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Therapeutic Discovery

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

Diana Ayan, Ren�e Maltais, Jenny Roy, and Donald Poirier

Abstract17b-Hydroxysteroid dehydrogenase type 1 (17b-HSD1) converts estrone (E1) into estradiol (E2) and is

expressed inmany steroidogenic tissues and breast cancer cell lines. Because the potent estrogen E2 stimulates

the growth and development of hormone-dependent diseases, inhibition of the final step of E2 synthesis is

considered a promising strategy for the treatment of breast cancer. On the basis of our previous study

identifying 16b-(m-carbamoylbenzyl)-E2 (CC-156) as a lead compound for the inhibition of 17b-HSD1, weconducted anumber of structuralmodifications to reduce its undesired residual estrogenic activity. The steroid

derivative PBRM [3-(2-bromoethyl)-16b-(m-carbamoylbenzyl)-17b-hydroxy-1,3,5(10)-estratriene] emerged asa potent inhibitor of 17b-HSD1 with an IC50 value of 68 nmol/L for the transformation of E1 into E2. Whentested in the estrogen-sensitive breast cancer cell lineT-47Dand inmice, PBRMshowednoestrogenic activity in

the range of concentrations tested. Furthermore, with the purpose of evaluating the bioavailability of PBRM

and CC-156 injected subcutaneously (2.3 mg/kg), we measured their plasmatic concentrations as a function

of time, calculated the area under the curve (AUC0–12h) and showed a significant improvement for PBRM

(772 ng�h/mL) compared with CC-156 (445 ng�h/mL). We next tested the in vivo efficiency of PBRM on the T-47Dxenograft tumormodel in female ovariectomized athymic nudemice.After a treatmentwith PBRM, tumor

sizes in mice stimulated with exogenous E1 were completely reduced at the control group level (without E1

treatment). As a conclusion, PBRM is a promising nonestrogenic inhibitor of 17b-HSD1 for the treatment ofestrogen-dependent diseases such as breast cancer. Mol Cancer Ther; 11(10); 2096–104. �2012 AACR.

IntroductionSteroid hormones play an important role in the devel-

opment and differentiation in several tissues (1). They aresynthesized by a combined action of enzymes from dif-ferent families including P450 (CYP) enzymes (2), theshort chain dehydrogenases/reductases (SDR) and thealdoketo reductases (AKR; refs. 3–6). 17b-Hydroxysteroiddehydrogenases (17b-HSDs) convert steroids at position17 to modulate their biologic potency as estrogens andandrogens. In fact, this enzyme family transforms theketo-forms of sexual steroids, usually inactive, into thehydroxyl-forms, active over their receptor (7). To date,there are 15 known isoforms of 17b-HSDs, which arecofactor-dependent (8), and all of these belong to the SDR

family except 17b-HSD5,which is anAKRenzyme (1). Theenzymatic activities associatedwith thedifferent isoformsof 17b-HSDs arewidespread in human tissues, not only inclassic steroidogenic tissues, such as the testis, ovary, andplacenta, but also in a large series of peripheral intracrinetissues (9).More importantly, each 17b-HSD isoformhas aspecific tissue distribution (10–12) anddisplays a selectivesubstrate affinity, andmoreover, in intact cells, its activityis unidirectional (reductive or oxidative; refs. 9, 13). Thesefindings indicate that selectivity of drug action could beachieved by targeting a particular 17b-HSD isoform withselective inhibitors. For cancer therapy, the inhibition ofoxidative 17b-HSDs, the transformation of the most pro-liferative cell form (hydroxyl) of hormone into a lesspotent form (ketone), is not suitable. In contrast, theselective inhibition of reductive 17b-HSDs involved inthe transformation of ketosteroids into hydroxysteroidsmust be encouraged (9). The most extensively character-izedof 17b-HSDs is type I (17b-HSD1),which catalyzes theNAD(P)H-dependent reduction of estrone (E1) into thepotent estrogen estradiol (E2; Fig. 1A; ref. 14). E1 only hasa low affinity for the estrogen receptor (ER) and has toundergo prereceptor activation by 17b-HSD1, a reductiontoE2, to bind to the ERwithhigh affinity (15). This enzymealso catalyzes the reduction of DHEA into 5-androstene-3b,17b-diol (5-diol), a weaker estrogen that becomesmore

Authors' Affiliation: Laboratory of Medicinal Chemistry, CHUQ (CHUL) -Research Center (Endocrinology and Genomic Unit) and Laval University(Faculty of Medicine), Quebec, Canada.

Note: Supplementary data for this article are available at Molecular CancerTherapeutics Online (http://mct.aacrjournals.org/).

Corresponding Author: Dr. Donald Poirier, Laboratory of MedicinalChemistry, CHUQ (CHUL)-Research Center, 2705 Laurier Boulevard,Quebec G1V 4G2, Canada. Phone: 418-654-2296; Fax: 418-654-2761;E-mail: [email protected]

doi: 10.1158/1535-7163.MCT-12-0299

�2012 American Association for Cancer Research.

MolecularCancer

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important after menopause (16). Expression of 17b-HSD1is increased in breast tumors of postmenopausal womenand the level of expression has prognostic significance(15, 17, 18). Inhibiting 17b-HSD1 activity could thus con-stitute a valuable way of reducing E2 level with the aim ofshrinking 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 17b-HSD1 inhibitors. In one of these studies,CC-156 (16b-(m-carbamoylbenzyl)-E2; Fig. 1B) was iden-tified as a lead compound for the inhibition of 17b-HSD1(19). Because the carbamoylbenzyl group can be found inthe nicotinamide moiety of 17b-HSD1 cofactor (NADPHor NADH), we hypothesized that it could generate a keyinteraction with an amino acid neighboring the catalyticsite. In fact, them-carbamoylbenzyl seems to be an impor-tant characteristic of this new class of 17b-HSD1 inhibitors(19, 29). Despite of its excellent inhibitory activity, it couldonly reduce 62% of the proliferative activity induced by aphysiologic concentration of E1 (0.1 nmol/L) in T-47Destrogen-sensitive (ERþ) breast cancer cells. The cellgrowth reduction was not 100% because a weak (38%)estrogenic activity was induced by CC-156 itself, an E2derivative having a residual estrogenic activity (19). Onthe basis of these results, we conducted a number ofstructural modifications at position 3 of CC-156 in anattempt to modulate interaction with important aminoacids belonging to the catalytic site and to reduce the

