selective androgen receptor modulator rad140 inhibits the ... · arn-509 (selleckchem) for 24...

$: Selective Androgen Receptor Modulator RAD140 Inhibits the ... · ARN-509 (Selleckchem) for 24 hours. ... The nuclear and cytoplasmic fractions of the cells were prepared using the$
Cancer Therapy: Preclinical

Selective Androgen Receptor Modulator RAD140Inhibits the Growth of Androgen/EstrogenReceptor–Positive Breast Cancer Models witha Distinct Mechanism of ActionZiyang Yu, Suqin He, Dannie Wang, Hitisha K. Patel, Chris P. Miller,Jeffrey L. Brown, Gary Hattersley, and Jamal C. Saeh

Abstract

Purpose: Steroidal androgens suppress androgen receptor andestrogen receptor positive (AR/ERþ) breast cancer cells and wereused to treat breast cancer, eliciting favorable response. Thecurrent study evaluates the activity and efficacy of the oral selectiveAR modulator RAD140 in in vivo and in vitro models of AR/ERþ

breast cancer.Experimental Design: A series of in vitro assays were used to

determine the affinity of RAD140 to 4 nuclear receptors andevaluate its tissue-selective AR activity. The efficacy and pharma-codynamics of RAD140 as monotherapy or in combination withpalbociclib were evaluated in AR/ERþ breast cancer xenograftmodels.

Results: RAD140 bound AR with high affinity and specificityand activatedAR inbreast cancer but not prostate cancer cells.Oraladministration of RAD140 substantially inhibited the growth of

AR/ERþ breast cancer patient-derived xenografts (PDX). Activa-tion of AR and suppression of ER pathway, including the ESR1gene, were seen with RAD140 treatment. Coadministration ofRAD140 andpalbociclib showed improved efficacy in theAR/ERþ

PDX models. In line with efficacy, a subset of AR-repressed genesassociated with DNA replication was suppressed with RAD140treatment, an effect apparently enhanced by concurrent admin-istration of palbociclib.

Conclusions: RAD140 is a potent AR agonist in breast cancercells with a distinct mechanism of action, including the AR-mediated repression of ESR1. It inhibits the growth of multipleAR/ERþ breast cancer PDX models as a single agent, and incombinationwith palbociclib. The preclinical data presented heresupport further clinical investigation of RAD140 in AR/ERþ breastcancer patients. Clin Cancer Res; 23(24); 7608–20. �2017 AACR.

IntroductionBreast cancer is the second leading cause of cancer-related death

in women, with an estimated 246,660 newly diagnosed cases and40,450deaths in theUnited States alone in 2016 (1). Breast canceris a heterogeneous disease categorized into several histopatho-logic subtypes based on the status of estrogen receptor a (ER),progesterone receptor (PR), and HER2 receptor. Although ER-positive (ERþ) breast cancers are now treated with standard-of-care agents targeting the ER axis, including tamoxifen, fulvestrant,and aromatase inhibitors (AI), and HER2-positive tumors aretreated with HER2 inhibitors, such as trastuzumab, novel thera-peutic approaches are still needed to address resistance emergingfrom these established regimens (2, 3). More recently, combinedadministration of ER-targeted therapies with the inhibitors ofcyclin-dependent kinase (CDK) 4/6 (4) ormTOR (5) have yieldedimproved therapeutic efficacy in ERþ breast cancer, and these

combination therapies now represent a new generation of stan-dard of care for this indication.

Recent histopathologic studies revealed that the androgenreceptor (AR) is themost commonly expressed hormone receptorin breast cancer, with 75% to 90%of ERþ and approximately 30%of ER� breast cancers expressing AR (6, 7). Although AR is widelypresent in breast cancer tissue, accumulating evidence has dem-onstrated that the role of AR in these tumors is subtype dependent(8–11). AR antagonists including bicalutamide and enzalutamidehave been shown to reduce the growth of AR-positive but ER�,including triple-negative, breast cancer in preclinical models(12–14) and in patients (11). In contrast, in ERþ breast cancers,AR has been considered antiproliferative and has been associatedwith a favorable prognosis. Clinically, until the 1970s, breastcancers were often treated with nonselective steroidal androgensincluding testosterone derivatives and danazol with responserates of 20% to 25% (9, 15–18). In line with these clinicalexperiences, preclinical studies have also shown treatment withclassic androgens, including DHT, reduced the growth of AR andERþ (AR/ERþ) breast cancer cells in vitro (19–21) and in vivo (22).Classic androgen-based therapy for breast cancer declined due toits virilizing effects, potential risk of further aromatization toestrogens, and the emergence of ER-targeted agents includingtamoxifen. However, it is worth noting that more recent clinicalevidence showed androgen treatment also led to remission inpatients who were progressing after ER-targeted therapy withobjective response rate around 17% to 39% (23, 24). Fulvestrant

Radius Health, Inc., Waltham, Massachusetts.

Note: Supplementary data for this article are available at Clinical CancerResearch Online (http://clincancerres.aacrjournals.org/).

Corresponding Authors: Ziyang Yu, Radius Health, Inc., 950 Winter Street,Waltham, MA 02451. Phone: 617-551-4065; Fax: 617-551-4701; E-mail:[email protected]; and Jamal C. Saeh, [email protected]

doi: 10.1158/1078-0432.CCR-17-0670

�2017 American Association for Cancer Research.

ClinicalCancerResearch

Clin Cancer Res; 23(24) December 15, 20177608

on April 10, 2020. © 2017 American Association for Cancer Research. clincancerres.aacrjournals.org Downloaded from

Published OnlineFirst October 3, 2017; DOI: 10.1158/1078-0432.CCR-17-0670

http://crossmark.crossref.org/dialog/?doi=10.1158/1078-0432.CCR-17-0670&domain=pdf&date_stamp=2017-11-20

http://clincancerres.aacrjournals.org/

at second line or later in large phase III trials elicited objectiveresponse rates of 2.1% to 11% (25–28).

The high prevalence of AR observed in ERþ breast cancers,along with the prior clinical efficacy demonstrated with classicandrogens, provides a strong rationale for the development of anew generation of oral, selective AR agonists and to furtherexploit their therapeutic benefits in AR/ERþ breast cancer. Here,we describe the tissue-selective AR agonist activity of RAD140,an oral nonsteroidal selective AR modulator (SARM), and itsdistinct AR-mediated mechanism of action including the sup-pression of ESR1. RAD140 exhibited potent antitumor activityin the in vivo models of AR/ERþ breast cancer both as a singleagent and in combination with the CDK4/6 inhibitor palbo-ciclib. RAD140 represents a new generation of tissue-selectiveAR agonists and potentially a novel therapeutic option forAR/ERþ breast cancer.

Materials and MethodsNuclear receptor binding assay

The PolarScreen nuclear receptor competitor assays (ThermoFisher Scientific) for AR, ER, PR, and glucocorticoid receptor (GR)were used to determine the binding affinity and specificity ofRAD140. Briefly, RAD140 and fluorescence-labeled ligands(Fluormones) for AR, ER, PR, or GR, respectively, were added toeach reaction well, and the plate was incubated for 4 hours atroom temperature in the dark. Polarization values for each wellweremeasured and plotted against the concentration of RAD140.As a reference, the binding affinity of the standard ligands DHT,17b-estradiol, progesterone, and dexamethasone (for AR, ER, PR,and GR, respectively) was also measured. The relative bindingaffinity of RAD140 versus the 4 standard ligands is reflected by theratios of IC50 (standard ligand)/IC50 (RAD140). A commerciallyavailable spectrum screen was performed for RAD140 (MDSPharma Services). The binding of RAD140 (1 mmol/L) to a panelof cellular targets was assessed (Supplementary Table S1). Appre-ciable binding was defined as a larger than 50% inhibition of thereference radio-ligand.

