Vecchi e nuovi “targets”, vecchi e nuovi farmaciVecchi e nuovi “targets”, vecchi e nuovi farmaci
Fortunato Ciardiello
Division of Medical Oncology,
Department of Clinical and Experimental Medicine,
Second University of Naples, Italy
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 LigandsThe EGFR (erbB) Family and Ligands
nucleus
EGFR EGFR
HER2/neu
Ligand-induced Receptor DimerizationLigand-induced Receptor Dimerization
Cell Membrane
TGF
HER2/
neu HER3TGF
EGFRHER4
tyrosinekinase
tyrosinekinase
EGFR
Receptor Dimerization is Essential for Receptor Dimerization is Essential for Intracellular SignalingIntracellular Signaling
Individual receptor pairings can consist of two molecules of the same type (homodimers), or two molecules of different types (heterodimers).
All possible homo- and heterodimeric receptor complexes between members of the EGFR family have been identified in living cells.
Formation of heterodimers can significantly affect the duration (different internalization rate) and the type (activation of different pathways) of signaling.
The EGFR/erbB Signaling NetworkThe EGFR/erbB Signaling Network
Yarden Y and Sliwkowski M. Nat Rev Mol Cell Biol 2001; 2: 127–37.
SrcCbl
PLC PI3KShp2 GAP
AktBad 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
Apoptosis Migration Growth Adhesion Differentiation
1 31
122 2
124 1 4 3 2
4 443 3 3
Inputlayer
Signal-processinglayer
Outputlayer
Ligands
Receptordimers
Adaptorsand enzymes
Cascades
Transcriptionfactors
HER2/HER2/neuneu: Role in Breast Cancer: Role in Breast Cancer
HER2/neu plays an important role in the development and progression of human breast cancer.
HER2/neu is overexpressed in 25-30% of human breast cancers. Protein overepression is generally due to gene amplification.
HER2/neu overexpression is generally associated with poor prognosis and with resistance to hormone therapy.
TGFTGF: Role in Breast Cancer: Role in Breast Cancer
Mitogen for mammary epithelial cells.
Estrogen-inducible in estrogen-dependentbreast cancer.
Expression increases from atypical hyperplasia tocarcinoma in situ, to invasive carcinoma.
Overexpression in 50-70% primary breast cancer.
EGFR: Role in Breast CancerEGFR: Role in Breast Cancer
Expressed in 35-60% primary breast cancers.
Overexpression correlates with multidrugresistance.
Inverse correlation with ER and PgR.
Overexpression correlates with resistance tohormonotherapy.
Overexpression is generally associated withpoor prognosis.
Co-expression of EGFR and ErbB-2Co-expression of EGFR and ErbB-2
Co-expression of EGFR and ErbB-2 has been observed in 10-30% primary human breast carcinomas.
Overexpression of both ErbB-2 and EGFR is associated with a poorer prognosis than overexpression of either receptor alone in breast cancer patients.
A recent study has demonstrated an adverse prognostic independent role of P-ErbB-2 and EGFR coexpression in a subset of radically resected early breast cancers (Di Giovanna et al., JCO, 23: 1152-1160, 2005).
Open clinical issues for the therapeutic use of Open clinical issues for the therapeutic use of EGFR-targeted drugsEGFR-targeted drugs
Appropriate selection of potentially responding patients to EGFR-targeted agents: EGFR expression is necessary. Is EGFR expression sufficient? “Gain of function” somatic EGFR gene mutations. Expression of ligands and receptors of the erbB family. Downstream signaling molecules activation (MAPK, AKT).
Timing and schedule for the combination of cytotoxic treatments and EGFR-targeted agents.
Combination with other signal transduction inhibitors and molecular targeted therapies.
Control of cancer cell resistance to EGFR-targeted agents.
EGFR inhibitors in pretreated NSCLC patients:EGFR inhibitors in pretreated NSCLC patients: possible interpretration of clinical resultspossible interpretration of clinical results
Non-Responders SD PR
10 - 15% 20 - 30% 55 - 70%
Apoptosis
Growth arrestNo effect on tumor growth
Non-EGFR-dependent GrowthEGFR-dependent Growth
TGF
Major clinicalbenefit: PR
EGFR mutations/gene amplification
TGF
Intermediate clinicalbenefit: SD
EGFR mutationsor
Wild-type EGFR+ HER2/HER3
+ EGFR ligands+ loss of Cbl
TGF
No clinicalbenefit: PD
Smokers with wild-type EGFR
(males?squamous?)
