A Novel Antiandrogen, Compound 30, Suppresses Castration-Resistant and MDV3100-Resistant Prostate...

Published OnlineFirst March 14, 2013.Mol Cancer Ther Hidetoshi Kuruma, Hiroaki Matsumoto, Masaki Shiota, et al.

In Vivo and In VitroCancer Growth Castration-Resistant and MDV3100-Resistant Prostate A Novel Antiandrogen, Compound 30, Suppresses

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Chemical Therapeutics

A Novel Antiandrogen, Compound 30, SuppressesCastration-Resistant and MDV3100-Resistant ProstateCancer Growth In Vitro and In Vivo

Hidetoshi Kuruma1, Hiroaki Matsumoto1, Masaki Shiota1, Jennifer Bishop, Francois Lamoureux1,Christian Thomas1, David Briere2, Gerrit Los2, Martin Gleave1, Andrea Fanjul2, and Amina Zoubeidi1

AbstractResistance to antiandrogen drugs, likeMDV3100, occurs in patientswith castration-resistant prostate cancer

(CRPC). Thus, preventing or treating antiandrogen resistance is a major clinical challenge. We identified a

novel antiandrogen, Compound 30, and compared its efficacy with MDV3100. We found that Compound 30

inhibits androgen receptor (AR) activity in LNCaP cells, C4-2 cells, as well as MDV3100-resistant cell lines.

ComparedwithMDV3100, Compound 30 treatment induces greater reduction inAR, prostate-specific antigen

(PSA), and AR transcriptional activity, and prevents AR nuclear translocation in AR-sensitive LNCaP cells.

Compound 30 has antiproliferative effects in LNCaP cells, in castrate-resistant C4-2 cells, and those resistant to

MDV3100. Compound 30 was equally as effective as MDV3100 in reducing tumor volume and PSA in vivo.

More importantly, Compound 30 is effective at inhibiting AR activity in MDV3100-resistant cell lines and

significantly prevented tumor growth and PSA increases in mice bearing MDV3100-resistant xenografts.

Together, our data show that Compound 30 strongly inhibitedAR activity and suppressed castration-resistant

LNCaP growth as well as MDV3100-resistant cell growth in vitro and in vivo. These data provide a preclinical

proof-of-principle that Compound 30 could be a promising next generation anti-AR agent, especially in the

context of antiandrogen-resistant tumors. Mol Cancer Ther; 12(5); 1–10. �2013 AACR.

IntroductionProstate cancer is the most common male cancer in

North America and second leading cause of cancer-relat-ed deaths (1). Androgens drive prostate cancer carcino-genesis and progression, regulating gene and signalingnetworks that promote cell survival through bindingwiththe androgen receptor (AR), a ligand-responsive tran-scription factor. Androgen deprivation therapy (ADT),consisting of castration combined with an antiandrogen,blocks the growth-promoting effects of androgens andactivates apoptosis in prostate cancer tumor cells (2),prolonging survival for patients with prostate cancer(3). Despite high initial response rates, remissions follow-ing ADT are temporary due to the emergence of castra-tion-resistant prostate cancer (CRPC), where AR reacti-vation occurs and tumors grow in the presence of low

levels of androgens. AR activation remains a centralmechanism driving CRPC progression (4), involving var-iable combinations of AR gene amplification, increasedAR sensitivity, promiscuousAR-bindingmutants, alteredexpression of coregulators, ligand-independent activa-tion by oncogenic signaling pathways, and increases inandrogen biosynthesis (5–8). These mechanisms likelywork in concert to drive CRPC; hence, targeting theAR remains a critical component of novel CRPC therapies(9, 10).

ADT prevents AR activation by reducing the levels ofandrogen production through castration or by targetingthe AR directly using AR inhibitors. Limited efficacy ofantiandrogens like bicalutamide has led to the develop-ment ofmore potent AR-targeting compounds.MDV3100is a second-generation AR inhibitor that has significantantitumor effects both in vitro and in vivo (11). A phase I/IItrial in patients with metastatic CRPC showed MDV3100treatment decreased serum prostate-specific antigen(PSA) by 50%and reduced or stabilizedmetastatic disease(12). Furthermore, the phase III AFFIRM trial (13), testingMDV3100 in postdocetaxel patients with CRPC, showedan overall survival advantage and a 37% reduction inmortality risk (14). Despite these results, MDV3100 treat-ment does not lead to complete CRPC regression, asprogression and death from drug-resistant disease occursin most patients (14). Therefore, the development ofMDV3100-resistant tumors in CRPC represents a

Authors' Affiliations: 1The Vancouver Prostate Centre, Vancouver, BritishColumbia, Canada; and 2Pfizer Oncology Research Unit, WorldwideResearch & Development, San Diego, California

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

Corresponding Author: Amina Zoubeidi, The Vancouver Prostate Centre,Department of Urologic Science University of British Columbia, 2660 OakStreet, Vancouver, BC V6H 3Z6, Canada. Phone: 604-809-4947; Fax: 604-875-5654: E-mail: [email protected]

doi: 10.1158/1535-7163.MCT-12-0798

�2013 American Association for Cancer Research.

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significant challenge in the treatment of advanced pros-tate cancer. As such, identifying novel compounds withactivity in MDV3100-resistant tumors is critical toimprove the future of prostate cancer therapy.

