The role of estrogens and estrogen receptor signaling pathways in cancer and infertility: the case of schistosomes

The

role

of

estrogens

and

estrogen

receptor

signaling

pathways

in

cancer

and

infertility:

the

case

of

schistosomes

Mo´nica

C.

Botelho

,

Helena

Alves

,

Alberto

Barros

,

Gabriel

Rinaldi

,

Paul

J.

Brindley

,

and

Ma´rio

Sousa

1

INSA,NationalInstituteofHealthDr.RicardoJorge,Porto,Portugal

2

IPATIMUP,InstituteofPathologyandMolecularImmunologyoftheUniversityofPorto,Portugal

3_{CentreforReproductiveGeneticsProf.AlbertoBarros,Porto,Portugal}

4_{DepartmentofGenetics,FacultyofMedicine,UniversityofPorto,Porto,Portugal} 5

DepartmentofMicrobiology,ImmunologyandTropicalMedicine,andResearchCenterforNeglectedTropicalandInfectious Diseases,SchoolofMedicine&HealthSciences,GeorgeWashingtonUniversity,Washington,DC20037,USA

6_{DepartmentofMicroscopy,LaboratoryofCellBiology,InstituteofBiomedicalSciencesAbelSalazar(ICBAS),Multidisciplinary}

UnitforBiomedicalResearch-UMIB,UniversityofPorto,Porto,Portugal

Schistosoma haematobium, a parasitic flatworm that infects more than 100 million people, mostly in the developing world, is the causativeagentof urogenital schistosomiasis,andisassociatedwithahighincidence ofsquamouscellcarcinoma(SCC)ofthebladder. Schis-tosomiasishaematobiaalsoappearstonegatively influ-encefertility,andisparticularlyassociatedwithfemale infertility. Giventhatestrogensandestrogenreceptors are key players in human reproduction, we speculate that schistosomeestrogen-likemoleculesmay contrib-utetoinfertilitythroughhormonalimbalances.Here,we review recent findings on the role of estrogens and estrogenreceptorsonbothcarcinogenesisandinfertility associatedwithurogenitalschistosomiasisanddiscuss thebasichormonalmechanismsthatmightbecommon incancerandinfertility.

Thecaseofschistosomiasis

Schistosomiasisisaneglectedtropicaldiseasetransmitted tohumansfromfreshwatersnails.Itiscausedbyablood flukeofthegenusSchistosoma.Schistosomiasisis consid-ered the most important of the helminthiases and the secondmostimportantparasitosis,aftermalaria,causing highratesofmorbidityandmortality.Schistosomesaffect at least 76 countries and 200 million people worldwide. Fromthese,20millionhaveseverediseaseand120million are considered symptomatic. Risk of infection affects 600 million others including travelers from developed countries[1].

Thisopinionfocusesonestrogenmetabolismand estro-gen receptor (ER) signaling pathways associated with cancer induction andfemale infertility in the context of Schistosoma haematobium infection. The present work attemptstointegrateavarietyofstudiesandexperimental approaches with S. haematobium models, while giving particular emphasis to the in vitro studies that have contributedtoexpandingourunderstandingofthe mech-anismsofactionofestrogenmetabolismandERsignaling pathways associated with schistosomiasis. In particular, we suggest that hormonal imbalance resulting from S.haematobiummaypromotecancerandinfertility. Urogenitalschistosomiasis

Three major species of schistosomes are the agents of human schistosomiasis – Schistosoma japonicum and Schistosomamansonicause intestinalschistosomiasisin EastAsia,Africa,SouthAmericaandtheCaribbean,while S.haematobium,occurringwidelythroughout Africaand theMiddleEast,causesurogenitalschistosomiasis.Recent recalibrationofhealthburdensrevealedthatintherange of 4.5–70 million disability adjusted life years (DALYs) arelosttoschistosomiasis.Morepeopleareinfectedwith S. haematobium than with the other schistosomes com-bined.Of112millioncasesofS.haematobiuminfection in sub-Saharan Africa, 70 million are associated with hematuria,18millionwithmajorbladderwallpathology, and 10 million with hydronephrosis leading to kidney damage[2–4].Inmanypatients,depositionofS. haema-tobium parasite ova eventually leads to squamous cell carcinoma(SCC)ofthebladder[5,6].Accordingly,S. hae-matobiumhasbeenclassifiedasaGroup1carcinogenby theInternational AgencyforResearchonCancer(IARC) [7,8].Inaddition,asmanyas75%ofwomeninfectedwith S.haematobiumsufferfromfemalegenitalschistosomiasis (FGS) of the lower genital tract [3]. FGS results from

1471-4922/

ß2015ElsevierLtd.Allrightsreserved.http://dx.doi.org/10.1016/j.pt.2015.03.005

Correspondingauthor:Botelho,M.C. ([email protected],

[email protected]).

