Meccanismo di azione e Meccanismo di azione e superamento della resistenza superamento della resistenza

ormonaleormonale

Giuseppe Naso, Roberta FerraldeschiGiuseppe Naso, Roberta Ferraldeschi

Oncologia Medica BOncologia Medica B

Università di Roma Sapienza Università di Roma Sapienza

Novel therapeutic strategies combining antihormonal and targeted approaches.

American Cancer Society. American Cancer Society. Breast Cancer Facts and FiguresBreast Cancer Facts and Figures 2003-2004 ; 2003-2004 ; Jemal et alJemal et al. . CA Cancer J Clin. CA Cancer J Clin. 2004 ; 54 : 8-29 ;2004 ; 54 : 8-29 ; Rhodes et al.Rhodes et al. J Clin Pathol. J Clin Pathol. 2000 ; 53 : 688-6962000 ; 53 : 688-696; ; Johnston et al.Johnston et al. Nat Rev Cancer. Nat Rev Cancer. 2003 ; 3 2003 ; 3 : : 821-831.821-831.

• Breast cancer tumors can be divided into Breast cancer tumors can be divided into two basic types :two basic types :– Endocrine-dependent tumorsEndocrine-dependent tumors– Endocrine-independent tumorsEndocrine-independent tumors

• About 75 % of breast cancers are estrogen receptor (ER) About 75 % of breast cancers are estrogen receptor (ER) and/or progesterone receptor (PgR) positive and and/or progesterone receptor (PgR) positive and therefore are suitable for endocrine therapytherefore are suitable for endocrine therapy

Endocrine Dependency in Breast Cancer

Mammosphere Culture system

Dontu et al, Gen &Dev, 2003

Mammary stem cell and Mammary stem cell and mammapoiesismammapoiesis

Visvander, Canc Res. 2006

LT-RC = long-term repopulating stem cell; ST-RC = short-term repopulating stem cell

GATA-3 directed fate of breast stem cells

GATA-3 is a transcription factor that

binds to the regulatory region (promoter)

of the gene encoding FoxA1. FoxA1

might interact with Erα and bind to the

ERα gene promoter to turn on the

transcription of ERα target genes, which

include FoxA1 and ERα.

Reduction of FoxA1 might hinder the

action of the ER, which is required for

ductal- and lobuloalveolar-cell

development and drives cell proliferation

in breast tumours

Tong et al: Nature 2007

GATA-3 influence in breast developmentGATA-3 influence in breast development

Impaired development in MMTV–cre;Gata-3f/f mammary glands

Asselin-Labat, Nature Cell Biol 2006

Struttura dei ER e loro parti funzionali

Review Breast Cancer Res (2004), 6: 39-52

Funzioni e meccanismi regolatori della funzione dei ERs.

Estrogen Receptors

ERs belog to a super family of nuclear hormone receptors that includes receptors for other steroid hormones:

Thyroid hormone, vitamina D and retinoic acid

ER e proliferazione cellulare

Endocrine-Related Cancer (2004), 11: 537–551.

ER e proliferazione cellulare

• Espressione costruttiva ER diminuisce nelle cellule in fase S

• ER inibisce il gene ciclina D1

• ER diminuisce l’attività trascrizionaledi ER con conseguente diminuzione della proliferazione cellulare

• Espressione ER > Tessuti normali

< Tessuti neoplastici

• Induzione di espressione ER in cellule ER positive di BC riduce la loro crescita

Estrogen ReceptorsEstrogen Receptors

(adipe, ovaie, mammelle…)

ER +

Estrone

Androstenedione

Testosterone

Estradiolo

AROMATASI

RECETTORI ESTROGENICI: ERRECETTORI ESTROGENICI: ERαα/ER/ERββ

ER-ER-ER-

Cellula mammaria tumorale

?