Cell culture and treatmentThe ZR-75-1, HCC1428, and T47D breast cancer cells and

LNCaP prostate cancer cells were purchased from ATCC andmaintained in medium and condition as recommended by thevendor. All cell lines involved in the study were tested negative formycoplasma contamination (MycoAlert Detection Kit) and sub-jected to annual authentication using STR profiling (ThermoFisher Scientific). All the cells were under passage 15 at the timeof experiments. HCC1428 long-term estrogen-depleted (LTED)cells were established by culturing these cells in medium with10% charcoal dextran-stripped serum (CSS) for 16weeks, and theexpressions of AR, ER, and PRwere confirmed byWestern blotting(Supplementary Fig. S1A). For proliferation assay, cells wereseeded in medium with 10% CSS at 30,000 cells per well in24-well plates and subjected to treatment with RAD140, DHT, orDMSO for 14 days with treatments being renewed every 3 days. Atthe endof the treatment period, trypsinized cellswere stainedwithTrypan blue before being subjected to live cell counting in aNexcelom Cellometer (Nexcelom Bioscience). The PC3-AR cellline, described previously (29), was a generous gift fromDr. SteveBalk of Beth Israel Deaconess Medical Center (Boston, MA) andDr. Changemeng Cai of University of Massachusetts (Boston,MA). For ZR-75-1 cell-based assays, cells were seeded and incu-bated in medium with 5% CSS for 48 hours. Cells were thentreated with 17b-estradiol at 1 nmol/L final concentration orvehicle (ethanol) before being incubated with DMSO, RAD140(Radius Health, Inc., as described previously; ref. 30), or DHT(Sigma Aldrich) in the presence or absence of enzalutamide orARN-509 (Selleckchem) for 24 hours. The experiments wereperformed in duplicate. The nuclear and cytoplasmic fractionsof the cells were prepared using the NE-PER Nuclear and Cyto-plasmic Extraction Kit (Thermo Fisher Scientific) following themanufacturer's instructions.

AR reporter gene assaysAR reporter gene assay was carried out using a Cignal Androgen

Receptor Reporter (luc) Kit (Qiagen). ZR-75-1 breast cancer cells,LNCaP, and PC3-AR prostate cancer cells were transfected with atandem androgen-responsive element (ARE)-driven firefly lucif-erase construct and a Renilla luciferase construct in RMPI1640media containing 5% CSS. Forty-eight hours later, transfectedcells were treated with DMSO, RAD140, or DHT. The luciferaseactivity was determined using the Dual-Luciferase Reporter Assay(Promega) as a representation of AR transcription activity andnormalized to the activity ofRenilla luciferase. Each set was carriedout in triplicate. The valueswere presented as average fold changes� SD over the vehicle control.

In vivo efficacy studyAll study protocols were reviewed by Institutional Animal Care

and Use Committees and Radius Health, Inc., and conducted incompliance with U.S. and European regulations of protection oflaboratory animals. The HBCx-22, HBCx-21, and HBCx-3 breastcancer patient-derived xenograft (PDX) models were establishedand evaluated at XenTech by implanting tumor fragments sub-cutaneously in the flank female athymic nude mice (Foxn1nu,Envigo RMS). The ST897 breast cancer PDX model was estab-lished in female athymic nude mice (Foxn1nu, Charles RiverLaboratories) and studies conducted at South Texas AcceleratedResearch Therapeutics. Thesemodelswere characterized as ER andPR positive and negative for HER2 overexpression based on IHC

Translational Relevance

The expression of the androgen receptor (AR) in estrogenreceptor (ER)–positive breast cancer has been well described.Androgens inhibit the proliferation of AR and ER-positive(AR/ERþ) breast cancer cells, and androgen-based treatmentshowed clinical benefit in breast cancer patients. However, theclinical utility of the classic steroidal androgen-based therapieshas been limited. We report here that RAD140, an oral, tissueselective androgen receptormodulator, exhibited potent activ-ity in inhibiting the growth of multiple AR/ERþ breast cancerxenograft models. The coadministration of RAD140 withCDK4/6 inhibitor elicited enhanced efficacy in these AR/ERþ

models. Mechanistically, RAD140 potently regulates AR targetgenes, while suppressing the ER targets and the ESR1 mRNA.These suggest a distinct AR-mediated mechanism of actioncompared with the traditional ER-targeted agents. RAD140may present a novel therapeutic approach for AR/ERþ breastcancer. Thefirst-in-human studyof RAD140 in recurrent breastcancer has been initiated.

SARM Inhibits AR/ERþ Breast Cancer

www.aacrjournals.org Clin Cancer Res; 23(24) December 15, 2017 7609




and later confirmed using Western blot analysis and qPCR (Sup-plementary Table S2; Supplementary Fig. S1). The efficacy study inT-47D breast cancer cell line–derived xenograft model, estab-lished in NOD/Shi-scid/IL-2Rgnull mice, was conducted at WuXiAppTec. These xenograft models were supplemented with exog-enous estradiol in drinking water (HBCx-3, HBCx-21, HBCx-22,and ST897) or subcutaneous pellet (0.18 mg/90-day release,Innovative Research America) implanted 3 days prior to tumorcell inoculation (T-47D). The PDX-bearing mice were random-ized once their tumor volumes reached 60 to 200 mm3, asdescribed previously (31). The cell line–derived xenografts withvolumes between 125 and 250 mm3 were enrolled in treatmentgroups. RAD140, DHT, or fulvestrant were administered as singleagent or in combination with palbociclib. RAD140, palbociclib,and the vehicle (0.5% carboxymethyl cellulose) were given orally.Fulvestrant (clinical formulation, AstraZeneca) was given as sub-cutaneous injection. DHT was administered as subcutaneouslyimplanted pellets (12.5 mg/60-day release, Innovative ResearchAmerica) from the day of randomization to the end of treatment(14). Tumor volume and body weight were measured twice aweek. Tumor growth inhibition (TGI %) was calculated on thebasis of the change of mean tumor volume from treatment day 1to the last day of assessment and presented as a percentage of thatof the vehicle control group [100� (1-DTVtreatment/DTVvehicle)], asan indication of antitumor efficacy. Statistical analyses wereperformed for the changes in individual tumor volume (DTV,from treatment day 1 to the last day of assessment) between thegroups compared.

IHCFormalin-fixed paraffin-embedded sections of xenograft

tumors underwent antigen retrieval and were then blocked using5% goat serum and avidin blocking solution. IHCwas performedin a Leica Bond III auto-stainer (LeicaBiosystems) according to themanufacturer's instructions. The primary rabbit mAbs against ER(SP1) and PR (SP42) were obtained from Cell Marque. RabbitmAbagainst Ki67 (D2H10) andphospho-retinoblastomaprotein(phospho-Rb) were from Cell Signaling Technology. Mouse anti-body against AR (441) was obtained from Dako. The AR IHCstaining protocol included the use of ImmunoDNA BackgroundBlocker (Bio SB). Sections shown in each figure were stained andphotographed under identical conditions. Staining and scoring ofthe samples were performed by a pathologist blinded to thetreatment groups.

Western blot analysisTumor lysates from the in vivo studies were prepared using Cell

Lysis Buffer with protease and phosphatase inhibitors (Cell Sig-naling Technology) in a FastPrep Sample Preparation System (MPBiomedicals). Cell lysates were prepared using Cell Lysis Buffer asdescribed above. Total protein was quantitated and subjected toWestern blot analysis using antibodies against AR (PG-21, EMDMillipore; Clone 441, Thermo Fisher Scientific), ERa, PR,GAPDH, b-tubulin, and HDAC2 (Cell Signaling Technology).

Gene expression analysisTotal RNA fromxenograft samples and cells was extracted using

the RNeasy Mini Kit (Qiagen), with a DNase incubation stepincluded to ensure complete removal of genomic DNA. Theexpression of genes was evaluated using qPCR and RNA sequenc-ing (RNA-seq). For qPCR, TaqMan primer and probe sets for AR,

KLK2, FKBP5, ZBTB16, ESR1, PGR, TFF1, GREB1, BLM, FANCI,LMNB1, MCM2, MCM4, MCM7, 18S, and GAPDH were pur-chased fromThermo Fisher Scientific. The PCR reactionwas set upwith TaqMan Fast Virus 1-Step Master Mix in a Quant Studio6 Flex qPCR system (Thermo Fisher Scientific). Samples from aminimum of 3 tumors from each treatment group or those fromaminimumof 2 replicated cell-based assays were included for theanalysis. The expression of genes of interest were normalized tothat of GAPDH or 18S and presented as the mean fold changecompared with the vehicle control group. For RNA-seq analysis,total RNA samples were converted into cDNA libraries using theTrueSeq Stranded mRNA Sample Preparation Kit (Illumina).Libraries are quantified, normalized, and pooled before beingsubjected to HiSeq 2 � 50 bp paired end sequencing on anIllumina sequencing platform. De-multiplexed FASTQ files werealigned to HG19 human genome using the STAR aligner version2.4 and genetic features were quantified using RSEM version1.2.14. Thedata discussed in this publication havebeendepositedin NCBI's Gene Expression Omnibus and are accessible throughGEO Series accession number GSE104177. The AR and ER targetgenes with significant changes in expression (FDR < 0.05) wereanalyzed and log2 fold changes presented in heatmap. Globalpathway analysis was performed for genes with significantchanges in expression (FDR < 0.001) using the functional anno-tation tools (Gene Ontology) available on the National HealthInstitute DAVID Bioinformatics Resources platform (https://david.ncifcrf.gov).

