importance of 5α-reductase gene polymorphisms on circulating … · 23/11/2013 · 125 is...

SRD5A polymorphisms and androgens in PCa

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Importance of 5α-Reductase Gene Polymorphisms on Circulating and 1

Intraprostatic Androgens in Prostate Cancer 2

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Éric Lévesque1,2, Isabelle Laverdière1, Louis Lacombe2, Patrick Caron1, Mélanie 4

Rouleau1, Véronique Turcotte1, Bernard Têtu2, Yves Fradet2 and Chantal Guillemette1† 5

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1Pharmacogenomics Laboratory, Centre Hospitalier Universitaire de Québec (CHU de 7

Québec) Research Center and Faculty of Pharmacy, Laval University, Québec, Canada. 8

2CHU de Québec Research Center and Faculty of Medicine, Laval University, Québec, 9

Canada. 10

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Running head: SRD5A polymorphisms and androgens in PCa 12

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†Correspondence: 14

Chantal Guillemette, Ph.D. 15

CHU de Québec Research Center, T3-48, 2705 Boul. Laurier, Québec, Canada, G1V 16

4G2. Tel. (418) 654-2296 Fax. (418) 654-2298 17

[email protected] 18

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Key words: prostate cancer, germline polymorphisms, SRD5A, 5-alpha-reductase, 20

circulating and intraprostatic hormone levels. 21

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Research. on December 15, 2020. © 2013 American Association for Cancerclincancerres.aacrjournals.org Downloaded from

Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on November 25, 2013; DOI: 10.1158/1078-0432.CCR-13-1100

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Authors’ disclosures of potential conflicts of interest: Eric Lévesque, Chantal 24

Guillemette, Louis Lacombe, and Yves Fradet have been named inventors on a patent 25

application owned by Laval University on a previous work related to this study. 26

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Number of: 28

Tables: 4 29

Figures: 2 30

Supplementary Tables: 2 31

References: 33 32

33

Number of words: 34

Abstract: 250 35

Introduction: 492 36

Methods: 829 37

Results: 489 38

Discussion: 1511 39

Body text: 3321 40

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Abbreviations 42

HR: hazard ratio; htSNP: haplotype-tagging SNP; SRD5A1: 5α-reductase type 1, 43

SRD5A2: 5α-reductase type 2, HSD17B: 17β−hydroxysteroid dehydrogenase; PCa: 44

prostate cancer; PSA: prostate-specific antigen; SNP: Single nucleotide polymorphism; 45

T: Testosterone; MS: mass spectrometry; TNM: Tumor/Nodes/Metastasis staging 46

system, CYP17A1; cytochrome P450 17α-hydroxylase and 17-20 lyase; CYP19A1: 47

aromatase, UGT: UDP-glucuronosyltransferase; 4-dione: androstenedione; ADT: 48

Androsterone; ADT-G: androsterone-glucuronide; 3α-diol-3G; androstane-3α, 17β-diol 49

3-glucuronide; 3α-diol-17G: androstane-3α, 17β-diol 17-glucuronide; DHEA: 50

dehydroepiandrosterone; DHEA-S: DHEA-sulfate; 5-diol: androst-5-ene-3β,17β-diol; E1-51

S: estrone-sulfate; E2: estradiol; androst-5-ene-3β,17β-diol, 3α-diol: androstane-3α, 17β-52

diol, Testo: testosterone, DHT: dihydrotestosterone. 53

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Study concept, design and supervision: Lévesque, Guillemette 56

Acquisition of data: Lévesque, Laverdière, Caron, Turcotte, Têtu, and Guillemette 57

Analysis and interpretation of data: Lévesque, Laverdière, Guillemette 58

Drafting of the manuscript: Lévesque, Guillemette 59

Critical revision of the manuscript for important intellectual content: All authors 60

Statistical analysis: Lévesque, Laverdière, Rouleau, Guillemette 61

Patients’ recruitment and clinical data: Têtu, Lacombe, Fradet 62

Obtaining funding: Lévesque, Lacombe, Fradet, Guillemette 63

Other (specify): None. 64

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Translational Relevance 67

Prostate cancer (PCa) is a heterogeneous disease at the molecular and clinical levels 68

requiring an individualized approach to patient care. The importance of androgen in 69

cancer initiation and progression is clearly established at all disease stages. Moreover, 70

5alpha-reductase inhibitors (5-ARI) have shown potential clinical efficacy as 71

chemopreventive medications. Likewise, SRD5A polymorphisms were recently 72

associated with altered risk of biochemical recurrence in localized PCa disease. To gain 73

insights into SRD5A biological effects, we examined the relationship between SRD5A 74

prognostic markers and endogenous sex-steroid levels measured by gold-standard mass 75

spectrometry methods in plasma and corresponding prostatic tissues of PCa patients. 76

Our findings sustain the significance of common inherited SRD5A polymorphisms on the 77

modulation of the androgenic milieu of cancer patients. The utility of understanding the 78

impact of SRD5A germline determinants on androgen exposure is that it could potentially 79

be used to identify subgroups of patients, with a particular androgen microenvironment 80

profile, that would most benefit from 5-ARI. Therefore, protective and high-risk germline 81

SRD5A alleles associated with changes in hormone levels may lead to more personalized 82

approaches to refining 5-ARI therapy, especially early in disease course. 83

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Abstract 85

Purpose. Polymorphisms in the genes SRD5A1 and SRD5A2 encoding androgen 86

biosynthetic 5-alpha-reductase enzymes have been associated with an altered risk of 87

biochemical recurrence after radical prostatectomy in localized prostate cancer (PCa). 88

