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EGFR and NF-kB: partners in cancer

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EGFR

and

NF-kB:

partners

in

cancer

Kateryna

Shostak

1,2,3

and

Alain

Chariot

1,2,3,4

1

InterdisciplinaryClusterforAppliedGenoproteomics(GIGA),CentreHospitalierUniversitaire(CHU),Sart-Tilman,Liege,Belgium

2LaboratoryofMedicalChemistry,UniversityofLiege,CHU,Sart-Tilman,Liege,Belgium

3

GIGA–SignalTransduction,UniversityofLiege,CHU,Sart-Tilman,Liege,Belgium

4

WalloonExcellenceinLifeSciencesandBiotechnology(WELBIO),Wavre,Belgium

Oncogenicproteinscooperatetopromotetumor devel-opmentandprogressionbysustainingcellproliferation, survival and invasiveness. Constitutive epidermal growth factor receptor (EGFR) and nuclear factor kb (NF-kB) activities are seen in multiple solid tumors and combine to provide oncogenic signals to cancer cells. Understanding how these oncogenic pathways areconnected is crucial, giventheir role inintrinsicor acquiredresistancetotargetedanticancertherapies.We reviewmolecularmechanismsbywhichbothEGFR-and NF-kB-dependentpathwaysestablishpositiveloopsto increasetheironcogenicpotential.Wealsodescribehow NF-kBpromotes resistancetoEGFRinhibitors.

ConstitutiveEGFRsignalinginsolidtumors

Oncogenicproteins(see Glossary)cooperate toefficiently drivetumordevelopmentandprogression.Cancercellsare indeed characterized by a powerful signaling network showingmultiple connections tosurvive, proliferateand to resist to targeted anticancer therapies. Constitutive signaling from the EGF receptor (EGFR/HER1/ERBB1), aproteinof170kDaandamemberoftheERBBfamilyof receptortyrosinekinases(RTKs;Box1),cruciallypromotes cellsurvival,proliferation,andinvasiveness[1].Avariety ofEGFpeptidestriggerEGFRdimerizationand phosphor-ylationofmultipletyrosineresiduesinitscytoplasmictail. Those phosphorylated EGFR residues provide docking sitesforcytoplasmicSRChomology2(SH2)and phospho-tyrosine-binding (PTB) domain-containing proteins to specifically trigger PKC, PI3K/AKT/mTOR, SRC, STAT andRAS/RAF/MEK1/ERK1/2activation(Figure1)[2,3].

Morethan 100EGFR-interacting proteinshavebeen describedsofar[4].Amongthemisgrowthfactor receptor-boundprotein 2(GRB2) which binds to phosphorylated tyrosines 1068, 1086, and 1148. RAS is subsequently activated byphosphorylation,a modificationthat relies onsonofsevenless(SOS).ActivatedRASbindstoRAF, and this interaction leads to mitogen-activated protein kinasekinase1(MEK1)followedbyextracellular signal-regulated kinases 1/2 (ERK1/2) phosphorylations [5,6]. RAS activation also relies on the recruitment of the SRC homology domain-containing adaptor protein C (SHC) tophosphorylated EGFR[7].The p85 regulatory

subunit of phosphatidylinositol-3-kinase (PI3K), the ki-nase SRC, and protein tyrosine phosphatases such as PTP1B, SHP1, and SHP2 also associate with distinct phosphorylated EGFR residues (Figure 1) [8]. EGFR directlyorindirectly(throughJAK)activatessignal trans-ducer and activator of transcription (STAT) members. EGFR phosphorylation also triggers STAT activation throughSRCaswellastheactivationofPI3Kthat subse-quentlypromotesAKTactivation.ActivatedAKTtargets multiplesubstrates,includingmammaliantargetof rapa-mycin (mTOR). Phosphoinositide-specific phospholipase Cg1 (PLCg1) binds to EGFR through its SH2 domain, becomingactivatedandhydrolyzingphosphatidylinositol 4,5-bisphosphate to diacylglycerol (DAG) and inositol trisphosphate(IP3).DAGthentriggerstheactivationof serine/threoninekinaseproteinkinaseC(PKC)(Figure1). NF-kB is also activatedthrough the IKKcomplex upon EGFRphosphorylation.

Overexpression and activating mutations of EGFR, which have beenreported in up to 30% of solidtumors (includingbreast,colorectal,lung,pancreatic,gastric,head and neck cancer,and glioblastomas), generallycorrelate withapoorprognosis[9].Avarietyofsolidtumors, includ-ing lung carcinomas, are indeed dependent upon EGFR activation,andthismakesthemsensitivetoEGFR inhi-bitors such as erlotinib or gefitinib (see [10] for a full descriptionofEGFRinhibitors)[11].Somepatients suffer-ing from lung cancer are highly responsive to gefitinib because ofactivating EGFRpointmutationsor in-frame deletions (Figure 1) [12,13]. These genetic alterations

Glossary

Erlotinib:apharmacologicalinhibitorthatbindsinareversiblefashiontothe ATPbindingsiteoftheEGFRreceptor.ThisEGFRinhibitorshowedasurvival benefitinthetreatmentoflungcancerinPhaseIIIclinicaltrials.Erlotinibis moreeffectiveinpatientswithEGFRactivatingmutations.

Gefitinib:thefirstEGFRinhibitorapprovedforthetreatmentofnon-smallcell lungcarcinoma.Similarlytoerlotinib,thisdrugbindsinareversiblefashionto theATP-bindingsiteoftheEGFRreceptor.

