Vecchi e nuovi targets, vecchi e nuovi farmaci

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
2
The 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
3
Ligand-induced Receptor Dimerization
TGFa
TGFa
HER2/ neu
HER3
HER4
EGFR
Cell Membrane
EGFR
EGFR
EGFR
tyrosine kinase
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
Yarden Y and Sliwkowski M. Nat Rev Mol Cell Biol
2001 2 12737.
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 1152-1160, 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
EGFR 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
12
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 20032228122822.
15
Reconstution of PTEN function restores
sensitivity to EGFR inhibitors in
EGFR-overexpressing MDA-468 breast cancer cells
Bianco R, et al. Oncogene 20032228122822.
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 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.

18
ZD6474a 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.
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Proposed 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
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
784-793.
21
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
784-793.
22
Characteristics of EGFR-targeted drugs resistant
GEO cells
Ciardiello F. et al. Clin Cancer Res 2004 10
784-793.
23
Acquired and constitutive resistance to EGFR
inhibitors Differential sensitivity to EGFR
inhibitors
24
Acquired 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
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
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
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
  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

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

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

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

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/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
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 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)

40
Evidence 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
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
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.

43
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
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.