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cancers
Article
DifferentialHIF2 ProteinExpressioninHumanCarotidBody
andAdrenalMedullaunderPhysiologicand
TumorigenicConditions
LucíaCelada1,2,TamaraCubiella1,2,JaimeSan-Juan-Guardado1,AndrésSanJoséMartínez1,
NuriaValdés1,3,PaulaJiménez-Fonseca1,4,IgnacioDíaz5,JoseMaríaEnguita5,AuroraAstudillo1,
EnolÁlvarez-González1,6,7,LuisaMaríaSierra1,6,7andMaría-DoloresChiara1,2,7,*
1InstituteofSanitaryResearchofthePrincipalityofAsturias,33011Oviedo,Spain;
******@(.);******@(.);******@(.-J.-G.);
andres.******@(.);nvaldes@(.);******@(.-F.);
******@(.);******@(E.Á.-G.);******@(.)
2CIBERONC(NetworkofBiomedicalResearchinCancer),28029Madrid,Spain
3DepartmentofInternalMedicine,SectionofEndocrinologyandNutrition,CabueñesUniversityHospital,
33394Gijón,Spain
4DepartmentofMedicalOncology,CentralUniversityHospitalofAsturias,33011Oviedo,Spain
5DepartmentofElectricalEngineering,UniversityofOviedo,33203Gijón,Spain;******@(.);
******@(.)
6DepartmentofFunctionalBiology,UniversityofOviedo,33006Oviedo,Spain
7InstituteofOncologyofthePrincipalityofAsturias,UniversityofOviedo,33006Oviedo,Spain
*Correspondence:mdchiara.******@;Tel.:+34-985-109-844
SimpleSummary:Thoraco-abdominalparagangliomaandpheochromocytoma(PPGL)arepathogenically
Citation:Celada,L.;Cubiella,T.;
-of-functionofSDHhasbeenassociatedwithstabilizationof
San-Juan-Guardado,J.;SanJosé
Martínez,A.;Valdés,N.;HIF2 ,whichotherwisewouldbedegradedviaoxygen-
Jiménez-Fonseca,P.;Díaz,I.;Enguita,alsopredisposetothedevelopmentofparagangliomasarisingatthecarotidbodyorotherheadand
.;Astudillo,A.;Álvarez-González,neckparaganglia(HNPGL).AlthoughPPGLandHNPGLsharesimilarfeatures,theyhavecertain
E.; expressioninHNPGLandPPGL,we
ExpressioninHumanCarotidBodyfoundthatfunctionalHIF2 isoverexpressedin80%ofPPGLs,includingthosewithSDHmutations
andAdrenalMedullaunderascomparedwithnon-,HIF2 isalreadyhighlyexpressedinthecarotid
PhysiologicandTumorigenicbodyunderphysiologicconditions,
,14, inhibitorsalreadyinclinicaltrialsmaybenefltawidespectrumofPPGL.
/
cancers14122986Abstract:Hypoxia-induciblefactors(HIF)2 and1 arethemajoroxygen-sensingmoleculesin
Received: hasbeenpathogenicallylinkedtoparagangliomaandpheochromocytoma
Accepted:15June2022(PPGL)arisinginsympatheticparagangliaortheadrenalmedulla(AM),,
Published:17June2022itsinvolvementinthepathogenesisofparagangliomaarisinginthecarotidbody(CB)orother
Publisher’sNote:MDPIstaysneutralparasympatheticgangliaintheheadandneck(HNPGL),weretrospec-
withregardtojurisdictionalclaimsintivelyanalyzedHIF2 byimmunohistochemistryin62PPGL/HNPGLandhumanCBandAM,
publishedmapsandinstitutionalaffll-andcomprehensivelyevaluatedtheHIF-relatedtranscriptomeof202publishedPPGL/
isbarelydetectedintheAM,butaccumulatesathighlevelsinPPGL,mostly(but
notexclusively)inthosewithloss-of-functionmutationsinVHLandgenesencodingcomponents
ofthesuccinatedehydrogenase(SDH)
andtheHIF2 -,HIF2 andHIF2 -regulated
Copyright:©,
LicenseeMDPI,Basel,Switzerland.
alsofoundthatHIF2 andHIF1
Thisarticleisanopenaccessarticle
distributedunderthetermsandaddition,HIF1 -targetgenesarealmostexclusivelyoverexpressedinVHL-mutatedHNPGL/PPGL.
conditionsoftheCreativeCommonsCollectively,thedatasuggestthatinvolvementofHIF2 inthephysiologyandtumorpathologyof
Attribution(CCBY)license(https://humanparagangliaisorgan-of-origin-dependentandHIF1 -independent.
