A New Nonestrogenic Steroidal Inhibitor of 17β-Hydroxysteroid Dehydrogenase Type I Blocks the Estrogen-Dependent Breast Cancer Tumor Growth Induced by Estrone

Steroid hormones play an important role in the development and differentiation in several tissues (1). They are synthesized by a combined action of enzymes from different families including P450 (CYP) enzymes (2), the short chain dehydrogenases/reductases (SDR) and the aldoketo reductases (AKR; refs. 3–6). 17β-Hydroxysteroid dehydrogenases (17β-HSDs) convert steroids at position 17 to modulate their biologic potency as estrogens and androgens. In fact, this enzyme family transforms the keto-forms of sexual steroids, usually inactive, into the hydroxyl-forms, active over their receptor (7). To date, there are 15 known isoforms of 17β-HSDs, which are cofactor-dependent (8), and all of these belong to the SDR family except 17β-HSD5, which is an AKR enzyme (1). The enzymatic activities associated with the different isoforms of 17β-HSDs are widespread in human tissues, not only in classic steroidogenic tissues, such as the testis, ovary, and placenta, but also in a large series of peripheral intracrine tissues (9). More importantly, each 17β-HSD isoform has a specific tissue distribution (10–12) and displays a selective substrate affinity, and moreover, in intact cells, its activity is unidirectional (reductive or oxidative; refs. 9, 13). These findings indicate that selectivity of drug action could be achieved by targeting a particular 17β-HSD isoform with selective inhibitors. For cancer therapy, the inhibition of oxidative 17β-HSDs, the transformation of the most proliferative cell form (hydroxyl) of hormone into a less potent form (ketone), is not suitable. In contrast, the selective inhibition of reductive 17β-HSDs involved in the transformation of ketosteroids into hydroxysteroids must be encouraged (9). The most extensively characterized of 17β-HSDs is type I (17β-HSD1), which catalyzes the NAD(P)H-dependent reduction of estrone (E1) into the potent estrogen estradiol (E2; Fig. 1A; ref. 14). E1 only has a low affinity for the estrogen receptor (ER) and has to undergo prereceptor activation by 17β-HSD1, a reduction to E2, to bind to the ER with high affinity (15). This enzyme also catalyzes the reduction of DHEA into 5-androstene-3β,17β-diol (5-diol), a weaker estrogen that becomes more important after menopause (16). Expression of 17β-HSD1 is increased in breast tumors of postmenopausal women and the level of expression has prognostic significance (15, 17, 18). Inhibiting 17β-HSD1 activity could thus constitute a valuable way of reducing E2 level with the aim of shrinking breast tumors (19–23).

Our group has previously reported the synthesis of a number of E2 derivatives modified at position 16 (19, 24–28) for use as 17β-HSD1 inhibitors. In one of these studies, CC-156 (16β-(m-carbamoylbenzyl)-E2; Fig. 1B) was identified as a lead compound for the inhibition of 17β-HSD1 (19). Because the carbamoylbenzyl group can be found in the nicotinamide moiety of 17β-HSD1 cofactor (NADPH or NADH), we hypothesized that it could generate a key interaction with an amino acid neighboring the catalytic site. In fact, the m-carbamoylbenzyl seems to be an important characteristic of this new class of 17β-HSD1 inhibitors (19, 29). Despite of its excellent inhibitory activity, it could only reduce 62% of the proliferative activity induced by a physiologic concentration of E1 (0.1 nmol/L) in T-47D estrogen-sensitive (ER⁺) breast cancer cells. The cell growth reduction was not 100% because a weak (38%) estrogenic activity was induced by CC-156 itself, an E2 derivative having a residual estrogenic activity (19). On the basis of these results, we conducted a number of structural modifications at position 3 of CC-156 in an attempt to modulate interaction with important amino acids belonging to the catalytic site and to reduce the undesired residual estrogenic activity. The steroid derivative PBRM [3-(2-bromoethyl)-16β-(m-carbamoylbenzyl)-17β-hydroxy-1,3,5(10)-estratriene (Fig. 1C)] emerged as a potent 17β-HSD1 inhibitor without residual estrogenic activity. After publishing the chemical synthesis and the preliminary data of in vitro assays with PBRM (30), these interesting results now require additional in vitro and in vivo studies. Because PBRM with its bromoethyl group at C3 is likely to be more hydrophobic (cLog P = 6.26) than CC-156 (cLogP = 4.82), the analog inhibitor with a hydroxyl group at C3, and that this may impinge the physicochemical properties, we assessed both inhibitors. In this article, we present the in vitro 17β-HSD1 inhibition and estrogenic activity in T-47D cells of this new C3/C16 derivative of E2, as well as the results of in vivo studies evaluating the plasma concentration, the estrogenicity and 17β-HSD1 inhibitory activity in a breast cancer xenograft model.