Oncogenes:genecandidatescodingforproteinsinvolvedintumor develop-ment.Manyoncogenesareamplifiedortargetedbyactivatingmutationstoact inageneticallydominantmanner.

Paronychia:abacterialorfungalinfectionofthehandorfootwherethenail andskinmeet.

Polyubiquitination:apost-translationalmodificationinwhichseveralcopiesof 7kDaubiquitinareboundtoaproteinsubstratetocreateapolyubiquitinchain. This covalent modification involves three sequential enzymatic reactions catalyzedbytheE1(ubiquitin-activating),E2(ubiquitin-conjugating),andE3 (ubiquitinligase)enzymes[78].

Xerosis:askindiseaseinvolvingtheintegumentarysystem.Symptomsinclude thepeelingoftheouterskinlayer,itching,andskincracking.

1471-4914/

ß2015ElsevierLtd.Allrightsreserved.http://dx.doi.org/10.1016/j.molmed.2015.04.001

Correspondingauthor:Chariot,A. (Alain.chariot@ulg.ac.be).

Keywords:EGFR;gefitinib;erlotinib;NF-kB;resistance;tumor-initiatingcells.

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targetthecytoplasmicdomainofEGFRinlung adenocar-cinomas, while, in contrast, mutations in glioblastomas showingconstitutiveEGFRsignalingtargetthe extracel-lular domain of this receptor [14,15]. Exons 18–21 of the tyrosine kinase domain of EGFR harbor all key mutations. About 40% of genetic alterations found in highly-responsive patients arein exon 21,andthe most

common is L858R. Some in-frame deletions in exon 19 (DE746-A750 as well as other deletions in this exon) accountforabout46%ofthereportedEGFRgenetic altera-tionsfoundinhighly-responsivepatients.Pointmutations in exon 18 (G719A, G719S, and G719C) have been described in about 1% of those patients. Less-frequent EGFRmutationsunderlyingdrugsensitivityorresistance havebeendescribedelsewhere[16,17].

The therapeutic effectiveness of EGFR inhibitors has been disappointing due to the emergence of resistant cancer cells. Virtually all patients who initially respond toEGFRinhibitorsbecomeresistanttothesedrugsas a resultofacquiredEGFRmutations[18].Themost clinical-ly relevant EGFR mutation found in 50% of the cases showingacquiredresistancetoEGFRinhibitors(gefitinib anderlotinib)is theT790M mutationlocated inexon 20 (Figure1)[19].Thismutation,whichislocatedwithinthe ATP-bindingsiteofthekinasedomain,causessteric hin-dranceforaccessoftheinhibitortothecleftowingtothe bulkiness of the methionine sidechain [20]. The use of irreversibleinhibitorsoftheEGFRkinaseactivitytotreat patients harboring this mutation is an attractive thera-peutic approach, and has prompted the search for new EGFRinhibitorsthatspecificallytargettheEGFRT790M mutation. Several molecules have been identified that aremorespecificforthismutatedEGFRthanforthewild typereceptor[21].

Box1.ERBBmembers

The ERBB receptors include the EGF receptor (EGFR, also named

HER1),ERBB2(HER2/Neu),ERBB3(HER3),andERBB4(HER4), and

belongtothefamilyoftypeIreceptortyrosinekinases(RTK)[79].

ERBB receptors are mainly expressed in epithelial, mesenchymal,

and neuronal cells, as well as in their progenitors. The receptors

share an extracellular ligand-binding domain, a single

membrane-spanning region, and a cytoplasmic domain that includes a

juxtamembrane domain, a region harboring an intrinsic tyrosine

kinase activity, as well as a C-terminal domain[80]. ERBB receptors

are bound by EGF-family peptides. These ligands include EGF,

transforming growth factor (TGF)-a, amphiregulin (AR), and epigen

(EPG) which bindto EGFR; b-cellulin (BTC), heparin-binding EGF

(HB-EGF),andepiregulin(EPR)whichbindtobothEGFRandERBB4;

and neuregulins (NRGs) such as NRG-1 and NRG-2 that are known

to bind to both ERBB3 and ERBB4, as well as NRG-3 and NRG-4

acting as ligands for ERBB4 only[79,80]. NRG-1 has several isoforms

(type I NRG-1,alsonamed ‘heregulin’,totype VINRG-1). ERBB2

does not directly bindto any of these peptides, whereas ERBB3

isdevoidofanystrongkinaseactivityandonlysignalswhenbound

to other ERBB members.

RAS MEK1 RAF DAG AKT IKK mTOR STAT PKC PI3K PLCγ ERK1/2 17 18 19 20 21 22 24 25 28 Mutaons Phosphorylated sites Recruited proteins Exons P P P P P P P P P Y703 Y845 Y920 Y992 Y1045 Y1068 Y1086 Y1148 Y1173 STAT1 SRC, STAT5 PI3K (p85) Cbl GRB2, STAT3 GRB2, STAT3 SHC, GRB2 SHC, SHP-1/2, PLCγ PLCγ G719C, G719S, G719A ΔE746-A750 T790M L858R TM Tyrosine kinase Autophosphorylaon P P P P P P P P P P P SHC GRB2 SOS P P Proliferaon, angiogenesis, survival, and invasion

EGFR

EGF, TGF-α, AR

JAK P SRC P

TRENDS in Molecular Medicine Figure1.EGFR-dependentsignalingpathwaysandEGFRmutationsinsolidtumors.LigandsofEGFRhomodimersincludeEGF,TGF-a,andAR.Examplesofproteins recruitedontyrosine-phosphorylatedEGFRresiduesarelistedontheleftandthemostcharacterizedsignalingpathwaysactivateduponEGFRphosphorylationare illustratedontheright.Themostfrequentmutationslinkedtodrugsensitivityandfoundindrugrespondersaredepictedingreen.ThemostclinicallyrelevantEGFR mutation(T790M)thatpromotesresistancetoEGFRinhibitorsisrepresentedinred.Abbreviations:AR,amphiregulin;EGF,epidermalgrowthfactor;EGFR,epidermal growthfactorreceptor;P,phosphorylation;TGF-a,transforminggrowthfactora;TM,transmembrane.

