Available online at www.sciencedirect.com Journal of Steroid Biochemistry & Molecular Biology 109 (2008) 158–167 Estrone sulfate (E1S), a prognosis marker for tumor aggressiveness in prostate cancer (PCa)ଝ Frank Giton a,∗, Alexandre de la Taille b, Yves Allory b, Herve´ Galons c, Francis Vacherot b, Pascale Soyeux b, Claude Clement´ Abbou b, Sylvain Loric b, Olivier Cussenot b, Jean-Pierre Raynaud d, Jean Fiet b a AP-HP CIB INSERM IMRB U841eq07, Henri Mondor, Facult´edeM´edecine, 94010 Cr´eteil, France b INSERM IMRB U841 eq07, CHU Henri Mondor, Facult´edeM´edecine, 94010 Cr´eteil, France c Service de Chimie organique, Facult´e de Pharmacie Paris V, 75006 Paris, France d Universit´e Pierre et Marie Curie, 75252 Paris, France Received 26 December 2006; accepted 26 October 2007 Abstract Seeking insight into the possible role of estrogens in prostate cancer (PCa) evolution, we assayed serum E2, estrone (E1), and estrone sulfate (E1S) in 349 PCa and 100 benign prostatic hyperplasia (BPH) patients, and in 208 control subjects in the same age range (50–74 years). E1 (pmol/L ± S.D.) and E1S (nmol/L ± S.D.) in the PCa and BPH patients (respectively 126.1 ± 66.1 and 2.82 ± 1.78, and 127.8 ± 56.4 and 2.78 ± 2.12) were significantly higher than in the controls (113.8 ± 47.6 and 2.11 ± 0.96). E2 was not significantly different among the PCa, BPH, and control groups. These assays were also carried out in PCa patients after partition by prognosis (PSA, Gleason score (GS), histological stage, and surgical margins (SM)). Significantly higher E1S levels were found in PCa with: PSA > 10 ng/L (3.05 ± 1.92) versus PSA ≤ 10 ng/mL (2.60 ± 1.55), stage pT3-T4 (2.99 ± 1.80) versus pT2 (2.58 ± 1.58), and positive (3.26 ± 1.95) versus negative margins (2.52 ± 1.48). E1 was higher in poor- than in better-prognosis PCa. E2 was significantly higher in PCa with GS ≥ 4 + 3 (109.5 ± 43.8) versus GS ≤ 3 + 4 (100.6 ± 36.5) and increased significantly when GS increased from 3 + 3 to 4 + 4. Estrogens, especially E1S appeared to be possible markers of PCa progression. Attempting to identify potential sources of E2 in PCa according to prognosis, as well as in BPH, we found a significant correla- tion coefficient between E1S and E2 (0.266–0.347) in poor-prognosis PCa and no correlation in BPH (0.026) and better-prognosis PCa (0.013–0.104). It is as though during progression of PCa from good to poor prognosis there were a shift in the E1 to E2 metabolic pathway from predominantly oxidative to predominantly reductive. © 2008 Elsevier Ltd. All rights reserved. Keywords: Estrone sulfate; Estrogens; Prostate cancer; Tumor aggressiveness 1. Introduction Although the prostate gland is the site of intense steroid metabolism [1], normal and pathological development of the Abbreviations: PCa, prostate cancer; BPH, benign prostatic hyperplasia; prostate depends on the blood hormone environment, i.e., on E1, estrone; E2, estradiol; E1S, estrone sulfate; TT, testosterone; A, 4- androstenedione; GS, Gleason score; HS, histological stage; SM, surgical the concentration of androgens and estrogens synthesized by margins. the gonads and adrenals, as well as on those that derive from ଝ This work has been presented in part at the 17th International Symposium peripheral metabolism in all tissues. Sensitive and specific of the Journal of Steroid Biochemistry and Molecular Biology, Seefeld, Austria, assays of blood androgen and estrogen concentrations could 31 May–3 June 2006. ∗ Corresponding author. Tel.: +33 1 49 81 35 58; fax: +33 1 49 81 35 52. therefore shed light on both normal and pathological prostate E-mail address: [email protected] (F. Giton). function. 0960-0760/$ – see front matter © 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.jsbmb.2007.10.005 F. Giton et al. / Journal of Steroid Biochemistry & Molecular Biology 109 (2008) 158–167 159 Over the years, numerous studies have attempted to evalu- Twelve-hour fasting blood samples were drawn between 8 ate the role of androgens, particularly testosterone, in PCa and and 10 am. Serum was separated from the plasma and divided BPH [2–19]. However, few studies have addressed the role of into several fractions. Each volunteer was subjected to clini- estrogens (estradiol and estrone) in these pathologies, due to dif- cal chemistry testing that included the following parameters: ficulties encountered in measuring their low circulating levels in blood glucose, creatinine, triglycerides, ␥-glutamyltransferase males. Some studies [8,9,17,19] suggested a reduction in the risk (␥-GT), alanine l-aminotransferase (ALAT), and l-aspartate of PCa with higher levels of estradiol (E2), whereas others did aminotransferase (ASAT). Volunteers with abnormal clin- not [6,7,12]. ical chemistry values were excluded from the study. Recent evidence suggests that estrogens significantly con- Serum samples were kept frozen until hormone and PSA tribute to the genesis of prostate cancer (PCa) [20].Inthe assays. presence of androgen, E2 has been shown to stimulate carci- Of the 539 selected healthy men aged 20–74, 268 