Breast cancer cell line T-47D was obtained from the American Type Culture Collection and maintained in a 175 cm² culture flask at 37°C in a humidified atmosphere at 5% CO₂. This cell line was not authenticated in the authors’ laboratory. Cells were grown in RPMI medium supplemented with 10% (v/v) fetal bovine serum (FBS), l-glutamine (2 nmol/L), penicillin (100 IU/mL), streptomycin (100 μg/mL), and estradiol (1 nmol/L).

T-47D cells were seeded in a 24-well plate (3000 cells/well) in 990 μL of medium supplemented with insulin (50 ng/mL) and 5% dextran-coated charcoal-treated FBS, which was used rather than untreated 10% FBS, to remove the remaining steroid hormones. Stock solutions of inhibitors CC-156 and PBRM were previously prepared in ethanol (EtOH) and diluted with culture medium to achieve appropriate concentrations before use. After 24 hours of incubation, 5 μL of the diluted solution was added to the cells to obtain a final concentration ranging from 1 to 10 μmol/L to determine the IC₅₀ value. The final concentration of EtOH in the well was adjusted to 0.1%. In addition, 5 μL of a solution of [¹⁴C]-estrone (American Radiolabeled Chemicals, Inc.) was added to obtain a final concentration of 60 nmol/L. Cells were incubated for 24 hours and each inhibitor was assessed in triplicate. After incubation, the culture medium was removed and labeled steroids (E1 and E2) were extracted with 1 mL of diethyl ether. The organic phases were evaporated to dryness with nitrogen. Residues were dissolved in dichloromethane and dropped on silica gel thin layer chromatography plates (EMD Chemicals Inc.) and eluted with toluene/acetone (4:1) as solvent system. Substrate [¹⁴C]-E1 and metabolite [¹⁴C]-E2 were identified by comparison with reference steroids (E1 and E2) and quantified using the Storm 860 system (Molecular Dynamics). The percentage of transformation and the percentage of inhibition were calculated as follows: % transformation = 100 × [¹⁴C]-E2/([¹⁴C]-E1 + [¹⁴C]-E2) and % of inhibition = 100 × (% transformation without inhibitor −% transformation with inhibitor)/% transformation without inhibitor (31, 32).

Quantification of cell growth was determined by using CellTiter 96 Aqueous Solution Cell Proliferation Assay (Promega) following the manufacturer's instructions. T-47D cells were resuspended with the medium supplemented with insulin (50 ng/mL) and 5% dextran-coated charcoal treated FBS rather than 10% FBS to remove remaining hormones. Aliquots (100 μL) of the cell suspension were seeded in 96-well plates (3000 cells/well). After 48 hours, the medium was changed with a new one containing an appropriate concentration of products to be tested and was replaced every 2 days. Cells were grown either in absence or presence of the compounds for 7 days. To determine the proliferative (estrogenic) activity, the estrogen-sensitive T-47D cells were grown in absence (basal cell proliferation was fixed as 100%) or presence of compounds to be tested at 0.5 to 10 μmol/L. The potent estrogen E2 was used as a reference control. To determine the inhibition of E1-induced cell proliferation, the T-47D cells were grown in the presence of E1 (0.1 nmol/L) without (control) or with the inhibitor at a concentration of 0.5, 1, 2.5, and 5 μmol/L. Stock solutions of PBRM and CC-156 inhibitors were previously prepared in EtOH and diluted with culture medium to achieve appropriate concentrations before use. The cell proliferation without E1 ± inhibitor (control) was fixed as 100%. To determine the potential antiestrogenic activity of inhibitor PBRM, the T-47D (ER⁺) cells were grown in the presence of estrogen E2 (0.1 nmol/L) and pure antiestrogen EM-139 (0.5 μmol/L; ref. 33] or inhibitor PBRM (0.5 μmol/L). The cell proliferation without E2 and tested compounds (control) were fixed as 100%.