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EGFRpointmutationsarenottheonlymechanismby which cancer cells are (or become) resistant to EGFR inhibitors. Activation of other RTKs such as ERBB2/ HER2 also occurs in cells resistant to cetuximab, an EGFR-targeting monoclonal antibody, which paves the way for the dual inhibition of both EGFRand HER2 to improvetheclinicalresponse[22].SignalingfromERBB3/ HER3isalsospecificallyactivatedinepithelial malignan-ciestreatedwithEGFRinhibitors[23–25].AlthoughHER3 lacks intrinsic kinase activity, it nevertheless strongly activates AKT signaling as a dimer with HER2 [26,27]. Therefore, a variety of pharmacological approaches, in-cluding HER3-blocking antibodies, have been recently developedtocircumventresistance[10,28].Itiscurrently unclearwhethertheuseofmultipleERBBinhibitorsisthe bestapproach,orwhetherothertypesofinhibitorshaveto becombinedwiththem.Inanycase,dissectingallrelevant oncogenicpathwaysisofparamountimportancetoidentify newmechanisms underlying resistance to EGFR inhibi-tors,andtodefinethebestcombinationofspecificdrugsto fight epithelial malignancies. This review will focus on molecular mechanismsby which thetranscription factor NF-kBisactivated uponEGFR activation,as wellas on NF-kB-dependent pathways underlying resistance to EGFRinhibitors.

MolecularmechanismslinkingEGFRsignalingtoNF-kB activation

GrowthfactorspromoteNF-kBactivationthroughERBB members, butthe underlying mechanisms are only now startingtobeelucidated.The familyofNF-kB transcrip-tion factors are typically activated by proinflammatory cytokines such as tumor necrosis factor (TNF)-a or IL-1b,aswellasbyToll-likereceptor(TLR)ligandsthrough extremely well defined signaling cascades (Box 2) [29]. Early studies demonstrated that EGF triggers NF-kB activationthroughtheproteasome-mediateddegradation oftheinhibitorymoleculeIkBainestrogenreceptor ERa-negative breast cancer cells and in lung cancer-derived cells [30,31]. Heregulin also triggers NF-kB activation through the IKK complex in ERa-negative and ERBB2-positivebreastcancercells[32].Inaddition, constitutive EGFRsignalingleadstoNF-kBactivationthroughIkBa phosphorylationonserines 32and36in prostatecancer cells[33].

AlthoughitisnowwellestablishedthatEGFactivates NF-kBthroughtheIKKcomplex(thatincludesboth cata-lytic subunits IKKa and IKKb as well as the scaffold proteinNEMO/IKKg),signalingmoleculesthatlinkEGFR activation to the IKK complex have only been recently characterized (Figure 2). Distinct pathways have been elucidatedindetail,butitremainsunclearwhetherthey areactivatedsimultaneouslyorinacellspecificmanner. EGF stimulation in prostate and breast cancer cells, as well as in EGFR-overexpressing glioblastoma-derived cells, triggers PKCemonoubiquitination at Lys 321 in a PLCg1-dependentmanner[34].PKCemonoubiquitination relies on the E3 ligase RINCK1, but not on the linear ubiquitin assembly complex (LUBAC) that includes HOIL-1L and HOIP. Monoubiquitinated PKCe recruits theIKKcomplextotheplasmadomainthroughaphysical

interaction with a ubiquitin-binding domain in the zinc finger of NEMO/IKKg. PKCe then activates NF-kB throughIKKb phosphorylationatSer177. Thispathway ultimatelydrivestumorgrowthbyinducingtheexpression ofpyruvatekinase2(PKM2),theenzymeinvolvedinthe rate-limitingfinalstepofglycolysis.The IKKb-phosphor-ylatingkinaseTGFb-activatingkinase1(TAK1)appears tobedispensableforthispathway,meaningthatEGFas wellasproinflammatorycytokinessuchasTNFaand IL-1b activate NF-kB through distinct signaling pathways thatwillneverthelessconvergeattheIKKcomplex[34].

EGF-dependent NF-kB activation in some breast and lungcancer-derivedcellsalsoreliesonthescaffoldprotein caspaserecruitmentdomain(CARD), membrane-associat-edguanylatekinase-likedomainprotein3(CARMA3;also referredtoasCARD10), andBcelllymphoma protein10 (BCL-10) [35]. Interestingly, both CARMA3 and BCL-10 alsopromoteGPCR-andPKC-dependentNF-kBactivation when complexedwithmucosa-associated lymphoid tissue lymphomatranslocationgene1(MALT1),butitiscurrently unclearwhetherMALT1 is actuallyrequired in EGF-de-pendentIKKphosphorylation[36,37].MALT1,asasubunit oftheCBM(CARD10–BCL-10–MALT1)complex,recruits theE3ligaseTRAF6,whichformsK63-linkedpolyubiquitin

Box2.TheNF-kBfamilyoftranscriptionfactors

NF-kB proteins, which include RelA (also named p65), RelB, and

c-Rel,shareaN-terminalRelhomologydomain(RHD)thatisrequired

for homo- and heterodimerization and for bindingto

sequence-specific DNA-binding sites in the promoters of 200target genes.

