Presentation on theme: "I recettori della famiglia erbB/HER come bersaglio terapeutico"— Presentation transcript:
1 I recettori della famiglia erbB/HER come bersaglio terapeutico Giampaolo TortoraDivisione di Oncologia Medicae Laboratori di Terapia Molecolare dei TumoriDipartimento di Endocrinologia e Oncologia Molecolare e ClinicaUniversità di Napoli “Federico II”
2 Autocrine and Paracrine growth regulation First evidence by Sporn, M. and Todaro, G. New England Journal of Medicine 1980; 383: 878Autocrine GrowthParacrine GrowthTGFaTGFaFibroblastTGFaTGFaVEGFVEGFVEGFEndothelial cellsTortora & Ciardiello, 2003
3 Increasing Complexity of Growth Factors and Receptors of the EGF Family in Evolution SpeciesReceptorsLigandsC. elegansLET-23LIN-3D. melanogasterDER I + II + III(splice variants)SpitzVeinGurkenArgos (inhibitor)H. sapiensEGFRHER2/neu (c-erbB-2)HER3 (c-erbB-3)HER4 (c-erbB-4)EGFTGFaAmphiregulinHB-EGF, Betacellulin, Epiregulin, NRG-1,2,3,4The complexity of the family of growth factors and receptors in the epidermal growth family has increased during evolution from worms (C. elegans) to flies (D. melanogaster) to humans (H. sapiens). From one known receptor and one ligand, there are now four receptors and at least 10 different ligands that can interact with different receptors.Yarden et al., 2002
4 The EGFR (erbB) Family and Ligands TGFaAmphiregulinb-cellulinHB-EGFEpiregulinNRG2NRG3Heregulinsb-cellulinHeregulinsCysteine-richdomains100448233365924487928The EGFR family consists of four members, HER1/erbB-1 through HER4/erbB-4. They all share the same structure – an extracellular domain that interacts with specific ligands, a short transmembrane domain and a tyrosine kinase domain within the cell, which is the activator of downstream signaling. Each receptor has some homology with the others but they vary in terms of ligand binding and tyrosine kinase activity.Tyrosine kinasedomainC-terminusHER1EGFRErbB-1HER2/neuErbB-2HER3ErbB-3HER4ErbB-4
5 Ligand-induced Receptor Dimerization TGFaTGFaHER2/neuHER3HER4EGFRCell MembraneEGFREGFREGFRtyrosinekinaseAfter ligand binding to each receptor, a complex process, the start of intracellular signaling, is activated. The first part of this process is formation of dimers of the receptor. Heterodimers form when two different receptors interact with each other and homodimers form when two of the same receptors dimerize. Receptor dimerization is essential for activation of tyrosine kinase activity and for downstream signaling, which leads to signal-dependent modulation of gene activation.tyrosinekinaseHER2/neunucleus
6 Signal transduction through EGFR, ErbB-2 and ErbB-3:heterodimer formation KLigandEGFRErbB-2LigandûErbB-2ErbB-3KLigandûEGFRErbB-3KNormanno et al., J Cell Physiol 2002
7 The HER/erbB Signaling Network SrcCblPLCgPI3KShp2GAPAktBadS6KPKCSosGrb2NckRas-GTPRas-GDPMAPKMEKRAFJNKJNKKPAKAblRacVavShcGrb7CrkJakCytokinesNRG3 (4)NRG2 (4)NRG1 (3,4)Amphi- regulin (1)HB-EGF (1,4)b-cellulin (1)Epiregulin (1,4)EGF (1)TGFa (1)LPA, thrombin ET, etc.NRG4 (4)ElkJunFosMycSp1Egr1Stat1324LigandsInput layerReceptor dimersSignallingcascadeA complex signaling network can be triggered upon ligand binding to each receptor. There is great complexity due to the formation of different dimers and the activity of different signaling pathways in the cell. The final output through activation of nuclear events is an effect, direct or indirect, on important cellular functions that are very relevant for cancer formation, such as apoptosis, migration, growth, cell adhesion and differentiation.Output layerTranscription factorsApoptosisMigrationGrowthAdhesionDifferentiationYarden Y and Sliwkowski M. Nat Rev Mol Cell Biol 2001; 2: 127–37.