In this study, we tested the anticancer activity of thenovel antiandrogen, Compound 30 (15), in in vitro and invivo models of prostate cancer. Compound 30 was devel-oped by optimizing AR ligand-binding efficiency of ary-loxy tetramethylcyclobutyl lead compounds identified ina high-throughput cell-based screen for pure, nonsteroi-dal, AR antagonists (15). In that study, Compound 30wasshown to more potently inhibit AR activity than bicalu-tamide and had significant antitumor effects in a mousemodel of CRPC (15). Likewise, we found that Compound30 potently inhibitedAR activity in LNCaP andC4-2 cells,and was as efficacious as MDV3100 in preventing tumorprogression and PSA increases in a mouse model ofCRPC. Importantly, using our newly developed modelof MDV3100 resistance, we showed that Compound 30has antiproliferative and AR-targeting effects in anMDV3100-resistant LNCaP cell line established fromMDV3100-resistant tumors. Overall, our results suggestthat Compound 30 may be a viable treatment for CRPCthat is resistant to AR pathway inhibitors.

Materials and MethodsCell culture

AR-positive LNCaP and C4-2 cells were provided byDr. Leland W. K. Chung (Cedar Sinai, Los Angeles, CA)tested and authenticated by whole-genome and whole-transcriptome sequencing on Illumina Genome AnalyzerIIx platform in July 2009. They were maintained in RPMI-1640 supplemented with 5% FBS (Invitrogen Life Tech-nologies). MR49F cells from MDV3100-resistant LNCaPxenografts were maintained in RPMI-1640 with 5% FBSand 10 mmol/L MDV3100.

Reagents and antibodiesThe synthetic androgen R1881 was from PerkinElmer,

Compound 30 was supplied by Pfizer Inc., and custom-ized synthetic MDV3100 was purchased from ShanghaiHaoyuan Chemexpress. AR, PSA, and cyclin D werepurchased from SantaCruz Biotechnology, PARP (CellSignaling Technology), vinculin (Sigma Chemical Co.),and Ki67 (Lab Vision Corporation).

Cell growth assayCells were treated with various doses of compounds or

dimethyl sulfoxide for the indicated amount of time, andgrowth rates were assessed with a crystal violet assay(described previously; ref. 16). Each assay was done intriplicate.

Reverse transcription and quantitative PCR analysisTotal RNA was extracted from cultured cells after 48

hours of treatment using TRIzol reagent (Invitrogen LifeTechnologies, Inc.). Quantitative real-time PCR (qRT-PCR) was conducted as described previously (17).

Transient transfection and luciferase assayLNCaP andMR49F cells were cotransfected with 0.6 mg

of probasin luciferase reporter and 0.6 mg of controlRenillaluciferase plasmids per 10 cm plate using Lipofectin(Invitrogen). Cellswere treatedwithdifferent compoundsfor 1 hour before treatment with 1 nmol/L of R1881.Luciferase activity was measured as previously reported(18).

Immunoblotting analysisTotal proteins were extracted as previously reported

using radioimmunoprecipitation assay buffer (17). A totalof 30 mg of total proteins were separated by SDS-PAGE)and transferred to polyvinylidene difluoride membranes.Western blot analyses were conducted as previouslyreported (17).

ImmunofluorescenceLNCaP andMR49F cellswere cultured in 12-well plates

containing coverslips and RPMIþ5% charcoal strippedserum (Invitrogen Life Technologies) for 48 hours. Cellswere then treated for 2 hours with indicated concentra-tions of compounds followed by 1 nmol/L of R1881treatment for 15 minutes. Immunofluorescence was con-ducted as previously described (18).

Cell-cycle analysisCells were incubated in the absence or the presence of

different concentrations of MDV3100 or Component 30and were stained with propidium iodide. Cell-cycle pop-ulation was analyzed by flow cytometry as previouslyreported (17).

Assessment of in vivo tumor growth for castration-resistant LNCaP xenografts andMDV3100-resistantxenograft transplantation

A total of 1 � 106 LNCaP cells with 0.1 mL Matrigel(Becton Dickinson Labware) were inoculated in bilateralflanksof 6- to 8-week-oldmale athymicnudemice (HarlanSprague Dawley Inc.). Tumor volume, body weight, andserum PSA levels were measured weekly. When serumPSA levels reached more than 50 ng/mL, castration wasconducted. When PSA recovered to precastration levels,mice were randomized into 3 treatment groups; vehicle,10 mg/kg of MDV3100 or 25 mg/kg of Compound 30and treated daily.

Generation of MDV3100-resistant tumorsMDV3100-resistant LNCaP xenografts were excised

and transplanted to castrated mice treated with10 mg/kg/daily of MDV3100. When tumors reached2,000 mm3, xenografts were harvested and seriallytransplanted into castrated male mice receiving 10mg/kg/daily of MDV3100. Third-generation trans-planted tumors were used as MDV3100-resistant xeno-grafts in this study. When PSA levels reached more than50 ng/mL, mice were randomized into 3 groups; (1)stop MDV3100 dosing and switch to vehicle, (2)

Kuruma et al.

Mol Cancer Ther; 12(5) May 2013 Molecular Cancer TherapeuticsOF2




continuance of MDV3100, and (3) stop MDV3100 andswitch to Compound 30.