These NF-kB proteins harbor a C-terminal transactivating domain

(TAD). NF-kB proteins also include p50 and p52, which are

generated from precursors NF-kB1/p105 and NF-kB2/p100. Both

p50andp52lackanyTADandthereforerelyonothermembersto

drive gene expression of NF-kB-target genes. In unstimulated cells,

NF-kB proteins are sequestered in the cytoplasm through binding to

inhibitory molecules whose prototype is IkBa[81]. Other inhibitory

moleculesincludep100,p105,IkBb,andIkBe,aswellasBCL-3,IkBz,

andIkBNS.IkBproteinsaswellasp100andp105 bindtoNF-kB

dimers through multiple ankyrin repeats. Stimulation with a variety

of stimuli, such as proinflammatory cytokines (e.g., TNFa and IL-1b),

Toll-like receptor ligands [e.g., lipopolysaccharide (LPS) and

double-strandedRNA(dsRNA)],triggersNF-kBactivationthroughthe

so-called ‘classical’ or ‘canonical’ pathway. This signaling pathway

leadstotheIKKcomplex,composedofbothkinasesIKKaandIKKb

assembled by the scaffold protein NEMO/IKKg. The IKK complex

phosphorylates IkBa on N-terminal serines, and this triggers its

degradative polyubiquitination through the proteasome. NF-kB

dimers(mainlyp50/p65andp50/c-Rel)areconsequently released

and translocated to the nucleus to drive gene transcription of

candidates involved in innate immunity, inflammation, proliferation,

and survival. Growth factors can also trigger the activation of the

IKKcomplexthroughsignalingpathwaysdescribedinFigure2in

maintext.

The ‘alternative’ or ‘non-classical’ NF-kB-activating pathway is

triggered by cytokines such as BAFF and lymphotoxin-b, and leads

to the activation of an IKKa homodimer, which phosphorylates

p100. This inhibitory molecule is subsequently processed to

generatep52.NF-kB dimers(p52/RelB) moveintothenucleus to

drive the expression of candidates involved in adaptive immunity,

as well as in lymphoid organogenesis. The activation of all NF-kB

signaling pathways relies on the sequential phosphorylation of

multipleproteins,aswellasonthepolyubiquitinationofkeyactors

throughseveraltypesofchains,themostcharacterizedbeingthe

K48-linkedchain,whichtriggersthedegradationofitssubstrateor

both linear and K63-linked chains, which enhance protein–protein

interactions[82].

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chains, to promote IKK activation through TAK1 in T lymphocytes [38].Because MALT1and BCL-10are poly-ubiquitinatedby TRAF6,they couldbeboundby NEMO/ IKKginaTAK1-independentmanner,amodelthatfitswith thereporteddispensableroleofTAK1 inEGF-dependent IKKbphosphorylation[34].ActivationoftheCBMcomplex relies onPKC activation,butthePKC isoformthatlinks EGFR activation to CARMA3 has not been identified. WhetherPKCefulfillsthisfunctionremainstobe demon-strated.

An additional pathway that links EGFRactivationto NF-kB involves the guanine nucleotide exchange factor SOS1 [39].Upon EGF stimulation, SOS1binds to phos-phorylated EGFR through the adaptor protein GRB2, which thentriggersRAS activation at theplasma mem-brane[40].Interestingly,itsGDP–GTPexchangeactivity, known to be crucial for EGF-dependent MAP kinases activation, is dispensable for NF-kB activation upon EGF stimulation, and this supports the notion that SOS1mayalsoactasascaffoldproteintotransmit onco-genicsignals[39].Nevertheless,signalingmoleculesthat link SOS1 to the IKK complex are totally unknown (Figure2).

These studies have convincingly demonstrated that growthfactorspromoteNF-kBactivationthrough signal-ingpathwayswhoseinitialstepsarelargelydistinctfrom thosetriggeredbyproinflammatorycytokines.These sig-naling cascades are believed to crucially contribute to

tumordevelopment andprogression through the expres-sionofNF-kB-dependentgenesthatpromotecell prolifer-ationandsurvival.

CrosstalkbetweenEGFR-andNF-kB-dependent pathwaysthroughthetranscriptionalinductionof targetgenes

While growth factors trigger NF-kB-activating cascades uponbindingtoERBBmembers,thetranscriptional induc-tionofsomeNF-kBtargetgenesalsofeedsbacktoimpact onEGFR-dependentsignaling pathways. In thiscontext, KIAA1199istranscriptionallyinducedbyNF-kBproteinsin transformed keratinocytes as well as in breast cancer-derivedcells(Figure3)[41,42].Theoncogenichuman pap-illomavirus(HPV)alsopositivelyregulatesKIAA1199gene transcriptionthroughBCL-3 incervical cancercells [41]. KIAA1199promotes EGFRstability bylimiting its EGF-dependentdegradationinlysosomes,andtherefore positive-lyregulatesEGFRsignaling[41].KIAA1199actuallylimits semaphorin3A-dependentcelldeathby promotingEGFR phosphorylationandaswellasEGF-dependentepithelial– mesenchymal transition (EMT) in cervical cancer cells [41].Assuch,KIAA1199linksNF-kB-dependentgene tran-scription to EGFR signaling to sustain cell survival and invasion [41]. Another example of positive correlation between NF-kB and EGFR activitieshas been described inheadandnecksquamouscellcarcinomas(HNSCCs) in which IKKa and/or b knockdown significantly decreased