ResultsRAD140 binds to AR with high affinity and high specificity

Thebinding affinity of RAD140was assessed usingfluorescencepolarization-based competitive binding assays for AR, ER, PR, andGR and compared against the standard ligands for each nuclearreceptor. As positive controls, assays evaluating the binding of thestandard ligands, including DHT, 17b-estradiol (E2), progester-one, and dexamethasone to AR, ER, PR, and GR, respectively,were performed. In the competitive binding assay against theAR-Fluoromone, RAD140 exhibited 1.6-fold lower AR affinitythan DHT (Fig. 1A). This suggests binding affinity of RAD140 toAR is slightly lower but comparablewith that of natural androgen.RAD140 exhibited 33-fold lower affinity to PR compared withprogesterone. Nobinding of RAD140 to ERorGRwas detected. Inaddition, a cellular target spectrum screen was performed todetermine the binding selectivity of RAD140 against a broadpanel of 165 molecular targets that included many pharmaco-logically relevant molecules including ion channels, G-protein–coupled receptors, enzymes, and nuclear hormone receptors(Supplementary Table S1).No appreciable interactionof RAD140with any targets screened, other than AR and PR, was detected.These results indicate RAD140 binds to AR with high affinityand specificity.

RAD140 is a potent tissue-selective AR agonistSARMs are selective AR ligands that have been shown to act as

agonists or antagonists in a tissue-context–dependent manner(32). We previously demonstrated that RAD140 exhibits differ-ential activity in prostate versus in the muscle (30). To furtherassess the activity of RAD140onAR transcription in tissue context,an AR reporter assay was performed in ZR-75-1, a human breastcancer cell line expressing endogenous AR and ER (21), and the

Yu et al.

Clin Cancer Res; 23(24) December 15, 2017 Clinical Cancer Research7610



https://david.ncifcrf.gov

https://david.ncifcrf.gov


AR-positive prostate cancer cell line LNCaP (29). Dose responsesby RAD140 were compared with the known AR agonist DHT.Treatment of steroid-depleted ZR-75-1 cells with 1, 10, and100 nmol/L RAD140 induced AR transcription activity by 1.9-to 2.9-fold, comparable with the induction seen with DHT at thesame concentrations (Fig. 1B, left). In contrast, in the low-passage,androgen-sensitive LNCaP cells treated with RAD140 at 0.1 to10nmol/L, no induction ofAR activitywas observedwhileDHT at

1 or 10 nmol/L led to more than a 10-fold induction of ARtranscription activity (Fig. 1B, right). To rule out the possibilitythat the attenuated AR activity of RAD140 is due to the expressionof a mutant AR T877A in LNCaP cells, we also performed ARreporter assay in PC3-AR cells, which express ectopic wild-type AR(29). Similar to the finding in LNCaP cells, RAD140 exhibitedmuch attenuated activity on ectopic wild-type AR in prostatecancer cells compared with DHT (Supplementary Fig. S2). These

Figure 1.

Binding affinity and specificity of RAD140 and its tissue-selective AR agonist activity. A, The binding affinity of RAD140 to AR, ER, PR, and GR wasdetermined using PolarScreen Competitive Binding Assays for each of these nuclear receptors. DHT, 17b-estradiol, progesterone, and dexamethasone wereincluded as positive control for the binding to AR, ER, PR, and GR, respectively. The relative affinity of RAD140, versus the standard ligands, to thesereceptors was calculated on the basis of the IC50 values using Prism. Data presented are an average of at least three independent experiments. B, ZR-75-1 breastcancer cells and LNCaP prostate cancer cells were transfected with ARE-luc and Renilla luciferase constructs in medium containing 5% CSS. Twenty-fourhours after the transfection, cells were treated with RAD140 or DHT at the indicated final concentrations. ARE-driven luciferase activity was measured andnormalized to that of Renilla luciferase. Data presented are an average of biological triplicates. C, Steroid-deprived ZR-75-1 cells were treated with ethanol (vehicle)or 1 nmol/L of 17b-estradiol (E2) for 2 hours, followed by treatment with RAD140 at 100 nmol/L in the presence or absence of ARN-509 (10 mmol/L) for overnight.Whole-cell lysates and nuclear fractions were prepared and subjected to Western blot analyses for AR. HDAC2 and b-tubulin were probed as loadingcontrols for nuclear and whole-cell extracts, respectively. D, HCC1428 LTED cells were maintained in steroid-depleted condition and treated with RAD140 orDHT at the indicated concentrations for 14 days, with medium and compounds replenished every 3 days. At the end of the treatment, cells were stained withTrypan blue before subjected to live-cell counting in a Nexcelom Cellometer. Average cell number � SD of biological triplicates is presented. � , P < 0.05 forcomparison with the DMSO-treated group.






results indicated that RAD140 is a tissue-specific AR agonist,with selective activity in breast cancer cells but not in prostatecancer cells.

Classic AR agonists are known to induce AR conformationchange and subsequent localization to the nuclei, while pure ARantagonist, such as enzalutamide and ARN-509 act partially viablocking AR nuclear localization (33, 34). We then examinedwhether RAD140 induces the nuclear expression of AR in breastcancer cells. Treatment of ZR-75-1 cells with RAD140 led toincreased AR expression in the nuclear fraction while the estrogen(E2) showed no apparent effect (Fig. 1C). The positive control,DHT, substantially increased the nuclear expression of AR. ARN-509 substantially reduced the nuclear expression induced byDHTandRAD140, suggesting a direct antagonism against these twoARagonists. This provided further evidence that RAD140 is an ARagonist in breast cancer cells.

RAD140monotherapy suppresses the growth of AR/ERþ breastcancer PDX models

It has been previously reported that DHT inhibits the prolif-erationof ERþbreast cancer cells driven by supplemental estradiol(21). We evaluated the activity of RAD140 and DHT in anestrogen-independent AR/ERþbreast cancer lineHCC1428 LTED,in which ER signaling appears to be active as judged by PRexpression (Supplementary Fig. S1). RAD140 exhibited inhibito-ry effect on the proliferation of these endocrine-resistant breastcancer cells at concentrations as low as 10 nmol/L, with anapparent maximal effect seen at 100 nmol/L (Fig. 1D). This wascomparable with the inhibitory effect seen with DHT in the 0.1 to10 nmol/L range. To further assess how the AR agonist activity ofRAD140 translates into antitumor activity in vivo, RAD140 wasevaluated as a single agent in AR/ERþ breast cancer PDX models.Oral administration of RAD140 induced significant TGI of 76%,59%, and 58%when compared with the vehicle control groups inHBCx-22, HBCx-3, and HBCx-21, respectively (Fig. 2A, Supple-mentary Fig. S3A and S3B). In the HBCx-22 model, fulvestrant, aselective ER downregulator (SERD) and standard of care for ERþ

breast cancer dosed at 1 mg weekly, exhibited a statisticallysignificant antitumor activity, as judged by a TGI of 59%, com-parable with a previous report of this model and indicative of itsdependency on ER (31). It has been previously determined thatthe 1 mg weekly administration of fulvestrant achieves exposureequivalent to the 500 mg monthly dose in human (35). Thechanges in tumor volume of the RAD140- or fulvestrant-treatedHBCx-22 xenografts appeared to be lower compared with thosetreated with vehicle (Fig. 2B). To further verify the concept ofinhibiting the growth of AR/ERþ breast cancer tumors with ARagonists, T-47D cell line–derived xenografts were treated withDHT, RAD140, or fulvestrant (Supplementary Fig. S3C). Treat-ment with DHT or RAD140 inhibited the growth of these T-47Dtumors to a comparable extent. Fulvestrant also elicited potentgrowth inhibition. In addition, the activity of the combination offulvestrant with RAD140 was evaluated in HBCx-22 model, butno appreciable improvement in tumor growth inhibition wasobserved, suggesting apotential overlap in thepathway inhibitioninduced by these two agents in this model (SupplementaryFig. S3D). This observation is consistent with previous clinicalexperience with anastrozole–fulvestrant combination and pre-clinical data of fulvestrant–RAD1901 combination, bothofwhichshowed such intense ERblockade failed to yield additional benefit(35, 36). Together, these results indicated that RAD140 as a single

agent is effective in inhibiting the growth of AR/ERþ breast cancerxenografts.