8 EGFR Activation by Other Receptor Signaling Pathways CytokinereceptorsIntegrinsVGCCGPCRCa++EndothelinAJAK2SrcPKCCa++Cell Membranetyrosinekinase??After ligand binding to each receptor, a complex process, the start of intracellular signaling, is activated. The first part of this process is formation of dimers of the receptor. Heterodimers form when two different receptors interact with each other and homodimers form when two of the same receptors dimerize. Receptor dimerization is essential for activation of tyrosine kinase activity and for downstream signaling, which leads to signal-dependent modulation of gene activation.?SurvivalProliferationMotilityTortora & Ciardiello, 2004
9 COX-2 is involved in EGFR-dependent signaling TGFαEGFRp21raspGRB2SOSraftyrosinekinaseyppyPKAIyMEKPLCγyPI-3KpMAPKCOX-2ProstaglandinsCell proliferationAngiogenesisTortora et al. , 2003
10 HP and EGFRHelicobacter pylori VacA toxin up-regulates VEGF expression in gastric cancer cells through an EGFR-, COX-2-dependent mechanism.HP VacA toxinEGFRGastric cancercellsCOX-2VEGFVEGFVEGFEndothelial cellsCaputo et al., 2003
11 Mechanisms of EGFR Dysregulation OverexpressLigandOverexpressReceptorsMutations ConferringConstitutive ActivityTyrosine kinase within the intracellular domain of the EGFR catalyzes transphosphorylation of the dimerized receptors, the initial reaction responsible for the activation of genes controlling cell replication.Several mechanisms of EGFR dysregulation have been demonstrated.Overexpression of growth factors results in increased EGFR signaling even at low levels of receptor.Overexpression of receptors results in an abundance of receptors available for activation by growth factor.EGFR gene mutations can result in constitutive tyrosine kinase activity, even in the absence of ligand binding or absence of the extracellular binding domain.The mechanisms that downregulate EGFR can be dysfunctional because of interference with internalization or degradation.These mechanisms of EGFR dysregulation all have the same outcome, high tyrosine kinase activity, which facilitates signal transduction into the nucleus resulting in properties characteristic of tumor cells.Reduced apoptotic potentialCell cycle progressionThere is no clear correlation between EGFR overexpression and tumor response.Khazaie K, Schirrmacher V, Lichtner RB. EGF receptor in neoplasia and metastasis.Cancer Metastasis Rev. 1993;12:Wiley HS, Burke PM. Regulation of receptor tyrosine kinase signaling by endocytictrafficking. Traffic. 2001;2:12-18.EGFRv.II/IIIEGFRv.IDefectiveInternalizationorDownregulation by PhosphataseTortora & Ciardiello, 2004 (Modified by Wiley and Burke. Traffic. 2001;2:12).
