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Endocrine Research

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The cellular and molecular effects of the androgenreceptor agonist, Cl-4AS-1, on breast cancer cells

Mamoun Ahram, Ebtihal Mustafa, Shatha Abu Hammad, Mariam Alhudhud,Randa Bawadi, Lubna Tahtamouni, Faisal Khatib & Malek Zihlif

To cite this article: Mamoun Ahram, Ebtihal Mustafa, Shatha Abu Hammad, Mariam Alhudhud,Randa Bawadi, Lubna Tahtamouni, Faisal Khatib & Malek Zihlif (2018): The cellular and moleculareffects of the androgen receptor agonist, Cl-4AS-1, on breast cancer cells, Endocrine Research,DOI: 10.1080/07435800.2018.1455105

To link to this article: https://doi.org/10.1080/07435800.2018.1455105

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The cellular and molecular effects of the androgen receptor agonist, Cl-4AS-1,on breast cancer cellsMamoun Ahrama, Ebtihal Mustafaa, Shatha Abu Hammada, Mariam Alhudhuda, Randa Bawadia,Lubna Tahtamounic, Faisal Khatiba, and Malek Zihlifb

aDepartment of Physiology and Biochemistry, School of Medicine, The University of Jordan, Amman, Jordan; bDepartment of Pharmacology,School of Medicine, The University of Jordan, Amman, Jordan; cDepartment of Biology and Biotechnology, Faculty of Science, HashemiteUniversity, Zarqa, Jordan

ABSTRACTPurpose: The androgen receptor (AR) has attracted attention in the treatment of breast cancer.Due to the undesirable side effects of AR agonists, attempts have been undertaken to developselective AR modulators. One of these compounds is Cl-4AS-1. This study examined this com-pound more closely at the cellular and molecular levels. Methods: Three different breast cancercell lines were utilized, namely the luminal MCF-7 cells, the molecular apocrine MDA-MB-453 cells,and the triple negative, basal MDA-MB-231 cells. Results: High and significant concordancebetween dihydrotestosterone (DHT) and Cl-4AS-1 in regulation of gene expression in MDA-MB-453 cells was found. However, some differences were noted including the expression of AR, whichwas upregulated by DHT, but not Cl-4AS-1. In addition, both DHT and Cl-4AS-1 caused a similarmorphological change and reorganization of the actin structure of MDA-MB-453 cells into amesenchymal phenotype. Treatment of cells with DHT resulted in induction of proliferation ofMCF-7 and MDA-MB-453 cells, but no effect was observed on the growth of MDA-MB-231 cells. Onthe other hand, increasing doses of Cl-4AS-1 resulted in a dose-dependent inhibition on thegrowth of the three cell lines. This inhibition was a result of induction of apoptosis whereby Cl-4AS-1 caused a block in entry of cells into the S-phase followed by DNA degradation.Conclusions: These results indicate that although Cl-4AS-1 has characteristics of classical ARagonist, it has dissimilar properties that may make it useful in treating breast cancer.

ARTICLE HISTORYReceived 22 October 2017Revised 3 March 2018Accepted 16 March 2018

KEYWORDSApoptosis; Cl-4AS-1; geneexpression; morphology;SARM

Introduction

Understanding the biology of breast cancer and theutilization of advanced molecular technology haveenabled better differentiation and classification ofthis disease paving the road for targeted therapy andimproving its treatment.1,2 For example, tamoxifen, aselective estrogen receptor modulator (SERM), hasbeen effective in reducing recurrence and progressionof hormone-responsive breast cancerwhile being ben-eficial for the treatment of osteoporosis.3,4 The use ofaromatase inhibitors that block the conversion ofandrogens into estrogens has been reported toincrease overall survival and progression among post-menopausal women.5,6 A breast cancer subtype that ismarked by amplification of human epidermal growthfactor receptor 2 (HER2), hence termed HER2-enriched breast cancer, is currently treated with

trastuzumab, a humanized monoclonal antibody thatinhibits the function of the receptor. Combinationaltherapy like trastuzumab and aromatase inhibitors hasshown to be effective in therapy.7 Nevertheless, resis-tance toward these treatments is a hallmark of breastcancer therapy.Mutations inmolecular component ofother pathways such as PI3K/AKT/mTOR or cellcycle regulators compromise endocrine therapy.8 Inaddition, it is challenging to treat triple-negative breastcancer (TNBC), which lacks the expression of estro-gen receptor (ER), progesterone receptor, and HER2,due to the absence of a target molecule. Making thisworse is the fact that TNBC is actually a heterogeneousdisease that is composed of multiple subtypes, eachwith a distinct molecular profile, behavior, andresponse to therapy.9,10

In recent years, attention has been directed towardalternative therapeutic targets. One such target is

CONTACT Mamoun Ahram m.ahram@ju.edu.jo; Dr.Ahram@gmail.com Department of Physiology and Biochemistry, School of Medicine, TheUniversity of Jordan, Amman 11942, Jordan

Supplemental data for this article can be accessed at publisher’s website.