We then examined the changes in common breast cancerbiomarkers in the HBCx-22 xenografts treated with RAD140 orfulvestrant using IHC.As shown inFig. 2C,HBCx-22 tumors in thecontrol group were positive for AR, ER, and PR, consistent withprior characterization of this model (Supplementary Fig. S1). Anapparently increased AR expression, predominantly localized inthe nuclei, was seen in RAD140-treated tumors. This demonstrat-ed that RAD140 possesses AR agonist properties, such as stabi-lizing AR and promoting its nuclear localization, which arecommonly seen with classic androgens (37, 38). Interestingly,RAD140 treatment led to substantially decreased expression of ERand its downstream target PR. A profound decrease in Ki67expression was also seen in these RAD140-treated tumors, indi-cating a suppressive effect on theproliferation of these tumor cells.Treatment with fulvestrant also led to substantial decreases in ER,PR, and Ki67 expression, to levels seemingly comparable withthose seenwith RAD140. Furthermore, the epithelial nature of theterminal tumor mass was confirmed using IHC assays for cyto-keratin (CK) 18 and vimentin (Supplementary Fig. S4). Thissuggests SARM and SERD both inhibit the ER pathway and breastcancer cell proliferation.

Pharmacodynamic analysis reveals RAD140-mediatedregulation of AR and ER target genes and ESR1

It has been proposed that androgens suppress ER signaling inAR/ERþ breast cancer cells, which may contribute to the anti-proliferative activity (19, 21). To better understand the mecha-nism of action of the SARM RAD140 in AR/ERþ breast cancermodels, we examined the pharmacodynamic changeswith a focuson the AR and ER pathways. In the HBCx-22 model, a profoundinduction of AR target genes, including FKBP5, KLK2, andZBTB16, was seen in RAD140-treated tumors (Fig. 3A, top) whilethe expression of the AR gene was not affected. Fulvestrant, asexpected, did not induce the expression of these AR target genes,although a slight increase in AR message was seen. The ER targetgenes PGR, TFF1, and GREB1 were found to be substantiallysuppressed in RAD140-treated tumors to levels comparable oreven lower than that seen with fulvestrant (Fig. 3A, bottom).Interestingly, in these RAD140-treated tumors, the mRNA expres-sion of ESR1, the coding gene of ER, was profoundly suppressed.In contrast, the fulvestrant treatment did not lead to appreciablechange in ESR1 gene expression. At protein level, both RAD140and fulvestrant exhibited a profound effect in decreasing theexpression of ER and PR in HBCx-22 xenografts (Fig. 3B, left),consistent with the IHC findings described above. Similardecrease in ER and PR expression was seen in HBCx-3 AR/ERþ

xenografts treated with RAD140 (Fig. 3B, middle). In the T-47Dxenografts, treatment with RAD140, DHT, or fulvestrant also ledto decreased ER and PR expression (Fig. 3B, right).

Next, themodulation of AR andER target genes byRAD140wasfurther examined in vitro. As determined in ARE-luc reporter assayand proliferation assay (Fig. 1B and D), RAD140 at 100 nmol/Lexhibited maximal effects, comparable with DHT at 10 nmol/L;therefore, 100 nmol/L was selected as the RAD140 concentrationfor further in vitro assays. In ZR-75-1 cells treated with RAD140,substantial induction of FKBP5 and ZBTB16 messages was seen(Fig. 3C), which was comparable with that seen with DHT. Theinduction of these AR targets by RAD140 or DHT was completelyblocked by the antagonist enzalutamide. The expression of theAR

Yu et al.





gene did not seem to be affected by RAD140 or DHT treatment.The ER target genes PGR and TFF1 were induced by 1 nmol/L ofE2. Treatment with either RAD140 or DHT was found to repressthe E2-induced expression of these genes. Similar to the effectsseen in xenograft models, treatment with RAD140 or DHTdecreased ESR1 mRNA. The effects of RAD140 and DHT on AR

pathway and ER pathway genes appear to be comparable, suggest-ing anAR-specific effect. Furthermore, competitive blockade of ARactivation by enzalutamide effectively reversed the AR and ERpathway modulation by RAD140 or DHT. The incomplete rever-sal of ESR1 suppression by enzalutamide may be due to therelatively lower affinity of this compound compared with AR

Figure 2.

The effect of oral administration ofRAD140 on the growth of AR/ERþ

breast cancer PDX. A, Mean tumorvolumes � SEM of HBCx-22 breastcancer xenografts treated withvehicle, RAD140 (100 mg/kg oncedaily), or fulvestrant (1 mg onceweekly) for the indicated period oftime (n ¼ 8/group). B, Changes intumor volume of individual HBCx-22xenografts from the beginning to theend of the treatment.C, IHC analysis ofHBCx-22 xenografts collected at theend of the study was performed usingantibodies against AR, ER, PR, andKi67. Representative images of threeindividual tumors from each treatmentgroup are shown.






Figure 3.

Expression of AR and ER pathway genes and ESR1 in RAD140-treated AR/ERþ breast cancer cells and xenografts. A, Tumor samples from the HBCx-22collected 6 hours after last treatment were subjected to qPCR analysis for the expression of AR, ER, and the indicated downstream target genes. B, Samplesfrom HBCx-22, HBCx-3, and T-47D xenograft models were collected 6 hours after last treatment and subjected to Western blot analysis for the expression ofAR, ER, and PR. C, Steroid-deprived ZR-75-1 cells were pretreated with 1 nmol/L of E2 for 2 hours and then treated with RAD140 (100 nmol/L) or DHT (10 nmol/L)in the presence or absence of enzalutamide (10 mmol/L) for 24 hours. RNA of the treated cells was isolated and subjected to qPCR analysis for theexpression of AR, ESR1, and the indicated downstream targets.

Yu et al.





agonists RAD140 and DHT. These in vivo and in vitro observationsfurther demonstrated the AR agonist activity of RAD140 inAR/ERþ breast cancer models. More importantly, RAD140 andDHT both inhibited ER downstream targets, as well as the ESR1gene, suggesting a novel mechanism of action of AR agonists ininhibiting the ER pathway in breast cancer cells.

Global modulation of signaling pathways by SARM in AR/ERþ

breast cancer xenograftsTo further understand the global effect of RAD140 on signaling

cascades in AR/ERþ breast cancer cells, RNA-seq analysis ofHBCx-22 xenografts was performed.We examined a broader range of ARand ER target genes with significantly altered expression inRAD140-treated tumors (Fig. 4A). The results indicated that in

addition to the targets examined by qPCR, RAD140-treatedtumors had higher levels of AR-activated genes in addition tothose shown in qPCR. A subset of the ER target geneswas found tobe suppressed in RAD140-treated tumors, consistent with theqPCR findings. Using a stringent filtering criteria (FDR < 0.001),we found that globally 86 genes were significantly upregulatedand 77 were downregulated by at least 2-fold in RAD140-treatedHBCx-22 xenografts (Fig. 4B; Supplementary Tables S3 andS4). Inthe Gene Ontology analysis, these upregulated genes were foundto be most significantly enriched in the metabolic process. Thiswas consistentwith theprevious report describing the role ofARasa master regulator of central metabolism and biosynthesis inprostate cancer cells (39). The downregulated genes inRAD140-treated tumors were found to be enriched in categories

Figure 4.

Global pathway modulation by RAD140 in the HBCx-22 PDX model. A, Heatmaps of mRNA expression fold change of the known AR and ER pathway genesin RAD140- versus vehicle-treated tumors (n ¼ 3/group), with downregulation represented by shades of blue and upregulation by shades of red. GeneOntology analyses of the significantly upregulated (B) or downregulated (C) genes in RAD140-treated tumors.






associated with cell division, DNA replication, and cell-cycleprogression. This negative regulation of gene transcription wasin line with previous reports on the transcription suppressor roleof AR (40, 41). These data further confirmed the regulation of ARand ER pathways by RAD140 and suggested a uniquemechanismof action of RAD140 via the AR-mediated transcription repres-sion. These data offer evidence for further uncovering the under-lying mechanism of RAD140 as a single agent in AR/ERþ breastcancer models.