Generation of MDV3100-resistant MR49F cellsThird-generationMDV3100-resistant xenograft tumors

were harvested into RPMIþ10% FBS. Adherent cells weremaintained in RPMIþ5%FBSþ10 mmol/L MDV3100.

ImmunohistochemistryA tissue microarray was constructed using a manual

tissue microarrayer (Beecher Instruments Inc.). Immuno-histochemical staining was conducted as we previouslyreported. All comparisons of staining intensities weremade at �200 magnification.

Statistical analysisDatawere analyzed statistically using a 1-wayANOVA

test, with P < 0.05 considered to indicate significance.Student t test (2 sided) was used to evaluate statisticallysignificant differences in all experiments. Results areexpressed as mean� SEwith at least 3 biologic replicates.A P < 0.05 was considered significant.

ResultsCompound 30 is more potent than MDV3100 ininhibiting AR transcriptional activityThe antiandrogen, Compound 30, is more potent than

the first-generation antiandrogen bicalutamide both invitro and in vivo (15); however, it has not been comparedwith new-generation antiandrogens. Here, we exploredthe efficacy of Compound 30 (chemical structure is shownin Fig. 1A) compared with the new-generation antiandro-gen,MDV3100, onARpathwayactivity, proliferation, andapoptosis in the prostate cancer cell line LNCaP. Wefound that Compound 30 decreased AR transcriptionalactivity, measured by probasin luciferase activity (Fig. 1B,top left), in a dose-dependent manner and with greaterpotency compared with MDV3100 [probasin 50% effec-tive concentration (EC50): 12.5 and 20 mmol/L, respective-ly; Supplementary Table S1]. Similar data were observedusing PSA luciferase (Fig. 1B, bottom left). Decreased ARactivity correlated with a decrease of AR at both mRNA(Fig. 1B, top right) and protein (Fig. 1C) levels, and thesedecreases were exaggerated in comparison with thoseinduced by MDV3100, especially at the 10 mmol/L dose.In accordancewith ourPSA luciferase data,weobserved adose-dependent decrease in PSA protein expression inLNCaP cells treated with Compound 30 (Fig. 1C), andCompound 30 induced an approximate 70% decrease inPSA mRNA levels at 0.1 mmol/L, compared with anapproximate 30% decrease induced by MDV3100 at thesame dose (Fig. 1B, right bottom). Finally, we found thatlike MDV3100, Compound 30 also inhibited AR nucleartranslocation, as shown by fluorescence microscopy (Fig.1D). Interestingly,we observed that although treatment ofLNCaP cells with MDV3100 also reduced AR proteinexpression,ARmRNA levelswere dramatically increasedby treatmentwith 1or 10mmol/LMDV3100. Thismaybea

compensatory mechanism to upregulate AR protein pro-duction after MDV3100 treatment that was not observedwith Compound 30.

Compound 30 potently inhibits cell proliferation andinduces apoptosis

Compound 30 was able to inhibit AR activity morepotently thanMDV3100 asmeasured by decreases in PSAand AR protein and mRNA expression. We found thatCompound 30 was significantly more effective thanMDV3100 in suppressing cell growth (Fig. 2A) in AR-positive LNCaP and its castration-resistant subline, C4-2cells. More importantly, Compound 30 was equally asefficacious in suppressing growth in both cell lines (IC50¼1.86 mmol/L for LNCaP and 2.02 mmol/L for C4-2),whereas MDV3100 had a stronger antiproliferative effectin androgen-sensitive LNCaP cells comparedwith andro-gen resistant C4-2 cells (IC50 ¼ 12.52 mmol/L for LNCaPand 34.90 mmol/L for C4-2; Fig. 2A and SupplementaryTable S2). Reduction in cellular proliferation induced byMDV3100 and Compound 30 was associated with dose-dependent arrest in the sub-G0 phase of cell cycle asassessed by flow cytometry (Fig. 2B), and these datashowed that at the 10 mmol/L dose, Compound 30 hada more substantial effect on cell-cycle arrest thanMDV3100. Furthermore, because MDV3100 has beenshown to induce PARP cleavage in VCaP cells, we inves-tigated the effects of Cpd30 treatment on PARP cleavagein LNCaP cells compared with MDV3100. Importantly,only Compound 30 was able to induce PARP cleavage inLNCaP cells in a dose-dependent fashion (Fig. 2C).

Compound 30 elicits anticancer activity in acastration-resistant in vivo model

As Compound 30 showed potent antiproliferativeactivity in vitro, we sought to test its efficacy comparedwithMDV3100 in an LNCaP CRPCmodel in vivo. LNCaPcells were xenografted into athymic male nude mice andmice were castrated when serum PSA reached approxi-mately 50 ng/mL. Treatment was initiated at the timewhen serum PSA reached precastration levels, whichrepresents the hallmark of CRPC in the LNCaP model(19). Mice with CRPC tumors were randomized to 3treatment groups: vehicle (8 mice), 10 mg/kg/dayMDV3100 (13 mice), and 25 mg/kg/day Compound 30(13 mice). As shown in Fig. 3A, both compounds signif-icantly suppressed tumor growth (left) and decreasedserum PSA (right) compared with vehicle controls. After5weeks of treatment, therewere no significant differencesbetween Compound 30 and MDV3100-treated tumorvolumes or PSA (change from baseline was þ488% invehicle, þ19% in MDV3100, and þ7% in Compound 30,and serum PSA change from baseline was þ619% invehicle, �32% in MDV3100, and �27% in Compound30). Waterfall plots of individual responses in theMDV3100-treatment group showed 5 (38%) animals hadreduced tumor volume from baseline (Fig. 3B, left) and9 (69%) animals had reduced PSA levels from baseline

