The role of SLIT–ROBO pathway in crucial cell processes during physiological and pathological conditions

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Review/Praca poglądowa

The role of SLIT–ROBO pathway in crucial cell processes during physiological and pathological conditions

Znaczenie szlaku SLIT-ROBO w kluczowych dla komórki procesach w warunkach ﬁzjologii i patologii

Aleksandra Gołos, Agnieszka Wierzbowska *

KatedraiKlinikaHematologiiUMwŁodzi,Kierownik:prof.drhab.n.med.TadeuszRobak,Łódź,Poland

Introduction

SLIT and their ROBO receptors are members of the axon guidancemoleculesfamilythathavebeenidentiﬁedtoplay a crucial role in developmentof nervous system of vertebrates and invertebrates (Table I). They function as

a repulsive cue with an evolutionarily conserved role in preventingaxonsfrommigratingtoinappropriatelocations.

Asthevascularandaxonnetworkshowsmanysimilarities in their structure (reviewed by Carmeliet and Tessier- Lavigne,2005)[1],researchersstartedtoinvestigatewhether there exist similar signaling pathways, too. As a result,in last 20 years, the axon guidance cues have widely been article info

Articlehistory:

Received:08.04.2014 Accepted:23.07.2014 Availableonline:4.08.2014

Keywords:

SLIT–ROBO

Angiogenesis

Cancer

Słowakluczowe:

SLIT–ROBO

Angiogeneza

Nowotwory

abstract

TheSLITglycoproteinsandtheirroundabout(ROBO)receptorswereoriginallyidentiﬁed asaxonguidancemoleculesthatpreventaxonsfromre-crossingthemidline.Inaddition, theSLIT–ROBOinteractionisinvolvedintheregulationofcellmigration,celldeathand angiogenesis. Furthermore, ithas a pivotal role duringthe morphogenesis,controlling thecorrectdevelopmentoflung,kidney,liverandbreast.ThefunctionswhichtheSLIT–

ROBOfulﬁllsduringtissuemorphogenesisareoftendysregulatedduringcancerdevelop- ment. Thereforeinactivation of certain SLITs andROBOs is associated with advanced tumorformationand progressionindisparate tissues.However, somestudiesrevealed thatSLIT–ROBOmay promote tumor angiogenesisand, consequently, its growth. This reviewisfocused onsummarizingthecurrent knowledge abouttheroleofSLIT–ROBO pathwayindevelopmentofdisparatetissuesinphysiologicalconditionsandinpathoge- nesisofneoplasms,includinghematologicalmalignancies.

*Correspondingauthorat:KlinikaHematologiiUM,ul.Ciołkowskiego2,93-510Łódź,Poland.Tel.:+48426895191;

fax:+48426895192.

E-mailaddress:agawierzbowska@wp.pl(A.Wierzbowska).

ContentslistsavailableatScienceDirect

Acta Haematologica Polonica

journalhomepage:www.elsevier.com/locate/achaem

http://dx.doi.org/10.1016/j.achaem.2014.07.003

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studiedoutsidethenervoussystem. Theirwidenetworkof downsignalling,inﬂuencingapoptosis,cellcycle,cellmigra- tion determines its multiple roles in organogenesis and tumorigenesis.There is evidencethat SLIT–ROBO signaling maytake partinthe pathogenesis of hematologicalmalig- nancies. The aim of this article is to present the current knowledgeabouttheroleofSLIT–ROBOpathwayindevelop- mentof disparatetissuesinboth,physiologicaland patho- logicalconditions.

SLIT–ROBO structure and signaling

SLITisasecretedproteinthatwasﬁrstdescribedasaligand for ROBO in 1999, by Brose et al. [2]. Invertebrates have singleslit protein whereas vertebrates have three of them named slit1, slit2, and slit3. All slits remain evolutionarily conserved.Itconcernstheirstructureaswellasfunction[2].

Theslit isaglycoprotein of about 200kDa thatconsists of an N-terminal signal peptide, four leucine-rich domains (LRR) termed D1–D4followed by sevento nine endothelial growthfactor-likedomains (EGF-like),a laminin-Gdomain, andaC-terminal cysteinknot(Fig.1)[3].Structural studies haveshownthattheLRRdomainseachcontainamotifthat creates a concave shape that might be important for Slit interaction with their receptors. Slits are proteolytically cleavedbetweentwoEGF-likedomains[2].

