CCR7 : Roles in cancer cell dissemination, migration and metastasis formation

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CCR7: Roles in cancer cell dissemination, migration and metastasis formation

Daniel F. Legler

^∗

, Edith Uetz-von Allmen, Mark A. Hauser

BiotechnologyInstituteThurgau(BITg)attheUniversityofKonstanz,Kreuzlingen,Switzerland

Keywords:

Chemokinereceptor Cancer

Metastasisformation Cellmigration Lymphnodehoming

a b s t r a c t

TheCC-chemokinereceptor7(CCR7)coordinatesthemigrationofcancercellsaswellasimmunecells towardslymphaticorganswhereitstwoligandsCCL19andCCL21areconstitutivelyexpressed.Here weprovideatopologicalmodelofCCR7,whichbelongstotheclassAofG-proteincoupled,seven- transmembranespanningreceptors,anddescribehowCCR7expressionisregulated.Wefocusonitsrole incancercellmigrationandmetastasisformationanddiscusshowcancercellscanutilizeCCR7orits ligandstoescapefromimmunesurveillance.

1. Introduction

Cellmigrationisanessentialprocessinthedevelopmentand maintenance of multicellular organisms and is defined by the orchestrated movement of cells in a particular direction to a specificlocation.Locallyproducedchemokinescanformgradients (Weberetal.,2013)andrepresentmajorguidancecuesthatcontrol directionalcellmigrationinbothhealthanddisease,e.g.during cancerdissemination and metastasisformation. Migrating cells sensethechemokinegradientviaspecializedchemokinereceptors, whichbelongtothefamilyofG-proteincoupledreceptors(GPCRs) (Venkatakrishnan et al., 2013). The CC-chemokine receptor 7 (CCR7)wasidentifiedin1993asthefirstlymphocytespecific,but orphanGPCRandnamedEpstein–Barrvirus-inducedgene1(EBI1) (Birkenbachetal.,1993).Thesamegenewassubsequentlyiden- tifiedinahomologyscreenforchemokinereceptorsinBurkitt’s lymphomatwoyearslaterandtermedBurkitt’slymphomarecep- tor2(BLR2)(Burgstahleretal.,1995).EBI1/BLR2wasrenamedto CCR7aftertheidentificationofitschemokineligandELC(Yoshida etal.,1997).AdditionalsynonymsareCC-CKR-7,CMKBR7,CD197 andCDw197.TwoligandsforCCR7,termedCCL19(ELC)andCCL21 (SLC),have beenidentified (Willimannet al.,1998).BothCCR7

∗Correspondingauthorat:BiotechnologyInstituteThurgau(BITg)attheUniver- sityofKonstanz,Unterseestrasse47,CH-8280Kreuzlingen,Switzerland.

Tel.:+41716785030;fax:+41716785021.

E-mailaddress:daniel.legler@bitg.ch(D.F.Legler).

ligandsarepredominantlyproducedconstitutivelybystromalcells withinprimaryandsecondarylymphoidorgansandaretherefore consideredtobehomeostaticchemokines.CCL21isadditionally expressed by lymphatic endothelial cells in peripheral tissues (Forsteretal.,2008).

2. Structure

The gene encoding CCR7is locatedon humanchromosome 17q12-21.2andis composedof threeexonsthat encodeapro- tein of 378 amino acids (Schweickart et al., 1994).The mouse homologisonchromosome11andtheproteinshows86%iden- titytohumanCCR7.Structurally,CCR7belongstothelargeclass AsubgroupofGPCRswhichalsoincludesrhodopsin,monoamine and␤-adrenergicreceptors(Venkatakrishnanetal.,2013).Based on the ␤1-adrenergic receptor crystal structure (Huang et al., 2013),wehavemodeledaputativestructureofCCR7(Fig.1).Sys- tematic analysisof recentlysolved crystal structuresof class A GPCRshaverevealedstructuralexplanationsforcharacteristicfea- turesofconservedsequencesofGPCRs(AudetandBouvier,2012;

Venkatakrishnanetal.,2013).Mostprominently,asequenceele- menttermedtheDRYmotif(DRYVAIVinCCR7,Fig.1,lightgreen box)attheendoftransmembranedomain(TM)3playsanessential roleincontrollingreceptoractivityandG-proteincoupling.Inaddi- tion,thepresenceofapolarinteractionbetweenthearginineofthe DRYmotifandaglutamateinTM6stabilizestheinactivestateofthe receptor,formingthesocalled“ioniclock”(Fig.1,darkgreenbox).

