Giuseppe Naso, Roberta Ferraldeschi Università di Roma Sapienza Meccanismo di azione e superamento della resistenza ormonale Novel therapeutic strategies combining antihormonal and targeted approaches. Giuseppe Naso, Roberta Ferraldeschi Oncologia Medica B Università di Roma Sapienza

Endocrine Dependency in Breast Cancer #1 Am. Cancer Soc. p 1, col 2, par 2, ln 1-4 #5 Jemal p 9, table 1 Breast cancer tumors can be divided into two basic types : Endocrine-dependent tumors Endocrine-independent tumors About 75 % of breast cancers are estrogen receptor (ER) and/or progesterone receptor (PgR) positive and therefore are suitable for endocrine therapy #6 Rhodes p 688, col 2, par 1, ln 1-17 #3 Johnston p 821, col 1, par 1, ln 1-4 #1 Am. Cancer Society p 1, col 2, par 2, ln 1-4 SPEAKER'S NOTES Breast cancers can be classified as one of two types: hormone dependent or hormone independent. Hormone-dependent tumors are ER-positive, PgR-positive, or both, and are generally considered to be hormone responsive. Hormone-independent tumors are negative for both receptors. Approximately 75% of breast cancers are hormone responsive and can be treated with hormone therapy. BACKGROUND NOTES In one study, based on more than 7000 breast cancers, data from immunohistochemical assays used for determining the ER and PgR status were analyzed between June 1996 and September 1998 to determine the frequency of receptor positivity. ER-positive/PgR-positive tumor frequency was 59.1%, ER-positive/ PgR-negative was 18.8%, ER- negative/PgR-positive was 2.9%, and ER-negative/PgR-negative was 19.1% . #3 Johnston p821, col 1, par 1, ln 1-4 #6 Rhodes p 688, col 2, par 1, ln 1-17 American Cancer Society. Breast Cancer Facts and Figures 2003-2004 ; Jemal et al. CA Cancer J Clin. 2004 ; 54 : 8-29 ; Rhodes et al. J Clin Pathol. 2000 ; 53 : 688-696; Johnston et al. Nat Rev Cancer. 2003 ; 3 : 821-831. #6 Rhodes p690, col 2, par 5, ln 1-7; p 691, table 5 References 1. American Cancer Society. Breast Cancer Facts and Figures 2003-2004. Atlanta, Ga: American Cancer Society, Inc.; 2003. 2. Jemal A, Tiwari RC, Murray T, et al. Cancer Statistics, 2004. CA Cancer J Clin. 2004;54:8-29. 3. Rhodes A, Jasani B, Balaton AJ, Barnes DM, Miller KD. Frequency of oestrogen and progesterone receptor positivity by immunohistochemical analysis in 7016 breast carcinomas: correlation with patient age, assay sensitivity, threshold value, and mammographic screening. J Clin Pathol. 2000;53:688-696. 4. Johnston SRD, Dowsett M. Aromatase inhibitors for breast cancer: lessons from the laboratory. Nat Rev Cancer. 2003;3:821-831. 2

Mammosphere Culture system Dontu et al, Gen &Dev, 2003

Mammary stem cell and mammapoiesis LT-RC = long-term repopulating stem cell; ST-RC = short-term repopulating stem cell Visvander, Canc Res. 2006

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 development Impaired development in MMTV–cre;Gata-3f/f mammary glands Asselin-Labat, Nature Cell Biol 2006

Estrogen Receptors Funzioni e meccanismi regolatori della funzione dei ERs. Struttura dei ER e loro parti funzionali ERs belog to a super family of nuclear hormone receptors that includes receptors for other steroid hormones: Thyroid hormone, vitamina D and retinoic acid Review Breast Cancer Res (2004), 6: 39-52

ER  e proliferazione cellulare ER  e proliferazione cellulare Estrogen Receptors ER  e proliferazione cellulare ER  e proliferazione cellulare > Tessuti normali Espressione ER  < Tessuti neoplastici Espressione costruttiva ER  diminuisce nelle cellule in fase S Induzione di espressione ER  in cellule ER  positive di BC riduce la loro crescita ER  inibisce il gene ciclina D1 ER  diminuisce l’attività trascrizionaledi ER  con conseguente diminuzione della proliferazione cellulare Endocrine-Related Cancer (2004), 11: 537–551.

