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SRD5A polymorphisms and androgens in PCa
1
Importance of 5α-Reductase Gene Polymorphisms on Circulating and 1
Intraprostatic Androgens in Prostate Cancer 2
3
Éric Lévesque1,2, Isabelle Laverdière1, Louis Lacombe2, Patrick Caron1, Mélanie 4
Rouleau1, Véronique Turcotte1, Bernard Têtu2, Yves Fradet2 and Chantal Guillemette1† 5
6
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
11
Running head: SRD5A polymorphisms and androgens in PCa 12
13
†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
19
Key words: prostate cancer, germline polymorphisms, SRD5A, 5-alpha-reductase, 20
circulating and intraprostatic hormone levels. 21
22
23
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SRD5A polymorphisms and androgens in PCa
2
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
27
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
41
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SRD5A polymorphisms and androgens in PCa
3
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
54
55
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SRD5A polymorphisms and androgens in PCa
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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
65
66
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SRD5A polymorphisms and androgens in PCa
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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
84
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SRD5A polymorphisms and androgens in PCa
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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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SRD5A polymorphisms and androgens in PCa
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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
110
111
112
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SRD5A polymorphisms and androgens in PCa
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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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SRD5A polymorphisms and androgens in PCa
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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
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SRD5A polymorphisms and androgens in PCa
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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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SRD5A polymorphisms and androgens in PCa
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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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SRD5A polymorphisms and androgens in PCa
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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
227
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SRD5A polymorphisms and androgens in PCa
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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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SRD5A polymorphisms and androgens in PCa
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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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SRD5A polymorphisms and androgens in PCa
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with an increase and decrease, respectively, in prostate volume at time of surgery (not 271
shown). 272
273
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SRD5A polymorphisms and androgens in PCa
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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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SRD5A polymorphisms and androgens in PCa
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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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SRD5A polymorphisms and androgens in PCa
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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
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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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SRD5A polymorphisms and androgens in PCa
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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
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SRD5A polymorphisms and androgens in PCa
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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
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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
532
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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.
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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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