ENDOCRINE RESEARCHhttps://doi.org/10.1080/07435800.2018.1455105

© 2018 Taylor & Francis

androgen receptor (AR). In fact, androgen therapywas a strategy of treating breast cancer decades agotill the emergence of targeted therapeutics such asSERMs9 and inhibitors of androgen aromatizationinto estrogen.11 There has recently been a revival ofutilizing antiandrogen therapy due to a number ofreasons. First of all, breast cancer is a heterogeneousdisease with multiple subclasses, one of which is char-acterized by active AR signaling pathway and, hence,known a luminal AR and is represented by molecularapocrine breast cancer.9 In addition, AR is expressedin all subtypes of breast cancer, although the extent ofexpression varies according to the type.12 For example,up to 90% of luminal breast cancer is found to be AR-positive, relative to 32% of basal-like breast cancer.12

In fact, AR expression is associated with better prog-nosis of the disease12,13, although it has been suggestedthat it can also act as an oncogene in ER-negativebreast cancers.14,15 It has been shown that treatmentof TNBC cells with AR antagonists reduces cellproliferation.16 On the other hand, contradictoryresults have been reported on the effect of AR agonistson breast cancer cells (for review, see11,17).Nevertheless, clinical trials have shown promisingresults in treating breast cancer patients.18

The undesirable effects of classicalAR agonists suchas testosterone or dihydrotestosterone (DHT) havegeared scientists toward developing AR ligands thatare tissue selective, hence termed, selective androgenreceptormodulators (SARMs). Thesemolecules act asmimics of AR agonists, but can regulate the activity ofAR in tissues selectively reducing undesirable sideeffects (for review, see19). Some success has beenreported whereby SARMs possess anabolic effects onmuscles without affecting other tissues.20–22 In addi-tion, one such SARM was recently shown to inhibitthe growth of TNBC cells.23 Cl-4AS-1 was firstdescribed by Tolman et al. as a potential SARM.24 Itwas later shown to have an agonistic effect similar toDHT both in vitro and in vivo25 and has recently beenshown to bind AR and the complex translocates intothe nucleus.26 Due to insufficient studies on this com-pound, we aimed to further analyze Cl-4AS-1 as analternative AR agonist. In this study, the effect of Cl-4AS-1 on, primarily, a luminal AR breast cancermodel system, MDA-MB-453 cells, was investigated.We reveal that Cl-4AS-1 possesses some functionalresemblance to DHT, but it regulates some genes

differently and induces cell death via blocking cellentry into the S phase of the cell cycle.

Methods

Cell lines

Three breast cancer cell lines, MDA-MB-453, MDA-MB-231, andMCF-7 cells were used in this study. Thecells weremaintained in a Leibovitz’s L-15, Dulbecco’smodified Eagle’s medium-high glucose, and RPMImedia, respectively, supplemented with 10% heat-inactivated fetal bovine serum (FBS), 1% L-glutamine,1% penicillin-streptomycin. MDA-MB-453 cells weremaintained in a humidified incubator at 37°C withoutCO2. MDA-MB-231 and MCF-7 cells were main-tained in a humidified incubator at 5%CO2 and 37°C.

Compounds

DHT was obtained from Tokyo Chemical Industry(Tokyo, Japan), whereas Cl-4AS-1 was purchasedfrom Tocris Inc. (Bristol, UK). Bicalutamide wasobtained from Santa Cruz Biotechnology(Heidelberg, Germany). All compounds were pre-pared in dimethyl sulfoxide (DMSO), aliquoted, andstored at −20°C. The concentrations used herein wereinitially based on the previous study of Schmidt et al.25

In general, experiments were performed within2 months of reconstitution of Cl-4AS-1 in DMSO.

Polymerase chain reaction (PCR) array

All reagents for the PCR arrays were obtained fromQiagen (Valencia, CA, USA). The PCR array wascustom-made containing genes from different path-ways including adhesion receptors, proteases and theirinhibitors, signaling molecules, and apoptosis(Supplemental Table 1S). A gene ontology analysiswas carried out using the web-based gene ontologydatabase application DAVID (http://david.abcc.ncifcrf.gov). A level 5 search in the “biological process”category was carried out in order to provide the high-est degree of specificity for target function.

MDA-MB-453 cells were plated onto 100-mmplates. Cells were treated with either 100 nM DHTor 1 µM Cl-4AS-1 for 24 and 72 h. Cells treatedwith DMSO were used as a negative control. Total

2 M. AHRAM ET AL.

cellular RNA was extracted using RNeasy Mini Kitand treated with DNase I digestion according tomanufacturer’s instructions. One microgram ofRNA was converted to cDNA by RT2 FirstStrand Kit according to manufacturer’s protocol.DNA amplification was performed using RT2

qPCR Master Mix. PCR amplification was per-formed in a Bio-Rad’s IQ Real-Time System(Hercules, CA, USA). The reaction conditionswere as follows: 95°C for 10 min, 40 cycles of 95°C for 15 s, and 60°C for 1 min. At the completionof the reaction, Ct values were determined, andΔΔCt and fold changes of expression were deter-mined using the RT2 Profiler PCR Array DataAnalysis web portal based on the average expres-sion of four housekeeping genes. Samples wereanalyzed in duplicates. Pearson correlation wasused to compare changes in gene expressioninduced by DHT and Cl-4AS-1.

Immunoblotting

Cells were lysed in ice-cold lysis buffer (2% sodiumdodecyl sulfate (SDS), 10 mM Tris pH 7.5, 10 mMNaF, 2 mM EDTA, 10 mM dithiothreitol). Proteinswere resolved by electrophoresis on a 7.5% SDS-poly-acrylamide gel and transferred onto 0.45-µm nitrocel-lulosemembranes. Primary antibodies, rabbit anti-AR(Santa Cruz Biotechnology) and rabbit anti-GAPDH(Abcam, UK), were used followed by appropriatehorseradish peroxidase (HRP)-conjugated-secondaryantibodies. Detection was performed usingLuminanta Crescendo Western HRP Substrate(Millipore, MA, USA). Images were captured usingC-Digit blot scanner (Licor Biosciences, Lincoln, NE,USA). Densitometric analysis was performed on theblots using Image Studio Digits v. 5.2 (LicorBiosciences).

