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I recettori della famiglia erbB/HER I recettori della famiglia erbB/HER
come bersaglio terapeuticocome 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”
Fibroblast
Autocrine and Paracrine growth regulation
VEGF
First evidence by Sporn, M. and Todaro, G. New England Journal of Medicine 1980; 383: 878
TGF
TGFTGF
Autocrine Growth Paracrine Growth
VEGF
VEGF
Endothelial cells
TGF
Tortora & Ciardiello, 2003
Increasing Complexity of Growth Factors and Increasing Complexity of Growth Factors and Receptors of the EGF Family in EvolutionReceptors of the EGF Family in Evolution
Species Receptors Ligands
C. elegans LET-23 LIN-3
D. melanogaster DER I + II + III(splice variants)
SpitzVeinGurkenArgos (inhibitor)
H. sapiens EGFRHER2/neu (c-erbB-2) HER3 (c-erbB-3)HER4 (c-erbB-4)
EGFTGF AmphiregulinHB-EGF, Betacellulin, Epiregulin, NRG-1,2,3,4
Yarden et al., 2002
EGFTGF
Amphiregulin-cellulinHB-EGF
Epiregulin Heregulins
NRG2NRG3
Heregulins-cellulin
Cysteine-richdomains
Tyrosine kinasedomain
HER1EGFRErbB-1
HER2/neuErbB-2
HER3ErbB-3
HER4ErbB-4
C-terminus
100
100
100
44
82
33
36
59
24
48
79
28
The EGFR (erbB) Family and Ligands
nucleus
EGFR EGFR
HER2/neu
Ligand-induced Receptor Dimerization
Cell Membrane
TGF
HER2/
neu HER3TGF
EGFRHER4
tyrosinekinase
tyrosinekinase
EGFR
Signal transduction through EGFR, ErbB-2 and ErbB-3:heterodimer formation
K K
Ligand
EGFR ErbB-2
Ligand
ErbB-2 ErbB-3
K
Ligand
EGFR ErbB-3
K
Normanno et al., J Cell Physiol 2002
The HER/erbB Signaling Network
Yarden Y and Sliwkowski M. Nat Rev Mol Cell Biol 2001; 2: 127–37.
SrcCbl
PLC PI3KShp2 GAP
Akt
Bad S6KPKC
Sos
Grb2 Nck
Ras-GTP
Ras-GDP
MAPKMEK
RAF
JNKJNKK
PAKAbl
Rac
Vav
ShcGrb7
CrkJak
CytokinesNRG3(4)
NRG2(4)
NRG1(3,4)
Amphi-regulin
(1)
HB-EGF(1,4)
-cellulin(1)
Epiregulin(1,4)
EGF(1)
TGF(1)
LPA,thrombinET, etc.
NRG4(4)
ElkJun
FosMycSp1 Egr1 Stat
1 31
122 2
124 1 4 3 2
4 443 3 3
Inputlayer
Outputlayer
Apoptosis Migration Growth Adhesion Differentiation
Ligands
Receptordimers
Signalling cascade
Transcriptionfactors
EGFR Activation by Other Receptor Signaling Pathways
Cell Membranetyrosinekinase
EGFR
GPCRIntegrins
Cytokine receptors VGCC
Ca++
Ca++
SrcPKC
JAK2
?
Survival MotilityProliferation
?
EndothelinA
Tortora & Ciardiello, 2004
PKAI
PI-3KPLCγ
py
tyrosinekinase
GRB2 SOSp21ras
raf
MEK
MAPK
y py p
EGF TGFα
y
EGFR
p
Cell proliferation Angiogenesis
Prostaglandins
COX-2
Tortora et al. , 2003
COX-2 is involved in EGFR-dependent signaling
HP and EGFR
Helicobacter pylori VacA toxin up-regulates VEGF expression in gastric cancer cells through an EGFR-, COX-2-dependent mechanism.
EGFRHP VacA toxin
COX-2
VEGF Endothelial cells
VEGF
VEGF
Gastric cancercells
Caputo et al., 2003
Mechanisms of EGFR Dysregulation
Overexpress Ligand
Overexpress Receptors
Mutations ConferringConstitutive Activity
EGFRv.II/III
EGFRv.I Defective
Internalizationor
DefectiveDownregulation by Phosphatase
Tortora & Ciardiello, 2004 (Modified by Wiley and Burke. Traffic. 2001;2:12).