Efficacy of Compound 30 in MDV3100-Resistant Prostate Cancer

www.aacrjournals.org Mol Cancer Ther; 12(5) May 2013 OF3




(Fig. 3B, right). In the Compound 30-treated group,6 (46%) animals had reduced tumor volume frombaseline(Fig. 3B, left) and 10 (77%) mice had reduced PSA levelsfrombaseline (Fig. 3B, right). Furthermore, staining for thecellular proliferation marker Ki67 in tumors fromMDV3100- and Compound 30-treated mice showed sim-ilar reduction in Ki67 expression compared with vehiclecontrol (Fig. 3D). However, because our in vitro datashowed that Compound 30 had a greater effect on cell

cycle repartition than MDV3100 (Fig. 2C), we furtherinvestigated the effects on cell-cycle progression by ana-lyzing the expression of cyclin D protein, which is knownas aG1phase cyclin that regulates the entry of cells intoG1,in Compound 30- and MDV3100-treated tumors. Consis-tent with our in vitro results, we observed lower cyclin Dprotein expression in 2 of 4 randomly selectedCompound30-treated tumors compared with those from MDV3100-treated mice (Fig. 3C).

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Figure 1. Compound 30 inhibitsactivity and expression of the AR.A, chemical structure ofCompound 30. B, left, 24 hoursafter transfection, LNCaP cellstransfected with probasin (top left)or PSA (bottom left) luciferase andcontrol Renilla plasmids weretreated for 24 hours with variousconcentrations of Compound 30 orMDV3100. Cells were harvestedand luciferase activity wasdetermined. Data represent meansof at least 3 independentexperiments done in triplicate.B, right, LNCaP cells were treatedwithCompound 30orMDV3100 for24 hours and RNA was extractedand qRT-PCR was conducted toevaluate the expression of AR(top right) and PSA (bottom right).C, LNCaP cells were treated withCompound 30 or MDV3100 for 24hours; AR and PSA were analyzedby Western blot analysis; vinculinwas used as a loading control.D, LNCaP cells were maintained inandrogen-deprived conditionsfor 24 hours and treated with10 mmol/L Compound 30 orMDV3100 for 2 hours followed byaddition of 1 nmol/L of R1881. After15 minutes incubation, cells werefixed and AR localization wasassessed by immunofluorescenceimaging.

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Compound 30 affects cell growth and ARtranscriptional activity in vitro in MDV3100-resistant MR49F cellsOur in vivo experiment confirmed that both MDV3100

andCompound 30 hadpotent anticancer activity inCRPCtumors. However, we also observed that tumors treatedwith MDV3100 for 8 weeks or longer eventually resumedgrowth, which we interpreted as the establishment ofMDV3100 resistance. Resistance to anticancer therapiesis a commonphenomenon observed in patients, therefore,we sought to develop a preclinical model of MDV3100-resistance. LNCaP-CRPC xenografts were treated with 10mg/kg/day MDV3100 until tumor volume and PSAnecessitated euthanasia and tumors were harvested andreimplanted in precastrated male nude mice subcutane-ously (first generation transplant). Mice were continu-ously treated with MDV3100 to ensure maintenance ofthe resistant phenotype. This procedure was repeated toobtain second and third transplant generations. Thirdgeneration transplanted MDV3100-resistant xenografttumors were harvested and single cell suspensionsgrown in in vitro culture under the constant pressure of10 mmol/L MDV3100 to generate MDV3100-resistantMR49F cells (Supplementary Fig. S1). Results from immu-noblotting analyses showed that MR49F cells expressedboth AR and PSA protein, and their expression was notaffected byMDV3100 treatment (Supplementary, Fig. S2).As expected,MR49F cells showed a significant decrease

in their response to MDV3100 treatment when comparedwith LNCaP parental cells, (IC50 ¼ 12.52 mmol/L inLNCaP vs. 33.89 mmol/L in MR49F; Fig. 4A, left, Supple-mentary Table S3). In addition, MDV3100 was no longerable to reduce PSA expression, measured byWestern blot

analysis (Fig. 4C) or inhibit AR translocation from cyto-plasm to nucleus upon androgen stimulation (Fig. 4D).Taken together, these results suggest that MR49F cells,which still express AR and PSA, resisted the effects ofMDV3100 on AR activity.

Once MDV3100-resistant cells were established, weassessed the anticancer activity of Compound 30 on thesecells. Importantly, although Compound 30 did notdecrease AR protein expression in MDV3100-resistanttumors to the same extent as in LNCaP cells, reductionin AR expressionwas observed at higher treatment doses,and it profoundly reduced the expression of PSAin MR49F cells at as low a dose as 1 mmol/L (Fig. 4C).In addition, Compound 30 suppressedAR transcriptionalactivity in MR49F cells with greater potency tothat obtained from LNCaP parental cells (EC50 ¼ 0.23mmol/L in MR49F and 2.0 mmol/L LNCaP cells; Fig.4B, Supplementary Tables S1 and S4) and, more impor-tantly, it was effective in its antiproliferative effects inMR49F cells (Fig. 4A right, Supplementary Table S5).Finally, AR nuclear translocation after androgen stimu-lation was inhibited by Compound 30 (Fig. 4D). Together,these results indicated that in the context of MDV3100resistance modeled in MR49F cells, Compound 30retained its ability to function as a potent AR antagonistand cell growth inhibitor.