Cell proliferation (MTT) assay

Cell proliferation assay (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide [MTT] assay) wasperformed according to themethod ofMosmannwithmodifications.27 Briefly, 7 × 103 viable cells wereseeded in the wells of a 96-well tissue culture platecontaining growth media supplemented with FBS.Cultures were treated at 24 h after seeding. Freshlyprepared MTT) salt (5 mg/ml in phosphate-buffered

saline (PBS)) was added to each well to give a finalconcentration of 0.5 μg/μl. The plates were incubatedfor 4 h and the formation of formazan crystals waschecked using an inverted microscope. Equal volumeof 1:1 (200 μl) of mix of DMSO and isopropanol wasadded to each well and incubated for 30–45 min. Cellviability was evaluated bymeasuring the absorbance at570 nm using Sunrise microplate reader (Tecan,Switzerland).

Morphology

For morphology assays, MDA-MB-453 cells wereseeded at 2 × 106 cells in 60-mm tissue culturedishes. Cells were grown overnight and then trea-ted with either 1 µM Cl-4AS-1 or 100 nM DHT for3 and 6 days in the presence or absence of 10 µMbicalutamide. Images were captured by DMIL LEDinverted microscope (Leica Microsystems,Wetzlar, Germany).

Immunofluorescence

Cells (10,000 cells/well) were cultured on cover-slips coated with 10 μg/ml collagen type 1 solution(Sigma, USA). Following treatment, cells werefixed by incubation with 4% paraformaldehydefor 1 h and permeabilized with 0.5% TritonX-100/PBS for 10 min. Actin was stained by incu-bation with Alexa Fluor 488 phalloidin(Invitrogen, USA) for 1 h. The coverslips weremounted with ProLong Gold Antifade Mountantin the presence of DAPI (Invitrogen).

Cell cycle analysis

The cells were seeded in 100-mmplate at 1 × 106 cells/dish in growthmedia. The cells were allowed to attachovernight and then treated with either 1 or 4 µM Cl-4AS-1 for 3 or 6 days. The collected cells were thenwashed in PBS, fixed in 75% ethanol, then stainedwith50 μg/mL propidium iodide containing 10 μg/mLRNase A. The cells were analyzed in a MiltenyiMACS Quant flow cytometer (Miltenyi Biotec,Bergisch Gladbach, Germany). This instrument ana-lyzes specified volume rather than recorded events.

ENDOCRINE RESEARCH 3

Results

Selective regulation of gene expression by DHTand Cl-4AS-1

A comparison of the regulation of gene expression byboth Cl-4AS-1 andDHTwas first carried out. For thispurpose, MDA-MB-453 cells were treated with Cl-4AS-1 (1 µM) or DHT (100 nM) for 24 and 72 h. Theconcentrations were used based on the previous studyof Schmidt et al.25 The expression of 85 genes relatedto adhesion receptors, proteases and their inhibitors,signaling molecules, and apoptosis was examined byPCR arrays. Changes of gene expression of more thantwofold for the 24-h treatment by either AR agonistwere considered, whereas differences of fivefold andhigher for the 72-h treatment were considered sincethe latter treatment would be expected to have sec-ondary or tertiary transcriptional effects. Upon asses-sing changes in the expression of genes, there was ahigh level of concordance of gene expression follow-ing treatment with either DHT or Cl-4AS-1 withPearson correlation coefficient (R) values of 0.90 and0.93 after 24 and 72 h of treatment, respectively. Thisconcordance was highly significant with a p-value of<0.001 for both correlations (Figure 1A and B). Suchconcordance is an indication of a similar effect of bothDHT and Cl-4AS-1 on gene expression. As shown inTable 1, 16 genes had an altered expression in cellstreated with either DHT or Cl-4AS-1. The fold changeof expression was even comparable between the twotreatments. For example, strong induction of geneexpression was observed for metalloprotease 13(MMP13) and integrin α1 (ITGA1) following 3 daysof treatment. Interestingly, tenascin C (TNC) was theonly gene that was consistently regulated by both

treatments at both time points, where it was down-regulated by five- to tenfold (Supplemental Table 1S,Table 2S).

Nevertheless, treatment-specific alterations of geneexpression were also noted whereby 14 genes had analtered expression by one treatment, but not the other.For example, integrin β6 (ITGB6) and MMP12 wereboth highly upregulated in cells treated by Cl-4AS-1only for 72 h. Interestingly, the effect of Cl-4AS-1 andDHT on the expression of MMP10 was the oppositewhereby the former compound downregulated theexpression of this gene after 72 h of treatment, whereasDHT induced its expression.

DHT, but not Cl-4AS-1, changes the expression ofAR

We aimed then to investigate if Cl-4AS-1 specificallychanges the expression of AR protein. MDA-MB-453cells were treated with either DHT (10 or 100 nM) orCl-4AS-1 (100 nM or 1 µM) for 24 or 72 h in thepresence or absence of the AR antagonist, bicaluta-mide. As shown in Figure 2 and based on densito-metric measurement, neither treatment altered thelevel of AR protein after 24 h. However, there was atwo- to threefold increase in the level of AR proteinupon treating the cells with DHT for 72 h, but not byCl-AS-1. Bicalutamide negated the DHT-inducedincrease in AR protein levels.

Both DHT and Cl-4AS-1 alter cell morphology

Interestingly, there was a dramatic change in themorphology of MDA-MB-453 cells with long-termtreatment with either Cl-4AS-1 or DHT. Normally,

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Figure 1. Concordant regulation of gene expression by Cl-4AS-1 and DHT. (A) MDA-MB-453 cells were treated with either Cl-4AS-1(1 μM) or DHT (100 nM) for 24 h (A) or 72 h (B). Only changes of two- or fivefold and higher were considered for the 24-h and the72-h treatments, respectively. Samples were done in duplicates.