Experimental Design. To gain potential insights into SRD5A biological effects, we 89

examined the relationship between SRD5A prognostic markers and endogenous sex-90

steroid levels measured by mass spectrometry in plasma samples and corresponding 91

prostatic tissues of PCa patients. Results. We report that five of the seven SRD5A 92

markers differentially affect sex-steroid profiles of dihydrotestosterone and its metabolites 93

in both the circulation and prostatic tissues of PCa patients. Remarkably, a 32% increase 94

in intraprostatic testosterone levels was observed in the presence of the high-risk SRD5A 95

rs2208532 polymorphism. Moreover, SRD5A2 markers were associated predominantly 96

with circulating levels of inactive glucuronides. Indeed, the rs12470143 SRD5A2 97

protective allele was associated with high circulating androstane-3α, 17β-diol 3-98

glucuronide (3α-diol-17G) levels as opposed to lower levels of both 3α-diol-17G and 99

androsterone-glucuronide observed with the rs2208532 SRD5A2 risk allele. Moreover, 100

SRD5A2 rs676033 and rs523349 (V89L) risk variants, in strong linkage disequilibrium, 101

were associated with higher circulating levels of 3α-diol-3G. The SRD5A2 rs676033 102

variant further correlated with enhanced intraprostatic exposure to 5α-reduced steroids 103

(dihydrotestosterone and its metabolite 3β-diol). Similarly, the SRD5A1 rs166050C risk 104

variant was associated with greater prostatic exposure to androsterone whereas no 105

association was noted with circulating steroids. Conclusions. Our data support the 106





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association of 5α-reductase germline polymorphisms with the hormonal milieu in PCa 107

patients. Further studies are needed to evaluate if these variants influence 5α-reductase 108

inhibitor efficacy. 109

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Introduction 113

Prostate cancer (PCa) is the most frequent malignancy in men and the second 114

leading cause of cancer death in Western countries (1). It is well established that 115

androgens play a central role in PCa progression even until it reaches advanced stages. 116

Indeed, the conversion of testosterone (T) by 5alpha-reductase (5-AR) enzymes (types 117

1 and 2) leads to dihydrotestosterone (DHT), the most potent androgen receptor agonist 118

in prostate cells. Under normal physiological conditions, SRD5A2, which encodes a 5-119

AR, is preferentially expressed over SRD5A1 in the prostate (2, 3). In PCa cells, 120

however, the balance in expression of these genes shifts towards predominant 121

expression of SRD5A1 (3, 4), supporting a role for both enzymes in DHT bioavailability 122

and carcinogenesis. In fact, 5-AR enzymes represent attractive targets for preventing 123

PCa development. In that context, the importance of androgens in early cancer initiation 124

is emphasized by the fact that finasteride, a 5-AR type 2 inhibitor, and dutasteride, a 125

dual 5-AR inhibitor targeting both 5-AR type 1 and type 2 enzymes, have been shown to 126

reduce by almost 23% the risk of PCa incidence (5, 6). More recently, the REDEEM trial 127

conducted with low-risk PCa patients showed that dutasteride also reduced by 10% the 128

likelihood of cancer progression (7). 129

However, PCa is clearly a heterogeneous disease, and prediction of response to 130

treatment and progression remains a major challenge for the field. In addition to 131

established tumor markers, certain patient genetic factors seem clearly associated with 132

cancer progression. For instance, germline genetic polymorphisms in SRD5A, UGT2B, 133





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and HSD17B were shown to be associated with progression in Caucasian and Asian 134

PCa patients after radical prostatectomy (RP) (8-10). In a previous study, four SRD5A2 135

single nucleotide polymorphisms (SNPs) (rs2208532, rs12470143, rs523349 (V89L), 136

and rs4952197) were associated with biochemical recurrence (BCR) after RP in 137

Caucasians and Asians (9). The strongest effect was conferred by the SRD5A2 V89L 138

nonsynonymous SNP (rs523349C allele) with a hazard ratio (HR) of 2.87 (95% 139

confidence interval [CI], 2.07–4.00; p = 4×10−10; 48% BCR). In addition, two SNPs, 140

rs518673T in SRD5A1 and rs12470143A in SRD5A2, were associated with a reduced 141

BCR rate (HR: 0.37; 95% CI, 0.19–0.71; p = 0.003 when combined; 16% BCR) 142

compared with non-carriers (38% BCR). Such results support a significant effect of 143

inherited genetic variations in the androgenic pathways on hormonal homeostasis and 144

PCa recurrence. Despite the identification of such potential biomarkers, however, there 145

is a clear lack of biological data explaining these findings. 146

It is hypothesized that polymorphisms in SRD5A genes affect the corresponding 147

exposure to sex-steroids of androgen-responsive cells, which may impact cell 148

proliferation and cancer progression. At present, the influence of SRD5A polymorphisms 149

linked to recurrence and progression in PCa patients on the systemic and prostatic sex-150

steroid hormonal environment remains undetermined. To gain potential insight into their 151

biological effects, we sought to evaluate the association of previously reported 152

prognostic markers in SRD5A1 and SRD5A2 with levels of circulating and prostatic sex-153

steroids in the same cohort of PCa patients. 154





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Patients and Methods 155

This study was based on a cohort of 526 Caucasians diagnosed with PCa recruited at 156

the CHU de Québec-Hotel-Dieu de Québec Hospital (Québec, Canada) between 1999 157

and 2002, as described (8, 9). For the specific purpose of this study, available plasma 158

and tissue samples were studied (numbers are indicated in Tables), and we excluded 159

patients who received hormonal treatment. Patients did not receive 5-AR inhibitors (5-160

ARI) in this study. Two high-volume surgeons performed all surgeries for these patients 161

