecula, CA) antibody was

88 used along with AR-441 and 100 nM DHT and SARM3 were used for treatment. One of the DHT input samples was lost during the assay so the average value of the remaining two was used in place of the missing value to maintain n=3.

ALT-1 and ALT-2 promoter primer sets (Table C.1 and Table C.2) were designed using IDT’s PrimerQuest (https://www.idtdna.com/Scitools/Applications/Primerquest/).

Specifically, ALT-1 primers were made to span 100 bp sections of genomic promoter

DNA separated by approximately 400 bp similar to Wang et al.[151]. ALT-2 primers were designed to flank in silico determined AREs. To ensure specific amplification, traditional end-point PCR reactions on LNCaP genomic DNA were performed along with melting curves at the end of each qRT-PCR run. Optimal primer concentrations were determined by running a primer matrix and choosing the greatest sensitivity (lowest Ct) in the absence of non-specfic amplification. All pairs were optimal between 200-300 nM. IP and input DNA were quantitated by the relative standard curve method using serial dilutions of LNCaP genomic DNA and 2x SYBR Green Master Mix qRT-PCR reagents on a 7300 Real Time PCR System (Applied Biosystems). Fold recruitment was reported as previously described (3.2.3).

4.2.7 Primary Rat Hepatocyte Gene Expression (251)

Cryo-preserved primary male Sprague-Dawley rat hepatocyte suspensions

(CellzDirect Durham, NC) were plated in collagen-coated 6 well plates (Beckton

Dickinson) in William’s E-medium supplemented with 5% FBS, 0.1 U/ml penicillin/streptomycin, 4 µg/ml bovine insulin, 1 µM dexamethasone, 17.5 mg/ml L- glutamine and 15mM HEPES in accordance with the vendor’s protocol (Supplement

89 Pack, CellzDirect). Four hours after plating, the media was replaced with phenol red-free

William’s E Media supplemented with 0.1 U/ml penicillin/streptomycin, 100 nM dexamethasone, 1x insulin-transferrin-selenium solution, 17.5mg/ml L-glutamine and 15 mM HEPES (Supplement Pack, CellzDirect). Twelve hours later, this media was replaced with media containing either 0.1% ethanol vehicle alone or 1 µM DHT or

SARM3 dissolved in 0.1% vehicle. Cells were treated for 48 hours. Following treatment, cells were harvested and total RNA extracted using Trizol. Reconstituted

RNA was concentration normalized and quantitative gene expression analyses were performed using a One-Step RT-PCR Kit (Applied Biosystems), .5 to 1 µg of sample

RNA, Taqman probes normalized to 18S and Applied Biosystem’s 7300 Real Time PCR system. Data was analyzed using the 2-∆∆Ct method[107] with previously described statistical analysis and representation (3.2.7).

4.2.8 Orchidectomized Rat Acute High Dose Study

Male Sprague-Dawley rats (12 weeks old) were orchidectomized (ORX) at the beginning of the study. ORX animals were randomized by pre-surgery body weight into groups of 5. One group of 5 sham-operated male rats was included as an intact control

(Table 4.1). The ORX animals were treated the next day following surgery. All treatments were via 200 µL subcutaneous injection in 80:20 (v:v) PEG300:DMSO. At the end of treatment, animals were weighed, anesthetized, and sacrificed within 8 hours after the last dose. Serum was collected via abdominal aortic puncture. Tissues of interest were harvested and weighed at sacrifice including prostate, seminal vesicles, soleus muscle, levator ani muscle, and liver. After weighing, tissues were flash frozen in

90 a dry ice ethanol bath and stored at -80°C prior to RNA extraction. Aliquots (50-100 mg) of preserved tissue samples were homogenized in Trizol using Lysing Matrix D (MP

Biomedicals) and a FastPrep FP120 homogenizer (Thermo Savant). Total RNA was then extracted, reconstituted in DEPC-treated molecular biology grade water, and diluted to

200 ng per ul. cDNA was generated using the High Capacity cDNA Kit with RNase inhibitor (Applied Biosystems) and 2 ug of RNA from each sample. Quantitative gene expression analyses were performed using Taqman probes normalized to 18S (liver and levator ani) or GAPDH (prostate) and Applied Biosystem’s 7300 Real Time PCR system.

Data was analyzed using the 2-∆∆Ct method[107] with previously described statistical analysis and representation (3.2.7).

4.3. Results

4.3.1 Putative Androgen Response Elements

ALT-1 has three very strongly conserved human, mouse and rat AREs at -5536, -

1373 and +1963 base pairs relative to the TSS (Table 4.2). ALT-2 has three slightly weaker AREs conserved between human and mouse only at -1827, -2002, and -2976 base pairs relative to the TSS (Table 4.3). The most distal ARE at -3977 is conserved in rat as well.

4.3.2 T877A-AR Trans-activation

The capacity of SARM3 to trans-activate the T877A-AR was measured in COS-1 cells at a 24 hour time point (Figure 4.1). Empty expression plasmid (pCR3.1) showed no activation and the requirement of AR expression in generating a response. In this 91 assay, SARM3 showed increased efficacy over DHT with similar potency. Maximal or near maximal activation was reached by each ligand at 100 nM.

4.3.3 ALT Gene Expression

The capacity for DHT and SARM3 to regulate ALT-1 and ALT-2 expression in

LNCaP cells was monitored at 24, 48, and 72 hours with 100 nM of each ligand (Figure

4.2). Robust induction of the positive control PSA was detected with both ligands that increased over time. SARM3 showed greater induction at all time points. At 24 hours both ALT-1 and ALT-2 showed modest but significant regulation by both ligands. At 48 hours, DHT showed slight repression of ALT-1 and only slight induction of ALT-2, though both were significantly different from vehicle. SARM3 showed no change in

ALT-1, but an induction of ALT-2 greater than 3 fold. By 72 hours, both ligands were slightly repressing ALT-1. However, only SARM3 showed continued strong induction of ALT-2.

A similar evaluation was performed in primary rat hepatocytes at 48 hours with 1 uM of each ligand (Figure 4.3). Similar to the results obtained in LNCaP cells, both ligands showed slight repression of ALT-1 and induction of ALT-2, though significance was not met due to large variability in the vehicle group.

4.3.4 ALT Promoter Scanning

ChIP reactions were employed to monitor AR loading on the proximal promoters of both ALT-1 and ALT-2 genes. ALT-1 primers were designed mainly around the area covered by the ChIP-DSL experiment (Chapter 3) to detect any binding this experiment

92 might have missed (Figure 4.4). Following treatment with 100 nM DHT for 2 hours, both AR and PolII ChIPs showed significant recruitment to the positive control PSA enhancer, though none of the eight ALT-1 sets picked up any significant TF recruitment

(Figure 4.5). Following 100 nM SARM3 treatment for 2 hours, both AR and PolII ChIPs showed significant recruitment to the positive control PSA enhancer. Only set 3 showed significant SARM3-mediated Pol II recruitment, whereas none of the other seven sets showed either AR or Pol II occupancy.

ALT-2 primers were designed to span the four conserved ARE’s located in the proximal promoter (Figure 4.7), all residing outside of the ChIP-DSL coverage area, to determine if ARE-mediated binding was responsible for the ChIP-DSL hit. None of the four sets evaluated showed any TF recruitment for either ligand (Figure 4.8).

4.3.5 ORX Rat Acute High Dose Study

Tissue ALT-1 and ALT-2 transcript levels were determined in both control and treated animals receiving 5 mg/day of either DHT or SARM3 subcutaneously. Castration alone, or followed by treatment with SARM3 or DHT had no effect on liver ALT expression (Figure 4.9). Castration resulted in repression of ALT-2 mRNA in levator ani tissue, but no effect on ALT-1(Figure 4.10). Treatment by either DHT or SARM3 resulted in a significant 6-fold induction of ALT-2 expression over castrated control animals. Androgen treatment also generated levator ani ALT-2 expression in significant excess of levels measured in intact animals. Treatment had no effect on ALT-1 expression. In prostate, castration resulted in an incredible, greater than 100-fold repression of ALT-2 expression, but again no effect on ALT-1 (Figure 4.11). Treatment

93 of castrated animals resulted in 100- to 200-fold increases in ALT-2 expression over

ORX controls. Importantly, neither ligands’ ALT-2 expression was different than intact control animals.

Body weight normalized tissue weights showed a 50% decrease in prostate weight following castration at 3 days in vehicle-treated animals (Figure 4.12). DHT treatment yielded prostates not different in weight from intact animals, whereas SARM3 treatment resulted in intermediate prostate weights that were less than intact, but still greater than those observed in castrate controls. Seminal vesicles showed a 60% reduction in weight following castration. DHT treatment for 3 days grew this tissue to greater than intact levels, whereas SARM3 only maintained seminal vesicle weights. Castration caused a

25% atrophy in levator ani muscle. Both DHT and SARM3 maintained the weight of this tissue at intact control levels after 3 days of treatment.

4.4. Discussion

The well-characterized increases in serum ALT following many androgen therapeutic regimens have long been attributed to hepatotoxicity. The genome-wide AR promoter binding experiment (Chapter 3) pointed to ALT-2 as a bone fide direct target of the AR, implying that AR action could contribute in part to the observed serum elevations of ALT. Bioinformatics searches of the promoter region of both ALT-1 and

ALT-2 yielded a number of conserved AREs (Table 4.2 and Table 4.3), suggesting that

ALT-1 might also be a direct target and that the regulation might be ARE-mediated.

Previous experiments confirmed ALT-2 regulation by DHT and SARM2 in the LNCaP prostate cancer cell model (Figure 3.10). In an effort to better characterize this effect,

94 gene expression following treatment with DHT and another aryl propionamide SARM,

SARM3, were studied in both LNCaP cells and primary rat hepatocytes.

SARM3 was compared to DHT in a simple transient transfection system reflecting the T877A mutant present in LNCaP, showing similar potency but greater efficacy than the endogenous androgen. SARM3 also showed a greater ability to induce

PSA in the LNCaP time course analysis (Figure 4.2), consistent with the increased efficacy in the transient transfection system. SARM3 showed maximal transcriptional activation at a concentration of 100 nM with DHT showing near maximal response at this exposure level. This data combined with previous ALT-2 regulation at the 100 nM level led to the selection of 100 nM of each ligand for the experiments in LNCaP.

ALT-1 and ALT-2 gene expression was evaluated in LNCaP after treating with

100 nM DHT and SARM3 for 24, 48 and 72 hours (Figure 4.2). Both ligands induced each ALT at 24 hours to a significant degree, though the small fold-change is of questionable biological relevance as 2-fold is a generally accepted cut-off. By 48 hours, induction of ALT-2 by both ligands was maintained, though DHT’s regulation was very marginal. SARM3 showed near 3-fold induction of ALT-2 that was extended through 3 days. Slight repression of ALT-1 by DHT was detected at 48 hours, with both ligands significantly down regulating ALT-1 by three days. At all time points, the PSA positive control transcript was up-regulated by both ligands. This experiment demonstrates two things; First, both ALTs appear to be androgen regulated. Second, ALT-2 is clearly induced by aryl propionamide SARMs in LNCaP as two ligands have shown similar responses.

95 To further evaluate AR regulation of the ALTs, promoter scanning via ChIP was employed in LNCaP cells. These experiments were designed to test two corollaries to the

LNCaP gene expression experiments. One, ALT-1 is a direct target of the androgen receptor. Two, AR binding to the ALT-2 promoter region is mediated by conserved

AREs. To address the first hypothesis, primer pairs were designed to span both conserved and non-conserved AREs mostly outside of the ChIP-DSL detection area

(Figure 4.4). These regions were chosen as ALT-1 was not reported as an AR direct target by the ChIP-DSL experiment. AR and RNA polymerase II loading onto the ALT-

1 promoter were both monitored as other nuclear hormone receptors have been shown to modulate Pol II recruitment in a ligand-dependent fashion, while their promoter occupancy/release remains unchanged[126]. Following 100 nM DHT stimulation, both

AR and Pol II were significantly recruited to the positive control PSA enhancer region

(Figure 4.5), corresponding nicely with the detected gene regulation at this concentration of ligand. None of the regions examined in the ALT-1 promoter showed either AR or Pol

II recruitment/release. It bears mentioning that both the ‘ON’ and the ‘OFF’ signal are of likely importance as initially DHT induces ALT-1 in a ligand dependent fashion, but by

72 hours repression had occurred (Figure 4.2). Ligand-mediated AR or Pol II motility could offer support that this regulation is a direct action of AR, but none was detected.

Similar results were found with 100 nM SARM3, including significant recruitment to the

PSA enhancer region and no significant AR recruitment to any of the ALT-1 regions tested (Figure 4.6). However, significant Pol II recruitment was detected by SARM3 using primer Set 3, the closest set tested to the TSS. This evidence corresponds with

SARM3’s early induction of ALT-1, but suggests that if ALT-1 is directly AR regulated, 96 AR is recruited to areas other than those examined. The lack of Pol II recruitment by

DHT, even though DHT’s induction of ATL-1 was similar to SARM3 at 24 hours, could be explained by the reduced efficacy of DHT in the mutant LNCaP AR and the sensitivity of the assay.

To address the second hypothesis, primer pairs were designed flanking conserved

AREs in the proximal promoter region of ALT-2 (Figure 4.7). No conserved AREs were found within the genomic region covered by the ChIP-DSL experiment, but two promising AREs were quite close (#2 and #3). Much like the ALT-1 promoter scan, both

AR and Pol II occupancy were monitored on the ALT-2 promoter. Neither AR nor Pol II showed any significant ligand-dependent recruitment or release (Figure 4.8) for 100 nM

DHT or SARM3. It is likely that the AR binding was mediated by ARE #1, which was not conserved and corresponds with the ChIP-DSL hit, though this region was not evaluated. LNCaP prostate cancer cells offered a convenient model to study AR- mediated regulation of the ALTs, but the regulation in a prostate cancer cell of gene products thought largely derived from normal liver is only weakly supportive of the larger reaching hypothesis. To this end, AR-mediated ALT regulation was evaluated in primary rodent hepatocytes and tissues from treated animals.

Primary hepatocytes are a challenging model to use in studying steroid hormone gene regulation for myriad reasons discussed at length in Chapter 5. Nevertheless, DHT and SARM3 regulation of the ALTs was evaluated in male rat primary hepatocytes

(Figure 4.3). 48 hours was chosen, as LNCaP cell had shown maximal response at two days and hepatocyte viability was questionable at 72 hours. 1 uM ligand was applied in the hopes that some signal would be detectable in the noisier primary system. No 97 significant regulation of either ALT-1 or ALT-2 was detected though both transcripts trended similarly in hepatocytes as they did in LNCaP with slight repression of ALT-1 and induction of ALT-2. Though not examined directly, the lower expression of AR in rat hepatocytes as compared to LNCaP cells could explain this result.

In vivo conformation of in vitro AR-mediated ALT regulation was evaluated using a castrated rat model. Male rats were castrated, then immediately treated with either vehicle or 5 mg/kg DHT or SARM3 subcutaneously for 3 days. The dose chosen was in great excess of the Emax dose in the hope that the increased tissue exposures would afford detection of potentially subtle mRNA regulation. The three day treatment duration was chosen to limit the potential of animals reaching a new “on-drug” homeostasis and to allow enough time for androgen-dependent tissues to respond to castration. This permitted useful comparison between both intact and castrated vehicle treated animals that would not have been possible in an immediate treatment one dose

‘pulse’ study design often employed to study in vivo direct gene regulation[83]. The animals were also sacrificed within several hours following the final dose, ensuring some drug was still present at the time tissues were harvested for mRNA analyses.

ALT regulation in liver tissue was surprising, as castration alone or in combination with androgen treatment had no effect (Figure 4.9). Given the ALT-2 trend in primary hepatocytes, hepatic regulation was expected. In the levator ani muscle, an anabolic tissue chosen for its AR expression[152], castration showed a clear repression in

ALT-2 (Figure 4.10). When animals were castrated and treated with either DHT or

SARM3, significant ALT-2 induction over castrate and even intact controls was apparent.

However, no ALT-1 regulation was detected in this tissue. When looking at ALT 98 expression in the prostate, an incredible 100-fold repression of ALT-2 resulted from castration (Figure 4.11). ALT-2 expression was maintained at intact control levels in treated animals and again ALT-1 showed no regulation at 3 days. These data are in clear support of AR mediated ALT-2 regulation in both the levator ani and prostate tissues.

The timing of regulation is also consistent with a direct drug effect, not a secondary result of androgens’ effects on the animal. The lack of acute liver regulation argues against AR action in this tissue contributing to elevated serum ALT, though a delayed response is not excluded by this study. The same could also be said of ALT-1 regulation in any of the tissues examined.

When body weight normalized tissue weights were analyzed, the incredible remodeling capacity for the prostate and seminal vesicles were evident as castration resulted in 50% weight reduction at only 3 days (Figure 4.12). Surprisingly, at nearly 5- fold the Emax dose, administered immediately following castration, rats on SARM3 still experienced a loss in prostate size when compared to both intacts and DHT-treated animals. A similar pattern was evident in seminal vesicles, though DHT actually increased the weight of this tissue. Castration also resulted in 20% atrophy of levator ani muscle that was prevented by treatment with either ligand at 3 days.

If the differences in DHT and SARM action in prostate were solely due to potency then maximal responses should be identical. It is reasonable to assume that at a dose nearly five times larger than the previously determined Emax, the true maximal response of both DHT and SARM3 fully replaced endogenous hormone signaling. At a maximal response, albeit for a very short time, SARM3 was still selective again arguing

99 qualitative differences between SARM and endogenous androgens’ actions in the prostate.

In conclusion, these studies provide sound evidence that ALT-2 is androgen regulated and up-regulated by the androgen treatment. Previous work had shown ALT-2 promoter recruitment of the AR. Though the actual location of AR binding was not determined, several conserved AREs were excluded. ALT-1 showed more subtle regulation in vitro ranging from weak induction to repression with no regulation detected in vivo. The actual contribution of AR-mediated ALT regulation to serum ALT levels was not determined by these works, though the opportunity for AR action to contribute to serum levels is clear. Also, these studies do not definitively argue the absence of liver toxicity following SARM therapy which must be addressed directly with liver histology and systematic toxicology studies. These studies do however; offer a plausible explanation for asymptomatic elevated serum ALT levels often observed following androgen administration.