4 M. AHRAM ET AL.

the cells appeared rounded forming colonies of cellclusters with cells growing on top of each other.Upon treating the cells with either 1 µM Cl-4AS-1or 100 nM DHT, the cell shape was altered drama-tically where, after 3 days of treatment, cell cluster-ing was reduced and the cells had a spindle shape(Figure 3A, C, and E). Treatment of cells with lowerconcentrations of Cl-4AS-1 (100 nM) or DHT(10 nM) also resulted in a similar change (data notshown). Prolonged treatment (6 days) caused amore pronounced change in cell morphology(Figure 3B, D, and F). At both periods, the changein cell morphology by DHT (Figure 3I and J) or Cl-

4AS-1 (Figure 3K and L) was inhibited by co-treat-ing cells with 10 µM of bicalutamide (Figure 3G andH). A combinational treatment of cells with both Cl-4AS-1 and bicalutamide reduced the change in cellmorphology at day 3, but not at day 6. During theprolonged treatment, though, there was an increasein cell clustering indicating an inhibitory effect ofbicalutamide on the function of Cl-4AS-1.

The alteration of cell morphology may havereflected actin reorganization. Indeed, cells treatedwith either 1 µM of Cl-4AS-1 or 100 ng of DHT hadan increase in filamentous actin compared to controlcells, which had actin concentrated at the cell cortexand regions of cell–cell contact (Figure 4).Additionally, cells treated with DHT showed a meta-stable cell phenotype. On the other hand, MDA-MB-453 cells exposed toCI-4AS-1 had intense filamentousactin staining and formation of lamellipodia. CI-4AS-1-treated cells were also spindle in shape and had afront–back polarity, characteristics of the mesenchy-mal phenotype.

Cl-4AS-1, but not DHT, suppresses cell growth

The effect of both DHT and Cl-4AS-1 on cell prolif-eration was investigated on MCF-7, MDA-MB-453,andMDA-MB-231 cells. The three cell lines expressedAR at variable levels with MDA-MB-453 expressingthe highest levels and MDA-MB-231 cells expressingthe least (data not shown). The cells were treated withincreasing concentrations of both DHT (1, 10, 100,and 1000 nM) andCl-4AS-1 (0.1, 1, 5, and 10 µM) andcell proliferation was measured after 3, 5, and 7 days.

Table 1. Alterations of gene expression following treatment ofMDA-MB-453 cells with either Cl-4AS-1 (1 µM) or DHT (100 nM)for either 24 or 72 ha.

24 h 72 h

Gene Cl-4AS-1 DHT Cl-4AS-1 DHT

Genes either concordantly upregulated or downregulated by bothtreatments at either durationITGA1 1.4 1.3 133.4 47.7ITGA5 −2.1 −2.4 −1.1 1.4ADAM9 −1.1 1.2 9.3 6.4ADAM17 −1.1 −1.3 18.5 14.2MMP13 −1.9 −1.7 340.1 117.4ADAMTS15 −1.5 −1.2 6.5 7.1EPHA4 −6.5 −7.0 1.6 −1.1ERBB2 −3.0 −3.6 −1.7 −2.0CADM2 −1.4 −1.2 27.1 27.4CTNNA1 −1.1 −1.1 5.3 5.4CXCR7 −2.7 −4.6 −2.6 −4.8FOXA1 −2.0 −2.1 1.0 −1.3ZEB1 LE LE 30.1 26.4FOXO1 −3.0 −2.0 2.6 1.8TNC −6.5 −9.6 −5.6 −6.6CASP9 −1.2 −1.3 5.7 6.6

Genes either upregulated or downregulated by one treatment onlyat either durationITGB5 1.9 2.4 −1.0 −1.3ITGB6 −1.4 −1.4 −78.2 2.4ADAM2 −1.5 −1.3 −13.4 3.6ADAM15 2.1 1.7 −1.6 −1.8MMP12 1.4 −1.1 69.1 LEMMP16 −1.9 −2.6 2.0 −1.3ADAMTS18 −2.1 −1.8 −17.0 3.4CSTB −1.1 1.2 6.3 3.7CDH1 1.1 −1.1 6.1 3.9CDH2 −2.6 −1.7 3.6 2.0CD44 LE LE 7.3 LEIGF2R 1.4 1.2 5.7 3.5CCL5 −1.4 −2.3 −6.2 −1.2CASP10 1.8 1.1 5.7 LE

Genes with opposite effects of treatments at either durationMMP10 1.3 −1.0 −5.2 5.0

aChanges of differential expression of more than two folds for eithertreatment at 24 h and more than five folds for either treatment at72 h were considered and are in bold.

LE = low expression (appearing at cycle >32) in both control andtreated samples.

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Figure 2. DHT, but not Cl-4AS-1, changes the expression of AR.Expression of AR in MDA-MB-453 cells treated with DHT (10 or100 nM) or Cl-4AS-1 (100 nM or 1 μM) in the presence orabsence of 10 µM of bicalutamide (Bic) for 24 or 72 h wasanalyzed. GAPDH was used as a loading control. Densitometricanalyses were performed as described in the “Methods” section.Experiment was performed in triplicate.

ENDOCRINE RESEARCH 5

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Figure 3. AR-dependent morphological alteration of MDA-MB-453 cell line by Cl-4AS-1 and DHT. MDA-MB-453 cells were eitheruntreated (A and B) or treated with 100 nM DHT (C and D), 1 µM Cl-4AS-1 (E and F), 10 µM bicalutamide only (G and H), 100 nM DHTand bicalutamide (10 µM; I and J), and Cl-4AS-1 (1 µM) and bicalutamide (10 µM; K and L) for 3 (A, C, E, G, I, and K) or 6 (B, D, F, H, J,and L) days. Magnification was set at 20×. Experiment was done in triplicate.

