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.