Disruptionofthe“ioniclock”ofthe␤2-adrenergicreceptorresults

Konstanzer Online-Publikations-System (KOPS) URL: http://nbn-resolving.de/urn:nbn:de:bsz:352-0-258852

Erschienen in: The International Journal of Biochemistry & Cell Biology ; 54 (2014). - S. 78-82 https://dx.doi.org/10.1016/j.biocel.2014.07.002

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Fig.1.CCR7:Topologicalorganizationandsignalingparadigm.ThreedimensionalillustrationoftheseventransmembranetopologicalconformationofCCR7.Themodel wascreatedusingPyMOL(www.pymol.org)andbasedonthe␤1-adrenergicreceptorcrystalstructure(accessionnumber4GPOinthePBDdatabase).Conservedstructural featuresofclassAGPCRsarehighlighted:DRYmotifinlightgreen,NPXXYmotifinred,ioniclockindarkgreen.Inaddition,thepositionoffourknownreceptoractivation residuesaremarkedinblue.(Forinterpretationofthereferencestocolorinthisﬁgurelegend,thereaderisreferredtothewebversionofthisarticle.)

inanoutwardmovementofTM6creatingacrevicefortheinterac- tionwiththeheterotrimericG-protein.Furthermore,thetyrosine residueinthewellconservedNPXXYmotifattheendofTM7(Fig.1, redbox)isrepositionedastheentiremotifmovesinwardinthe activestateofthereceptorasdemonstratedforopsin.Thisreposi- tioningpreventsthereversemovementofTM6andhencestabilizes theopenconformationofthereceptorthatformsthecradleforthe G-protein.Althoughchemokinereceptorsgenerallydonotsignalin theabsenceofligands,severalsinglepointmutations–primarily atthecytoplasmicendsofTM3andTM6–resultinginconstitu- tiveactivereceptorswithinvolvementincancerformationhave beenreported(Han,2014).Apartfromhighconservationamong certainsequencefeaturesandTMdomainsofGPCRs,substantial differencesarefoundintheextracellularloop(EL)domains,espe- ciallyinEL2,providingspeciﬁcityforligandsenteringthereceptor bindingpocket.Principally,chemokinesarethoughttobindtotheir receptorsbya“two-step/two-site”mechanism(Crumpetal.,1997).

First,theN-terminusofthereceptorbindstotheextendedloop ofthechemokine(site1)followedbythechemokineN-terminus bindingtoasecondsite (pocket)ofthereceptorcausingrecep- toractivation.For CCR7, fourreceptor activationresidueswere identiﬁedtobecrucialforCCL19/CCL21-mediatedreceptoracti- vation, but not for highafﬁnity ligand-binding(Fig. 1, in blue) (Ottetal.,2004).Suchstructure–function-relationsarekeyforthe rationaldesignofnoveltherapeuticdrugs.Suchanapproachhas successfullybeenpersuitedtodevelopCCR5antagonists(likeTAK- 779,maravirocandpiperidinecorecontainingleadcompounds)to interferewithCCL5-mediatedprostatecancerproliferationand/or invasion(Arnattetal.,2013).

3. Expression,activationandturnover

CCR7 is naturally a homeostatic chemokine receptor and expressedonvarioussubtypesof immune cellsthat migrateto andwithinlymphoidorgans(Comerfordetal.,2013;Forsteretal., 2008).CCR7-expressingimmune cells encompassnaïve,central memoryand regulatoryTcells, Bcells, NKs,subsets of thymo- cytes and (semi-)mature dendritic cells (DCs). Generally, Tcell subsets constitutivelyexpress CCR7 enabling them to circulate between thebloodstream, lymphatics and secondary lymphoid organs.Incontrast,DCsacquireCCR7expressionuponpathogen encountering,whichenableshomingtolymphnodesandantigen- presentationtocognateTcells.Ofnote,thisCCR7-mediatedhoming ofDCsiscrucialfortheinitiationofanadaptiveimmuneresponse.