6 M. AHRAM ET AL.

Disparate effect of DHTwas observed on the three celllines (Figure 5A, C, and E). Whereas it stimulatedgrowth of MCF-7 cells at increasing doses(Figure 5A), it induced the proliferation of MDA-MB-453 cells at all concentrations at a similar extent(Figure 5C), and did not have any effect on the growthof MDA-MB-231 cells (Figure 5E). The effect of DHTon the growth of cells started at day 3, but it becamemore appreciable at longer treatments. On the otherhand, increasing concentrations of Cl-4AS-1 sup-pressed the proliferation of the three cell lines similarlywith complete inhibition at a concentration of 10 µM(Figure 5B, D, and F). The SARM also had a similarinhibitory effect on two other breast cancer cell lines,ZR75-1 and T47D (data not shown). The effect of Cl-4AS-1 on cell growthwas initially apparent on day 3. Itwas also noted that as preparations of the compoundbecame older, its effect became less. However, themolecular and cellular effects sustained.

Cl-4AS-1 induces apoptosis via blocking entryinto S phase

We decided to elucidate the mechanism of celldeath induced by Cl-4AS-1. Cell cycle analysiswas carried out on MDA-MB-453 cells treatedwith 1 and 4 μM of Cl-4AS-1 for 3 and 6 days.As illustrated in Figure 6, a block in the early Sphase was observed in cells treated with 4 µM Cl-4AS-1 for 3 days relative to both the control cellsand those treated with 1 µM (Figure 6A–C). Alarge fraction of the cells treated with 4 µM Cl-4AS-1 was detected in the sub-G phase indicatingDNA degradation and cell death (Figure 6C). After6 days of treatment, cells treated with 1 µM Cl-4AS-1 had a similar block in the S phase and anincrease in apoptotic cells (Figure 6E).

Discussion

The AR agonist, Cl-4AS-1, was previously shownto act in a similar manner as DHT in terms of itseffects on gene expression, bone formation, anduterus weight.25 The latter study has also illu-strated that DHT and Cl-4AS-1-induced altera-tions in gene expression in MDA-MB-453 cellscorrelated by approximately 90%. We similarlyfound a strong correlation in changes in geneexpression between Cl-4AS-1 and DHT in MDA-MB-453 cells. However, both this study and that ofSchmidt et al.25 have found a degree of differentialeffect in modulating gene expression by both ARagonists necessitating caution when interpretingresults using Cl-4AS-1. The change in cell mor-phology upon treating MDA-MB-453 cells witheither AR agonist also illustrates that they functionvia a common mechanism. Such an effect isobserved despite the fact that the AR gene pos-sesses a mutation that reduces its transcriptionalactivity.28 The transformation in cell morphologywas reversed by co-treating the cells with the ARinhibitor, bicalutamide. However, pronouncedeffect of Cl-4AS-1 on the actin cytoskeleton andthe lesser inhibitory effect of the antagonist on thisSARM could be due to the use of a smaller ratio ofcompound:inhibitor in case of Cl-4AS-1 (1:10)than that of DHT (1:100). Unfortunately, using alarger ratio of Cl-4AS-1:bicalutamide wouldrequire the use higher concentration of the antago-nist, which was toxic to the cells.

Whereas DHT has a stimulatory effect on prolif-eration of MCF-7 and MDA-MB-453, Cl-4AS-1inhibited proliferation of all breast cancer cell lines,including two other ones (T47D and ZR75-1),regardless of the subtype. It is not clear whether Cl-4AS-1 acts via the same pathway that suppresses cell

A B C

Figure 4. Both DHT and Cl-4AS-1 alter the structure of the actin cytoskeleton. MDA-MB-453 cells were grown on collagen-coatedsurface and were either treated with DMSO (A), 100 nM DHT (B), or 1 µM Cl-4AS-1 (C) for 3 days. Actin cytoskeleton was stained withFITC conjugated phalloidin. Magnification was set at 40×. Experiment was performed at least twice.

ENDOCRINE RESEARCH 7

growth in the different breast cancer cell types or not.SARMs, similar to SERMs, have a tendency to selec-tively bind coregulators.29 Such selectivity is gov-erned by the conformational change induced by thenatural ligand versus the tissue-selective modulator,the coactivator:corepressor ratio in cells, and theexistence of external stimuli. In a previous study,we have shown that Cl-4AS-1 differentially altersexpression of microRNA molecules in breast cancercell lines including two of the same subtype.30

Unfortunately, no other molecular study has beenconducted on this compound.

Cl-4AS-1 loses its apoptotic efficacy with longerstorage, although its behavior as a DHT analog ispreserved. We observed that at a concentration of10 μM, the compound was completely lethal whenfreshly prepared. This concentration was used bySchmidt et al. when testing its transactivation ofAR.25 Apparently, Cl-4AS-1 lost its lethal effect inthe aforementioned study at the time of perform-ing the experiment. It is not known how Cl-4AS-1loses its killing properties. It can be hypothesizedthat the modifying group at carbon 1724 is cleavedmaking it structurally similar to DHT. This group

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8 M. AHRAM ET AL.

is linked to the D ring of Cl-4AS-1 via an amidebond, which was shown in other SARMs to behydrolyzed.31 Further investigation is warrantedto analyze any structural modification of Cl-4AS-1.

The mechanism of action of Cl-4AS-1 in inhibit-ing cell proliferation appeared to be blocking the cellcycle at the early S phase inducing DNA degradationand apoptosis with continuing blocking. It is notclear, however, what the molecular mechanism ofthis SARM exactly is, but the arrest could be due toa stalemate at any of the processes of DNA replica-tion such as inhibition of histone synthesis, enzymeactivities like DNA polymerases and topoisomerases,and uncoiling of chromatin. Such phenomenon doesnot seem to be restricted to Cl-4AS-1. Yu et al. haverecently reported that the SARM, RAD140, down-regulated the expression of genes associated with celldivision, DNA replication, and cell cycleprogression.32 In addition, a SARM, MK04541, haspreviously been reported to induce cell death speci-fically in AR-positive prostate cancer cells via induc-tion of caspase 3.33 Another SARM, termed GTx-024, was found to inhibit the growth of AR-expres-sing MDA-MB-231 cells in vitro and in vivo.23 Thelatter SARM also reduced the release of two

invasion-promoting factors, interleukin 6 andmatrixmetalloproteases 13 (MMP13), in correlation withreduced cell invasion and metastasis. Interestingly,we have recently reported that the expression ofMMP13 is upregulated in MDA-MB-453 cells trea-ted with DHT.30 The SERM, raloxifene, inducesapoptosis in prostate cell lines following modulationof both transcriptional regulation and non-genomicsignals via activation of ERβ and antagonism ofERα.34