Modified from an original concept of Carlos Arteaga
Target loss in cancer cells.
Loss/inactivation of downstream signaling molecules.
Activation of downstream signaling pathways through EGFR-independent mechanisms: Other cell membrane growth factor receptors (IGF-I R); PTEN-PI3K-AKT pathway; Raf-ras-MEK-ERK pathway; Pro-angiogenic growth factors (VEGF) production; Bcl-2/Bcl-xL pathway.
Molecular changes in cancer cells which affect EGFR inhibitor uptake.
Possible mechanisms of resistance to Possible mechanisms of resistance to EGFR inhibitorsEGFR inhibitors
Resistance to gefitinib in EGFR-overexpressing MDA-468 Resistance to gefitinib in EGFR-overexpressing MDA-468 breast cancer cells with mutant PTEN and constitutive Akt breast cancer cells with mutant PTEN and constitutive Akt
activationactivation
Bianco R, et al. Oncogene 2003;22:2812–2822.
Reconstution of PTEN function restores sensitivityReconstution of PTEN function restores sensitivityto EGFR inhibitors in EGFR-overexpressing MDA-468to EGFR inhibitors in EGFR-overexpressing MDA-468
breast cancer cellsbreast cancer cells
Bianco R, et al. Oncogene 2003;22:2812–2822.
Combination with other tumor cell-directed signal transduction inhibitors: Bcl-2/Bcl-xL PKA-I COX-2 MDM-2 MAPK AKT
Combination with anti-angiogenic treatment modalities: VEGF signaling inhibitors (VEGF antisense oligos; VEGF neutralizing
antibodies; VEGFR blocking antibodies; VEGFR small molecule inhibitors);
Other angiogenesis inhibitors (endostatin).
Strategies to overcome resistance to EGFR Strategies to overcome resistance to EGFR inhibitorsinhibitors
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 and Rational basis for combination of EGFR and VEGF inhibitorsVEGF inhibitors
ZD6474:ZD6474:a VEGFR and EGFR inhibitora VEGFR and EGFR inhibitor
ZD6474 is a quinazoline, an orally bioavailable small molecule, that inhibits the tyrosine kinase domain of the VEGF Receptor 2 (KDR/FLK-1).
ZD6474 is a potent angiogenesis inhibitor.
ZD6474 is in phase II clinical development. ZD6474 also inhibits the EGFR tyrosine kinase.
HN
N
N
O
O
Cl
F
N
O
ZD6474
Gefitinib
Wedge SR, et al. Cancer Res 2002;62:4645–4655. Ciardiello F, et al. Clin Cancer Res 2003;9:1546–1556.
ras raf
MEK
MAPK
EGFR
Cyclin D1
Block of cancer cell proliferation
VEGF
Endothelial cellCancer cell
TGF
KDR
ZD6474
Proposed antitumor activity of ZD6474Proposed antitumor activity of ZD6474
Block of endothelial cell proliferation
Days
0 20 40 60 80 100 120 140 1600,0
0,5
1,0
1,5
2,0
2,5
3,0
Control C225 (continous treatment)ZD1839 (continous treatment)C225 (4 weeks of treatment)ZD1839 (4 weeks of treatment)ZD6474 (continous treatment) ZD6474 (4 weeks of treatment)
Tu
mo
r V
olu
me
(c
m3)
Development of resistant GEO colon cancer Development of resistant GEO colon cancer xenografts following chronic treatment with xenografts following chronic treatment with Cetuximab or Gefitinib, but not with ZD6474Cetuximab or Gefitinib, but not with ZD6474
Ciardiello F. et al. Clin Cancer Res 2004; 10: 784-793.
EGFR expressionin GEO cells resistant to Cetuximab or to Gefitinib
Effect of Gefitinib on EGFR phosphorylation in GEO cells resistant to Cetuximab or to Gefitinib
Ciardiello F. et al. Clin Cancer Res 2004; 10: 784-793.
Characteristics of EGFR-targeted drugs Characteristics of EGFR-targeted drugs resistant GEO cellsresistant GEO cells
Ciardiello F. et al. Clin Cancer Res 2004; 10: 784-793.
