New Selective Progesterone Receptor Modulators and Their Impact on the RANK/RANKL Complex Activity

molecules

Article New Selective Progesterone Receptor Modulators and Their Impact on the RANK/RANKL Complex Activity

Katarzyna Błaszczak-Swi´ ˛atkiewicz

Department of Applied Pharmacy, Medical University of Lodz, Muszynskiego 1, 90-151 Lodz, Poland; [email protected]; Tel.: +48-426779240

Academic Editors: Simona Collina and Mariarosaria Miloso Received: 8 February 2020; Accepted: 11 March 2020; Published: 13 March 2020

Abstract: Breast cancer depends on women’s age. Its chemotherapy and hormone therapy lead to the loss of bone density and disruption of the skeleton. The proteins RANK and RANKL play a pivotal role in the formation of osteoclasts. It is also well established that the same proteins (RANK and RANKL) are the main molecules that play an important role in mammary stem cell biology. Mammary stem cells guarantee diﬀerentiation of the epithelial mammary cells, the growth of which is regulated by the progesterone-induced RANKL signaling pathway. The crosstalk between progesterone receptor, stimulated by progesterone and its analogues results in RANKL to RANK binding and activation of cell proliferation and subsequently unlimited expansion of the breast cancer cells. Therefore downstream regulation of this signaling pathway is desirable. To meet this need, a new class of selective estrogen receptor modulators (SPRMs) with anti- and mesoprogestin function were tested as potential anti-RANK agents. To establish the new feature of SPRMs, the impact of tested SPRMs on RANK-RANKL proteins interaction was tested. Furthermore, the cells proliferation upon RANKL stimulation, as well as NFkB and cyclin D1 expression, induced by tested SPRMs were analyzed. Conducted experiments proved NFkB expression inhibition as well as cyclin D1 expression limitation under asoprisnil and ulipristal treatment. The established paracrine anti-proliferative activity of antiprogestins together with competitive interaction with RANK make this class of compounds attractive for further study in order to deliver more evidence of their anti-RANK activity and potential application in the breast cancer therapy together with its accompanied osteoporosis.

Keywords: RANK-RANKL complex; antiprogestins; mesoprogestins; osteoporosis; breast cancer

1. Introduction Breast cancer is a disorder which depends on the women’s age and hormonal system activity [1]. Estradiol determines the growth of mammary stem cells. It is well established that receptor activator of nuclear factor κB (RANK, the receptor for RANKL) and receptor activator of nuclear factor kappa-B ligand (RANKL) are the main molecules that play an important role in mammary stem cell biology. Differentiation of epithelial mammary cells is regulated by the progesterone-induced RANKL signaling pathway. Crosstalk between progesterone receptor, stimulated by progesterone, is expressed by the interaction between the progesterone receptor and RANKL, results in RANKL to RANK binding and activates cell proliferation and subsequently unlimited expansion of the breast cancer cells. Moreover, the same RANK-RANKL complex plays a pivotal role in osteoclastogenesis [2]. Osteoporosis often accompanies breast cancer treatment with selective estrogen receptor modulators (SERMs) and is a common disease, especially among menopausal women [3]. Estradiol deficiency during menopause or surgical removal of the ovaries is responsible for the loss of bone density in the female body. Although a similar bone density reduction effect is observed in correlation to low testosterone levels in the male body, osteoporosis is more common for women than men. The statistical analysis proves about 15% of