Compound 30 has anticancer activity in MDV3100-resistant tumors in vivo

As described above, our in vitro studies indicated thatCompound 30 was still effective in inhibiting AR activityin the context ofMDV3100 resistance. Therefore, to test theefficacy of Compound 30 in suppressingMDV3100 tumor

Figure 2. Compound 30 inhibits cellgrowth and induces apoptosis. A,LNCaP (left) and C4-2 cells (right)were treated with Compound 30 orMDV3100 for 72 hours and cellgrowth was evaluated using crystalviolet. B, LNCaP cells were treatedwith of Compound 30 or MDV3100for 72 hours. Cells were stained withpropidium iodide and cell-cyclepopulations were analyzed by flowcytometry. C, LNCaP cells weretreated with Compound 30 orMDV3100 for 72 hours and totalproteins were analyzed for theexpression of cleavedPARP; vinculinwas used as a loading control.

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growth in vivo, we implanted third generation MDV3100-resistant tumors into 34 mice under continuous treatmentwith 10mg/kg/day MDV3100. When tumors reached300 mm3 or approximately 50 ng/mL PSA, mice wererandomly divided into 3 groups. At this time, MDV3100treatment ceased, except for those animals assignedto MDV3100-treated group (11 animals). The other 2groups received treatment with either vehicle (10 mice)or 25 mg/kg/day Compound 30 (13 mice; Fig. 5A). Con-sistent with the resistant nature of MR49F cells, we foundthat MDV3100-resistant tumors grew rapidly in theabsence or presence of MDV3100, whereas those treatedwith Compound 30 showed significantly lower tumorvolume and PSA. After 3 weeks of treatment, tumorvolume change from baseline was 324% in vehicle-,389% in MDV3100- and 138% Compound 30-treatedgroups (Fig. 5A, left), and serum PSA change from base-line was þ708% in vehicle-, þ1,013% in MDV3100- andþ485%, in Compound 30-groups (Fig. 5A, right). Micewere euthanized when tumor volume exceeded 10% oftheir body weight and therefore at 3 weeks of treatment 1mouse in vehicle-, 5 mice in MDV3100- and 2 mice inCompound 30-treated groups required euthanasia (Fig.5B). Nevertheless, the group treated with Compound 30survived significantly longer than vehicle and MDV3100treatment groups (Fig. 5C). In addition, results from

tumor immunohistochemistry (IHC) showed a marginaldecrease Ki67 staining in Compound 30-treated tumorscompared with vehicle and MDV3100-treated tumors(Fig. 5D) suggesting that Compound 30 had an impacton cell proliferation in vivo.

DiscussionAndrogen ablation remains the most effective therapy

for patients with advanced prostate cancer. Althoughmost patients initially respond, progression to CRPCfrequently occurs within 18 to 36months. Blocking ligandbinding to the AR is a complimentary approach that hasbeen used clinically for nearly 3 decades. Although firstgeneration antiandrogens, such as bicalutamide, do inhib-it AR activity, they can induce mutated or overexpressedAR,which limits their clinical application inCRPC,whereAR variants and overexpression occur frequently (20). Infact, even after castration,more than 80%ofCRPC expressAR and androgen-responsive genes (4, 21–23). Further-more, it is well known that AR activity is a key driver ofCRPC progression (24–26). Hence, identifyingmore effec-tive AR pathway inhibitors in CRPC should enhancesurvival and slow disease progression.

Several new classes ofARpathway inhibitors are now indevelopment and or approved for treatment of CRPC,

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Tumor volume Serum PSAFigure 3. Compound 30 prevents invivo tumor growth and PSAincreases in castration-resistantLNCaP xenografts. LNCaP cellswere xenografted into athymicmale nude mice, followed bycastration when serum PSAreached 50 ng/mL. Treatmentswere initiated at the time whenserum PSA reached precastrationlevels. Mice with CRPC tumorswere randomized into 3 groupsreceiving vehicle control (8 mice),10mg/kgMDV3100 daily (13mice),or 25 mg/kg Compound 30 daily(13 mice). A, mean tumor volume(right) and circulating PSA (left) foreach treatment group are reported.B, waterfall plots represent percentchange in tumor volume (left) andPSA (right) after 5 weeks oftreatment for individual mice ineach treatment group. C, totalproteins were extracted fromtumors from 4 randomly mice andanalyzed for the expression ofcyclin D; vinculin was used as aloading control. D, tumors werecollected at the end of thetreatment and Ki67 positivity wasevaluated by IHC.

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includingmorepotentARantagonists (e.g.,MDV3100) andinhibitors of intratumoral steroidogenesis (e.g., abirater-one; refs. 13, 20, 27), as well as AR chaperone inhibitors(18, 28–30). MDV3100 is a potent, second-generation ARantagonist that binds to the ligand-binding domain of theAR with approximately 8-fold higher affinity than bicalu-tamide and impairs AR nuclear translocation and DNAbinding (11).MDV3100 inhibitsgrowthofCRPCxenograftsand has efficacy in phase III clinical trials (31) and has beenrecently approved. However, despite its promising effectsin some patients with CRPC, resistance to MDV3100 even-tually occurred. The heterogeneity of responses of patientswith CRPC toMDV3100 treatment and the eventual tumorprogression, indicating MDV3100 resistance, suggest thatAR targetingagents that areeffective inMDV3100-resistanttumors will be required to treat CRPC.

