Investigating Eph/Ephrinsignaling duringMelanomaMetastasisin vivo
ABSTRACT
Currently,the lifetimeriskfordevelopingcanceris justover40%and itispredictedthat nearly600,000 Americanswill die ofcancerthisyear.Importantly,itisestimatedthat90%ofall cancer-relateddeathscan be ascribedtometastasis. Metastasishighlightsa complex,dynamic relationship between thetumorand itsmicroenvironment.Microenvironmentalsignalsthat regulateorinhibitmetastatic behaviorshave beenslowto discoverduetochallenges associatedwith studyingthemetastaticprocessinvivo.
Ourlaboratoryhasdeveloped an in vivochickembryo modelto studycomplexmolecular and behavioral traitsassociated with metastasis. Ipresentpreliminarydata suggesting that guidancereceptors,namelyEph receptortyrosinekinases,maybe involved in themetastatic step ofintravasation andthattheirexpressionpatternsmaydefineametastaticsignature.The affirmationofourhypothesiscarries both prognosticandtherapeutic implications.
SPECIFIC GOALSANDSIGNIFICANCE
Mylong-termresearchgoalistoidentifysignatures ofmetastaticprogressionthat predictprecisecell behaviorsfrom geneexpression profiles. Asan NIH RuthKirschsteinPostdoctoralFellow(2010-2013),Ideveloped thechickembryo asamulti-facetedmodel system,usingin vivodynamicimagingand novelgeneprofilingtechniques,to visualize metastaticmelanomabehaviorsand definemolecularsignatures ofsmallnumbersof transplantedmelanomacellsintothechickembryonicneural crestmicroenvironment.
Thefocusofmystudy isto investigatetherolesofEphreceptortyrosinekinase
receptors(and ligands)insensingandresponding tomicroenvironmentalcuesthat influence metastasis.The ratification ofmyhypothesiscarriessignificantprognosticandtherapeutic implications. First,expression patternsofthese neuralcrestguidance receptorsmayuniquely identifycellswith metastatic potential,providingnovelprognostictoolstoclinicians. Second, themolecularmechanismsassociatedwith guidance receptorfunction mayelucidate specific processesofthemetastatic cascade amenable to therapeutictargeting.
BACKGROUNDAND PRELIMINARY DATA
Melanomarepresentsone of themostaggressive solid tumors and occursfollowingtheneoplastic transformationofmelanocytes. Melanocytes derive froma highly invasive and multi-potentembryonic cell population called theneural crest.Assuch,we tested the hypothesisthatmelanoma (acancer ofmelanocytes)isintrinsically predisposedto aggressive,
metastaticbehaviorsresulting from itsancestralrelationshiptothe neural crest(Figure1).Totest
Figure1.Thisgraphical abstractrepresentsourhypothesisthat
melanomametastasis ispromoted by the aberrant expression of neural-crest-related genes.
this,we transplantedsmall clustersofC8161 humanmetastaticmelanomacellsintothe chick embryonicneuralcrestmicroenvironment.Weobserved thatmelanomacellsrespondedtohost embryonicneuralcrestcuesbymigrating alongcanonicalneural crestmigratoryroutesand populatingneuralcresttargettissues (Kulesaetal.,2006). Sincethattime,we have been working tounderstandthe powerful effectsoftheembryonicmicroenvironmentin dictating melanomacell migratorybehaviorsandgeneexpression patterns.
As these initialfindings supportedourhypothesisthat melanomacellsexploitan intrinsic,neuralcrest-related migrationprogramtodrive metastasis,we nextanalyzed gene expression patternsofanumberofneuralcrest-relatedgenes in C8161 cellsfollowingtransplantation intothe neuralcrest microenvironment (citation).Weobserved that C8161 cellsresponded to theneural crestmicroenvironment byup-regulatingseveralclassesofcell surfacereceptors responsibleforsensing and responding tomicroenvironmental cues.Thesegenefamilies included EphsandEphrins, Plexins and Semaphorins,Neuropilins,SlitsandRobos. In contrast,primarymelanocytes did notup-regulateanyofthese genes(citation). All ofthesegenefamilies are involved in cell guidanceduring embryonicdevelopment,but theirroles in adult tissuesorcancerpathogenesishave been less characterized.