undesired residual estrogenic activity. The steroid deriv-ative PBRM [3-(2-bromoethyl)-16b-(m-carbamoylbenzyl)-17b-hydroxy-1,3,5(10)-estratriene (Fig. 1C)] emerged as apotent 17b-HSD1 inhibitor without residual estrogenicactivity. After publishing the chemical synthesis and thepreliminary data of in vitro assays with PBRM (30), theseinteresting results now require additional in vitro and invivo studies. Because PBRMwith its bromoethyl group atC3 is likely to be more hydrophobic (cLog P ¼ 6.26) thanCC-156 (cLogP¼ 4.82), the analog inhibitorwith a hydrox-yl group at C3, and that this may impinge the physico-chemical properties, we assessed both inhibitors. In thisarticle, we present the in vitro 17b-HSD1 inhibition andestrogenic activity in T-47D cells of this new C3/C16derivative of E2, as well as the results of in vivo studiesevaluating the plasma concentration, the estrogenicityand 17b-HSD1 inhibitory activity in a breast cancer xeno-graft model.

Materials and MethodsIn vitro studies

Cell culture. Breast cancer cell line T-47Dwas obtainedfrom the American Type Culture Collection and main-tained in a 175 cm2 culture flask at 37�C in a humidifiedatmosphere at 5%CO2. This cell linewas not authenticatedin the authors’ laboratory. Cells were grown in RPMImedium supplementedwith 10% (v/v) fetal bovine serum(FBS), L-glutamine (2 nmol/L), penicillin (100 IU/mL),streptomycin (100 mg/mL), and estradiol (1 nmol/L).

17b-HSD1 inhibition assay. T-47D cells were seededin a 24-well plate (3000 cells/well) in 990 mL of mediumsupplemented with insulin (50 ng/mL) and 5% dextran-coated charcoal-treated FBS, which was used rather thanuntreated 10% FBS, to remove the remaining steroidhormones. Stock solutions of inhibitorsCC-156 andPBRMwere previously prepared in ethanol (EtOH) and dilutedwith culture medium to achieve appropriate concentra-tions before use. After 24 hours of incubation, 5 mL of thediluted solution was added to the cells to obtain a finalconcentration ranging from 1 to 10 mmol/L to determinethe IC50 value. The final concentration of EtOH in thewellwas adjusted to 0.1%. In addition, 5 mL 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 wasassessed in triplicate. After incubation, the culture medi-um was removed and labeled steroids (E1 and E2) wereextracted with 1 mL of diethyl ether. The organic phaseswere evaporated to dryness with nitrogen. Residues weredissolved in dichloromethane and dropped on silica gelthin layer chromatography plates (EMD Chemicals Inc.)and eluted with toluene/acetone (4:1) as solvent system.Substrate [14C]-E1 andmetabolite [14C]-E2were identifiedby comparison with reference steroids (E1 and E2) andquantified using the Storm 860 system (MolecularDynamics). The percentage of transformation and thepercentage of inhibition were calculated as follows: %

Figure 1. A, 17b-Hydroxysteroid dehydrogenase type I (17b-HSD1)transforms estrone (E1) to estradiol (E2) in presence of cofactor NAD(P)H.B, 17b-HSD1 steroidal inhibitor CC-156 (16b-(m-carbamoylbenzyl)-E2.C, 17b-HSD1 nonestrogenic steroidal inhibitor PBRM (3-(2-bromoethyl)-16b-(m-carbamoylbenzyl)-17b-hydroxy-1,3,5(10)-estratriene).

A New 17b-HSD1 Inhibitor Blocks E1-Sensitive Tumor Growth

www.aacrjournals.org Mol Cancer Ther; 11(10) October 2012 2097




transformation ¼ 100 � [14C]-E2/([14C]-E1 þ [14C]-E2)and % of inhibition ¼ 100 � (% transformation withoutinhibitor �% transformation with inhibitor)/% transfor-mation without inhibitor (31, 32).

Cell proliferation assays (17b-HSD1 inhibitory, estro-genic, and antiestrogenic activities). Quantification ofcell growth was determined by using CellTiter 96 Aque-ous SolutionCell ProliferationAssay (Promega) followingthe manufacturer’s instructions. T-47D cells were resus-pended with the medium supplemented with insulin(50 ng/mL) and 5% dextran-coated charcoal treated FBSrather than 10% FBS to remove remaining hormones.Aliquots (100 mL) of the cell suspension were seeded in96-well plates (3000 cells/well). After 48 hours, the medi-um was changed with a new one containing an appro-priate concentration of products to be tested and wasreplaced every 2 days. Cells were grown either in absenceor presence of the compounds for 7 days. Todetermine theproliferative (estrogenic) activity, the estrogen-sensitiveT-47D cellswere grown in absence (basal cell proliferationwas fixed as 100%) or presence of compounds to be testedat 0.5 to 10 mmol/L. The potent estrogen E2 was used as areference control. To determine the inhibition of E1-induced cell proliferation, the T-47D cells were grown inthe presence of E1 (0.1 nmol/L) without (control) or withthe inhibitor at a concentration of 0.5, 1, 2.5, and 5 mmol/L.Stock solutions of PBRM and CC-156 inhibitors werepreviously prepared in EtOH and diluted with culturemedium to achieve appropriate concentrations beforeuse.The cell proliferation without E1� inhibitor (control) wasfixed as 100%. To determine the potential antiestrogenicactivity of inhibitor PBRM, the T-47D (ERþ) cells weregrown in the presence of estrogen E2 (0.1 nmol/L) andpure antiestrogenEM-139 (0.5mmol/L; ref. 33] or inhibitorPBRM (0.5 mmol/L). The cell proliferationwithout E2 andtested compounds (control) were fixed as 100%.