Keywords:schistosomiasis;estrogenreceptor;cancer;infertility.

depositionofschistosomeeggsintheuterus,cervix, vagi-na, and/or vulva, with ensuing inflammatory responses; it also increases susceptibility of the woman to HIV [9–11].TheresultingFGSisassociatedwithcontact bleed-ing,discharge,painonintercourse,aswellasdiminished fertility,besidesbeingasourceofshameandstigma[12]. The cellular and molecular mechanisms linking S.haematobium infection eitherwith bothcancer induc-tion and female infertility remain to be deciphered [12,13].However,estrogen-derivedmoleculesandestrogen receptorsignalingpathwayshavebeendescribedforboth associations.Accordingly,wereviewanddiscussthe gen-eralmolecularmechanisms underlyingestrogen metabo-lism,focusing onthehormonesandreceptorsinvolved. Molecularmechanismunderlyingestrogenmetabolism Estrogensare steroid hormones produced in theovaries, adrenal glands, and placenta during pregnancy. The hypothalamus secretes gonadotropin-releasing hormone (GnRH),whichstimulatestheanteriorpituitarytorelease follicle-stimulating hormone (FSH) and luteinizing hormone(LH).FSHandLHinducetheproductionof estro-genintheformofestradiolandestronebytheovaries.These estrogensbindtoERsintargettissuesofthebreast,uterus, brain,bone,liver,andheart[14].Whentheestrogen mole-culebindstoitsreceptor,aconformationalchangeintheER permitsitsinteractionwithaspecificregulatorysequenceof the ER gene(estrogen responsive element), inducingthe transcriptionofthistargetcodingsequence.Theresulting ERproteinpromoteschangesinthecellaccordingtotissue typeandunderlyingconditions.Thecycleiscompletedwhen highlevelsofestrogeninthebloodsendnegativefeedbackto thehypothalamustosuppressthereleaseofGnRH[14].

Bythe1950s,mostofthebasicactionsoftheestrogenic hormoneswererecognized,suchastheirstimulationonthe growthand functionoftissuesofthefemalereproductive tract.However,thebiochemicalprocessesinvolvedwerenot entirelyclear[15].Thegenerallyacceptedhypothesiswas thatthe17-hydroxylgroupofestradiolunderwent enzymat-icoxidationfromacholesterolmoleculeusingonecoenzyme (NADH), and the resulting estrone was reduced using another(NADPH)[15].TheidentificationoftheERprovided a mechanism to describe the target site specificity of estrogenactionintheuterus,vagina,pituitarygland,and breasttissue[16].Mostimportantly,atestwasestablished topredict theoutcomeofantihormonaltherapy inbreast cancer,andatargetwasidentifiedtodevelopnewdrugsfor thetreatmentandpreventionofbreastcancer[16]. ERsandactionof estrogen

Nuclearhormonereceptorsbelongtoafamilyof hormone-activatedtranscriptionfactorsthatcaninitiateorenhance the transcription of genes containing specific hormone response elements [17]. The human ER, which belongs to this family, was cloned and sequenced from MCF-7 human breast cancer cells [17]. The human ER locusis located on chromosome 6q sub-band 25.1 [18] and the mouseER is located onchromosome 10[19,20].The ER consists of 595 amino acids with a molecular mass of 66kDaandincludessix functionaldomains[20–22];two ofthedomainsarehighlyconservedamongthemembersof

the nuclear hormone receptor superfamily [20–22]. Two zinc fingers at the DNA-binding domain (DBD) of ER mediate receptor bindingto hormone-response elements inthepromoterregionsofhormone-responsivegenes.The hormone-binding domain (HBD), located at the ER C terminus,exhibitstworegionsofsequencehomologywith other hormone receptors. These regions confer hormone specificityandselectivitytoER[22–26].

More recently, another sequence belonging to the nuclearhormonereceptorsuperfamilywasclonedfroma rat prostate cDNA library [27,28]. This sequence was namedERb(asopposedtoERa).ERbcontains485amino acid residues and has a molecular weight of 54.2kDa (Figure 1). There is a high homology between ERa and ERb,mainly inthe DBD(95%) andthe HBD(55%),and bothproteins bindestrogenwithhigh affinity,bestowing functional homology.The latterhas beendeterminedby the activation of transcription of a vitellogenin A2, an estrogen-response element (ERE)-containing reporter plasmidinthepresenceandabsenceofestrogen[20,27].

Themechanismoftargetsitespecificityandselectivity seen with anti-estrogens, such as raloxifene, could be explainedby theexistenceoftwodifferentERs[29].The receptor-specific regions are probablyresponsiblefor the differencesseenbetweenERaandERb,inspiteofthehigh homologyintheconservedregionsofbothERs[20].