As well as slit, robo was first discovered in Drosophila melanogaster [4]. There had been three homolog proteins described, robo1, robo2, and robo3. Subsequently, another protein, robo4 (also termed as magic roundabout), was identified in vertebrates and was initially thought to be endothelial specific [5]. Robo1, robo2 and robo3 share the same extracellular domain structure. This region contains five immunoglobulins-like (Ig) domainsand three fibronec- tintype3(FN3)repeats[4]. Theintracellularpartof robo1– robo3 is comprised of four conserved cytoplasmic motifs (CC0,CC1,CC2,CC3).ROBO3lacksofCC1domain[4].Robo4, which shows the lowest homology withother Robos, contains only two Ig domains, a FN3 and CC2 domain [5]

(Fig.1). TheD2domain of theslit and Ig2 domainof robo are crucial for ligand–receptor interaction [6]. The slit–robo interactionleadstotheactincytoskeletonreorganizationin the target cells. Slit binding to robo is involved in a great amount of cell functions, mainly concerning cytoskeletal dynamicsand cellcycle(Fig.2)[3].Todirectthemotilityof cells, slit–robo requires several adaptor proteins which

conductthesignaltotheeffectorproteinsthatsubsequently canchangethestructureofcytoskeleton.TheRacfamilyof small GTPases (srGAP – Slit Robo Rho GTPase activating protein) are crucial for the downstream signaling of SLIT– ROBO [7]. The mostestablished functionof slit–robo is its pro- or anti-migratory activity. Slit–robo can stimulate the interaction between E-cadherin and b-catenin and so pro- motescelladhesion[8].Thiswasobservedduringmammary glanddevelopment[9].Nevertheless, duringtheheartmor- phogenesis, slit–robo inhibits the E-cadherin/b-catenin- mediatedcelltocelladhesion.Thisallowstoformtheheart lumen[10].Aswellasmigrationandadhesion,slit–robocan regulate other processes involved in cell growth. Through direct blocking of cdc42 (cell division control protein 42) activity, the cell cycle is blocked [11]. In addition, slit and robo control these functions independently. Slit induces apoptosis through binding to netrin-1, which disables its interaction with DCC (deleted in colorectalcancer). Conse- quently,caspases3and9canbeactivatedbyDCC.Whatis more,robobindstoDCCtooandinturncausesdissociation of Netrin-1 from DCC and activation of caspases [12]. Slit inhibitscellcyclebyblockingSDF-1,WNTandHGFsignaling [8]. Through themultiplepathways, SLIT–ROBO isinvolved inmorphogenesis,angiogenesisandtumorigenesis.

The role of SLIT–ROBO in CNS development

Robo receptorsand theirligands Slitform oneof the most crucial pairings among the axon guidance molecules. robo gene was identiﬁed in Drosophila as one that controls the midline crossing of commissural axons [4]. Similarly, slit was described as a protein secreted by midline glia cells [13]. The migrating axons are chemotactically attracted to the midline.After crossing it,isstarted tobeexpressed on theirsurface.Consequently,thehighconcentrationofslitin midline glia cells and the expression of robo prevent recrossing the line and sothe axons ﬁndtheir way tothe destination place [14]. In robo /robo Drosophila embryos, commissuralaxonscrossedthemidlinemultipletimes[14].

Furthermore, slit–robo functions in many developmental processes in CNS outside the midline. These include: the formation of the olfactory tract [15], optic tract and optic chiasm[16], andmotor axonpathﬁnding inthe hindbrain [17].

The role of SLIT–ROBO in organogenesis

Inadditiontoaxonguidancefunctionat theCNS,slitsand robosarealsoimplicatedinotherdevelopmentalprocesses.