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TheThe The The statisticalstatistical statistical statistical analysisanalysis analysis analysis provesproves proves proves aboutabout about about 15%15% 15% 15% ofof of people peopleof people people inin in their theirin their their 5050 50 ss50 andsand sand and 70%70% 70% 70% ofof of those thoseof those those women [3]. Estradiol deficiency during menopause or surgical removal of the ovaries is responsible moleculesoveroveroverover 8080 80 80have goeshave have have throughosteoporosisosteoporosis osteoporosis osteoporosis two biological [4].[4]. [4]. [4]. TheThe The The expressi responseexpressi expressi expressionon pathways:on onofof of RANK-RANKLofRANK-RANKL RANK-RANKL RANK-RANKL directly by moleculesmolecules interactionmolecules molecules goesgoes withgoes goes throughthrough RANKthrough through/ RANKLtwotwo two two for the loss of bone density in the female body. Although a similar bone density reduction effect is andbiologicalbiologicalbiological indirectlybiological responseresponse response byresponse expression pathways:pathways: pathways: pathways: of thedirectlydirectly directly progesteronedirectly byby by byinteractioninteraction interaction interaction receptor. withwith with Thewith RANK/RANKLRANK/RANKL firstRANK/RANKLRANK/RANKL way is based andand and and onindirectlyindirectly indirectly theindirectly activity byby by by of observed in correlation to low testosterone levels in the male body, osteoporosis is more common for osteoprotegerinexpression of (OPG)the progesterone as a decoy receptor. receptor The for first RANKL. way is The based second on the one activity is an interactionof osteoprotegerin between expressionexpressionexpression womenofof thetheof the progesteroneprogesteronethan progesterone men. 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[4]. and TheTheThe The progesterone.The secondsecondexpressi second second oneoneon one one of isis is TheanRANK-RANKLan is an interactionaninteraction osteoprotegerininteraction interaction betweenbetweenmolecules between between expression thethe goesthe the progesteroneprogesterone progesterone throughprogesterone is induced two byreceptorreceptor femalereceptorreceptor (PR)(PR) sex (PR)biological (PR) hormonesligandligand ligand ligand responseandand and andand progesterone.progesterone. progesterone. progesterone. inhibitspathways: the ThedirectlyThe The development The osteoprotegerinosteoprotegerin osteoprotegerin osteoprotegerinby interaction and expression expression activation withexpression expression RANK/RANKL is ofis is inducedinduced osteoclasts,is induced induced and byby by byfemaleindirectlyfemale whichfemale female sexsex in sexby sex the longhormoneshormoneshormoneshormones run results andandexpression and and inhibitsinhibits in inhibits inhibits the of inhibitionthe thethe the the development developmentprogesterone development development of bone receptor. andand loss.and and activationactivation activation OPGactivationThe first is ofof the wayof osteoclasts, osteoclasts,of osteoclasts, main osteoclasts,is based regulator onwhichwhich which thewhich activityin ofin in the thein the the long long crosstalkof long osteoprotegerinlong runrun run run resultsresults betweenresults results estrogenininin thethein the the inhibitioninhibition andinhibition inhibition(OPG) RANKL ofasof of aboneboneof decoybone expression bone loss.loss. receptorloss. loss. OPGOPG OPG referringOPG for isis RANKL.is thetheis the the mainmain tomain mainThe the regulatorregulator secondregulator bone regulator destructionone ofof of is thetheof anthe the crosstinteractioncrosst crosst crosst repressionalkalkalk alk between betweenbetween between between [2]. theestrogenestrogen Toestrogen progesteroneestrogen prevent andand and and the overexpressionRANKLRANKLRANKLRANKL expressionexpression expressionreceptor expression of osteoclasts (PR)referringreferring referring referring ligand to into toand the osteoporosis,theto the the progesterone.bonebone bone bone destructiondestruction destruction destruction the The new repressionosteoprotegerinrepression repression target—RANK—andrepression [2].[2]. [2]. [2]. 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OPG is the main regulator of the crosstalk between estrogen and progesteroneprogesteroneprogesteroneprogesterone receptorreceptor receptor receptor andand and and theirtheir their their biologicalbiological biological biological funcfunc func functiontiontiontion shouldshould should should bebe be beaddressedaddressed addressed addressed asas as asaa a newnew anew new aspectaspect aspect aspect ofof of of prevention ofRANKL women’s expression health disordersreferring to duringthe bone their destruction postmenopausal repression [2]. and Tohormone-depend prevent the overexpression time of of life. preventionpreventionpreventionprevention ofof of women’s women’sof women’s women’s healthhealth health health disordersdisorders disorders disorders duringduring during during theirtheir their their postmenopausalpostmenopausal postmenopausal postmenopausal andand and and hormone-dependhormone-depend hormone-depend hormone-depend timetime time time ofof of of Some alternativeosteoclasts ways in of osteoporosis, RANK-RANKL the new signaling target—RANK—and have been discovered RANK/RANKL and describedcomplex as in well the [as5,6 ]. life. Some alternative ways of RANK-RANKL signaling have been discovered and described in the Transcriptionallife.life.life. SomeSome Some alternativealternativeprogesterone alternative NF-κB waysways factor receptor ways ofof normally RANK-RANKL RANK-RANKLof and RANK-RANKL their exists biological insignsignthe signalingaling func cytoplasmaling havetionhave have should beenbeen asbeen discovered discoveredanbe discovered inactiveaddressed andand complex asand describeddescribed a describednew with aspect inin nuclear the thein of the [5–6]. Transcriptional NF-κB factor normally exists in the cytoplasm as an inactive complex with factor[5–6].[5–6].[5–6]. of TranscriptionalTranscriptional kappa Transcriptionalprevention light polypeptide of NF-NF- women’s NF-κκBB κfactorfactorB healthfactor gene normallynormally enhancerdisordersnormally existsexists during inexists B-cells inin their thethein inhibitor,the postmenopausalcytoplasmcytoplasm cytoplasm alpha asas anan asand (I κinactiveaninactive Bhormone-depend αinactive). Phosphorylation complexcomplex complex timewithwith with of of nuclearnuclear factor factor of of kappa kappa light light polypeptide polypeptide ge genene enhancer enhancer in in B-cells B-cells inhibitor, inhibitor, alpha alpha (I κ(IBκαB).