Under certain pathological conditions, CCR7-dependentnaïveT cellmigrationtochronicallyinﬂamednon-lymphoidtissuescon- tributestothedevelopmentof autoimmunediseases, including rheumatoidarthritis(Comerfordetal.,2013).

An association of CCR7 with cancer was recognized in T cell leukemia patients where T cell leukemia cells from adult patientswithlymphoidorganinfiltrationexpressedsignificantly higherlevelsofCCR7thannormalTcellsorleukemiacellsfrom patientswithoutlymphoidorganinvolvement (Hasegawaetal., 2000).Subsequently,ahallmarkstudyidentifiedacrucialrolefor chemokinereceptors,includingCCR7,inbreastcancermetastasis formation (Muller et al., 2001). In fact, expression of a single chemokinereceptorgeneincancercells,asexemplifiedbyCCR7 onmelanoma,resultsinasignificantincreaseinmetastasisfor- mationindraininglymphnodes,suggestingthatcancercellscan

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adoptnormalmechanismsoflymphnodehoming(Wileyetal., 2001).ExpressionofCCR7ontumorcellshasmainlybeenreported forbreastcancer,melanoma,non-smallcelllungcancer,prostate cancer, head-and-neck cancer, chronic lymphocytic leukemia, non-Hodgkin’slymphoma,Tcellleukemia,stomachcancer, and colorectalcancer(Zlotniketal.,2011).

ThecorrelationbetweenaberrantCCR7expressionandmore aggressive,metastatictumorsassociatedwithdecreasedsurvival hasencouragedresearchonhowCCR7expressionandfunctionis regulatedbothundernormalconditionsandduringdisease.Onthe transcriptionallevel,anumberoftranscriptionfactorsandregula- toryelementsintheCCR7promoterregionhavebeendescribed.

SeveralbindingsitesforNF-␬Bhavebeenidentiﬁed inthepro- moterregionandconstitutiveNF-␬Bactivitywasassociatedwith theup-regulationofCCR7expressiononHodgkin’sdisease-derived cells(Hopkenetal.,2002;Mburuetal.,2012).Moreover,ithasbeen shownthatNF-␬BandAP1cooperativelyregulateCCR7expression inmetastaticsquamouscellcarcinomaoftheheadandneck(Mburu etal.,2012).

Inadditiontotranscriptionalregulation,CCR7expressionwas alsoshowntobemodulatedbylipidderivatives.Forinstance,over- expressionofCOX-2, anenzymethat convertsarachidonicacid (derivedfromphospholipids)toprostaglandins,aswellastreat- mentwithprostaglandin(PG)E₂ wasshowntoup-regulateCCR7 expressionin breast cancercells, thereby increasing theirlym- phaticinvasion(Panet al.,2008).COX-2/PGE₂-dependentCCR7 expressioninthesecellswas,atleastinpart,mediatedbyAKT- inducedactivationofthetranscriptionfactorSp1(Chuangetal., 2013). Notably, PGE₂ also signiﬁcantly enhances DC migration towardsCCR7ligands(Legleretal.,2006)(seealsoSection5).

Onceexpressed,CCR7seemstoberatherlong-lived(Oteroetal., 2006)andinformationonhowCCR7expressionisdown-regulated issparse.However,Runx3 wasfoundtoberequiredfor TGF␤- mediatedtranscriptionalinhibitionofCCR7inDCs(Fainaruetal., 2005).Moreover,tumor-derivedoxysterolwasshowntointerfere withCCR7expressiononDCs(Villablancaetal.,2010)(seealso Section5).

Meanwhile,othermechanismshavebeenidentiﬁedthatrender cellsnon-responsivetoCCR7ligands.Oneofthesemechanisms involvesscavengingofchemokinesbyso-calleddecoyreceptors.

CCR7ligandscanbescavengedbyACKR4(CCX-CKR)andACKR5 (CRAM-B)(Comerfordetal.,2013).Knock-outanimalsforeither oneofthetwodecoyreceptorsrevealaroleinadaptiveimmunity and are prone todevelop EAE more rapidly.A further mecha- nismisreceptordesensitization.Interestingly,CCR7stimulationby CCL19,butmuchlessbyCCL21,leadstorobustreceptordesensiti- zation,whichismediatedbyCCR7phosphorylationand␤-arrestin bindingresultinginareducedchemotacticcellresponse(Kohout etal.,2004).Finally,analternativemechanismofrenderingcells non-respondingtochemokinesisthroughligand-mediatedrecep- torendocytosis.CCR7wasshowntoberapidlyendocytosedvia clathrin-coatedpitspredominantlyuponbindingofCCL19(Otero etal.,2006).Ofnote,inendosomes,CCR7dissociatesfromCCL19 and recyclesvia theTGNbacktothe plasmamembrane tore- participateinchemotaxis,whereasCCL19issortedforlysosomal degradation(Oteroetal.,2006;Schaeubleetal.,2012).