ERa binding assay. A competitive binding assayusing a purified full-length recombinant humanERa (LifeTechnologies) was done as previously described (34, 35).Briefly, each reaction consisted of 1.2 nmol/L rhERa and2.5 nmol/L [3H]-estradiol in assay buffer (10 mmol/LTris, 1.5 mmol/L EDTA, 1 mmol/L dithiothreitol, 10%glycerol, 1 mg/mL bovine serum albumin, pH 7.5) withdifferent concentrations of the inhibitors or untritiatedestradiol (E2) in a total reaction volume of 100 mL. Stocksolutions of PBRMandCC-156 inhibitorswere previouslyprepared inEtOHand10mLadded to the reactionmixtureto achieve appropriate concentrations. Nonspecific bind-ing was determined by incubation with an excess of E2(1mmol/L).After anovernight incubation at 4�C, 100mLofcold 50% hydroxyapatite slurry was added to bind thereceptor/ligand complex. After 15 minutes, 1 mL of washbuffer (40 mmol/L Tris, 1 mmol/L EDTA, 1 mmol/LEGTA, 100 mmol/L KCl, pH 7.4) was added and thetubes were centrifuged at 4,500 rpm for 5 minutes at 4�C.Thewashing stepwas repeated twice. The radioactivity ofthe pellet was extracted by incubation with 1 mL of EtOHfor 1 hour at room temperature. The suspension was then

put into 10 mL of Biodegradable Counting Scintillant andthe radioactivity counted with a Wallac 1411 Liquid Scin-tillation Counter. IC50 values were obtained using Graph-Pad Prism 5 and relative binding affinity (RBA) valueswere obtained by using the following equation: (IC50 of17b-E2/IC50 of tested compound) � 100.

In vivo studiesAnimals. All animals were acclimatized to the envi-

ronmental conditions (temperature, 22 � 3�C; humidity,50 � 20%; 12-hour light/dark cycles, lights on at 07:15hours) for at least 3 days before starting the experiment.The animals were allowed free access to water and acertified commercial rodent food (Rodent Diet #T.2018.15,Harlan Teklad) and randomized according to their bodyweight. The experiments with animals were conducted inan animal facility approved by the Canadian Council onAnimal Care (CCAC) and the Association for Assessmentand Accreditation of Laboratory Animal Care. The studywas conducted in accordance with the CCAC Guide forCare and Use of Experimental Animals. Institutionalapproval was obtained.

Plasmatic concentration of inhibitor after a singlesubcutaneous injection. Six week-old male Sprague-Dawley rats (Crl:CD(SD)Br VAF/Plus) weighing approx-imately 220 g were obtained from Charles River, Inc. Theanimals were housed as 3 per cage. A pharmacokineticstudy was carried out following one subcutaneous injec-tion of the inhibitor at one concentration (2.3 mg/kg ofbody weight in 0.5 mL of vehicle fluid). The inhibitor wasfirst dissolved in EtOH and thereafter, we added propyl-ene glycol (PG) to obtain a final concentration of EtOH of8%. During this experiment, the rats were housed indi-vidually and were fasted for 8 hours before inhibitorinjection but allowed free access to water. Blood samplesfor determination of inhibitor plasma concentration werecollected at the jugular vein (0.4 mL by animals) at targetintervals of 3, 7, 12, and24hours afterdose forPBRMand3and 12 hours for CC-156, from 3 rats per time point. Afterthe collection at 7 hours, a replacement fluid (0.9% sodiumchloride injection USP) was injected in the rat. Bloodsamples were collected into Microvette potassium-EDTA(ethylenediamine tetraacetic acid)-coated tube (Sarstedt,Aktiegesellchaft&Co.) and centrifugedat 3,200 rpmfor 10minutes at 4�C. The plasma was collected and stored at�80�Cuntil analyzed by liquid chromatography/tandemmass spectrometry (LC/MS/MS) analysis.

Measurement of plasma concentrations. The concen-tration of the inhibitors (CC-156 and PBRM) was deter-mined by LC/MS/MS analysis using a proceduredeveloped at CHUQ (CHUL) - Research Center (Qu�ebec,Canada). Briefly, for extraction from serum, 100 mL ofserumsample is transferred to individual tubesand600mLof ammonium acetate (1mmol/L) is added. Amethanolicsolution (50 mL) containing a steroidal internal standard(compound 48 in ref. 36) is then added to each tube.Samples are transferred on Strata-X SPE columns (Phe-nomenex), which have been conditioned with 2 mL of

Ayan et al.

Mol Cancer Ther; 11(10) October 2012 Molecular Cancer Therapeutics2098




methanol and 2mL ofwater. Each column is washedwith2 mL of methanol:water (10:90, v/v). The inhibitor is theneluted with 5 mL of methanol containing 5 mmol/Lammoniumacetate.Methanol is evaporated at 45�Cunderinert atmosphere and the residue dissolved in 100 mL ofmethanol:water (85:15, v/v). Calibration standard curvesfor CC-156 and PBRMwere prepared in serum by extract-ing the steroidal inhibitor as reported above for samples.For steroid analysis, the high-performance liquid chro-matography system uses a 75 � 4.6-mm, 3-mm reversed-phaseLunaPhenyl-Hexyl column (Phenomenex) at aflowrate of 0.8 mL/minute. The inhibitor is detected using anAPI 3000 mass spectrometer equipped with TurboIon-Spray (Applied Biosystems). ESI in positive ionmodewasused. The area under the curve (AUC) was calculatedusing the linear trapezoidal rule.In vivo estrogenicity assay. Female ovariectomized