α ). IκnuclearBαnuclearby IKKfactor life. complexfactor Someof ofkappa alternative (IkappaκB kinase,light lightways polypeptide IKK, ofpolypeptide RANK-RANKL is an enzyme ge nege ne enhancer complex)sign enhanceraling havein leads inB-cellsbeen toB-cells discovered complex inhibitor, inhibitor, degradation and alpha described alpha (Iκ and Bin(Iακ the).B free α ). PhosphorylationPhosphorylationPhosphorylationPhosphorylation[5–6]. Transcriptionalofof of I Iκofκ IBBκ IααBκ αBbyby αby IKKbyIKK IKK IKKNF- complexcomplex complexκ complexB factor (I(I κκ(Inormally BBκ(I Bkinase,κkinase, Bkinase, kinase, exists IKK,IKK, IKK, IKK, in isis is theanan is an enzymeenzymeancytoplasm enzyme enzyme complex)complex) complex)as complex) an inactive leadsleads leads leads complextoto to complex complexto complex complex with NFkB migrates to the nucleus and triggers DNA. Transcription of specific genes ensures i.a osteoclast degradationdegradationdegradationdegradationnuclear andand and and freefree freefactor free NFkBNFkB NFkB NFkB of migrates migrates kappamigrates migrates light toto to thethe to thepolypeptide the nucleusnucleus nucleus nucleus andand andge and triggerstriggersne triggers triggersenhancer DNA.DNA. DNA. DNA. in TranscriptionTranscription TranscriptionB-cells Transcription inhibitor, ofof of specificspecific of specificalpha specific genesgenes(I genesκ genesBα ). formation and also protects cells against apoptosis like in breast cancer expansion [7–13]. Given the ensuresensuresensuresensures i.ai.a i.a Phosphorylation i.aosteoclastosteoclast osteoclast osteoclast formationformation formation formationof IκBα byandand and IKKand alsoalso complexalso also protectsprotects protects protects (IκB kinase,cellscells cells cells againstagainst IKK,against against is anapoptosisapoptosis apoptosis enzymeapoptosis complex) likelike like like inin in leadsbreastinbreast breast breast to cancercomplexcancer cancer cancer NF-κB function approach as well as its relation to osteoporosis and breast cancer, it is obvious why the expansionexpansionexpansionexpansion [7–13].[7–13].degradation [7–13]. [7–13]. GivenGiven Given Given and thethe the free the NF-NF- NF- NFkB NF-κκBBκ Bfunctionκfunction migratesBfunction function approachapproach to approach theapproach nucleus asas as wellwell as and well well asastriggers as itsits as its relation relationits relation DNA. relation toTranscriptionto to osteoporosisosteoporosis to osteoporosis osteoporosis of specific andand and and breastbreast breast genes breast recognition ofensures the NF- i.a κosteoclastB activity formation in the crossway and also betweenprotects cells RANK against/RANKL apoptosis and like SPRMs in breast is crucial cancer to cancer,cancer,cancer,cancer, itit itisis it is obviousisobvious obvious obvious whywhy why why thethe the the recognitionrecognition recognition recognition ofof of ofthethe the the NF-NF- NF- NF-κκBBκ Bκactivityactivity Bactivity activity inin in inthethe the the crosswaycrossway crossway crossway betweenbetween between between expansion [7–13]. Given the NF-κB function approach as well as its relation to osteoporosis and breast provideRANK/RANKLRANK/RANKLRANK/RANKLRANK/RANKL the evidence andand and and of SPRMsSPRMs SPRMs useful SPRMs isis SPRMs is crucial crucialis crucial crucial as toto anto provideprovideto provide anti-RANKL provide thethe the the evidenceevidence evidence andevidence antiproliferative ofof of usefulusefulof useful useful SPSP SPRMs RMsSPRMs agentsRMs asas as ananas an into anti-RANKLananti-RANKL anti-RANKL anti-RANKL osteoporosis cancer, it is obvious why the recognition of the NF-κB activity in the crossway between andand breast antiproliferative cancer treatment. agents The into aim osteoporosis of planed and studies breast was cancer to initiate treatment. the experiments The aim of planed referring studies to the andandand antiproliferativeantiproliferative antiproliferativeRANK/RANKL agentsagents agents and intointo SPRMs into ososteoporosisteoporosis os isteoporosis crucial andandto provideand breastbreast breast cancerthecancer cancerevidence treatment.treatment. treatment. of useful TheThe SPThe aimaimRMs aim ofof as planedplaned of an planed anti-RANKL studiesstudies studies role of tested compounds into NFkB function, especially in breast cancer and osteoporosis progress. waswaswaswas toto to initiatetoinitiate initiate andinitiate antiproliferative thethe the the experimentsexperiments experiments experiments agents referringreferring referring intoreferring os teoporosis toto to thtoth the e th erolerole eandrole role ofofbreast of testedoftested tested cancertested compoundscompounds compounds treatment.compounds The intointo into aiminto NFkBNFkB NFkBof NFkB planed function,function, function, function, studies Ulipristalespeciallyespeciallyespeciallyespecially and ininwas in breast breast Asoprisnilin breast tobreast initiate cancercancer cancer cancer havethe andand and experimentsand been osteoporosisosteoporosis osteoporosis osteoporosis tested referring as progreprogre potentialprogre progre toss.ss. ss.th Ulipristalss.Ulipristale Ulipristal ligands roleUlipristal of testedand forand and and the AsoprisnAsoprisn Asoprisncompounds RANKAsoprisnilil il receptor.havehave il haveinto have beenbeen NFkBbeen been In testedtested thistestedfunction, tested study,asas as as thepotentialpotentialpotential epotentialffect of ligandsligands SPRMsespeciallyligands ligands forfor onfor in forthethe RANKL-inducedbreastthe the RANKRANK RANK RANKcancer receptor.receptor. receptor.and receptor. osteoporosis signaling InIn In thisthisIn this this study, study,progre hasstudy, study, been ss.thethe the Ulipristal the effecteffect examined. effect effect ofof andof SPRMsSPRMsof SPRMsAsoprisn TheSPRMs structuresonon on il onRANKL-induced RANKL-inducedhave RANKL-induced RANKL-induced been of thetested tested as compoundssignalingsignalingsignalingsignaling hashas arepotentialhas has beenbeen shownbeen been examined.examined. ligandsexamined. examined. in Table for ThTh 1the Th.e eTh structuresestructuresRANK estructures structures receptor. ofof of the theof the In the testedtested testedthis tested study, compcomp comp comp oundsoundstheounds oundseffect areare are of are shownshown shownSPRMs shown inin inon Table Tablein TableRANKL-induced Table 1.1. 1. 1. signaling has been examined. The structures of the tested compounds are shown in Table 1. TableTableTableTable 1.1. 1. StructureStructure 1.Structure Structure ofof of of testedtested testedof tested tested progestinsprogestins progestins progestins progestins andand andand and SPRMs.SPRMs. SPRMs. SPRMs. Table 1. Structure of tested progestins and SPRMs. ProgestinsProgestinsProgestinsProgestinsProgestins SPRMsSPRMsSPRMs SPRMsSPRMs Progestins SPRMs ProgesteroneProgesterone PromegestonePromegestone Mifepristone Mifepristone Ulipristal Ulipristal Asoprisnil Asoprisnil ProgesteroneProgesteroneProgesterone Promegestone PromegestonePromegestone Promegestone Mifepristone Mifepristone Mifepristone Mifepristone Ulipristal Ulipristal Ulipristal Asoprisnil Asoprisnil