4. Biologicalfunctions

TheCCR7-CCL19/CCL21axisiswellcharacterizedforitscrucial roleintheformationofsecondarylymphoidstructuresunderphys- iologicalconditionsmainlythroughorchestratingtherecruitment ofimmunecellstothesestructures(Comerfordetal.,2013;Forster etal.,2008).MicelackingeitherCCR7(bygenetargeting)orits ligands(intheplt/pltmice,aspontaneousmutantstrainlacking

CCL19andthelymphoidformofCCL21)showanalteredarchitec- tureoflymphoidorgansduetoimpairedhomingoflymphocytes andDCs.Consequently,thesemicehavedeﬁcitsinmultipleaspects ofdevelopmentandexecutionofanadaptiveimmunity.Moreover, CCR7-deﬁcientmicearepronetodevelopgeneralizedmulti-organ autoimmunityalthoughtheyhaveanormallifespananddonotsuf- ferfromclinicallyapparentautoimmunedisease(Comerfordetal., 2013;Forsteretal.,2008).

Incancer,chemokinesingeneralcanfulﬁllseveralfunctions.

Inparticular,chemokinesarepartofthenetworkofinﬂammatory mediators.Theyarealsoimportantforrecruitingtumor-inﬁltrating immunecellsandcanactasangiogenicfactors(Balkwill,2012).

Thechemokine receptor CCR7,however,fulfills adistinct function.TheCCR7-CCL19/CCL21axisisprimarilyresponsibleforlymph nodemetastasisformationbyrecruitingtumorcellstotheTcell zoneoflymphnodes(Rehmetal.,2011;Zlotniketal.,2011).The chemokineCCL21isimmobilizedonlymphaticendothelialcellsvia itsheparin-bindingdomain(Fig.2)andtherebyformsagradient thatgraduallydecreasesinthedirectionoftheinterstitium(Weber etal.,2013).Cancercellsofseveraltumorsup-regulateCCR7and disseminatefromtheprimarytumorpresumablybysensingthe immobilizedCCL21gradientandactivelymigratetowardsthenext lymphaticvessel(Fig.2).Moreover,metastaticcancercellswere showntoproduceCCR7ligandsthat,underinterstitialflow,create anautologous,transcellularchemokinegradientpermittingcan- cercellstomigratetowardsdraining lymphatics(Shieldsetal., 2007).Remarkably, CCL21 released from lymphaticendothelial cellsalsopromotesadirectedgrowthofmelanomacellstowards thelymphatics(Emmettetal.,2011).Interestingly,underinflam- matoryconditions,CCL19andCCL21werefoundtobeup-regulated in endothelialcells of theblood–brainbarrier (Altetal., 2002) enablingacuteTcellleukemiacells toinfiltrateintothecentral nervoussystemina CCR7-dependentmanner(Buonamicietal., 2009).Furthermore,CCR7alsocontributestotumorcellsurvival byactivatingthePI3K/Aktsignalingpathway(Wangetal.,2005).