PKAI
ras raf
MEK
PI-3KPLC py
tyrosinekinase
GRB2 SOSy py p
EGF TGF
y
EGFRerbB2erbB3erbB4
p
Cyclin D1
CDK
Rb
Proliferazione Cellulare
AKTPTEN
Effetti pleiotropici dell’attivazione di EGFR
MAPK
Bcl-2
Angiogenesi
Invasione,metastasi
SopravvivenzaAnti-apoptosi
Resistenza a Chemioterapia,Resistenza a Chemioterapia,Radioterapia e OrmonoterapiaRadioterapia e Ormonoterapia
VEGFCOX-2
Tortora & Ciardiello, 2003
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%
0 NA
bladder 35-86% 9-50% 30-56%
30%
ovary 35-70%
8-32%
85% 93%
Type of cancer EGFR ErbB-2 ErbB-3
ErbB-4
head and neck 36-100% 17-53% 81%
28-69%
Normanno, Endocr Relat Cancer 2003
Grandis JR et al. J Natl Cancer Inst 1998; 90: 824–832.
EGFR
Low
Medium
Highp=0.0001
1.0
0.8
0.6
0.4
0.2
0.0Pro
po
rtio
n s
urv
ivin
g w
ith
NE
D
0 1 2 3 4 5 6Years after surgery
TGF
p=0.0001
Low
Medium
High
1.0
0.8
0.6
0.4
0.2
0.0Pro
po
rtio
n s
urv
ivin
g w
ith
NE
D
0 1 2 3 4 5 6
Years after surgery
Disease-free Survival According to EGFR and TGF Levels in HNSCC
58 consecutive prostate patients treated with radical prostatectomy
Di Lorenzo et al. Clin Cancer Res 2002; 8: 3438-3444.
EGFR Expression and Disease-freeSurvival in Operable Prostate Cancer
0 10 20 30 40 50 60
0.0
0.2
0.4
0.6
0.8
1.0
EGFR negative
EGFR positive
p<0.00004
Dis
eas
e fr
ee
su
rviv
al
Months
EGFR Expression and disease features
Metastasis, advanced disease
Poor outcome
Resistance to chemotherapy, hormonotherapy, radiotherapy
Overexpression associated with:
Reprinted with permission from Slamon DJ et al. Science 1987; 235: 177-182. Copyright 1987. American Association for the Advancement of Science.
HER2 oncoprotein overexpression
HER2/neu oncogeneamplification
Shortened Median Survival
HER2/neu overexpressing
3 years
HER2/neu normal 6-7 years
Role of HER2/Role of HER2/neuneu in Breast Cancer in Breast Cancer
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).
Ciardiello & Tortora, 2002
THERAPEUTIC STRATEGYTHERAPEUTIC STRATEGY
ChemotherapyRadiotherapy
TUMOR INHIBITION(apoptosis + tumor dormancy)
RTK inhibitors Survival signals
(RTK)
CELL DAMAGECell Death(apoptosis)
CELL PROLIFERATION
Tortora & Ciardiello, 2004
Signal transduction
Signal transduction
MAbs
TKIs
Ligand
KK KK
Ligand
Anti-EGFR Approaches
Modified after Tabernero, 2003
Omnitarg and Trastuzumab PossessDistinct Epitope Specificity for HER2
HER2
Ligand-binding domain(inactive)
Cell membrane
Tyrosine kinase domain
Omnitarg
Trastuzumab
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
--
Differences between MAbs and small molecules TKI
Courtesy of C. Sessa
Small molecules inhibitors of EGFR-TKSmall molecules inhibitors of EGFR-TK
Agent EGFR IC50 (nM)
EGFR-specific Inhibition Development stage
Gefitinib 23 yes reversible phase III
Erlotinib 20 yes reversible phase III
CI-1033 2 no irreversible phase I
PKI-166 0.7 yes reversible phase I
GW-2016 9.2 no reversible phase I
EKB-569 38.5 yes irreversible phase I
PDI-168393 0.7 no irreversible preclinical
AG-1478 <3 no irreversible preclinical
CGP-59326A 27 yes reversible preclinical
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 EGFR Fully humanized Advanced EGFR+ cancer
Phase I clinical trials
EMD 55900 EGFR Murine MAb Malignant glioma Phase II trials
EMD 72000 EGFR Fully humanized Advanced EGFR+ cancer
Phase I/II clinical trials
TheraCIM EGFR Fully humanized Breast, SCHN, lung and brain cancer