• Attività genomica classicaAttività genomica classica (nucleare) – attività

NISS (Nuclear Initiated Steroid Signaling)

• Attività genomica non classicaAttività genomica non classica (nucleare)

• Attività non genomica o non trascrizionaleAttività non genomica o non trascrizionale

(membrana) – attività MISS (Membrane Initiated

Steroid Signaling)

RECETTORI ESTROGENICI: ERα

ER Genomic Function - Classical ER Genomic Function - Classical

Cell Growth&

Differentiation

Cell Growth&

Differentiation

mRNAmRNA

Target GeneTarget GeneEREERE

EE EE

ERER ERER

AF1AF1AF2AF2 AIB1AIB1

EE EEEE

EE

EEEEEE

N-CorN-Cor

AIB1AIB1N-CorN-Cor

ER Genomic Function - non Classical ER Genomic Function - non Classical

ERER ERER

EE EEEE

EE

EEEE

TumorGrowthTumorGrowthmRNAmRNA

Target GeneTarget GeneAP-1AP-1

Fos

Fos

Fos

Fos Jun

JunJunJun

ER Non Genomic - DirectER Non Genomic - Direct

AKT

Grb2 Sos Ras

Raf

MAPKK

MAPK

CblCblP85 P110

EGFR/HER2 EGFR/HER2 RecRec

ER ER

ER ER

Ligand-induced conformations of the Ligand-induced conformations of the receptorsreceptors

Modulation of receptor by ligand and helix 12 position

Helix 12 covers the ligand pocket after it has bound, and form the landing platform for coregulators, that drive all the subsequent reactions

Different ligands can induce different conformation changes, different coregulation recruitment and different functions

BRCA1 and HSP 90 exert a strong BRCA1 and HSP 90 exert a strong

control on Ercontrol on Er

GATA-3 turn on the ErGATA-3 turn on the Er transcription transcription

BRCA-1 and ERBRCA-1 and ER

•The only known enzymatic activity associated with

BRCA1 is its activity as a ubiquitin protein ligase

•Attachment of ubiquitin to substrates is a versatile method

of regulation involved in practically all aspects of cell

biology. Substrate ubiquitination involves several steps

and a well-known trio of enzymes called ubiquitin

activating (E1), ubiquitin conjugating (E2), and ubiquitin

ligase (E3).

Irminger-Finger et al. Nature Reviews Cancer 6, 382–391 (May 2006) | doi:10.1038/nrc1878

Ligand-induced estrogen receptor α degradation by the proteasome

The molecular chaperone heat shock protein (hsp)-90

maintains estrogen receptor (ER)-a in an active

conformation, allowing to bind 17h-estradiol (E2) and

transactivate genes, including progesterone receptor

(PR)-h and the class IIB histone deacetylase HDAC6. The

latter, in turn, exert its own control on (hsp)-90

Hsp-90 and ERHsp-90 and ER

•Hyperacetylation of hsp90

inhibits its chaperone

function, thereby depletingh

hsp90 client proteins

Hsp-90 and ERHsp-90 and ER

Warren Fiskus, Clin Cancer Res 2007

Inhibition of Inhibition of histone deacetylase HDAC6 determines the Hyperacetylation of hsp90

Antiestrogen TherapiesAntiestrogen Therapies

• Reduction of estradiol levels in host and tumor

– LHRH superagonists, aromatase inhibitors

(anastrozole, letrozole, exemestane)

• Selective ER modulators (SERMs) with agonistic and

antagonistic activity

– Tamoxifen

• ER modulators with pure antagonistic activity

(receptor downregulation)