100 4.5. Acknowledgments

The ORX Rat sudy was designed with help from Dr. Jeff Kearbey and executed with help from GTx Animal Resources and Amanda Jones.

101 AR Transactivation in COS-1 - 24 Hr. Tx 0.8 DHT SARM3

0.6

0.4 RLU

0.2

0.0 eh M eh M M M M M M V u V n n n n n n 1 01 .1 1 10 00 00 . 1 10 GRE-LUC + pCR3.1 GRE-LUC T877A-AR

Figure 4.1 AR Transactivation in COS-1 – T877A-AR Dose response curves of both DHT and SARM3 in a transient transfection system reflecting LNCaP mutation. SARM3 shows both increased efficacy with similar potency when compared to DHT.

102 LNCaP Gene Expression - Time Course 50 24 Hours 48 Hours 72 Hours *

40 PSA *

30

*

20 PSA FC vs. Vehicle vs. FC PSA

10

0 e 3 e 3 e 3 icl HT M icl HT M icl HT M eh D AR eh D AR * eh D AR V nM S V nM S V nM S * 3 00 nM 00 nM 00 nM 1 00 1 00 1 00 1 1 1 24 Hours* 2 *

1

0

ALTs FC vs Vehicle vs FC ALTs -1

* -2 * ALT-1 * ALT-2

-3 e 3 e 3 e 3 icl HT M icl HT M icl HT M eh D AR eh D AR eh D AR V nM S V nM S V nM S 00 nM 00 nM 00 nM 1 00 1 00 1 00 1 1 1 * p<.05 Versus corresponding vehicle (Fisher's LSD)

Figure 4.2 LNCaP ALT-1/2 Gene Expression Values are represented as mean + SD (n=3), as described in the methods section, fold change versus vehicle group. Control line represents no change.

103 Primary Rat Hepatocytes - Gene Expression 3

ALT-1 ALT-2 2

1

0 FC vs. Vehicle FC vs.

-1

-2 Veh 1 uM DHT 1 uM SARM3

48 Hour Treatment

Figure 4.3 Primary Rat Hepatocytes – ALT-1/2 Gene Expression Values are represented as mean + SD (n=3), as described in the methods section, fold change versus vehicle group. Control line represents no change.

104 Set 8 Set 7

10 - 6.5 kb -6.0 kb - 5.5 kb

Set 6 Set 5

9 8 7 - 5.5 kb - 5.0 kb - 4.5 kb

- 4.5 kb -4.0 kb -3.5 kb

- 3.5 kb -3.0 kb - 2.5 kb

-2.5 kb Set 4 - 2.0 kb - 1.5 kb

ChIP-DSL

6 5 - 1.5 kb -1.0 kb - 0.5 kb

ChIP-DSL

4 3 - 0.5 kb TSS + 0.5 kb

ChIP-DSL Set 3 Set 2

+ 0.5 kb 1.0 kb + 1.5 kb

Set 1

2 1 + 1.5 kb + 2.5 kb 2.0 kb

Figure 4.4 ALT-1 9kb Promoter Region Schematic Conserved AREs are depicted with diamonds and numbered according to Table 4.2. Non-conserved AREs are depicted with ovals. ChIP primer sets are represented by the solid blue lines and numbered according to Table C.2. The ChIP-DSL coverage is depicted by the dashed red line.

105 ALT-1 Promoter Scan - DHT

PSA Enh. AR Pol II *

6

4

Set 1 Set 2 Set 3 Set 4

2 FC vs Vehicle (ChIP/Input) vs FC

0 PSA Enh. eh T eh T eh T eh T eh T V DH V DH V DH V DH V DH M M M M M 0 n 0 n 0 n 0 n 0 n * 10 10 10 10 10

6

4

Set 5 Set 6 XSet Data 7 Set 8

2 FC vs Vehicle (ChIP/Input) Vehicle vs FC

0 eh T eh T eh T eh T eh T V DH V DH V DH V DH V DH M M M M M 0 n 0 n 0 n 0 n 0 n 10 10 10 10 10

*p<.05 Versus corresponding vehicle (Fisher's LSD)

Figure 4.5 ALT-1 Promoter Scan – DHT Results AR and RNA Pol II loading on ALT-1 promoter regions in LNCaP following a 2 hour treatment with 100 nM DHT. The PSA-Enhancer region is presented as a positive control. Each value is represented as mean+SD (n=3).

106 ALT-1 Promoter Scan - SARM3

PSA Enh. AR Pol II * 5

4

Set 1 Set 2 Set 3 Set 4 3 *

2 FC vs Vehicle (ChIP/Input)FC vs 1

0 h h 3 h 3 h 3 PSAh Enh.3 e nM e M e M e M e M V 0 V R V R V R V R 10 SA SA SA SA M M M M * 5 0 n 0 n 0 n 0 n 10 10 10 10

Set 5 Set 6 XSet Data 7 Set 8 4

3

2 FC vs Vehicle (ChIP/Input) vs FC 1

0 h 3 h 3 h 3 h 3 h 3 Ve M Ve M Ve M Ve M Ve M AR AR AR AR AR S S S S S nM nM nM nM nM 00 00 00 00 00 1 1 1 1 1 *p<.05 Versus corresponding vehicle (Fisher's LSD)

Figure 4.6 ALT-1 Promoter Scan – SARM3 Results AR and RNA Pol II loading on ALT-1 promoter regions in LNCaP following a 2 hour treatment with 100 nM SARM. The PSA-Enhancer region is presented as a positive control. Each value is represented as mean+SD (n=3). 107 - 5.5 kb - 6.5 kb -6.0 kb

7 6 -5.5 kb -5.0 kb - 4.5 kb

Set 4

5 -4.5 kb -4.0 kb -3.5 kb

Set 3

4 -3.5 kb -3.0 kb -2.5 kb Set 2 Set 1

3 2 - 2.5 kb - 1.5 kb -2.0 kb

ChIP-DSL

-1.5 kb - 1.0 kb -0.5 kb

ChIP-DSL

1 -0.5 kb TSS + 0.5 kb

ChIP-DSL

+ 0.5 kb 1.0 kb + 1.5 kb

+ 1.5 kb + 2.5 kb 2.0 kb

Figure 4.7 ALT-2 9kb Promoter Region Schematic Conserved AREs are depicted with diamonds and numbered according to Table 4.3. Non-conserved AREs are depicted with ovals. ChIP primer sets are represented by the solid blue lines and numbered according to Table C.2. The ChIP-DSL coverage is depicted by the dashed red line.

108 ALT-2 Promoter Scan

7 AR Pol II PSA Enh.

6 * 5

4 Set 1 Set 2 Set 3 Set 4

3

- FC vs. Vehicle (ChIP/Input) Vehicle vs. FC - 2

1 SARM3

0 h h h h PSAh Enh. 7 e nM e nM e nM e nM e nM V 0 V 0 V 0 V 0 V 0 10 10 10 10 10 * 6

5

4 Set 1 Set 2 Set 3 Set 4

3 FC vs. Vehicle (ChIP/Input) Vehicle FC vs.

- 2

DHT DHT 1

0

eh nM eh nM eh nM eh nM eh M V 0 V 0 V 0 V 0 V n 10 10 10 10 00 1 * p<.05 Versus corresponding vehicle (Fisher's LSD)

Figure 4.8 ALT-2 Promoter Scan Results AR and RNA Pol II loading on ALT-2 promoter regions in LNCaP following a 2 hour treatment with 100 nM DHT or SARM3. The PSA-Enhancer region is presented as a positive control. Each value is represented as mean+SD (n=3). 109 ORX Rat High Dose Study - Liver Gene Expression 3 ALT-1 ALT-2 2

1

0

-1 FC vs. ORX Vehicle ORX vs. FC

-2

-3 le T 3 eh hic DH RM V Ve SA act Int

ORX

Figure 4.9 ORX Rat Acute High Dose – Liver Gene Expression Values represented as mean+SD (n=5), as described in the methods section, FC versus ORX vehicle. Control line represents no change. Treated animals received 5 mg/day subcutaneously for 3 days.

110 ORX Rat High Dose Study - Levator Ani Gene Expression 10 ALT-1 3 days S.C. ALT-2 0 I 0 0 I 0 I 0 I I 8

6

0 0 4 FC vs ORX Vehicle ORX vs FC

2 I I

0 RX HT M3 eh O D R t V SA ac Int ORX 0 p<.05 vs. ORX Veh, Fisher's LSD I p<.05 vs. Intact Veh, Fisher's LSD

Figure 4.10 ORX Rat Acute High Dose Study – Levator Ani Gene Expression Values represented as mean+SD (n=5), as described in the methods section, FC versus ORX vehicle. Solid control line represents no change from ORX and dashed line represents no change from Intact. Treated animals received 5 mg/day subcutaneously for 3 days.

111 ORX Rat High Dose Study - Prostate Gene Expression

700 0 0 p<.05 vs. ORX Veh, Fisher's LSD 600 ALT-1 I p<.05 vs. Intact Veh, Fisher's LSD 500 ALT-2 400 300 0 200 0 100

I

2 FC vs. ORX Vehicle

0

-2 cle HT M3 eh hi D R t V Ve SA ac Int ORX

Figure 4.11 ORX Rat Acute High Dose Study – Prostate Gene Expression Values represented as mean+SD (n=5), as described in the methods section, FC versus ORX vehicle. Solid control line represents no change from ORX and dashed line represents no change from Intact. Treated animals received 5 mg/day subcutaneously for 3 days.

112 ORX Rat High Dose Study - 3 Day Tissue Weights 500 ORX 0 DHT I SARM3 400 Intact 0 0 300 0 0

0 200 I 0 00 % of ORX Vehicle of ORX % I I I 100

0 i te les n sta ic r A ro es to P l V va na Le mi Se I p< .05 vs. Intact Control (Fisher's LSD) 0 p< .05 vs. ORX Control (Fisher's LSD)

Figure 4.12 ORX Rat Acute High Dose – 3 Day Tissue Weights Values are normalized to individual animal bodyweights and then represented as percentage of mean body weight normalized ORX control tissue weight at Day 3 (mean+SD, n=5).

113 Gonadal Status Drug Treatment Time Dose Intact Vehicle 3 days NA ORX Vehicle 3 days NA ORX DHT 3 days 5 mg/day ORX SARM3 3 days 5 mg/day

Table 4.1 ORX Rat Acute High Dose Treatment Groups All animals were dosed subcutaneously.

114 Gene Location PWM Species Symbol RefSeq (∆ TSS) Strand Chr. Score Sequence ARE-1 Human GPT NM_005309 +1963 Plus 8 .958 AGGACAACGTGTACG ARE-1 Mouse GPT NM_182805 +1631 Plus 15 .914 GGCACACAGAGTGGT ARE-1 Rat GPT1 NM_031039 +1698 Plus 7 .909 AGGACAACGTGTATG ARE-5 Human GPT NM_005309 -1373 Plus 8 .906 GGCACAATGTGTCAG ARE-5a Mouse GPT NM_182805 -710 Minus 15 .912 AGAACAGGAAGTGAG ARE-5b -531 Plus .958 AGGGCACCAAGTCTT ARE-5a Rat GPT1 NM_031039 -877 Minus 7 .872 GGGACAGAGGGATCA ARE-5b -551 Plus .958 AGAACTAGCTGTCCC ARE-10 Human GPT NM_005309 -5536 Minus 8 .953 AGAACAGGCCGTGCT

ARE-10 Mouse GPT NM_182805 -6006 Plus 15 .862 AGGGCACCAAGTCTT ARE-10 Rat GPT1 NM_031039 -5375 Minus 7 .909 AGAACTAGCTGTCCC

Table 4.2 ALT-1 Conserved AREs ARE number corresponds to Figure 4.4.

115 Gene Location PWM Species Symbol RefSeq (∆ TSS) Strand Chr. Score Sequence ARE-2 Human GPT2 NM_133443 -1827 Minus 16 .908 TGTGCTTGTTGTTCT ARE-2 Mouse GPT2 NM_173866 -1437 Plus 8 .945 GGCACAGTTGGTGCT ARE-3 Human GPT2 NM_133443 -2002 Minus 16 .934 AATACTGCCTGTGCT ARE-3 Mouse GPT2 NM_173866 -1437 Plus 8 .945 GGCACAGTTGGTGCT ARE-4 Human GPT2 NM_133443 -2976 Minus 16 .932 GAAACTGTATGTTCT

ARE-4 Mouse GPT2 NM_173866 -2978 Minus 8 .917 AGCACTGATTGCTCT

ARE-5 Human GPT2 NM_133443 -3977 Minus 16 .912 CTCACTTTCTGTTCT

ARE-5 Mouse GPT2 NM_173866 -4535 Plus 8 .984 GGGACAAGCTGTTCT

ARE-5 Rat GPT2 XM_001052974 -4334 Plus 19 .926 GGTACACGCGGTCCA

Table 4.3 ALT-2 Conserved AREs ARE number corresponds to Figure 4.7.

116 CHAPTER 5

5. ANDROGEN REGULATION OF SERUM LIPIDS

5.1. Introduction

Androgens’ effects on serum lipids as they pertain to cardiovascular disease

(CVD) are complex and controversial. Observational studies link low serum testosterone

(T) in men, but elevated serum T in women, to visceral obesity, reduced circulating high density lipoprotein cholesterol (HDL-C) and elevated low density lipoprotein cholesterol

(LDL-C) and triglycerides[72, 153]. However, exogenous steroidal androgen administration to men and women reduces visceral fat mass while concomitantly decreasing HDL-C with neutral effect or only slight changes in LDL-C[72]. Age, gender, steroidal androgen administered, route of administration, the health and body mass index

(BMI) of the patient, and the dose(sub-, physiological or supra-physiological) all contribute to an individual’s serum lipid response to androgen administration[72]. In light of the confounding pro- and anti-atherogenic changes associated with steroidal androgen therapy, leading clinicians have recently stated; “Based on current evidence, the therapeutic use of T in men need not be restricted by concerns regarding cardiovascular side effects”[72]. That said, currently absent, large prospective, controlled studies are 117 needed to truly evaluate the cardiovascular risks associated with exogenous steroidal androgen administration.

Previous work comparing oral steroidal androgen administration to parenteral dosage forms have shown consistently greater reductions in serum cholesterol following oral administration on acute time scales (1-3 months) [76, 154]. Testosterone and its ester derivatives have very poor oral bioavailability due to high rates of first pass metabolism and are traditionally administered either intramuscularly or transdermally[2].

Orally available steroidal androgens, such as stanozolol and methyltestosterone, are capable of achieving efficacious systemic plasma concentrations, but at the cost of greater hepatic exposure compared to their parenteral counterparts. A potential explanation for the route of administration specific serum lipid effect is a pharmacologic signal initiated in the liver, triggered by the increased hepatic exposure to steroidal androgen following an oral dose. This effect remains to be characterized concerning either non-steroidal AR agonists or SARMs. With this hypothesis in mind, androgen- mediated gene expression was studied in both in vitro liver models and in liver tissue of androgen-treated animals. Also, a multiple route of administration and divided dose study were performed to evaluate the impact of hepatic exposure and peak liver concentrations on androgen mediated-reductions in serum lipids.

5.1.1 HDL-C Homeostasis Target Genes

Steroidal androgen administration affects wholesale changes in mammalian lipid systems. Dramatic remodeling of both HDL-C and LDL-C subfractions has been reported after as little as three weeks of treatment[74]. In general, total serum lipid

118 content is reduced with a skew of the HDL-C/LDL-C ratio[72]. Given the larger, better characterized, androgen-mediated HDL-C flux and the HDL-C centric lipid physiology of rodent and canine model systems, HDL-C was the primary focus of these works. A number of genes involved in HDL-C homeostasis are highly expressed in both rodent and human hepatic tissues. As it is currently unknown whether androgens affect HDL-C synthesis or metabolism, gene targets important in both were evaluated.

Increases in plasma hepatic lipase (LIPC) activity during steroidal androgen administration are well characterized [74, 76, 155]. LIPC induction is consistent with the selective reduction in large buoyant HDL-C particles seen in some studies[83-85]. The promoter of the human LIPC gene has an ARE conserved across three species, increasing the likelihood of direct AR regulation. If steroidal androgens and SARMS are affecting

HDL-C metabolism, this could be explained in part by hepatic LIPC induction at the mRNA level. The class B scavenger receptor type I (SRB1) has been characterized as an

HDL-C specific receptor whose hepatic over-expression resulted in decreased plasma

HDL-C in mice [156]. Though SRB1 contains no conserved promoter AREs, its expression in macrophages is regulated by testosterone[157]. Like LIPC, SRBI induction in liver tissue would point towards androgens and SARMS increasing HDL-C clearance.

Reductions in plasma levels of the primary protein components of HDL-C molecules, apolipoprotein A1 (APOA1) and apolipoprotein A2 (APOA2), following steroidal androgen administration are also well characterized [76, 154, 155, 158].

APOA1 has a conserved ARE in its promoter region, but APOA2 does not. If androgens and SARMs are affecting HDL-C synthesis, this could be explained in part by APOA1 and/or APOA2 mRNA repression in liver. Also, ATP-binding cassette protein A1 119 (ABCA1) in both liver and intestine controls the rate-limiting step in HDL-C synthesis by transferring intracellular cholesterol to helical apolipoprotein, thus creating nascent HDL-

C[159]. Naturally occurring mutations in ABCA1 are causal in syndromes of HDL-C deficiency such as Tangier’s disease[160]. Further, liver-specific ABCA1-KO mice display an 80% reduction in serum cholesterol when compared to wild-type littermates, as well as reduced circulating APOA1[161]. ABCA1 was previously shown as AR regulated and a potential direct target of the AR in LNCaP cells (Table A.5, Figure 2.1) containing three conserved proximal AREs. Like the apolipoproteins, ABCA1 repression in liver would suggest androgens and SARMS are affecting HDL-C synthesis.