2. Results and2. Results Discussion and Discussion 2.2. 2. ResultsResults2. Results Results andand and and DiscussionDiscussion Discussion Discussion 2.1. Proliferation2.1. Proliferation Assay Assay 2.1.2.1.2.1.2.1. ProliferationProliferation Proliferation Proliferation AssayAssay Assay Assay Hormone replacementHormone replacement therapy therapy (HRT) with(HRT) combination with combination therapy therapy of of estradiol estradiol andand progesterone bringsHormoneHormone breastHormoneHormonebrings cancer replacementreplacement replacement breast replacement risk cancer for therapytherapy women therapyrisk therapy for (HRT) (HRT) [women 13(HRT) (HRT)]. Therefore withwith [13].with with combinationcombinationTherefore combination combination to eliminate to eliminate therapytherapy therapy therapy the the ofof negative of estradiol negativeestradiolof estradiol estradiol aspect aspect andand and and progesterone progesteroneof ofprogesterone thisprogesterone this therapy, therapy, brings breast cancer risk for women [13]. Therefore to eliminate the negative aspect of this therapy, thebringsbrings rolebrings breast ofbreast progesterone—thebreastthe cancercancer role cancer of risk riskprogesterone risk forfor forwomenwomen main women – the factor[13].[13]. main [13]. ThereforeTherefore that factorTherefore triggers that toto triggerseliminate eliminateto RANKLeliminate RANKL thethe expression the negativenegative expression negative aspectaspect and andaspect breast ofbreastof this thisof this cancer cancertherapy,therapy, therapy, cells cells thethe role role of of proliferation—needsprogesterone progesterone – –the the tomain mainbe limited. factor factor Thethat that first triggers triggers test th RANKLat RANKL allowed expression usexpression to estimate and and the breast breastinfluence cancer cancer of steroidcells cells proliferation—needsthe therole role of progesteroneof progesterone to be – limited.the – themain mainThe factor factor first that test that triggers that triggers allowed RANKL RANKL us expression to expression estimate and the and breast influence breast cancer cancer of cells steroid cells proliferation—needsproliferation—needsproliferation—needsproliferation—needsligands and toto OPGto be beto be limited. limited.beinto limited. limited. RANKL TheThe The The inducedfirstfirst first first testtest test proliferationtest thth thatat that allowedallowedat allowed allowed of T47Dusus us totous to cells estimate estimateto estimate estimate was WST thethe the the influenceassay.influence influence influence The ofcontrolof of steroid steroidof steroid steroid cells ligands and OPG into RANKL induced proliferation of T47D cells was WST assay. The control ligandsligandsligandsligands andand and andproliferation OPGOPG OPG OPG intointo into into RANKLRANKL underRANKL RANKL RANKLinducedinduced induced induced stimulation proliferationproliferation proliferation proliferation was ofof of T47D T47Ddeof T47Dtected T47D cellscells cells cellsand waswas was comparedwas WSTWST WST WST assay.assay. assay. assay.with TheThe Thenegative The controlcontrol control control control cellscells cells cells cells proliferation under RANKL stimulation was detected and compared with negative control proliferationproliferationproliferationproliferation(RANKL-). under underunder under RANKLRANKL OsteoprotegerinRANKL RANKL stimulationstimulation stimulation stimulation (OPG) wasis was wasthewas de demainde tectedtecteddetected tectedregulator and andand and compared comparedofcompared thecompared crosstalk withwith with withbetween negative negativenegative negative estrogen control controlcontrol control and (RANKL-).RANKL Osteoprotegerin expression therefore (OPG) isOPG the was main used regulator as a positive of control. the crosstalk When the between OPG level estrogen goes down, and (RANKL-).(RANKL-).(RANKL-).(RANKL-). OsteoprotegerinOsteoprotegerin Osteoprotegerin Osteoprotegerin (OPG)(OPG) (OPG) (OPG) isis is thetheis the the mainmain main main rere regulatorgulator regulatorgulator ofof of thetheof the the crosstalkcrosstalk crosstalk crosstalk betweenbetween between between estrogenestrogen estrogen estrogen andand and and RANKL expressionthe RANKL therefore activity OPGincreases was and used results as ainpositive cells proliferation. control. The When test thesystem OPG was level treated goes by OPG down, RANKLRANKLRANKLRANKL expressionexpression expression expression thereforetherefore therefore therefore OPGOPG OPG OPG waswas was was usedused used used asas as aaas positiveapositive apositive positive control.control. control. control. WhenWhen When When thethe the the OPGOPG OPG OPG levellevel level level goesgoes goes goes down,down, down, down, thethethethe RANKL the RANKLRANKL RANKL RANKL activity activityactivity activity activity increases increasesincreases increases increases andand and and results results results results inin in cells cells cellsin cells cells proliferation.proliferation. proliferation. proliferation. proliferation. TheThe The The testtest test test systemsystem system system system waswas was waswas treatedtreated treated treatedtreated byby by OPG byOPG OPG OPG and increased concentration of mifepristone and progesterone (the highest tested concentration is shown in Figure1). The tested cell viability upon OPG stimulation proved their proliferation inhibition in the presence of RANKL. A similar effect was obtained using mifepristone. Although mifepristone Molecules 2020, 24, x 3 of 9 and increased concentration of mifepristone and progesterone (the highest tested concentration is shownMolecules 2020in Figure, 25, 1321 1). The tested cell viability upon OPG stimulation proved their proliferation3 of 9 inhibition in the presence of RANKL. A similar effect was obtained using mifepristone. Although mifepristone showed antiproliferation activity within RANKL- cells, its anti-RANKL activity towards showed antiproliferation activity within RANKL- cells, its anti-RANKL activity towards RANKL+ RANKL+ cells is significant and opposite to the strong proliferation of progesterone (Figure 1). A cells is significant and opposite to the strong proliferation of progesterone (Figure1). A positive aspect positive aspect of antiproliferation activity of mifepristone in RANKL-induced cells opened a new of antiproliferation activity of mifepristone in RANKL-induced cells opened a new perspective for perspective for SPRMs function. Therefore subsequent tests were extended to new anti- and SPRMs function. Therefore subsequent tests were extended to new anti- and mesoprogestin in order to mesoprogestin in order to compare their activity. compare their activity. cells viability [cells/well] viability cells