– Fulvestrant

17ESTRADIOLO

AF1

AF2

TAMOXIFENETAMOXIFENEERE

ERE

ATTIVAZIONE PARZIALEATTIVAZIONE PARZIALEDELLA TRASCRIZIONEDELLA TRASCRIZIONE

(solo AF1)(solo AF1)ANTAGONISTA

AGONISTA

INIBITORI DELLE INIBITORI DELLE AROMATASIAROMATASI

BLOCCO COMPLETO DELLA BLOCCO COMPLETO DELLA TRASCRIZIONE, TRASCRIZIONE, PERSISTENZA PERSISTENZA

DELLA VIA RECETTORIALE DELLA VIA RECETTORIALE ESTROGENICAESTROGENICA

FULVESTRANTFULVESTRANTBLOCCO COMPLETO DELLA BLOCCO COMPLETO DELLA TRASCRIZIONE, TRASCRIZIONE, ABOLIZIONE ABOLIZIONE

COMPLETA DEL SEGNALE COMPLETA DEL SEGNALE ESTROGENICOESTROGENICO

RE

AF1

AF2

ERE

ERE

RE

AF1

AF2

RE

ERE

ERE

DIFFERENTE MECCANISMO D’AZIONE: DIFFERENTE MECCANISMO D’AZIONE: SERM, IA, SERDSERM, IA, SERD

17ESTRADIOLO

17ESTRADIOLO

What are the mechanisms of resistance to hormonal

therapies?

Estrogen Estrogen dependent dependent

breast breast cancercancer

HYPOTHESIS:HYPOTHESIS:

Adaptation Adaptation or clonal or clonal selectionselection

1)Kinases activation 2)Estrogen deprivation

Modified by Santen RJ, SABCS 2006

Cell with enhanced estrogens

sensitivity by ERiperstimula

tion

LTER

Uno dei meccanismi alla base della resistenza Uno dei meccanismi alla base della resistenza

ormonale al Tamoxifene e agli Inibitori delle ormonale al Tamoxifene e agli Inibitori delle

Aromatasi sembra essere la capacità delle chinasi Aromatasi sembra essere la capacità delle chinasi

attivate da numerosi recettori di membrana (EGFR, attivate da numerosi recettori di membrana (EGFR,

HER2/NEU, IGFR, etc) di amplificare il segnale di HER2/NEU, IGFR, etc) di amplificare il segnale di

trascrizione del recettore per gli estrogenitrascrizione del recettore per gli estrogeni

RESISTENZA ORMONALE: RESISTENZA ORMONALE: IPOTESIIPOTESI

P

Ras

p8

5 p110 ERE

E

ERE

P

P

P

Transcription

ErbB ErbB

ER-Responsive Element

P

MAPKAkt

Ligand

Pathway Activation-Other Receptors

Pancholi S, SABCS 2007

La resistenza agli inibitori delle aromatasi potrebbe La resistenza agli inibitori delle aromatasi potrebbe

essere dovuta ad un aumentata sensibilità essere dovuta ad un aumentata sensibilità

all’estradiolo durate la deprivazione a lungo termine all’estradiolo durate la deprivazione a lungo termine

da estrogeni (LTED)da estrogeni (LTED)

RESISTENZA ORMONALE RESISTENZA ORMONALE IPOTESIIPOTESI

Model of adptation to estrogen Model of adptation to estrogen deprived conditionsdeprived conditions

MCF-7

> 6 MONTHS

ESTROGEN DEPRIVED MEDIA

LTED

Chan CM et al. J Ster Biochem & Mol Biol 2002

ER, oestrogen receptorPgR, progesterone receptor No. of weeks in oestrogen-deprived medium

0

5

10

15

20

25Number of cells/mL(x105)

Wild type (FBS)

1 1–20 >20–50

MCF-7 cells – oestradiol

Increased basal proliferation (>20 weeks)

Quiescent (<15 weeks)

ER

PgR PgR

In vitroIn vitro model of long-term oestrogen model of long-term oestrogen deprived (LTED) cellsdeprived (LTED) cells