The inhibitory effect of treating breast cancercells with anti-AR has resulted in re-gaininginterest in targeting AR in breast cancer. This isparticularly true considering that some types ofbreast cancer are stimulated to grow in the pre-sence of AR agonists35, and that treatment ofbreast cancer cells with AR antagonists results indecreased cell growth.16,36 Future use of ARantagonists in treating breast cancer is potentiallypromising for two types of breast cancers. Thefirst class is ER-positive cancers that developresistance to neoadjuvant endocrine therapy.Although the high expression of AR is associatedwith having better response toward ER-basedtherapies37, the overexpression of AR has been

D E F

A B C

Sub-G G1 S G2/M

G0: 5.4%

G1: 66.9%

S: 14.0%

G2/M: 13.7%

G0: 6.8%

G1: 63.0%

S: 14.6%

G2/M: 15.6%

G0: 8.0%

G1: 68.7%

S: 10.1%

G2/M: 13.2%

G0: 49.8%

G1: 36.9%

S: 7.6%

G2/M: 5.6%

G0: 16.2%

G1: 42.3%

S: 28.0%

G2/M: 13.5%

G0

: 49.9%

G1: 38.5%

S: 6.3%

G2/M: 5.3%

Figure 6. Induction of apoptosis by Cl-4AS-1 in MDA-MB-453 cells via blocking cell entry into the S phase. Cell cycle analysis wascarried out on MDA-MB-453 cells without treatment (A and D) or after treatment with either 1 μM Cl-4AS-1 (B and E) or 4 μM (C andF) for 3 (A, B, and C) or 6 (D, E, and F) days. Distribution of the cycles is indicated in (A). The straight arrows in (C) and (E) indicate theblock in the early S phase, whereas the dashed arrows in (C), (E) and (F) indicate DNA degradation and apoptotic cells. Theexperiment was done twice in duplicates.

ENDOCRINE RESEARCH 9

found to increase resistance of cells towardtamoxifen.38 In addition, a recent study hasshown that taking into consideration both ARand ER expressions in breast cancer cells can bea more accurate method for determiningresponse to tamoxifen treatment.39 In this study,overexpression of AR relative to ER is associatedwith failure to respond to tamoxifen and poorsurvival in ER-positive breast cancers. The samestudy also illustrates that treatment of this tumortype with AR antagonists results in decreased cellproliferation. Specifically, one antagonist, enzalu-tamide, inhibits both ER- and AR-stimulated cellgrowths in vivo and in vitro. In our case, DHTinduces the growth of ER-positive MCF-7 cellssimilar to what has been shown before40–44 indi-cating that, even in cells that have not developedresistance toward endocrine therapy, inhibitionof AR signaling can be beneficial.

The second type of breast cancers that can betargeted by AR antagonists is TNBC, particularly,those known as molecular apocrine tumors thatare characterized by being ER-negative, AR-posi-tive cells. In this breast cancer subtype and usingMDA-MB-453 cell line as a model system, ARtends to have the same oncogenic effect of ERvia promoting cell proliferation40 and binding tochromatin regions that are characteristic of ER-binding sites.14 In this study, we have observed anincrease in the proliferation of the same cell lineupon treating them with DHT. Blocking AR sig-naling via treatment of these cells with bicaluta-mide or reducing the expression of AR results inslower growth and the formation colonies.14

Interestingly, targeting AR with inhibitors suchas bicalutamide in TNBC has shown inhibitoryeffect on cell growth and tumorigenecity, eventhough they do not have an expression profileof active AR signaling.16,45 A number of clinicaltrials based on blocking the action of AR orandrogen synthesis in AR-positive breast cancerare currently underway.46

Conclusion

Collectively, Cl-4AS-1 can be used as an alterna-tive agonist to classical AR agonists such as DHT.However, it is recommended to be used at lowerconcentrations to prevent induction of apoptosis.

Our results also indicate a novel mechanism bywhich Cl-4AS-1 induces death of breast cancercells regardless of their subtypes. This effectcould be induced via interaction with differentco-regulators than DHT or at different affinitiesresulting in modulating divergent signaling path-ways and gene expression profiling. Hence, themolecular mechanism of action of this SARMneeds to be elucidated. However, the stability ofthe compound needs to be improved in order togenerate reproducible results and to be consideredfor in vivo studies. In addition, the fact that DHTcauses a stimulation of some AR-expressing breastcancer cells signifies the utilization of AR antago-nists in the treatment of this cancer type.

Acknowledgments

The authors would like to extend their gratitude to Dr AdeebZoubi and Ms Mahasen Zalloum of Stem Cells Arabia fortheir assistance in the flow cytometry. We would also like tothank Prof. Mohammad El-Khateeb, Ms Khadijah Al-Rashed,Ms Razan Abu Khashabeh, and Ms Ala’a Arafat at theNational Center for Diabetes, Endocrinology and Geneticsfor their technical assistance.

Funding

This work has been funded by the Deanship of ScientificResearch, The University of Jordan grant number [1436/2012] and by the Scientific Research Support Fund,Ministry of Higher Education and Scientific Research grantnumber [MPH/1/06/2012].

Declaration of interest

The authors report no conflict of interest. The authors aloneare responsible for the content and writing of the paper.