My workhas identifiedalteredexpression patternsfor Eph/Ephrinfamilymembersin C8161 cellscomparedto
primarymelanocytes (citation).Among these,I showed thatEphB6gene expression wassilenced in C8161
cellsbutexpressedbyprimarymelanocytes. Sinceloss of EphB6 is associatedwith ahigherrateofmetastasisand poor clinicaloutcomeinseveraldifferent cancertypes,I
investigatedwhethertheloss ofEphB6affectsmelanomacell invasion and metastasis. First,Ire-expressedEphB6 in
Figure 2.Re-expression of EphB6 in C8161melanoma cells results in aberrant directional migrationwithout affecting migratoryability. EphB6- expressingmelanomacells deviatedfromthetypical neural crestmigratoryroute,suggesting an altered cell-microenvironment interaction.
C8161 cellsandtransplantedthesecellsinto thechickembryoneural crestmicroenvironment (hindbrain at rhombomere 4(r4),10-somitestage).WhereasparentalC8161 cellstypically
migratealong the canonicalneural crestmigratorystreamlateraltor4, EphB6+cellswere
observed streamingenmassetowardthearea adjacenttor5, an areatypically inhibitoryto neuralcrestmigration (Figure
2A-D). Interestingly,total migratory lengthwas unaffected (Figure2E-F).
Followingtheseobservations,we nexttested whether EphB6 re-expression affectedmetastasis. Using a CAM metastasisassaythatspecificallyassessestherate of intravasation,Iobserved a significantreduction inmetastatic potentialin EphB6+ C8161 cells(Figure 3).
Lossofcell directionalityand reducedmetastatic potentialarguefora distinctroleforEphB6during metastasis.Ipostulatethat re-expression ofEphB6forces thetumorcelltorecognize inhibitorysignalspresentwithin themicroenvironment. Myproposed studywilltestthe
Figure 3.TheCAMmetastasis assaydemonstrates thatre- expression of EphB6 inC8161 melanoma cellscausesa significant loss ofmetastatic potential. Usingthisassay, tumors canbe grown on the vascularCAM tissue, visualized for celldynamics, and evaluated formetastatic potentialusing 2-photonmicroscopy andhighlysensitive qPCR.
followinghypothesis: metastaticcells are guidedand drivenby dysregulatedEph/Ephrin signaling thatallowstumorcells
to overcome inherentmicroenvironmentalobstacles.
The success ofmyfellowshipcarries both prognosticandtherapeuticimplications.First,expression patterns ofguidancereceptorsimplicated in themetastatic
processmayuniquely identifycellswith metastaticpotential,
providingnovel diagnosticandprognostictoolstoclinicians. Second,themolecularmechanismsassociatedwith
guidancereceptorfunction mayelucidatespecificprocesses ofthemetastaticcascade amenable to therapeutictargeting.
In additiontothepossible clinicalimplications,the proposedworkwill also benefitthe
scientificcommunitybydevelopingandmodernizing techniques,includingthe chorioallantoic membrane(CAM)metastasisassay,to improve the visualization and investigation ofcomplex tumorcell behaviorsinvivo.
RESEARCHDESIGN
Specific Aim1– Develop thechorioallantoicmembrane(CAM)assayfor2-photonmulti- spectralmicroscopyto visualizeearlysteps ofthemetastatic cascade. Currentanimal modelsofmetastasiscan betime-consuming,expensive,andarenottypicallyamenable to visualizationwith singlecell resolution.TheembryonicchickCAMprovides ahighlyvascularin vivo substratesimilartoa humanmicroenvironmentandaccessibleto observation ofthe complextumorcell behaviorsassociated with themetastaticcascade. Developmentofthis assaywould overcome thetechnicalroadblocktovisualize the metastaticstepsofdelamination, invasion throughtheextracellularmatrix,and intravasation (transendothelialmigration)intothe hostvasculatureofsingle metastaticmelanomacells.Thus,mygoalforthis aimis toenhance a 2-photon non-linearoptics-basedplatformto visualizecomplextumorcellbehaviorson theCAM(in vivo)with unprecedentedresolution.Thein vivodynamicimaging platformisbuilt around aZeiss780microscope equipped witha Coherent2-photon laser,an environmental chamber,andhigh sensitivitydetectors thatallowfordeeptissue,multi-spectralimaging (Fig. 4).Ihave been intimately involved in developingand using thisplatformformymelanoma metastasisstudies,and togetherwith myPI(x),have recentlypublished aprotocol and reviewsoflivein ovoimaging thatdemonstratethe innovation and
noveltyofthisapproach [citation]
The successfulcompletionofAim1willenhance our state-of-the-art dynamicinvivoimagingplatform toinclude
visualizationofcomplex3D metastatictumorcell dynamics inthe
highlyvascularized CAM.
