Title: Vecchi e nuovi targets, vecchi e nuovi farmaci
1Vecchi e nuovi targets, vecchi e nuovi farmaci
Fortunato Ciardiello Division of Medical
Oncology, Department of Clinical and Experimental
Medicine, Second University of Naples, Italy
2The EGFR (erbB) Family and Ligands
EGF TGFa Amphiregulin b-cellulin HB-EGF Epiregulin
NRG2 NRG3 Heregulins b-cellulin
Heregulins
Cysteine-rich domains
100 100 100
44 82 33
36 59 24
48 79 28
Tyrosine kinase domain
C-terminus
HER1 EGFR ErbB-1
HER2/neu ErbB-2
HER3 ErbB-3
HER4 ErbB-4
3Ligand-induced Receptor Dimerization
TGFa
TGFa
HER2/ neu
HER3
HER4
EGFR
Cell Membrane
EGFR
EGFR
EGFR
tyrosine kinase
tyrosine kinase
HER2/neu
nucleus
4Receptor Dimerization is Essential for
Intracellular 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.
5The EGFR/erbB Signaling Network
Yarden Y and Sliwkowski M. Nat Rev Mol Cell Biol
2001 2 12737.
6HER2/neu 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.
7TGFa Role in Breast Cancer
- Mitogen for mammary epithelial cells.
- Estrogen-inducible in estrogen-dependent
- breast cancer.
- Expression increases from atypical hyperplasia to
- carcinoma in situ, to invasive carcinoma.
- Overexpression in 50-70 primary breast cancer.
8EGFR Role in Breast Cancer
Expressed in 35-60 primary breast
cancers. Overexpression correlates with
multidrug resistance. Inverse correlation with
ER and PgR. Overexpression correlates with
resistance to hormonotherapy. Overexpression is
generally associated with poor prognosis.
9Co-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).
10Open clinical issues for the therapeutic use of
EGFR-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.
11EGFR inhibitors in pretreated NSCLC patients
possible interpretration of clinical results
Non-Responders
SD
PR
10 - 15 20 - 30 55
- 70
No effect on tumor growth
Apoptosis
Growth arrest
Non-EGFR-dependent Growth
EGFR-dependent Growth
12Major clinical benefit PR
Intermediate clinical benefit SD
No clinical benefit PD
EGFR mutations or Wild-type EGFR ? HER2/HER3
? EGFR ligands loss of Cbl
Smokers with wild-type EGFR (males? squamous?)
EGFR mutations/ gene amplification
Modified from an original concept of Carlos
Arteaga
13Possible mechanisms of resistance to EGFR
inhibitors
- 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.
14Resistance to gefitinib in EGFR-overexpressing
MDA-468 breast cancer cells with mutant PTEN and
constitutive Akt activation
Bianco R, et al. Oncogene 20032228122822.
15Reconstution of PTEN function restores
sensitivity to EGFR inhibitors in
EGFR-overexpressing MDA-468 breast cancer cells
Bianco R, et al. Oncogene 20032228122822.
16Strategies to overcome resistance to EGFR
inhibitors
- 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).
17Rational basis for combination of EGFR and VEGF
inhibitors
- Activation of EGFR by EGF or TGFa can up-regulate
the production of VEGF in cancer cells. - EGFR inhibition reduces VEGF production.
- Resistance to EGFR inhibitors is associated with
VEGF overexpression.
18ZD6474a 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.
Gefitinib
ZD6474
Wedge SR, et al. Cancer Res 20026246454655.
Ciardiello F, et al. Clin Cancer Res
2003915461556.
19Proposed antitumor activity of ZD6474
TGF?
ZD6474
EGFR
raf
ras
Endothelial cell
Cancer cell
KDR
MEK
VEGF
MAPK
Block of endothelial cell proliferation
Cyclin D1
Block of cancer cell proliferation
20Development of resistant GEO colon cancer
xenografts following chronic treatment with
Cetuximab or Gefitinib, but not with ZD6474
Ciardiello F. et al. Clin Cancer Res 2004 10
784-793.
21EGFR expression in 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.