In this study, we report for the first time that a new ARantagonist, Compound 30 (15), exhibits potent AR inhi-bition not only in CRPC, but also in MDV3100-resistantLNCaP derived cells in vitro and in vivo. In LNCaP cells,Compound 30 suppressedAR transcriptional activity andPSA expression levels in a dose-dependent manner. Sim-ilar to MDV3100 (11), Compound 30 prevented AR trans-location to the nucleus, highlighting an important level ofcontrol over AR activity. However, Compound 30 alsoenhanced the reduction in AR protein expression com-pared with MDV3100, providing an alternative mecha-nism for reducing AR activity. The more modest reduc-tion in AR protein expression induced by MDV3100 maybe hindered by compensatory synthesis of AR, as evi-denced by dramatically increased AR mRNA expressionafter MDV3100 treatment. Interestingly, this was not

Figure 4. Compound 30 affects cellgrowth and AR transcriptionalactivity in vitro inMDV3100-resistantMR49F cells. A, LNCaP andMDV3100-resistant cells (MR49F)were treated with MDV3100 (left) orcompound 30 (right) for 72 hours andcell growth was evaluated usingcrystal violet (left). B, 24 hours aftertransfection, MR49F cellstransfected with probasin luciferaseand control Renilla plasmids weretreated for 24 hours with Compound30 or MDV3100. Cells wereharvested and luciferase activity wasdetermined. Data represent mean ofat least 3 independent experimentsdone in triplicate. C, MR49F cellswere treated with Compound 30 orMDV3100 for 24hours. Total proteinswere extracted and analyzed for theexpression of AR and PSA; vinculinwas used as a loading control. D,MR49F cells were maintained inandrogen-deprived conditions for24 hours and treated with 10 mmol/LCompound 30 or MDV3100 for 2hours followed by addition of1 nmol/L of R1881. After 15 minutesof incubation, cellswere fixed andARlocalization was assessed byimmunofluorescence.

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observed after treatment with Compound 30, possiblyindicating an advantage of this drug over MDV3100. Onepossible mechanism for the downregulation of both ARmRNA and protein levels in Cpd30-treated cells may be afeed forward loop, whereby reduced levels of AR proteinexpression and translocation to the nucleus inhibit AR-dependent transcription factors in promoting the tran-scription and translation of AR.

Importantly, our in vivo studies pointed to an unmetapplication for the use ofCompound30 in the treatment ofanti-AR–resistant CRPC. When we compared the antitu-mor activity of Compound 30 and MDV3100 in micebearing castration-resistant xenograft tumors, we foundboth compounds significantly prevented increases intumor volume and PSA. However, after long-term treat-ment with MDV3100 or Compound 30 (� 8 weeks), evenwell-controlled tumors recurred and PSA recovered, sug-gesting that resistance to both antiandrogens occurs.Indeed, despite efficacy of MDV3100 and its imminentapproval for the treatment of CRPC (31), most patients

treated with MDV3100 will eventually recur, providingclinical evidence for anti-AR resistance. In light of ourdatashowing tumor recurrence withMDV3100, we developeda model of MDV3100 resistance in vivo and in vitro andwere able to generate LNCaP-derivedMDV3100-resistantxenograft tumors and cell lines that still expressedARandPSA. We used these resistant cells to test the efficacy ofCompound 30. Importantly, although our in vivo studiesshowed similar anticancer activity betweenCompound30and MDV3100 in CRPC xenografts, our in vitro resultsshowed significant antiproliferative effects of Compound30 in MDV3100-resistant cells. Accordingly, we foundCompound 30 suppressed progression of MDV3100-resistant tumors in castrated mice and significantly pro-longed survival in mice bearing MDV3100-resistanttumors. Furthermore, in vitro studies using the MR49FMDV3100-resistant cell line showed that Compound 30effectively inhibited AR transcriptional activity and ARtranslocation to the nucleus, and potently suppressed cellproliferation.

Log-rank test: Vehicle vs. Cpd 30, P = 0.0088, MDV3100 vs. Cpd 30, P = 0.036

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Figure 5. Compound 30 hasanticancer activity in MDV3100-resistant tumors in vivo. A,MDV3100-resistant tumors weretransplanted to castrated mice andmaintained under MDV3100pressure (10 mg/kg). When PSAreached 50–75 ng/mL, mice wererandomly divided into 3 groups andtreated with vehicle (10 mice), 10mg/kg/day MDV3100 (11 mice),and 25 mg/kg/day Compound 30(13 mice). A, mean tumor volume(right) and circulating PSA (left) foreach treatment group are reported.B, waterfall plots represent percentchange in tumor volume (left) andPSA (right) after 3 weeks oftreatment for individual mice ineach treatment group. C, cancer-specific survival was comparedbetween the 3 groups over theperiod of the treatment and isrepresented by a Kaplan–Meiercurve. D, tumors were collected atthe end of the treatment and Ki67positivity was evaluated betweendifferent groups by IHC.