A competitive binding assay using a purified full-length recombinant human ERα (Life Technologies) was done as previously described (34, 35). Briefly, each reaction consisted of 1.2 nmol/L rhERα and 2.5 nmol/L [³H]-estradiol in assay buffer (10 mmol/L Tris, 1.5 mmol/L EDTA, 1 mmol/L dithiothreitol, 10% glycerol, 1 mg/mL bovine serum albumin, pH 7.5) with different concentrations of the inhibitors or untritiated estradiol (E2) in a total reaction volume of 100 μL. Stock solutions of PBRM and CC-156 inhibitors were previously prepared in EtOH and 10 μL added to the reaction mixture to achieve appropriate concentrations. Nonspecific binding was determined by incubation with an excess of E2 (1 μmol/L). After an overnight incubation at 4°C, 100 μL of cold 50% hydroxyapatite slurry was added to bind the receptor/ligand complex. After 15 minutes, 1 mL of wash buffer (40 mmol/L Tris, 1 mmol/L EDTA, 1 mmol/L EGTA, 100 mmol/L KCl, pH 7.4) was added and the tubes were centrifuged at 4,500 rpm for 5 minutes at 4°C. The washing step was repeated twice. The radioactivity of the pellet was extracted by incubation with 1 mL of EtOH for 1 hour at room temperature. The suspension was then put into 10 mL of Biodegradable Counting Scintillant and the radioactivity counted with a Wallac 1411 Liquid Scintillation Counter. IC₅₀ values were obtained using GraphPad Prism 5 and relative binding affinity (RBA) values were obtained by using the following equation: (IC₅₀ of 17β-E2/IC₅₀ of tested compound) × 100.

All animals were acclimatized to the environmental conditions (temperature, 22 ± 3°C; humidity, 50 ± 20%; 12-hour light/dark cycles, lights on at 07:15 hours) for at least 3 days before starting the experiment. The animals were allowed free access to water and a certified commercial rodent food (Rodent Diet #T.2018.15, Harlan Teklad) and randomized according to their body weight. The experiments with animals were conducted in an animal facility approved by the Canadian Council on Animal Care (CCAC) and the Association for Assessment and Accreditation of Laboratory Animal Care. The study was conducted in accordance with the CCAC Guide for Care and Use of Experimental Animals. Institutional approval was obtained.

Six week-old male Sprague-Dawley rats (Crl:CD(SD)Br VAF/Plus) weighing approximately 220 g were obtained from Charles River, Inc. The animals were housed as 3 per cage. A pharmacokinetic study was carried out following one subcutaneous injection of the inhibitor at one concentration (2.3 mg/kg of body weight in 0.5 mL of vehicle fluid). The inhibitor was first dissolved in EtOH and thereafter, we added propylene glycol (PG) to obtain a final concentration of EtOH of 8%. During this experiment, the rats were housed individually and were fasted for 8 hours before inhibitor injection but allowed free access to water. Blood samples for determination of inhibitor plasma concentration were collected at the jugular vein (0.4 mL by animals) at target intervals of 3, 7, 12, and 24 hours after dose for PBRM and 3 and 12 hours for CC-156, from 3 rats per time point. After the collection at 7 hours, a replacement fluid (0.9% sodium chloride injection USP) was injected in the rat. Blood samples were collected into Microvette potassium-EDTA (ethylenediamine tetraacetic acid)-coated tube (Sarstedt, Aktiegesellchaft & Co.) and centrifuged at 3,200 rpm for 10 minutes at 4°C. The plasma was collected and stored at −80°C until analyzed by liquid chromatography/tandem mass spectrometry (LC/MS/MS) analysis.