0

0.5

1.0

1.5

2.0

2.5

C

Oestradiol (M)10-15 10-13 10-11 10-9 10-8

Cells

(x1

06) Wt

LTED

Martin et al, JBC 2003

ER IS ACTIVATED IN MCF-7 CELLS ER IS ACTIVATED IN MCF-7 CELLS RESISTANT TO LTEDRESISTANT TO LTED

0

0.5

1.0

1.5

2.0

2.5

C

Oestradiol (M)10-15 10-13 10-11 10-9 10-8

Cells

(x1

06) Wt

LTED

Martin et al, JBC 2003

ER IS ACTIVATED IN MCF-7 CELLS ER IS ACTIVATED IN MCF-7 CELLS RESISTANT TO LTEDRESISTANT TO LTED

Hypersensitivity to E2 induced proliferation

0

0.5

1.0

1.5

2.0

2.5

C

Oestradiol (M)10-15 10-13 10-11 10-9 10-8

Wt LTED

ERa Ser118

ERa

perbB2

pEGFR

EGFR

erbB2

0

2.5

5.0

7.5

10

Wt-MCF7 LTED

Cell line

Fold

act

ivit

y

Basal transcription

Cells

(x1

06) Wt

LTED

Martin et al, JBC 2003

ER IS ACTIVATED IN MCF-7 CELLS ER IS ACTIVATED IN MCF-7 CELLS RESISTANT TO LTEDRESISTANT TO LTED

RECETTORI ESTROGENICI: ERRECETTORI ESTROGENICI: ER

RECETTORE ESTROGENICO a

Cdk 2 p38RSK

PKA

AKT c-src familyMAPK

ER AF-1 DBD LBD/AF-2

NEW STRATEGIES TO OVERCOME NEW STRATEGIES TO OVERCOME ENDOCRINE RESISTANCEENDOCRINE RESISTANCE

1)1) Integrating targeted therapies with Integrating targeted therapies with

hormonal therapieshormonal therapies

2)2)SERD: SERD: FulvestrantFulvestrant

NEW STRATEGIES TO OVERCOME NEW STRATEGIES TO OVERCOME ENDOCRINE RESISTANCEENDOCRINE RESISTANCE

1)1) Integrating targeted therapies with Integrating targeted therapies with

hormonal therapieshormonal therapies

2)2)SERD: SERD: FulvestrantFulvestrant

1 2

K KSHC

PI3KGRB2

SOS

AKT

RAS

PTEN

mTOR FKHR GSK-3 BAD

Cell cicle

progression

Survival

RAF

MEK 1/2

MAP

Anti ErbB1-2 receptors Mabs(Trastuzumab, 2C4, C225)

HER1,HER2,HER4, Tyrosine Kinase Inhibitors (ZD 1839, OSI 774, EKB 559,

GW 2016, CI 1033)

Ras farnesyl transferase Inhibitors (BMS 214662, R115777)

RAF inhibithorsmTOR inhibitors (CCI 779)

Mek inhibitors (CI 1040)

Trastuzumab Effect on Trastuzumab Effect on HER2+XenograftsHER2+Xenografts

ED= Estrogen deprivation

Osborne CK, SABCS 2007

Superiority of multidrug anti-HER Superiority of multidrug anti-HER therapy in Xenograft Modelstherapy in Xenograft Models

Osborne CK, SABCS 2007

TANDEM:TANDEM:Anastrazole +/- Herceptin trial designAnastrazole +/- Herceptin trial design

Anastrazole +/- Herceptin in HER2+, ER+, MBC (n=202)

AnastrazoleAnastrazole

Herceptin

HER2 inhibitorsHER2 inhibitors

TANDEM:TANDEM:Clinical BenefitClinical Benefit

0

10

20

30

40

50

Pati

en

ts (

%)

Clinical Benefit

Anastazole (n =104)

Anastazole + herceptin (n= 103)

27,927,9

42,742,7P=0.026

Machej JR, SABCS 2006

NEW STRATEGIES TO OVERCOME NEW STRATEGIES TO OVERCOME ENDOCRINE RESISTANCEENDOCRINE RESISTANCE

1)1) Integrating targeted therapies with Integrating targeted therapies with

hormonal therapieshormonal therapies

2)2)SERD: FulvestrantSERD: Fulvestrant

0

10

20

30

40

50

60

70

80

90

100

1 5 10 50 100 300 1000 3000 10000

Concentrazione (nM)

Perc

en

tuae d

i in

ibiz

ion

e

E2

Faslodex

Tam

SELETTIVITASELETTIVITA’’ DEL LEGAME 3H- DEL LEGAME 3H-ESTRADIOLO/REESTRADIOLO/RE

Estradiolo vs Faslodex vs TamEstradiolo vs Faslodex vs Tam

0

20

40

60

80

100

120

Mea

n ±

1S

EM

Placebo(n=29)

50mg'Faslodex'

(n=31)

125mg'Faslodex'

(n=32)

250mg'Faslodex'

(n=32)

18mg SA s/c(*n=12)

J Robertson et al, Cancer Research 2001DeFriend DJ, et al. Cancer Res. 1994;54:408-414.