M P OPG Control

Figure 1. +RANKL and -RANKL induced T47D cells proliferation upon mifepristone (M, 1200 nM), Figure 1. +RANKL and -RANKL induced T47D cells proliferation upon mifepristone (M, 1200 nM), progesterone (P, 1200 nM) and OPG treatment. Data represent the mean SD (n = 3). p values progesterone (P, 1200 nM) and OPG treatment. Data represent the mean ±± SD (n = 3). P values correspond to controls with statistically significant differences: * p 0.05, ** p 0.01, *** p 0.001 correspond to controls with statistically significant differences: * p ≤0.05,≤ ** p ≤0.01,≤ *** p ≤0.001≤ (two- (two-way ANOVA variance, multiple comparison of the tested groups vs. control group, separately for way ANOVA variance, multiple comparison of the tested groups vs. control group, separately for +RANKL and -RANKL conditions). +RANKL and -RANKL conditions). 2.2. NFkB Expression Assay 2.2. NFkB Expression Assay The receptor activator of NF-kB-ligand (RANKL) is involved in osteoclast differentiation in the courseThe receptor of osteoporosis activator of and NF-kB-ligand required for (RANKL) mammary is involved cancer development. in osteoclast differentiation The engagement in the of progesteronecourse of osteoporosis in cell proliferation and required takes place for throughmammary two cancer pathways: development. an endocrine The one engagement via interaction of withprogesterone progesterone in cell receptor proliferation in PR takes+ cells place and through a paracrine two pathways: one via elicitation an endocrine of a mitogenicone via interaction effect in withPR- cellsprogesterone with the receptor activity in of PR+ tumor cells necrosis and a paracrine factor (TNF) one via family elicitation member of a –mitogenic receptor effect activator in PR- of cellsNF-κ withB-ligand the activity (RANKL). of tumor RANKL necrosis induces factor the (TNF) proliferation family member of PR- cells – receptor and ensures activator the of PR- NF- cellsκB- phenotypeligand (RANKL). survival. RANKL Therefore induces the searchthe proliferation for new potential of PR- cells inhibitors and ensures of RANK the/ RANKLPR- cells signaling phenotype of survival.both genomic Therefore and non-genomic the search pathwayfor new potential is desired inhibitors [14]. of RANK/RANKL signaling of both genomicThis and genomic non-genomic mechanism pathway of action is desired was [14]. tested on PR-positive T47D cells stably transfected withThis NFkB genomic vector mechanism linked to LUC of action reporter was vector. tested Theon PR-positive luminescence T47D signal cells wasstably measured transfected so aswith to NFkBassess vector NFkB expressionlinked to LUC upon reporter SPRMs induction.vector. The Mifepristone luminescence appeared signal was as ameasured strong anti-RANK so as to assess agent withNFkB 44-fold expression inhibition upon SPRMs of NFkB induction. expression. Mifepristo Also,ne ulipristal appeared proved as a strong its inhibition anti-RANK towards agent NFkB with 44-foldexpression. inhibition Its anti-RANK of NFkB expressi activityon. was Also, weaker ulipristal than pr mifepristoneoved its inhibition but almost towards seven NFkB times expression. stronger Itsthan anti-RANK promegestone activity (Figure was2B). weaker Both of thesethan antiprogestinsmifepristone showedbut almost dose-depend seven times inhibition stronger of NFkB than promegestoneexpression (Figure (Figure2A). 2B). Mifepristone Both of these and ulipristalantiprogestins e fficacy showed compared dose-depend to promgestone inhibition at ECof 70NFkBwas expressionequal to 5% (Figure and 27%, 2A). respectively. Mifepristone These and ulipri outcomesstal efficacy proved compared the potential to promgestone use of antiprogestins at EC70 was as equalanti-RANK to 5% and agents. 27%, Also respectively. a mesoprogestin—asoprisnil—presents These outcomes proved the potential potential use activity of antiprogestins as an anti-RANK as anti- RANKagent. itsagents. efficacy Also for a ECmesoprogestin—asoprisnil—presents70 although it was strong but still weaker potential than activity for promegestone as an anti-RANK (resulting agent. in its62% efficacy of the promegestonefor EC70 although effi cacy,it was Figure strong2B). but still weaker than for promegestone (resulting in 62% of the promegestone efficacy, Figure 2B).

Molecules 2020, 25, 1321 4 of 9 Molecules 2020, 24, x 4 of 9

A B P M A U

EC50 nM 3.2 139.4 6.01 21.18 Relative Potency [EC Test 50 1 43.6 1.88 6.62 Compound /EC50 Promegestone] Efficacy [% max. signal NE 5 62 27 EC70 Promegestone] NE – no effect.

Figure 2. NFkB expression analysis upon SPRM activity in transcription and transactivation manner Figure 2. NFkB expression analysis upon SPRM activity in transcription and transactivation manner (A) graph presents mean SD, n = 3; (B) EC50 and EC70 and efficacy parameters), P-Promegestone, (A) graph presents mean± ± SD, n = 3; (B) EC50 and EC70 and efficacy parameters), P-Promegestone, M-Mifepristone, A-Asoprisnil, U-Ulipristal. M-Mifepristone, A-Asoprisnil, U-Ulipristal. 2.3. Cyclin D1 Expression Assay 2.3. Cyclin D1 Expression Assay Progesterone-induced cell proliferation might occur as cyclin D1 depending on the mechanism. Progesterone-induced cell proliferation might occur as cyclin D1 depending on the mechanism. CyclinCyclin D1 inD1 positive in positive PR +PR+phenotype phenotype of T47Dof T47D cells cells represents represents the the target target gene gene for for progestins. progestins. PR PR ligated to progestinligated to resultsprogestin in results cyclin in D1 cyclin expression D1 expression linking linking to the intracellularto the intracellular signaling signaling with with mitogenic cellmitogenic cycle machinery cell cycle activationmachinery [acti15–vation17]. Therefore [15–17]. Therefore to estimate to estima its overexpressionte its overexpression inhibition inhibition in T47D cells,in testedT47D cells, cells tested were cells treated were treated with increasing with increa concentrationsing concentration of of SPRMs. SPRMs. Inactivation of of cyclin cyclin D1 expressionD1 expression as well as well as the as breastthe breast cancer cancer cells cells formation formation repression repression were were tested. tested. Immunoassay Immunoassay with with FACSFACS analysis analysis of cellsof cells population population with with endogenous endogenous cyclin cyclin D1 D1 expression expression was was performed. performed. Cells Cells treated withtreated promegestone with promegestone showed ashowed dose-dependent a dose-dependent induction induction of cyclin of cyclin D1 expression. D1 expression. Despite the fact asoprisnil showed a cyclin D1 expression induction tendency similar to that of promegestone, a dose-response curve revealed a weaker stimulation in the whole tested range of concentrations. No induction was observed for mifepristone with EC50 = 151.8 nM. Its potency relative to promegestone resulted in a factor of 1.6. A similar factor of 1.3 was obtained for ulipristal although its efficacy was equaled 78% of the promegestone activity. Efficacy parameters and EC50 values for mifepristone and ulipristal provide evidence that in the paracrine pathway antiprogestins act opposite to progestins in the cyclin D1 expression approach. Asoprisnil represents another trend. In spite of its low EC50 = 14.17 nM and strong efficacy (69.05%), Asoprisnil did not reach the full response of promegestone which may suggest a weaker than progesterone impact on cyclin D1 expression and breast cancer cell proliferation (Figure3 and Table2).