(Drs Lacombe and Fradet). A fragment of fresh prostatic tissue was selected by the 162

pathologist from the RP specimen in the area containing tumor tissue. Microdissection of 163

tumors was not performed. Each specimen was then immediately snap-frozen and kept 164

at –80°C. The remaining prostatic tissue was fixed and submitted in its entirety for 165

histologic examination. Gleason score and pTNM stage were evaluated with paraffin 166

sections. Detailed clinical information was available from a preoperative evaluation and 167

medical records. All participants provided written consent before surgery for the analysis 168

of their genome and corresponding hormone levels. The local research ethics committee 169

approved the research protocol. Analyses described below were conducted blinded. 170

Genotyping 171

Genomic DNA was prepared from peripheral blood mononuclear cells collected from 172

patients on the morning of a preoperative ambulatory clinical visit. All samples were kept 173

frozen at –80°C until the time of study. Genomic DNA was extracted using the QIAamp 174

DNA Blood mini kit (QIAGEN Inc., Mississauga, Ontario, Canada) and stored at –80°C. 175





11

For genotyping, PCR was performed using Sequenom iPLEX matrix-assisted laser 176

desorption/ionization-time-of-flight mass spectrometry (MS), as described (9). 177

Analyses of steroid levels in plasma and prostatic tissue samples 178

Dehydroepiandrosterone (DHEA), 5-androsten-3β, 17β-diol (5-diol), testosterone (testo), 179

dihydrotestosterone (DHT), androsterone (ADT), androstane-3β-17β-diol (3β-diol), 180

estrone (E1), estradiol (E2), 4-androstenedione (4-dione), ADT-glucuronide (ADT-G), 181

androstane-3α, 17β-diol 3-glucuronide (3α-diol-3G), androstane-3α, 17β-diol-17 182

glucuronide (3α-diol-17G), DHEA-sulfate (DHEA-S), and E1-Sulfate (E1-S) were 183

purchased from Steraloids (Newport, RI). Deuterated isotopes of hormones, namely 184

DHEA-d3, testo-d3, DHT-d3, E1-d4, E2-d4, ADT-d3, 3β-diol-d3, 4-dione-d7, androstane-185

3α, 17β-diol 17-glucuronide-d3 (3α−diol-17G-d3), E1-S d4, and ADT-S d2, were 186

purchased from C/D/N Isotopes (Montréal, Qc, Canada). Plasma steroid measures 187

were based on a procedure previously described (11, 12). Steroids measured in plasma 188

were DHEA-S, DHEA, 5-diol, testo, DHT, 3β-diol, 4-dione, ADT, E1-S, E1, E2, ADT-G, 189

3α-DIOL-3G, and 3α-DIOL-17G. For tissue, a 50-mg frozen tissue section of prostate 190

was transferred to a 12 × 75 mm glass tube containing 250 µL of 12.5 mM ammonium 191

bicarbonate and homogenized. To assess total steroids, namely total (unconjugated and 192

conjugated) T, DHEA, 5-diol, DHT, 3β-diol, and ADT, 50 µL of the deuterated internal 193

standards were added to the homogenized samples, following by the addition of 0.5 mL 194

of freshly prepared hydrolysis buffer containing β-glucuronidase/sulfatase enzyme (Helix 195

pomatia, type HP-2, ≥500 U β-glucuronidase and ≥37.5 U of sulfatase activity) as 196





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described (13). Samples were incubated overnight at 37°C for hydrolysis and then 197

extracted with 4 mL of chlorobutane:ethyl acetate (3:1) mixture. The organic layer was 198

transferred to a clean glass tube, evaporated to dryness at 35°C under nitrogen gas, 199

and dissolved with 100 µL of sodium bicarbonate (pH 9.0) and 100 µL of dansyl chloride 200

(1 mg/mL in acetone). Then, 3 mL methanol:water (1:4) mixture was added to the 201

sample, and solid-phase extraction was performed with a Strata-X Reversed SPE Phase 202

Sorbents 60-mg columns (Phenomenex, Torrance, CA) previously conditioned with 1 203

mL methanol followed by 2 mL of water. The loaded cartridges were then sequentially 204

washed with 3 mL of water and 3 mL of 55% methanol. The cartridges were dried under 205

full vacuum. The androgenic compounds were eluted with 3 mL of 90% methanol and 206

evaporated to dryness at 45°C under nitrogen gas. Next, androgens were derivatized 207

and analyzed by gas chromatography–MS as described (8). The sulfatase and beta-208

glucuronidase mixture from H. pomatia was tested for steroid-converting enzymatic 209

activities, and it was noted that this specific preparation presents some HSD3B activity 210

(<3%, data not shown). Lower limit of quantification (LLOQ) for T, DHEA, 5-diol, DHT, 211

3β-diol and ADT were 300 pg/g, 1000 pg/g, 500 pg/g, 50 pg/g, 100 pg/g, and 250 pg/g, 212

respectively. 213

Statistical analyses 214

To adjust for differences in the absolute levels of sex-steroid hormones, we calculated 215

residuals of the natural logarithm of the hormone level regressed on age at blood 216

donation and smoking status. We assessed the association of SNPs with variation in 217

hormone levels by performing the regression of hormone residuals on each SNP 218





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independently for two models: recessive and dominant with one degree of freedom. We 219

considered the association of a SNP with variation in hormone levels to be significant if 220

the p value was ≤0.05. To facilitate comparisons between groups, we displayed the 221

hormone level as untransformed data by using the geometric mean and standard error 222

of the mean. Statistical analyses were performed using SAS Statistical Software version 223

9.2 (SAS Institute, Cary, NC) and using PASW statistics version 17 (SPSS Inc., 224

Chicago, IL). Owing to the exploratory nature of this study on androgen metabolism in 225

patients with PCa, no correction was made for multiple testing. 226

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Results 228

Characteristics and descriptive statistics of the study cohort are listed in Table 1. The 229

cohort was mostly composed of white men who underwent RP at L’Hôtel-Dieu de 230

Québec Hospital (Québec, Canada) between February 1999 and December 2002 (8). 231