Acquired and constitutiveAcquired and constitutive resistance to EGFR inhibitors:resistance to EGFR inhibitors:
Differential sensitivity to EGFR inhibitorsDifferential sensitivity to EGFR inhibitorsCell line PTEN
statusEGFR levels
Gefitinib C225 ZD6474
MDA-468 mutated high low very low high
GEO normal moderate high high high
GEO-Gef-R normal moderate very low very low high
GEO-Cet-R normal moderate very low very low high
PC3 deleted moderate high very low high
PC3-Gef-R deleted moderate very low very low high
Acquired and constitutiveAcquired and constitutive resistance to EGFR inhibitors:resistance to EGFR inhibitors:Role of optimal pAKT inhibitionRole of optimal pAKT inhibition
MAPK
pMAPK
pAKT
PC3-Gef-RPC3-Gef-R
AKT
PC3PC3Ctr Cet
uxim
abG
efiti
nibZD64
74
Ctr Gef
itinib
ZD6474
ZD6474 is an orally available, small molecule tyrosine kinase inhibitor that blocks both the VEGFR-2 (FLK-1/KDR) and the EGFR.
ZD6474 in addition to inhibiting endothelial cell proliferation by blocking VEGF-induced signaling could inhibit cancer cell growth by blocking EGFR autocrine signaling.
ZD6474 produces a dose-dependent inhibition of tumour growth in a range of human xenograft models.
Long-term treatment of GEO xenografts with selective EGFR inhibitors results in the development of EGFR inhibitor-resistant cancer cells. Growth of EGFR inhibitor-resistant tumors can be inhibited by ZD6474.
Inhibition of VEGF signaling by ZD6474 is a potential anti-cancer strategy in tumors that become resistant to EGFR inhibitors.
ZD6474 inhibits AKT activation and cell proliferation in a panel of human cancer cell lines with intrinsic or acquired resistance to gefitinib or cetuximab.
ZD6474: Summary of preclinical dataZD6474: Summary of preclinical data
Increase in activated EGFR/HER2 dimers inIncrease in activated EGFR/HER2 dimers intamoxifen-resistant breast cancer cellstamoxifen-resistant breast cancer cells
Knowlden et al., Endocrinology 2003
Increase in activated EGFR/HER2 dimers in Increase in activated EGFR/HER2 dimers in tamoxifen-resistant breast cancer cellstamoxifen-resistant breast cancer cells
Knowlden et al., Endocrinology 2003
ER-HER2 cross talk in ER/HER2 positive ER-HER2 cross talk in ER/HER2 positive breast cancerbreast cancer
Chou et al., JNCI 2004
ER-HER2 cross talk in ER/HER2 positive ER-HER2 cross talk in ER/HER2 positive breast cancerbreast cancer
Chou et al., JNCI 2004
HER2 overexpression
Increased active EGFR/HER2 dimers with MAPK activation
phosphorylation and activation of CoA-AIB1
levels of activated CoA-AIB1 increase estrogen agonist activity of complex “Tamoxifen-ER”
resistance to tamoxifen
HER2 overexpression
Increased active EGFR/HER2 dimers with MAPK activation
phosphorylation and activation of CoA-AIB1
levels of activated CoA-AIB1 increase estrogen agonist activity of complex “Tamoxifen-ER”
restored sensitivity to tamoxifen
EGFR and/orHER2 inhibitors
ER-HER2 cross talk in ER/HER2 positiveER-HER2 cross talk in ER/HER2 positivebreast cancer: Summarybreast cancer: Summary
EGFR-HER2 and ER pathways are linked in breast cancer cells overexpressing HER2 with cross-phoshorylation of both ER and EGFR and HER2, activation of AKT, MAPK and AIB1 by estrogen treatment.
Elevated active EGFR-HER2 heterodimers are formed after continous exposure and development of tamoxifen resistance in breast cancer cells.
Breast cancer cells overexpressing HER2 are growth stimulated by tamoxifen which behaves as an estrogen agonist in this situation.
Treatment of breast cancer cells overexpressing HER2 with the EGFR selective small molecule tyrosine kinase inhibitor Gefitinib and/or with the anti-HER2 monoclonal antibody Trastuzumab could block ER/EGFR-HER2 cross-talk, eliminate tamoxifen’s agonistic effects and restore its antitumor activity.