References

1. Mohamed A, Krajewski K, Cakar B, Ma CX. Targetedtherapy for breast cancer. Am J Pathol. 2013;183(4):1096–1112.

2. De Abreu FB, Schwartz GN, Wells WA, Tsongalis GJ.Personalized therapy for breast cancer. ClinicalGenetics. 2014;86(1):62–67.

3. Cuzick J, Forbes JF, Sestak I, et al. Long-term results oftamoxifen prophylaxis for breast cancer—96-monthfollow-up of the randomized IBIS-I trial. J NatlCancer Inst. 2007;99(4):272–282. doi:10.1093/jnci/djk049.

10 M. AHRAM ET AL.

4. Maximov PY, Lee TM, Jordan VC. The discovery anddevelopment of selective estrogen receptor modulators(SERMs) for clinical practice. Curr Clin Pharmacol.2013;8(2):135–155.

5. Goss PE, Ingle JN, Ales-Martinez JE, et al. Exemestanefor breast-cancer prevention in postmenopausalwomen. N Engl J Med. 2011;364(25):2381–2391.doi:10.1056/NEJMoa1103507.

6. Mauri D, Pavlidis N, Polyzos NP, Ioannidis JP. Survivalwith aromatase inhibitors and inactivators versus stan-dard hormonal therapy in advanced breast cancer:meta-analysis. J Natl Cancer Inst. 2006;98(18):1285–1291.

7. Kaufman B, Mackey JR, Clemens MR, et al.Trastuzumab plus anastrozole versus anastrozolealone for the treatment of postmenopausal womenwith human epidermal growth factor receptor 2-posi-tive, hormone receptor-positive metastatic breast can-cer: results from the randomized phase III TAnDEMstudy. J Clinical Oncology: Official Journal Am Soc ClinOncol. 2009;27(33):5529–5537. doi:10.1200/JCO.2008.20.6847.

8. Toss A, Cristofanilli M. Molecular characterization andtargeted therapeutic approaches in breast cancer.Breast Cancer Research: BCR. 2015;17:60.

9. Lehmann BD, Bauer JA, Chen X, et al. Identification ofhuman triple-negative breast cancer subtypes and pre-clinical models for selection of targeted therapies. JClin Invest. 2011;121(7):2750–2767.

10. Burstein MD, Tsimelzon A, Poage GM, et al.Comprehensive genomic analysis identifies novel sub-types and targets of triple-negative breast cancer. ClinCancer Res: an off J American Assoc Cancer Res.2015;21(7):1688–1698. doi:10.1158/1078-0432.CCR-14-0432.

11. Garay JP, Park BH. Androgen receptor as a targetedtherapy for breast cancer. Am J Cancer Res. 2012;2(4):434–445.

12. Collins LC, Cole KS, Marotti JD, Hu R, Schnitt SJ,Tamimi RM. Androgen receptor expression in breastcancer in relation to molecular phenotype: results fromthe Nurses’ Health Study. Modern Pathol: Off J UnitedStates Can Academy Pathol, Inc. 2011;24(7):924–931.

13. Gonzalez LO, Corte MD, Vazquez J, et al. Androgenreceptor expresion in breast cancer: relationship withclinicopathological characteristics of the tumors, prog-nosis, and expression of metalloproteases and theirinhibitors. BMC Cancer. 2008;8:149.

14. Robinson JL, Macarthur S, Ross-Innes CS, et al.Androgen receptor driven transcription in molecularapocrine breast cancer is mediated by FoxA1. Embo J.2011;30(15):3019–3027. doi:10.1038/emboj.2011.216.

15. Hickey TE, Robinson JL, Carroll JS, Tilley WD.Minireview: the androgen receptor in breast tissues:growth inhibitor, tumor suppressor, oncogene? MolEndocrinology. 2012;26(8):1252–1267.

16. Barton VN, D’Amato NC, Gordon MA, et al. Multiplemolecular subtypes of triple-negative breast cancer cri-tically rely on androgen receptor and respond to enza-lutamide in vivo. Mol Cancer Ther. 2015. doi:10.1158/1535-7163.MCT-14-0926.

17. Traish AM, Fetten K, Miner M, Hansen ML, Guay A.Testosterone and risk of breast cancer: appraisal ofexisting evidence. Horm Mol Biol Clin Investig. 2010;2(1):177–190.

18. Narayanan R, Dalton JT. Androgen receptor: a com-plex therapeutic target for breast cancer. Cancers.2016;8:12.

19. Zhang X, Sui Z. Deciphering the selective androgenreceptor modulators paradigm. Expert Opin DrugDiscov. 2013;8(2):191–218.

20. Akita K, Harada K, Ichihara J, Takata N, Takahashi Y,Saito K. A novel selective androgen receptor modula-tor, NEP28, is efficacious in muscle and brain withoutserious side effects on prostate. Eur J Pharmacol.2013;720(1–3):107–114.

21. Cozzoli A, Capogrosso RF, Sblendorio VT, et al.GLPG0492, a novel selective androgen receptor mod-ulator, improves muscle performance in the exercised-mdx mouse model of muscular dystrophy. PharmacolRes: Official J Italian Pharmacol Soc. 2013;72:9–24.

22. Dalton JT, Barnette KG, Bohl CE, et al. The selectiveandrogen receptor modulator GTx-024 (enobosarm)improves lean body mass and physical function inhealthy elderly men and postmenopausal women:results of a double-blind, placebo-controlled phase IItrial. J Cachexia Sarcopenia Muscle. 2011;2(3):153–161.doi:10.1007/s13539-011-0034-6.

23. Narayanan R, Ahn S, Cheney MD, et al. Selectiveandrogen receptor modulators (SARMs) negativelyregulate triple-negative breast cancer growth andepithelial: mesenchymal stem cell signaling. PloS One.2014;9(7):e103202. doi:10.1371/journal.pone.0103202.