FirstdataabouttheimportanceofSlitandRoboinorgano- genesis comefrom experiments on mutantmice[18]. Mice withdeletionofRobo1diedshortlyafteritsbirthbecauseof respiratoryfailure.Furtherinvestigationsshowedthatthese mice had severe lung defects, such as abnormal and torturous bronchiole [18]. Grieshammer et al. described kidneyabnormalitiesinSlit2and Robo2mutantmicewhich consequentlyledthemtoquickdeath [19].Similarﬁndings were made in Slit3 mutant mice. Besides kidney defects, TableI–Neuronalguidancemoleculesandtheirrecep-

tors

TabelaI–Neuronalneczynnikiprzewodzeniaiichreceptory

Ligand Receptor

Semaphorins:Class3/class4–7 Neuropilins Integrins Plexin–B/C/D

EphrinsA/B EphA/BReceptors

Netrin1/2/4 DCC,UNC5A-D

Delta-like(Dll-1/3/4) Notch1/2/3/4

Serrate-like(JAG1,JAG2) Notch1/2/3/4

Slit1/2/3/ Robo1/2/3/4

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increased rate of diaphragmal hernia and enlarged right ventricleof heartwereobserved[20]. Theslit–robointerac- tionseemstoplayanimportantroleinthedevelopmentof the heart. InDrosophila, slit incooperation with robo1and robo2directs cardioblastsmigration,andthroughinhibition ofE-cadherin,controlstheiradhesion.Inturn,thelumenof the heart originates [10]. Mounting evidence conﬁrms the greatsigniﬁcanceofslit–roboinreproductivesystem.While studyingmammaryglanddevelopment,Slit–Robowascon- sidered as an adhesion molecule [21]. Dickinson et al.

showed that SLIT2, SLIT3, ROBO1, ROBO2, and ROBO4 are expressed in corpus luteum (CL) in human ovary [22].

Interestingly, the concentration rate of thecues was rising with the duration timeof luteal phase, reachingits topin the late phase, when CL is starting to regress [22]. Con- versely, however, when hCG (human chorionic gonadotro- pin) is released inorder toprotect CLfrom luteolysis, the concentration ofSLIT2, SLIT3,andROBO2decreasesrapidly [22]. These data suggest that SLITs and ROBOs promote luteolysis and that their expression may be regulated Fig.1–SlitandRobostructure,[3]

Ryc.1–BudowaSlitiRobo,[3]

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hormonally.SLITsandROBOsnotonlytakepartinphysiolo- gicaldevelopment,buttheir activityisalso knowninmany pathologiccircumstances.ExpressionofSLIT2correlatedwith increasedmicrovasculardensity(MVD)inendometriosisand was higher in recurrent endometriomas in comparison to non-recurrent [23]. Overall, during organogenesis, theSLIT–

ROBOpathwaycontrolsnumerousprocessesthatseemtobe vitalinthedevelopmentofdifferenttissues.

The SLIT–ROBO interaction in angiogenesis

Bloodvesselsoftengoalongsidethenervesinthebodyand the vascularand neuralnetworks look similarin itsstruc- ture.Indeed,thereareseveralparallelsinthedevelopment ofbothsystems(reviewedbyCarmelietandTessier-Lavigne

[1]). Oneof thecommonfactorsisinvolvinginvasculogen- esis all the members of the axon guidance molecules includingSlitandRobo.Robo4wasthefirsttobeassociated with angiogenesis. It was discovered by data mining for searchingfornewendothelialspecificgenes[24].Expression ofRobo4wasdetectedinmouseplacentalbloodvessels[24], heart, liver, kidney, and lung [25]. However, there was no expression foundinthebrain,skeletal muscle,spleen,and testis [25]. Suchting et al. reported an inhibitory effect of solublechimericreceptorRobo4onangiogenesisinvitroand invivo[26].TheyalsosuggestedthatRobo4doesnotbindto anyoftheSlits[26].However,thetheorywasnotconfirmed inanyofthesubsequentstudies.

Slit2,byactivatingitsreceptorRobo4,wasreportedasan inhibitor factor for vascular-endothelial growth factor (VEGF)-induced migration of mouse endothelial lung cells Fig.2–SLIT–ROBOfunction,[3]

Ryc.2–SzlakisygnałoweSLIT-ROBO,[3]