Enhanced antitumor activity of combined administrations ofRAD140 with CDK4/6 inhibitor

Given the observed effect of RAD140 on cell cycle and DNAreplication–related genes, we hypothesized that combinedadministration of this SARM and a CDK4/6 inhibitor, whichinhibits the phosphorylation and subsequent degradation of Rb,thus blocking cell-cycle entry (42–44), may produce improvedantitumor activity in AR/ERþ breast cancer models. Palbociclib, aCDK4/6 inhibitor recently approved for breast cancer treatment incombination with ER-targeted agents in as early as first line oftherapy (45–47), was selected for this study. Two AR/ERþ PDXmodels, HBCx-3 (Fig. 5A) and ST897 (Fig. 5B), were treated withRAD140, palbociclib, or a combination of RAD140 with palbo-ciclib. Compared with the vehicle-treated group, RAD140 asmonotherapy produced statistically significant inhibition oftumor growth in these models compared with vehicle control.Palbociclib as a single agent also inhibited the growth of thesexenografts to a comparable level. Importantly, RAD140 andpalbociclib when administered together elicited improved effica-cy over palbociclib or RAD140 monotherapies. Similarly,RAD140 and palbociclib combination produced improved effi-cacy compared with palbociclib alone in an additional AR/ERþ

PDX model, HBCx-22 (Supplementary Fig. S5A). An additionalTGI analysis performed for HBCx-3 xenografts treated withRAD140 andpalbociclibwith lower starting volume (�108mm3)versus those with higher starting volume (>108 mm3) did notreveal appreciable difference between the two subgroups, suggest-ing the combination treatment had similar effect in tumors ofdifferent starting size. As the combination of RAD140 withpalbociclib induced apparent regression in ST897 tumors, weexamined the expression of apoptoticmarker cleaved caspase-3 inST897 and HBCx-3 tumors treated with RAD140, palbociclib, orcombination but did not observe clear signs of apoptosis (Sup-plementary Fig. S5B), suggesting a cytostatic but not cytotoxiceffect. Together, these results indicate that the combination ofRAD140 with palbociclib led to improved antitumor activitycompared with each of these agents given as single agent.

The expressions of AR, ER, PR, Ki67, and phospho-Rb in theHBCx-3 PDX were assessed using IHC (Supplementary Fig. S6).Similar to that seen in HBCx-22 (Fig. 2C), RAD140 treatment ledto apparent enrichment of AR in the nuclei, suggesting AR acti-vation. ER andPR expressionwas profoundly decreased in tumorstreated with a RAD140-containing regimen. Palbociclib alonealso appeared to decrease the expression of ER and PR, a findingsimilar to the decrease of ER in breast cancer cells treated with apan-CDK inhibitor roscovitine (48). The phosphorylation of Rbwas found to be substantially suppressed in tumors treated withpalbociclib as a single agent and in combination with RAD140,while RAD140 alone did not seem to affect phospho-Rb level.Ki67 expression was suppressed in tumors treated with RAD140or palbociclib, and this suppression appeared to be further

enhanced in tumors that received both agents. These resultsfurther confirmed the inhibitory effect of RAD140 on ER signalingcascade. Gene expression analysis of the HBCx-3 xenograftstreated with RAD140, palbociclib, or their combination showedthat the AR target genes FKBP5 andZBTB16were robustly inducedin RAD140-treated tumors, while no appreciable effect on the ARgene was seen (Fig. 5C, top). The ER target genes PGR and TFF1were substantially suppressed in the tumors of the RAD140-treated groups (Fig. 5C, middle). Notably, a profound suppres-sion of the ESR1mRNA level was also observed in these RAD140-treated HBCx-3 xenografts. Palbociclib alone, in contrast, did nothave an apparent effect on AR target genes but did seem to repressPGR and induce TFF1 and ESR1 to a relatively modest degreecompared with the changes induced by RAD140 treatment.

We next explored the underlying mechanism for the improvedefficacy and enhanced inhibition of cell proliferation seen withthe RAD140–palbociclib combination. It has been recentlyreported that in castration-resistant prostate cancer cells, activatedAR recruits hypophosphorylated Rb to the loci of genes implicatedin DNA replication and suppress their transcription (41). Wehypothesized that AR activated by RAD140 inhibits DNA repli-cation–related genes in breast cancer cells, and this effect isenhanced by coadministration of palbociclib. Indeed, theHBCx-3 tumors treatedwith RAD140 alone had decreased expres-sion of the DNA replication–related genes including Bloomsyndrome recQ like helicase (BLM), Fanconi anemia complemen-tation group gene (FANCI), laminin B1 (LMNB1), and minichro-mosome maintenance genes 2, 4, and 7 (MCM2, MCM4, andMCM7; Fig. 5C, bottom). These decreases were comparable withthose seen with palbociclib alone. In tumors treated with bothRAD140 and palbociclib, the expression of these genes appearedto be further suppressed. Together, the enhanced effect ofRAD140–palbociclib combination on the expression of DNAreplication–related genes may have contributed to the enhancedinhibition on tumor growth.

DiscussionThis study demonstrates for the first time that RAD140, an

orally available SARM, is an AR agonist in breast cancer cells andsuppresses the growth and proliferation of multiple AR/ERþ

breast cancer cell line and xenograft models. The AR pathwaywas found to be activated in RAD140-treated breast cancer cellsand xenografts, while genes within the ER pathway, includingESR1, were suppressed. In addition, RAD140 treatmentwas foundto decrease the expression of DNA replication–related genes inbreast cancer cells, consistentwith previous report that these geneswere suppressed in androgen-treated prostate cancer cells. Com-bined administration of RAD140 with the CDK4/6 inhibitorpalbociclib was more efficacious compared with either of theagents used alone. These findings suggest a distinct mechanism ofaction of RAD140, which includes the AR-mediated suppressionof ESR1 in inhibiting AR/ERþ breast cancer growth.

Accumulating evidence in the recent years has further definedthe role of AR in breast cancer and has led to renewed interests inevaluating AR-targeted agents for breast cancer. The high preva-lence of AR in the predominant ERþ subtype of breast cancers (6),clinical benefit rates as high as 39% seenwith androgen therapy inbreast cancer patients progressing on ER-targeted treatments(23, 24), along with prior experience with steroidal androgensin breast cancer together lend support to the development of a

Yu et al.





Figure 5.

Combined administration of RAD140 with CDK4/6 inhibitor inhibited AR/ERþ PDX growth. A, Mean tumor volumes � SEM of HBCx-3 breast cancerxenografts treated with vehicle, RAD140 (100 mg/kg twice daily), palbociclib (75 mg/kg once daily), or a combination of RAD140 and palbociclib for theindicated period of time. n ¼ 7/group. Intergroup comparisons were carried out using Student t test based on changes in tumor volume (DTV) from thebeginning to the end of the study. For the comparison between RAD140 and vehicle groups, P < 0.05. � , the combination group versus palbociclib, P < 0.05;the combination group versus RAD140, P < 0.01. B, Mean tumor volumes � SEM of ST897 breast cancer xenografts treated with vehicle, RAD140 (100 mg/kgtwice daily), palbociclib (75 mg/kg once daily), or a combination of RAD140 and palbociclib for the indicated period of time. n ¼ 8/group. Statistical analysiswas performed as in A. For the comparison between RAD140 and vehicle groups, P < 0.001. � , combination group versus RAD140 group, P ¼ 0.0012; �� ,combination group versus palbociclib group, P ¼ 0.0027; C, Tumor samples from the HBCx-3 model, as described in A, were collected 6 hours after lasttreatment and subjected to qPCR analysis for the expression of AR, ESR1, and their downstream targets, and the genes implicated in DNA replicationincluding BLM, FANCI, LMNB1, MCM2, MCM4, and MCM7. Expression of these genes was normalized to that of ribosomal RNA 18S. Mean normalizedexpression � SD from three individual tumors of each group is presented.






newgeneration of oral, nonsteroidal AR agonists for the treatmentof AR/ERþ breast cancer.