Specific Aim2 – DeterminethespecificEph/Ephrininteraction(s) induced byEphB6 thatregulatetheintravasation process. Followingengagementwith an Ephrin ligand,Ephreceptoractivation typically inducesrepulsion awayfromthe Ephrin-expressingcell.I postulatethatthere-expression of EphB6 inducesarepulsive interactionbetweenthe tumorcell andthe chickendothelium comprisingthe CAMvasculature,effectivelyblocking themetastatic step ofintravasation. Mygoalsforthis aimaretwofold:a)todetermine thespecificEph/Ephrin interaction(s)induced byEphB6thatregulatetheintravasation processandb)evaluate whetherEph/Ephrin expression patterns defineasignature ofmetastasis. Pursuanttothecompletion ofthisaim,Iwill be able to addressthe following questions:
- WhichEphsandEphrinsexpressed bythechickendothelial cellspromotetherepulsive response?
- DoesEphB6,which iskinase-null,drive the anti-metastatic response inconjunction with orindependentofotherkinase-
activeEph receptors?
- Can Eph/Ephringeneexpression patternspredictcellswith the
Figure 4.Theimaging platform currentlyused for dynamicin ovoimaging. 2-Photon excitationprovidesdeep tissue,multi-spectral capabilitieswith high resolution optics.
abilityto intravasate(andtherefore predictmetastasis)?
Toanswerthesequestions,Iwill use acombination ofimaging,molecular,andbiochemical techniques. First, Iwill employan RT-qPCR approach toprofiletheexpression patternsofEphs
andEphrins onthechickCAMendothelium. Iwillthen comparetheEph/Ephrin expression profileofchickCAMendothelialcellstothatofthe C8161 cells(previouslypublished,citation)and identifyputative receptor-ligand interactionsthatmaybe involved in repellingthe tumorcellsawayfrom the endothelialcells.
Next,I will utilize the imagingplatformdescribedunderSpecificAim1tospecifically
visualizethe interactionbetween tumorcellsandthe chickCAMendothelium. To identify possible protein interactionsand dimerizations between EphB6 and otherEphsand/orEphrins,I will employco-immuno-precipitation,Westernblotting,andimmunofluorescensemicroscopy. Putative protein-protein interactionswill be verified byForsterResonanceEnergyTransfer (FRET)microscopy.Westernblotting with phospho-specificantibodies will allowme to evaluate Ephkinase activation andfunction.
Followingthe identification ofcandidatereceptor-ligand interactionsaltered bythere- expression ofEphB6,Iwill use knock-in andknock-down strategiesto confirm gene
involvement. Using myimaging platform,Iwill beable tovisualize,and thus confirm,specific
Eph/Ephrinfunction inregulating themetastaticprocessofintravasation.Thisprocesswill eventuallyallowme to identifya signature ofmetastasisthatcanthenbeused to interrogate
othermelanomas and,inthefuture,othersolid tumors.
Thesestudieswill bethefirstto investigatetherole ofEphB6 and Eph/Ephrin signaling in regulating themetastaticprocess ofintravasation. Ipostulatethat EphB6formsa heteromericcomplexwith one ormoreEphreceptorsand eitheractivatesoraltersitsspecific attractive/repulsive response. Iexpectthatthe successfulcompletion ofAim2will demonstrate avitalrole forEphB6in regulating intravasationoftumorcellsintoblood or lymph vessels. Thismaybe directlyattributableto attractive and/orrepulsive cues mediated bycell-cellEph/Ephrininteractionsand mayyieldnoveltherapeuticstrategies focused ondisruptingthe metastaticprocess
SUMMARY
In summary,Ipostulatethatmetastasisisfacilitated bydysregulatedcell surface receptorsresponsibleforsensingandresponding tothemicroenvironment. Specifically,loss of EphB6 allowsa permissive interaction between the tumorcell andthe endothelium, allowing intravasation,while the re-expression ofEphB6 restoresrepulsion andblocks intravasation (Figure5 –model).IpredictthatspecificEph/Ephrin expression patternsmaydefinea metastaticsignatureformelanoma.Itisalso possible thatthesemechanismsareconserved and thatsimilarsignatures maybepresentinothermetastatictumortypes.
Figure 5.A model depicting the developmentanduseof the CAMassayto visualizeand investigatecomplex tumorcell behaviorsin vivo. The modeldepictsmyhypothesisthat EphB6re-expression on metastaticmelanoma cellsblocksintravasationbyinducing arepulsive Eph/Ephrinsignalingresponse.