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Despite the positive effect of Compound 30 onMDV3100-resistant cells, we did observe indications ofresistance to Compound 30 in both LNCaP and MR49Fxenografts. This is not surprising, as the likelihood that apatient will become resistant to various treatments can bevery high, depending on the therapy and type of cancer.Furthermore, we observed that inMDV3100-resistant celllines, resistance to Compound 30 emerged 4 to 6 weeksafter treatment. Although our data indicates that AR isstill active, the emergence of tumors that are resistant toboth Compound 30 andMDV3100 suggests that targetingAR with a drug against ligand binding domain providesonly a short responding period before tumors becomerefractory to treatment. Possibly, targeting the AR withsimilar inhibitors, as in the case of MDV3100 and Com-pound 30, activates a resistance "memory" program,allowing cells to respond fasterwith survivalmechanismsthat were effective in protecting them from second-linetherapy. However, given the maintained anticancereffects on MDV3100-resistant cells in vitro and significantincrease in survival in vivo in Compound 30-treated micewith MDV3100-resistant tumors, Compound 30 may stillbe a viable alternative treatment offered to patients whofail MDV3100 therapy. In addition, our results do notexclude the possibility that Compound 30 may be moreeffective in some patients with CRPC as a second-linehormone therapy, or improve the efficacy of MDV3100 ifused in combination with this drug. Future studies inves-tigating the sequential and combinatorial use of thesedrugs are necessary.In summary, our results suggest that Compound 30 is

able to inhibit the continued AR activity that is associatedwith anti-AR resistance and may be a viable treatmentstrategy for MDV3100-resistant tumors in vivo. Further-more, our data provide a preclinical proof-of-principlethat novel antiandrogens, while they may have similar

efficacy against CRPC as other drugs, may be developedas an alternative in patients resistant to MDV3100.

Disclosure of Potential Conflicts of InterestA. Zoubeidi has commercial research grant from Pfizer and is a

consultant/advisory boardmember of Johnson and Johnson. No potentialconflicts of interest were disclosed by the other authors.

Authors' ContributionsConception and design: H. Kuruma, D. Briere, G. Los, A. Fanjul, A.ZoubeidiDevelopment of methodology: H. Kuruma, H. Matsumoto, F. Lamour-eux, D. Briere, A. ZoubeidiAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): H. Kuruma, H. Matsumoto, M. Shiota, F.Lamoureux, C. Thomas, M.E. GleaveAnalysis and interpretation of data (e.g., statistical analysis, biostatis-tics, computational analysis): H. Kuruma, M. Shiota, F. Lamoureux, D.Briere, G. Los, M.E. Gleave, A. Fanjul, A. ZoubeidiWriting, review, and/or revision of the manuscript: H. Kuruma, J.L.Bishop, M.E. Gleave, A. Fanjul, A. ZoubeidiAdministrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases): D. BriereStudy supervision: D. Briere, A. Zoubeidi

AcknowledgmentsTheauthors thankVirginiaYago, SoojinKim, andEstelle Li for technical

assistance.

Grant SupportThis work was supported by funding from Pfizer Global Research &

Development (United States), Prostate Cancer Foundation USA andMichael Smith for Health Research (A. Zoubeidi), Deutsche Forschungs-gemeinschaft German Research Foundation grant TH 1482/2-1 (T. Cor-donnier), and l’Association pour la Recherche sur le Cancer (France;F. Lamoureux).

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received August 2, 2012; revised February 6, 2013; accepted March 5,2013; published OnlineFirst March 14, 2013.

References1. Jemal A, Siegel R, Ward E, Murray T, Xu J, Smigal C, et al. Cancer

statistics, 2006. CA Cancer J Clin 2006;56:106–30.2. Gleave M, Goldenberg SL, Bruchovsky N, Rennie P. Intermittent

androgen suppression for prostate cancer: rationale and clinical expe-rience. Prostate Cancer Prostatic Dis 1998;1:289–96.

3. Eisenberger MA, Blumenstein BA, Crawford ED, Miller G, McLeodDG, Loehrer PJ, et al. Bilateral orchiectomy with or without fluta-mide for metastatic prostate cancer. N Engl J Med 1998;339:1036–42.

4. Snoek R, Cheng H, Margiotti K, Wafa LA, Wong CA,Wong EC, et al. Invivo knockdown of the androgen receptor results in growth inhibitionand regression of well-established, castration-resistant prostatetumors. Clin Cancer Res 2009;15:39–47.

5. GuoZ,YangX,SunF, JiangR, LinnDE,ChenH, et al. A novel androgenreceptor splice variant is up-regulated during prostate cancer pro-gression and promotes androgen depletion-resistant growth. CancerRes 2009;69:2305–13.

6. Shiota M, Yokomizo A, Naito S. Increased androgen receptortranscription: a cause of castration-resistant prostate cancerand a possible therapeutic target. J Mol Endocrinol 2011;47:R25–41.

7. Sun S, Sprenger CC, Vessella RL, Haugk K, Soriano K, Mostaghel EA,et al. Castration resistance in human prostate cancer is conferred by a

frequently occurring androgen receptor splice variant. J Clin Invest2010;120:2715–30.

8. Locke JA, Guns ES, Lubik AA, Adomat HH, Hendy SC,WoodCA, et al.Androgen levels increase by intratumoral de novo steroidogenesisduring progression of castration-resistant prostate cancer. CancerRes 2008;68:6407–15.