RECETTORI ESTROGENICI: ERα/ERβ Androstenedione Testosterone AROMATASI (adipe, ovaie, mammelle…) Estrone Estradiolo ? In condizioni fisiologiche nella donna in pre-menopausa la stimolazione esercitata dagli estrogeni sulla cellula sana avviene su ambedue i recettori e comporta un equilibrio tra le vie attivanti i segnali mitogenici e le vie pro-apoptotiche. La risultante di tale stimolazione è un bassissimo indice di crescita per le cellule recettori positive, che però producono citochine che stimolano a loro volta la crescita delle cellule negative per i recettori ormonali. ER-a ER-b ER + Cellula mammaria tumorale 9

RECETTORI ESTROGENICI: ERα Attività genomica classica (nucleare) – attività NISS (Nuclear Initiated Steroid Signaling) Attività genomica non classica (nucleare) Attività non genomica o non trascrizionale (membrana) – attività MISS (Membrane Initiated Steroid Signaling) I recettori estrogenici esercitano la loro attività attrverso tre meccanismi principali di attivazione

ER Genomic Function - Classical N-Cor AF2 AIB1 Cell Growth & Differentiation mRNA AF1 N-Cor AIB1 ERE Target Gene 11

ER Genomic Function - non Classical Tumor Growth mRNA Target Gene AP-1 Fos Jun 12

ER Non Genomic - Direct EGFR/HER2 Rec Cbl Grb2 Sos Ras P85 P110 Raf MAPKK AKT MAPK ER ER ER Non Genomic - Direct 13

Ligand-induced conformations of the receptors 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 control on Era GATA-3 turn on the Era transcription BRCA1 and HSP 90 exert a strong control on Era

BRCA-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

Hsp-90 and ER 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 ER Inhibition of histone deacetylase HDAC6 determines the Hyperacetylation of hsp90 Hyperacetylation of hsp90 inhibits its chaperone function, thereby depletingh hsp90 client proteins Warren Fiskus, Clin Cancer Res 2007

Antiestrogen 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

DIFFERENTE MECCANISMO D’AZIONE: SERM, IA, SERD AGONISTA AF1 AF2 ERE ATTIVAZIONE PARZIALE DELLA TRASCRIZIONE (solo AF1) TAMOXIFENE 17ESTRADIOLO RE ANTAGONISTA ERE INIBITORI DELLE AROMATASI AF1 AF2 ERE BLOCCO COMPLETO DELLA TRASCRIZIONE, PERSISTENZA DELLA VIA RECETTORIALE ESTROGENICA 17ESTRADIOLO Il 17beta estradiolo agisce attivando completamente il recettore attraverso la stimolazione delle vie AF1 ed AF-2. Il Tamoxifene, un modulatore selettivo del recettore estrogenico, lega in maniera competitiva il recettore determinando però solo una parziale inattivazione della trascrizione (blocco della via AF-2 , effetto antagonista) e mantenendo una stimolazione simil-estrogenica della via AF1 (effetto agonista) Gli inibitori delle aromatasi, impedendo la sintesi degli estrogeni, determinano un blocco completo della trascrizione mediata dal recettore estrogenico venendo meno lo stimolo del ligando. Tuttavia la via recettoriale persiste e conserva la sua potenzialità di fungere come substrato per la trasduzione del segnale mitogenico proveniente dalla stimolazione di altre vie recettoriali non estrogeniche (EGFR, HER2 neu, IGF RI etc.. ) Il fulvestrant esercita la sua azione mediante il blocco completo del recettore per gli estrogeni e la sua degradazione. Rispetto al Tamoxifene dunque non presenta alcuna attività agonista e stimolando la degradazione del recettore impedisce l’amplificazione trascrizionale attivata a partire da altre vie. RE ERE AF1 AF2 BLOCCO COMPLETO DELLA TRASCRIZIONE, ABOLIZIONE COMPLETA DEL SEGNALE ESTROGENICO ERE FULVESTRANT 17ESTRADIOLO RE ERE

What are the mechanisms of resistance to hormonal therapies?