Approval granted for phase I/II trials
ANTI-EGFR MABs IN CANCER THERAPY
Ciardiello F. et al. Clin Cancer Res 2000; 6: 3739-3747
Growth factors and angiogenesis in GEO colon Growth factors and angiogenesis in GEO colon cancer xenografts treated with Cetuximabcancer xenografts treated with Cetuximab
Tumor size (cm3)
Ki67 (%)
b-FGF (%)
VEGF (%)
TGFα (%)
MVD*(FVIII-RA)
Control 1.98 60 55 60 75 21 ± 3
Cetuximab 0.4 20 30 30 25 8 ± 2
MVD = Microvessel density
Combination of Cetuximab with cytotoxic therapiesCombination 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
Days
0 10 20 30 40 50 60 70 80 90 100 110 120 130
Tum
or
Vol
ume
(cm
3 )
0
1
2
3
Effects of RT in combination with C225in human GEO cancer xenografts
Control
RT
C225
Combination
treatment
Fan et al. Cancer Res 1993; 53: 4637-4642
Antitumor activity of Cetuximab in combination with Antitumor activity of Cetuximab in combination with cisplatin in squamous tumorscisplatin in squamous tumors
0 20 40 60 80 180
100
50
0
6
4
2
0
Tum
or s
ize
(cm
3)
C225
Cisplatin
0 5 15 25 35Days Days
Sur
viva
l (%
)
Control
C225
Cisplatin
C225 + cisplatin
C225 + cisplatin
Control
Cisplatin
C225
Combined effects of C225 + cisplatin in A431 xenografts
• Vie di fuga e acquisizione di resistenza a inibitori di EGFR
• Mutazioni nel dominio TK di EGFR
Problemi emergenti
PKAI
PI3KPLCp
y
tyrosinekinase
GRB2 SOS p21ras
Raf
MEK
MAPK
y py p
EGF TGF
y
EGFRerbB2erbB3erbB4
p
Cyclin D1
CDK
RbCell Proliferation
E2F
p14ARF
mdm2
p53
PKC
CANCER CELLS HAVE HYPERACTIVE SIGNALLING
PATHWAYS
AKTPTEN
PKAI
ras raf
MEK
PI-3KPLC py
tyrosinekinase
GRB2 SOSy py p
EGF TGF
y
EGFRerbB2erbB3erbB4
p
Cyclin D1
Cell Proliferation
AKTPTEN
Main escape pathways
MAPK
Bcl-2
Angiogenesis
Invasionmetastasis
Survival, anti-apoptosis
VEGF
Tortora & Ciardiello,2003
COX-2
Mutated EGFR isoforms Mutated EGFR isoforms in NSCLC patientsin NSCLC patients
Lynch et al., NEJM 350: 2004
Exons 18-21
EGFR L858R•Frequent in Japanese NSCLC
•More frequent in women
•More frequent in adenocarcinomas
•Non-smokers or former smokers
•Gefitinib-sensitive
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
Role of EGFR in the response to EGFR inhibitorsRole of EGFR in the response to EGFR inhibitors
EGFR Expression
Skin rash
Tarceva phase II trial in NSCLC:survival by grade of rash
Su
rviv
al d
istr
ibu
tio
n f
un
ctio
n
Months
Grade 2/3 (n=17)
Grade 1 (n=26)
No rash (n=14)
Median survival (95% CI)
No rash 1.5 (1–2.2)
Grade 1 8.5 (4.8–14.8) p<0.0001*
Grade 2/3 19.6 (10.8–22.1+) p<0.0001*
0 5 10 15 20 25 30
1.00
0.75
0.50
0.25
0.00
*vs no rash
Pérez-Soler R, et al. Lung Cancer 2003;41(Suppl. 2):S246 (Abs. P-611)
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)
Phase II: H&N
1. EGFR expression and its efficient inhibition
2. Pharmacodynamic markers (surrogates of response)
3. Signalling downstream to EGFR
PARAMETERS THAT MAY AFFECT/PREDICT CLINICAL RESPONSE TO EGFR-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
Rational basis for combination of EGFR Rational basis for combination of EGFR and VEGF inhibitorsand VEGF inhibitors
THERAPEUTIC STRATEGIESTHERAPEUTIC STRATEGIES
ChemotherapyRadiotherapy
RTK inhibitors
Genomic signals Survival signals
GENE DAMAGE
Selective Inhibitors
Cell Death(apoptosis)
Tortora & Ciardiello, 2004
TUMOR INHIBITION(apoptosis + tumor dormancy)Cell Proliferation