Tables 2–4 present data for prostatic and circulating steroid levels in samples from 232

patients heterozygous or homozygous for the minor allele in comparison with levels 233

measured in samples from homozygous carriers of the major allele. 234

235

SRD5A1 rs166050, which is associated with higher risk of BCR, was associated with 236

higher levels of numerous prostatic steroids, particularly DHEA (60%, 25.94 vs. 15.76 237

ng/g; p=0.045) and ADT (100%, 13.51 vs. 6.77 ng/g; p=0.015), in homozygous carriers 238

for the minor allele rs166050C (Table 2). No significant change in the concentrations of 239

steroid hormones was observed in the circulation in relation to this SNP (Supplementary 240

Table 1), both in circulating blood and prostatic tissues (Tables 2–4). 241

242

Five SNPs in SRD5A2 were evaluated, of which four (rs2208532, rs4952197, rs523349 243

(V89L) and rs676033) were significantly associated with a higher risk of BCR, and one 244

marker (rs12470143) with lower risk of BCR in Caucasians and Asians (9). Levels of 245

prostate DHT and 3β-diol were particularly affected by one SRD5A2 polymorphism 246

(rs676033). Indeed, patients homozygous for the risk allele rs676033A, in close genetic 247

linkage with rs523349 (V89L), displayed 30% higher prostatic levels of DHT (p=0.04) 248

and 40% higher 3β-diol (p=0.03) compared with GG/AG. In addition, plasma ADT levels 249





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were also significantly higher (17%; p=0.04) (Table 3) in the presence of this 250

polymorphism. Interestingly, tissue levels of 5-diol were higher for most unfavorable 251

SNPs, reaching significance for rs4952197 (37% higher; p=0.012) and rs523349 (30%; 252

p=0.030) but not attaining significance for rs2208532 (38%; p=0.052) and rs676003 253

(26%; p=0.058). Remarkably, we found a significant association between the SRD5A 254

rs2208532 SNP and intraprostatic testosterone levels. Indeed, carriers of this high-risk 255

allele had a 32% increase in prostatic testosterone levels (p=0.038). An apparent lower 256

level (16%) of prostatic testosterone was observed with the protective rs12470143 257

allele, but this difference was not statistically significant (p=0.13). Table 3 lists details of 258

the comprehensive effects of SRD5A2 SNPs on prostatic steroid hormone levels. 259

260

Of 14 steroids measured in plasma, significant associations were observed for 261

glucuronide (-G) metabolites of DHT in relation to SRD5A2 markers (Figure 1, Table 4 262

and Supplementary Table 2). The protective marker rs12470143A was associated with 263

significantly higher 3α-diol-17G levels (15% for homozygotes; p=0.048), whereas the 264

opposite was observed for carriers of an unfavorable allele of that gene. In particular, 265

rs2208532G was associated with significantly lower concentrations (10–20%) of ADT-G 266

(p=0.028) and 3α-diol-17G (p<0.001). Interestingly, the risk alleles rs676033 and 267

rs523349, in strong linkage disequilibrium in Caucasians (r2=0.90), were both associated 268

with elevated levels of 3α-diol-3G (20% for rs523349CC, p=0.036; 24% for rs676033AA, 269

p=0.039). Two SRD5A2 markers (rs12470143 and rs2208532) were also associated 270





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with an increase and decrease, respectively, in prostate volume at time of surgery (not 271

shown). 272

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Discussion 274

SRD5A genes encode rate-limiting enzymes involved in DHT formation in the prostate 275

and several other tissues, particularly the liver and skin. Inhibition of these enzymes is 276

the theoretical basis for chemoprevention strategies (5, 6) and was also recently shown 277

to reduce PCa progression in low-risk disease (14). In agreement with these previous 278

findings, inherited germline variations in SRD5A1 and SRD5A2 were positively 279

associated with BCR after RP in localized disease in both Caucasians and Asians (9). 280

These results suggest that SRD5A genes may represent potential biomarkers of disease 281

progression and response to treatment. 282

283

Our data suggest that SRD5A polymorphisms affect androgen metabolism in PCa 284

patients as reflected by significant changes in circulating steroid glucuronide metabolites 285

of the potent hormone DHT. Indeed, four of the five previously identified SRD5A2 286

markers (rs12470143, rs2208532, rs523349, rs676033) affect circulating glucuronide 287

levels. The SRD5A2 rs12470143 protective marker is associated with higher levels of 288

3α-diol-17G in PCa patients, whereas the SRD5A2 rs2208532 risk allele is associated 289

with lower levels of both 3α-diol-17G and ADT-G. Also, the genetically linked rs676033 290

and rs523349 risk variants are associated with increased levels of androgen 291

glucuronides, namely 3α-diol-3G. Thus, these SRD5A markers significantly affect sex-292

steroid profiles of DHT metabolites in circulation of PCa patients. Indeed, T and DHT are 293

rapidly transformed in the human prostate by several metabolic enzymes (15, 16). 294

Moreover, DHT and its metabolites are further metabolized by UDP-295





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glucuronosyltransferases (UGTs), namely UGT2B15, UGT2B17 and UGT2B28, to yield 296

their downstream inactive metabolites such as 3α-diol-3G, 3α-diol-17G, and ADT-G that 297

are subsequently released into the circulation (Figure 2) (17-19). These major inactive 298

steroids are considered biomarkers for intraprostatic DHT synthesis and exposure (20). 299

Therefore, the presence of SRD5A2 prognostic markers impacts androgen formation in 300

these men while additional steroidogenic enzymes would be involved in DHT 301

biotransformation prior to their inactivation by UGTs. In support of our findings, results 302

obtained with the SRD5A2 rs12470143 and rs2208532 SNPs are in complete 303

agreement with previous findings indicating an impact of these key genetic variations on 304

circulating androgen metabolites rather than on circulating SRD5A substrates (21, 22). 305