HYPOTHESIS: 1)Kinases activation 2)Estrogen deprivation Cell with enhanced estrogens sensitivity by ERa iperstimulation Estrogen dependent breast cancer Adaptation or clonal selection LTER Modified by Santen RJ, SABCS 2006

RESISTENZA ORMONALE: IPOTESI Uno dei meccanismi alla base della resistenza ormonale al Tamoxifene e agli Inibitori delle Aromatasi sembra essere la capacità delle chinasi attivate da numerosi recettori di membrana (EGFR, HER2/NEU, IGFR, etc) di amplificare il segnale di trascrizione del recettore per gli estrogeni

ER-Responsive Element Ligand ErbB ErbB E P P p85 p110 ER E P Ras Akt MAPK ER E Transcription ER-Responsive Element

Pathway Activation-Other Receptors

Pancholi S, SABCS 2007

RESISTENZA ORMONALE IPOTESI La resistenza agli inibitori delle aromatasi potrebbe essere dovuta ad un aumentata sensibilità all’estradiolo durate la deprivazione a lungo termine da estrogeni (LTED)

Model of adptation to estrogen deprived conditions MCF-7 LTED > 6 MONTHS ESTROGEN DEPRIVED MEDIA

In vitro model of long-term oestrogen deprived (LTED) cells PgR No. of weeks in oestrogen-deprived medium Number of cells/mL (x105) Wild type (FBS) 1 1–20 >20–50 MCF-7 cells – oestradiol Increased basal proliferation (>20 weeks) Quiescent (<15 weeks) Several in vitro models have been developed to study the molecular changes associated with long-term estrogen deprivation (LTED) . Based on the data from these models, there is evidence to suggest that resistance to AI treatment is due to enhanced sensitization to low estradiol levels . In one such model using the MCF-7 cell line, cells deprived of estrogen for 90 weeks passed through three distinct phases; quiescent (LTEDQ), hypersensitive (LTED-H), and an apparent estrogen- independent phase (LTED-I) . The LTED-Q phase lasted 15 weeks and during this time the mean ER protein levels increased approximately two-fold, whilst the progesterone receptor (PgR) decreased to almost undetectable levels when compared with wild-type (WT) cells . After 20 weeks, the cells entered the LTED-H phase, where an increase in the basal growth rate was observed in parallel with an increase in estradiol sensitivity. Maximal growth in these cells was stimulated with a significantly lower concentration of estradiol compared with WT cells (10)13 M versus 9 M estradiol, respectively). This shift to the left in the bell-shaped estrogen sensitivity dose–response curve meant the LTED cells became sensitized to the growth suppressive effect at physiological concentrations of estradiol (10)10 M). The mean ER protein levels were also elevated approximately five-fold, compared with WT cells grown in the presence of fetal bovine serum (FBS) . Therefore, the hypersensitivity to estradiol in LTED-H cells appeared to correlate with elevated levels of ERa phosphorylated on serine118.Culturing MCF-7 LTED cells for more than50 weeks (LTED-I cells) rendered the cells apparentlyestradiol independent, where the addition of estradiol had no effect on cell growth [9]. However, exclusion of insulin from the cell culture medium resulted in an approximately 50% drop in basal growth rate of LTEDI cells and restoration of estrogen hypersensitivity, suggesting LTED-I cells were super-sensitized to residual stradiol in the cell culture medium [27]. Characterization of LTED-I cells also revealed that ER levelsduring this time increased to approximately seven-fold, whereas levels of ERa phosphorylated on serine118 hadstabilized at approximately two- to three-fold compared with the control (Figures 1a and 2) [9]. The striking increase in phosphorylation of ERa on serine118 at the beginning of the LTED-H phase could, therefore, be a critical step in the acquisition of estrogen hypersensitivity. ER, oestrogen receptor PgR, progesterone receptor Chan CM et al. J Ster Biochem & Mol Biol 2002

ER IS ACTIVATED IN MCF-7 CELLS RESISTANT TO LTED 0.5 1.0 1.5 2.0 2.5 Wt Cells (x106) LTED C Oestradiol (M) 10-15 10-13 10-11 10-9 10-8 Our previously published studies demonstrated that MCF-7 cells deprived of E2 for over 80 weeks passed through three distinct phases: quiescent (LTED-Q) followed by a hypersensitive phase (LTED-H), where basal cell growth was stimulated by the addition of E2 at concentrations below 1013 M, and finally an apparent independent phase (LTED-I) in which exogenous E2 no longer affected their growth. Our studies also revealed that the LTED MCF-7 cells expressed elevated levels of ER that was phosphorylated on Ser118 in the absence of exogenous E2 suggesting this was a contributing factor to the acquired hypersensitivity of these cells (6). Martin et al, JBC 2003