24. Tolman RL, Sahoo SP, Bakshi RK, et al. 4-Methyl-3-oxo-4-aza-5alpha-androst-1-ene-17beta-N-aryl-carbox-amides: an approach to combined androgen blockade[5alpha-reductase inhibition with androgen receptorbinding in vitro]. J Steroid Biochem Mol Biol. 1997;60(5–6):303–309. doi:10.1016/S0960-0760(96)00199-9.

25. Schmidt A, Harada S, Kimmel DB, et al. Identificationof anabolic selective androgen receptor modulatorswith reduced activities in reproductive tissues andsebaceous glands. J Biol Chem. 2009;284(52):36367–36376. doi:10.1074/jbc.M109.049734.

26. Chiu CL, Patsch K, Cutrale F, et al. Intracellularkinetics of the androgen receptor shown by multimo-dal image correlation spectroscopy (mICS). Sci Rep.2016;6:22435.

27. Mosmann T. Rapid colorimetric assay for cellulargrowth and survival: application to proliferationand cytotoxicity assays. J Immunol Methods.1983;65(1–2):55–63.

ENDOCRINE RESEARCH 11

28. Moore NL, Buchanan G, Harris JM, et al. An androgenreceptor mutation in the MDA-MB-453 cell line modelof molecular apocrine breast cancer compromisesreceptor activity. Endocr Relat Cancer. 2012;19(4):599–613. doi:10.1530/ERC-12-0065.

29. Narayanan R, Coss CC, Dalton JT. Development ofselective androgen receptor modulators (SARMs). MolCell Endocrinol. 2017;465:134–142.

30. Ahram M, Mustafa E, Zaza R, et al. Differential expres-sion and androgen regulation of microRNAs andmetalloprotease 13 in breast cancer cells. Cell Biol Int.2017;41(12):1345–1355. doi:10.1002/cbin.10841.

31. Gao W, Wu Z, Bohl CE, Yang J, Miller DD, Dalton JT.Characterization of the in vitro metabolism of selectiveandrogen receptor modulator using human, rat, anddog liver enzyme preparations. Drug Metab Dispos.2006;34(2):243–253.

32. Yu Z, He S, Wang D, et al. Selective androgen receptormodulator RAD140 inhibits the growth of androgen/estrogen receptor-positive breast cancer models with adistinct mechanism of action. Clin Cancer Res: an off JAmerican Assoc Cancer Res. 2017;23(24):7608–7620.doi:10.1158/1078-0432.CCR-17-0670.

33. Schmidt A, Meissner RS, Gentile MA, et al.Identification of an anabolic selective androgen recep-tor modulator that actively induces death of androgen-independent prostate cancer cells. J Steroid BiochemMol Biol. 2014;143:29–39.

34. Rossi V, Bellastella G, De Rosa C, et al. Raloxifeneinduces cell death and inhibits proliferation throughmultiple signaling pathways in prostate cancer cellsexpressing different levels of estrogen receptor alphaand beta. J Cell Physiol. 2011;226(5):1334–1339.doi:10.1002/jcp.22461.

35. Birrell SN, Hall RE, Tilley WD. Role of the androgenreceptor in human breast cancer. J Mammary GlandBiol Neoplasia. 1998;3(1):95–103.

36. Naderi A, Liu J. Inhibition of androgen receptor andCdc25A phosphatase as a combination targeted ther-apy in molecular apocrine breast cancer. Cancer Lett.2010;298(1):74–87.

37. Park S, Park HS, Koo JS, Yang WI, Kim SI, Park BW.Higher expression of androgen receptor is a significantpredictor for better endocrine-responsiveness in estro-gen receptor-positive breast cancers. Breast Cancer ResTreat. 2012;133(1):311–320.

38. De Amicis F, Thirugnansampanthan J, Cui Y, et al.Androgen receptor overexpression induces tamoxifenresistance in human breast cancer cells. Breast Cancer ResTreat. 2010;121(1):1–11. doi:10.1007/s10549-009-0436-8.

39. Cochrane DR, Bernales S, Jacobsen BM, et al. Role ofthe androgen receptor in breast cancer and preclinicalanalysis of enzalutamide. Breast Cancer Research: BCR.2014;16(1):R7. doi:10.1186/bcr3599.

40. Birrell SN, Roder DM, Horsfall DJ, Bentel JM, TilleyWD. Medroxyprogesterone acetate therapy inadvanced breast cancer: the predictive value of andro-gen receptor expression. J Clinical Oncology: OfficialJournal Am Soc Clin Oncol. 1995;13(7):1572–1577.

41. Hackenberg R, Hofmann J, Holzel F, Schulz KD.Stimulatory effects of androgen and antiandrogen onthe in vitro proliferation of human mammary carcinomacells. J Cancer Res Clin Oncol. 1988;114(6):593–601.

42. Lippman M, Bolan G, Huff K. The effects of androgensand antiandrogens on hormone-responsive humanbreast cancer in long-term tissue culture. Cancer Res.1976;36(12):4610–4618.

43. Maggiolini M, Donze O, Jeannin E, Ando S, Picard D.Adrenal androgens stimulate the proliferation of breastcancer cells as direct activators of estrogen receptoralpha. Cancer Res. 1999;59(19):4864–4869.

44. Zava DT, McGuire WL. Androgen action throughestrogen receptor in a human breast cancer cell line.Endocrinology. 1978;103(2):624–631.

45. Cuenca-Lopez MD, Montero JC, Morales JC, Prat A,Pandiella A, Ocana A. Phospho-kinase profile of triplenegative breast cancer and androgen receptor signal-ing. BMC Cancer. 2014;14:302.

46. Anestis A, Karamouzis MV, Dalagiorgou G,Papavassiliou AG. Is androgen receptor targeting anemerging treatment strategy for triple negative breastcancer? Cancer Treat Rev. 2015;41(6):547–553.

12 M. AHRAM ET AL.