Figure 3. The tested compounds induced cyclin D1 expression in T47D cells during treatment for 24 Table 2. Cyclin D1 expression induced by SPRMs in T47D cells (EC50, relative potency and efficacy h; A: FACS analysis of cells population with cyclin D1 expression, data represent three separate parameters). experiments, n = 3, mean ± SD with statistic significant **** p ≤ 0.0001 (two-way ANOVA for comparison of tested groups against each other,Promegestone mean volumes for Mifepristone each concentration Asoprisnil were analyzed, Ulipristal data for the control group although statistically significant, there was not published) ; B: normalized % of cells withEC cyclin50 nM D1 expression to maximal % 56.22 of cells expressing 113 cyclin D1 upon 20.25 Promegestone 94.33 stimulation.Relative Potency 1 2.0 0.36 1.67 [EC50 Test Compound/EC50 Promegestone] Despite the Efactfficacy asoprisnil showed a cyclin D1 expression induction tendency similar to that of promegestone, a dose-response curve revealed a NEweaker stimulation 23 in the whole 82 tested range 27 of [% max. signal EC50 Promegestone] concentrations. No induction was observed for mifepristone with EC50 = 151.8 nM. Its potency relative NE-no effect. to promegestone resulted in a factor of 1.6. A similar factor of 1.3 was obtained for ulipristal although its efficacy was equaled 78% of the promegestone activity. Efficacy parameters and EC50 values for

Molecules 2020, 24, x 4 of 9

A B P M A U

EC50 nM 3.2 139.4 6.01 21.18 Relative Potency [EC Test 50 1 43.6 1.88 6.62 Compound /EC50 Promegestone] Efficacy [% max. signal NE 5 62 27 EC70 Promegestone] NE – no effect.

Figure 2. NFkB expression analysis upon SPRM activity in transcription and transactivation manner

(A) graph presents mean ± SD, n = 3; (B) EC50 and EC70 and efficacy parameters), P-Promegestone, M-Mifepristone, A-Asoprisnil, U-Ulipristal.

2.3. Cyclin D1 Expression Assay Progesterone-induced cell proliferation might occur as cyclin D1 depending on the mechanism. Cyclin D1 in positive PR+ phenotype of T47D cells represents the target gene for progestins. PR ligated to progestin results in cyclin D1 expression linking to the intracellular signaling with mitogenic cell cycle machinery activation [15–17]. Therefore to estimate its overexpression inhibition in T47D cells, tested cells were treated with increasing concentration of SPRMs. Inactivation of cyclin D1 expression as well as the breast cancer cells formation repression were tested. Immunoassay with Molecules 2020, 25, 1321 5 of 9 FACS Moleculesanalysis 2020 of , cells24, x population with endogenous cyclin D1 expression was performed. Cells 5 of 9 treated with promegestone showed a dose-dependent induction of cyclin D1 expression. mifepristone and ulipristal provide evidence that in the paracrine pathway antiprogestins act opposite to progestins in the cyclin D1 expression approach. Asoprisnil represents another trend. In spite of its low EC50 = 14.17 nM and strong efficacy (69.05%), Asoprisnil did not reach the full response of promegestone which may suggest a weaker than progesterone impact on cyclin D1 expression and breast cancer cell proliferation (Figure 3 and Table 2).

Table 2. Cyclin D1 expression induced by SPRMs in T47D cells (EC50, relative potency and efficacy parameters).

Promegestone Mifepristone Asoprisnil Ulipristal

EC50 nM 56.22 113 20.25 94.33 Relative Potency [EC50 Test Compound/EC50 1 2.0 0.36 1.67 FigureFigure 3. The 3.testedThe compounds tested compounds induced induced cyclin D1 cyclin expres D1sion expression in T47D incells T47D during cells treatment during treatment for 24 for Promegestone] h; A: FACS24 h; ( Aanalysis): FACS of analysis cells population of cells population with cyclin with D1 cyclin expression, D1 expression, data represent data represent three separate three separate experiments,experiments, n = 3n, =mean3, meanEfficacy ± SDSD with with statistic statistic significant significant ******** p p 0.0001≤ 0.0001 (two-way (two-way ANOVA ANOVA for comparison for ± ≤ comparisonof tested of tested groups groups[% against max. against eachsignal other, each other, mean mean volumesNE volumes for each for concentration each23 concentration were82 analyzed,were analyzed,27 data for the data forcontrol the control group groupEC although50 Promegestone] although statistically statistically significant, significant, there there was notwas published); not published) (B): normalized; B: normalized % of cells % of cellswith with cyclin cyclin D1 expression D1 expression to maximal to maximal % of cells% NE-noof expressingcells effect.expressing cyclin cyclin D1 upon D1 Promegestoneupon Promegestone stimulation. stimulation. 2.4. GST-Pull Down Analysis of RANK-RANKL Complex Destabilization 2.4. GST-Pull Down Analysis of RANK-RANKL Complex Destabilization Despite the fact asoprisnil showed a cyclin D1 expression induction tendency similar to that of TheThe presented presented work work raises raises one one more more aspect aspect of of SPRMs’ SPRMs’ application application as as a a new new treatment. treatment. Their Their new new promegestone, a dose-response curve revealed a weaker stimulation in the whole tested range of featurefeature as as inhibitors inhibitors of of the the RANK-RANKL RANK-RANKL complex complex were were studied. studied. This This activity activity was was tested tested based based on on concentrations. No induction was observed for mifepristone with EC50 = 151.8 nM. Its potency relative thethe protein-ligand protein-ligand interaction interaction according according to to the the manufacturer’s manufacturer’s instructions instructions of of a a GST-pull GST-pull down down assay assay to promegestone resulted in a factor of 1.6. A similar factor of 1.3 was obtained for ulipristal although (Thermo(Thermo Scientific, Scientific, Warsaw, Warsaw, Poland). Poland. To To analyze analyze the the complex complex RANK-RANKL RANK-RANKL binding binding destruction, destruction, the its efficacy was equaled 78% of the promegestone activity. Efficacy parameters and EC50 values for competitivethe competitive incubation incubation of the of RANK the RANK receptor receptor with antiprogestinwith antiprogestin was performed was performed for 24 for h. The24 h. next The day, the capture affinity of RANK by RANK-ligand previously immobilized on the agarose gel column next day, the capture affinity of RANK by RANK-ligand previously immobilized on the agarose gel wascolumn determined. was determined. The complex The visualization complex visualization was performed was on performed SDS-PAGE gelon duringSDS-PAGE electrophoresis gel during andelectrophoresis Coomassie Blue and staining.Coomassie As Blue shown staining. in Figure As sh4,own the complexin Figure of 4, RANKthe complex and GST–RANKL of RANK and were GST– elutedRANKL from were glutathione eluted from beads glutathione in both test beads and controlin both systemtest and (lane control D, part system I and (lane lane D, C, part I II), and while lane noneC , part of the II), RANK while none protein of moleculethe RANK was protein bound molecule with GST- was RANKL bound with (lane GST- C, part RANKL I) during (lane incubation C, part I) withduring antiprogestin. incubation with antiprogestin.