306

Moreover, SRD5A variants are associated with sex-steroid exposure in the prostate of 307

cancer patients. Remarkably, the rs2208532 SNP was linked with a 32% increase of 308

intraprostatic testosterone levels, combined with reduced circulating glucuronide levels, 309

suggesting that this high-risk allele is associated with reduced 5α-reductase enzyme 310

efficiency. In the same line of thought, in the presence of the low-risk allele rs12470143, 311

a non-significant reduction (16%) in prostatic testosterone levels combined with higher 312

glucuronide levels suggest that this SNP is associated with high enzyme activity. 313

Interestingly, the non-synonymous V89L variant of SRD5A2 (rs523349), previously 314

associated with aggressive forms of the disease (23), has been described as a low-315

activity allele in vitro (24). This coding SRD5A2 variant has been reported to impact sex-316

steroid concentrations in different ethnic groups whereas an ethnic-specific distribution 317





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and linkage with other SRD5A2 alleles also has been observed (21, 24-27). 318

Measurement of steroid levels in the prostate revealed that individuals carrying a tightly 319

linked variation to rs523349 (e.g., rs676033) display significantly higher intraprostatic 320

DHT and 3β-diol levels, higher circulating levels of ADT (a 5α-reduced metabolite of 321

DHT; Figure 2), and no significant alteration in circulating T or DHT levels. According to 322

these observations, we hypothesize that the V89L rs523349 SNP or another variation in 323

linkage disequilibrium such as rs676033 located in the promoter region of the gene 324

would modulate 5α-reductase activity/expression within prostate cells. Further studies 325

are definitely required to evaluate the molecular impact of the rs523349/rs676033 in 326

target cells of cancer patients. 327

328

Regarding SRD5A1 markers, no significant changes were observed in circulating sex-329

steroid levels. Among the statistically significant results, our data reveal an accumulation 330

of ADT without changes in steroid glucuronide levels, as observed for SRD5A2 markers. 331

These higher levels of ADT indicate that this variation might enhance enzyme activity, 332

also hypothetically favoring BCR in these patients. As previously suggested (28), this 333

accumulation of ADT combined with the absence of significant changes in T levels in 334

SRD5A carriers may also indicate that the alternate route of androgen biosynthesis not 335

involving T may perhaps predominate in these individuals. Additionally, high levels of 336

ADT in these patients could theoretically fuel DHT synthesis by the concerted action of 337

other pathways such as the HSD17B3 and HSD17B6 enzymes, supporting androgen 338

formation and conceivably recurrence of elevated prostate-specific antigen. 339





20

Unfavorable SNPs in SRD5A2 are also linked to an elevation at the target cell level of 340

other potent steroid hormones such as 5-diol secreted by the adrenals. The mechanism 341

leading to enhanced exposure of 5-diol in these individuals may be a result of additional 342

and complex interplay between 5α-reductases and other key steroidogenic enzymes 343

expressed in the prostate, e.g., CYP17A, HSD3B, and HSD17B. If further confirmed in 344

additional independent studies, this enhanced exposure to 5-diol may be of biological 345

significance in the context of PCa progression because this adrenal precursor was 346

elegantly shown to be a natural hormone with androgenic properties in human PCa cells 347

(29, 30). Indeed, 5-diol can activate the androgen receptor without being metabolized to 348

T or DHT, especially in the presence of the co-activator ARA70 that notably enhances 349

its androgenic properties (29). Moreover, 5-diol also has estrogenic activity at 350

physiological concentrations and can bind the estrogen receptor alpha (ERα), although 351

at low levels in the normal prostate, and trigger an estrogenic response (31). Thus, 352

additional studies of this sex-steroid hormone are warranted in the context of cancer 353

progression at different stages of the disease. Moreover, the level of 3β-diol, an 354

important metabolite of DHT, is also altered in the presence of SRD5A prognostic 355

markers (rs166050 and rs676033) and may potentially impact cancer progression as 356

well because it is the endogenous ligand of ERβ in human prostate (32). ERβ is the 357

most abundant estrogenic receptor in prostatic basal cells and is involved in major 358

cellular pathways including inflammation processes, differentiation, and apoptosis, thus 359

supporting a role for this non-aromatized estrogenic steroid in the prostate (32, 33). 360

361





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Overall, inherited SRD5A variations seem to have noteworthy physiopathological 362

consequences. Indeed, any subtle and persistent variation in sex-steroid hormone levels 363

and/or their relative ratios may modify the course of a slow-progressing disease such as 364

PCa over several years. Data further reinforce the significance of SRD5A genes in 365

hormone metabolism and the biological relevance of previous associations observed 366

between this pathway and clinical outcomes. The assessment of SRD5A markers based 367

on individual patients' germline genetic variations may also lead to a better patient 368

stratification in future 5-ARI clinical trials, targeting therapeutic interventions to optimize 369

hormonal manipulation in patients with a high risk of recurrence and helping to identify 370

patients who would more likely benefit from treatment. However, it is currently unknown 371

if any SRD5A genotypes, such as the low-risk SRD5A2 allele rs12470143 or any of the 372

high-risk variants (rs2208532, rs676033, rs523349), may modify 5-ARI response. The 373

impact of these common germline polymorphisms should certainly be addressed in 374

future studies. 375

376

To our knowledge, this is the first report of sex-steroid measurements in both the 377

circulation and prostatic tissues of PCa patients in relation to inherited genetic markers 378

associated with recurrence. The strengths of the study include the use of gold-standard 379

MS-based sex-steroid assays, fasting blood sampling on the morning of the surgery for 380

all patients with paired prostatic tissues collected at prostatectomy, the selection of 381

SRD5A markers (n=7) associated with biochemical recurrence (9) and the biological 382

plausibility of the association. Limitations are related to 1) the availability of only single 383