BCL-10 MALT1 PKC PLCγ1 DAG RINCK1 EGFR EGF Ub Ub Ub Ub Ub Ub IκBα p50 p50 p65 P P P P P P IκBα p50 p50 p65 p65 p65 P P P P Ub Ub Ub Ub Ub Ub Ub Ub Ub Ub Ub Ub CARMA3 SOS ? ? Nucleus κB site P IKKβ IKKα NEMO NEMO P IKKβ IKKα NEMO NEMO GRB2 RAS PKCε Ub P TRAF6 P SHC P

TRENDS in Molecular Medicine

Figure2.MolecularmechanismsbywhichEGFRactivatesNF-kB.EGFbindingtriggersEGFRphosphorylationanditsassociationwiththeSOS–GRB2proteincomplex.RAS issubsequentlyrecruitedandactivatedatthecellmembranetopromoteMEK1andERK1/2activation(notdepicted),apathwaythatreliesontheguaninenucleotide exchangecatalyticactivityofSOS.ConstitutiveEGFR-dependentNF-kBactivationreliesonSOS,whichtriggersIKKbactivation,apathwaythatdoesnotrequirethe catalyticactivityofSOS[39].EGFRphosphorylation(P)alsotriggersPLCg1activation,followedbyDAGproductionandPKCemonoubiquitination(Ub)bytheE3ligase RINGfingerproteinthatinteractswithCkinase1(RINCK1)boundtoHOIL-1LandHOIP.MonoubiquitinatedPKCeassociateswithaubiquitin-bindingdomainwithinthe zinc-fingermotifofNEMOtorecruittheIKKcomplextotheplasmamembrane.PKCecanthenphosphorylateIKKbtotriggerNF-kBactivation[34].EGF-dependentNF-kB activationincancercellsalsoreliesontheCBMcomplexthatincludesCARMA3andBCL-10.ThiscomplexalsoincludesMALT1,butitiscurrentlyunclearwhetherthis proteinisrequiredforNF-kBactivation.TheCBMcomplexactivatesIKKbthroughTRAF6,anE3ligaseknowntogenerateK63-linkedpolyubiquitinchainsboundbyNEMO. SignaltransductionthroughtheCBMcomplexreliesonPKCactivation,butthePKCisoformthattriggersEGF-dependentIKKphosphorylationthroughthiscascadehasnot been characterized[35].IKKbactivation isrequiredfor IkBaphosphorylation,polyubiquitination, and degradationthroughtheproteasome.NF-kB subsequently translocatestothenucleustodrivetheexpressionofitstargetgenes.

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EGFRmRNA and proteinlevels [43]. In contrastto this NF-kBsignalingpathwaythatpositivelyregulatesEGFR signaling, EGFR expression is negatively regulated at the transcriptional level by receptor-interacting kinase (RIPK1),whichis typicallyactivated byproinflammatory andNF-kB-activatingcytokinessuchasTNFa[44].RIPK1 indeedappearstointerferewithSp1activity,a transcrip-tion factor that promotes EGFR mRNA synthesis [44]. Therefore,multiplefeedbackloopsinvolvingthe transcrip-tionalinductionoftargetgenesthatlinkbothEGFRand NF-kB-dependent pathways havebeen described, evenif they do not systematically lead to the establishment of positiveloops.

NF-kBactivationasamechanismforresistancetoEGFR inhibitors

NF-kB-activatingcascades promoteresistance to chemo-therapythroughmultiplemechanisms,includingthe tran-scriptional induction of multidrug resistance gene-1 (MDR1)incoloncancercells[45].Recentstudieshavealso definedmechanisms by which resistance occursthrough crosstalk between EGFR- and NF-kB-dependent path-ways. The tyrosine kinase FER, known to be activated byEGFRandPDGFRuponligandengagement,promotes

resistance to quinacrine, a drug with antimalarial and anticancereffects,whenoverexpressedinprostatecancer cells [46–48]. Mechanistically, FER binds to EGFR to enhance its phosphorylation ontyrosine residues, which leads to NF-kB activation through an AKT-independent pathway[46].

Anunbiasedscreenforoncogenicpathwaysunderlying resistancetoEGFRinhibitorsledtothe identificationof multiple candidates involved in NF-kB signaling [49]. Indeed, an unbiased short hairpin RNA (shRNA)-based high-throughputscreencarriedoutinlungcancer-derived H1650 cells insensitive to EGFR inhibitors led to the identificationofseveralcandidates,manyofwhichactin NF-kB-activatingcascades[49].Consistentwith aroleof NF-kBin the resistancetoEGFR inhibitors, thegenetic orpharmacologicinhibitionofNF-kBincreasedthe sensi-tivitytoerlotinibinseveralmodelsofEGFR-mutatedlung cancers.Moreover, decreasedexpressionoftheinhibitory IkBaproteinisassociatedwithresistancetoerlotiniband ispredictiveofworseprogression-freesurvivalinpatients suffering from lung cancer [49]. Consistent with a role ofNF-kBasamediatorofresistancetoEGFRinhibitors, quinacrine overcomes resistance to erlotinib, at least by decreasing the level ofSSRP1, an activesubunit of the

HPV CYLD Plexin A2 NRP1 P P mRNA KIAA1199 RAS MEK1 ERK1/2 SRC P P P EGF Sema 3A EMT P P Ub Ub Ub Ub Ub Ub p50 p50 BCL-3 P P Ub Ub Ub Ub Ub Ub p50 p50 BCL-3 Survival EGFR Apoptosis KIAA1199

