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I recettori della famiglia erbB/HER come bersaglio terapeutico
Giampaolo Tortora Divisione di Oncologia Medica e Laboratori di Terapia Molecolare dei Tumori Dipartimento di Endocrinologia e Oncologia Molecolare e Clinica Università di Napoli “Federico II”
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Autocrine and Paracrine growth regulation
First evidence by Sporn, M. and Todaro, G. New England Journal of Medicine 1980; 383: 878 Autocrine Growth Paracrine Growth TGFa TGFa Fibroblast TGFa TGFa VEGF VEGF VEGF Endothelial cells Tortora & Ciardiello, 2003
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Increasing Complexity of Growth Factors and Receptors of the EGF Family in Evolution
Species Receptors Ligands C. elegans LET-23 LIN-3 D. melanogaster DER I + II + III (splice variants) Spitz Vein Gurken Argos (inhibitor) H. sapiens EGFR HER2/neu (c-erbB-2) HER3 (c-erbB-3) HER4 (c-erbB-4) EGF TGFa Amphiregulin HB-EGF, Betacellulin, Epiregulin, NRG-1,2,3,4 The 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
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The EGFR (erbB) Family and Ligands
TGFa Amphiregulin b-cellulin HB-EGF Epiregulin NRG2 NRG3 Heregulins b-cellulin Heregulins Cysteine-rich domains 100 44 82 33 36 59 24 48 79 28 The 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 kinase domain C-terminus HER1 EGFR ErbB-1 HER2/neu ErbB-2 HER3 ErbB-3 HER4 ErbB-4
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Ligand-induced Receptor Dimerization
TGFa TGFa HER2/ neu HER3 HER4 EGFR Cell Membrane EGFR EGFR EGFR tyrosine kinase 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. tyrosine kinase HER2/neu nucleus
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Signal transduction through EGFR, ErbB-2 and ErbB-3:heterodimer formation
K Ligand EGFR ErbB-2 Ligand û ErbB-2 ErbB-3 K Ligand û EGFR ErbB-3 K Normanno et al., J Cell Physiol 2002
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The HER/erbB Signaling Network
Src Cbl PLCg PI3K Shp2 GAP Akt Bad S6K PKC Sos Grb2 Nck Ras-GTP Ras-GDP MAPK MEK RAF JNK JNKK PAK Abl Rac Vav Shc Grb7 Crk Jak Cytokines NRG3 (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) Elk Jun Fos Myc Sp1 Egr1 Stat 1 3 2 4 Ligands Input layer Receptor dimers Signalling cascade A 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 layer Transcription factors Apoptosis Migration Growth Adhesion Differentiation Yarden Y and Sliwkowski M. Nat Rev Mol Cell Biol 2001; 2: 127–37.
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EGFR Activation by Other Receptor Signaling Pathways
Cytokine receptors Integrins VGCC GPCR Ca++ EndothelinA JAK2 Src PKC Ca++ Cell Membrane tyrosine kinase ? ? 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. ? Survival Proliferation Motility Tortora & Ciardiello, 2004
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COX-2 is involved in EGFR-dependent signaling
TGFα EGFR p21 ras p GRB2 SOS raf tyrosine kinase y p p y PKAI y MEK PLCγ y PI-3K p MAPK COX-2 Prostaglandins Cell proliferation Angiogenesis Tortora et al. , 2003
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HP and EGFR Helicobacter pylori VacA toxin up-regulates VEGF expression in gastric cancer cells through an EGFR-, COX-2-dependent mechanism. HP VacA toxin EGFR Gastric cancer cells COX-2 VEGF VEGF VEGF Endothelial cells Caputo et al., 2003
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Mechanisms of EGFR Dysregulation
Overexpress Ligand Overexpress Receptors Mutations Conferring Constitutive Activity Tyrosine 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 potential Cell cycle progression There 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 endocytic trafficking. Traffic. 2001;2:12-18. EGFR v.II/III EGFR v.I Defective Internalization or Downregulation by Phosphatase Tortora & Ciardiello, 2004 (Modified by Wiley and Burke. Traffic. 2001;2:12).