5.1.2 Androgen Responsive Liver Control Genes

In the hopes of bridging multi-species cellular and animal models, four liver targets known to be androgen regulated were chosen as controls. Induction of the ligand- free transcriptional repressor SHP-1 and consummate repression of its target CYP7A1 have been reported in both primate and rodent liver tissue following DHT administration[83]. Hepatic estrogen sulfotransferase (STE) and dehydropiandrosterone sulfotransferase (SULT2A1) expression are both gender- and age-specific in rodents.

STE is not normally expressed in female rodent liver, but is induced by DHT treatment[162]. SULT2A1 expression is readily repressed following androgen treatment[163]. SULT2A1 levels are undetectable in pubertal and young adult male rats, due to high levels of endogenous androgen, but can be detected in both pre-pubertal and senescent males. In females of all ages, basal expression levels are comparable to pre- pubescent males and are down-regulated by DHT treatment[162]. Though not as well

120 characterized, it is thought that these enzymes’ expression is similarly controlled in primates.

5.2. Materials and Methods

5.2.1 Materials

HepG2 hepatocellular carcinoma cells were purchased from ATCC (Manassas,

Virginia). Trypsin (0.25%)-EDTA, DMEM and RPMI-1640 medium were purchased from MediaTech (Manassas, Virginia). Trizol® reagent, penicillin/streptomycin and were purchased from Invitrogen Corp. (Carlsbad,California). Charcoal-treated fetal bovine serum (cFBS) was purchased from Atlanta Biologicals (Atlanta, Georgia). DHT was purchased from Sigma-Aldrich (St. Louis, Missouri).

5.2.2 Animals

Sprague-Dawley rats were purchased from Harlan Biosciences (Indianapolis, IN).

Female beagle dogs were purchased from Marshall Farms (North Rose, NY). Animals were maintained on a 12-hour light-dark cycle with food and water available ad libitum.

The animal protocol was reviewed and approved by the Institutional Laboratory Animal

Care and Use Committee of The University of Tennessee.

5.2.3 Orthologous Promoter Androgen Response Element (ARE) Search

The ARE search was performed as previously described (3.2.6) except the search was extended from a 7kb region to a 12kb region from(-10kb to +2kb) and the window

121 surrounding the human ARE was expanded to 800 bp in each direction when considering

ARE’s conserved between human and rodent species.

5.2.4 Primary Rat and Human Hepatocyte Experiments

Fresh primary male Sprague-Dawley rat hepatocyte suspensions (CellzDirect

Durham, NC) were plated in collagen-coated 6 well plates (Beckton Dickinson) in

DMEM supplemented with 5% FBS, 0.1 U/ml penicillin/streptomycin, 4 µg/ml bovine insulin, 1 µM dexamethasone, 17.5 mg/ml L-glutamine and 15mM HEPES in accordance with the vendor’s protocol (Supplement Pack, CellzDirect). Four hours after plating, the media was replaced with phenol red free William’s E Media supplemented with 0.1 U/ml penicillin/streptomycin, 100 nM dexamethasone, 1x insulin-transferrin-selenium solution,

17.5mg/ml L-glutamine and 15 mM HEPES (Supplement Pack, CellzDirect). One day later, this media was replaced with media containing either 0.1% ethanol vehicle alone or

100 nM SARM2 dissolved in 0.1% vehicle. Cells were treated for 24, 48 and 72 hours.

Following treatment, cells were harvested and total RNA extracted using Trizol.

Reconstituted RNA was concentration normalized and quantitative gene expression analyses were performed using a One-Step RT-PCR Kit (Applied Biosystems), .5 to 1 µg of sample RNA, Taqman probes normalized to 18S and Applied Biosystem’s 7300 Real

Time PCR system. Data was analyzed using the 2-∆∆Ct method[107] with previously described statistical analysis and representation (3.2.7).

Fresh primary Human Hepatocytes from a 63 year old non-smoking male donor

(CellzDirect) were handled similarly to rat hepatocytes, differing only in treatment times

(6, 12, 24 and 48 hours). DHT was used as the AR ligand.

122 5.2.5 HepG2 Transient Transfection Experiments

HepG2 hepatocellular carcinoma cells were subcultured in accordance with

ATCC recommendations. The lowest passage HepG2 available, p72-78, were transiently transfected with 1 µg cmv-hARwt plasmid[102] per million cells via electroporation using a Nucleofector II (Amaxa). The vendor-established protocol and recommended nucleofection kit (Kit V) were used in accordance with manufacturer’s guidelines. Two million transfected HePG2 cells were plated per well in a 6 well plate in phenol red-free

DMEM supplemented with 5%cFBS. Twelve hours later, the media was replaced with phenol red-free DMEM supplemented with 5% cFBS also containing 0.1% ethanol vehicle or 1 µM SARM2 dissolved in 0.1% ethanol vehicle. In the case of the control experiment, no vehicle or ligand was added to the media. Cells were harvested at 24, 48 or 72 hours using Trizol and total RNA was extracted. Reconstituted RNA was concentration normalized and quantitative gene expression analyses were performed using a One-Step RT-PCR Kit (Applied Biosystems), .5 to 1 µg of sample RNA, Taqman probes normalized to 18S and Applied Biosystem’s 7300 Real Time PCR system. Data was analyzed using the 2-∆∆Ct method[107] with previously described statistical analysis and representation(3.2.7).

5.2.6 Orchidectomized Rat Time Course Study

Male Sprague-Dawley rats (12 weeks old) approximately 200 grams in body weight were orchidectomized (ORX) at the beginning of the study. ORX animals were randomized by pre-surgery body weight into groups of 5. Two groups of 5 sham-

123 operated male rats were included as intact controls (Table 5.2). The ORX animals were treated the next day following surgery. All treatments were administered as 100 µL subcutaneous injections in 80:20 (v:v) PEG300:DMSO daily for the 28 day duration of the study. At the end of treatment, animals were weighed, anesthetized, and sacrificed within 24 hours after the last dose. Serum was collected via abdominal aortic puncture.

Serum total cholesterol was measured using an enzymatic/colormetric assay performed by Antech Diagnostic Services (South Haven, MS). Statistical analysis of serum cholesterol values were performed by one-way ANOVA on each treatment day followed by pair-wise, two-tailed Student’s t-tests between each treatment group and ORX control

(Fisher’s LSD). A threshold of p <.05 was set for significant differences. Tissues of interest were harvested and weighed at sacrifice including prostate, seminal vesicles, soleus muscle, gastrocnemius muscle, levator ani muscle, and liver. Statistical analysis of tissue weights were performed following per animal body weight normalization by one-way ANOVA on each treatment at day 28 followed by pair-wise, two-tailed

Student’s t-tests between each treatment group and ORX and intact controls (Fisher’s

LSD). A threshold of p <.05 was set for significant differences. After weighing, tissues were placed immediately into RNAlater (Ambion) for storage prior to RNA extraction.

Aliquots (50-100 mg) of preserved tissue samples were homogenized in Trizol using

Lysing Matrix D (MP Biomedicals) and a FastPrep FP120 homogenizer (Thermo

Savant). Total RNA was then extracted, reconstituted in DEPC-treated molecular biology grade water, and diluted to 300 ng per ul. cDNA was generated using the High

Capacity cDNA Kit with RNase inhibitor (Applied Biosystems) and 3 ug of RNA from each sample. Quantitative gene expression analyses were performed using Taqman 124 probes normalized to 18S and Applied Biosystem’s 7300 Real Time PCR system. Data was analyzed using the 2-∆∆Ct method[107] with previously described statistical analysis and representation(3.2.7).

5.2.7 Ovariectomized Rat Extended Treatment Study

Female Sprague-Dawley rats (23 weeks old) were ovariectomized (OVX) at the beginning of the study. OVX animals were randomized by pre-surgery body weight into

6 groups of 10 (Table 5.3). OVX animals were treated with their first dose the next day following surgery via oral gavage in 80:20 (v:v) PEG300:DMSO. At the end of 42 days of treatment, animals were anesthetized and sacrificed within 24 hours after the last dose.

Serum was collected via abdominal aortic puncture. Serum total cholesterol was measured using an enzymatic/colormetric assay performed by Antech Diagnostic

Serivces (South Haven, MS). Statistical analysis of serum cholesterol values were performed by one-way ANOVA on OVX or intact treatment groups followed by pair- wise, two-tailed Student’s t-tests between each treatment group and its vehicle control

(Fisher’s LSD). A threshold of p <.05 was set for significant differences. Distal femurs were excised and trabecular bone was analyzed by µCT (SKYSCAN, Belgium). Bone volume as a percentage of total volume (BV/TV) was calculated similarly to Muller et al.[164]. Briefly, once the bone had been scanned an area of interest was selected approximately 1.5 mm distal to the growth plate specifically excluding cortical bone.

The image of this area was categorized based on a gray scale cut-off into bone or not bone and the same area of interest was evaluated in 10 sections spanning approximately

2.5 mm in the distal direction. The total number of “bone” pixels were reported as a ratio

125 of the total number of pixels evaluated. Statistical analyses of BV/TV values were performed identically to serum cholesterol values. Liver tissues were harvested at sacrifice and placed immediately into RNAlater (Ambion) for storage prior to RNA extraction. Aliquots (50-100 mg) of preserved tissue samples were homogenized in

Trizol using Lysing Matrix D (MP Biomedicals) and a FastPrep FP120 homogenizer

(Thermo Savant). Total RNA was then extracted, reconstituted in DEPC-treated molecular biology grade water, and diluted to 300 ng per ul. cDNA was generated using the High Capacity cDNA Kit with RNase inhibitor (Applied Biosystems) and 3 ug of

RNA from each sample. Quantitative gene expression analyses were performed using

Taqman probes normalized to 18S and Applied Biosystem’s 7300 Real Time PCR system. Data was analyzed using the 2-∆∆Ct method[107] with previously described statistical analysis and representation(3.2.7).

5.2.8 Intact Female Beagle Dog Study

Blood samples were obtained twice weekly from sixteen intact female beagle dogs ranging from 2-4 years in age and 9-13 kg in body weight by jugular vein blood draw. Animals were maintained on a normal chow diet (approx. 20g total fat per day) ad libitum with 12 hour fasts prior to blood draws for the duration of the study. Following 4 weeks of baseline blood draws, animals were randomized by average baseline total serum cholesterol into 4 groups of 4 animals each (Table 5.4). Animals in the once daily oral dose group (Oral QD) received a 15 g ball of ID soft food (1.3 g of fat per day) laced with .01 mg/kg SARM2 in 100 uL of an aqueous micro-emulsion at 7:00 AM and ID food balls laced with vehicle alone at 3:00 PM and 11:00 PM for the first 14 days. The

126 dose was adjusted to 0.03 mg/kg from 15 days through day 56. Animals in the three times daily oral group (Oral Q8) received 15 g ID food balls laced with 0.0033 mg/kg

SARM2 in 100 uL of an aqueous micro-emulsion at 7:00 AM, 3:00 PM and 11:00 PM for the first 14 days. The dose was adjusted to 0.01 mg/kg from 15 days through day 56.

Animals in the once daily subcutaneous group (S.C. QD) received a 100 uL injection of

.01 mg/kg SARM2 dissolved in 80% PEG300 and 20% DMSO at 7:00 AM and vehicle injections at 3:00 PM and 11:00 PM for the first 14 days. The dose was adjusted to .03 mg/kg from 15 days through day 56. Animals in the three times daily subcutaneous (S.C.

Q8) group received a 100 uL injection of .0033 mg/kg SARM2 dissolved in 80%

PEG300 and 20% DMSO at 7:00 AM, 3:00 PM and 11:00 PM for the first 14 days. The dose was adjusted to .01 mg/kg from 15 days through day 56. All subcutaneously injected animals received a 15 g ball of ID food following injection to control for oral vehicle contributions to total dietary fat. All animals were dosed after weekly blood draws. Dose levels were adjusted to group average body weight and updated bi-monthly.

Serum total cholesterol was measured weekly while the animals were on drug using an enzymatic/colormetric assay performed by Antech Diagnostic Services (South Haven,

MS). Statistical analysis of serum cholesterol values were performed by one-way

ANOVA on all groups per day followed by pair-wise, two-tailed Student’s t-tests.

Significance was determined by a p <.05. Serum aliquots were also analyzed using lipoprotein gel electrophoresis apparatus (Helena Laboratories Beaumont, TX) by Antech

Diagnostics (Indianapolis, IN).

127 5.3. Results

5.3.1 Putative Androgen Response Elements

Putative AREs were detected in a number of genes integral to HDL metabolism/homeostasis as well as control genes (Table 5.1). Hepatic Lipase (LIPC),

ABCA1 and APOA1 all had one or more conserved AREs, whereas APOA2 and SRB1 had no conserved AREs within the 12kb region surrounding the annotated TSS. ABCA1 was also detected as a potential direct AR target in the cDNA micro-array experiment

(Chapter 2) and the AR binding potential of two of its conserved AREs evaluated by

EMSA (Figure 2.5). Concerning control genes, SHP-1 had four conserved AREs within the 12kb surrounding the TSS and STE had two conserved AREs. There were no conserved AREs detected in the promoters of either CYP7A1 or SULT2A1.

5.3.2 Gene Expression Analyses in Primary Hepatocytes

Androgen regulation of genes integral to HDL metabolism/homeostasis were studied in both human and rat male primary hepatocytes. In human, LIPC was significantly down regulated at 12, 24, and 48 hours when compared to vehicle following

100 nM DHT treatment (Figure 5.1). Also, SRB1 was marginally up-regulated by DHT at 6 hours and APOA1 was down regulated by DHT at 48 hours. No significant changes were detected in ABCA1 or APOA2 at any time point examined after treatment with

DHT. Amongst the control genes, CYP7A1 was up-regulated by DHT at 6 hours and down regulated to a similar degree at 48 hours (Figure D.1). SULT2A1 was also marginally up-regulated by DHT at the 48 hour time point. In an analogous experiment

128 in rat hepatocytes with SARM2, only APOA2 was marginally up-regulated at 48 hours when compared to vehicle following 100 nM SARM2 treatment (Figure 5.2). LIPC,

ABCA1, SRB1 and APOA2 showed no changes at any time point examined. Amongst the control genes examined, SULT2A1 was not expressed and no significant regulation of either SHP-1, CYP7A1, or STE was detected (Figure D.2).

5.3.3 Transient Transfection in HepG2 Hepatocellular Carcinoma Cells

Though AR is expressed at low levels in HepG2 cells, transient transfections are commonly used to study AR function in this ubiquitous liver cell model[165]. Following transfection by electroporation, AR expression in HepG2 was up-regulated approximately

100,000 fold and remained at that lvel for up to three days. (Figure D.5). Once AR expression was confirmed, cells were treated with 1 uM SARM2 for 24, 48 and 72 hours.

A large but variable increase in LIPC expression was seen at 24 hours, though significance was not achieved due to high variability in the vehicle group (Figure 5.3). A significant down regulation of ABCA1 was detected at 72 hours, whereas SRB1,

APOA1, and APOA2 remained unchanged. Concerning control genes, CYP7A1, STE, and SULT2A1 were all down-regulated at 72 hours (Figure D.6). SULT2A1 expression was detected, implying species-specific expression of this gene.

5.3.4 Orchidectomized Rat Time Course Study

Serum total cholesterol was monitored in castrated rats receiving vehicle or a single high dose (1 mg/day) of either SARM 2, 3, or 4 (Figure 5.4). Significant reductions from the ORX control group were detectable at day 7 for SARM2 and

129 SARM3. By day 10, all treated groups had significant reductions. However, a slight but insignificant rebound for SARM2 and 4 was evident at 14 days. At 28 days, all groups had realized a near 30% reduction in total serum cholesterol at this dose.

Prostate and seminal vesicle tissue weights were monitored as a measure of androgenic activity. At day 3, prostate weights were reduced to 25% of intact controls in the vehicle-treated animals, whereas all SARM-treated animals had prostate weights that were near 50% of intact controls (Figure 5.5). The vehicle-treated animals’ prostates decreased over time to 10% of intact controls by day 28, whereas SARMs partially maintained prostate weight at 70%, 80% and 50% of intact controls for SARM2,

SARM3, and SARM4, respectively. The trend was similar in seminal vesicle tissue weight, though SARMs showed a greater effect in this tissue (Figure 5.6). For example

SARM3, superceded intact controls by day 10. As a measure of anabolic activity, levator ani weight was measured. At day 3 ORX control animals had a 25% reduction in levator ani weight (Figure 5.7). This tissue continued to atrophy to 40% of intact controls by day

21. SARM-treated animals dropped to 85% at day 3, matched intact controls at day 7, and by day 14, the levator ani weight of all SARM-treated animals had superseded intact controls. At day 28, SARMs had increased levator ani weight to 120%, 122% and 110% for SARMs 2, 3 and 4 respectively. No changes were detected in either soleus or gastrocnemius weights at any time (Figure D.9, Figure D.10). Statistical analyses of day

28 tissue weights showed that SARM2- and SARM4-, but not SARM3-treated animals were significantly different from intact controls in prostate weight (Figure 5.8). When considering seminal vesicle weights, only SARM4-treated animals were significantly different from intact controls at 28 days. Finally, both SARM2- and SARM3- treated 130 animals had levator ani weights in excess of intact vehicle treated control animals at the conclusion of the study.

Liver tissue from each animal was assayed for gene expression differences in genes involved in HDL metabolism/homeostasis and control gene targets. In ORX vehicle-treated control animals, LIPC and APOA2 trended upward and ABCA1, SRB1 and APOA1 trended downward. However, none of the five genes of interest were significantly regulated at any time when compared to ORX animals at day 0 (Figure 5.9).

When looking at the control genes, only CYP7A1 was significantly down-regulated at day 3 (Figure D.11) with no significant change detected in SHP-1 or STE. SULT2A1 mRNA was not detected in any of the liver tissues in the study. In SARM2-treated animals, LIPC was significantly up-regulated at day 10 and day 28 when compared to

SARM2-treated animals at day 3 (Figure 5.10). APOA2 was significantly up-regulated at days 7, 10, 14, 21 and 28, whereas APOA1 was significantly down-regulated at days 10,

14 and 21. No significant changes were detected in either ABCA1 or SRB1 transcripts in

SARM2-treated animals. CYP7A1 was the only significantly regulated gene of the control genes and was down-regulated at day 10 (Figure D.12). In SARM3-treated animals none of the genes of interest were down-regulated (Figure 5.11). When looking at the control genes in the SARM3-treated animals (Figure D.13), CYP7A1 was significantly down-regulated at days 7, 14 and 21 with SHP-1 also down regulated at day

14. STE was not significantly regulated. In SARM4-treated animals, LIPC and APOA2 were significantly up-regulated at days 10, 14, 21, and 28 when compared to SARM4- treated animals at day 3 (Figure 5.12). SRB1 was also significantly up-regulated at day

28. APOA1 and ABCA1 showed no regulation at any time point. When looking at the 131 control genes in SARM4-treated animals, CYP7A1 trended downward though no changes of significance were detected due to large variability between animals (Figure

D.14). Neither SHP-1 nor STE showed any regulation in SARM4 treated animals.