(OVX) BALB/c mice weighing approximately 20 g wereobtained from Charles River, Inc. The animals werehoused 5 per cage. Groups of 5 mice were treated withE1 (0.02 mg/0.1 mL s.c.) or 17b-HSD1 inhibitor at 10, 50,and250mg (0.1mL s.c.) daily for 7days. The inhibitor or E1was first dissolved in EtOH and thereafter, we added PGto obtain the appropriate concentration in vehicle fluid(8% EtOH/92% PG). Animals were killed 24 hours afteradministration of the last dose of compound anduteri andvagina were removed, excised of fat, and weighed. Totalbody weights of mice were also recorded.Inhibition of E1-stimulated T-47D tumor growth in

nude OVX mice (xenograft model). Female OVXBALB/c athymic nude mice weighing approximately20 g were obtained from Charles River, Inc. The animalswere housed 5 per cage. For the inhibition of T-47D tumorgrowth, 24 hours after a predose of E1 [0.1 mg/0.1 mL ofvehicle (8% EtOH/92% PG)] injected subcutaneously permouse,micewere inoculated subcutaneouslywith 1� 107T-47D cells in 50 mL Matrigel (BD Biosciences) into bothflanks of each mouse. T-47D tumor growth was stimulat-ed using E1 (0.1 mg/0.1mL vehicle s.c.) permouse per dayfor 15 days. From day 16, animals with tumors wererandomized in function of tumor volume and separatedinto 3 groups. Group 1 (control mice) was treated 32 dayssubcutaneously with 0.1 mL of vehicle alone (8% EtOH/92% PG) per mouse per day. Group 2 (E1 0.1 mg) wastreated 32 days subcutaneously with E1 (0.1 mg/0.1 mLvehicle s.c.) per mouse per day. Group 3 (E1 0.1 mg þPBRM 250 mg) was treated with E1 (0.1 mg) and PBRM(250 mg) in a combined subcutaneous injection (0.1 mL ofvehicle) permouse per day for 32 days. The inhibitor of E1was first dissolved in EtOH and thereafter, we added PGto obtain the appropriate concentration in vehicle fluid(8% EtOH/92% PG). The mice were weighed at start, andthe volumes of tumors were determined by externalcaliper twice a week and the greatest longitudinal diam-eter (length) and the greatest transverse diameter (width)weredetermined. Tumor area (mm2)was calculatedusingthe formula 1/2 (length�width)� p. The areameasuredon the first day of treatment was taken as 100%, and

changes in tumor size were expressed as a percentage ofinitial tumor area. At the end of the studies, themice wereterminally anaesthetized, and final body weights andtumor sizes were determined. Uteri and vagina wereremoved, excised of fat, and weighed (38–40).

Statistical analysisStatistical significance was determined according to the

multiple range test of Duncan–Kramer (41). P values,which were less than 0.05, were considered as statisticallysignificant.

Results and Discussion17b-HSD1 inhibitory activity

The IC50 values of PBRM and CC-156 were determinedusing breast cancer T-47D cell line (Fig. 2), which exertsstrong endogenous expression of 17b-HSD1 (23). We cansee that PBRM has a good inhibitory effect on 17b-HSD1with IC50 value of 68 nmol/L. As a reference, inhibitorcompound CC-156 already synthesized by our researchteam, inhibited the enzymewith an IC50 of 27 nmol/L. ThisIC50 value is in agreement with the previous value of44 nmol/Lobtainedusing the same cell line, but a differentlot of cellsandalsowithadifferentnumberofpassages (19).PBRM is, thus, only 2.5 times less effective in inhibiting theenzyme than CC-156. In fact, the presence of a bromoethylat position C3 produces a slight decrease in the potency ofPBRM to inhibit the 17b-HSD1 activity. This suggests thatthe 3-bromoethyl chain generates another kind of interac-tionwith the catalytic site of the enzyme rather than theOHof CC-156 or E1, the natural substrate of the enzyme.

Inhibition of E1-stimulated cell proliferationWe investigated the effectiveness of PBRM and CC-156

to block the proliferative effect induced by E1 in estrogen-

Figure 2. Inhibitory potency of PBRM and CC-156 in T-47D intact cells.Breast cancer cells expressing 17b-HSD1 were incubated with variousconcentrations of inhibitors for 24 hours in presence of labeled [14C]-E1(60 nmol/L). IC50 represents the concentration that inhibited 17b-HSD1activity by 50%. Results are the mean (� SEM) of a triplicate.






sensitive breast cancer cell line T-47D.We tested the abilityof these 17b-HSD1 inhibitors to inhibit the cell growthinduced by the transformation of E1 (0.1 nmol/L) intopotent estrogen E2. This concentration of E1 is close to theintracellular concentration in breast cancer cells (42). Eventhough CC-156 inhibitor exerts some estrogenic effectswhen tested in the absence of E1, we decided to use it asa reference compound. PBRMwas able to inhibit the proli-ferative effect induced by E1 in a concentration-dependentmanner (Fig. 3A). At concentrations of 0.5 and 1 mmol/L,CC-156 showed a stronger effect than PBRM (221% vs.269% and 218% vs. 243%, respectively), which is in accor-dancewith its lower IC50 (27 and 68 nmol/L, respectively).At higher concentrations, however, the inhibitory effect ofCC-156 is probably counterbalanced by its residual estro-genic-like proliferation effect on ERþ cells and neverreached the basal level (100%) of cell proliferation. On theother hand, PBRM reduced the cell growth from 250% to156% and 125%, at 2.5 and 5 mmol/L, respectively.

The reduction of E1-induced cell proliferation obtainedwhen using inhibitor PBRM could also be the result of anantiestrogenic activity of this E2 derivative. Indeed, anantiestrogenic compound will block the proliferative(estrogenic) effect of E2 mediated by its action on the ER.We, thus, verified theantiestrogenicproperties ofPBRMtoconfirm that the observed inhibition of T-47D cell prolif-eration was due to the inhibition of 17b-HSD1 and not itsaction onER.As illustrated inFig. 3B, the enzyme inhibitorPBRMdoesnot reverse the proliferative effect onERþ cellsof E2 (0.1 nmol/L) like the pure antiestrogen EM-139 (33)does. This result suggests that PBRM does not work as anantiestrogenic compound, but acts instead as an inhibitorof E1 into E2 transformation catalyzed by 17b-HSD1.

Estrogenic activity onT-47D (ERþ) cell line and ERa-binding affinity

To detect any undesirable estrogenic activity of 17b-HSD1 inhibitors, cell proliferative assays were carried outon the T-47D cell line, which is known to express the ERþ

(43). Proliferative activity of compounds PBRM and CC-156 was evaluated at 0.5, 1, 2.5, and 5 mmol/L (Fig. 4A).