ER IS ACTIVATED IN MCF-7 CELLS RESISTANT TO LTED 0.5 1.0 1.5 2.0 2.5 Wt Hypersensitivity to E2 induced proliferation Cells (x106) LTED C Oestradiol (M) 10-15 10-13 10-11 10-9 10-8 Martin et al, JBC 2003

ER IS ACTIVATED IN MCF-7 CELLS RESISTANT TO LTED 0.5 1.0 1.5 2.0 2.5 Wt Wt LTED Cells (x106) ERa Ser118 LTED ERa C Oestradiol (M) 10-15 10-13 10-11 10-9 10-8 perbB2 pEGFR EGFR erbB2 2.5 5.0 7.5 10 Wt-MCF7 LTED Cell line Fold activity Basal transcription Martin et al, JBC 2003

RECETTORI ESTROGENICI: ERa RECETTORE ESTROGENICO a Cdk 2 p38 RSK PKA AKT c-src family MAPK ER a AF-1 DBD LBD/AF-2 Gli estrogeni ed il progesterone sono ormoni streroidei che regolano molteplici aspetti della patologia dell’epitelio ghiandolare mammario (crescita, differenziazione, sopravvivenza). Esistono due tipi di recettore estrogenico: alfa e beta. Sia il recettore alfa che il recettore beta contengono 4 domini funzionali. All’estremità N-terminale troviamo il dominio A/B che contiene la FUNZIONE DI ATTAVAZIONE 1 (AF-1), che contribuisca all’attivazione della trascrizione. Nella regione centrale il dominio C che contiene una regione responsabile del legame al DNA: DNA BINDING DOMAIN (DBD). Di seguito è presente il dominio D e il dominio E che contiene il sito di legame del ligando: LIGAND-BINDING DOMAIN (LBD) , siti regolatori per il legame con cofattori, il sito di transattivazione 2 (AF-2) e di localizzazione nucleare. Alcune regioni sono altamente conservate come per esempio il DBD, (96% di identicità) altre meno conservate come il LBD (56%) indicando come i recettori abbiano la stessa capacità nel legare le sequenze di riconoscimento sul DNA ma differente affinità per i ligandi. Inoltre un’altra regione scarsamente conservata è quella AF-1 e AF-2 suggerendo che differenti proteine possono interagire nel complesso trascrizionale potenziando la trascrizione di specifici targets.

NEW STRATEGIES TO OVERCOME ENDOCRINE RESISTANCE Integrating targeted therapies with hormonal therapies SERD: Fulvestrant

NEW STRATEGIES TO OVERCOME ENDOCRINE RESISTANCE Integrating targeted therapies with hormonal therapies SERD: Fulvestrant

Anti ErbB1-2 receptors Mabs (Trastuzumab, 2C4, C225) HER1,HER2,HER4, Tyrosine Kinase Inhibitors (ZD 1839, OSI 774, EKB 559, GW 2016, CI 1033) K K SHC GRB2 PI3K SOS Ras farnesyl transferase Inhibitors (BMS 214662, R115777) RAS PTEN AKT RAF RAF inhibithors mTOR inhibitors (CCI 779) Mek inhibitors (CI 1040) MEK 1/2 mTOR FKHR GSK-3 BAD MAP Cell cicle progression Survival

Trastuzumab Effect on HER2+Xenografts ED= Estrogen deprivation Osborne CK, SABCS 2007

Superiority of multidrug anti-HER therapy in Xenograft Models Osborne CK, SABCS 2007

TANDEM: Anastrazole +/- Herceptin trial design HER2 inhibitors TANDEM: Anastrazole +/- Herceptin trial design Anastrazole +/- Herceptin in HER2+, ER+, MBC (n=202) Anastrazole Herceptin Anastrazole

TANDEM: Clinical Benefit 50 Anastazole (n =104) 40 P=0.026 42,7 Anastazole + herceptin (n= 103) 30 27,9 20 Patients (%) 10 Clinical Benefit Machej JR, SABCS 2006

NEW STRATEGIES TO OVERCOME ENDOCRINE RESISTANCE Integrating targeted therapies with hormonal therapies SERD: Fulvestrant

SELETTIVITA’ DEL LEGAME 3H-ESTRADIOLO/RE Estradiolo vs Faslodex vs Tam 100 90 80 70 E2 60 Percentuae di inibizione Faslodex 50 Tam 40 30 20 10 1 5 10 50 100 300 1000 3000 10000 Concentrazione (nM)