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[27].Moreover,SLIT2inhibitedmigrationofendothelialcells ofseveralcelllines,suchashumanumbilicalcordvascular endothelial cell (HUVEC) [28], or human microvascular endothelialcell (HMVEC)[29]. Besidesthis,SLIT2decreased migration of human aortic smooth muscle cells [30]. In another study, HEK (human embryonic kidney) cells were initially transfected with ROBO4, then placed in SLIT2- conditionedmedium. Thisresultsininhibitionofmigration of the HEK cells [25]. Similar effects were observed with HMVEC [25]. These results implicate SLIT as an inhibitory angiogenic factor. Nonetheless, some experiments showed discordantconclusions.HUVECcelllineswhichweretreated withmediumcontainingSLIT2presenteddirectionalmigra- tion [31]. The effectwas blockedby treatingthe cells with anantibodyagainstROBO1[31]. Inaddition,human malig- nant melanomacells(A375), whichnormally induceangio- genesis,producedfewervesselswhentreatedwithananti- ROBOantibody[31].Takentogether,SLIT–ROBOseemstobe involved in endothelial cell migration but the discordant results in studies conﬁrm the complexity of the process.

Therefore,studiesonthedownstreamsignalingoftheSLIT– ROBOwillbecrucialtounderstandthedifferences.

SLIT–ROBO in tumor formation

ThecellularfunctionsthatarecontrolledbytheSLIT–ROBO pathway during tissue morphogenesis are dysregulated during cancerdevelopment. Deletionsor epigeneticmodifi- cation ofthe slit–robo geneshasrecently been describedin many tumors.There is mountingevidence indicating that SLIT 1, 2, 3, ROBO1, and 2 are candidate genes for tumor suppressorgenes.Thehypermethylationofthesegeneswas describedinnumerous tumortypes includingcervical cancer,breast,non-smallcelllungandovarian[32–35].Whatis more, Singh et al. suggested poorer prognosis for patients withcervicalcancerpatientsinwhichthedeletionofSLIT2 locuswasconfirmed[32].AdecreasedexpressionofSLIT1,2, 3,and ROBO 1 wasreported inglioma, kidney,breast, and lung carcinoma [36]. In addition, another study revealed lowerexpressionofSLIT2,3,ROBO1,2and4inepithelialcell ovarian carcinoma [37]. Although the findings had showed no clear correlation with tumor clinical stage, subsequent studies on many cancer cell lines revealed that the reex- pressionofSLIT2greatly inhibitstheproliferationof cancer cells, which suggests the role of Slit in tumor progression [36]. The investigation of germinal cell ovarian carcinoma showed deletion of locus for SLIT3 [38]. In one study, conductedbyXianetal.,atargetedmutationof Robo1gene wasgeneratedinmice.ThemajorityofRobo1heterozygotes developed neoplasms, including carcinomas and lympho- mas [39]. Interestingly, however, these candidate genes showednocleartrendinothertumors,beingupregulatedin prostatecarcinoma[40]and inmanytumorcelllines, such as melanoma, bladder squamous carcinoma, neuroblas- toma, small cell lung carcinoma [31]. Expression of SLIT3 correlated with areas of increased microvessel density in tumors and SLIT2-ROBO1 induced proangiogenic pathways [31]. The activity was blocked by an antibody to the ectodomain ofROBO–ROBON.Theambiguousresultswere

obtained in some independent studies concerning SLIT– ROBO activity in metastasis, too. Prasadet al. providedan evidenceofanti-metastaticactivityofSLIT2inbreastcancer and melanomacells [41].They suggestedamodelinwhich SLIT2inhibitedtheSDF-1inducedchemotaxisofT-cells(for details, seeFig.2)[41]. Onthecontrary,findingsof Schmid etal.revealedSLIT2asachemoattractantfor breastcancer cells thatpromoted brainmetastases[42].Furthermore,the expression of SLIT2 correlated with the clinical stage of endometriumcarcinoma,beingsignificantlyhigherinrecur- rent phase [43]. On the contrary, in mice bearing breast carcinoma,theinjectionofexogenicSlit2reducedthetumor sizebyoverahalf[8]. SLIT–ROBO mayinduceapoptosis of cellsand thepathwayisalsoinvolvedincancerbiology.In SLIT2-transfectedfibrosarcomasand squamosouscell carcinoma there was a higher number of apoptotic cells and a lower rate of proliferation [44]. Additionally, SLIT–ROBO induced programmed cell death through caspase-3 activation in ovarian tumor [37]. Overall, these findings indicate that the SLIT–ROBO pathway mainly suppresses tumor formation and growth by regulating processes including invasion, migration, proliferation, and apoptosis. However, more experiments are needed to explain the differences betweenpro-andanti-cancerousactivityofSLIT–ROBO.