/
/).
Cancers2022,14,:///cancers14122986:.
Cancers2022,14,29862of22
Keywords:paraganglioma;pheochromocytoma;carotidbody;adrenalmedulla;hypoxiainducible
factor;VonHippelLindau;succinatedehydrogenase
Biochemicalpathwaysinvolvedinthecellularresponsetohypoxiahavekeyroles
oncancerdevelopmentandmetastasis[1].Themajorhubwheretheoxygen-sensing
pathwaysconvergeisrepresentedbythehypoxiainduciblefactor,HIF,composedbyany
oftheHIF subunits(HIF1 ,HIF2 ,HIF3 )andHIF .HIF1 andHIF2 ,encodedby
HIF1AandEPAS1genes,respectively,arethemostwidelystudiedHIF
proteinsaredegradedintheproteasomeundernormoxicconditionsbyamechanism
requiringactiveprolylhydroxylase(PHD)enzymesandsubsequentHIF interaction
withtheVonHippelLindau(VHL)protein,acomponentoftheproteincomplexwith
ubiquitinligaseE3activity[2].HypoxiarepressesPHDactivitiesleadingtothestabilization
tumorcells’proliferationexpandsbeyondthecapacityofthetumortoincreasenewblood
vesselformation.
AlargenumberofreportshaveshownthathypoxiaandHIF1 accumulationinthe
growingtumorshaveanegativeimpactonpatientprognosisandresponsetotherapies
becauseofthetranscriptionalactivationofgenesinvolvedinangiogenesis,proliferation,
metabolismreprogramming,invasionandmetastasis[3].Morerecentdiscoverieshavealso
emphasizedthatHIF ,germline
geneticdefectsaffectingthehypoxiasignalingpathwayspredisposetothedevelopment
ofcertainneoplasia,mainlyclearcellrenalcellcarcinomas(ccRCC),paragangliomasand
pheochromocytomas,viamechanismslikelyinvolvingHIF factors[4,5].
Paragangliomasandpheochromocytomasareraretumorsarisingatneural-crest-
derivedtissuesofparasympatheticorsympatheticorigin,suchasthecarotidbody(CB)
andtheadrenalmedulla(AM),whicharetheprototypicaloxygenandstressorgansensors
paragangliaoftheheadandneckregion(hereafterreferredtoasHNPGL).Sympathetic
paragangliomadevelopintheparagangliaofthesympatheticnervoussystematthethorax
orabdomen;tumorsarisingattheAMareknownaspheochromocytoma,andtogether
withsympatheticthoraco-abdominalparaganglioma,
thephysiologicallevel,studiesinmicemodelshaveshownthatHIF2 hasprominent
functionsinthedevelopmentoftheCBanditsresponsivenesstoacutehypoxia[6–8].
Itisalsoessentialforthegrowth,differentiation,andfunctionofthesympathoadrenal
lineage[9–12].However,therolesofHIF1 andHIF2 inHNPGLandPPGLdevelopment
havebeenmorepoorlydeflned.
PPGLarehereditaryinabout40%ofcasesaffecting,amongothergenes,activators
oftheoxygen-sensingpathways,mainlyVHLandgenesencodingforcomponentsof
thesuccinatedehydrogenase(SDH)complex(SDHB,SDHD,SDHC,SDHAandSDHAF2
abbreviatedhereafterasSDH)[13].Inaddition,gain-of-functionmutationsinEPAS1have
beenrecentlyidentifled[14–18].TheactivationoftheHIF-signalingpathwayhasbeen
reportedtooccurinPPGLwithSDH,VHLorEPAS1mutations,aconditionthathasbeen
termedpseudohypoxia,giventhatitoccursinhighlyvascularized,non-hypoxictumors.