Post-treatment Mean ER H-scores 120 100 1ng/ml 80 2.5ng/ml 5ng/ml Mean ± 1SEM 60 40 7ng/ml 23ng/ml 20 500mg LA = 14ng/ml ? 50mg 'Faslodex' (n=31) 125mg 'Faslodex' (n=32) 250mg 'Faslodex' (n=32) 6mg SA s/c (*n=6) 18mg SA s/c (*n=12) Placebo (n=29) J Robertson et al, Cancer Research 2001 DeFriend DJ, et al. Cancer Res. 1994;54:408-414.

Post-treatment Mean PgR H-scores 20 40 60 80 100 NS p=0.003 p=0.0002 p=0.0001 Placebo (n=28) 50mg Fulvestrant (n=29) 125mg 250mg Tamoxifen (n=21) Mean ± 1SEM Overall treatment effect p=0.0001 Three different doses of fulvestrant each produced a lower mean PgR H-score compared with placebo This was significant for the higher two doses Tamoxifen produced an increase in PgR H-score The difference between tamoxifen and fulvestrant was significant for all doses As PgR expression is under the control of the ER, this is evidence of both the partial agonist activity of tamoxifen and the lack of estrogen agonist activity of fulvestrant NS = not significant Robertson JFR et al. Cancer Res 2001; 61: 6739–6746.

Fulvestrant inhibits the growth of long-term oestrogen-deprived (LTED) MCF-7 cells Fold change in cell growth compared with control 1.4 LTED Wild type + 10-9M oestradiol 1.2 1.0 Fulvestrant is an effective inhibitor of LTED cell growth Consistent with ER signalling mechanism and supersensitisation of cells to oestradiol 0.8 0.6 0.4 Emerging data suggest altered growth factor signaling cross-talk with the ER plays a central role in the development of acquired resistance to estrogen deprivation. Fulvestrant antagonizes estradiol-stimulated transcription in both WT and LTED-I cells, with a reduction in basal transcription of more than 50% in LTED cells compared with 20% in WT cells [27]. Increasing concentrations of fulvestrant markedly decreased the growth of LTED-I MCF-7 cells relative to WT cells grown in the presence of estradiol . In contrast, 10)12–10)6 M tamoxifen had no effect on the growth of MCF-7 cells deprived of estrogen for >20 weeks [9], suggesting that fulvestrant is a more appropriate alternative to tamoxifen following AI resistan 0.2 0.0 Cont 10-12 10-10 10-9 10-8 10-7 10-6 Fulvestrant (M) Martin LA et al. J Biol Chem 2003; 278: 30458–30468

Drug concentration (M) LTED-R cells are hypersensitive to E2, refractory to tamoxifen, but sensitive to fulvestrant 3.0 E2 2.0 Cells (x106) Tamoxifen 1.0 Fulvestrant 10-12 C 10-13 10-11 10-10 10-9 10-8 Drug concentration (M) ER ER Tam Fulvestrant Martin et al, JBC 2003

Fulvestrant: Summary of Preclinical Data Downregulates estrogen receptors in breast cancer cells No estrogenic activity Completely blocks estrogen action A summary of key actions of fulvestrant, an Estrogen Receptor Downregulator

MCF7-F = FULVESTRANT RESISTENT MCF7-T = TAM RESISTENT MCF7-F = FULVESTRANT RESISTENT Fan M, Clin Cancer Res 2006

Fan M, Clin Cancer Res 2006

Fan M, Clin Cancer Res 2006

EGFR ERBB3 EGFR P p85 P p85 p110 p110 AKT AKT raptor-mTOR raptor-mTOR HIF-activation cell-growth

mTOR inhibitors: Dual inhibition of mTOR and ER signaling enhances cell death in breast cancer cells Fulvestrant+Rad 001 Fulvestrant Rad 001 Beeram M, Ann Oncol 2007

CONCLUSION I CONVERSELY Acquired resistence to TAMOXIFEN correlates with maintenance of the Era-positive phenotype and ERa pathways (AF-1; AF-2) CONVERSELY Acquired resistence to FULVESTRANT is caracterized by up-regulation of Era pathways resulting in a Estrogen receptor a (Era) independent proliferation

Grazie per l’attenzione!!!!!