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Effetti pleiotropici dell’attivazione di EGFR
erbB2 erbB3 erbB4 EGF TGFa Effetti pleiotropici dell’attivazione di EGFR ras raf p SOS tyrosine kinase y GRB2 p y PKAI p MEK y y PLCg PI-3K p MAPK VEGF Cyclin D1 COX-2 PTEN AKT CDK Bcl-2 Angiogenesi Rb Sopravvivenza Anti-apoptosi Proliferazione Cellulare Invasione, metastasi Resistenza a Chemioterapia, Radioterapia e Ormonoterapia Tortora & Ciardiello, 2003
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Type of cancer EGFR ErbB-2 ErbB-3 ErbB-4 lung 40-80% 18-60% 25-85% NA
Type of cancer EGFR ErbB-2 ErbB-3 ErbB-4 lung 40-80% 18-60% 25-85% NA breast 14-91% 9-39% 22-90% 82% stomach 33-74% 8-40% 35-100% NA colon 25-77% 11-20% 65-89% NA esophagus 43-89% 7-64% 64% NA liver 47-68% 0-29% 84% 61% pancreas 30-50% 19-45% 57-63% 81% prostate 40-80% 40-80% 22-96% NA kidney 50-90% 0-40% NA bladder 35-86% 9-50% 30-56% 30% ovary 35-70% 8-32% 85% 93% head and neck 36-100% 17-53% 81% 28-69% Normanno, Endocr Relat Cancer 2003
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Disease-free Survival According to EGFR and TGFa Levels in HNSCC
Low Medium High p=0.0001 1.0 0.8 0.6 0.4 0.2 0.0 Proportion surviving with NED TGFa p=0.0001 Low Medium High 1.0 0.8 0.6 0.4 0.2 0.0 Proportion surviving with NED Years after surgery This 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 disease Years after surgery Grandis JR et al. J Natl Cancer Inst 1998; 90: 824–832.
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EGFR Expression and Disease-free Survival in Operable Prostate Cancer
58 consecutive prostate patients treated with radical prostatectomy 1.0 0.8 EGFR negative 0.6 Disease free survival 0.4 EGFR expression was highly correlated with disease recurrence in these patients with operable prosate cancer. 0.2 EGFR positive p< 0.0 10 20 30 40 50 60 Months Di Lorenzo et al. Clin Cancer Res 2002; 8:
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EGFR Expression and disease features
Overexpression associated with: Metastasis, advanced disease Poor outcome Resistance to chemotherapy, hormonotherapy, radiotherapy EGFR 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.
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Shortened Median Survival
Role of HER2/neu in Breast Cancer HER2/neu oncogene amplification Shortened Median Survival HER2/neu overexpressing 3 years HER2/neu normal 6-7 years This 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 overexpression Reprinted with permission from Slamon DJ et al. Science 1987; 235: Copyright American Association for the Advancement of Science.
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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
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(apoptosis + tumor dormancy)
THERAPEUTIC STRATEGY Chemotherapy Radiotherapy Cell Death (apoptosis) CELL DAMAGE Survival signals (RTK) RTK inhibitors TUMOR INHIBITION (apoptosis + tumor dormancy) CELL PROLIFERATION Tortora & Ciardiello, 2004
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Anti-EGFR Approaches MAbs TKIs Signal transduction Signal transduction
Ligand Ligand K K TKIs K K There 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. Signal transduction Signal transduction Modified after Tabernero, 2003
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Ligand-binding domain
Omnitarg and Trastuzumab Possess Distinct Epitope Specificity for HER2 HER2 Ligand-binding domain (inactive) Omnitarg Trastuzumab Cell membrane Tyrosine kinase domain
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receptor internalization Drug-drug interactions
Differences between MAbs and small molecules TKI Small-Molecule Antibody Target TK domain external domain Specificity +++ ++++ Binding reversible receptor internalization Activity Dosing oral, daily IV, weekly Toxicity rash, diarrhea rash Drug-drug interactions -- Courtesy of C. Sessa
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Small molecules inhibitors of EGFR-TK
Agent EGFR IC50 (nM) EGFR-specific Inhibition Development stage Gefitinib 23 yes reversible phase III Erlotinib 20 CI-1033 2 no irreversible phase I PKI-166 0.7 GW-2016 9.2 EKB-569 38.5 PDI preclinical AG-1478 <3 CGP-59326A 27
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ANTI-EGFR MABs IN CANCER THERAPY
Antibody Target Degree of humanization Indication Status Trastuzumab HER2 Fully humanized Metastatic breast cancer Licensed in 1998 USA, Switzerland, parts of Latin America Cetuximab EGFR Chimeric SCHN, NSCLC, breast, colorectal, pancreatic, esophageal, renal-cell and ovarian cancer Phase II/III clinical trials ABX-EGF Advanced EGFR+ cancer Phase I clinical trials EMD 55900 Murine MAb Malignant glioma Phase II trials EMD 72000 Phase I/II clinical trials TheraCIM Breast, SCHN, lung and brain cancer Approval granted for phase I/II trials