12 Effetti pleiotropici dell’attivazione di EGFR erbB2erbB3erbB4EGFTGFaEffetti pleiotropicidell’attivazione di EGFRrasrafpSOStyrosinekinaseyGRB2pyPKAIpMEKyyPLCgPI-3KpMAPKVEGFCyclinD1COX-2PTENAKTCDKBcl-2AngiogenesiRbSopravvivenzaAnti-apoptosiProliferazione CellulareInvasione,metastasiResistenza a Chemioterapia, Radioterapia e OrmonoterapiaTortora & Ciardiello, 2003
13 Type of cancer EGFR ErbB-2 ErbB-3 ErbB-4 lung 40-80% 18-60% 25-85% NA Type of cancerEGFRErbB-2ErbB-3ErbB-4lung40-80%18-60%25-85%NAbreast14-91%9-39%22-90%82%stomach33-74%8-40%35-100%NAcolon25-77%11-20%65-89%NAesophagus43-89%7-64%64%NAliver47-68%0-29%84%61%pancreas30-50%19-45%57-63%81%prostate40-80%40-80%22-96%NAkidney50-90%0-40%NAbladder35-86%9-50%30-56%30%ovary35-70%8-32%85%93%head and neck36-100%17-53%81%28-69%Normanno, Endocr Relat Cancer 2003
14 Disease-free Survival According to EGFR and TGFa Levels in HNSCC LowMediumHighp=0.00011.00.80.60.40.20.0Proportion surviving with NEDTGFap=0.0001LowMediumHigh1.00.80.60.40.20.0Proportion surviving with NEDYears after surgeryThis study looked at the prognostic role of EGFR and TGFα expression detected by immunohistochemistry in head and neck squamous cell carcinoma (HNSCC). It was found that overall survival is significantly affected by the level of expression of each. Patients with high levels of expression of both proteins have the worst prognosis, as shown on the graphs by low, medium and high expression levels.NED: no evidence of diseaseYears after surgeryGrandis JR et al. J Natl Cancer Inst 1998; 90: 824–832.
15 EGFR Expression and Disease-free Survival in Operable Prostate Cancer 58 consecutive prostate patients treated with radical prostatectomy1.00.8EGFR negative0.6Disease free survival0.4EGFR expression was highly correlated with disease recurrence in these patients with operable prosate cancer.0.2EGFR positivep<0.0102030405060MonthsDi Lorenzo et al. Clin Cancer Res 2002; 8:
16 EGFR Expression and disease features Overexpression associated with:Metastasis, advanced diseasePoor outcomeResistance to chemotherapy, hormonotherapy, radiotherapyEGFR is generally expressed in a wide range of human solid tumors. Expression is very variable due to the different detection assays used, since there is no standardized method of measuring EGFR expression.
17 Shortened Median Survival Role of HER2/neu in Breast CancerHER2/neu oncogene amplificationShortened Median SurvivalHER2/neu overexpressing3 yearsHER2/neu normal6-7 yearsThis slide shows the original report by Slamon 15 years ago about the relevance of HER2/neu gene amplification and HER2/neu protein overexpression in human breast cancer in terms of the prognostic indication of shorter survival.HER2 oncoprotein overexpressionReprinted with permission from Slamon DJ et al. Science 1987; 235:Copyright American Association for the Advancement of Science.
18 Basi razionali per il blocco di segnali mitogenici in combinazione con trattamenti standard In risposta a farmaci citotossici o radiazioni ionizzanti le cellule tumorali cercano di sfuggire all’apoptosi attivando segnali mitogenici regolati da fattori di crescita e proteine anti-apoptotiche.Il blocco dei segnali mitogenici e antiapoptotici mediante inibitori selettivi aumenterebbe l’attività di trattamenti convenzionali, forzando le cellule tumorali a entrare in apoptosi.Molte cellule tumorali sono particolarmente sensibili all’inibizione di EGFR rispetto a quelle normali. Pertanto il blocco di EGFR potrebbe inibirne la proliferazione (J. Mendelsohn, 1982).La scoperta di alcuni meccanismi molecolari della crescita tumorale e del ruolo di EGFR ha generato la seguente ipotesi di strategia terapeutica. Cellule tumorali trattate con farmaci citotossici o con radioterapia cercano di sfuggire alla morte apoptotica attivando una serie di segnali di sopravvivenza, veicolati da proteine cruciali tra cui EGFR. Il blocco di tali segnali mediante inibitori selettivi dovrebbe potenziare l’effetto dei trattamenti convenzionali forzando le cellule tumorali a morire per apoptosi.Ciardiello & Tortora, 2002