TheseflndingsinspiredthehypothesisthatHIF subunitshaveanoncogenicroleinPPGL
,therearestillmanyuncertaintiesthathamperunderstandingof
thephysiopathologicalsigniflcanceofpseudohypoxiainPPGLandHNPGL,aswellas
,
mostpreviouslyreportedstudieshavefocusedonPPGL,whereastheassociationofpseu-
dohypoxiaandSDH-
andHNPGLsharesimilarfeatures,theyhavecertainclinicalandgeneticpeculiarities.
Forinstance,metastasisandsecretorytumorsaremorefrequentlyfoundinpatientswith:.
Cancers2022,14,29863of22
PPGLthanHNPGL[19].Atthegeneticlevel,mutationsinVHLarefrequentlyassociated
withPPGL,buttheyareextremelyrareinHNPGL,whicharemorefrequentlyassociated
withSDHmutations[20].Moreover,gain-of-functionmutationsinEPAS1identifledin
,giventheheterogeneitiesas-
sociatedwithtumorgenotypeandlineageoforigin,itisstilluncertainwhetherthisis
reflectedinadifferentialexpressionofHIF
,manyofthecanonical
hypoxia-relatedgeneswerenotfoundtobeoverexpressedinHNPGL,suggestingthat
theHIF-relatedgeneticmodulehascertainspeciflcfeaturesinparasympathetictumors
versussympathetictumors[21].Furthermore,theassociationofSDHmutationswith
activationofHIF1 orHIF2 ,orofbothtranscriptionfactorstogether,hasbeenasubject
ofdebatewithconflictingpublisheddataincelllinesandtumortissues[22–24].Thefact
thatthesetwotranscriptionfactorshavespeciflcanduniquetargetsindifferentcelltypes
maypartiallyexplainthediscrepancies,-
fore,acomprehensiveknowledgeofwhichhypoxia/HIF-relatedgenes,amongtheentire
constellationidentifledsofar,aretheonesthatareoverexpressedinpseudohypoxicPPGL
andHNPGL,,unravelingwhetheritisHIF1 or
HIF2 thatisactivatedinpseudohypoxicHNPGLandPPGL,orwhetheritisbothproteins
together,couldalsoaddscientiflcknowledgerelevanttothetherapeuticmanagementof
,severalrecentlydevelopedpharmacologicalstrategiesspeciflcally
targetinganyofthetwoHIF subunitsareprovidingpromisingresultsinsyndromes
causedbyVHLorEPAS1mutations[25,26].
Inthisreport,weaimedtodeflneandcomparethepseudohypoxicproflleofPPGLand
HNPGLbyrelatingthemtotheirgenotypicorigin,cancer-associatedhypoxicphenotype
andHIF2
data,andthenusedthisresourcetoidentifytranscriptionalfeaturesofPPGLandHNPGL
tocorrelatephenotypicdiversitywithgenotypicheterogeneityandorgan-of-origin.
Surgicallyresectedspecimensofformalin-flxedandparaffln-embedded(FFPE)tis-
sueswereretrospectivelyobtainedfrom50patientswithPPGL/HNPGLwhoweredi-
agnosedandtreatedbetween2009and2020attheCentralUniversityHospitalofAs-
turias(Oviedo,Spain).Tumorsamplesincluded55primarytumorsand7metastases
(SupplementaryTableS1).
methodswerecarriedoutinaccordancewiththeapprovedguidelinesandtheprinciples
’
[27].
SequencingoftumorDNAwasperformedwithanin-house-developedtargetedsequenc-
ingpanel,includingallknownPPGL--tumoralcarotidbodies
hadbeenobtainedfromorgandonors,asdescribedpreviously[21].Non-tumoraladrenal
medullafromorgandonorswerenewlyobtainedforthecurrentstudy.
PC12cellsweregrowninDulbecco’smodifledEagle’smediumsupplementedwith
15%fetalbovineserum,100units/mLpenicillinand100g/
wereexposedto1mMwater-solublemono-methylhydrogensuccinate(Sigma-Aldrich,
,MO,USA)fordifferenttimes(1–48h).Whereindicated,cellswerepretreated
with1mMmono-,5MPT2385(Selleck
Chemicals,Munich,Germany)
AppendixAfordetailsontheSCC38celllinecultureconditions.
RNA-seqdatawereretrievedfromTheCancerGenomeAtlas(TCGA)dataportal
((accessedon15February2020)).ForHNPGL,we:.
Cancers2022,14,29864of22
usedpublishedmicroarraydat