The concentration of the inhibitors (CC-156 and PBRM) was determined by LC/MS/MS analysis using a procedure developed at CHUQ (CHUL) - Research Center (Québec, Canada). Briefly, for extraction from serum, 100 μL of serum sample is transferred to individual tubes and 600 μL of ammonium acetate (1 mmol/L) is added. A methanolic solution (50 μL) containing a steroidal internal standard (compound 48 in ref. 36) is then added to each tube. Samples are transferred on Strata-X SPE columns (Phenomenex), which have been conditioned with 2 mL of methanol and 2 mL of water. Each column is washed with 2 mL of methanol:water (10:90, v/v). The inhibitor is then eluted with 5 mL of methanol containing 5 mmol/L ammonium acetate. Methanol is evaporated at 45°C under inert atmosphere and the residue dissolved in 100 μL of methanol:water (85:15, v/v). Calibration standard curves for CC-156 and PBRM were prepared in serum by extracting the steroidal inhibitor as reported above for samples. For steroid analysis, the high-performance liquid chromatography system uses a 75 × 4.6-mm, 3-μm reversed-phase Luna Phenyl-Hexyl column (Phenomenex) at a flow rate of 0.8 mL/minute. The inhibitor is detected using an API 3000 mass spectrometer equipped with TurboIonSpray (Applied Biosystems). ESI in positive ion mode was used. The area under the curve (AUC) was calculated using the linear trapezoidal rule.

Female ovariectomized (OVX) BALB/c mice weighing approximately 20 g were obtained from Charles River, Inc. The animals were housed 5 per cage. Groups of 5 mice were treated with E1 (0.02 μg/0.1 mL s.c.) or 17β-HSD1 inhibitor at 10, 50, and 250 μg (0.1 mL s.c.) daily for 7 days. The inhibitor or E1 was first dissolved in EtOH and thereafter, we added PG to obtain the appropriate concentration in vehicle fluid (8% EtOH/92% PG). Animals were killed 24 hours after administration of the last dose of compound and uteri and vagina were removed, excised of fat, and weighed. Total body weights of mice were also recorded.

Female OVX BALB/c athymic nude mice weighing approximately 20 g were obtained from Charles River, Inc. The animals were housed 5 per cage. For the inhibition of T-47D tumor growth, 24 hours after a predose of E1 [0.1 μg/0.1 mL of vehicle (8% EtOH/92% PG)] injected subcutaneously per mouse, mice were inoculated subcutaneously with 1 × 10⁷ T-47D cells in 50 μL Matrigel (BD Biosciences) into both flanks of each mouse. T-47D tumor growth was stimulated using E1 (0.1 μg/0.1 mL vehicle s.c.) per mouse per day for 15 days. From day 16, animals with tumors were randomized in function of tumor volume and separated into 3 groups. Group 1 (control mice) was treated 32 days subcutaneously with 0.1 mL of vehicle alone (8% EtOH/92% PG) per mouse per day. Group 2 (E1 0.1 μg) was treated 32 days subcutaneously with E1 (0.1 μg/0.1 mL vehicle s.c.) per mouse per day. Group 3 (E1 0.1 μg + PBRM 250 μg) was treated with E1 (0.1 μg) and PBRM (250 μg) in a combined subcutaneous injection (0.1 mL of vehicle) per mouse per day for 32 days. The inhibitor of E1 was first dissolved in EtOH and thereafter, we added PG to obtain the appropriate concentration in vehicle fluid (8% EtOH/92% PG). The mice were weighed at start, and the volumes of tumors were determined by external caliper twice a week and the greatest longitudinal diameter (length) and the greatest transverse diameter (width) were determined. Tumor area (mm²) was calculated using the formula 1/2 (length × width) × π. The area measured on the first day of treatment was taken as 100%, and changes in tumor size were expressed as a percentage of initial tumor area. At the end of the studies, the mice were terminally anaesthetized, and final body weights and tumor sizes were determined. Uteri and vagina were removed, excised of fat, and weighed (38–40).

Statistical significance was determined according to the multiple range test of Duncan–Kramer (41). P values, which were less than 0.05, were considered as statistically significant.

The IC₅₀ values of PBRM and CC-156 were determined using breast cancer T-47D cell line (Fig. 2), which exerts strong endogenous expression of 17β-HSD1 (23). We can see that PBRM has a good inhibitory effect on 17β-HSD1 with IC₅₀ value of 68 nmol/L. As a reference, inhibitor compound CC-156 already synthesized by our research team, inhibited the enzyme with an IC₅₀ of 27 nmol/L. This IC₅₀ value is in agreement with the previous value of 44 nmol/L obtained using the same cell line, but a different lot of cells and also with a different number of passages (19). PBRM is, thus, only 2.5 times less effective in inhibiting the enzyme than CC-156. In fact, the presence of a bromoethyl at position C3 produces a slight decrease in the potency of PBRM to inhibit the 17β-HSD1 activity. This suggests that the 3-bromoethyl chain generates another kind of interaction with the catalytic site of the enzyme rather than the OH of CC-156 or E1, the natural substrate of the enzyme.