20 Anti-EGFR Approaches MAbs TKIs Signal transduction Signal transduction LigandLigandKKTKIsKKThere are various mechanisms by which the EGFR can be blocked. Anti-EGFR monoclonal antibodies bind extracellularly to the ligand-binding domain of the EGFR and prevent binding of the endogenous ligands, EGF and TGF-. Extracellular blockade prevents receptor dimerization and autophosphorylation from occurring, thus inhibiting activation of the tyrosine kinase signaling pathways.Tyrosine kinase (TK) inhibitors act directly on the cytoplasmic domain of the EGFR, inhibiting the activity of the EGFR pathway. A class of quinazoline derivatives has been developed that is more specific for the EGFR than earlier TK inhibitors, which exhibited varying specificity.Ligand conjugates, in which EGFR ligands are conjugated to toxins (such as ricin, genistein, and Pseudomonas exotoxin), bind exclusively to the EGFR in an extra-cellular fashion similar to monoclonal antibodies. In this method, a toxic and lethal compound is delivered to the cell after internalization of the ligand-toxin conjugate, resulting in cell death.Antisense oligonucleotides targeted to the EGFR prevent the translation of mRNA into protein. EGFR antisense therapy is in very early stages of development, and the clinical utility of this form of EGFR blockade is under study.SignaltransductionSignaltransductionModified after Tabernero, 2003
21 Ligand-binding domain Omnitarg and Trastuzumab PossessDistinct Epitope Specificity for HER2HER2Ligand-binding domain(inactive)OmnitargTrastuzumabCell membraneTyrosine kinase domain
22 receptor internalization Drug-drug interactions Differences between MAbs and small molecules TKISmall-MoleculeAntibodyTargetTK domainexternal domainSpecificity+++++++Bindingreversiblereceptor internalizationActivityDosingoral, dailyIV, weeklyToxicityrash, diarrhearashDrug-drug interactions--Courtesy of C. Sessa
23 Small molecules inhibitors of EGFR-TK AgentEGFR IC50 (nM)EGFR-specificInhibitionDevelopment stageGefitinib23yesreversiblephase IIIErlotinib20CI-10332noirreversiblephase IPKI-1660.7GW-20169.2EKB-56938.5PDIpreclinicalAG-1478<3CGP-59326A27
24 ANTI-EGFR MABs IN CANCER THERAPY AntibodyTargetDegree of humanizationIndicationStatusTrastuzumabHER2Fully humanizedMetastatic breast cancerLicensed in 1998 USA, Switzerland, parts of Latin AmericaCetuximabEGFRChimericSCHN, NSCLC, breast, colorectal, pancreatic, esophageal, renal-celland ovarian cancerPhase II/III clinical trialsABX-EGFAdvanced EGFR+ cancerPhase I clinical trialsEMD 55900Murine MAbMalignant gliomaPhase II trialsEMD 72000Phase I/II clinical trialsTheraCIMBreast, SCHN, lung and brain cancerApproval granted for phase I/II trials
25 Growth factors and angiogenesis in GEO colon cancer xenografts treated with Cetuximab Tumor size (cm3)Ki67 (%)b-FGF (%)VEGF (%)TGFα (%)MVD* (FVIII-RA)Control1.9860557521 ± 3Cetuximab0.42030258 ± 2MVD = Microvessel densityCiardiello F. et al. Clin Cancer Res 2000; 6:
26 Combination of Cetuximab with cytotoxic therapies Increased apoptosis and enhanced antitumor activity demonstrated in preclinical models with Cetuximab in combination with:5-FUIrinotecan, topotecanCisplatin, carboplatin, oxaliplatinPaclitaxel, docetaxelGemcitabineVinorelbineDoxorubicinRadiotherapy