TRENDS in Molecular Medicine

Figure3.TheNF-kB-inducedproteinKIAA1199promotesEGFRstabilityandsignalingandprotectsfromsemaphorin3A-mediatedcellapoptosis.Humanpapillomavirus (HPV)infectioninkeratinocytesinhibitsCYLD,aubiquitinC-terminalhydrolase.Asaresult,thenon-degradativeK63-linkedpolyubiquitinationofBCL-3,ap50-binding protein,isenhanced,leadingtoitsnucleartranslocation.NuclearBCL-3drivesKIAA1199genetranscription.KIAA1199bindstoplexinA2tolimitsemaphorin3A-dependent celldeath,andalsostabilizesEGFRtopromoteEGF-dependentSRCandERK1/2activation,andsubsequentepithelial–mesenchymaltransition(EMT)[41].

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facilitates chromatin transcription (FACT) complex that promotesNF-kBtranscriptionalactivity[50].

The crosstalk between EGFR- and NF-kB-activating cascadesisparticularlyrelevantintumor-initiatingcells, whichcruciallypromotedrugresistance[51,52].Integrin avb3-expressing tumor-initiating cellsfrom breast, lung,

andpancreaticcarcinomasare resistanttoEGFR inhibi-tors [53].NF-kBactivationcontributes tothisphenotype because integrin avb3 drives tumor stemness and

resis-tancetoEGFRinhibitorsbyinteractingwithKirstenrat sarcomaviraloncogenehomologGTPase(KRAS)through galectin-3 to promote the sequential activation of both GTPase v-Ral simian leukemia oncogene homolog B GTPase (RALB) and TANK-binding kinase-1 (TBK1), whichthentargets c-Rel,aNF-kBprotein[53]. Interest-ingly, this pathway does not require the bindingof any ligand tointegrin avb3,demonstrating thatresistanceto

EGFRinhibitorscanbecellautonomous.

Cell intrinsic mechanisms of resistance to anticancer therapies have alsobeendescribedin glioblastomas, the mostcommon malignantprimary braincancerofadults. 40–50% ofglioblastomas show EGFRgene amplification and/ormutations,which,inbothcases,causeconstitutive EGFRsignaling[15].ThemostcommonactivatingEGFR mutation(EGFRvIII)resultsfromadeletionofexons2–7 of the EGFRgene, whichcauses an in-framedeletion of 267 amino acids from the receptor in its extracellular

domain.Asaconsequence,EGFRvIIIcannotbindtoany ligandandisconstitutivelyactive[14].EGFRvIIItriggers mTORC2 activation, a complex composed of the kinase mTOR bound to unique regulatory proteins, including RictorandSIN1[54].mTORC2signalstoNF-kBthrough an AKT-independent pathway to promote proliferation, survival, and cisplatin resistance in glioblastomas [55] (Figure4). Although itiscurrently unclearwhetherthis pathway is specifically induced in glioma cellsresistant to EGFR inhibitors, this study nevertheless shows that NF-kB also acts as a central player in chemotherapy resistancein glioblastoma cellsharboring constitutively-active EGFR.EGFRvIII also activates NF-kBin glioma cellsbyassemblingasignalingplatformwithTNF recep-tor-associatedprotein2(TRAF2),RIPK1,boththecIAP1 andcIAP2 (cellularinhibitorofapoptosis1/2)E3ligases, as wellas TAK1 [56]. RIPK1 becomes polyubiquitinated inaK63-linked,non-degradativemannertotriggerTAK1 andsubsequentIKKbphosphorylation.

Allthesepathwaysoccurinacellautonomousmanner, butitisnowincreasinglyobviousthat the microenviron-ment also provides NF-kB-activatingsignals to promote resistancetoEGFRinhibitors.TNFa,mainlysynthesized by tumor-associated macrophages upon activation of TLR-dependent pathways, acts as a paracrine signal to triggerNF-kBactivationingliomacells[57].Thispathway involves the activation of the serine/threonine kinase

Par6 TAK1 p62 2 1 RIP1K cIAP TRAF2 IκBα mTORC2 aPKC aPKC

ERK1/2P NEMONEMO

ERK1/2P P P P P P P p65 P IκBα P P p65 P CD44, MMP9,... CD44, MMP9,... TNFα, IL-1β, IL-8 TNFα, IL-1β, IL-8 Bcl-xl, Cyclin D1 IL-6 ? Nucleus cFoscJunp50p65 Nucleus cFoscJun p50p65 Glioma cells TNFR1 TNFα TNFα TNFα TNFα EGFRvIII TLRs EGFR EGF EGF Ub Ub Ub Ub Ub Ub Ras PTEN P Macrophages