Figure 3. A, cell growth of T-47D cells induced by a physiologicconcentration of E1 (0.1 nmol/L) in the presence or absence of inhibitorsPBRM and CC-156 at various concentrations. Control (CTR) is fixed at100%.Results are expressedasmean (�SEM) of triplicate. a,P

From the data collected, it is clear that inhibitor CC-156exerts significant proliferative activities at all concentra-tions, which is in agreement with our previous studies(19, 30). On the other hand, it is clear that PBRM was notestrogenic at any concentration tested, which underlinesthe importance of the bromoethyl chain to remove theundesired estrogenicity.Having assessed the in vitro estrogenic activity of PBRM

andCC-156onERþ cell proliferation,wenext investigatedtheir affinity for ERa (Fig. 4B), the predominant receptorisoform involved in estrogenic effect. The concentration atwhich the unlabeled natural ligand (E2) displaces half thespecific binding of [3H]-17b-E2 on ERa (IC50) was deter-mined by computer fitting of the data using nonlinearregression analysis and the RBA then calculated. The RBAof E2 was established as 100%, whereas the RBA forinhibitor CC-156 was 1.5%. Although low, this bindingaffinity for ERa can explain the proliferative (estrogenic)activitywehavemeasured in theT-47Destrogen-sensitivecell line. Contrary to CC-156, however, no bindingaffinity was detected for PBRM, the second generation of17b-HSD1 inhibitor. Thus, these results are clearly inagreement with the findings generated from the in vitroproliferation tests with ERþ cells.

Estrogenic activity of inhibitors in miceTo verify that the lack of estrogenicity of PBRMobserved

in vitro in the T-47D cell proliferation assay translates intothe in vivo setting, the estrogenicity of PBRM was investi-gated using the OVX mouse model by measuring theweight of the uterus (Fig. 5A) and vagina (Fig. 5B), 2ERþ tissues. For the OVX mice control group (OVX-CTR),a low weight of 22 mg was observed for the uterus. How-ever,when administrated subcutaneously toOVXmice, E1(0.02 mg/mouse/d) is converted into E2 by 17b-HSD1 andwe observed a 2.5 times increase in uterine weight com-paredwithOVX-CTR (22mgvs. 55mg;P < 0.01).We testedCC-156 as reference at a single dose of 50 mg/mouse/day,because we already know that this compound was estro-genic invitroandweexpectedasimilaraction invivo. In fact,we could see that at a 50 mg/mouse/day dose uterinegrowth is stimulated from 22 mg for the OVX-CTR groupto 29 mg for OVX-CC-156 group (P < 0.05). In counterpart,weights of the uterus from all PBRM dose groups (10, 50,and250mg/mouse/day),werenot significantlydifferent tothose of the OVX-CTR group after 7 days of treatment (25,24, and 23mg, respectively). Thus, these results confirmedthat PBRM is nonestrogenic in vivo. The measurement ofvagina weights clearly showed the same tendency forPBRM as previously observed with the uterus.

Plasma concentration of inhibitorsA single subcutaneous injection (2.3 mg/kg) of inhibi-

tors PBRM and CC-156 was given to 2 different groups ofrats to determine the inhibitor bioavailability andwhetherthe structural modification in the substituent at positionC3 (bromoethyl vs. OH) increases the plasma concentra-tion. Themean plasma concentrations of inhibitors PBRM

andCC-156 at different times and the correspondingAUCare presented in Fig. 6A. At first, we found that themaximum plasma concentration (Cmax) was attained at3 hours following injection for both inhibitors. Even ifPBRM showed a nonsignificant different Cmax comparedwith CC-156 (73.4 ng/mL vs. 65.7 ng/mL), the plasmaconcentration for the 2 inhibitors declineddifferently. CC-156 had almost disappeared in blood 12 hours after theinjection (11.3 ng/mL), suggesting that the dose of2.3 mg/kg is too weak to be administrated only once aday. For PBRM, however, values of 73.8 and 50.7 ng/mLwere found after 7 and 12 hours of injection, respectively;the last one was significantly different from the concen-tration found at the same time for CC-156 (P < 0.05).AUC0–12h values indicate that PBRM was 1.7 timesmore available than CC-156 (772 ng�h/mL vs. 445ng�h/mL; P < 0.01). After 24 hours, a plasma concentra-tion of 11.7 ng/mLwasmeasured for PBRM, and thus, weobtained an AUC0–24h of 1146 ng

�h/mL for it.

Inhibition of E1-stimulated T-47D tumor growth inOVX nude mice

After we established that PBRM inhibitor was found inplasma after a one-day single subcutaneous injection, wedecided to study the efficacy of PBRM in vivo. FemaleOVXBalb/c nude mice were inoculated with 1 � 107 T-47D(ERþ) human breast cancer cells in Matrigel as describedin the procedure by Day and colleagues (23), except thatthe inoculation was made into both flanks of mouse. The

Figure 5. Effect of inhibitors PBRM and CC156 on uterine (A) and vagina(B) weight of ovariectomized (OVX)mice treated for 7 days. �,P < 0.05 and��, P < 0.01, experimental vs. OVX control animals (CTR).






mice receivedE1 (0.1mg/d),which after its transformationto E2 by 17b-HSD1 stimulates tumor growth. Only micewith tumors that were well established after 15 days oftreatment with 0.1 mg E1/mouse s.c. were selected tocontinue the study. We used the dose of 250 mg/mouse ofPBRMbecause thiswas thehighest dose tested in the in vivoestrogenicity assay that proved to be nonestrogenic. Figure6B shows the effect of PBRM on the growth of tumorsstimulated with 0.1 mg E1/mouse/day. In the first 18 daysof treatment, the tumors were not actively growing andmaintained their initial size at the beginning of treatment.From day 19, the size of tumors in the control (CTR) groupbegan to decrease until they reached approximately the74% of the initial size after 28 days and continued at thesame 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 thetumors was inhibited (74%), decreasing to the level of theCTR group at the end of treatment (P < 0.01 at days 28 and32,E1-PBRMvs.E1).Clearly,PBRMblocks the formationof

E2 in the tumor through the inhibition of 17b-HSD1 andthus, the tumor growth.