Estrogendiffusesthroughtheplasmamembraneofcells where it binds to the ER. Once estrogen binds to the inactive ER, the receptor is activated, a conformational change andhomodimerization occurs,andtwo receptor– ligand monomers dimerize and bind to the ERE. Once bound to the ERE, the ER uses activation functions (AFs) (AF-1 and AF-2) to stimulate transcription from

Homology rat ERα and ERβ

rERα Chromosome

6

AF-1 DNA binding

domain DNA binding domain Homology Ligand binding domain AF-2 Ligand binding domain AF-2 rERβ Chromosome 14 66 kDa 595 a.a. 54.2 kDa 485 a.a. AF-1 16.5 % 95.5 % 28.9 % 53.5 % TRENDS in Parasitology

Figure1.Comparisonoftherat(r)ERaandrERbproteinsandpercentaminoacid homologyinthefunctionalregions(Adaptedfrom[20]).Abbreviation:ER,estrogen receptor.

the promoter[20,30].TheERcontains twofunctional do-main areascalledAFs:AF-1islocatedintheN-terminal regionoftheERandAF-2islocatedintheC-terminalregion intheligand-bindingdomain (LBD)oftheER.Theseare synergistic when the ER is activated by estrogen. Using mammalian cells, it was shown that the AF-1 and AF-2 regions,whenexpressedasseparatepolypeptides, function-ally interact in response to estrogen and anti-estrogens, thussuggestingthatestrogenbindingtotheERfacilitates aconformationalchangethatbringsAF-1andAF-2indirect associationwithoneanother,leadingtosynergythatresults in transcriptional activation. These findings explained mechanisticallytheroleofthetwoAFsinmediating hor-mone-regulatedtranscription[20,28].TheEREsare consti-tuted by 13-bp palindromic sequences upstream the transcriptional startsite. BindingoftheER tothe corre-spondingEREenhancesthetranscriptionalrateofthegene inthetargettissue,forexample,breast[20,30].

ER mediates the biological effects of estrogens in a varietyoftargettissues.LigandbindingtoERstimulates gene transcription via interaction with EREs. ERs are knowntomediateimportantphysiologicalfunctions,such asreproduction,metabolism,maintenanceofbonedensity and growth of estrogen-responsive tumors, including breastandendometrialcancers.Estrogensarealsoknown to have a mitogenic effect in estrogen-responsive cells [31].ProgressintheregulationoftheERinbreastcancer andanti-estrogentherapyhasbeenreviewed[20]. S. haematobium-associatedbladder cancer

SCCisamalignant,poorlydifferentiatedneoplasm.SCC isthecommonformofbladdercancerinruralAfricawhere S. haematobium is prevalent [32,33]. By contrast, the majority of bladder cancer in developing countries and regions not endemic for urogenital schistosomiasis is transitional cell carcinoma(TCC),which arisesfrom the transitionalepitheliumliningofthebladder.Theparasite eggstrappedinthebladderwallreleaseantigensandother metabolites (presumably evolved to expedite egress to the urine, and henceto the external environment). The phenomenonleadstohematuriaandtochronic inflamma-tion,inturnincreasingtheriskofSCCofthebladder.The epidemiological association between SCC of the bladder with schistosomiasis haematobia is based both on case control studiesand on the correlation of bladder cancer incidence with prevalence of S. haematobium infection within diverse geographic locations. The incidence of urogenital schistosomiasis-associated SCC is estimated in3–4casesper100000[34].Schistosomiasishaematobia is a chronic infection. The adult, egg-producing schisto-somesliveformanyyears,re-infectionsfrequentlyoccur, andschistosomiasisassociatedbladderSCCappears rela-tivelyearly,oftenbythemid-decadesoflife(TCCusually presentsin thelaterdecades oflife).Inits recent mono-graph, IARC confirmed that chronic infection with S.haematobiumcausescanceroftheurinarybladder[8]. While addressing schistosomiasis-induced hypogonad-ism in patients infected with S. haematobium and S.mansoni,Botelhoetal.observedanoteworthyelevation inserumlevelsofestradiol,whereasthoseofLHandFSH remained normal, and hypothesized that the excess

estradiol could be external to the host [13]. In fact, we foundthatthemoleculeresponsiblefortheeffectwasanS. haematobium-derived estradiol-like molecule that is an antagonistofestradiol andthusrepressedthe transcrip-tionalactivityoftheER.Moreover,newestrogenic mole-culeswereidentifiedinS. haematobiumtotal antigenas well as in the serum of infected individuals with this parasiticdiseasethatseemtobeproducedbythisparasite [35].ERtranscriptionalactivitywassuppressedin urothe-lial cells andER expression was also suppressed in the bladdersofmiceinresponsetoS.haematobium[36].