were over noma in situ and adenocarcinoma in Noble rats [21,22]. The 50 years of age. Among these 268 subjects, 60 were excluded administration of E2 in conjunction with testosterone to Noble for having moderate symptoms of BPH or a PSA > 3 ng/mL, rats results in a higher incidence of PCa than does adminis- abnormal gonadotrophins, low testosterone (<8.67 nmol/L), or ® tration of testosterone alone [23,24]. Toremifene , a selective E1S < 5th or >95th percentiles of the overall population included estrogen receptor modulator (SERM), suppressed the develop- in the study. ment of high-grade of prostatic intraepithelial neoplasia (PIN), In addition, 51 healthy volunteers from among the laboratory and decreased the incidence of adenocarcinoma in the transgenic staff, aged 40–73, provided two blood samples during the same mouse prostate model [25]. day, the first one between 8 and 10 am in the fasting state, and the Estrogen metabolites have been proposed as causative factors second one between 4 and 6 pm, to assess a potential circadian in neoplastic transformation of prostatic epithelium [26,27].To rhythm of serum androgens and estrogens. obtain insight into the possible role of estrogens in the evolu- tion of PCa, we assayed unconjugated serum estrogens (E2,E1) 2.2. Patients and estrone sulfate (E1S) – an estrogen which is not directly active but whose plasma level is the highest – in all consecutive Patients who underwent radical prostatectomy had untreated untreated and newly diagnosed PCa patients who underwent rad- prostate cancer (PCa) diagnosed on the basis of prostate biopsy, ical prostatectomy in our urology service, starting in July 2004 negative computerized tomography (CT-scan) results, and neg- (and still ongoing). The same estrogens were simultaneously ative bone scan. All were treated for clinically localized prostate assayed in patients with BPH (and who underwent surgery for cancer. it), and in controls recruited during the same period. Androgens BPH patients underwent prostatectomy or transurethral pro- such as testosterone (TT) and 4-androstenedione (A) were also static resection for acute urinary retention or severe or moderate measured in PCa and BPH, since they are possible precursors urinary symptoms. None was treated with Finasteride® or for estrogens. Dunasteride®. The patients recruited for this prospective study were con- secutive, but only those with a BMI ≤ 29 kg/m2 (nPCa = 349, 2. Populations studied nBPH = 100) were retained. Blood samples were drawn on the day before surgery at 4 and 6 pm, as per the practice at our 2.1. Healthy volunteers hospital. PCa patients were classified as having a good or a poor prog- Volunteer men aged 20–74 were recruited in a health nosis according to histological criteria: Gleason score (GS), care center (Institut Inter-regional´ pour la Sante´ (IRSA), histological stage (HS), surgical margins (SM), and serum PSA Tours, France). All volunteers with a body mass index (BMI) levels. All participants signed a written informed consent docu- >29 kg/m2 or suffering from chronic disease (diabetes, can- ment. cer, renal disease, liver disease, chronic inflammatory disease, heart or pulmonary disease, and myocardial infarction dur- 3. Methods ing the preceding 3 months) were excluded from the study, as were men under treatment with one or more drugs known 3.1. Histological methods to influence the hypothalamic/pituitary/gonadal axis, such as androgens, gonadotropin-releasing hormone (GnRH) agonists, All specimens obtained from patients who underwent radi- gonadotropins, antiandrogens, corticoids, Synacthen®, thyroid cal prostatectomy were analyzed in our Pathology Department drugs, and pharmacological agents that could elicit hyperpro- laboratory. Each of the 349 surgically removed prostates was lactinemia. weighed, stained, and fixed in 10% formalin. After fixation, a 1- These subjects underwent digital rectal examination for cm piece of tissue was cut from the proximal and distal surgical prostate tumor detection and responded to questions concern- margins (base and apex) and transected into multiple sections. ing their urinary symptoms. None of them was under treatment The remaining prostate gland was cut into sequential sections for benign prostate hyperplasia (BPH) or prostate disease. They from the right and left lobes, from apex to base. The remainder all had no or only moderate symptoms of BPH (n = 10). of the prostate was then embedded in paraffin. The seminal vesi- 160 F. Giton et al. / Journal of Steroid Biochemistry & Molecular Biology 109 (2008) 158–167 cles were cut and separately embedded at the junction of entry for evaporation. The dried residues were re-dissolved in 0.5 mL to the prostate gland. of methanol/water, 25/75 (v/v), for chromatography. Extracapsular extension was diagnosed if tumor was found The second, chromatographic step was carried out using the in the periprostatic soft tissues or was seen penetrating through a VisiprepTM VacuumManifold (Supelco®, Bellefonte, PA16823, fibromuscular capsule and exiting the other side.