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blood and tissue samples for measurement of steroids and 2) quantification of steroids 384

in available PCa samples containing surrounding peritumoral tissues, because no tumor 385

microdissection was performed. The latter may have resulted in potential 386

underestimation of the impact of these germline variants on overall androgen 387

metabolism in cancer samples. Hence, although this is the first report of sex-steroids 388

measured in human prostatic tissues in relation to germline SNPs in key androgenic 389

genes, one must bear in mind that the number of cases assessed was limited owing to 390

the infrequency of acquiring samples from which a relatively large quantity of fresh 391

frozen PCa tissue could be obtained for steroid measurements. Therefore, these 392

analyses are exploratory, with no attempt to correct for multiplicity. Additional studies 393

analyzing both circulating and prostatic tissue levels of a wide range of sex-steroid 394

hormones in PCa patients are clearly necessary to gain important information on steroid 395

biotransformation and PCa progression. 396

397

We conclude that the assessment of host genetic variants in key steroidogenic 398

pathways, such as those governed by SRD5A genes, represents additional indications 399

that the inherited genetic background influences the hormonal microenvironment to 400

which cancer cells are exposed. Our results support that SRD5A genetic variations 401

modify sex-steroid exposure to potentially promote cancer growth and proliferation. 402

Further studies are required to fully characterize at the molecular level the impact of 403

functional variations in SRD5A genes in both normal and PCa cells. Moreover, markers 404

in SRD5A genes, especially the SRD5A1 rs166050, SRD5A2 rs12470143, rs2208532 405





23

and rs676033/rs523349 SNPs, may ultimately represent clinically relevant PCa 406

indicators and lead to more personalized management of the most prevalent cancer in 407

men, especially early in the course of the disease. 408

409





24

Acknowledgements 410

We gratefully thank Dr Alain Bélanger for helpful discussion. The authors would also like 411

to thank the biostatistics services of the clinical research platform (CHUQ research 412

center) for their support as well as Michele Orain, Johanne Ouellette, Étienne Audet-413

Walsh, and Christine Flageole for their technical support in the preparation of tissue 414

samples prior to extraction and measurement of hormones by MS. This work was 415

supported by Canadian research grants from the Cancer Research Society (C.G.), 416

Fonds de recherche du Québec - Santé (FRQS), and the Canada Research Chair 417

Program (C.G.). E.L is recipient of a Prostate Cancer Canada rising star award 418

(RS2013-55). C.G. holds the Canada Research Chair in Pharmacogenomics (Tier II). 419

I.L. and M.L. are both recipients of a Frederick Banting and Charles Best Canada 420

Graduate Scholarship award from CIHR. 421

422





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531

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Table 1. Clinical and pathological characteristics of the study population.

Characteristics Localized

PCa (n=526)

Age at diagnosis (years) Mean SD Range

63.3 6.8

43.5-80.7 Median follow up (months) 88.8 Number (%) Biochemical recurrence 130 (24.7) PSA at diagnosis (ng/mL) ≤10 >10-20 >20

362 (69) 103 (20) 56 (11)

Pathologic Gleason score ≤6 7 ≥8

158 (31) 244 (48) 107 (21)

Pathologic T stage pT = T2 pT = T3a pT ≥ T3b

313 (60) 131 (25) 77 (15)

Nodal invasion N0 N+

481 (92)

44 (8) Neoadjuvant hormonotherapy Yes No

31 (6)

495 (94) Adjuvant hormone therapy Yes No

30 (6)

496 (94) Margin status Negative Positive

368 (70) 154 (30)

D’Amico risk classification Low Intermediate High

187 (36) 208 (40) 122 (24)

PSA, prostate-specific antigen; tumor (T); node (N); Standard deviation (SD).

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Table 2. Prostatic steroid levels in relation to SRD5A1 markers (n=247). Gene Hormone SNP Major/ Secondary model (Dominant) Secondary model (Recessive) P values

Minor Homozygote major ≥ 1 minor allele ≤ 1 minor allele Homozygote minor Dom. Reces.

allele Mean ± SEM* Mean ± SEM* Mean ± SEM* Mean ± SEM*

SRD5A1 DHEA rs518673 C / T 16.49 ± 1.74 16.06 ± 1.74 16.13 ± 1.34 17.46 ± 2.70 0.711 0.710 (ng/g) rs166050 T / C 16.50 ± 1.58 15.93 ± 1.97 15.76 ± 1.27 25.94 ± 5.05 0.647 0.045 5-diol rs518673 C / T 6.43 ± 2.08 5.40 ± 0.72 5.78 ± 1.04 6.07 ± 1.67 0.122 0.689 (ng/g) rs166050 T / C 6.27 ± 1.60 5.29 ± 0.78 5.74 ± 1.01 6.71 ± 1.58 0.134 0.652 Testo rs518673 C / T 619.35 ± 173.61 599.69 ± 125.74 617.34 ± 112.11 527.56 ± 189.12 0.762 0.456 (pg/g) rs166050 T / C 617.40 ± 137.84 584.96 ± 149.90 601.69 ± 100.36 604.77 ± 601.91 0.749 0.930 DHT rs518673 C / T 2.20 ± 0.24 2.08 ± 0.11 2.13 ± 0.13 2.09 ± 0.27 0.467 0.900 (ng/g) rs166050 T / C 2.12 ± 0.12 2.13 ± 0.21 2.10 ± 0.10 2.65 ± 1.09 1.000 0.159 ADT rs518673 C / T 6.77 ± 2.55 7.27 ± 1.91 7.01 ± 1.65 7.51 ± 3.90 0.612 0.752 (ng/g) rs166050 T / C 7.40 ± 2.59 6.62 ± 0.99 6.77 ± 1.59 13.51 ± 4.21 0.386 0.015 3b-diol rs518673 C / T 0.94 ± 0.14 0.91 ± 0.16 0.92 ± 0.12 1.00 ± 0.29 0.664 0.569 (ng/g) rs166050 T / C 1.02 ± 0.18 0.82 ± 0.08 0.92 ± 0.11 1.04 ± 0.13 0.029 0.565

Total steroid levels (geometric mean ± SEM). Data are presented for individuals we had genotyping and tissue assay data.