SLIT–ROBO in hematological malignancies

Although theSLIT–ROBOroleinsolidtumorshave recently beenexamined extensively,thestudiesdedicatedtohema- tological malignancies have started few years agoand are represented poorly. The background to research of ROBO4 expressioninleukemiasnoticedSmith-Berdanetal.in2011 [45]. They demonstrated the expression of the protein in hematopoietic stem cells (HSC) and described its role in hematopoietic nichereachingbyHSC. Leukemiaisbelieved to derive from leukemia stem cell (LSC) and there exists evidencethattheregulationofLSCissimilartotheHSC.Up todate,onlytwostudiesconcerningtherole of SLIT–ROBO in leukemias were conducted. The ﬁrst determined the methylation status of SLIT2 gene in 64 blood marrow samplesofpediatricacutelymphoblasticleukemia(ALL),30 blood samplesofadultchroniclymphocyticleukemia(CLL), bothof themobtained atthe timeof diagnosis,and inten leukemiacelllines.Thestudyalsorevealedthemethylation status of the genes after treatment with a demethylating agent.Allof leukemiacelllineshad completelymethylated SLIT2. Additionally,inpediatricALL, 83%of T-ALLand58%

of B-ALL had methylated SLIT2. However, the mutational status of SLIT2 had no impact on clinical data in ALL.

Interestingly, SLIT2 expression was restored after treating ALLlineswith5-aza-2dC(5-aza-2-deoxycytidine,decitabine).

80% of CLL samples demonstrated SLIT2. However, no correlation wasfoundbetween themethylation statusand IGVH orTP53mutationalstatus.Besidesthis,therewasno associationbetweenSLIT2methylationand CLLprogression [46]. In another study, Wellbrock et al. determined mRNA expression statusof ROBO1,4,andSLIT2genesinquantita- tivepolymerasechainreaction(PCR)analysisin104patients with newly diagnosed acute myeloid leukemia (AML). The

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study excluded t(15;17) samples. They found that ROBO4 mRNAwas expressedin83% patients.ROBO1was detected in 27% and SLIT2 in 18% of AML patients. The ﬁndings revealed a negative correlation of high ROBO4 expression with overall survival (OS) and event-free survival (EFS).

Whatismore,thecompleteremission(CR)ratewas54%for thehighROBO4expressiongroupcomparedwith71%inthe low ROBO4 expression group [47]. Interestingly, for ROBO1 and SLIT2 no signiﬁcant impacton survival was observed.

Toconclude,theseﬁndingsare promising forsearchingfor newprognostic factorsand therapeutic aimsof leukemias, particularlyinAML.Nevertheless,itrequiresmoreresearch toconﬁrmtheroleof SLIT–ROBOinleukemogenesisandto determine any association with patientsurvival and other pathologicalfeatures.

Summary

Despite the considerable body of knowledge of the role of SLIT–ROBO inaxonguidance, neuronalmigrationand axon branching, theinformation onthe effectsof SLIT–ROBOon normalandneoplasticcellsisstillfragmentary.Ithasbeen suggestedthatthepathwayseemstoregulateproliferation, apoptosis, adhesion and angiogenesis. Recent studies impliedits key role during organ development, tumorigenesis and physiology. Up to date, deletions or epigenetic modiﬁcationof the SLIT–ROBO geneshasbeen describedin many tumors which might suggest their role as a tumor suppressor gene. However, some contradictory statements aboutitsroleinmetastasisshowthattheSLIT–ROBOpath- wayisextremely importantbut highlycomplex.Therefore, new studies are needed to give more details about its activityanddetermineitsroleintumorbiology.

Authors' contributions/Wkład autorów

AG – study design, manuscript preparation, literature search.AW–manuscriptpreparation.

Conﬂict of interest/Konﬂikt interesu

Nonedeclared.

Financial support/Finansowanie

Nonedeclared.

Ethics/Etyka

Thework described inthis article has been carriedout in accordance with The Code of Ethics of the World Medical Association(Declaration of Helsinki)for experimentsinvol- ving humans; EU Directive 2010/63/EU for animal experiments;UniformRequirementsformanuscriptssubmittedto Biomedicaljournals.

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