At the end of the study, the body weights of the micewere recorded and the ERþ tissues (uterus and vagina)were taken for analysis. Therewas no effect of either E1 orPBRM onmouseweight over the 32-day treatment period(Supplementary Fig. S1A), indicating that there is noapparent toxicity of PBRM at 250 mg/day/mouse (s.c.).Although uterine and vagina weights were increasedsignificantly in both of the E1-treated groups (P < 0.01,E1 and E1-PBRM vs. CTR), treatment with PBRM had noeffect on the E1-stimulated uterine and vaginal weightincrease (Supplementary Fig. S1B and S1C). Our resultsare in agreement with those obtained by other researchgroups (23, 38), which have shown that despite the factthat the 17b-HSD1 inhibitor was used at a concentrationthat produces a decrease in tumor volume (by inhibitingthe human 17b-HSD1 in xenograft), the doses of E1(0.1 mmol/L) used stimulated uterine weight gain thatcould not be reduced at the end of the study. In fact, themain expression of 17b-HSD1 is in the ovary of femalerodents (44), and low levels are detected in the uterus onlyby real-time PCR (45), but not by in situ hybridization (46).Day and colleagues (23) suggested that the lack of effect of17b-HSD1 inhibitor on uterine and vaginal weight maytherefore bedue to thehigher sensitivity of the uterus thanthe tumor to circulating estrogens. Because the murine17b-HSD1 is an ortholog of the human 17b-HSD1 andpresents in xenografted tumor (47), it is also possible thatPBRM — always tested on human 17b-HSD1— did notinhibit the murine enzyme present in the uterus. Interest-ingly, our results obtained with the T-47D xenografttumor model revealed that 17b-HSD1 inhibitor PBRMcompletely blocks the tumor growth induced by E1, theprecursor of potent estrogen E2.

ConclusionSeveral groups are working towards the development

of 17b-HSD1 inhibitors for clinical use in the treatment ofhormone-dependent breast cancer (48). However, to date,only the groups ofDay and colleagues (23) andHusen andcolleagues (38) have shown the efficacy of an inhibitor inthe in vivo treatment of E1-stimulated breast tumors innudemice, but to our knowledge, no inhibitor is currentlyin the clinical trial. In our study, we describe the in vitroand in vivo evaluation of a new inhibitor of 17b-HSD1.Known as PBRM, this steroid (E2) derivative has 2 char-acteristic elements: a carbamoylbenzyl chain at positionC16 for 17b-HSD1 inhibition and a 2-bromoethyl sidechain at position C3 for removing the residual estrogenicactivity associated with its E2 nucleus (30). PBRM has anIC50 value of 68 nmol/L in the whole T-47D cell assay, isnonestrogenic both in vitro and in vivo, and gave a higherplasma concentration when compared with the referenceinhibitor CC-156 after one-day subcutaneous injection of2.3 mg/kg. Most importantly, after 32 days of treatment,tumor sizes of OVXmice treated with E1 and PBRMwere

Figure 6. A, plasma concentration of PBRM and CC-156 as a function oftime following subcutaneous injection of 2.3 mg/kg in Sprague-Dawleyrats. B, effect of PBRM, an inhibitor of 17b-HSD1, on the growth of E1(s.c.)-stimulated T-47D tumors (xenograft) in ovariectomized (OVX) nudemice. �, P < 0.05 and ��, P < 0.01, E1-PBRM and OVX control animals(CTR) vs. E1.

Ayan et al.





completely reduced at the control group level (without E1treatment). It is noteworthy that for the first time aninhibitor of 17b-HSD1 is able to decrease the final tumorsize by 100%. These results strongly imply that in theexperimental setting the tumor growth was mainly medi-ated via E2, produced by the action of 17b-HSD1expressed in the T-47D cells, and that the remaining E1seems not to be able to sustain the tumor growth. Anotherinteresting aspect of inhibiting 17b-HSD1 came from thefact that this enzymealso transformsDHEAinto 5-diol.Asshown by Poulin and Labrie (49), 5-diol at physiologicconcentrations, acts as a genuine estrogen in ERþ breastcancer cells through its direct interaction with ER. Thus,inhibiting the transformation of DHEA into 5-diol isanotherway to deprive breast cancer cells of an estrogenicstimulus that becomes more important after menopause(16). Our in vitro and in vivo results clearly highlightedthe potential use of PBRM, a new 17b-HSD1 inhibitor, forthe treatment of breast cancer, which could be usedalternately or sequentially with other drugs against ER-dependent diseases.

Disclosure of Potential Conflict of InterestR.Maltais has ownership interest (including patents) in PCT/CA2012/

000316 and D. Poirier has ownership interest (including patents) in patentapplication. No potential conflicts of interest were disclosed by the otherauthors.

Authors' ContributionsConception and design: D. Ayan, R. Maltais, J. Roy, D. PoirierDevelopment of methodology: D. Ayan, R. Maltais, J. RoyAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): D. Ayan, D. PoirierAnalysis and interpretation of data (e.g., statistical analysis, biostatis-tics, computational analysis): D. AyanWriting, review, and/or revision of themanuscript:D.Ayan, R.Maltais, J.Roy, D. PoirierAdministrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases): D. AyanStudy supervision: D. Poirier

AcknowledgmentsThe authors thank Ren�e B�erub�e from the CHUQ (CHUL)-Bioana-

lytical Service for the plasma inhibitor concentration determinationand Sonia Francoeur and France L�etourneau for their assistance in thein vivo experiments. The authors also thank Charles Ouellet whoconducted the estrogen receptor binding assay (Fig. 4B). Carefulreading of the manuscript by Ms. Micheline Harvey is also greatlyappreciated.

Grant SupportThisworkwas supported by a grant (MOP-43994 toD. 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 markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received March 23, 2012; revised July 9, 2012; accepted July 15, 2012;published OnlineFirst August 20, 2012.