5.3.5 Ovariectomized Rat Extended Treatment Study

Total serum cholesterol was monitored in both intact and OVX rats receiving 1 mg/day of either SARM2 or SARM4. After 6 weeks of treatment, intact animals had significant reductions of 25% and 15% in total serum cholesterol when treated with

SARM2 and SARM4, respectively (Figure 5.13). Total cholesterol was significantly elevated in the OVX control animals versus the intact animals receiving vehicle only, but treatment reduced total serum cholesterol by 30% in both SARM2 and SARM4 OVX groups.

Following sacrifice, distal femurs were analyzed for trabecular bone volume as a percentage of total scanned volume (BV/TV). Intact animals had nearly 35% BV/TV that was unchanged by SARM treatment (Figure 5.14). OVX control animals lost approximately 40% of the trabecular bone in their distal femurs after 6 weeks, with the

BV/TV of vehicle treated animals declining to 20%. SARM2 and SARM4 treatment restored BV/TV to near intact levels with no significant differences detected between the

OVX-treated and intact control groups detected.

Liver tissue was assayed for expression differences in five genes important in

HDL metabolism/homeostasis as well as control gene targets. In the intact animals,

ABCA1 was down regulated by both SARM treatments with no significant regulation of

LIPC, SRB1, APOA1 or APOA2 (Figure 5.15). None of the genes examined showed

132 regulation in OVX animals. Striking induction of STE was detected in all treated animals, regardless of gonadal status, with intact animals realizing better than 5000-fold and 1000-fold increases in this enzyme’s expression in intact and OVX animals, respectively (Figure D.15). Smaller but still significant down-regulation of CYP7A1 in intact SARM-treated animals and up-regulation of SHP-1 in OVX SARM treated animals was also detected.

5.3.6 Intact Female Beagle Dog Study

Sixteen intact female beagle dogs were given SARM2 via 4 different administration paradigms at .01 mg/kg/day for 2 weeks and then .03 mg/kg/day for the following eight weeks. Fasting total serum cholesterol was assayed weekly. All four administration paradigms, Oral QD, Oral Q8, S.C. QD and S.C. Q8 saw reductions at 14 days ranging from 8% to 18% of baseline in the S.C. Q8 and Oral Q8 groups, respectively (Figure 5.16). Following the second week the dose was increased 3x to .03 mg/kg/day and cholesterol continued to decrease across all groups with maximal reductions of approximately 30% in the Oral QD and S.C. Q8 groups, and 40% in the

Oral Q8 and S.C. QD groups. No difference was detected between any of the groups at any time, due in part to the large variability between individual dogs’ serum lipids.

Serum lipids were also fractionated by lipoprotein electrophoresis and trended nearly identically to the total cholesterol parameter (Figure 5.17).

133 5.4. Discussion

5.4.1 In Vitro Models

Initial in vitro experiments in male human hepatocytes showed a downward trend in LIPC expression over time following 100 nM DHT treatment (Figure 5.1). Given the known increases in plasma LIPC activity following androgen treatment, this gene was expected to be induced by DHT. However, APOA1 was moderately down-regulated at

48 hours in agreement with APOA1 reductions seen in human. CYP7A1 was down regulated at 48 hours as expected, but SHP-1 showed no induction and the measurable induction of CYP7A1 at 6 hours escapes ready explanation (Figure D.1). Due to the great expense of primary human hepatocytes and their poor viability in media conditions typically required to detect subtle hormonal gene regulation, this model was abandoned for primary rat hepatocytes which also offered the added benefit of species correspondence to in vivo models.

Gene expression in male rat primary hepatocytes was compared for 100 nM

SARM2 versus vehicle at 1, 2, and 3 days of treatment. Only APOA2 showed modest induction at 48 hours (Figure 5.2). SHP-1 expression was not induced by SARM2, nor was CYP7A1 repressed (Figure D.2). SULT2A1 expression was not detected which was expected for male rats with androgen responsive liver tissue. This experiment was also designed to examine the effect of the hepatocyte’s reduced viability over time on gene expression. Surprisingly, all control genes were increasingly down-regulated after plating without treatment (Figure D.3). Also, all target genes showed time dependent ligand independent regulation (Figure D.4). The ligand independent down-regulation of

134 LIPC over time in rat raised questions as too the legitimacy of the LIPC repression detected in human hepatocytes, albeit with a different androgen, as the human hepatocyte experiment had only one vehicle group at 24 hours. Such large changes in target gene expression, regardless of ligand, are likely to negatively affect one’s chances of detecting ligand-dependent regulation. Primary hepatocytes were thus also abandoned in favor of immortal hepatocellular carcinoma cells in the hopes that target gene expression would be more stable over time.

HepG2 hepatocellular carcinoma cells are a common in vitro liver model derived from an adolescent human male that lost AR expression once transformed. Transfected

AR expression was stable through 72 hours (Figure D.5) but the only significant regulation detected following 1 uM SARM2 treatment was a modest repression of

ABCA1 at 72 hours (Figure 5.3). LIPC displayed the anticipated induction at 24 hours, though significance was not achieved due to large variability in the vehicle group.

Ligand-independent target gene expression over-time was successfully stabilized by utilizing a transformed cell model (Figure D.7), though a significant induction of

CYP7A1 was detected at 72 hours (Figure D.8). Repression of SULT2A1 at 72 hours of

1 uM SARM2 versus vehicle combined with CYP7A1 repression, likely falling short of significance due to the aforementioned induction over time, suggest this model was working.

5.4.2 In Vivo Models

HDL-C accounts for 50% of total serum lipids in rodents[166] compared to 25 % in most [167], offering in theory a very sensitive model to study androgen effects

135 on HDL-C homeostasis. Also, several of the target genes monitored extensively in vitro perform their biological functions in the vascular space as structural proteins, enzymes, ligands or some combination of the three. It is not unlikely that an in vivo system is necessary for proper liver-HDL-C signaling and to detect any androgen-mediated perturbation therein.

Young male rats were castrated and were dosed subcutaneously with 1 mg/day of three separate aryl propionamide SARMs for 4 weeks. 1.0 mg/day (approximately 4-5 mg/kg/day) is an Emax dose[45] and was chosen not to examine any dose relationships between the three SARMs, but to ensure an anabolic effect and study mechanism. All three SARMs significantly reduced total serum cholesterol in only 10 days (Figure 5.4).

Interestingly, removal of endogenous androgen by surgical castration did not increase total serum cholesterol in male rats, whereas chemical castration in humans results in both total C and HDL-C increases[168]. Androgen-dependent tissues atrophied over time following castration in vehicle-treated animals whereas sub-maintenance to anabolism were detected in SARM-treated animals (Figure 5.5, Figure 5.6, Figure 5.7). At day 28,

SARMs 2 and 4 displayed the tissue selectivity for which they are named by failing to restore prostate to intact control levels while fully restoring, or even increasing, levator ani tissue weight. SARM3 was anabolic in levator ani but less sparing in prostate or seminal vesicles.

No significant liver target gene regulation was detected in the castrated control animals (Figure 5.9) and CYP7A1 was repressed at day 3 as the only regulated control gene. SULT2A1 was not expressed in any animal, indicating the rats were of an appropriate age for liver androgen responsiveness. In animals given SARM2 or SARM4, 136 significant LIPC and APOA2 induction were detected at 10 days that increased with time

(Figure 5.10, Figure 5.12). APOA1 was repressed in SARM2-treated, but not SARM4- treated animals, and no target gene regulation was evident in the SARM3 group (Figure

5.11). Mixed and noisy CYP7A1 repression was detected across all three treatment groups, though no induction of SHP-1 was seen (Figure D.12, Figure D.13, Figure D.14).

SHP-1 is a known target of the farsenoid X receptor, to which the testosterone metabolite androsterone has been a reported agonist[169]. It is possible that the reported SHP-1 induction following DHT treatment is actually mediated by a metabolite that is unavailable following non-steroidal androgen treatment.

LIPC and APOA2 regulation were detected on the day cholesterol reductions became significant. However, the timing of this regulation makes it as equally likely the result of the serum lipid changes as the cause. Interestingly, SARM3 lacked tissue selectivity at the dose given and failed to show any target gene regulation, though the serum cholesterol reductions were arguably the largest in this group. Determining whether or not there is a relationship between selectivity, target liver gene regulation, and serum cholesterol effects requires further study. Finally, removal of endogenous androgen by castration showed no lipid effects and no target gene regulation. Taken together, these data argue against these liver targets as the source of androgen-mediated serum lipid effects.

OVX and intact female rats were also treated with an Emax dose of 1 mg/day orally for 6 weeks with SARMs 2 and 4. Oral dosing ensured maximal liver exposure while maintaining systemic exposure with both SARMs having bioavailibilties nearing

100%. At 42 days, SARM treatment reduced serum cholesterol regardless of gonadal 137 status with a slightly larger reduction in OVX animals (Figure 5.13). Interestingly, ovariectomy resulted in an increase in cholesterol, an effect not seen in the ORX male at

28 days. Also, at the 1 mg/day dose level, both SARM2 and SARM4 successfully maintained trabecular bone mass in OVX animals (Figure 5.14).

When looking at target gene expression in the liver, ABCA1 was modestly down regulated, indicative of impaired HDL-C synthesis, but only in the intact animals. The expected induction of STE was apparent, but SULT2A1 was undetectable. Strangely,

CYP7A1 was only repressed in intact animals and SHP-1 induced in OVX animals. The consistency of the serum cholesterol reduction coupled with the inconsistency of target gene regulation again argue against liver regulation of these targets as causal in androgen mediated serum lipid effects.

5.4.3 Route vs. Rate – Female Beagle Dog Study

Previous toxicology work had shown the female beagle dog an extremely sensitive model in studying androgen-mediated serum lipid effects. This is not surprising given that HDL-C accounts for 75% of total serum cholesterol in canines[170]. With the knowledge of a clinically efficacious dose of 0.1 mg/kg/day, scaled from human to dog based on exposure, and a known cholesterol reducing dose of .05 mg/kg/day, a dose escalation study was designed beginning at 0.01 mg/kg/day of SARM2. 10% of the goal exposure was chosen as a starting point in the hopes that escalating doses of .03, .1, and

.3 mg/kg/day would move through the lower dose response range and afford detection of route (oral vs. subcutaneous) and rate (once daily vs. divided dose) specific cholesterol effects. It is likely that these effects would be harder to separate at an Emax dose if the

138 route specific cholesterol effects are due to either increased liver exposure (oral vs. subcutaneous) and/or peak liver concentrations (once daily vs. divided dose).

If in fact reduced liver exposure and peak concentrations could ameliorate androgen-mediated cholesterol reduction, the oral once daily group should have had the greatest effect and conversely the subcutaneous divided dose the least. In fact all dosage paradigms were equally effective in reducing total cholesterol and HDL-C in the female beagle dog at the lowest dose tested (Figure 5.16, Figure 5.17). The dose escalation was stopped at .03 mg/kg/day when the cholesterol reductions approached 50%. These data argue against reducing liver exposure or peak concentration as a means to limit the androgen-mediated serum cholesterol effects.

5.4.4 Conclusions and Future Directions

While target gene regulation was detected in several models, very little correspondence in expression profiles was seen. Anabolic doses in both male and female rats resulted in serum cholesterol reductions and target liver gene regulation but of different genes. Also, in time course analysis, expression changes were only detected concomitantly with serum cholesterol reductions but not before. It is possible that androgen-mediated lipid effects are initiated in the liver non-genomically. This situation is not excluded by the studies performed, though others have evaluated both primary hepatocytes and liver tissue extensively concluding the signal is not hepatic in origin[83].

Perhaps if an even lower dose of SARM2 was used in the female beagle dog study, route and rate effects could have been seen but that dose level would fall well below what was determined to be clinical relevant.

139 A possible complication of the analyses performed is species-specific lipid physiology. Enzymes important in primate HDL-C homeostasis are not expressed in lower mammals, namely cholesterol esterase transfer protein (CETP). CETP induction is consistent with the reductions in total HDL-C as well as HDL particle size seen following androgen treatment[171]. When CETP is trans-genetically over-expressed in mice, which lack endogenous CETP, ApoA1 and HDL-C were reduced with a concomitant reduction in fat mass[172]. Most convincingly, CETP mRNA induction in primate adipose tissue following DHT treatment has been documented[83]. However, CETP cannot stand alone as the androgen-regulated mediator of serum cholesterol reduction.

Neither rat nor dog expresses this protein and they clearly maintain the capacity to lower serum cholesterol in response to androgen. It is likely the mechanism behind androgen mediated HDL-C reductions is species specific.

Reduced liver exposure offers a rationale explanation for the route specific lipid effects of androgen therapy, though the studies presented argue against this as a factor.

As previously mentioned, recent work in cynomoglus monkeys showed rapid DHT- mediated induction in of CETP expression in adipose, where no rapid changes of any lipid homeostasis genes were detected in liver[83]. Like the work in rodents presented here, LIPC induction and APOA2 induction were only detectable after several days of therapy, again suggesting this regulation is the result of changes in lipid homeostasis not the cause. Also, recent characterization of an adipose specific AR-KO mouse with altered lipid profiles suggest AR function in adipose could contribute to the effect in question[173].

140 Another potential explanation is the contribution of estradiol (E2) via aromatization of testosterone (T). Physiological levels of E2 are thought to maintain favorable lipid profiles in women as the onset of menopause, and the accompanying abrogation of E2 production, results in lower HDL-C, higher LDL-C and increases in cardiovascular disease (CVD)[174]. Also, estrogen administration in a post-menopausal population has been shown to raise HDL-C 15%[175]. Studies directly comparing oral androgen therapy to parenteral dosage forms in a randomized, placebo controlled, double-blind trial setting have administered testosterone analogs parenterally that are susceptible to aromatization [76, 154]. Also, in studies using LDL-KO mice, the anti- atherogenic properties of testosterone were partially reversed by co-administration of an aromatase inhibitor[176]. More convincingly, a similar result was achieved in lean healthy young men receiving testosterone enanthate (TE) intramuscularly alone or in conjunction with the oral aromatase inhibitor testacolone[154], arguing the importance of aromatase in the route specific serum cholesterol effect. Oddly, trans-dermal administration of non-aromatizable DHT in older androgen deficient men resulted in lower total and LDL cholesterol, but no effects on HDL-C[177]. And, in a study of healthy older men on a long acting GnRH agonist also receiving varied IM doses of TE, dose dependent reductions in HDL-C were reported as testosterone levels entered the supra-physiological range[178]. These studies point to first, the likelihood of age dependency in estradiol’s ability to reduce androgen-mediated HDL-C effect as the E2 benefit is relegated to mostly young men and second, the potential need for a physiological level of E2 to combat the androgen-mediated HDL-C reductions. Though

E2 levels were not monitored by Bahsin et al, supra-physiological serum testosterone 141 levels are likely accompanied by serum E2 levels well above what was seen in studies where parenteral androgens showed no HDL-C effect. Also the confounding factor of

LH shutdown at all doses by a long acting GnRH agonist complicate suppositions of serum E2 levels in this study. It is likely however, that the LH shutdown following non- aromatizable androgen administration contributes to the HDL-C effect by negatively affecting endogenous estrogen production.

Though all aryl propionamide SARMs tested reduced serum cholesterol in models of varied species, sex, hormonal status, duration of treatment and route of administration, flexibility in the pharmacophore may afford the discovery of a clinically efficacious non- steroidal androgen with no or limited HDL-C effects[26, 34, 45]. The varied dosage forms examined displayed little success but transdermal and oral extended release formulations were only modeled by divided subcutaneous and oral dosing. The body of work on steroidal androgens suggests that a window might exist where efficacious systemic exposure is achieved while limiting liver exposure given proper formulation.

Either way, the opportunity to circumvent SARM mediated HDL-C effects remains in light of the studies presented.

142 5.5. Acknowledgments

The ORX Rat study was executed by Amanda Jones with help from GTx Animal

Resources including; Drs. Jeff Kearbey and Jeetendra Eswaraka, Terry Costello, Matt

Bauler, Stacy Lindsey and Kati Kail. The OVX Rat Study was executed by Dr. Jeff

Kearbey and Deanna Parke with help from GTx Animal Resources. The female beagle dog study was only possible due to the generous after-hours help provided by Dr.

Jeetendra Eswaraka, Terry Costello, Matt Bauler, Stacey Lindsey and Deanna Parke.

143 Primary Human Hepatocyte - Gene Expression 4

* 2

0

-2

* * * -4 LIPC ABCA1

FC vs. Vehicle (24 Hours) (24 Vehicle vs. FC SRB1 -6 APOA1 APOA2

* -8 Vehicle (24 Hrs) 6 Hrs 12 Hrs 24 Hrs 48 Hrs 100 nM DHT *p<.05 Fisher's LSD vs Vehicle

Figure 5.1 Primary Human Hepatocyte Time Course – Gene Expression HDL metabolism/homeostasis genes’ of interest expression in male primary human hepatocytes. Values represented as mean+SD (n=3), as described in the methods section, FC versus vehicle at 24 hours.

144 Primary Rat Hepatocyte - Gene Expression

2.0

LIPC 1.5 ABCA1 * SRB1 APOA1 1.0 APOA2

0.5

0.0

-0.5 FC vs. Vehicle vs. FC

-1.0

-1.5 *p<.05 Fisher's LSD vs. corresponding Vehicle -2.0

eh M2 eh M2 eh M2 V AR V AR V AR S S S nM nM nM 00 00 00 1 1 1

24 hours 48 hours 72 hours

Figure 5.2 Primary Rat Hepatocyte Gene Expression – Time Course HDL metabolism/homeostasis genes’ of interest expression in male primary rat hepatocytes. Values represented as mean+SD (n=3), as described in the methods section, FC versus corresponding vehicle.