We investigated the effectiveness of PBRM and CC-156 to block the proliferative effect induced by E1 in estrogen-sensitive breast cancer cell line T-47D. We tested the ability of these 17β-HSD1 inhibitors to inhibit the cell growth induced by the transformation of E1 (0.1 nmol/L) into potent estrogen E2. This concentration of E1 is close to the intracellular concentration in breast cancer cells (42). Even though CC-156 inhibitor exerts some estrogenic effects when tested in the absence of E1, we decided to use it as a reference compound. PBRM was able to inhibit the proliferative effect induced by E1 in a concentration-dependent manner (Fig. 3A). At concentrations of 0.5 and 1 μmol/L, CC-156 showed a stronger effect than PBRM (221% vs. 269% and 218% vs. 243%, respectively), which is in accordance with its lower IC₅₀ (27 and 68 nmol/L, respectively). At higher concentrations, however, the inhibitory effect of CC-156 is probably counterbalanced by its residual estrogenic-like proliferation effect on ER⁺ cells and never reached the basal level (100%) of cell proliferation. On the other hand, PBRM reduced the cell growth from 250% to 156% and 125%, at 2.5 and 5 μmol/L, respectively.

The reduction of E1-induced cell proliferation obtained when using inhibitor PBRM could also be the result of an antiestrogenic activity of this E2 derivative. Indeed, an antiestrogenic compound will block the proliferative (estrogenic) effect of E2 mediated by its action on the ER. We, thus, verified the antiestrogenic properties of PBRM to confirm that the observed inhibition of T-47D cell proliferation was due to the inhibition of 17β-HSD1 and not its action on ER. As illustrated in Fig. 3B, the enzyme inhibitor PBRM does not reverse the proliferative effect on ER⁺ cells of E2 (0.1 nmol/L) like the pure antiestrogen EM-139 (33) does. This result suggests that PBRM does not work as an antiestrogenic compound, but acts instead as an inhibitor of E1 into E2 transformation catalyzed by 17β-HSD1.

To detect any undesirable estrogenic activity of 17β-HSD1 inhibitors, cell proliferative assays were carried out on the T-47D cell line, which is known to express the ER⁺ (43). Proliferative activity of compounds PBRM and CC-156 was evaluated at 0.5, 1, 2.5, and 5 μmol/L (Fig. 4A). From the data collected, it is clear that inhibitor CC-156 exerts significant proliferative activities at all concentrations, which is in agreement with our previous studies (19, 30). On the other hand, it is clear that PBRM was not estrogenic at any concentration tested, which underlines the importance of the bromoethyl chain to remove the undesired estrogenicity.

Having assessed the in vitro estrogenic activity of PBRM and CC-156 on ER⁺ cell proliferation, we next investigated their affinity for ERα (Fig. 4B), the predominant receptor isoform involved in estrogenic effect. The concentration at which the unlabeled natural ligand (E2) displaces half the specific binding of [³H]-17β-E2 on ERα (IC₅₀) was determined by computer fitting of the data using nonlinear regression analysis and the RBA then calculated. The RBA of E2 was established as 100%, whereas the RBA for inhibitor CC-156 was 1.5%. Although low, this binding affinity for ERα can explain the proliferative (estrogenic) activity we have measured in the T-47D estrogen-sensitive cell line. Contrary to CC-156, however, no binding affinity was detected for PBRM, the second generation of 17β-HSD1 inhibitor. Thus, these results are clearly in agreement with the findings generated from the in vitro proliferation tests with ER⁺ cells.