FigureFigure 4. 4.SDE-PAGE SDE-PAGE analysis analysis of of RANK-RANKL RANK-RANKL complex complex stabilization. stabilization. I testI test system: system: Line Line A—RANKL A—RANKL proteinprotein control, control, Line Line B—RANKL B—RANKL after after column column washing; washing; Line Line C—complex C—complex destruction destruction after after 24 24h h incubationincubation of of RANK RANK with with antiprogestin, antiprogestin, Line Line D—complex D—complex binding binding after after 24 24 h h incubation incubation of of RANKL RANKL andand RANK; RANK; Line Line E—RANK E—RANK protein protein after after column column washing; washing; Line Line F—RANK F—RANK protein protein control. control. II II control control system:system: Line Line A—RANKL A—RANKL protein protein control, control, Line Line B—RANKL B—RANKL after after column column washing; washing; Line Line C—complex C—complex bindingbinding after after 24 24 h h incubation incubation of of RANKL RANKL with with RANK, RANK, Line Line D—RANK D—RANK protein protein after after washing washing column; column; LineLine E—RANK E—RANK protein protein control. control. The The protein protein ladder ladder comes comes from from separate separate run. run.

TheThe results results demonstrate demonstrate RANK-RANKL RANK-RANKL associated associated blocking blocking by by antiprogestin. antiprogestin. Additionally, Additionally, the the parallelparallel performed performed control control proved proved complex complex stability stability during during the the incubation incubation without without the the presence presence of of antiprogestin in a tested complex binding environment. The experiment carried out proved that

Molecules 2020, 25, 1321 6 of 9 antiprogestin in a tested complex binding environment. The experiment carried out proved that antiprogestin destroys the RANK-RANKL complex. This class of SPRMs appears as potential anti-RANK agents with a new perspective for the problem of osteoporosis in postmenopausal women but also with potential benefits for women during breast cancer therapy that occurs with bone density loss.

3. Materials and Methods

3.1. Tested Compounds Some SPRMs were selected so as to evaluate their activity as anti-RANK and antiproliferation agents via NFkB-cyclin D1 axis inhibition. Promegestone with agonistic activity towards progesterone receptor as well as mifepristone with antagonistic activity to progesterone receptor were used as a control. The main aim of the presented study is to estimate the potential use of new mesoprogestin (asoprisnil) and antiprogestin (ulipristal) for eﬀective treatment of menopausal and postmenopausal symptoms like osteoporosis and breast cancer.

3.2. Reagents The human breast cancer cell line T47D was obtained from Sigma-Aldrich (Poznan, Poland). The plasmids: pGL4.27 [luc2P/minP/Hygro] vector coding reporter gene of luciferase luc2P (control vector) and pGL4.32 [luc2P/NF-κB-RE/Hygro] vector coding an NF-κB response element (NF-κB-RE) were purchased from Promega (Tychy, Poland). Cell culture medium composition: DMEM; 10% FBS, 2 mM L-glutamine, 100 U/mL penicillin, 0.1 mg/mL streptomycin, 50 mg/mL higromycin were purchased from Thermo Fisher (Warsaw, Poland), Biowest (Zgierz, Poland) and Sigma-Aldrich, respectively. Ulipristal, asoprisnil, mifepristone, promegestone, and progesterone were purchased in Sigma-Aldrich. The transfection reagent Lipofectamine was obtained from Thermo Fisher Scientiﬁc. Lysate reagents M-PER Mammalian Protein Extraction Reagent and Health Protease Inhibitor Cocktail were from Thermo Fisher Scientiﬁc. The antibodies for cyclin D1 expression immunoassay analysis—mouse monoclonal IgG2-PE cyclin D1 and normal mouse IgG2a-PE antibody—came from Santa Cruz Biotechnology (Dallas, Texas, USA). In the GST-pull down analysis pure RANK and RANKL proteins were generated by Gencript (Piscataway, NJ, USA). For the WST test RANKL mouse recombinant protein and OPG human recombinant protein were purchased from Sigma-Aldrich.

3.3. Cell Culture T47D cells were cultured in phenol-free DMEM medium supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine, and 100 U/mL penicillin/streptomycin. Cultures were fed every 2–3 days by replacing with fresh medium.