SARMs are tissue-selective AR agonists by design andmay offera novel approach to inhibit the growth of AR/ERþ breast cancers,with substantially attenuated side effects commonly seen withclassic nontissue selective androgens (32, 49). The activity ofRAD140 are AR specific, as evidenced by the similar effectsobserved with DHT and RAD140 on AR and its downstreamtargets, along with the reversal of these effects by AR antagonists(Figs. 1C and 3C). In addition, due to its nonsteroidal structure,the SARM RAD140 is not subject to further conversion to estro-gens by CYP19 aromatase, or to DHT by 5a-reductase, thusreducing the potential risk of stimulating ERþ tumor growth orincreasing virilization. As a proof-of-concept study, we report herethat RAD140 treatment inhibited the growth of the breast cancerxenograft models supplemented by exogenous estrogen. Of note,RAD140and its classic androgen comparator, DHT, also inhibitedthe proliferation of an endocrine-resistant breast cancer cell linemodel, HCC1428 LTED, suggesting AR agonists may inhibit thegrowth of ERþ breast cancer models with or without estrogensupplementation. Furthermore, in multiple PDX models,RAD140 inhibited tumor growth as a single agent, and this effectwas enhanced by combining with palbociclib. PDX models havebeen demonstrated to closely recapitulate human tumors withregards to tumor heterogeneity and response to standard-of-careagents (31, 50). Indeed, heterogeneity was observed in the PDXmodels used in this study, as indicated by variable levels of AR, ER,andPR expression, suggesting a good representation of the patienttumors with a spectrum of expression levels of these nuclearreceptors. Also, the growth pattern and response to RAD140 seenwith HBCx-3 and HBCx-22 PDX models, each evaluated in twoindependent studies (Figs. 2A and 5A; Supplementary Figs. S3Aand S5A), seemed to be consistent. This suggests good reproduc-ibility of these studies. Therefore, the efficacy seenwithRAD140 inthese PDX models may be suggestive of the potential therapeuticbenefit in breast cancer patient population. It is worth noting thatalthough RAD140 and fulvestrant led to similar tumor growthinhibition in HBCx-22 tumors, further studies are needed toevaluate the efficacy of RAD140 relative to that of fulvestrant ortamoxifen in hormone-dependent models to predict its value infirst-line setting. It is interesting to note that although HBCx-3(Supplementary Fig. S6) andHBCx-22 (Fig. 2) xenografts containcomparable percentage of AR-positive cells, RAD140 inducedhigher degree of inhibition in growth and Ki-67 expression inHBCx-22 than in HBCx-3. This may be due to the presence ofhigher percentage of ERþ cells inHBCx-22 tumors than inHBCx-3(Supplementary Table S2). As summarized previously, AR ago-nists exhibit inhibitory effect on AR/ERþ breast cells but not onARþ/ER� cells (9, 51). It is conceivable that the varying degree ofgrowth inhibition seen with SARMmay be attributed to differentER positivity of these models.

The tissue selectivity of RAD140 is evidenced by potent andro-gen-like effects in bone and muscle, with much attenuated effectsin other androgen-responsive tissues, such as prostate and sem-inal vesicles (30). Notably, when administered along with tes-tosterone, RAD140 was found to partially antagonize the growtheffect of testosterone in prostate and seminal vesicles, suggestingthat RAD140 acts as a competitive AR ligand with attenuatedactivity. Here, we demonstrated for the first time that the SARMRAD140 is a potent AR agonist in breast cancer cells. It exhibitedmuch attenuated activity on AR in prostate cancer cells, a finding

consistent with the previously described attenuated effect onprostate growth (30). It has beenproposed that the tissue-selectiveAR activity of SARMs is due to the differential allosteric activationof AR compared with classic androgens, which impacts therecruitment of coactivators needed for AR-mediated gene tran-scription (52, 53). An earlier study on the selective estrogenreceptor modulator tamoxifen showed that its tissue selectivitymay be attributed to the differential expression of ER coactivatorssuch as SRC-1 in breast versus endometrial cancer cells (54).Similarly, it is conceivable that AR upon binding to RAD140assumes a conformation that only allows the interaction with asubset of coregulators that are uniquely expressed in breastepithelial cells but not in the prostate.

Despite the preclinical evidence and favorable clinical outcomeseen with androgens in ERþ breast cancer (9, 10), improvedunderstanding of the underlying mechanism is needed to furtherimprove the design and development of new AR agonist-basedtherapy. It has been proposed that activated AR suppresses ERsignaling by competing with ER for transcriptional coactivators ordirectly competing for binding sites and subsequently leads toinhibited cell proliferation (55, 56), suggesting a functionalinterference between these two nuclear receptors. Here, we showAR agonists, including RAD140 andDHT, activate AR target genesin breast cancer cells in vitro and in vivo, while suppressing a subsetof ER target genes. Importantly, we found that treatment withthese AR agonists also led to substantial suppression of ER at bothmRNA and protein levels. This suggests that AR may negativelyregulate ER signaling by twomechanisms: while ARmay competewith ER for coactivators (functional interference), it may directlydownregulate ER expression (direct suppression). The reductionof ER expression by AR agonists has not been extensively docu-mented but can be seen in a study by Poulin and colleagues (57),in which DHT treatment in ZR-75-1 cells for 8 or 12 days led todecreases of ER message by 50% or 65%, respectively. Anotherstudy also showed reduced ER protein level in ZR-75-1 cellstreated with DHT, along with decreased expression of classic ERtargets PGR and TFF1 (56), although the mechanism for thedecrease in ER expression had remained to be further elucidated.Our data showboth RAD140 andDHT treatment led to decreasedESR1 expression at as early as 24 hours, whichmaypresent a novelmechanism by which RAD140 suppresses ER signaling and dif-ferentiates from traditional ER-targeted agents that act via inhibit-ing ER protein function or leading to its degradation. This maytranslate into unique therapeutic benefit of SARMs in overcomingresistance to ER-targeted therapy seen in tumors with reactivatedER signaling, either via posttranslational modification or enrich-ment of ESR1 alterations. The inhibitory effect of RAD140 on theproliferation of HCC1428 LTED cells shed light on its efficacy insuch E2-independent, albeit ER-active, models but further studiesare needed to fully evaluate the activity of SARMs in similarmodels in vivo. In addition to the suppression of ER signaling,our RNA-seq analysis suggests that RAD140 may inhibit tumorgrowth via other AR-mediated mechanisms. Of note, RAD140treatment suppressed genes implicated in DNA replication andcell-cycle progression, which were further suppressed by concur-rent CDK4/6 inhibition. Gao and colleagues (41) described therole of AR as a tumor suppressor in castration-resistant prostatecancer (CRPC) cells by inhibiting DNA replication–related genesvia the recruitment of hypophosphorylated Rb. In addition, theclinical and preclinical efficacy seen with high-dose androgentherapy in CRPC was also attributed to androgen-induced

Yu et al.





stabilization of AR, which prevents the relicensing of DNA forreplication and subsequent cell-cycle progression (58, 59). In thecurrent study, the AR-mediated suppression of ESR1 and itsdownstream pathway, along with the suppression of DNA rep-lication- and cell cycle–related genes seenwith RAD140 treatmentsuggest a distinct mechanism of action of this SARM in AR/ERþ

breast cancer cells. The potent antitumor activity and tissue-selective AR activity, along with overall tolerability in animalmodels and oral availability together lend support to furtherclinical investigation of RAD140 inAR/ERþbreast cancer patients.

Disclosure of Potential Conflicts of InterestC.P. Miller and G. Hattersley hold ownership interest (including patents) in

Radius Health. No potential conflicts of interest were disclosed by the otherauthors.

Authors' ContributionsConception and design: Z. Yu, S. He, J.L. Brown, G. Hattersley, J.C. SaehDevelopment of methodology: Z. Yu, S. He, H.K. PatelAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): Z. Yu, S. He, H.K. Patel

Analysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): Z. Yu, S. He, D. Wang, J.L. Brown, G. Hattersley,J.C. SaehWriting, review, and/or revision of the manuscript: Z. Yu, S. He, D. Wang,C.P. Miller, J.L. Brown, G. Hattersley, J.C. SaehAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): Z. Yu, J.L. BrownStudy supervision: Z. Yu, J.L. Brown, J.C. Saeh

AcknowledgmentsThe authors would like to acknowledge Dr. Steven P. Balk of Beth Israel

Deaconess Medical Center and Dr. Changmeng Cai of University of Massachu-setts Boston for their comments on the manuscript. The authors thank Drs.Fiona Garner and Teeru Bihani, and Margaret Ward, of Radius Health, Inc., fortheir comments and suggestions on the project and themanuscript. The authorsthank Dr. Jim Humphreys and the histopathology team at Precision Pathology(San Antonio, TX) for the IHC work.

The costs of publication of this articlewere defrayed inpart by the payment ofpage charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

ReceivedMarch 7, 2017; revised July 14, 2017; accepted September 28, 2017;published OnlineFirst October 3, 2017.