5. CCR7atthecross-roadbetweencancerandimmunecells

Sinceimmunecellscanrecognizeandkilltumorcellsdepending ontheirantigenproﬁle,tumorshavedevelopedwaysofescaping theimmunesurveillance,whichinvolvestheCCR7-CCL19/CCL21 axis.Forinstance,melanomatumorshavebeenproposedtocre- ate an immunotolerant microenvironment by releasing CCL21 andtherebyinducingalymphoid-likereticularstromalnetwork (Shieldsetal.,2010).Thistumor-associatedmicroenvironmentis rich inTGF␤ and inﬁltratedby regulatoryTcells and myeloid- derivedsuppressorcells.Othertumorcells havebeenshownto produceoxysterolsthatactivateliverXreceptor␣(LXR␣)onDCs (Villablancaetal.,2010).LXR␣activationonDCscausesthedown- regulationofCCR7andconsequentlyimpairsDChoming,thereby preventingtheinductionofananti-tumorimmuneresponse.How- ever,tumorcellsalsoproducePGE₂(Fig.2).Inthisscenario,PGE₂ isrequiredfortheformationofnewbloodvesselssupplyingsolid tumorswithnutrition,aswellasforthesecretionofmatrixmetallo- proteinasesMMP2andMMP9crucialfortumorcellinvasion(Wang andDubois,2010).Notably,PGE2notonlyinducesCCR7expression ontumorcells(Panetal.,2008),butalsoonDCs(Legleretal.,2006).

Moreover,PGE₂counteractsoxysterol-mediateddown-regulation ofCCR7onDCs,restoring–atleastinpart–theabilityofDCsto migrateandhometolymphnodes(Bruckneretal.,2012).

In summary, the CCR7-CCL19/CCL21 axis is simultaneously involvedincancercelldisseminationandmetastasisformationas wellasinadaptiveimmunecellhomingtolymphoidorgans.Its ambiguousroleincellhomingtolymphoidorgansseemstomake it difﬁculttopharmaceutically target CCR7in cancertherapies.

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Fig.2.CCR7:Centralrolesintumorcelldissemination,migrationandmetastasisformation.Cancermetastasisisaprocesshighlydependentontheinteractionsbetween tumorandhoststromalcells.CCL21promotesmigrationandinvasionofcancercellsviaCCR7.Cyclooxygenase2(COX2)expressedintumorcellsgeneratesprostaglandin E2(PGE2)thatampliﬁestumorcellproliferationandextracellularmatrix(ECM)degradationbymetalloproteinase2(MMP2)andMMP9.InresponsetoCCL21tumorcells polarize,resultinginaleadingedgethatprotrudesoutward,coupledwithcontractileforcesatthebackandsidesofthecellthatleadstomovementtowardstheCCL21 source.CancercellsmigratealongtheCCL21gradientuntiltheyreachthesiteforsecondarycolonization,e.g.theTcellzoneoflymphnodes.

However,accumulatingevidenceisemergingthatboththetumor and the immune system simultaneously evolve strategies to interfereeachotherbymeansoftheCCR7-CCL19/CCL21axis.Thus, co-evolutionmight beinherited to develop novel strategies to preventtumorcellmigrationwhilstleavingimmunecellmigration unaffected.FurtherstudiesonCCR7,particularlyonitsregulation ofexpressionandondistinctsignalingpathways,arethusrequired forabetterunderstandingofthisfundamentalprocessofhoming.

Acknowledgments

WethankSarahVanesforcommentsonthemanuscript.This workwassupportedbyresearchfundingfromtheSwissNational Science Foundation (SNF 31003A-143841/1), the Thurguische Krebsliga,theNovartisFoundationformedial-biologicalresearch, theThurgauischeStiftungfürWissenschaftundForschung,andthe SwissStateSecretariatforEducationandResearchtoD.F.L.M.A.H.

holdsascholarshipfromtheResearchTrainingGroup1331founded bytheGermanResearchFoundation.

References

Alt C, Laschinger M, Engelhardt B. Functional expression of the lymphoid chemokinesCCL19(ELC)andCCL21(SLC)attheblood–brainbarriersuggests theirinvolvementinG-protein-dependentlymphocyterecruitmentintothe centralnervoussystemduringexperimentalautoimmuneencephalomyelitis.

EurJImmunol2002;32:2133–44.

ArnattCK,ZaidiSA,ZhangZ,LiG,RichardsonAC,WareJL,etal.Design,syntheses,and characterizationofpharmacophorebasedchemokinereceptorCCR5antagonists asantiprostatecanceragents.EurJMedChem2013;69:647–58.

AudetM,BouvierM.RestructuringG-protein-coupledreceptoractivation.Cell 2012;151:14–23.

BalkwillFR.Thechemokinesystemandcancer.JPathol2012;226:148–57.

BirkenbachM,JosefsenK,YalamanchiliR,LenoirG,KieffE.Epstein-Barrvirus- inducedgenes:ﬁrstlymphocyte-speciﬁcGprotein-coupledpeptidereceptors.