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Growth factors and angiogenesis in GEO colon cancer xenografts treated with Cetuximab
Tumor size (cm3) Ki67 (%) b-FGF (%) VEGF (%) TGFα (%) MVD* (FVIII-RA) Control 1.98 60 55 75 21 ± 3 Cetuximab 0.4 20 30 25 8 ± 2 MVD = Microvessel density Ciardiello F. et al. Clin Cancer Res 2000; 6:
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Combination of Cetuximab with cytotoxic therapies
Increased apoptosis and enhanced antitumor activity demonstrated in preclinical models with Cetuximab in combination with: 5-FU Irinotecan, topotecan Cisplatin, carboplatin, oxaliplatin Paclitaxel, docetaxel Gemcitabine Vinorelbine Doxorubicin Radiotherapy
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Effects of RT in combination with C225 in human GEO cancer xenografts
Control RT C225 Combination Un 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
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Antitumor activity of Cetuximab in combination with cisplatin in squamous tumors
Combined effects of C225 + cisplatin in A431 xenografts C225 Cisplatin C225 + cisplatin 6 4 2 100 50 Cisplatin Control Tumor size (cm3) Survival (%) C225 Cisplatin C225 C225 + cisplatin Control Days Days Fan et al. Cancer Res 1993; 53:
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Problemi emergenti Vie di fuga e acquisizione di resistenza a inibitori di EGFR Mutazioni nel dominio TK di EGFR
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HYPERACTIVE SIGNALLING
EGF TGFa CANCER CELLS HAVE HYPERACTIVE SIGNALLING PATHWAYS EGFR erbB2 erbB3 erbB4 tyrosine kinase p GRB2 SOS y p21 ras Raf p y PKAI p y MEK PI3K PLCg y p MAPK PKC PTEN AKT p14ARF Cyclin D1 mdm2 E2F CDK p53 Cell Proliferation Rb
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Main escape pathways EGFR erbB2 erbB3 erbB4 Cell Proliferation raf p y
TGFa EGFR erbB2 erbB3 erbB4 Main escape pathways ras raf p SOS tyrosine kinase y GRB2 p y PKAI p MEK y y PLCg PI-3K p MAPK Cyclin D1 VEGF PTEN AKT Bcl-2 COX-2 Angiogenesis Survival, anti-apoptosis Cell Proliferation Invasion metastasis Tortora & Ciardiello,2003
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Mutated EGFR isoforms in NSCLC patients
EGFR L858R Frequent in Japanese NSCLC More frequent in women More frequent in adenocarcinomas Non-smokers or former smokers Gefitinib-sensitive Exons 18-21 Lynch et al., NEJM 350: 2004
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EGFR gene sequence analysis in mCRC patients
treated with Cetuximab after failing standard treatments PR 18 All wt SD 8 6 wt / 2 mut PD 9 9 wt Mutants are of a novel type: heterozygous P753L in exon 19 and heterozygous V689 in exon 18 Lenz et al., ASCO 2004
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Role of EGFR in the response to EGFR inhibitors
EGFR Expression Skin rash
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Tarceva phase II trial in NSCLC: survival by grade of rash
1.00 0.75 0.50 0.25 0.00 Grade 2/3 (n=17) *vs no rash Survival distribution function Grade 1 (n=26) No rash (n=14) Key words: Tarceva (erlotinib) Tarceva (erlotinib): monotherapy Tarceva (erlotinib): daily Phase II Lung cancer: NSCLC: second line/third line Adverse events: rash Survival Surrogate marker Months Pérez-Soler R, et al. Lung Cancer 2003;41(Suppl. 2):S246 (Abs. P-611)
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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 CRC Phase II: CRC Phase II: + Cisplatin in H&N cancer Phase II: + Gemcitabine in pancreatic cancer Phase III: single agent vs. CPT-11 in CRC ERLOTINIB (Clark et al., ASCO 2003) Phase II: NSCLC Phase II: H&N Phase II: ovarian GEFITINIB (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 treated Gender: well balanced in the two treatment arms but no significant difference in RR KPS: only 3 patients with KPS <80, no significant difference between the treatment arms for KPS>80 EGFR 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
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PARAMETERS THAT MAY AFFECT/PREDICT CLINICAL RESPONSE TO EGFR-INHIBITORS
EGFR expression and its efficient inhibition Pharmacodynamic markers (surrogates of response) Signalling downstream to EGFR
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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 cells EGFR inhibition reduces VEGF production Resistance to EGFR inhibitors is associated with VEGF overexpression
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THERAPEUTIC STRATEGIES
Chemotherapy Radiotherapy Cell Death (apoptosis) GENE DAMAGE Genomic signals Survival signals Selective Inhibitors RTK inhibitors TUMOR INHIBITION (apoptosis + tumor dormancy) Cell Proliferation Tortora & Ciardiello, 2004
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