PAMPs, dying cells PAMPs, dying cells

Glioblastoma

p50 IKKα IKKβ

P

p50

TRENDS in Molecular Medicine Figure4.MechanismsbywhichNF-kBpromotesresistancetochemotherapyinglioblastomas.(1)TheproductionofTNFabymacrophagespromotesresistancetoEGFR inhibitorsthroughNF-kBactivationingliomacells.Tumor-associatedmacrophagessensedyingcellsandpathogen-associatedmolecularpatterns(PAMPs)throughTLRs totriggerIKK-dependentNF-kBandERK1/2activation.ThesepathwayspromoteTNFasynthesis,whichactsinaparacrinefashiontoactivateNF-kBthrough TNFR1-dependentaPKCphosphorylationonresidues403/410ingliomacells[57].aPKC,whoseactivationrequiresthescaffoldproteinp62,triggersIKKb(notillustrated)andIkBa phosphorylationtopromotethenucleartranslocationofNF-kBsubunitsp50andp65.Thissignalingcascadeultimatelyinducestheexpressionofproinflammatory cytokines(TNFa,IL-1b,IL-8),whichestablishapositivelooptofurtheractivateNF-kBingliomacells.ItisimportanttonotethataPKCactivationalsooccursuponEGFR activation,butthescaffoldproteininvolvedinthispathwayisPar6andthetargetgenesdownstreamofthiscascadearenotproinflammatorycytokinesbutEGF-induced candidatessuchasCD44andthematrixmetalloproteinaseMMP9.Whether,andhow,thesecandidatespreciselypromoteresistancetoEGFRinhibitorsingliomacells remainstobeclarified.(2)AsecondpathwayunderlyingresistancetochemotherapyinvolvesanEGFRvIII-dependent(butAkt-independent)signalingpathwaythatleadsto mTORC2activationingliomacells.SignalingfromtheEGFRvIIImutanttriggersmTORC2phosphorylation,apathwaynegativelyregulatedbyPTEN.mTORC2activation ultimatelytargetsIkBaphosphorylationandNF-kBactivationthroughapoorlydefinedsignalingcascade,whichwillultimatelydrivetranscriptionofNF-kBtargetgenes suchasBcl-xl,cyclinD1,andIL-6.EGFRvIIIexpressionalsopromotescellsurvivalingliomacellsthroughK63-linkedpolyubiquitinationofRIPK1byc-IAPsboundtoTRAF2. ThissignalingcomplexculminatesinNF-kBactivationthroughTAK1-dependentIKKbphosphorylation[56].

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atypical protein kinase C (aPKC) bound to the scaffold proteinp62 toinduce the expressionofproinflammatory cytokines through NF-kB. Interestingly, another pool of aPKCinteractingwiththescaffoldproteinPar6is specifi-cally activated upon EGF stimulation, and drives the expression of another set of genes such as CD44 and MMP9(Figure4). TargetingaPKCactivitycausestumor regressioninEGFRkinaseinhibitor-resistant glioblasto-ma[57].

CombiningEGFRandNF-kBinhibitors:thewaytogo? ThedemonstrationthatNF-kBcontributestoresistanceto EGFRinhibitorssuggeststhatthesimultaneousinhibition ofbothEGFRandNF-kBactivitymightbeatherapeutic strategy to circumvent resistance. While tempting, it is currentlyunclearwhetherpatientswouldbenefitfromthis combination. Preclinical studies to treat HNSCCs have beenconductedtoaddressthisissuebecausebothEGFR andNF-kBactivityareenhancedinahighpercentageof thesemalignancies [58]. TheEGFR-targeting cetuximab usedincombinationwithradiationprolongedoverall sur-vivalbutfailedtoreducetheincidenceofmetastasis,and theresponseratewithcetuximabadministeredaloneisnot above13%[59].Onemechanismunderlyingresistanceto cetuximab results from the expression of the EGFRvIII mutant,whichhasatruncatedligand-bindingdomain,and signalstoLyn,amemberoftheSRCfamilyofkinasesand toSTAT3inupto42%casesofHNSCC[60,61].Although the EGFR inhibitor gefitinib showed some response in clinical trials of HNSCC by decreasing EGFR, MEK1, andNF-kBp65phosphorylation,itfailedtointerferewith these oncogenic pathways in nonresponder patients [62].Therefore, therewasaneedtocombineEGFR inhi-bitorswithothertargetedtherapies.

Bortezomib,a proteasome inhibitor, triggers cell apo-ptosisbyblockingNF-kBactivationthroughIkBa stabili-zation[63].However, bortezomib showed limitedclinical efficacyinHNSCCbecauseNF-kBactivationthroughthe noncanonicalpathwayremainedunchanged[64,65]. More-over,bortezomibdidnotimpactonotherprosurvival path-ways driven by STAT3 and ERK1/2 phosphorylation [64].Therefore,thecombinationofbortezomibwithEGFR inhibitorswasexpectedtoimprovetheclinicalresponse.A PhaseIclinicaltrialwascarriedoutwithpatients suffer-ingfromHNSCCtoassesspreclinicalevidencefor combin-ingthe EGFR inhibitor cetuximab with bortezomib and radiationtherapy[66].Thiscombinationshowed unexpect-edly early progression due to EGFR stabilization, in-creased prosurvival signaling, and enhanced cytokine expression. Bortezomib attenuated the cytotoxic effects ofcetuximabandradiationbylimitingEGFRdegradation throughtheproteasome[66].Proteasomeinhibition actu-allyenhancedMAPK,AKT,andSTAT3prosurvival path-ways through both EGFR-dependent and -independent mechanisms [67].These studies highlighted the need to design new combinatorial approaches by using a more-specificNF-kBinhibitorthanbortezomib,whichblocksthe degradationofIkBaaswellasofnumerousotherproteins, includingkeyoncogenicproducts[68].Interestingly,both IKKaandIKKbareaberrantlyactivatedinHNSCC, act as mediators of NF-kB activation, and enhance EGFR

signaling in HNSCC [43]. Consistently, wedelactone, a dual IKKa/b inhibitor, more effectively inhibited NF-kB activation than MLN120b, a specific IKKb inhibitor [43,69].Moreover, 17-DMAG, a geldanamycinderivative that inhibits the heat shock protein 90kDa (HSP90), a molecularchaperoneinvolvedinbothIKKa/b-and EGFR-dependent signalingcascades,was moreeffectiveat trig-geringcelldeaththanMLN120balone[43,70].Therefore, these data demonstrated why specific IKKb inhibitors have not ledtoexpected efficaciesin preclinicalstudies, andsuggestthatcombiningdualIKKa/bandEGFR inhi-bitors would be more successful, at least for epithelial malignancies.