JVirol1993;67:2209–20.

BrucknerM,DickelD,SingerE,LeglerDF.ConverseregulationofCCR7-drivenhuman dendriticcellmigrationbyprostaglandinE(2)andliverXreceptoractivation.

EurJImmunol2012;42:2949–58.

BuonamiciS,TrimarchiT,RuoccoMG,ReavieL,CathelinS,MarBG,etal.CCR7 signallingasanessentialregulatorofCNSinﬁltrationinT-cellleukaemia.Nature 2009;459:1000–4.

BurgstahlerR,KempkesB,SteubeK,LippM.Expressionofthechemokinereceptor BLR2/EBI1isspeciﬁcallytransactivatedbyEpstein–Barrvirusnuclearantigen2.

BiochemBiophysResCommun1995;215:737–43.

ChuangCW,PanMR,HouMF,HungWC.Cyclooxygenase-2up-regulatesCCR7 expressionviaAKT-mediatedphosphorylationandactivationofSp1inbreast cancercells.JCellPhysiol2013;228:341–8.

ComerfordI,Harata-LeeY,BuntingMD,GregorC,KaraEE,McCollSR.Amyriadof functionsandcomplexregulationoftheCCR7/CCL19/CCL21chemokineaxisin theadaptiveimmunesystem.CytokineGrowthFactorRev2013;24:269–83.

CrumpMP,GongJH,LoetscherP,RajarathnamK,AmaraA,Arenzana-SeisdedosF, etal.Solutionstructureandbasisforfunctionalactivityofstromalcell-derived factor-1;dissociationofCXCR4activationfrombindingandinhibitionofHIV-1.

EMBOJ1997;16:6996–7007.

EmmettMS,LanatiS,DunnDB,StoneOA,BatesDO.CCR7mediatesdirectedgrowth ofmelanomastowardslymphatics.Microcirculation2011;18:172–82.

FainaruO, ShseyovD,HantisteanuS,GronerY.Acceleratedchemokinerecep- tor 7-mediated dendriticcellmigration inRunx3 knockoutmice andthe spontaneousdevelopmentofasthma-likedisease.ProcNatlAcadSciUSA 2005;102:10598–603.

ForsterR,Davalos-MisslitzAC,RotA.CCR7anditsligands:balancingimmunityand tolerance.NatRevImmunol2008;8:362–71.

Han X. Constitutively active chemokine CXC receptors. Adv Pharmacol 2014;70:265–301.

HasegawaH,NomuraT,KohnoM,TateishiN,SuzukiY,MaedaN,etal.Increased chemokinereceptorCCR7/EBI1expressionenhancestheinﬁltrationoflymphoid organsbyadultT-cellleukemiacells.Blood2000;95:30–8.

HopkenUE,FossHD,MeyerD,HinzM,LederK,SteinH,etal.Up-regulationof thechemokinereceptorCCR7inclassicalbutnotinlymphocyte-predominant Hodgkindiseasecorrelateswithdistinctdisseminationofneoplasticcellsin lymphoidorgans.Blood2002;99:1109–16.

HuangJ,ChenS,ZhangJJ,HuangXY.Crystalstructureofoligomericbeta1-adrenergic Gprotein-coupledreceptorsinligand-freebasalstate.NatStructMolBiol 2013;20:419–25.

KohoutTA, NicholasSL,Perry SJ,Reinhart G,JungerS,StruthersRS.Differen- tialdesensitization,receptorphosphorylation,beta-arrestinrecruitment,and ERK1/2activationbythetwoendogenousligandsfortheCCchemokinereceptor 7.JBiolChem2004;279:23214–22.

(5)

82

LeglerDF,KrauseP,ScandellaE,SingerE,GroettrupM.ProstaglandinE2isgenerally requiredforhumandendriticcellmigrationandexertsitseffectviaEP2andEP4 receptors.JImmunol2006;176:966–73.

MburuYK,EgloffAM,WalkerWH,WangL,SeethalaRR,vanWaesC,etal.Chemokine receptor7(CCR7)geneexpressionisregulatedbyNF-kappaBandactivatorpro- tein1(AP1)inmetastaticsquamouscellcarcinomaofheadandneck(SCCHN).

JBiolChem2012;287:3581–90.