References1. Jansson A. 17Beta-hydroxysteroid dehydrogenase enzymes and

breast cancer. J Steroid Biochem Mol Biol 2009;114:64–7.2. MillerWL.Molecular biologyof steroid hormone synthesis. Endocr Rev

1988;9:295–318.3. Penning TM. Hydroxysteroid dehydrogenases and pre-receptor reg-

ulation of steroid hormone action. Hum Reprod Update 2003;9:193–205.

4. Hyndman D, Bauman DR, Heredia VV, Penning TM. The aldo-ketoreductase superfamily homepage. Chem Biol Interact 2003;143–144:621–31.

5. Moeller G, Adamski J. Multifunctionality of human 17beta-hydroxys-teroid dehydrogenases. Mol Cell Endocrinol 2006;248:47–55.

6. Meier M, Moller G, Adamski J. Perspectives in understanding the roleof human 17beta-hydroxysteroid dehydrogenases in health and dis-ease. Ann N Y Acad Sci 2009;1155:15–24.

7. Mindnich R, Moller G, Adamski J. The role of 17 beta-hydroxysteroiddehydrogenases. Mol Cell Endocrinol 2004;218:7–20.

8. Luu-The V, Belanger A, Labrie F. Androgen biosynthetic pathways inthe human prostate. Best Pract Res Clin Endocrinol Metab 2008;22:207–21.

9. Poirier D. New cancer drugs targeting the biosynthesis of estrogensand androgens. Drug Devel Res 2008;69:304–18.

10. Pelletier G, Luu-The V, Li S, Labrie F. Localization of type 5 17beta-hydroxysteroid dehydrogenase mRNA in mouse tissues as studied byin situ hybridization. Cell Tissue Res 2005;320:393–8.

11. Pelletier G, Luu-The V, Li S, Labrie F. Localization of type 8 17beta-hydroxysteroid dehydrogenase mRNA in mouse tissues as studied byin situ hybridization. J Histochem Cytochem 2005;53:1257–71.

12. Pelletier G, Luu-The V, Li S, Labrie F. Localization of type 7 17beta-hydroxysteroid dehydrogenase in mouse tissues. In situ hybridizationstudies. J Steroid Biochem Mol Biol 2005;93:49–57.

13. Luu-TheV, ZhangY,Poirier D, Labrie F.Characteristics of human types1, 2 and317beta-hydroxysteroid dehydrogenase activities: oxidation/reduction and inhibition. J Steroid Biochem Mol Biol 1995;55:581–7.

14. Brozic P, Lanisnik Risner T, Gobec S. Inhibitors of 17beta-hydroxys-teroid dehydrogenase type 1. Curr Med Chem 2008;15:137–50.

15. Purohit A, Tutill HJ,Day JM,ChanderSK, LawrenceHR,AllanGM, et al.The regulation and inhibition of 17beta-hydroxysteroid dehydroge-nase in breast cancer. Mol Cell Endocrinol 2006;248:199–203.

16. Simard J, Vincent A, Duchesne R, Labrie F. Full oestrogenic activity ofC19-delta 5 adrenal steroids in rat pituitary lactotrophs and somato-trophs. Mol Cell Endocrinol 1988;55:233–42.

17. Miyoshi Y, Ando A, Shiba E, Taguchi T, Tamaki Y, Noguchi S. Involve-ment of up-regulationof 17beta-hydroxysteroiddehydrogenase type1inmaintenance of intratumoral high estradiol levels in postmenopausalbreast cancers. Int J Cancer 2001;94:685–9.

18. Oduwole OO, Li Y, Isomaa VV, Mantyniemi A, Pulkka AE, Soini Y, et al.17beta-hydroxysteroid dehydrogenase type 1 is an independent prog-nostic marker in breast cancer. Cancer Res 2004;64:7604–9.

19. Laplante Y, Cadot C, Fournier MA, Poirier D. Estradiol and estrone C-16 derivatives as inhibitors of type 1 17beta-hydroxysteroid dehydro-genase: blocking of ERþ breast cancer cell proliferation induced byestrone. Bioorg Med Chem 2008;16:1849–60.

20. Poirier D. Inhibitors of 17 beta-hydroxysteroid dehydrogenases. CurrMed Chem 2003;10:453–77.

21. Sasano H, Suzuki T, Nakata T, Moriya T. New development in intra-crinology of breast carcinoma. Breast Cancer 2006;13:129–36.

22. Vicker N, Lawrence HR, Allan GM, Bubert C, Smith A, Tutill HJ, et al.Focused libraries of 16-substituted estrone derivatives and modifiede-ring steroids: inhibitors of 17beta-hydroxysteroid dehydrogenasetype 1. Chem Med Chem 2006;1:464–81.

23. Day JM, Foster PA, Tutill HJ, Parsons MF, Newman SP, Chander SK,et al. 17beta-hydroxysteroid dehydrogenase type 1, and not type 12, isa target for endocrine therapy of hormone-dependent breast cancer.Int J Cancer 2008;122:1931–40.

24. Berube M, Poirier D. Synthesis of simplified hybrid inhibitors of type 117beta-hydroxysteroid dehydrogenase via cross-metathesis andSonogashira coupling reactions. Org Lett 2004;6:3127–30.






25. Ciobanu LC, Poirier D. Synthesis of libraries of 16beta-aminopropylestradiol derivatives for targeting two key steroidogenic enzymes.Chem Med Chem 2006;1:1249–59.

26. Poirier D, Boivin RP, Tremblay MR, Berube M, Qiu W, Lin SX.Estradiol-adenosine hybrid compounds designed to inhibit type 117beta-hydroxysteroid dehydrogenase. J Med Chem 2005;48:8134–47.

27. Poirier D, Ciobanu LC, Berube M. A multidetachable sulfamate linkersuccessfully used in a solid-phase strategy to generate libraries ofsulfamate and phenol derivatives. Bioorg Med Chem Lett 2002;12:2833–8.