145 HepG2 - AR Transfection 6 LIPC ABCA1 SRB1 4 APOA1 APOA2

2

0 FC vs. Vehicle vs. FC

-2 *

*p<.05 Fisher's LSD vs. Vehicle -4 e 2 e 2 e 2 icl M icl M icl M eh AR eh AR eh AR V S V S V S uM uM uM 1 1 1 24 hours 48 hours 72 hours

Figure 5.3 HepG2 AR Transfection – Gene Expression HDL metabolism/homeostasis genes’ of interest expression in HepG2 cell transiently transfected with human AR. Values represented as mean+SD (n=3), as described in the methods section, FC versus corresponding vehicle.

146 ORX Rat Time Course Study

10

0

-10

-20 from ORX Day 0 (mg/dL) from Day ORX

-30 * o + -40 Vehicle + SARM2 + -50 SARM3 SARM4 mean + SE -60 Total Plasma Change C - 0 5 10 15 20 25 30 Days + p<.05 All SARMS vs ORX Control * p<.05 SARM3 and SARM4 vs ORX Control o p<.05 SARM3 only vs ORX Control

Figure 5.4 Total Plasma Cholesterol – ORX Rat Time Course Total plasma cholesterol as determined by colorimetric/enzymatic assay represented as mean + SE (n=5).

147 ORX Rat Time Course - Prostate Weights 140 ORX SARM2 120 SARM3 SARM4 100

80

60

% of Intacct Control 40

20

0 Day 3 Day 7 Day 10 Day 14 Day 21 Day 28

Figure 5.5 Prostate Weights – ORX Rat Time Course Values are normalized to individual animal bodyweights and then represented as percentage of mean body weight normalized intact control tissue weight at Day 28 (mean+SD, n=5).

148 ORX Rat Time Course - Seminal Vesicle Weights 140 ORX 120 SARM2 SARM3 SARM4 100

80

60

% of Intacct Control 40

20

0 Day 3 Day 7 Day 10 Day 14 Day 21 Day 28

Figure 5.6 Seminal Vesicle Weights – ORX Rat Time Course Values are normalized to individual animal bodyweights and then represented as percentage of mean body weight normalized intact control tissue weight at Day 28 (mean+SD, n=5).

149 ORX Rat Time Course - Levator Ani Weights 140 ORX SARM2 120 SARM3 SARM4 100

80

60

% of Intacct Control 40

20

0 Day 3 Day 7 Day 10 Day 14 Day 21 Day 28

Figure 5.7 Levator Ani Weights – ORX Rat Time Course Values are normalized to individual animal bodyweights and then represented as percentage of mean body weight normalized intact control tissue weight at Day 28 (mean+SD, n=5).

150 ORX Rat Time Course - 28 day Tissue Weights 160 ORX 140 O II SARM2 OO SARM3 O SARM4 120 O O Intact O O 100 O

I 80 O I I O 60 O I % of Control Intact 40

20 I I 0 e s i s s at le An eu u st sic r ol mi ro e ato S ne P l V ev oc na L str mi a Se G o - p<.05 vs. ORX, Fisher's LSD Test I - p<.05 vs. Intact, Fisher's LSD Test

Figure 5.8 28 Day Tissue Comparisons – ORX Rat Time Course Values are normalized to individual animal bodyweights and then represented as percentage of mean body weight normalized intact control tissue weight at Day 28 (mean+SD, n=5).

151 ORX Rat Time Course - Liver Gene Expression 8

6

4

2

0

-2 FC vs ORX Day 0

-4 LIPC ABCA1 SRB1 -6 APOA1 APOA2 -8 Day 0 Day 3 Day 7 Day 10 Day 14 Day 21 Day 28

ORX Control Animals

Figure 5.9 ORX Rat Time Course – ORX Animal Liver Gene Expression HDL metabolism/homeostasis genes’ of interest expression in liver tissue of vehicle treated ORX rats. Values represented as mean+SD (n=5), as described in the methods section, FC versus corresponding vehicle.

152 ORX Rat Time Course - Liver Gene Expression 15 * LIPC ABCA1 SRB1 10 APOA1 APOA2 *

5 * * ** * FC vs Day 3 0

* * * -5

Day 3 Day 7 Day 10 Day 14 Day 21 Day 28 SARM2 1 mg/day * p<.05 Fisher's LSD vs Day 3

Figure 5.10 ORX Rat Time Course – SARM2 Treated Liver Gene Expression HDL metabolism/homeostasis genes’ of interest expression in liver tissue of SARM2 treated ORX rats. Values represented as mean+SD (n=3), as described in the methods section, FC versus SARM2 treated at Day 3.

153 ORX Rat Time Course - Liver Gene Expression 4

2

0 *

-2 FC vs Day 3 -4 LIPC ABCA1 SRB1 -6 APOA1 APOA2

-8 Day 3 Day 7 Day 10 Day 14 Day 21 Day 28 SARM3 1 mg/day

Figure 5.11 ORX Rat Time Course – SARM3 Treated Liver Gene Expression HDL metabolism/homeostasis genes’ of interest expression in liver tissue of SARM3 treated ORX rats. Values represented as mean+SD (n=3), as described in the methods section, FC versus SARM3 treated at Day 3.

154 ORX Rat Time Course - Liver Gene Expression 4 * * * LIPC * * 3 ABCA1 * SRB1 2 APOA1 * APOA2 * * * 1

0

FC vs Day 3 -1

-2

-3

-4 Day 3 Day 7 Day 10 Day 14 Day 21 Day 28 SARM4 1 mg/day * p<.05 Fisher's LSD vs Day 3

Figure 5.12 ORX Rat Time Course – SARM4 Treated Liver Gene Expression HDL metabolism/homeostasis genes’ of interest expression in liver tissue of SARM4 treated ORX rats. Values represented as mean+SD (n=3), as described in the methods section, FC versus SARM4 treated at Day 3.

155 OVX Rat Extended Treatment 160

140 *

120 & & 100 * * 80

60

Total Cholesterol (mg/dl) Total Cholesterol 40

20

0 e 2 4 e 2 4 icl M M icl M M eh AR AR eh AR AR V S S V S S Intact OVX * p<.05 vs. Intact vehicle (Fisher's LSD) & p<.001 vs. OVX vehicle (Fisher's LSD)

Figure 5.13 Total Serum Cholesterol – OVX Rat Extended Treatment Total serum cholesterol as determined by enzymatic assay. Each compound was administered orally for 42 days at 1 mg/day. Values represented as mean+SD(n=10).

156 OVX Rat Extended Treament 40 mean+SE

& & 30

# 20

10 Percent bone volume (BV/TV) 2D µCT

0 e 2 4 e 2 4 icl M M icl M M eh AR AR eh AR AR V S S V S S

Intact OVX & p<.05 vs. OVX vehicle (Fisher's LSD) # p<.05 vs. Intact Vehicle (Fisher's LSD)

Figure 5.14 Percent Bone Volume – OVX Rat Extended Treatment BV/TV as determined by 2D µCT. Each compound was administered orally for 6 weeks at 1 mg/day. Values represented as mean+SE (n=10).

157 OVX Rat Extended Treatment - Liver Gene Expression 3

2

1

0

-1

-2 LIPC ABCA1

FC vs. Vehicle (Intact or OVX) or (Intact Vehicle vs. FC * * -3 SRB1 APOA1 APOA2 -4 e 2 4 e 2 4 icl M M icl M M eh R R h V SA SA Ve SAR SAR

Intact OVX * p<.05 (Fisher's LSD) compared to Intact Vehicle

Figure 5.15 OVX Rat Extended Treatment – Liver Gene Expression

HDL metabolism/homeostasis genes’ of interest expression in OVX rat liver tissue. Values represented as mean+SD (n=8), as described in the methods section, FC versus vehicle of corresponding gonadal status.

158 Intact Female Beagle Dog Study 20

0

-20

-40 Oral QD

Total C - %Change from BL C - %Change from Total Oral Q8 S.C. QD S.C. Q8 mean+SE -60 012345678

.01 mg/kg .03 mg/kg

Weeks

Figure 5.16 Total Serum Cholesterol – Intact Female Beagle Dog Total serum cholesterol as determined by enzymatic assay (n=5). No differences were detected between groups at any day.

159 Intact Female Beagle Dog Study 20

0

-20

-40 Oral QD Oral Q8 HDL-C - %Change from BL S.C. QD S.C. Q8 mean+SE -60 012345678

.01 mg/kg .03 mg/kg Weeks

Figure 5.17 Serum HDL-C – Intact Female Beagle Dog Absolute values of HDL calculated using percentage of total serum lipids determined HDL by lipoprotein electrophoresis and total serum cholesterol. Values are represented as a percentage change from average baseline (n=5).

160 Gene Name Code RefSeq Location Strand Chr. PMW Sequence LIPC HMR NM_000236 -3145 Minus 15 0.91 GATACATGTTGTCCC ABCA1 HM NM_005502 -6017 Minus 9 0.923 AGAGCTATCTGTACT ABCA1 HR NM_005502 -7276 Minus 9 0.927 TGAACTTTGTGTGCA ABCA1 HR NM_005502 -7624 Plus 9 0.907 AGGACACAATGTAGC SRB1 - NM_001082959 - - 12 - - APOA1 HM NM_000039 -8639 Plus 11 0.911 GGGACAGCGTGACCT APOA1 HM NM_000039 -8639 Minus 11 0.921 GGGACAGCGTGACCT APOA2 - NM_001643 - - 1 - -

SHP-1 HMR NM_021969 -8183 Plus 1 0.926 TGGACACAGTGTTCC SHP-1 HMR NM_021969 -8183 Minus 1 0.968 TGGACACAGTGTTCC SHP-1 HMR NM_021969 -4725 Minus 1 0.931 ATCACTGTCTGTGCT SHP-1 HR NM_021969 -2892 Plus 1 0.906 GGTTCAAGCGGTTCT SHP-1 HR NM_021969 1256 Plus 1 0.965 GGGACAAGAAGTTCC CYP7A1 - NM_000780 - - 8 - - STE HM NM_005420 -1945 Plus 4 0.925 GGAGCATGATGTTCT STE HM NM_005420 -1945 Minus 4 0.92 GGAGCATGATGTTCT STE HR NM_005420 96 Plus 4 0.9 GGCACATATAGTGAG SULT2A1 HM NM_003167 -9414 Minus 19 0.942 AGAACTGCTTGAACT

Table 5.1 Conserved AREs – HDL Metabolism/Homeostasis and Control Genes

161 Drug Treatment Time Gonadal Status Drug Treatment Time Gonadal Status SARM 2,3,4 3 days ORX Vehicle 0 days Intact SARM 2,3,4 7 days ORX Vehicle 0 days ORX SARM 2,3,4 10 days ORX Vehicle 3 days ORX SARM 2,3,4 14 days ORX Vehicle 7 days ORX SARM 2,3,4 21 days ORX Vehicle 10 days ORX SARM 2,3,4 28 days ORX Vehicle 14 days ORX Vehicle 21 days ORX Vehicle 28 days ORX Vehicle 28 days Intact

Table 5.2 Orchidectomized Rat Time Course Study – Treatment Groups All doses were 1 mg/day and administered via subcutaneous injection.

162 Drug Gonadal Status Dose Time Vehicle Intact - 42 days SARM2 Intact 1 mg/day 42 days SARM4 Intact 1 mg/day 42 days Vehicle OVX - 42 days SARM2 OVX 1 mg/day 42 days SARM4 OVX 1 mg/day 42 days

Table 5.3 Ovariectomized Rat Time Course Study – Treatment Groups

163 Dose (Day 1-14) Dose (Day 15-56) Group 7:00 AM 3:00 PM 11:00 PM mg/kg mg/kg Oral QD 0.01 0.03 Dose Vehicle Vehicle Oral Q8 0.0033 0.01 Dose Dose Dose Subcutaneus QD 0.01 0.03 Dose Vehicle Vehicle Subcutaneus Q8 0.0033 0.01 Dose Dose Dose

Table 5.4 Female Beagle Dog Study – Treatment Groups All animals were treated with SARM2.

164 CHAPTER 6

6. SUMMARY AND CONCLUSIONS

The main objectives of these studies included: 1) Determining if differences in potency between SARMs and DHT can explain SARM pharmacology in prostate; 2)

Identifying ligand-specific AR regulation as well as direct AR transcriptional targets; 3)

Determining if ALT-1 and ALT-2 are AR regulated; 4) Determining the mechanism by which androgens reduce serum HDL-C.

The specific investigations herein showed that:

1) Potency alone cannot explain SARM mechanism. At extended treatment times and high concentrations, SARM1 superseded DHT-mediated LNCaP growth. In LNCaP, the transcriptional capacities of these ligands were identical, but their maximal response, in terms of cell growth, were varied at 72 hours. Gene expression profiles in LNCaP between DHT and SARM1 were largely overlapping but distinct at concentrations of ligand equi-efficacious in terms of cells growth. AR promoter binding profiles in LNCaP between SARM2 and DHT were largely overlapping but distinct at ligand concentrations saturating receptor binding and maximal in terms of transcriptional activation. Finally, the apparent maximal response of SARM3 in prostate at 3 days is less than both intact

165 and DHT-treated animals. The combined interpretation of these data is that a qualitative difference exists between aryl propionamide SARM and DHT action in prostate.

2) Ligand specific regulation occurs as a function of both time and concentration.

The equi-efficacious conditions of 1 nM ligand and 24 hour treatment chosen for gene expression analyses resulted in very similar profiles between SARM1 and DHT. When analyses were expanded to four time points and three concentrations, clear differences were evident. AR promoter occupancy at two hours following ligand stimulation proved a poor predictor of transcriptional regulation. Of all the well characterized genes shown to recruit the AR in response to either DHT, SARM2 or both, less then 10% proved to be regulated. In these studies, acute AR promoter release was at least as predictive of regulation as occupancy. Conserved proximal promoter AREs are not requisite in AR recruitment or transcriptional regulation. When comparing genes regulated by DHT and

SARM1, only 37% contained a bioinformatically determined conserved ARE. When evaluating promoters biologically determined to bind the AR, only 50% contained conserved AREs. Given these relaxed search criteria, the same percentage of conserved

ARE positive promoters were found when all human promoters were assayed. ALT-2, a bone fide direct target of AR also demonstrating reliable mRNA regulation, showed neither AR nor RNA Pol II recruitment to any of its conserved proximal promoter AREs.

Several AR regulated transcripts, and direct targets were determined in LNCaP, though their causality in SARM tissue selectivity requires further analyses.

166 3) ALT-2 is an androgen regulated gene. Many studies with different androgens showed reliable induction of ALT-2 in LNCaP cells. ALT-2 was also repressed in androgen dependent tissues following castration in an effect that was reversible by androgen administration. ALT-1 showed weak effects in vitro that did not reproduce in vivo. A direct link to elevated serum ALT levels was not made. However, AR induction of ALT-2 in muscle and prostate offer an explanation other than xenobiotic-induced hepatotoxicity.

4) The role of liver tissue in androgen-mediated serum lipid effects remains unknown. No molecular target was reliably regulated at the mRNA level by androgens.

Numerous genes were evaluated in a plethora of systems to no avail. Androgen-mediated reductions in serum HDL-C appear linked to anabolic activity. Lipid effects were seen in several rodent models of mixed gender and gonadal status with concurrent muscle and bone anabolism. Fully efficacious, and therefore anabolic, doses were selected as all studies were mechanistic in nature and not intended to determine “cholesterol effect free” levels of androgen. However, in time course analyses, cholesterol effects worsened over time, trending with increased anabolism. Varied route of administration and divided dose studies in female beagle also argue against the importance of liver in this effect as all dosing regimens showed similarly large reductions in serum cholesterol following androgen treatment.

167 One byproduct of these studies was showing the importance of the T877A mutation in dictating aryl propionamide SARM response in LNCaP. Though this mutation created a convenient model system, correcting for potency discrepancies between many aryl propionamide SARMs and DHT, use of this model in SARM mechanistic work carries a heavy caveat- it simply does not reflect the wild type situation. Some believe ligand specific AR conformation drives SARM mechanism in prostate [92]. T877A not only affects potency but receptor conformation as well, changing the types of co-activator interactions possible upon ligand binding[179]. These studies show great care should be used in drawing conclusions based on SARM mechanistic work performed in LNCaP cells.

The discovery of tissue selective androgens greatly expanded the therapeutic opportunities for anabolic agents. A more complete understanding of the biology underlying SARM selectivity will undoubtedly lead to enhanced SARM development.

Evaluation of non-genomic actions and complex co-activator interactions are called for as potency alone cannot explain aryl propionamide action.

Deviation from a steroidal pharmacophore in the development AR agonists resulted in numerous advantages over existing androgens. Regardless of the developmental challenge, flexibly in the chemophore of efficacious non-steroidal AR agonists offers hope towards new SARM therapies with improved activity and reduced side effects.

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185 APPENDICES

186

7. APPENDIX A

SUPPLEMENTS RELEVANT TO CHAPTER 2

187 Gene Expression Validation 12

10

8

6

4

Fold Change qRT-PCR y=2.32x-1.042

2 r ²=0.9794

0 012345

Fold Change cDNA Array

Figure A.1 qRT-PCR Validation of cDNA Microarray Results LNCap cells were cultured exactly as in the cDNA microarray experiment and re-treated. 8 genes corresponding to different types of regulation were (Figure 2.2) assayed using traditional qRT-PCR. (●) DHT vs. Control. (●) SARM1 vs. Control.