References1. Siegel RL, Miller KD, Jemal A. Cancer statistics, 2016. CA Cancer J Clin

2016;66:7–30.2. Ma CX, Reinert T, Chmielewska I, Ellis MJ. Mechanisms of aromatase

inhibitor resistance. Nat Rev Cancer 2015;15:261–75.3. Mohamed A, Krajewski K, Cakar B, Ma CX. Targeted therapy for breast

cancer. Am J Pathol 2013;183:1096–112.4. Turner NC, Ro J, Andre F, Loi S, Verma S, Iwata H, et al. Palbociclib in

hormone-receptor-positive advanced breast cancer. N Engl J Med2015;373:209–19.

5. Baselga J, CamponeM, Piccart M, Burris HA III, RugoHS, Sahmoud T, et al.Everolimus in postmenopausal hormone-receptor-positive advancedbreast cancer. N Engl J Med 2012;366:520–9.

6. Collins LC, Cole KS, Marotti JD, Hu R, Schnitt SJ, Tamimi RM. Androgenreceptor expression in breast cancer in relation to molecular phenotype:results from the Nurses' Health Study. Mod Pathol 2011;24:924–31.

7. Vera-Badillo FE, Templeton AJ, de Gouveia P, Diaz-Padilla I, Bedard PL,Al-Mubarak M, et al. Androgen receptor expression and outcomes in earlybreast cancer: a systematic review and meta-analysis. J Natl Cancer Inst2014;106:djt319.

8. Chia K, O'Brien M, Brown M, Lim E. Targeting the androgen receptor inbreast cancer. Curr Oncol Rep 2015;17:4.

9. Lim E, Ni M, Cao S, Hazra A, Tamimi RM, Brown M. Importance of breastcancer subtype in the development of androgen receptor directed therapy.Curr Breast Cancer Rep 2014;6:71–8.

10. McNamara KM, Moore NL, Hickey TE, Sasano H, Tilley WD. Complexitiesof androgen receptor signalling in breast cancer. Endocr Relat Cancer2014;21:T161–81.

11. Proverbs-Singh T, Feldman JL, Morris MJ, Autio KA, Traina TA. Targetingthe androgen receptor in prostate and breast cancer: several new agents indevelopment. Endocr Relat Cancer 2015;22:R87–R106.

12. Barton VN, D'Amato NC, Gordon MA, Lind HT, Spoelstra NS, Babbs BL,et al. Multiple molecular subtypes of triple-negative breast cancer criticallyrely on androgen receptor and respond to enzalutamide in vivo. MolCancer Ther 2015;14:769–78.

13. Cochrane DR, Bernales S, Jacobsen BM, Cittelly DM, Howe EN, D'AmatoNC, et al. Role of the androgen receptor in breast cancer and preclinicalanalysis of enzalutamide. Breast Cancer Res 2014;16:R7.

14. Ni M, Chen Y, Lim E, Wimberly H, Bailey ST, Imai Y, et al. Targetingandrogen receptor in estrogen receptor-negative breast cancer. Cancer Cell2011;20:119–31.

15. Current status of hormone therapy of advanced mammary cancer. J AmMed Assoc 1951;146:471–7.

16. Dowsett M, Murray RM, Pitt P, Jeffcoate SL. Antagonism of aminoglu-tethimide and danazol in the suppression of serum free oestradiol in breastcancer patients. Eur J Cancer Clin Oncol 1985;21:1063–8.

17. Goldenberg IS. Testosterone propionate therapy in breast cancer. JAMA1964;188:1069–72.

18. Kennedy BJ. Fluoxymesterone therapy in advanced breast cancer. N Engl JMed 1958;259:673–5.

19. Cops EJ, Bianco-Miotto T,Moore NL, Clarke CL, Birrell SN, Butler LM, et al.Antiproliferative actions of the synthetic androgen, mibolerone, in breastcancer cells are mediated by both androgen and progesterone receptors.J Steroid Biochem Mol Biol 2008;110:236–43.

20. Ortmann J, Prifti S, Bohlmann MK, Rehberger-Schneider S, Strowitzki T,Rabe T. Testosterone and 5 alpha-dihydrotestosterone inhibit in vitrogrowth of human breast cancer cell lines. Gynecol Endocrinol 2002;16:113–20.

21. Poulin R, Baker D, Labrie F. Androgens inhibit basal and estrogen-inducedcell proliferation in the ZR-75-1 human breast cancer cell line. BreastCancer Res Treat 1988;12:213–25.

22. Dauvois S, Geng CS, Levesque C, Merand Y, Labrie F. Additive inhibitoryeffects of an androgen and the antiestrogen EM-170 on estradiol-stimulated growth of human ZR-75-1 breast tumors in athymic mice.Cancer Res 1991;51:3131–5.

23. Boni C, Pagano M, Panebianco M, Bologna A, Sierra NM, Gnoni R, et al.Therapeutic activity of testoterone in metastatic breast cancer. AnticancerRes 2014;34:1287–90.

24. Manni A, Arafah BM, Pearson OH. Androgen-induced remissions afterantiestrogen and hypophysectomy in stage IV breast cancer. Cancer1981;48:2507–9.

25. Chia S, Gradishar W, Mauriac L, Bines J, Amant F, Federico M, et al.Double-blind, randomized placebo controlled trial of fulvestrant com-pared with exemestane after prior nonsteroidal aromatase inhibitortherapy in postmenopausal women with hormone receptor-positive,advanced breast cancer: results from EFECT. J Clin Oncol 2008;26:1664–70.

26. Cristofanilli M, Turner NC, Bondarenko I, Ro J, Im SA, Masuda N, et al.Fulvestrant plus palbociclib versus fulvestrant plus placebo for treatment ofhormone-receptor-positive, HER2-negative metastatic breast cancer thatprogressed on previous endocrine therapy (PALOMA-3): final analysis ofthemulticentre, double-blind, phase 3 randomised controlled trial. LancetOncol 2016;17:425–39.

27. Di Leo A, JerusalemG, Petruzelka L, Torres R, Bondarenko IN, Khasanov R,et al. Results of the CONFIRM phase III trial comparing fulvestrant 250mg






with fulvestrant 500 mg in postmenopausal women with estrogen recep-tor-positive advanced breast cancer. J Clin Oncol 2010;28:4594–600.

28. Di LeoA, Keun S, Ciruelos E, Lønning P, JanniW,O'Regan R, et al. BELLE-3:a phase III study of buparlisib þ fulvestrant in postmenopausal womenwith HRþ, HER2-, aromatase inhibitor-treated, locally advanced or met-astatic breast cancer, who progressed on or after mTOR inhibitor-basedtreatment. In: Proceedings of the 2016 San Antonio Breast Cancer Sym-posium; 2016Dec 6–10; San Antonio, TX. Philadelphia (PA): AACR; 2017.Abstract nr S4–07.

29. Yu Z, Cai C, Gao S, Simon NI, Shen HC, Balk SP. Galeterone preventsandrogen receptor binding to chromatin and enhances degradation ofmutant androgen receptor. Clin Cancer Res 2014;20:4075–85.

30. Miller CP, Shomali M, Lyttle CR, O'Dea LS, Herendeen H, Gallacher K,et al. Design, synthesis, and preclinical characterization of the selectiveandrogen receptor modulator (SARM) RAD140. ACS Med Chem Lett2011;2:124–9.

31. Cottu P, Marangoni E, Assayag F, de Cremoux P, Vincent-Salomon A,Guyader C, et al. Modeling of response to endocrine therapy in a panel ofhuman luminal breast cancer xenografts. Breast Cancer Res Treat 2012;133:595–606.

32. Mohler ML, Bohl CE, Jones A, Coss CC, Narayanan R, He Y, et al.Nonsteroidal selective androgen receptor modulators (SARMs): dissociat-ing the anabolic and androgenic activities of the androgen receptor fortherapeutic benefit. J Med Chem 2009;52:3597–617.

33. CleggNJ,Wongvipat J, Joseph JD, TranC,Ouk S,Dilhas A, et al. ARN-509: anovel antiandrogen for prostate cancer treatment. Cancer Res 2012;72:1494–503.

34. TranC,Ouk S, CleggNJ, Chen Y,Watson PA, Arora V, et al. Development ofa second-generation antiandrogen for treatment of advanced prostatecancer. Science 2009;324:787–90.

35. Bihani T, Patel HK, Arlt H, Tao N, Jiang H, Brown J, et al. Elacestrant(RAD1901), a selective estrogen receptor degrader (SERD), has anti-tumoractivity in multiple ERþ breast cancer patient-derived xenograft models.Clin Cancer Res 2017;23:4793–804.