1ng/ml1ng/ml

2.5ng/ml2.5ng/ml

5ng/ml5ng/ml

23ng/ml23ng/ml

6mg SA s/c(*n=6)

7ng/ml7ng/ml

500mg LA = 14ng/ml500mg LA = 14ng/ml

??

Post-treatment Mean ER H-scoresPost-treatment Mean ER H-scores

Robertson JFR et al. Cancer Res 2001; 61: 6739–6746.

Post-treatment Mean PgR H-scoresPost-treatment Mean PgR H-scores

NS = not significantNS = not significant

0

20

40

60

80

100

NS

p=0.003

p=0.0002

p=0.0001p=0.0001

p=0.0001

Placebo(n=28)

50mgFulvestrant

(n=29)

125mgFulvestrant

(n=29)

250mgFulvestrant

(n=29)

Tamoxifen(n=21)

Mea

n ±

1S

EM

Overall treatment effect p=0.0001

Wild type + 10-9M oestradiolLTED

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

Cont 10-12 10-10 10-9 10-8 10-7 10-6

Fulvestrant (M)

Fold changein cell growth comparedwith control

Fulvestrant is an effective inhibitor of LTED cell growth

Consistent with ER signalling mechanism and supersensitisation of cells to oestradiol

Martin LA et al. J Biol Chem 2003; 278: 30458–30468

Fulvestrant inhibits the growth of Fulvestrant inhibits the growth of long-term oestrogen-deprived (LTED) long-term oestrogen-deprived (LTED)

MCF-7 cellsMCF-7 cells

C

Cells (

x10

6)

Drug concentration (M)10-13 10-12 10-11 10-10 10-9

0

1.0

2.0

3.0

10-8

E2

Tamoxifen

Fulvestrant

ER

ER

Tam Fulvestrant

LTED-R cells are hypersensitive to E2LTED-R cells are hypersensitive to E2, , refractory to tamoxifen, but sensitive torefractory to tamoxifen, but sensitive to

fulvestrantfulvestrant

Martin et al, JBC 2003

Fulvestrant: Fulvestrant: Summary of Preclinical DataSummary of Preclinical Data

• Downregulates estrogen receptors in breast cancer Downregulates estrogen receptors in breast cancer

cells cells

• No estrogenic activityNo estrogenic activity

• Completely blocks estrogen actionCompletely blocks estrogen action

MCF 7MCF 7

MCF7-T = TAM RESISTENTMCF7-T = TAM RESISTENT

MCF7-F = FULVESTRANT RESISTENTMCF7-F = FULVESTRANT RESISTENT

Fan M, Clin Cancer Res 2006

Fan M, Clin Cancer Res 2006

Fan M, Clin Cancer Res 2006

p85

p110

AKT

raptor-mTOR

EGFR

p85

p110

AKT

raptor-mTOR

EGFR ERBB3

PP

HIF-activation cell-growth

Dual inhibition of mTOR and ER signaling enhancescell death in breast cancer cells

mTOR inhibitors:mTOR inhibitors:

Fulvestrant+Rad 001 Fulvestrant Rad 001

Beeram M, Ann Oncol 2007

Acquired resistence to TAMOXIFEN correlates with

maintenance of the Er-positive phenotype and ER

pathways (AF-1; AF-2)

CONVERSELYCONVERSELY

Acquired resistence to FULVESTRANT is caracterized by up-regulation of Er pathways resulting in a Estrogen

receptor a (Er) independent proliferation

CONCLUSION ICONCLUSION I

Grazie per l’attenzione!!!!!Grazie per l’attenzione!!!!!