188 Gene Information Species Region Position Score Sequence Predicted protein KIAA0146 Human Enhancer -4172 0.926 A G A G C TtggTG T T CC Abbreviation KIAA0146 Mouse Enhancer -4006 0.900 C C A A C AgaaTG T A CC Human Accession U00948 Mouse Accession BC046558 Human Enhancer -4398 0.946 A A AACAattT G T G C T 24 hr DHT up 1.72x Mouse Enhancer -4006 0.900 C C AACAgaaT G T A C C 24 hr SARM up 2.3x Human Promoter -585 0.910 A GAACActtGGTATT Mouse Promoter -266 0.902 T GAACTgccTGGGCC ATPase family Human Enhancer -3992 0.923 AGAGCT gtcTGT A CT Abbreviation ATAD2 Mouse Enhancer -3825 0.917 AGAGCA gttAGT G CT Human Accession BC019909 Mouse Accession BC032211 24 hr DHT up 1.94x 24 hr SARM up 2.04x Bone marrow protein BM039 Human Promoter -229 0.961 G G CACAgtgA G T C CA Abbreviation BM039 Mouse Promoter -70 0.905 A G GGCAgaaA G T T CA Human Accession BC008972 Mouse Accession BC058243 Human Promoter -229 0.961 G G C ACAgtgAGT C C A 24 hr DHT up 1.99x Mouse Promoter -189 0.918 T A T ACTcctTGT G C T 24 hr SARM up 2.68x RAS oncogene family Human Enhancer -2383 0.909 A GAACCacaT G T A C A Abbreviation RAB27A Mouse Enhancer -2429 0.936 A ACACTgacT G T T C T Human Accession U57094 Mouse Accession BC009656 24 hr DHT up 1.89x 24 hr SARM up 2.47x FK506 binding protein 5 Human Enhancer -3839 0.917 G GAG CTc c g T GTTCT Abbreviation FKBP5 Mouse Enhancer -4116 0.927 T GAA CTg c t T GACCT Human Accession X97300 Mouse Accession U36220 Human Enhancer -2752 0.906 T C T ACTtcaTGTTCC 24 hr DHT up 1.96x Mouse Enhancer -2574 0.905 A G A ACAcacAGAGCC 24 hr SARM up 3.18x H+/peptide transporter Human Intron 1 454 0.907 A G A ACTgtaTG T GAT Abbreviation SLC15A2 Mouse Intron 1 733 0.907 G T T ACTaagTG T CCC Human Accession S78203 Mouse Accession BC051199 Human Intron 1 454 0.907 AGAACTgtgTG TGAT 24 hr DHT up 1.48x Mouse Intron 1 615 0.907 AGAACTgtgTG TGAT 24 hr SARM up 2.01x Human Intron 1 570 0.916 GTTACTat gTG TTCC Mouse Intron 1 733 0.907 GTTACTaa gTG TCCC

Human Intron 1 570 0.916 G T T ACTa ttTG T TCC Mouse Intron 1 615 0.907 A G A ACTg ttTG T GAT

Table A.1 Conserved AREs in Genes 2x Up-Regulated in SARM Only Blue and yellow shading indicate the invariant C and G bases, respectively, making up the PWM core. Red shading indicates identity between mouse and human whereas green shading indicates either conserved purine or pyrimidine bases. Positions are reported relative to TSS with negative oriented upstream. Locations -1 to -2000 were designated promoter and -2001 to -5000 as enhancer.

189 Gene InformationSpecies Region Position Score Sequence Snail homolog 2 Human Intron 2 1679 0.908 AGGGCAcaaAG TGCT Abbreviation SNAI2, SLUG Mouse Intron 2 1427 0.985 AGAACAgcaAG TGCT Human Accession BC014890 Mouse Accession U97059 24 hr DHT up 2.34x 24 hr SARM up 8.96x Kallikrein 2, prostatic Human Promoter -182 0.978 G G AAC A g caAGT G C T Abbreviation KLK2 Mouse Exon 1 90 0.914 A G CTC T g tcTGT T C C Human Accession S39329 Mouse Accession X04574 24 hr DHT up 2.26x 24 hr SARM up 3.15x Ribonucleotide reductase M2 Human Enhancer -3176 0.926 T T A ACAgagT G T C CT Abbreviation RRM2 Mouse Enhancer -3181 0.938 A A C ACAggaT G T T CT Human Accession X59618 Mouse Accession M14223 24 hr DHT up 2.13x 24 hr SARM up 2.00x Glycine N-methyltransferase Human Intron 1 ** 211 0.912 G G C ACT g g gTGAGCC Abbreviation GNMT Mouse Exon 1 17 0.958 T G G ACA g c gTGTACC Human Accession BC032627 Mouse Accession D89664 Human Exon 1 17 0.958 TGGACAgcgTG TACC 24 hr DHT up 2.12x Mouse Exon 1 17 0.958 TGGACAgcgTG TACC 24 hr SARM up 2.06x Catenin Human Enhancer -4200 0.925 G G AACAgac CGT A C G Abbreviation CTNNAL1 Mouse Enhancer -3961 0.922 C T AACAagc TGT G C T Human Accession U97067 Mouse Accession BC050070 Human Promoter -1773 0.910 A A TACCct c T G T T C T 24 hr DHT up 2.77x Mouse Enhancer -2031 0.916 A C TACAtt t T G T C C C 24 hr SARM up 3.97x Human Promoter -1773 0.910 A A T A C CctcTG TTCT Mouse Promoter -1426 0.911 G G T G C AtacTG TTCT Serine transmembrane protease Human Enhancer -4430 0.906 AGAA CAtctCGT TGT Abbreviation TMPRSS2 Mouse Enhancer -4637 0.932 AGAT CAgaaTGT GCT Human Accession U75329 Mouse Accession BC054348 Human Enhancer -4657 0.923 G G GACAatgAGT CAT 24 hr DHT up 2.76x Mouse Enhancer -4637 0.932 A G ATCAgaaTGT GCT 24 hr SARM up 3.64x Hydroxyprostaglandin dehydrogenase 15 Human Intron 2 1634 0.967 G G A ACAt a t T G T A C C Abbreviation HPGD Mouse Intron 2 1948 0.938 A G C ACAa a a T G T T C A Human Accession X82460 Mouse Accession U44389 24 hr DHT up 2.03x 24 hr SARM up 3.20x

Table A.2 Conserved AREs in Genes 2x Up-Regulated in SARM and DHT Blue and yellow shading indicate the invariant C and G bases, respectively, making up the PWM core. Red shading indicates identity between mouse and human whereas green shading indicates either conserved purine or pyrimidine bases. Positions are reported relative to TSS with negative oriented upstream. Locations -1 to -2000 were designated promoter and -2001 to -5000 as enhancer.

190 Gene Information Species Region Position Score Sequence Absent in melanoma 1 Human Intron 1 1612 0.913 A G TTCAagcGGT TCT Abbreviation AIM1 Mouse Intron 1 1793 0.911 G G CACAtcaAGT CTC Human Accession BC062788 Mouse Accession AF397913 24 hr DHT up 2.30x 24 hr SARM up 1.93x

Table A.3 Conserved AREs in Genes 2x Up-Regulated in DHT Only Blue and yellow shading indicate the invariant C and G bases, respectively, making up the PWM core. Red shading indicates identity between mouse and human whereas green shading indicates either conserved purine or pyrimidine bases. Positions are reported relative to TSS with negative oriented upstream. Locations -1 to -2000 were designated promoter and -2001 to -5000 as enhancer.

191 Gene InformationSpecies Region Position Score Sequence Transmembrane protein 38B Human Enhancer -2999 0.936 GGA ACTcaa TGAT C T Abbreviation TMEM38B Mouse Enhancer -3266 0.903 GGG ACAtca GGTT T T Human Accession BC031938 Mouse Accession BC011072 24 hr DHT down 2.03x 24 hr SARM down 2.59x

Table A.4 Conserved AREs in Genes 2x Down-Regulated in DHT and SARM Blue and yellow shading indicate the invariant C and G bases, respectively, making up the PWM core. Red shading indicates identity between mouse and human whereas green shading indicates either conserved purine or pyrimidine bases. Positions are reported relative to TSS with negative oriented upstream. Locations -1 to -2000 were designated promoter and -2001 to -5000 as enhancer.

192 Gene InformationSpecies Region Position Score Sequence Pre-B-cell leukemia TF 1 Human Enhancer -3779 0.909 T G T ACAatc AGTGC C Abbreviation PBX1 Mouse Enhancer -4094 0.917 A G C ACTtgc TGCTC T Human Accession M86546 Mouse Accession BC057879 24 hr DHT down 1.35x 24 hr SARM down 2.00x ATP-binding cassette (A) Human Enhancer -3386 0.923 T G GAC TctaT G T T C A Abbreviation ABCA1 Mouse Enhancer -3041 0.911 A G TGC AgctT G T C C C Human Accession AK130814 Mouse Accession X75926 Human Enhancer -2945 0.920 T G ATCAt t g T G T T C T 24 hr DHT down 1.54x Mouse Enhancer -3041 0.911 A G TGCAg c t T G T C C C 24 hr SARM down 2.01x Plasticity related gene 3 Human Enhancer -4407 0.934 AGT ACActcTGAGCC Abbreviation PRG-3 Mouse Enhancer -4477 0.932 AGA ACAaagAGTTTA Human Accession BC022465 Mouse Accession AY345342 24 hr DHT down 1.41x 24 hr SARM down 2.35x Phospholipase C, beta 4 Human Enhancer -3982 0.907 AGGTC cccgAGTGC G Abbreviation PLCB4 Mouse Enhancer -3748 0.907 AGCAC TaatTGGAC T Human Accession L41349 Mouse Accession U50959 24 hr DHT down 1.49x 24 hr SARM down 2.17x Non-muscle Myosin HC Human Promoter -1697 0.918 TGTACTcc aTGTGCA Abbreviation MYH10 Mouse Enhancer -2056 0.925 TGTACAgc gAGTGCT Human Accession U34304 Mouse Accession BC059863 Human Enhancer -2337 0.905 TGA ACTTC CTGCCCT 24 hr DHT down 1.63x Mouse Enhancer -2056 0.925 TGT ACAGC GAGTGCT 24 hr SARM down 2.03x Pellino homolog 2 Human Enhancer -2453 0.905 GGT ACAtagAGT CTA Abbreviation PELI2 Mouse Enhancer -2265 0.949 GGA ACAattGGT GCT Human Accession BC009476 Mouse Accession BC027062 24 hr DHT down 1.63x 24 hr SARM down 2.03x Sex determining region Y-box 11 Human Enhancer -2274 0.906 G G T TCAcctTGT G C T Abbreviation SOX11 Mouse Enhancer -2092 0.903 A G C TCAcaaGGT T C C Human Accession U23752 Mouse Accession AF009414 24 hr DHT down 1.81x 24 hr SARM down 2.51x

Table A.5 Conserved AREs in Genes 2x Down-Regulated in SARM Only Blue and yellow shading indicate the invariant C and G bases, respectively, making up the PWM core. Red shading indicates identity between mouse and human whereas green shading indicates either conserved purine or pyrimidine bases. Positions are reported relative to TSS with negative oriented upstream. Locations -1 to -2000 were designated promoter and -2001 to -5000 as enhancer. (Continued on next page)

193 Gene InformationSpecies Region Position Score Sequence Midline 1 (Opitz/BBB syndrome) Human Promoter 1204 0.957 C G A ACTttc TGT C C C Abbreviation MID1 Mouse Promoter 1350 0.976 A G G ACAgcc AGT G C T Human Accession Y13667 Mouse Accession BC053704 24 hr DHT down 1.87x 24 hr SARM down 3.19x Nucleobindin 2 Human Enhancer -3724 0.918 TGC ACActgT GACCT Abbreviation NUCB2 Mouse Enhancer -3882 0.900 TGA ACAcaCT GCCCC Human Accession X76732 Mouse Accession M96823 Human Promoter -1283 0.961 G GCACA cagAGTCC A 24 hr DHT down 1.88x Mouse Promoter -994 0.917 A GCACT catTGCTC T 24 hr SARM down 2.31x Human Enhancer -2300 0.906 AGA ACCactT GAACC Mouse Enhancer -2572 0.910 AGC ACTgacT GCTCC Ras-like without CAAX 2 Human Enhancer -4252 0.923 A G A ACC cac T GATC T Abbreviation RIT2 Mouse Enhancer -4049 0.959 G G G ACA cag T GTCC C Human Accession Y07565 Mouse Accession U71205 24 hr DHT down 1.84x 24 hr SARM down 2.24x Cbp/p300-interacting transactivator Human Exon 2 1429 0.910 CGGACTtcgTG TGCA Abbreviation CITED2 Mouse Exon 2 1485 0.910 CGGACTtcgTG TGCA Human Accession U65093 Mouse Accession U86445 Human Enhancer -2308 0.901 A G G ACAtt cAGTGGA 24 hr DHT down 1.35x Mouse Enhancer -2542 0.929 G G A ACTct tTGATCC 24 hr SARM down 2.04x Suppression of tumorigenicity 7 Human Enhancer -4726 0.978 AGCA C AattT G T A CT Abbreviation ST7 Mouse Enhancer -4903 0.912 AGCT C TggcT G T C CT Human Accession BC030954 Mouse Accession BC060630 Human Enhancer -4621 0.975 T G GAC Aag g T G T T CT 24 hr DHT down 1.35x Mouse Enhancer -4903 0.912 A G CTC Tgg c T G T C CT 24 hr SARM down 2.04x Human Enhancer -4899 0.904 AGCTCAccaCGT G CT Mouse Enhancer -4903 0.912 AGCTCTggcTGT C CT Hypothetical protein FLJ11155 Human Enhancer -2945 0.988 AGA ACAtgcA G T ACT Abbreviation FLJ11155 Mouse Enhancer -2794 0.922 AGT ACAtaaA G T TAA Human Accession BC060875 Mouse Accession BC018493 24 hr DHT down 1.42x 24 hr SARM down 2.11x Tetraspanin 8 Human Intron 1 181 0.922 TCAACTtctTG TTCT Abbreviation TM4SF3 Mouse Intron 1 149 0.922 TCAACTtctTG TTCT Human Accession M35252 Mouse Accession BC034198 Human Exon 2 266 0.940 A G AACAgacT G T A C A 24 hr DHT down 1.93x Mouse Intron 1 149 0.922 T C AACTtctT G T T C T 24 hr SARM down 2.65x

Table A.6 (Continued) Conserved AREs in Genes 2x down-regulated in SARM only

194 8. APPENDIX B

SUPPLEMENTS RELEVANT TO CHAPTER 3

195 MA plots: LNCaP Chip-DSL

Vehicle

DHT

SARM2

Figure B.1 MA Plots of LNCaP ChIP-DSL Experiment MA plots showing labeled oligo intensities across all ChIP-DSL replicates. Red (AR- ChIP) probes located in the tope right of each plot are putative AR binders. The spread of normalized intensities are highly similar between replicates and treatments indicating reproducible hybrization.

196 Gene Name Accession Description ARE Taqman Probe Regulated Carbamoyl-phosphate synthetase 2, aspartate CAD NM_004341 YES Hs00983188_m1 NO transcarbamylase, and dihydroorotase ENm009 TL_000353 - - - - ENm009 TL_000056 - - - - ENm009 TL_000348 - - - - EOMES NM_005442 homolog (Xenopus laevis) NO Hs00172872_m1 NO Coagulation factor IX (plasma thromboplastic F9 NM_000133 YES Hs00609168_m1 NO component, Christmas disease, hemophilia B) FBXL11 NM_012308 F-box and leucine-rich repeat protein 11 NO Hs00367034_m1 NO Fasciculation and elongation protein zeta 1 FEZ1 NM_005103 YES Hs00192714_m1 NO (zygin I) FGF18 NM_003862 Fibroblast growth factor 18 NO Hs00818572_m1 NO Glycosylphosphatidylinositol specific GPLD1 NM_177483 YES Hs00412832_m1 YES phospholipase D1 GREB1_145 TL_000605 - - - - GREB1_234 TL_000623 - - - - JUND NM_005354 Jun D proto-oncogene NO Hs00534289_m1 NO KAI1_30 TL_000240 - - - Potassium voltage-gated channel, subfamily H KCNH6 NM_030779 NO Hs00229215_m1 NO (eag-related), member 6 Centrobin, centrosomal BRCA2 interacting LIP8 NM_053051 NO Hs00378791_m1 NO protein LOC170242 XM_093201 - - - - LOC220416 XM_166971 - - - - LOC253883 XM_171154 - - - - LOC286017 XM_208371 - - - - MIR16 NM_016641 Glycerophosphodiester phosphodiesterase 1 NO Hs00213347_m1 NO Myxovirus (influenza virus) resistance 1, MX1 NM_002462 YES Hs00182073_m1 NO interferon-inducible protein p78 (mouse) PANK3 NM_024594 Pantothenate kinase 3 YES Hs00388175_m1 NO RARB_359 TL_000228 Data not found - - - RXRA NM_002957 , alpha NO Hs01067640_m1 NO SFRP4 NM_003014 Secreted frizzled-related protein 4 NO Hs00180066_m1 NO SKIIP NM_012245 SNW domain containing 1 YES Hs00273351_m1 NO SRRM2 NM_016333 Serine/arginine repetitive matrix 2 - Hs00249492_m1 NO TAC1 NM_003182 Tachykinin, precursor 1 YES Hs00243227_m1 NO TEF NM_003216 Thyrotrophic embryonic factor NO Hs00162657_m1 NO Transient receptor potential cation channel, TRPM3 NM_020952 NO Hs00326297_m1 NO subfamily M, member 3 Ubiquitin-conjugating enzyme E2D 3 (UBC4/5 UBE2D3 NM_181893 YES Hs00704312_m1 NO homolog, yeast)

Table B.1 ChIP-DSL AR Binding Promoters – Group A Tabulation of genes in the AR ON – DHT only group. If gene expression analyses were performed, a Taqman Probe number is listed along with detected regulation. If the gene had sufficient information to be searched for AREs, the result of the search is listed.

197 Gene Name Accession Description ARE Taqman Probe Regulated IHPK1 NM_153273 Inositol hexaphosphate kinase 1 NO Hs00384812_m1 NO pbr322_141_50 NC_050311 - - - - RNA binding protein, autoantigenic (hnRNP- RALY NM_007367 YES Hs00247124_m1 NO associated with lethal yellow homolog (mouse)) RASGRF1 NM_002891 Ras protein-specific guanine nucleotide-releasing NO Hs00182314_m1 NO SLAMF8 NM_020125 SLAM family member 8 YES Hs00252301_m1 NO UAP1 NM_003115 UDP-N-acteylglucosamine pyrophosphorylase 1 NO Hs00268394_m1 NO

Table B.2 ChIP-DSL AR Binding Promoters – Group B Tabulation of genes in the AR ON – SARM2 only group. If gene expression analyses were performed, a Taqman Probe number is listed along with detected regulation. If the gene had sufficient information to be searched for AREs, the result of the search is listed.