To verify that the lack of estrogenicity of PBRM observed in vitro in the T-47D cell proliferation assay translates into the in vivo setting, the estrogenicity of PBRM was investigated using the OVX mouse model by measuring the weight of the uterus (Fig. 5A) and vagina (Fig. 5B), 2 ER⁺ tissues. For the OVX mice control group (OVX-CTR), a low weight of 22 mg was observed for the uterus. However, when administrated subcutaneously to OVX mice, E1 (0.02 μg/mouse/d) is converted into E2 by 17β-HSD1 and we observed a 2.5 times increase in uterine weight compared with OVX-CTR (22 mg vs. 55 mg; P < 0.01). We tested CC-156 as reference at a single dose of 50 μg/mouse/day, because we already know that this compound was estrogenic in vitro and we expected a similar action in vivo. In fact, we could see that at a 50 μg/mouse/day dose uterine growth is stimulated from 22 mg for the OVX-CTR group to 29 mg for OVX-CC-156 group (P < 0.05). In counterpart, weights of the uterus from all PBRM dose groups (10, 50, and 250 μg/mouse/day), were not significantly different to those of the OVX-CTR group after 7 days of treatment (25, 24, and 23 mg, respectively). Thus, these results confirmed that PBRM is nonestrogenic in vivo. The measurement of vagina weights clearly showed the same tendency for PBRM as previously observed with the uterus.

A single subcutaneous injection (2.3 mg/kg) of inhibitors PBRM and CC-156 was given to 2 different groups of rats to determine the inhibitor bioavailability and whether the structural modification in the substituent at position C3 (bromoethyl vs. OH) increases the plasma concentration. The mean plasma concentrations of inhibitors PBRM and CC-156 at different times and the corresponding AUC are presented in Fig. 6A. At first, we found that the maximum plasma concentration (C_max) was attained at 3 hours following injection for both inhibitors. Even if PBRM showed a nonsignificant different C_max compared with CC-156 (73.4 ng/mL vs. 65.7 ng/mL), the plasma concentration for the 2 inhibitors declined differently. CC-156 had almost disappeared in blood 12 hours after the injection (11.3 ng/mL), suggesting that the dose of 2.3 mg/kg is too weak to be administrated only once a day. For PBRM, however, values of 73.8 and 50.7 ng/mL were found after 7 and 12 hours of injection, respectively; the last one was significantly different from the concentration found at the same time for CC-156 (P < 0.05). AUC_0–12h values indicate that PBRM was 1.7 times more available than CC-156 (772 ng*h/mL vs. 445 ng*h/mL; P < 0.01). After 24 hours, a plasma concentration of 11.7 ng/mL was measured for PBRM, and thus, we obtained an AUC_0–24h of 1146 ng*h/mL for it.

After we established that PBRM inhibitor was found in plasma after a one-day single subcutaneous injection, we decided to study the efficacy of PBRM in vivo. Female OVX Balb/c nude mice were inoculated with 1 × 10⁷ T-47D (ER⁺) human breast cancer cells in Matrigel as described in the procedure by Day and colleagues (23), except that the inoculation was made into both flanks of mouse. The mice received E1 (0.1 μg/d), which after its transformation to E2 by 17β-HSD1 stimulates tumor growth. Only mice with tumors that were well established after 15 days of treatment with 0.1 μg E1/mouse s.c. were selected to continue the study. We used the dose of 250 μg/mouse of PBRM because this was the highest dose tested in the in vivo estrogenicity assay that proved to be nonestrogenic. Figure 6B shows the effect of PBRM on the growth of tumors stimulated with 0.1 μg E1/mouse/day. In the first 18 days of treatment, the tumors were not actively growing and maintained their initial size at the beginning of treatment. From day 19, the size of tumors in the control (CTR) group began to decrease until they reached approximately the 74% of the initial size after 28 days and continued at the same level until day 32 (77%). In the E1-treated group, however, tumors grew reaching 136% of their initial size, whereas in the mice-treated E1-PBRM, the growth of the tumors was inhibited (74%), decreasing to the level of the CTR group at the end of treatment (P < 0.01 at days 28 and 32, E1-PBRM vs. E1). Clearly, PBRM blocks the formation of E2 in the tumor through the inhibition of 17β-HSD1 and thus, the tumor growth.