3.4. The proliferation Assay T47D cells were seeded in 96-well plates at a density of 8 103 cells per well with three replicates × and stimulated with RANKL (50 ng mL 1). Three days later, cells were treated with the increasing · − concentrations of SPRMs (1.17–1200 nM) and 100 ng mL 1 of OPG for 24 h post-culture. The medium · − was refreshed into one with charcoal FBS. On the 5th day, WST reagent was added and the absorbance was measured. Based on the previously determined calibration curve, the number of cells per well was calculated and expressed in concentration dependent manner.

3.5. Estimation of NFkB Expression

3.5.1. Cell Test System The initial cells density (0.5 103 cells per well) were grown on 6-well plates in transfection medium × w/o antibiotics. Only confluent cells were used for transfection with NFkB DNA using Lipofectamine according to the manufacturer’s instruction. Next, the stable transfectants were cultured in the growth Molecules 2020, 25, 1321 7 of 9 medium containing antibiotic (hygromycin 600 µg/mL). The selection of stably transfected cells was carried out based on the resistance to hygromycin, which came to the transfected cells together with the NFkB DNA. It meant, that only cells growing in such a medium were able to carry a gene of selection [18].

3.5.2. Transactivation Assay Briefly, cells were seeded into 96-well plates and maintained in cell culture media (phenol-free DMEM and charcoal FBS) for 24 h. Cells were then pre-treated with the increasing concentrations of SPRMs (1–1200nM) (ulipristal U and asoprisnil—A) and reference compounds mifepristone (M), and promegestone (P) for 24 h, with or without RANKL stimulation (50 ng mL 1), respectively. Luciferase activity was · − measured using the Promega Luciferase Assay System and normalized to that of the vehicle control.

3.6. Flow Cytometry Analysis of Cyclin D1 Expression

Cell Test System T47D cells (0.5 106 cells/well) were cultured in 6-well plates in a medium. Twenty-four h later, the × attached cells were treated with tested compounds. Compounds were used at six different concentrations: 1.17, 19, 75, 300, 600 and 1200 nM. After 24 h cells were detached and transferred into centrifuge tubes (1–2 106 sample) and spun at 400 g for 8 min at room temperature. Next cells were fixed in 22.5 µL of × × PBS with 157.5 µL of 80% ethanol (cold, chilled at 20 °C, final conc. of ethanol was 70%) and incubated − overnight at 4 °C. After washing them in PBS, 150 µL of 3% BSA/PBS was added to the sample and cells were incubated for 30 min at 4 °C. Next immunolabeling was performed. Incubation of 100 µL of previously prepared antibody recognizing cyclin D1 (anti cyclin D1 Ab – PE conjugated, 1:50 dilution in 3% BSA/PBS) with 1 106 cells/sample lasted 30 min at 4 °C and was protected from direct light. For × negative control, 100 µL of isotype control IgG – PE conjugated (diluted the same way as first antibody) was used. Gently resuspended cells were incubated for 30 min at 4 °C, sample was protected from light as well. Next cells were washed two times in 300 µL of PBS and finally, cells were resuspended in 500 µL of DPBS and flow cytometry measurement was performed. A CytoFLEX flow cytometer, (Beckmann Coulter, Warsaw, Poland) was used for analysis with the following settings: 10,000–30,000 gated cells were analyzed for each sample. The expression of the cyclin D1 for each sample was analyzed by the Kaluza Analysis Software, ver. 1.5a (Beckmann Coulter).

3.7. GST-Pull Down Assay for RANK/RANKL Complex Binding The binding interaction of RANK and RANKL was tested in a GST-pull down assay. Firstly 100 nM 0.1% DMSO stock solution of antiprogestin was prepared. Prepared samples of RANK and RANKL contained 100 µg of each protein. The incubation of RANK and antiprogestin mixture lasted 24 h. First, RANKL fused with GST was immobilized on glutathione agarose resin at 4 ◦C for 60 min in a Pierce spin column. Next, 800 µL of prepared RANK solution potentially bonded with tested antiprogestin was added to the column containing the immobilized RANKL and incubated for the next 120 min with gentle rocking motion on rotating platform to let the complex building or its destruction occur. After washing of unassociated RANK, the bounded RANK and RANKL were eluted by addition elution buﬀer. The eluted proteins as well as the complex was run on 12% SDS–PAGE and stained with Coomassie Blue.

4. Conclusions The RANK/RANKL factors are still being intensively studied. Nowadays their function in bone metabolism and breast cancer proliferation is well documented. The mechanism of RANK/RANKL stimulation is known and widely described in many papers. Progesterone is the ligand of the progesterone receptor (PR) and it is known to trigger RANK expression, which enhances the proliferation of different cells such as osteoclasts and epithelial breast cells. In this way, the therapy based on the administration of the agonistic ligands of the sex hormone receptors leads to different strong and Molecules 2020, 25, 1321 8 of 9 serious sides effects [18–24]. A new class of compounds which would be used as anti-RANK agents are selective progesterone modulators – mesoprogestins and antiprogestins. Asoprisnil was tested in both the endocrine and paracrine pathways with NFkB and cyclin D1 expression- stimulated cells division with the same force (62% and 68% of promegestone efficacy, respectively). This data confirms its weaker interaction either with progesterone and RANK receptor. Ulipristal at EC70 = 21 nM limited NFkB expression via an endocrine pathway with effectiveness equal to 27% of the efficacy of promegestone and subsequently reduced cyclin D 1 expression in a paracrine pathway. Based on the obtained results, the main achievement of the presented work is to propose the new generation of antiprogestins as promising candidates for the effective treatment of over proliferative tissues like breast cancer and osteoclasts in osteoporosis. Additionally, based on the performed positive first attempt to prove a direct interaction of antiprogestin with RANK with regards to RANKL competitive binding to RANK, antiprogestins arise as interesting compounds that may play an important role in the treatment of osteoporosis in hormonal-dependent diseases. Furthermore, their unique mechanism of downstream RANK-RANKL signaling might guarantee the prevention of breast cancer and diminish the metastasis of breast cancer to bone.

Funding: Research was funded by the Medical University of Lodz, Poland. Acknowledgments: The study was supported by the Medical University of Lodz, Poland. Conﬂicts of Interest: The author declares no conﬂict of interest.

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