Acquired resistance mechanisms to gefitinib in Acquired resistance mechanisms to gefitinib in breast cancer and features of the phenotype:breast cancer and features of the phenotype:
Mediated by the compensatory upregulation / activation of other growth factor receptor pathways to maintain cell growth i.e. IGF-1R signalling
TAM-R TAM/TKI-R
Plus 1 M gefitinib(6 mths)
Generation of MCF-7 breast cancer Generation of MCF-7 breast cancer cells resistant to gefitinib:cells resistant to gefitinib:
Characterization of gefitinib (1Characterization of gefitinib (1M) resistant breast cells M) resistant breast cells (1)(1)
Moderate expression of EGFR
No detectable basal pEGFR and low levels of ERK1/2
Jones et al., Endocrine-related Cancer 11: 1-22, 2004
0
50
100
150
200
250
300
350
EGF
TGF-IG
F-I
IGF-II
Her-
bFGF
PDGF
Cellgrowth(% of
control)
* **
*
*Significant at p<0.05
IGF, insulin-like growth factor; bFGF, basic fibroblast growth factor
TGF, transforming growth factor; PDGF, platelet-derived growth factor
pEGFR1173
pERK1/2
Total EGFR
TAM-RTAM/TKI-R
-actin
Growth stimulation by IGF-I, IGF-II, heregulin- and bFGF
0
20
40
60
80
100
120
0 1 5 10
AG1024 (M)
*
*
*
Cellgrowth(% ofcontrol)
TAM-RTAM/TKI-R
Characterization of gefitinib-resistant breast cells (2)Characterization of gefitinib-resistant breast cells (2)
Production of IGF-II by RT-PCR
IGF-II
TAM-RTAM/TKI-R
-actin
Small reduction in IGF-1R expression but elevation in pIGF-1R Increased sensitivity to growth inhibition by the IGF-1R inhibitor
AG1024
pIGF-1R
Total IGF-1R
-actin
TAM-R TAM/TKI-RpPKC
pAKT
-actin
• Gefitinib resistant breast cells show elevated basal levels of activated AKT and PKC
• Phosphorylation of AKT and PKC was reduced in the presence of the IGF-1R inhibitor AG1024
42 kD
0 1 5 M AG1024
60 kDpAKT
-actin
77 kDpPKC
TAM/TKI-R
Downstream targets of IGF-1R signallingDownstream targets of IGF-1R signalling
Despite elevated HER-2, TAM/TKI-R cells were Despite elevated HER-2, TAM/TKI-R cells were insensitive to challenge with trastuzumabinsensitive to challenge with trastuzumab
* Significant at p<0.05
0
20
40
60
80
100
120
140
0 1 5 10 50 100
Trastuzumab (nM)
*
* * * *
Cell growth(% of control)
TAM-RTAM/TKI-R
Total HER-2
pHER-2
-actin
-actin
TAM-RTAM/TKI-R
Other studies have shown that:Other studies have shown that:
IGF-1R signalling is central in modulating the
responses to trastuzumab in MCF7/HER-18 cells (Lu
et al., JNCI 2001;93(24):1852)
HER-2 can be activated by the IGF-1R which involves a
physical association of the two receptors (Balana et al.,
Oncogene 2001;20:34)
IGF-1R / HER-2
IGF-1R HER-2
Evidence of physical association between IGF-1R and Evidence of physical association between IGF-1R and HER-2, and co-localisation at tumour cell membranesHER-2, and co-localisation at tumour cell membranes
IP, immunoprecipitation WB, Western blot
IP: pIGF-1R
WB: total HER-2
IP: total HER-2
WB: pIGF-1R
TAM/TKI-R cells
Investigating the possible interaction Investigating the possible interaction between IGF-1R and HER-2between IGF-1R and HER-2
Concentrations of AG1024 that blocked IGF-1R
phosphorylation also inhibited HER-2 phosphorylation
0 10 20
1 h
AG1024 (M)
TAM/TKI-R cells
pIGF-1R
pHER-2
0 10 20
24 h
SummarySummary
Type II receptors i.e. IGF1-R and/or InsR are important in both
acquired and de novo gefitinib resistance in breast cancer and
other cancer types.
Via EGFR blockade, gefitinib can faciliate Type II receptor
signalling which in turn can modulate EGFR phosphorylation.
EGFR expression/activation can increase with long-term
exposure to gefitinib which in turn may contribute to growth.
2053
Treatment period (weeks)
1253
100
50
0
Gefitinib (1M)
Gefitinib + AG1024 (5M)
0
TAM-R cell Growth rate as
% of controlTotal cell kill
Strategies to improve gefitinib responseStrategies to improve gefitinib response
Delay gefitinib resistance in breast cancer: combine gefitinib plus the resistance mechanism inhibitore.g. Treat tamoxifen resistant breast cancer cells with gefitinib and an IGF-1R inhibitor
ConclusionsConclusions
Single blockade of EGFR (or any growth factor) signalling through monotherapy is unlikely to be sufficient for maximum anti-tumour activity.
Identification of components involved in resistance mechanisms is essential and the subsequent co-targeting of these elements in combinatorial strategies.