At the end of the study, the body weights of the mice were recorded and the ER⁺ tissues (uterus and vagina) were taken for analysis. There was no effect of either E1 or PBRM on mouse weight over the 32-day treatment period (Supplementary Fig. S1A), indicating that there is no apparent toxicity of PBRM at 250 μg/day/mouse (s.c.). Although uterine and vagina weights were increased significantly in both of the E1-treated groups (P < 0.01, E1 and E1-PBRM vs. CTR), treatment with PBRM had no effect on the E1-stimulated uterine and vaginal weight increase (Supplementary Fig. S1B and S1C). Our results are in agreement with those obtained by other research groups (23, 38), which have shown that despite the fact that the 17β-HSD1 inhibitor was used at a concentration that produces a decrease in tumor volume (by inhibiting the human 17β-HSD1 in xenograft), the doses of E1 (0.1 μmol/L) used stimulated uterine weight gain that could not be reduced at the end of the study. In fact, the main expression of 17β-HSD1 is in the ovary of female rodents (44), and low levels are detected in the uterus only by real-time PCR (45), but not by in situ hybridization (46). Day and colleagues (23) suggested that the lack of effect of 17β-HSD1 inhibitor on uterine and vaginal weight may therefore be due to the higher sensitivity of the uterus than the tumor to circulating estrogens. Because the murine 17β-HSD1 is an ortholog of the human 17β-HSD1 and presents in xenografted tumor (47), it is also possible that PBRM — always tested on human 17β-HSD1— did not inhibit the murine enzyme present in the uterus. Interestingly, our results obtained with the T-47D xenograft tumor model revealed that 17β-HSD1 inhibitor PBRM completely blocks the tumor growth induced by E1, the precursor of potent estrogen E2.

Several groups are working towards the development of 17β-HSD1 inhibitors for clinical use in the treatment of hormone-dependent breast cancer (48). However, to date, only the groups of Day and colleagues (23) and Husen and colleagues (38) have shown the efficacy of an inhibitor in the in vivo treatment of E1-stimulated breast tumors in nude mice, but to our knowledge, no inhibitor is currently in the clinical trial. In our study, we describe the in vitro and in vivo evaluation of a new inhibitor of 17β-HSD1. Known as PBRM, this steroid (E2) derivative has 2 characteristic elements: a carbamoylbenzyl chain at position C16 for 17β-HSD1 inhibition and a 2-bromoethyl side chain at position C3 for removing the residual estrogenic activity associated with its E2 nucleus (30). PBRM has an IC₅₀ value of 68 nmol/L in the whole T-47D cell assay, is nonestrogenic both in vitro and in vivo, and gave a higher plasma concentration when compared with the reference inhibitor CC-156 after one-day subcutaneous injection of 2.3 mg/kg. Most importantly, after 32 days of treatment, tumor sizes of OVX mice treated with E1 and PBRM were completely reduced at the control group level (without E1 treatment). It is noteworthy that for the first time an inhibitor of 17β-HSD1 is able to decrease the final tumor size by 100%. These results strongly imply that in the experimental setting the tumor growth was mainly mediated via E2, produced by the action of 17β-HSD1 expressed in the T-47D cells, and that the remaining E1 seems not to be able to sustain the tumor growth. Another interesting aspect of inhibiting 17β-HSD1 came from the fact that this enzyme also transforms DHEA into 5-diol. As shown by Poulin and Labrie (49), 5-diol at physiologic concentrations, acts as a genuine estrogen in ER⁺ breast cancer cells through its direct interaction with ER. Thus, inhibiting the transformation of DHEA into 5-diol is another way to deprive breast cancer cells of an estrogenic stimulus that becomes more important after menopause (16). Our in vitro and in vivo results clearly highlighted the potential use of PBRM, a new 17β-HSD1 inhibitor, for the treatment of breast cancer, which could be used alternately or sequentially with other drugs against ER-dependent diseases.

R. Maltais has ownership interest (including patents) in PCT/CA2012/000316 and D. Poirier has ownership interest (including patents) in patent application. No potential conflicts of interest were disclosed by the other authors.

Conception and design: D. Ayan, R. Maltais, J. Roy, D. Poirier

Development of methodology: D. Ayan, R. Maltais, J. Roy

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): D. Ayan, D. Poirier

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): D. Ayan

Writing, review, and/or revision of the manuscript: D. Ayan, R. Maltais, J. Roy, D. Poirier

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): D. Ayan

Study supervision: D. Poirier

The authors thank René Bérubé from the CHUQ (CHUL)-Bioanalytical Service for the plasma inhibitor concentration determination and Sonia Francoeur and France Létourneau for their assistance in the in vivo experiments. The authors also thank Charles Ouellet who conducted the estrogen receptor binding assay (Fig. 4B). Careful reading of the manuscript by Ms. Micheline Harvey is also greatly appreciated.

This work was supported by a grant (MOP-43994 to D. Poirier) from the Canadian Institutes of Health Research (CIHR).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.