1. Vecchi e nuovi “targets”, vecchi e nuovi farmaci
Fortunato Ciardiello
Division of Medical Oncology,
Department of Clinical and Experimental Medicine,
Second University of Naples, Italy 1 1

2. 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

3. 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

4. Receptor 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.

5. The EGFR/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
Apoptosis
Migration
Growth
Adhesion
Differentiation 1 3 2 4
Input layer
Signal- processing layer
Output layer
Ligands
Receptor dimers
Adaptors and enzymes
Cascades
Transcription factors
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.
Yarden Y and Sliwkowski M. Nat Rev Mol Cell Biol 2001; 2: 127–37.

6. HER2/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.

7. TGFa: 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.

8. EGFR: 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.

9. Co-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: , 2005).

10. Open 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.

11. No effect on tumor growth
EGFR inhibitors in pretreated NSCLC patients: possible interpretration of clinical results PR
Non-Responders SD
% % %
Apoptosis
No effect on tumor growth
Growth arrest
EGFR-dependent Growth
Non-EGFR-dependent Growth

12. Intermediate clinical
TGFa
TGFa
TGFa
Major 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

13. Possible 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.

14. Resistance to gefitinib in EGFR-overexpressing MDA-468 breast cancer cells with mutant PTEN and constitutive Akt activation
Bianco R, et al. Oncogene 2003;22:2812–2822.

15. Reconstution of PTEN function restores sensitivity
to EGFR inhibitors in EGFR-overexpressing MDA-468
breast cancer cells
Bianco R, et al. Oncogene 2003;22:2812–2822.

16. Strategies 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).

17. 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.

18. ZD6474: a 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 O Cl F
Gefitinib
ZD6474
Wedge SR, et al. Cancer Res 2002;62:4645–4655.
Ciardiello F, et al. Clin Cancer Res 2003;9:1546–1556.

19. Proposed antitumor activity of ZD6474
TGF
ZD6474
EGFR
ras
raf
Endothelial cell
Cancer cell
KDR
MEK
VEGF
MAPK
Block of endothelial cell proliferation
Cyclin D1
Block of cancer cell proliferation

20. Development 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:

21. in GEO cells resistant to Cetuximab or to Gefitinib
EGFR 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:

22. Characteristics of EGFR-targeted drugs resistant GEO cells
Ciardiello F. et al. Clin Cancer Res 2004; 10:

23. Acquired and constitutive resistance to EGFR inhibitors:
Differential sensitivity to EGFR inhibitors
Cell line
PTEN status
EGFR levels
Gefitinib
C225
ZD6474
MDA-468
mutated
high
low
very low
GEO
normal
moderate
GEO-Gef-R
GEO-Cet-R
PC3
deleted
PC3-Gef-R

24. PC3 PC3-Gef-R Acquired and constitutive resistance to EGFR inhibitors:
Role of optimal pAKT inhibition
PC3
PC3-Gef-R
Cetuximab
Gefitinib
ZD6474
Gefitinib
ZD6474
Ctr
Ctr
pAKT
AKT
pMAPK
MAPK

25. ZD6474: 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.

26. Increase in activated EGFR/HER2 dimers in
tamoxifen-resistant breast cancer cells
Knowlden et al., Endocrinology 2003

27. Increase in activated EGFR/HER2 dimers in tamoxifen-resistant breast cancer cells
Knowlden et al., Endocrinology 2003

28. ER-HER2 cross talk in ER/HER2 positive breast cancer
Chou et al., JNCI 2004

29. ER-HER2 cross talk in ER/HER2 positive breast cancer
Chou et al., JNCI 2004

30. 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”
resistance to tamoxifen

31. restored sensitivity 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/or
HER2 inhibitors

32. ER-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 tamoxifen’s 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

34. Generation of MCF-7 breast cancer cells resistant to gefitinib:
TAM-R TAM/TKI-R
Plus 1 mM gefitinib
(6 mths)

35. Characterization 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
pEGFR1173
pERK1/2
Total EGFR
TAM-R
TAM/ TKI-R
b-actin 50
100
150
200
250
300
350
EGF
TGF-a
IGF-I
IGF-II
Her-b
bFGF
PDGF
Cell growth (% 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
Jones et al., Endocrine-related Cancer 11: 1-22, 2004

36. Characterization 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
IGF-II
TAM-R
TAM/ TKI-R
b-actin 20 40 60 80
100
120 1 5 10
AG1024 (mM) *
Cell growth (% of control)
TAM-R
TAM/TKI-R
pIGF-1R
Total IGF-1R
b-actin

37. Downstream 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
42 kD
mM AG1024
60 kD
pAKT
b-actin
77 kD
pPKCd
TAM/TKI-R
TAM-R TAM/TKI-R
pPKCd
pAKT
b-actin

38. Despite elevated HER-2, TAM/TKI-R cells were insensitive to challenge with trastuzumab
TAM-R
TAM/TKI-R
TAM/ TKI-R
140
TAM-R
Total HER-2
120
Cell growth (% of control)
100 *
b-actin 80 * * 60 * * 40
pHER-2 20
b-actin 1 5 10 50
100
Trastuzumab (nM)
* Significant at p<0.05

39. 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)

40. Evidence of physical association between IGF-1R and HER-2, and co-localisation at tumour cell membranes
IP: pIGF-1R
WB: total HER-2
IP: total HER-2
WB: pIGF-1R
TAM/TKI-R cells
IGF-1R
HER-2
IP, immunoprecipitation WB, Western blot
IGF-1R / HER-2

41. Investigating 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
AG1024 (mM)
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.

43. TAM-R cell Growth rate as % of control Treatment period (weeks)
Strategies 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 3 5 20 3 5 12
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.