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Table 3. Prostatic steroid levels in relation to SRD5A2 markers (n=247).

Gene Hormone SNP Major/ Secondary model (Dominant) Secondary model (Recessive) P values Minor Homozygote major ≥ 1 minor allele ≤ 1 minor allele Homozygote minor

Dom. Reces.allele Mean ± SEM* Mean ± SEM* Mean ± SEM* Mean ± SEM*

SRD5A2 DHEA rs12470143 G / A 15.75 ± 2.53 16.50 ± 1.37 16.49 ± 1.41 15.49 ± 2.61 0.677 0.712 (ng/g) rs2208532 A / G 15.91 ± 1.73 16.42 ± 1.64 17.26 ± 1.39 12.94 ± 2.70 0.886 0.020 rs4952197 G / A 16.25 ± 1.41 16.39 ± 2.35 16.41 ± 1.23 14.36 ± 8.75 0.839 0.838 rs523349 G / C 15.84 ± 1.44 16.02 ± 2.14 16.20 ± 1.29 12.25 ± 3.59 0.823 0.288 rs676033 G / A 16.28 ± 1.55 16.21 ± 2.00 16.25 ± 1.29 16.25 ± 4.51 0.991 0.778 5-diol rs12470143 G / A 6.81 ± 2.59 5.36 ± 0.57 6.20 ± 1.21 4.71 ± 0.85 0.066 0.059 (ng/g) rs2208532 A / G 4.93 ± 0.76 6.33 ± 1.39 5.49 ± 0.53 7.19 ± 4.08 0.052 0.089 rs4952197 G / A 5.18 ± 0.58 7.08 ± 2.39 5.80 ± 1.01 5.84 ± 1.38 0.012 0.731 rs523349 G / C 5.16 ± 0.62 6.73 ± 2.17 5.77 ± 1.03 5.43 ± 1.37 0.030 0.969 rs676033 G / A 5.24 ± 0.65 6.62 ± 2.00 5.62 ± 0.98 7.96 ± 3.43 0.058 0.056 Testo rs12470143 G / A 665.13 ± 156.55 564.01 ± 132.88 634.14 ± 114.98 505.79 ± 213.22 0.236 0.138 (pg/g) rs2208532 A / G 501.26 ± 149.70 663.12 ± 132.09 583.70 ± 107.85 670.62 ± 257.55 0.038 0.416 rs4952197 G / A 567.02 ± 127.94 659.71 ± 165.85 591.76 ± 108.04 738.06 ± 224.91 0.268 0.428 rs523349 G / C 561.37 ± 135.73 690.06 ± 169.13 594.29 ± 114.51 813.07 ± 212.95 0.138 0.217 rs676033 G / A 560.20 ± 131.37 663.81 ± 158.92 584.53 ± 112.34 753.39 ± 165.85 0.181 0.263 DHT rs12470143 G / A 2.08 ± 0.17 2.15 ± 0.15 2.11 ± 0.14 2.18 ± 0.16 0.653 0.722 (ng/g) rs2208532 A / G 2.09 ± 0.12 2.14 ± 0.16 2.13 ± 0.13 2.09 ± 0.23 0.785 0.818 rs4952197 G / A 2.08 ± 0.14 2.22 ± 0.20 2.11 ± 0.12 2.55 ± 0.37 0.378 0.237 rs523349 G / C 2.12 ± 0.15 2.10 ± 0.19 2.08 ± 0.12 2.51 ± 0.39 0.922 0.214 rs676033 G / A 2.14 ± 0.16 2.10 ± 0.17 2.07 ± 0.12 2.70 ± 0.39 0.820 0.040 ADT rs12470143 G / A 7.25 ± 3.59 6.95 ± 1.40 7.24 ± 1.96 6.46 ± 1.16 0.843 0.506 (ng/g) rs2208532 A / G 7.03 ± 2.17 7.05 ± 2.02 7.23 ± 1.68 6.39 ± 3.60 0.866 0.385 rs4952197 G / A 7.59 ± 2.18 6.31 ± 1.83 7.02 ± 1.58 8.34 ± 6.05 0.135 0.515 rs523349 G / C 7.48 ± 2.06 6.39 ± 2.37 6.96 ± 1.62 7.80 ± 5.26 0.192 0.642 rs676033 G / A 7.44 ± 2.10 6.56 ± 2.19 6.90 ± 1.58 8.63 ± 5.41 0.275 0.315 3b-diol rs12470143 G / A 0.97 ± 0.30 0.91 ± 0.07 0.96 ± 0.14 0.84 ± 0.07 0.507 0.291 (ng/g) rs2208532 A / G 0.95 ± 0.07 0.92 ± 0.16 0.92 ± 0.12 0.95 ± 0.25 0.734 0.777 rs4952197 G / A 0.91 ± 0.07 0.98 ± 0.27 0.94 ± 0.11 0.88 ± 0.27 0.554 0.938 rs523349 G / C 0.92 ± 0.07 0.93 ± 0.25 0.92 ± 0.11 0.93 ± 0.24 0.890 0.858 rs676033 G / A 0.95 ± 0.08 0.90 ± 0.23 0.90 ± 0.10 1.26 ± 0.52 0.534 0.031

Total steroid levels (geometric mean ± SEM). Data are presented for individuals we had genotyping and tissue assay data. The SNP associated with reduced risk of BCR is underlined; the others are associated with increased risk of BCR.

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Table 4. Levels of circulating steroid glucuronides in relation to SRD5A1 and SRD5A2 markers (n=495).