28. TremblayMR, Poirier D. Overviewof a rational approach to design typeI 17beta-hydroxysteroid dehydrogenase inhibitors without estrogenicactivity: chemical synthesis and biological evaluation. J Steroid Bio-chem Mol Biol 1998;66:179–91.

29. Poirier D. Contribution to the development of inhibitors of 17beta-hydroxysteroid dehydrogenase types 1 and 7: Key tools for studyingand treating estrogen-dependent diseases. J Steroid Biochem MolBiol 2011;125:83–94.

30. Maltais R, Ayan D, Poirier D. Crucial role of a 3-bromoethyl side-chainin removing the undesirable estrogenic activity of potent 17b-hydro-xysteroid dehydrogenase type 1 inhibitor 16b-(m-carbamoybenzyl)estradiol. ACS Med Chem Lett 2011;2:678–81.

31. Tremblay MR, Boivin RP, Luu-The V, Poirier D. Inhibitors of type 117beta-hydroxysteroid dehydrogenasewith reduced estrogenic activ-ity: modifications of the positions 3 and 6 of estradiol. J Enzyme InhibMed Chem 2005;20:153–63.

32. Cadot C, Laplante Y, Kamal F, Luu-The V, Poirier D. C6-(N,N-butyl-methyl-heptanamide) derivatives of estrone and estradiol as inhi-bitors of type 1 17beta-hydroxysteroid dehydrogenase: Chemicalsynthesis and biological evaluation. Bioorg Med Chem 2007;15:714–26.

33. Levesque C, Merand Y, Dufour JM, Labrie C, Labrie F. Synthesis andbiological activity of new halo-steroidal antiestrogens. J Med Chem1991;34:1624–30.

34. Davis DD, Díaz-Cruz ES, Landini S, Kim YW, Brueggemeier RW.Evaluation of synthetic isoflavones on cell proliferation, estrogenreceptor binding affinity, and apoptosis in human breast cancer cells.J Steroid Biochem Mol Biol 2008;108:23–31.

35. Arcaro KF, Yi L, Seegal RF, Vakharia DD, Yang Y, Spink DC, et al.2,20,6,60-Tetrachlorobi-phenyl is estrogenic in vitro and in vivo. J CellBiochem 1999;72:94–102.

36. Poirier D, Maltais R, Roy J, inventors; Universit�e Laval, assignee.Inhibitors of 17b-HSD1, 17b-HSD3 and 17b-HSD10. Internationalpatent application PCT/CA2012/00316 filed March 26, 2012.

37. Dauvois S, Geng CS, L�evesque C, M�erand Y, Labrie F. Additiveinhibitory effects of an androgen and the estrogen EM-170 on estra-diol-stimulated growth of human ZR-75-1 breast tumors in athymicmice. Cancer Res 1991;51:3131–5.

38. Husen B, Huhtinen K, Poutanen M, Kangas L, Messinger J, Thole H.Evaluation of inhibitors for 17beta-hydroxysteroid dehydrogenasetype 1 in vivo in immunodeficient mice inoculated with MCF-7 cellsstably expressing the recombinant human enzyme. Mol Cell Endocri-nol 2006;248:109–13.

39. Husen B, Huhtinen K, Saloniemi T, Messinger J, Thole HH, PoutanenM. Human hydroxysteroid (17-beta) dehydrogenase 1 expressionenhances estrogen sensitivity of MCF-7 breast cancer cell xenografts.Endocrinology 2006;147:5333–9.

40. Messinger J, Hirvela L, Husen B, Kangas L, Koskimies P, PentikainenO, et al. New inhibitors of 17beta-hydroxysteroid dehydrogenase type1. Mol Cell Endocrinol 2006;248:192–8.

41. Kramer C. Extension of multiple range tests to group with uniquenumbers of replications. Biometrics 1956;12:307–10.

42. Pasqualini JR, Cortes-Prieto J, Chetrite G, Talbi M, Ruiz A. Concen-trations of estrone, estradiol and their sulfates, and evaluation ofsulfatase and aromatase activities in patients with breast fibroade-noma. Int J Cancer 1997;70:639–43.

43. Keydar I, Chen L, Karby S, Weiss FR, Delarea J, Radu M, et al.Establishment and characterization of a cell line of human breastcarcinoma origin. Eur J Cancer 1979;15:659–70.

44. Peltoketo H, Luu-The V, Simard J, Adamski J. 17beta-hydroxysteroiddehydrogenase (HSD)/17-ketosteroid reductase (KSR) family; nomen-clature and main characteristics of the 17HSD/KSR enzymes. J MolEndocrinol 1999;23:1–11.

45. Nokelainen P, Puranen T, Peltoketo H, Orava M, Vihko P, Vihko R.Molecular cloning of mouse 17 beta-hydroxysteroid dehydrogenasetype 1 and characterization of enzyme activity. Eur J Biochem 1996;236:482–90.

46. Pelletier G, Luu-The V, Li S, Ren L, Labrie F. Localization of 17beta-hydroxysteroid dehydrogenase type 1 mRNA in mouse tissues. J MolEndocrinol 2004;33:459–65.

47. Moller G, Husen B, Kowalik D, Hirvela L, Plewczynski D, Rychlewski L,et al. Species used for drug testing reveal different inhibition suscep-tibility for 17beta-hydroxysteroid dehydrogenase type 1. PLoS One2010;5:e10969.

48. Poirier D. 17beta-Hydroxysteroid dehydrogenase inhibitors: a patentreview. Expert Opin Ther Pat 2010;20:1123–45.

49. Poulin R, Labrie F. Stimulation of cell proliferation and estrogenicresponse by adrenal C19-delta 5-steroids in the ZR-75-1 humanbreast cancer cell line. Cancer Res 1986;46:4933–7.

Ayan et al.





2012;11:2096-2104. Published OnlineFirst August 20, 2012.Mol Cancer Ther Diana Ayan, René Maltais, Jenny Roy, et al. Cancer Tumor Growth Induced by EstroneDehydrogenase Type I Blocks the Estrogen-Dependent Breast

-HydroxysteroidβA New Nonestrogenic Steroidal Inhibitor of 17

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