9. Feldman BJ, Feldman D. The development of androgen-independentprostate cancer. Nat Rev Cancer 2001;1:34–45.

10. Scher HI, Buchanan G, Gerald W, Butler LM, Tilley WD. Targeting theandrogen receptor: improving outcomes for castration-resistant pros-tate cancer. Endocr Relat Cancer 2004;11:459–76.

11. Tran C, Ouk S, Clegg NJ, Chen Y, Watson PA, Arora V, et al. Devel-opment of a second-generation antiandrogen for treatment ofadvanced prostate cancer. Science 2009;324:787–90.

12. Scher HI, Beer TM, Higano CS, Anand A, Taplin ME, Efstathiou E, et al.Antitumour activity of MDV3100 in castration-resistant prostate can-cer: a phase 1-2 study. Lancet 2010;375:1437–46.

13. Scher HI, Fizazi K, Saad F, Taplin ME, Sternberg CN, Miller K, et al.Increased survival with enzalutamide in prostate cancer after chemo-therapy. N Engl J Med 2012;367:1187–97.

14. Adamo V, Noto L, Franchina T, Chiofalo G, Picciotto M, Toscano G,et al. Emerging targeted therapies for castration-resistant prostatecancer. Front Endocrinol 2012;3:73.






15. Guo C, Linton A, Kephart S, Ornelas M, Pairish M, Gonzalez J, et al.Discovery of aryloxy tetramethylcyclobutanes as novel androgenreceptor antagonists. J Med Chem 2011;54:7693–704.

16. Kiyama S, Morrison K, Zellweger T, Akbari M, Cox M, Yu D, et al.Castration-induced increases in insulin-like growth factor-bindingprotein 2 promotes proliferation of androgen-independent humanprostate LNCaP tumors. Cancer Res 2003;63:3575–84.

17. Lamoureux F, ThomasC, YinMJ, KurumaH, Fazli L,GleaveME, et al. Anovel HSP90 inhibitor delays castrate-resistant prostate cancer with-out altering serum PSA levels and inhibits osteoclastogenesis. ClinCancer Res 2011;17:2301–13.

18. Zoubeidi A, Zardan A, Beraldi E, Fazli L, Sowery R, Rennie P, et al.Cooperative interactions between androgen receptor (AR) and heat-shock protein 27 facilitate AR transcriptional activity. Cancer Res2007;67:10455–65.

19. GleaveME, Hsieh JT, WuHC, von Eschenbach AC, Chung LW. Serumprostate specific antigen levels inmicebearinghumanprostate LNCaPtumors are determined by tumor volume and endocrine and growthfactors. Cancer Res 1992;52:1598–605.

20. Chen Y, Clegg NJ, Scher HI. Anti-androgens and androgen-depletingtherapies in prostate cancer: new agents for an established target.Lancet Oncol 2009;10:981–91.

21. Linja MJ, Savinainen KJ, Saram€aki OR, Tammela TL, Vessella RL,Visakorpi T. Amplification and overexpression of androgen receptorgene in hormone-refractory prostate cancer. Cancer Res 2001;61:3550–5.

22. Holzbeierlein J, Lal P, LaTulippe E, Smith A, Satagopan J, Zhang L,et al. Gene expression analysis of human prostate carcinomaduring hormonal therapy identifies androgen-responsive genes

and mechanisms of therapy resistance. Am J Pathol 2004;164:217–27.

23. Stanbrough M, Bubley GJ, Ross K, Golub TR, Rubin MA, Penning TM,et al. Increased expression of genes converting adrenal androgens totestosterone in androgen-independent prostate cancer. Cancer Res2006;66:2815–25.

24. Hobisch A, Culig Z, Radmayr C, Bartsch G, Klocker H, Hittmair A.Androgen receptor status of lymph node metastases from prostatecancer. Prostate 1996;28:129–35.

25. Edwards J, Krishna NS, Grigor KM, Bartlett JM. Androgen receptorgene amplification and protein expression in hormone refractoryprostate cancer. Br J Cancer 2003;89:552–6.

26. Zegarra-Moro OL, Schmidt LJ, Huang H, Tindall DJ. Disruption ofandrogen receptor function inhibits proliferation of androgen-refrac-tory prostate cancer cells. Cancer Res 2002;62:1008–13.

27. Fenner A. Prostate cancer: abiraterone increases overall survival in menwith castration-resistant prostate cancer. Nat Rev Urol 2011;8:351.

28. Zoubeidi A, Zardan A, Wiedmann RM, Locke J, Beraldi E, Fazli L, et al.Hsp27 promotes insulin-like growth factor-I survival signaling in pros-tate cancer via p90Rsk-dependent phosphorylation and inactivation ofBAD. Cancer Res 2010;70:2307–17.

29. Lamoureux F, Thomas C, Yin MJ, Kuruma H, Beraldi E, Fazli L, et al.Clusterin inhibition using OGX-011 synergistically enhances Hsp90inhibitor activity by suppressing the heat shock response in castrateresistant prostate cancer. Cancer Res 2011;71:5838–49.

30. Whitesell L, Lindquist SL. HSP90 and the chaperoning of cancer. NatRev Cancer 2005;5:761–72.

31. PetrylakDP.Current clinical trials in castrate-resistant prostate cancer.Curr Urol Rep 2011;12:173–9.

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