DoesNF-kBplayany roleinthetoxicityofEGFR inhibitors?

AlthoughNF-kB activationlimitsthe clinicalresponseto EGFRinhibitors,itiscurrentlyunclearwhethertheside effects commonly reported with these drugs would still occurbytargetingNF-kB.Cutaneoussideeffectsareoften describedinpatientstreatedwithEGFRinhibitors (mono-clonalantibodiesorsmall-moleculeinhibitors)[71,72].They includehairloss,acneiformeruption,paronychia,and xero-sis.Mechanistically,thesesymptomsresultfromthe inflam-mation-proneresponseofkeratinocytestoearlyinfiltration of macrophages and mast cells into the skin, as well as from an increasedpercentage of circulating granulocytes and platelets, but decreased percentage of lymphocytes in the plasma [73,74]. The genetic inactivation ofEGFR intheepidermismimicsthosesymptoms,suggestingthat EGFR expression maintains skin immune homeostasis [73]. EGFR-deficient keratinocytes overexpress a variety of chemokines and cytokines (such as IL-1b, TNFa, and IL-6)thatareknowntoberegulatedbyNF-kB-dependent pathways.However,thegeneticinactivationofsingle proin-flammatory pathways (e.g., TNFR1/R2, Myd88) did not reversetheinductionandmaintenanceoftheskin pheno-typeintheEGFR-deficientmousemodel[73,74].Therefore, it is unlikely that targeting NF-kB alone will improve cutaneoustoxicityseenwithEGFRinhibitors.

Concludingremarksandfutureperspectives

Recentstudieshaveelucidatedthemolecularmechanisms bywhichtargetedtherapiesleadtoresistanceinepithelial malignanciesdisplayingconstitutivesignalingfromERBB receptors. The simultaneous administration of specific inhibitors is the most logical approach to treat tumors with intrinsic or acquired resistance. Combining ERBB andNF-kBinhibitorsisverypromising,giventhekeyrole ofNF-kBintumorsresistanttoEGFRinhibitors;however, key issues remain unresolved (Box 3). New irreversible EGFRinhibitorsarecurrently being testedtotreat lung tumorsharboringtheT790Mmutation.Giventhegenomic plasticityofmanyaggressivesolidtumors,includinglung carcinomas,itislikelythatnewoncogenicmutationsand/ oractivationofsignalingpathwayswilloccurinpatients treatedwiththeseirreversibleEGFRinhibitors.Whether NF-kB activation contributes or not to this resistance remainstobeclarified.Inaddition,itiscurrentlyunclear whichNF-kBinhibitorshouldbeusedincombinationwith ERBBinhibitorsforthebestclinicalresponse.Avarietyof

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IKKinhibitorshavebeendevelopedoverthepast15years, andsomehavebeenusedinclinicaltrials[75–77]. Howev-er,noneofthemhavebeenapprovedsofar,atleastbecause somedoubtsontheinterestoftargetingIKKbitselfwere raised[77].AsrecentstudiescarriedoutinHNSCC indi-catedthatspecificIKKbinhibitorsdonottotallyblock NF-kBactivation,itislikelythatotherNF-kBinhibitorswill needtobetestedincombinationwithERBBinhibitorsin allepithelialmalignanciesshowingconstitutivesignaling from ERBB receptors [43]. Being over-specific (i.e., by targetingonlyIKKb)maynotbethebeststrategy.Finally, combiningEGFRorHER2inhibitorswithHER3blocking antibodies tocircumvent resistancehold promisefor the future, but it is also likely that some cancer cells will ultimately escape from this therapeutic strategy. If so, the potential role of NF-kB in these resistant cells will needtobeexplored.

Acknowledgments

Our laboratory issupportedbygrantsfrom theFondsNational dela

Recherche Scientifique (FNRS), TELEVIE, the University of Liege

[ConcertedResearchActionProgram(BIO-ACET)andFonds Spe´ciaux

(C-11/03)], the Centre Anti-Cance´reux, the Leon FredericqFondation

(University ofLiege),as wellasbyWELBIO.A.C. isSenior Research

AssociateattheFNRS.

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Box3.Outstandingquestions

What is the best combination of EGFR and NF-kB inhibitors to use

intheclinic?

Does NF-kB promoteresistance in solidtumors harboring the

T790M mutation?

Does the simultaneous inhibition of HER2 and HER3 lead to

resistance, and, if so, does NF-kB play a role?

Does the simultaneous inhibition of both NF-kB and

EGFR-dependent signaling pathways in patients improve cutaneous

side effects typically seen with EGFR inhibitors?

The ligand-induced versus constitutive EGFR-dependent signaling

may rely on partially distinct signaling complexes, but how can

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Figure

Figure 1. EGFR-dependent signaling pathways and EGFR mutations in solid tumors. Ligands of EGFR homodimers include EGF, TGF-a, and AR
Figure 2. Molecular mechanisms by which EGFR activates NF-kB. EGF binding triggers EGFR phosphorylation and its association with the SOS–GRB2 protein complex
Figure 3. The NF-kB-induced protein KIAA1199 promotes EGFR stability and signaling and protects from semaphorin 3A-mediated cell apoptosis
Figure 4. Mechanisms by which NF-kB promotes resistance to chemotherapy in glioblastomas

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