Estrogenic molecules present in the egg extract of S. haematobium were also identified and characterized by liquid chromatography-mass spectroscopy (LC-MS). The majority of these compounds are catechol estrogens [1]. Catechol estrogens are formed by hydroxylation on the steroid aromatic ring A. Hydroxylation of both C-2 and C-3 on a steroid ring was apparent and suffered furtheroxidationintoanestradiol-2,3-quinone.The geno-toxiceffectsofestrogenmetabolitesmightbeattributedto oxidation of catechol estrogens to quinones, followed by redoxcyclingandformationofreactiveoxygenspeciesthat inturnreactwithDNA[37,38](Figure2).

Given the context of the unarguable link between S. haematobiuminfectionandbladdercancer, the pres-ence of putative carcinogenic molecules in S. haemato-bium eggs hopefully may have practical consequences fornewapproachestodiseasecontrol[1,35].Metabolism of estrogens and the production of depurinating estrogen–DNAadducts can be implicated in apathway underlying S. haematobium-promoted host cell DNA damage, leading eventually to cell transformation. The carcinogenic effect of this estrogen–DNA adduct-mediatedpathwaycouldexplainthelinkbetween chron-ic schistosomiasis haematobia and SCC of the bladder [1]. We anticipate that these findings will contribute tounderstanding howschistosomiasishaematobialeads toSCC ofthe bladder.

Schistosomahaematobiumandinfertility

Infertilityisacommonmedicalcondition,affectingonein sixcouples(15–20%)worldwide [39].Humanandanimal models have unraveled an association between estrogen insufficiency with abnormal spermatogenesis and male infertility[40–42].AnimalmodelsrevealthatERa knock-out (ERaKO)anddouble ERa/ERb knockout(ERa/bKO) miceareinfertilefrompuberty,anddisplayatrophyofthe testesandseminiferoustubuledysmorphogenesis,likelyto leadtodecreasedspermatogenesisandspermmotility[43]. Bycontrast,ERbknockout(ERbKO)micearefertileand havenoapparentreproductivealterationsormorphologic changes[40,43].

Hormonal disturbances in women with FGS may be linked to infertility and suboptimal fecundity [11,44]. Recently, estrogen-like metabolites were detected by LC–MSinurineofS.haematobium-infectedwomen.These metabolites are similar to those identified previously in the adult worm and egg stages of S. haematobium [1].Thepresenceofestrogen-likemetabolitesduringFGS was statistically associated with self-reported infertility [11]. These electrophilic compounds can react with DNA 248

toformdepurinatingadducts.Itisnotinconceivablethat apurinicsitesinchromosomal DNA thatresultfrom this reactiongeneratemutationsthatmightunderlieinfertility [11,44].

Concludingremarksandfutureperspectives

Studies are necessary to identify and characterize production of these estradiol-like moieties in schisto-somes and ascertain the functions of the hormone in thedevelopmentalcycleofthebloodfluke.Indeed, block-ingthebindingofthismoleculetoitsreceptor,withthe useofantihormonaltherapysuchasICI182,780,apotent anti-estrogenwiththeabilitytoinhibitanddownregulate ER[45],couldbeexploredasacomplementarytherapyto schistosomiasis.

Afunctionalestrogentransmembrane receptor,G pro-tein-coupledreceptor(GPR)30,modulatesbothrapid non-genomic events and genomic transcriptional events of estrogen.GPR30promotestheprogressofestrogen-related tumorsthroughmitogen-activatedproteinkinase(MAPK) signalingpathways.EffectsmediatedbyGPR30are main-tainedwhenclassicERsareabsentorblocked.Inaddition, GPR30isinvolvedindrugresistance,whichisoften occur-ringduringcancertreatment.Hence,simultaneous block-ingbothGPR30andclassicERsmaybeabetterstrategy for the treatment of schistosomiasis-related cancer and infertility[46].

Itwillbeinformativetoassessthespecificeffectsofthe estrogenic molecules identified in the lysates of schisto-somesandtoevaluateactivitiesofspecificcatechol estro-gens identified in the schistosome eggs, either by using catecholestrogenspurified from eggsofS.haematobium and/orsyntheticversionsoftheseputativecarcinogens.In addition,giventhatthegenomeandtranscriptomeofeggs, female and male adult worms of S. haematobium are

available, studies utilizing RNAi to silence components of estrogen catabolism pathways such as schistosome estradiol 17-b dehydrogenase andother candidategenes shouldbeinformative[47–49].

Acknowledgments

UMIBisfundedbyNationalFundsthroughFCT-FoundationforScience andTechnology,underthe‘‘Fcomp-01-0124-FEDER-015893’’project.

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