198 Gene Name Accession Description ARE Taqman Probe Regulated AD-017 NM_152932 Glycosyltransferase 8 domain containing 1 YES - - ARHGEF3 NM_019555 Rho guanine nucleotide exchange factor (GEF) 3YES- - ASXL2 NM_018263 Additional sex combs like 2 (Drosophila) - - - Bile acid Coenzyme A: amino acid N-acyltransferase BAAT NM_001701 YES - - (glycine N-choloyltransferase) C20orf27 NM_017874 - NO - - CASP6 NM_001226 Caspase 6, apoptosis-related cysteine peptidase NO Hs00154246_m1 NO CHERP NM_006387 Calcium homeostasis endoplasmic reticulum protein NO - - COMMD7 NM_053041 COMM domain containing 7YES-- COXVIB2 NM_144613 Cytochrome c oxidase subunit VIb polypeptide 2 (testis) - - - CTHRC1 NM_138455 Collagen triple helix repeat containing 1YES- - ENTPD2 NM_001246 Ectonucleoside triphosphate diphosphohydrolase 2 YES - - FHR-4 NM_006684 Complement factor H-related 4 - - - FLJ10781 NM_018215 PNMA-like 1 YES - - FMO4 NM_002022 Flavin containing monooxygenase 4 NO - - GPSM3 NM_022107 G-protein signaling modulator 3 (AGS3-like, C. elegans) YES - - HEL308 NM_133636 DNA helicase HEL308 YES - - HNRPL NM_001533 Heterogeneous nuclear ribonucleoprotein L YES - - KIAA0586 NM_014749 - YES - - KIAA1117 NM_015018 Dopey family member 1 NO - - KIRREL3 NM_032531 Kin of IRRE like 3 (Drosophila) YES - - LAMB1 NM_002291 Laminin, beta 1 NO - - LDHB NM_002300 Lactate dehydrogenase B - - - LOC387743 XM_373488 - - - - LOC388567 XM_371200 - - - - LOC389080 XM_371598 - - - - MAN1A1 NM_005907 Mannosidase, alpha, class 1A, member 1 YES - - MCOLN2 XM_371263 - - - - MK-STYX NM_016086 Serine/threonine/tyrosine interacting-like 1 NO - - NCOA6 NM_014071 Nuclear receptor coactivator 6 YES Hs00204160_m1 NO Optic atrophy 3 (autosomal recessive, with chorea and OPA3 NM_025136 YES - - spastic paraplegia) PC4 NM_006713 SUB1 homolog (S. cerevisiae) - - - Protein kinase, cAMP-dependent, regulatory, type I, PRKAR1A NM_002734 YES - - alpha (tissue specific extinguisher 1) PTCRA NM_138296 Pre T-cell antigen receptor alpha NO - - RP1L1 NM_178857 Retinitis pigmentosa 1-like 1 YES - - Ribose 5-phosphate isomerase A (ribose 5-phosphate RPIA NM_144563 YES - - epimerase) RPS27 NM_001030 Ribosomal protein S27 (metallopanstimulin 1) - - - Sema domain, transmembrane domain (TM), and SEMA6D NM_020858 NO - - cytoplasmic domain, (semaphorin) 6D SKIL NM_005414 SKI-like oncogene YES - - STRA13 NM_144998 Stimulated by retinoic acid 13 homolog (mouse) -- - TITF1 NM_003317 NK2 1 YES - - TM4SF6 NM_003270 Tetraspanin 6 NO - - TMPRSS3 NM_032401 Transmembrane protease, serine 3 YES Hs00225161_m1 YES TTLL1 NM_012263 Tubulin tyrosine ligase-like family, member 1 NO - - TUBB2 NM_006088 Tubulin, beta 2C NO - - YAP NM_018253 - YES - -

Table B.3 ChIP-DSL AR Binding Promoters – Group C Tabulation of genes in the AR OFF – DHT and SARM2 group. If gene expression analyses were performed, a Taqman Probe number is listed along with detected regulation. If the gene had sufficient information to be searched for AREs, the result of the search is listed.

199 Gene Name Accession Description ARE Taqman Probe Regulated ACBD5 NM_145698 Acyl-Coenzyme A binding domain containing 5 NO - - ANKRD15 NM_015158 KN motif and domains 1 YES - - Asp (abnormal spindle) homolog, microcephaly ASPM NM_018136 YES - - associated (Drosophila) ATP1A3 NM_152296 ATPase, Na+/K+ transporting, alpha 3 YES - - BC-2 NM_198426 Chromatin modifying protein 2A NO - - C11orf16 NM_020643 open reading frame 16 NO - - C18orf24 NM_145060 Chromosome 18 open reading frame 24 NO - - C20orf22 NM_015600 Abhydrolase domain containing 12 YES - - CARM1 NM_199141 Coactivator-associated arginine YES - - CDP-diacylglycerol synthase (phosphatidate CDS2 NM_003818 -- - cytidylyltransferase) 2 CRAT NM_000755 Carnitine acetyltransferase YES - - DDEF2 NM_003887 Development and differentiation enhancing NO - - DKFZp761B0514 NM_032289 Pleckstrin and Sec7 domain containing 2 NO - - ENG NM_000118 Endoglin (Osler-Rendu-Weber syndrome 1) YES Hs00164438_m1 NO ENm009 TL_000070 - - - - FGF6 NM_020996 Fibroblast growth factor 6 NO Hs00173934_m1 NO FLJ11029 NM_018304 Proline rich 11 NO - - FLJ12606 NM_024804 Zinc finger protein 669 - - - FLJ20643 NM_017916 PIH1 domain containing 1 NO - - FLJ39647 NM_173625 Chromosome 17 open reading frame 78 NO - - FLJ40172 NM_173649 open reading frame 61 NO - - GYLTL1B NM_152312 Glycosyltransferase-like 1B NO - - HT036 NM_031207 Hydroxypyruvate isomerase homolog (E. coli) - - - IL22RA1 NM_021258 Interleukin 22 receptor, alpha 1 YES - - ITGA11 NM_012211 - YES - - ITSN2 NM_006277 Intersectin 2 YES - - KAI1_104 TL_000679 - - - - KCNK10 NM_138318 Potassium channel, subfamily K, member 10 YES - - KIAA0103 NM_014673 Tetratricopeptide repeat domain 35 NO - - KIAA1604 XM_034594 - - - - KIAA1991 XM_171476 - - - - KLF3 NM_016531 Kruppel-like factor 3 (basic) YES - - KLK6 NM_002774 Kallikrein-related peptidase 6 YES Hs00160519_m1 NO

Table B.4 ChIP-DSL AR Binding Promoters – Group D Tabulation of genes in the AR OFF – SARM2 only group. If gene expression analyses were performed, a Taqman Probe number is listed along with detected regulation. If the gene had sufficient information to be searched for AREs, the result of the search is listed.

200 Gene Name Accession Description ARE Taqman Probe Regulated LOC255809 XM_172995 open reading frame 38 YES - - LOC387840 XM_373526 - - - - LOC392964 XM_374636 - - - - LOH12CR1 NM_058169 Loss of heterozygosity, 12, chromosomal region NO - - MGC16384 NM_053048 - - - - MGC20806 NM_144999 Leucine rich repeat containing 45 NO - - MGC33212 NM_152773 Transmembrane 4 L six family member 19 YES - - MSH5 NM_002441 MutS homolog 5 (E. coli) YES - - P21/Cdc42/Rac1-activated kinase 1 (STE20 PAK1 NM_002576 YES - - homolog, yeast) PDPK1 NM_002613 3-phosphoinositide dependent protein kinase-1 - - - PPIL2 NM_014337 Peptidylprolyl isomerase (cyclophilin)-like 2 - - - Protein phosphatase 3 (formerly 2B), catalytic PPP3CB NM_021132 YES - - subunit, beta isoform PSEN1 NM_000021 Presenilin 1 (Alzheimer disease 3) NO - - RARB_12 TL_000233 - - - - RPL18A NM_000980 Ribosomal protein L18a - - - SAM domain, SH3 domain and nuclear SAMSN1 NM_022136 -- - localization signals 1 SCYL1 NM_020680 SCY1-like 1 (S. cerevisiae) NO - - SF3A1 NM_005877 Splicing factor 3a, subunit 1, 120kDa YES - - SLC19A1 NM_003056 - NO - - SLC22A17 NM_016609 Solute carrier family 22, member 17 YES - - Solute carrier family 4 (anion exchanger), SLC4A1AP NM_018158 NO - - member 1, adaptor protein SPPL2B NM_020172 - - - - TAS2R8 NM_023918 Taste receptor, type 2, member 8 - - - TCF1 NM_000545 HNF1 homeobox A YES - - TCTEL1 NM_006519 Dynein, light chain, Tctex-type 1 - - - TEB4 NM_005885 Membrane-associated ring finger (C3HC4) 6 YES - - TFF_122 TL_000031 - - - - TXNRD2 NM_006440 Thioredoxin reductase 2 - - - ZNF444 NM_018337 Zinc finger protein 444 NO - -

Table B.5 ChIP-DSL AR Binding Promoters – Group D (Continued) Tabulation of genes in the AR OFF – SARM2 only group. If gene expression analyses were performed, a Taqman Probe number is listed along with detected regulation. If the gene had sufficient information to be searched for AREs, the result of the search is listed.

201 Gene Name Accession Description ARE Taqman Probe Regulated ALK NM_004304 Anaplastic lymphoma receptor tyrosine kinase NO Hs00608289_m1 YES C10orf107 NM_173554 open reading frame 107 YES Hs00416785_m1 NO CASP7_19 TL_000693 - - - - UQCRC1 NM_003365 Ubiquinol-cytochrome c reductase core protein I YES Hs00163415_m1 NO

Table B.6 ChIP-DSL AR Binding Promoters – Group E Tabulation of genes in the AR ON – DHT and SARM2 group. If gene expression analyses were performed, a Taqman Probe number is listed along with detected regulation. If the gene had sufficient information to be searched for AREs, the result of the search is listed.

202 Gene Name Accession Description ARE Taqman Probe Regulated C8orf20 NM_025232 Receptor accessory protein 4 YES Hs00228780_m1 NO FLJ14827 NM_032848 Chromosome 12 open reading frame 52 YES - - IRLB NM_005848 DENN/MADD domain containing 4A NO Hs00400648_m1 NO KAI1_45 TL_000673 - - - - PCP2 XM_058956 - - - - Phosphatidylinositol glycan anchor biosynthesis, PIGS NM_033198 YES Hs00264209_m1 NO class S

Table B.7 ChIP-DSL AR Binding Promoters – Group F Tabulation of genes in the AR OFF – DHT only group. If gene expression analyses were performed, a Taqman Probe number is listed along with detected regulation. If the gene had sufficient information to be searched for AREs, the result of the search is listed.

203 Gene Name Accession Description ARE Taqman Probe Regulated ADK NM_006721 Adenosine kinase NO - - ADNP NM_181442 Activity-dependent neuroprotector homeobox YES - - ALAD NM_000031 Aminolevulinate, delta-, dehydratase - - - ANKRD22 NM_144590 Ankyrin repeat domain 22 NO - - AP3S2 NM_005829 Adaptor-related protein complex 3, sigma 2 subunit YES - - Androgen receptor (dihydrotestosterone receptor; AR NM_000044 testicular feminization; spinal and bulbar muscular YES Hs00171172_m1 YES atrophy; Kennedy disease) BCL9 NM_004326 B-cell CLL/lymphoma 9 YES - - C13orf24 NM_006346 Progesterone immunomodulatory binding factor 1 NO - - C20orf144 NM_080825 Chromosome 20 open reading frame 144 - - - C20orf147 NM_152667 N-acetylneuraminic acid phosphatase NO - - C20orf40 NM_144703 LSM14B, SCD6 homolog B (S. cerevisiae) NO - - C6orf208 NM_025002 - - - - Core-binding factor, runt domain, alpha subunit 2; CBFA2T3 NM_175931 YES - - translocated to, 3 CD36 NM_000072 CD36 molecule (thrombospondin receptor) YES - - CDK8 NM_001260 Cyclin-dependent kinase 8 YES - - CFDP1 NM_006324 Craniofacial development protein 1 NO - - COG7 NM_153603 Component of oligomeric golgi complex 7 YES - - COL23A1 NM_173465 Collagen, type XXIII, alpha 1 YES - - COLM NM_181789 Gliomedin YES - - CSRP2 NM_001321 Cysteine and glycine-rich protein 2 YES Hs00426717_m1 NO DKFZp451J011 NM_175852 Taxilin alpha NO - - 8 DKFZp547A023 NM_018704 CTTNBP2 N-terminal like YES - - DMD NM_004013 Dystrophin (muscular dystrophy, Duchenne and Becker NO - - DNAJB9 NM_012328 DnaJ (Hsp40) homolog, subfamily B, member 9 YES - - DPEP1 NM_004413 Dipeptidase 1 (renal) NO - - Dolichyl-phosphate mannosyltransferase polypeptide 2, DPM2 NM_003863 -- - regulatory subunit DYRK2 NM_003583 Dual-specificity tyrosine-(Y)-phosphorylation regulated YES - - EFA6R NM_018422 - YES - - ELP4 NM_019040 Elongation protein 4 homolog (S. cerevisiae) YES - - ENm009 TL_000241 - - - - ENm009 TL_000299 - - - - ENm009 TL_000167 - - - - ENm009 TL_000016 - - - - ENm009 TL_000214 - - - -

Table B.8 ChIP-DSL AR Binding Promoters – Group G Tabulation of genes in the AR ON – ALL group. If gene expression analyses were performed, a Taqman Probe number is listed along with detected regulation. If the gene had sufficient information to be searched for AREs, the result of the search is listed.

204 Gene Name Accession Description ARE Taqman Probe Regulated ENPEP NM_001977 Glutamyl aminopeptidase (aminopeptidase A) -- - FAM20C NM_020223 Family with sequence similarity 20, member C - - - FBXL5 NM_012161 F-box and leucine-rich repeat protein 5 - - - FBXO23 XM_030295 - - - - FGFR1OP NM_007045 FGFR1 oncogene partner NO - - FLJ10055 NM_017983 WD repeat domain, phosphoinositide interacting 1YES - - FLJ10748 NM_018203 Kelch domain containing 8A NO - - FLJ10808 NM_018227 Ubiquitin-like modifier activating enzyme 6 NO - - FLJ11342 NM_018394 Abhydrolase domain containing 10 - - - FLJ11588 NM_024603 open reading frame 165 NO - - FLJ12700 NM_024910 Zinc finger family member 767 - - - FLJ13456 XM_038291 - - - - FLJ20125 NM_017676 Gypsy retrotransposon integrase 1 NO - - FLJ21865 NM_022759 - YES - - FLJ22671 NM_024861 Chromosome 2 open reading frame 54 NO - - FLJ25476 NM_152493 Zinc finger protein 362 YES - - FLJ30277 NM_153008 - - - - FLJ30430 NM_153009 - - - - FLJ32154 NM_152502 - - - - FLJ33814 NM_173510 Coiled-coil domain containing 117 NO - - FLJ34283 NM_182612 Parkinson disease 7 domain containing 1NO- - FLJ35630 NM_152618 Bardet-Biedl syndrome 12 NO - - FLJ36208 XM_208927 - - - - FLJ40342 NM_152347 Chromosome 17 open reading frame 57 NO - - FLJ45645 NM_198557 RNA binding motif protein 43 NO - - FLJ90022 NM_153690 Family with sequence similarity 43, member A -- - GABARAPL2 NM_007285 GABA(A) receptor-associated protein-like 2 NO - - GABPA NM_002040 GA binding protein transcription factor, alpha subunit YES - - GABPB2 NM_002041 GA binding protein transcription factor, beta subunit 2 NO - - Glyceraldehyde-3-phosphate dehydrogenase, GAPDS NM_014364 NO - - spermatogenic GNAS NM_000516 GNAS complex locus - - - GPT2 NM_133443 Glutamic pyruvate transaminase (alanine YES Hs00370287_m1 YES GREB1_173 TL_000563 - - - - GREB1_63 TL_000642 - - - -

Table B.9 ChIP-DSL AR Binding Promoters – Group G (Continued) Tabulation of genes in the AR ON – ALL group. If gene expression analyses were performed, a Taqman Probe number is listed along with detected regulation. If the gene had sufficient information to be searched for AREs, the result of the search is listed.

205 Gene Name Accession Description ARE Taqman Probe Regulated GSK3A NM_019884 Glycogen synthase kinase 3 alpha NO - - GUK1 NM_000858 Guanylate kinase 1 NO - - HIPK1 NM_152696 Homeodomain interacting protein kinase 1 YES - - HMG20B NM_006339 High-mobility group 20B NO - - HRMT1L2 NM_001536 Protein arginine methyltransferase 1 NO - - HSF2BP NM_007031 Heat shock transcription factor 2 binding protein YES - - IGFBPL1 XM_294567 - - - - IPO8 NM_006390 Importin 8 NO - - ITGB1BP1 NM_004763 Integrin beta 1 binding protein 1 YES - - KAI1_77 TL_000675 - - - - Potassium voltage-gated channel, shaker-related KCNA1 NM_000217 YES - - subfamily, member 1 (episodic ataxia with myokymia) KIAA0553 XM_290758 - - - - KIAA0664 NM_015229 KIAA0664 NO - - KIAA1365 NM_020794 Leucine rich repeat containing 7YES-- KRTAP18-11 NM_198692 Keratin associated protein 10-11 - - - KUB3 NM_033276 XRCC6 binding protein 1 YES - - L3MBTL3 XM_027074 - - - - LOC114990 NM_138440 Vasorin YES - - LOC115861 NM_138454 Nucleoredoxin-like 1 YES - - LOC145741 XM_096852 - - - - LOC169834 XM_095965 - - - - LOC284767 XM_209367 - - - - LOC286513 XM_210082 - - - - LOC342918 XM_292779 Similar to mCG134545 NO - - LOC345557 XM_293875 - - - - LOC388114 XM_373626 - - - - LOC388930 XM_373975 - - - - LOC388947 XM_373984 - - - - LOC389259 XM_374105 - - - - LOC389635 XM_374253 - - - - LOC390851 XM_372694 - - - - LOC56926 NM_020170 Nicalin homolog (zebrafish) YES - - LRRN1 NM_020873 Leucine rich repeat neuronal 1 NO - - LSM10 NM_032881 LSM10, U7 small nuclear RNA associated - - -

Table B.10 ChIP-DSL AR Binding Promoters – Group G (Continued) Tabulation of genes in the AR ON – ALL group. If gene expression analyses were performed, a Taqman Probe number is listed along with detected regulation. If the gene had sufficient information to be searched for AREs, the result of the search is listed.