22Characteristics of EGFR-targeted drugs resistant
GEO cells
Ciardiello F. et al. Clin Cancer Res 2004 10
784-793.
23Acquired and constitutive resistance to EGFR
inhibitors Differential sensitivity to EGFR
inhibitors
24Acquired and constitutive resistance to EGFR
inhibitors Role of optimal pAKT inhibition
PC3-Gef-R
PC3
Cetuximab
Gefitinib
ZD6474
Gefitinib
ZD6474
Ctr
Ctr
pAKT
AKT
pMAPK
MAPK
25ZD6474 Summary of preclinical data
- 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.
26Increase in activated EGFR/HER2 dimers
in tamoxifen-resistant breast cancer cells
Knowlden et al., Endocrinology 2003
27Increase in activated EGFR/HER2 dimers in
tamoxifen-resistant breast cancer cells
Knowlden et al., Endocrinology 2003
28ER-HER2 cross talk in ER/HER2 positive breast
cancer
Chou et al., JNCI 2004
29ER-HER2 cross talk in ER/HER2 positive breast
cancer
Chou et al., JNCI 2004
30HER2 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
31HER2 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/or HER2 inhibitors
32ER-HER2 cross talk in ER/HER2 positive breast
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
tamoxifens agonistic effects and restore its
antitumor activity.
33- Acquired resistance mechanisms to gefitinib in
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
34Generation of MCF-7 breast cancer cells resistant
to gefitinib
TAM-R TAM/TKI-R
Plus 1 mM gefitinib (6 mths)
35Characterization of gefitinib (1mM) resistant
breast cells (1)
- Moderate expression of EGFR
- No detectable basal pEGFR and low levels of ERK1/2
- Growth stimulation by IGF-I, IGF-II, heregulin-b
and bFGF
Jones et al., Endocrine-related Cancer 11 1-22,
2004
36Characterization of gefitinib-resistant breast
cells (2)
- Production of IGF-II by RT-PCR
- Small reduction in IGF-1R expression but
elevation in pIGF-1R
- Increased sensitivity to growth inhibition by the
IGF-1R inhibitor AG1024
37Downstream targets of IGF-1R signalling
- Gefitinib resistant breast cells show elevated
basal levels of activated AKT and PKCd
- Phosphorylation of AKT and PKCd was reduced in
the presence of the IGF-1R inhibitor AG1024
TAM-R TAM/TKI-R
pPKCd
pAKT
b-actin
38Despite elevated HER-2, TAM/TKI-R cells were
insensitive to challenge with trastuzumab
TAM/TKI-R
140
TAM-R
120
Total HER-2
Cell growth( of control)
100
80
b-actin
60
40
pHER-2
20
0
b-actin
0
1
5
10
50
100
Trastuzumab (nM)
Significant at plt0.05
39Other studies have shown that
- IGF-1R signalling is central in modulating the
responses to trastuzumab in MCF7/HER-18 cells (Lu
et al., JNCI 200193(24)1852)
- HER-2 can be activated by the IGF-1R which
involves a physical association of the two
receptors (Balana et al., Oncogene 20012034)
40Evidence of physical association between IGF-1R
and HER-2, and co-localisation at tumour cell
membranes
IGF-1R
HER-2
IP, immunoprecipitation WB, Western blot
IGF-1R / HER-2
41Investigating the possible interaction between
IGF-1R and HER-2
- Concentrations of AG1024 that blocked IGF-1R
phosphorylation also inhibited HER-2
phosphorylation
TAM/TKI-R cells
1 h
24 h
0 10 20
AG1024 (mM)
0 10 20
pIGF-1R
pHER-2
42- Summary
-
- 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.
43Strategies to improve gefitinib response
- Delay gefitinib resistance in breast cancer
- combine gefitinib plus the resistance mechanism
inhibitor - e.g. Treat tamoxifen resistant breast cancer
cells with gefitinib and an IGF-1R inhibitor
Gefitinib (1mM)
Gefitinib AG1024 (5mM)
100
TAM-R cell Growth rate as of control
50
Total cell kill
0
20
5
3
12
5
3
0
Treatment period (weeks)
44- Conclusions
-
- 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.