MullerA,HomeyB,SotoH,GeN,CatronD,BuchananME,etal.Involvementof chemokinereceptorsinbreastcancermetastasis.Nature2001;410:50–6.

OteroC,GroettrupM,LeglerDF.OppositefateofendocytosedCCR7anditsligands:

recyclingversusdegradation.JImmunol2006;177:2314–23.

OttTR, Pahuja A, Nickolls SA, Alleva DG, Struthers RS. Identiﬁcation of CC chemokinereceptor7residuesimportantforreceptoractivation.JBiolChem 2004;279:42383–92.

PanMR,HouMF,ChangHC,HungWC.Cyclooxygenase-2up-regulatesCCR7via EP2/EP4receptorsignalingpathwaystoenhancelymphaticinvasionofbreast cancercells.JBiolChem2008;283:11155–63.

RehmA,MensenA,SchradiK,GerlachK,WittstockS,WinterS,etal.Cooperative functionofCCR7andlymphotoxinintheformationofalymphoma-permissive nichewithinmurinesecondarylymphoidorgans.Blood2011;118:1020–33.

SchaeubleK,HauserMA,RipplAV,BrudererR,OteroC,GroettrupM,etal.Ubiquity- lationofthechemokinereceptorCCR7enablesefﬁcientreceptorrecyclingand cellmigration.JCellSci2012;125:4463–74.

SchweickartVL,RaportCJ,GodiskaR,ByersMG,EddyRLJr,ShowsTB,etal.Cloning ofhumanandmouseEBI1,alymphoid-speciﬁcG-protein-coupledreceptor encodedonhumanchromosome17q12-q21.2.Genomics1994;23:643–50.

ShieldsJD,FleuryME,YongC,TomeiAA,RandolphGJ,SwartzMA.Autologous chemotaxisasamechanismoftumorcellhomingtolymphaticsviainterstitial ﬂowandautocrineCCR7signaling.CancerCell2007;11:526–38.

ShieldsJD,KourtisIC,TomeiAA,RobertsJM,SwartzMA.Inductionoflymphoid- likestromaandimmuneescapebytumorsthatexpressthechemokineCCL21.

Science2010;328:749–52.

VenkatakrishnanAJ,DeupiX,LebonG,TateCG,SchertlerGF,BabuMM.Molecular signaturesofG-protein-coupledreceptors.Nature2013;494:185–94.

VillablancaEJ,RaccostaL,ZhouD,FontanaR,MaggioniD,NegroA,etal.Tumor- mediatedliverXreceptor-alphaactivationinhibitsCCchemokinereceptor-7 expressionondendriticcells anddampensantitumorresponses.Nat Med 2010;16:98–105.

WangD,DuboisRN.Eicosanoidsandcancer.NatRevCancer2010;10:181–93.

WangJ,ZhangX,ThomasSM,GrandisJR,WellsA,ChenZG,etal.Chemokine receptor7activatesphosphoinositide-3kinase-mediatedinvasiveandprosur- vivalpathwaysinheadandneckcancercellsindependentofEGFR.Oncogene 2005;24:5897–904.

WeberM,HauschildR,SchwarzJ,MoussionC,deVriesI,LeglerDF,etal.Inter- stitialdendriticcellguidance byhaptotacticchemokinegradients. Science 2013;339:328–32.

WileyHE,GonzalezEB,MakiW,WuMT,HwangST.ExpressionofCCchemokine receptor-7andregionallymphnodemetastasisofB16murinemelanoma.JNatl CancerInst2001;93:1638–43.

WillimannK,LeglerDF,LoetscherM,RoosRS,DelgadoMB,Clark-LewisI,etal.The chemokineSLCisexpressedinTcellareasoflymphnodesandmucosallym- phoidtissuesandattractsactivatedTcellsviaCCR7.EurJImmunol1998;28:

2025–34.

YoshidaR,ImaiT,HieshimaK,KusudaJ,BabaM,KitauraM,etal.Molecularcloningof anovelhumanCCchemokineEBI1-ligandchemokinethatisaspeciﬁcfunctional ligandforEBI1,CCR7.JBiolChem1997;272:13803–9.

ZlotnikA,BurkhardtAM,HomeyB.Homeostaticchemokinereceptorsandorgan- speciﬁcmetastasis.NatRevImmunol2011;11:597–606.