27 Effects of RT in combination with C225 in human GEO cancer xenografts ControlRTC225CombinationUn potente effetto cooperativo è stato osservato anche con inibitori di EGFR e radioterapia. L’esempio accanto dimostra come la radioterapia a dosi subottimali, combinata con una dose standard di C225, produca un potente effetto antitumorale in topi nudi xenotrapiantati con un tumore colorettale umano. Su questa base sono stati avviati studi clinici in diversi tipi di tumore.treatment
28 Antitumor activity of Cetuximab in combination with cisplatin in squamous tumors Combined effects of C225 + cisplatin in A431 xenograftsC225CisplatinC225 + cisplatin64210050CisplatinControlTumor size (cm3)Survival (%)C225CisplatinC225C225 + cisplatinControlDaysDaysFan et al. Cancer Res 1993; 53:
29 Problemi emergentiVie di fuga e acquisizione di resistenza a inibitori di EGFRMutazioni nel dominio TK di EGFR
31 Main escape pathways EGFR erbB2 erbB3 erbB4 Cell Proliferation raf p y TGFaEGFRerbB2erbB3erbB4Main escape pathwaysrasrafpSOStyrosinekinaseyGRB2pyPKAIpMEKyyPLCgPI-3KpMAPKCyclinD1VEGFPTENAKTBcl-2COX-2AngiogenesisSurvival,anti-apoptosisCell ProliferationInvasionmetastasisTortora & Ciardiello,2003
32 Mutated EGFR isoforms in NSCLC patients EGFR L858RFrequent in Japanese NSCLCMore frequent in womenMore frequent in adenocarcinomasNon-smokers or former smokersGefitinib-sensitiveExons 18-21Lynch et al., NEJM 350: 2004
33 EGFR gene sequence analysis in mCRC patients treated with Cetuximab after failing standard treatmentsPR18All wtSD86 wt / 2 mutPD99 wtMutants are of a novel type: heterozygous P753L in exon 19 and heterozygous V689 in exon 18Lenz et al., ASCO 2004
34 Role of EGFR in the response to EGFR inhibitors EGFR ExpressionSkin rash
35 Tarceva phase II trial in NSCLC: survival by grade of rash 1.000.750.500.250.00Grade 2/3 (n=17)*vs no rashSurvival distribution functionGrade 1 (n=26)No rash (n=14)Key words:Tarceva (erlotinib)Tarceva (erlotinib): monotherapyTarceva (erlotinib): dailyPhase IILung cancer: NSCLC: second line/third lineAdverse events: rashSurvivalSurrogate markerMonthsPérez-Soler R, et al. Lung Cancer 2003;41(Suppl. 2):S246 (Abs. P-611)
36 Clinical trials with anti-EGFR agents showing a relationship between rash and survival CETUXIMAB (Cunningham et al., NEJM 2004; Saltz et al., ASCO 2003)Phase II: + CPT-11 in CRCPhase II: CRCPhase II: + Cisplatin in H&N cancerPhase II: + Gemcitabine in pancreatic cancerPhase III: single agent vs. CPT-11 in CRCERLOTINIB (Clark et al., ASCO 2003)Phase II: NSCLCPhase II: H&NPhase II: ovarianGEFITINIB (Cohen et al., JCO 2003)These are the only subsets which show some difference: Age and Tumor type in might be of interest in correlation to EGFR mutations (more frequent in Adeno-Ca and younger (female) patients. LDH?Race: only one subset treatedGender: well balanced in the two treatment arms but no significant difference in RRKPS: only 3 patients with KPS <80, no significant difference between the treatment arms for KPS>80EGFR expression: no response correlation to staining intensity or % stained cells. We discussed during our meeting the reason for this (sensitivity of ICH?).Disease stage: only 7 patients with stage IIIb
37 PARAMETERS THAT MAY AFFECT/PREDICT CLINICAL RESPONSE TO EGFR-INHIBITORS EGFR expression and its efficient inhibitionPharmacodynamic markers (surrogates of response)Signalling downstream to EGFR
38 Rational basis for combination of EGFR and VEGF inhibitors Activation of EGFR by EGF or TGFα can up-regulate the production of VEGF in cancer cellsEGFR inhibition reduces VEGF productionResistance to EGFR inhibitors is associated with VEGF overexpression