36. Johnston SRD, Kilburn LS, Ellis P, Dodwell D, Cameron D, Hayward L,et al. Fulvestrant plus anastrozole or placebo versus exemestane alone afterprogression on non-steroidal aromatase inhibitors in postmenopausalpatients with hormone-receptor-positive locally advanced or metastaticbreast cancer (SoFEA): a composite, multicentre, phase 3 randomised trial.Lancet Oncol 2013;14:989–98.

37. Lee DK, Chang C. Endocrine mechanisms of disease: Expression anddegradation of androgen receptor: mechanism and clinical implication.J Clin Endocrinol Metab 2003;88:4043–54.

38. Yuan X, Cai C, Chen S, Chen S, Yu Z, Balk SP. Androgen receptor functionsin castration-resistant prostate cancer andmechanisms of resistance to newagents targeting the androgen axis. Oncogene 2014;33:2815–25.

39. Massie CE, Lynch A, Ramos-Montoya A, Boren J, Stark R, Fazli L, et al. Theandrogen receptor fuels prostate cancer by regulating central metabolismand biosynthesis. EMBO J 2011;30:2719–33.

40. Cai C,HeHH,Chen S, Coleman I,WangH, FangZ, et al. Androgen receptorgene expression in prostate cancer is directly suppressed by the androgenreceptor through recruitment of lysine-specific demethylase 1. Cancer Cell2011;20:457–71.

41. Gao S, Gao Y, He HH, Han D, Han W, Avery A, et al. Androgen receptortumor suppressor function is mediated by recruitment of retinoblastomaprotein. Cell Rep 2016;17:966–76.

42. Finn RS, Crown JP, Lang I, Boer K, Bondarenko IM, Kulyk SO, et al. Thecyclin-dependent kinase 4/6 inhibitor palbociclib in combination

with letrozole versus letrozole alone as first-line treatment of oestrogenreceptor-positive, HER2-negative, advanced breast cancer (PALOMA-1/TRIO-18): a randomised phase 2 study. Lancet Oncol 2015;16:25–35.

43. Finn RS, Dering J, Conklin D, Kalous O, Cohen DJ, Desai AJ, et al. PD0332991, a selective cyclin D kinase 4/6 inhibitor, preferentially inhibitsproliferation of luminal estrogen receptor-positive human breast cancercell lines in vitro. Breast Cancer Res 2009;11:R77.

44. Thangavel C, Dean JL, Ertel A, Knudsen KE, Aldaz CM,Witkiewicz AK, et al.Therapeutically activating RB: reestablishing cell cycle control in endocrinetherapy-resistant breast cancer. Endocr Relat Cancer 2011;18:333–45.

45. Finn RS, Martin M, Rugo HS, Jones S, Im SA, Gelmon K, et al. Palbocicliband letrozole in advanced breast cancer. N Engl J Med 2016;375:1925–36.

46. Hortobagyi GN, Stemmer SM, Burris HA, Yap YS, Sonke GS, Paluch-Shimon S, et al. Ribociclib as first-line therapy for HR-positive, advancedbreast cancer. N Engl J Med 2016;375:1738–48.

47. Ma CX, Gao F, Luo J, Northfelt DW, Goetz M, Forero A, et al. NeoPalAna:neoadjuvant palbociclib, a cyclin-dependent kinase 4/6 inhibitor, andanastrozole for clinical stage 2 or 3 estrogen receptor-positive breast cancer.Clin Cancer Res 2017;23:4055–65.

48. Nair BC, Vallabhaneni S, Tekmal RR, Vadlamudi RK. Roscovitine conferstumor suppressive effect on therapy-resistant breast tumor cells. BreastCancer Res 2011;13:R80.

49. Miller CP, Bhaket P, Muthukaman N, Lyttle CR, Shomali M, Gallacher K,et al. Synthesis of potent, substituted carbazoles as selective androgenreceptor modulators (SARMs). Bioorg Med Chem Lett 2010;20:7516–20.

50. Matthews SB, Sartorius CA. Steroid hormone receptor positive breastcancer patient-derived xenografts. Horm Cancer 2017;8:4–15.

51. Lim E, Tarulli G, Portman N, Hickey TE, Tilley WD, Palmieri C. Pushingestrogen receptor around in breast cancer. Endocr Relat Cancer 2016;23:T227–T41.

52. Baek SH, Ohgi KA, Nelson CA, Welsbie D, Chen C, Sawyers CL, et al.Ligand-specific allosteric regulation of coactivator functions of androgenreceptor in prostate cancer cells. ProcNatl Acad SciU SA2006;103:3100–5.

53. Kazmin D, Prytkova T, Cook CE, Wolfinger R, Chu TM, Beratan D, et al.Linking ligand-induced alterations in androgen receptor structure to dif-ferential gene expression: a first step in the rational design of selectiveandrogen receptor modulators. Mol Endocrinol 2006;20:1201–17.

54. Shang Y, Brown M. Molecular determinants for the tissue specificity ofSERMs. Science 2002;295:2465–8.

55. Lanzino M, De Amicis F, McPhaul MJ, Marsico S, Panno ML, Ando S.Endogenous coactivator ARA70 interacts with estrogen receptor alpha(ERalpha) and modulates the functional ERalpha/androgen receptorinterplay in MCF-7 cells. J Biol Chem 2005;280:20421–30.

56. Need EF, Selth LA, Harris TJ, Birrell SN, Tilley WD, Buchanan G. Researchresource: interplay between the genomic and transcriptional networks ofandrogen receptor and estrogen receptor alpha in luminal breast cancercells. Mol Endocrinol 2012;26:1941–52.

57. Poulin R, Simard J, Labrie C, Petitclerc L, DumontM, Lagace L, et al. Down-regulation of estrogen receptors by androgens in the ZR-75-1 human breastcancer cell line. Endocrinology 1989;125:392–9.

58. Isaacs JT, D'Antonio JM, Chen S, Antony L, Dalrymple SP, Ndikuyeze GH,et al. Adaptive auto-regulation of androgen receptor provides a paradigmshifting rationale for bipolar androgen therapy (BAT) for castrate resistanthuman prostate cancer. Prostate 2012;72:1491–505.

59. Schweizer MT, Antonarakis ES, Wang H, Ajiboye AS, Spitz A, Cao H, et al.Effect of bipolar androgen therapy for asymptomatic men with castration-resistant prostate cancer: results from a pilot clinical study. Sci Transl Med2015;7:269ra2.


Yu et al.




2017;23:7608-7620. Published OnlineFirst October 3, 2017.Clin Cancer Res Ziyang Yu, Suqin He, Dannie Wang, et al. Models with a Distinct Mechanism of Action

Positive Breast Cancer−Growth of Androgen/Estrogen Receptor Selective Androgen Receptor Modulator RAD140 Inhibits the

Updated version

10.1158/1078-0432.CCR-17-0670doi:

Access the most recent version of this article at:

Material

Supplementary

http://clincancerres.aacrjournals.org/content/suppl/2017/10/03/1078-0432.CCR-17-0670.DC1

Access the most recent supplemental material at:

Cited articles

http://clincancerres.aacrjournals.org/content/23/24/7608.full#ref-list-1

This article cites 58 articles, 19 of which you can access for free at:

Citing articles

http://clincancerres.aacrjournals.org/content/23/24/7608.full#related-urls

This article has been cited by 1 HighWire-hosted articles. Access the articles at:

E-mail alerts related to this article or journal.Sign up to receive free email-alerts

Subscriptions

Reprints and

[email protected]

To order reprints of this article or to subscribe to the journal, contact the AACR Publications Department at

Permissions

Rightslink site. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC)

.http://clincancerres.aacrjournals.org/content/23/24/7608To request permission to re-use all or part of this article, use this link



http://clincancerres.aacrjournals.org/lookup/doi/10.1158/1078-0432.CCR-17-0670

http://clincancerres.aacrjournals.org/content/suppl/2017/10/03/1078-0432.CCR-17-0670.DC1

http://clincancerres.aacrjournals.org/content/23/24/7608.full#ref-list-1

http://clincancerres.aacrjournals.org/content/23/24/7608.full#related-urls

http://clincancerres.aacrjournals.org/cgi/alerts

mailto:[email protected]

http://clincancerres.aacrjournals.org/content/23/24/7608


selective androgen receptor modulator rad140 inhibits the ... · arn-509 (selleckchem) for 24...

Documents