Gene Hormone SNP Major/ Secondary model (Dominant) Secondary model (Recessive) P values Minor Homozygote major ≥ 1 minor allele ≤ 1 minor allele Homozygote minor

Dom. Reces. allele Mean ± SEM* Mean ± SEM* Mean ± SEM* Mean ± SEM*

SRD5A1 ADT-G rs518673 C / T 29.57 ± 1.46 31.00 ± 2.24 30.26 ± 1.47 31.24 ± 4.78 0.397 0.738 (ng/mL) rs166050 T / C 31.36 ± 2.18 29.14 ± 1.51 30.21 ± 1.46 33.11 ± 4.92 0.178 0.344 3a-diol-3G rs518673 C / T 1.65 ± 0.08 1.62 ± 0.09 1.65 ± 0.07 1.53 ± 0.16 0.787 0.443 (ng/mL) rs166050 T / C 1.66 ± 0.09 1.61 ± 0.09 1.63 ± 0.06 1.72 ± 0.34 0.534 0.420 3a-diol-17G rs518673 C / T 2.98 ± 0.17 3.19 ± 0.13 3.13 ± 0.11 2.85 ± 0.34 0.246 0.351 (ng/mL) rs166050 T / C 3.24 ± 0.15 2.94 ± 0.14 3.12 ± 0.11 2.94 ± 0.37 0.095 0.691

SRD5A2 ADT-G rs12470143 G / A 29.76 ± 3.20 30.68 ± 1.41 29.81 ± 1.69 32.52 ± 2.13 0.611 0.185 (ng/mL) rs2208532 A / G 32.44 ± 1.77 29.40 ± 1.90 31.01 ± 1.25 28.19 ± 4.65 0.028 0.104 rs4952197 G / A 31.09 ± 1.48 29.13 ± 2.78 30.30 ± 1.48 30.73 ± 2.63 0.200 0.865 rs523349 G / C 31.01 ± 1.62 29.57 ± 2.51 30.18 ± 1.10 33.16 ± 14.20 0.401 0.359 rs676033 G / A 31.35 ± 1.66 29.27 ± 2.34 30.25 ± 1.10 31.80 ± 11.16 0.160 0.534 3a-diol-3G rs12470143 G / A 1.63 ± 0.14 1.64 ± 0.07 1.63 ± 0.08 1.66 ± 0.10 0.892 0.772 (ng/mL) rs2208532 A / G 1.66 ± 0.08 1.63 ± 0.09 1.64 ± 0.06 1.62 ± 0.19 0.509 0.689 rs4952197 G / A 1.66 ± 0.07 1.61 ± 0.13 1.62 ± 0.07 1.93 ± 0.19 0.454 0.106 rs523349 G / C 1.66 ± 0.07 1.61 ± 0.12 1.61 ± 0.06 2.00 ± 0.52 0.462 0.036 rs676033 G / A 1.66 ± 0.07 1.61 ± 0.11 1.61 ± 0.06 1.94 ± 0.43 0.458 0.039 3a-diol-17G rs12470143 G / A 2.87 ± 0.17 3.23 ± 0.13 3.02 ± 0.12 3.46 ± 0.23 0.057 0.048 (ng/mL) rs2208532 A / G 3.57 ± 0.21 2.90 ± 0.11 3.18 ± 0.12 2.88 ± 0.18 0.0004 0.118 rs4952197 G / A 3.20 ± 0.15 2.98 ± 0.14 3.10 ± 0.11 3.22 ± 0.34 0.203 0.693 rs523349 G / C 3.21 ± 0.16 2.96 ± 0.13 3.06 ± 0.11 3.61 ± 0.37 0.151 0.174 rs676033 G / A 3.22 ± 0.16 2.98 ± 0.13 3.10 ± 0.11 3.18 ± 0.33 0.141 0.770

Total steroid levels (geometric mean ± SEM). Data are presented for individuals we had genotyping and plasma assay data. SNPs associated with reduced risk of BCR are underlined; the others are associated with increased risk of BCR.

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Figure Legends

Figure 1. Positive associations observed for SRD5A2 markers and circulating

androgen metabolite levels. Graphic representation of variation associated with four

SNPs in SRD5A2 (rs2208532, rs4952197, rs523349 (V89L), and rs676033; gray tone

histograms), which were significantly associated with a higher risk of biochemical

recurrence (BCR), and one SNP (rs12470143; white histograms) with lower risk of BCR

in this cohort of patients. Positive associations were observed with circulating levels of

3α-diol-3G (A), 3α-diol-17G (B), and ADT-G (C). Variation (%) compared to

homozygotes of the major allele is shown on the y axis.

Figure 2. Schematic representation of sex-steroid biosynthesis pathways in PCa

patients. SRD5A2 markers significantly affect the levels of prostatic androgens and

circulating androgen metabolites (ADT-G, 3α-DIOL-3G, 3α-DIOL-17G) whereas the

SRD5A1 rs166050C risk variant is correlated with greater prostatic exposure to DHT.

UGT: UDP-glucuronosyltransferase; 4-dione: androstenedione; ADT: Androsterone;

ADT-G: androsterone-glucuronide; 3α-diol-3G; androstane-3α, 17β-diol 3-glucuronide;

3α-diol-17G: androstane-3α, 17β-diol 17-glucuronide; DHEA: dehydroepiandrosterone;

5-diol: androst-5-ene-3β,17β-diol, 3α-diol: androstane-3α, 17β-diol, Testo: testosterone,

DHT: dihydrotestosterone.




Published OnlineFirst November 25, 2013.Clin Cancer Res Eric Levesque, Isabelle Laverdiere, Louis Lacombe, et al. Circulating and Intraprostatic Androgens in Prostate Cancer

-Reductase Gene Polymorphisms onαImportance of 5

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