206 Gene Name Accession Description ARE Taqman Probe Regulated LTB NM_002341 Lymphotoxin beta (TNF superfamily, member 3) YES - - MAD NM_002357 MAX dimerization protein 1 NO - - MAEA NM_005882 Macrophage erythroblast attacher YES - - V-maf musculoaponeurotic fibrosarcoma oncogene MAFF NM_012323 YES - - homolog F (avian) MAP2K7 NM_005043 - YES - - MARCKS NM_002356 Myristoylated alanine-rich protein kinase C substrate NO - - MAS1L NM_052967 MAS1 oncogene-like - - - MASA NM_021204 Enolase-phosphatase 1 NO - - MEIS2 NM_020149 - NO - - MGC10854 NM_032300 Trichoplein, keratin filament binding YES - - MGC13114 NM_032366 open reading frame 13 NO - - MGC16824 NM_020314 Chromosome 16 open reading frame 62 NO - - MGC3731 NM_024313 Nucleolar protein 12 NO - - MGC40368 NM_152772 T-complex 11 (mouse)-like 2 YES - - MGC90512 XM_058581 - - - - MGC9712 NM_152689 - YES - - MLYCD NM_012213 Malonyl-CoA decarboxylase YES - - MRPL35 NM_016622 Mitochondrial ribosomal protein L35 NO - - MSI1 NM_002442 Musashi homolog 1 (Drosophila) YES - - NAP1L4 NM_005969 Nucleosome assembly protein 1-like 4 YES - - NOXO1 NM_144603 NADPH oxidase organizer 1 YES - - NUP93 NM_014669 Nucleoporin 93kDa NO - - NYREN18 NM_016118 Negative regulator of ubiquitin-like proteins 1 NO - - OR1G1 NM_003555 Olfactory receptor, family 1, subfamily G, member 1 - - - OR52D1 XM_372359 - - - - Platelet-activating factor acetylhydrolase, isoform Ib, PAFAH1B1 NM_000430 YES - - alpha subunit 45kDa PCNP NM_020357 PEST proteolytic signal containing nuclear protein - - - PDE5A NM_033431 - - - - PERLD1 NM_033419 Per1-like domain containing 1YES-- PEX16 NM_004813 Peroxisomal biogenesis factor 16 NO - - PGD NM_002631 Phosphogluconate dehydrogenase NO - - PHF17 NM_199320 PHD finger protein 17 YES - - PIK3R1 NM_181524 Phosphoinositide-3-kinase, regulatory subunit 1 (alpha) YES - - PODN NM_153703 Podocan - - - Processing of precursor 1, ribonuclease P/MRP POP1 NM_015029 YES - - subunit (S. cerevisiae)

Table B.11 ChIP-DSL AR Binding Promoters – Group G (Continued) Tabulation of genes in the AR ON – ALL group. If gene expression analyses were performed, a Taqman Probe number is listed along with detected regulation. If the gene had sufficient information to be searched for AREs, the result of the search is listed.

207 Gene Name Accession Description ARE Taqman Probe Regulated POU3F2 NM_005604 POU class 3 homeobox 2 NO - - PPFIBP1 NM_003622 PTPRF interacting protein, binding protein 1 (liprin beta NO - - PPIA NM_021130 Peptidylprolyl isomerase A (cyclophilin A) -- - RAB8B NM_016530 RAB8B, member RAS oncogene family YES - - RARB NM_000965 , beta NO - - RARB_299 TL_000322 - - - - RARB_424 TL_000230 - - - - RNF166 NM_178841 Ring finger protein 166 YES - - SCAP2 NM_003930 Src kinase associated phosphoprotein 2 YES - - SCGB1A1 NM_003357 Secretoglobin, family 1A, member 1 (uteroglobin) -- - SERPINC1 NM_000488 Serpin peptidase inhibitor, clade C (antithrombin), NO - - SERPINC1 NM_000488 Serpin peptidase inhibitor, clade C (antithrombin), NO - - SIP NM_014412 Calcyclin binding protein NO - - SKP2 NM_005983 S-phase kinase-associated protein 2 (p45) NO - - Solute carrier family 10 (sodium/bile acid cotransporter SLC10A4 NM_152679 NO - - family), member 4 SLC2A10 NM_030777 Solute carrier family 2 (facilitated glucose transporter), YES - - SLK NM_014720 STE20-like kinase (yeast) YES - - SORCS2 NM_020777 Sortilin-related VPS10 domain containing receptor 2 YES - - SPATA5 NM_145207 Spermatogenesis associated 5 - - - SPC18 NM_014300 SEC11 homolog A (S. cerevisiae) -- - SPTBN1 NM_003128 Spectrin, beta, non-erythrocytic 1 NO - - STCH NM_006948 Stress 70 protein chaperone, microsome-associated, YES - - STX1B2 NM_052874 Syntaxin 1B YES - - TAF6 RNA polymerase II, TATA box binding protein TAF6 NM_005641 YES - - (TBP)-associated factor, 80kDa TCERG1 NM_006706 Transcription elongation regulator 1 NO - - TFF_143 TL_000083 - - - - TMPO NM_003276 Thymopoietin - - - TMPRSS7 XM_293599 - - - - TRHDE NM_013381 Thyrotropin-releasing hormone degrading enzyme YES - - TRIM4 NM_033017 Tripartite motif-containing 4--- TXNL4 XM_371120 - NO - - UBD NM_006398 Ubiquitin D - - - UBE1 NM_003334 Ubiquitin-like modifier activating enzyme 1 YES - - UNG2 NM_021147 Cyclin O YES - - VPS52 NM_022553 Vacuolar protein sorting 52 homolog (S. cerevisiae) YES - - WDR13 NM_017883 WD repeat domain 13 NO - - WFDC2 NM_006103 WAP four-disulfide core domain 2 - - - WISP3 NM_130396 WNT1 inducible signaling pathway protein 3 YES - - ZCCHC2 NM_017742 Zinc finger, CCHC domain containing 2- - - ZNF135 NM_003436 Zinc finger protein 135 - - - ZNF189 NM_003452 Zinc finger protein 189 YES - - ZNF291 NM_020843 S phase cyclin A-associated protein in the ER YES - - ZNF34 NM_030580 Zinc finger protein 34 - - - ZNF395 NM_018660 Zinc finger protein 395 - - - ZNF629 XM_034819 - - - -

Table B.12 ChIP-DSL AR Binding Promoters – Group G (Continued) Tabulation of genes in the AR ON – ALL group. If gene expression analyses were performed, a Taqman Probe number is listed along with detected regulation. If the gene had sufficient information to be searched for AREs, the result of the search is listed.

208 9. APPENDIX C

SUPPLEMENTS RELEVANT TO CHAPTER 4

209 ALT-1 Promoter Scan Set ∆ TSS Foreward Primer (5'-3') Reverse Primer (5'-3') Length 1 +1848 TTCGCCTTCGAAGAGCGGCTCTTT CCATGAGCACCTTCTTGAATGAGTGGAACT 169 2 +1356 TGGACGTGGCCGAGCTTCACC GGTGGGGTTGCCAGGGTTGATGAC 91 3 +814 AGGGCGGAGCGCATCTTGC TCTCCTCAGCCCTCCCAAGGGA 102 4 -1401 AGTTGGCCACTCTGGCTCTGA ACAAAGCCGCCCACCATGAACAGA 124 5 -4610 CCTTTACCTTTAACACAGAGGGACGTAGA ACCTCTCTCCCACTACAGATG 109 6 -5147 GCTCACGTGGCACGGTTT CACTCACTGTCACAGGCATTGATGTT 101 7 -5571 AGGTGTGGGATTGTTGGAGTGCTG CAGAAGCACCAGGCATCTGTC 145 8 -6180 TGGGACCAGCCACCTCTGT GCACAGGTGAGGAGAAGAGAGT 121

Table C.1 ALT-1 ChIP Promoter Scan Primers

210 ALT-2 Promoter Scan Set ∆ TSS Foreward Primer (5'-3') Reverse Primer (5'-3') Length 1 -1847 GAGCACACATAAGAAGGCTGTGTGCTTG ACCCACGTGTCAGTGTACTATGCTCT 141 2 -2069 GCACTGGGTTAGAAATCACAATTCCTGCTC ACCCGTCTCTCTGAGATTGCAT 200 3 -3008 TAATCACGTGTGATGACCTCCGCCTT TGAGGCTGAGTTCTGGGACTACAAAGAG 100 4 -4063 AGACCAGGCATGATCTGGCATTCT TGGCGTAGGTAAGTATGCTAAGGG 193

Table C.2 ALT-2 ChIP Promoter Scan Primers

211 10. APPENDIX D

SUPPLEMENTS RELEVANT TO CHAPTER 5

212 Primary Human Hepatocyte - Gene Expression 8 * 6

4

* 2

0

-2

-4

FC vs. VehicleFC vs. (24 Hours) -6 SHP-1 STE CYP7A1 -8 SULT2A1 * -10 Vehicle (24 Hrs) 6 Hrs 12 Hrs 24 Hrs 48 Hrs 100 nM DHT *p<.05 Fisher's LSD vs Vehicle

Figure D.1 Primary Human Hepatocyte Time Course – Control Gene Expression Control genes’ of interest expression in male primary human hepatocytes. Values represented as mean+SD (n=3), as described in the methods section, FC versus vehicle at 24 hours.

213 Primary Rat Hepatocytes - Gene Expression 4 SHP-1 CYP7A1 3 STE SULT2A1 2

1

0 FC vs. Vehicle FC vs.

-1

-2

-3

eh M2 eh M2 eh M2 V R V R V AR SA SA S nM nM nM 00 00 00 1 1 1

24 Hours 48 Hours 72 Hours

Figure D.2 Primary Rat Hepatocytes Control Gene Expression – Time Course A time course of control gene regulation in primary male rat hepatocytes. SULT2A1 was not expressed. Values represented as mean+SD (n=3), as described in the methods section, FC versus corresponding vehicle.

214 Primary Rat Hepatocytes - Gene Expression 100

0 * * * * * -100 *

-200 *

* -300

-400 SHP-1 FC vs. Vehicle (0 hours) FC vs. CYP7A1 STE -500 SULT2A1

*p<.05 Fisher's LSD vs Vehicle 0 hrs. * -600 rs rs rs rs ou ou ou ou H H h h 0 24 48 72

Figure D.3 Primary Rat Hepatocyte Control Gene Expression – Time Course (No Ligand) Control genes’ expression in male primary rat hepatocytes solely as a function of time. Values represented as mean+SD (n=3), as described in the methods section, FC versus vehicle at time 0.

215 Primary Rat Hepatocyte - Gene Expression

* *

0

* * * * * * LIPC ABCA1 -5 SRB1 FC vs. Vehicle (0 hours) FC vs. APOA1 APOA2 *

-15 -20 *p<.05 Fisher's LSD vs. corresponding Vehicle -25 * 0 Hrs 24 hrs 48 Hrs 72 Hrs

Figure D.4 Primary Rat Hepatocyte Gene Expression – Time Course (No Ligand) HDL metabolism/homeostasis genes’ of interest expression in male primary rat hepatocytes solely as a function of time. Values represented as mean+SD (n=3), as described in the methods section, FC versus vehicle at time 0.

216 HepG2 AR Gene Expression 2.5e+5

2.0e+5

1.5e+5

1.0e+5 FC vs. Blank vs. FC

5.0e+4

0.0 k R k R k R lan A lan A lan A B ug B ug B ug .5 .5 .5 2 2 2

24 Hrs. 48 Hrs. 72 Hrs.

Figure D.5 HepG2 AR Expression Time Course Values are represented as mean+SD of three replicates each compared to an untransfected control.

217 HepG2 - AR Transfection 6

SHP-1 CYP7A1 4 STE SULT2A1

2

0

FC vs. Vehicle FC vs. -2

-4 * *p<.05 Fisher's LSD vs. Vehicle -6 * * e 2 e 2 e 2 icl M icl M icl M eh AR eh AR eh AR V S V S V S uM uM uM 1 1 1

24 hours 48 hours 72 hours

Figure D.6 HepG2 AR Transfection – Control Gene Expression Control genes’ of interest expression in HepG2 cell transiently transfected with human AR. Values represented as mean+SD (n=3), as described in the methods section, FC versus corresponding vehicle.

218 HepG2 - AR Transfection 4

3

2

1

0

-1 FC vs. Vehicle (24 Hrs) Vehicle (24 FC vs. LIPC ABCA1 SRB1 -2 APOA1 APOA2 -3 24 Hrs 48 Hrs 72 Hrs

Figure D.7 HepG2 AR Transfection Target Gene Expression – Time Course (No Ligand) HDL metabolism/homeostasis genes’ of interest expression in HepG2 cells transfected with AR solely as a function of time. Values represented as mean+SD (n=3), as described in the methods section, FC versus vehicle at time 24 hours.

219 HepG2 - AR Transfection 14 *p<.05 Fisher's LSD vs. Vehicle (24 Hrs) SHP-1 12 STE * CYP7A1 SULT2A1 10

8

6

4 FC vs. Vehicle (24 hours) (24 Vehicle FC vs.

2

0 24 Hrs 48 Hrs 72 Hrs

Figure D.8 HepG2 AR Transfection Control Gene Expression – Time Course (No Ligand) Control genes’ expression in HepG2 cells transfected with AR solely as a function of time. Values represented as mean+SD (n=3), as described in the methods section, FC versus vehicle at time 24 hours.

220 ORX Rat Time Course - Soleus Muscle Weights 160 ORX 140 SARM2 SARM3 SARM4 120

100

80

60 % of Intacct Control Intacct of % 40

20

0 Day 3 Day 7 Day 10 Day 14 Day 21 Day 28

Figure D.9 Soleus Muscle Weights – ORX Rat Time Course Values are normalized to individual animal bodyweights and then represented as percentage of Intact Control Tissue weight at Day 28 (mean+SD).

221 ORX Rat Time Course - Gastrocnemius Weights

140 ORX SARM2 120 SARM3 SARM4

100

80

60

% of Intacct Control 40

20

0 Day 3 Day 7 Day 10 Day 14 Day 21 Day 28

Figure D.10 Gastocnemius Weights – ORX Rat Time Course

Values are normalized to individual animal bodyweights and then represented as percentage of Intact Control Tissue weight at Day 28 (mean+SD).

222 ORX Rat Time Course - Liver Gene Expression

5

0

-5 *

FC vs ORX Day 0 -10 SHP-1 CYP7A1 STE -15 SULT2A1

Day 0 Day 3 Day 7 Day 10 Day 14 Day 21 Day 28 ORX Control Animals

* p<.05 Fisher's LSD vs ORX Day 0

Figure D.11 ORX Rat Time Course – ORX Animal Liver Control Gene Expression Control genes’ of interest expression in liver tissue of vehicle treated ORX rats. Values represented as mean+SD (n=5), as described in the methods section, FC versus ORX Day 0. SULT2A1 was not expressed.

223 ORX Rat Time Course - Liver Gene Expression

4

2

0

-2

-4 FC vs Day 3 *

-6 SHP-1 CYP7A1 -8 STE SULT2A1

-10 Day 3 Day 7 Day 10 Day 14 Day 21 Day 28 SARM2 1mg/day

* p<.05 Fisher's LSD vs Day 3

Figure D.12 ORX Rat Time Course – SARM2 Treated Liver Control Gene Expression Control genes’ of interest expression in liver tissue of SARM2 treated ORX rats. Values represented as mean+SD (n=5), as described in the methods section, FC versus Day 3 SARM2 treated animals. SULT2A1 was not expressed.

224 ORX Rat Time Course - Liver Gene Expression 5

0

-5

-10 *

* -15 FC vs Day 3 * -20 SHP-1 CYP7A1 -25 STE * SULT2A1

-30 Day 3 Day 7 Day 10 Day 14 Day 21 Day 28 SARM3 1mg/day

* p<.05 Fisher's LSD vs Day 3

Figure D.13 ORX Rat Time Course – SARM3 Treated Liver Control Gene Expression Control genes’ of interest expression in liver tissue of SARM3 treated ORX rats. Values represented as mean+SD (n=5), as described in the methods section, FC versus corresponding vehicle.

225 ORX Rat Time Course - Liver Gene Expression 5

0

-5

-10 FC vs Day 3

SHP-1 -15 CYP7A1 STE SULT2A1

-20 Day 3 Day 7 Day 10 Day 14 Day 21 Day 28 SARM4 1mg/day

Figure D.14 ORX Rat Time Course – SARM4 Treated Liver Control Gene Expression Control genes’ of interest expression in liver tissue of SARM4 treated ORX rats. Values represented as mean+SD (n=5), as described in the methods section, FC versus Day 3 SARM4 treated animals.

226 OVX Rat Extended Treatment - Liver Gene Expression

* * 10000 SHP-1 CYP7A1 7500 STE SULT2A1 5000

2500 * *

* *

0

* FC vs Vehicle (Intact or OVX) or (Intact Vehicle vs FC

*

e 2 4 e 2 4 icl M M icl M M eh AR AR eh AR AR V S S V S S

Intact OVX *p<.05 Fisher's LSD vs Vehicle of corresponding gonadal status

Figure D.15 OVX Rat Extended Treatment – Liver Control Gene Expression Control genes’ expression in OVX rat liver tissue. Values represented as mean+SD (n=8), as described in the methods sections, FC versus vehicle of corresponding gonadal status.

227