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Meccanismo di azione e superamento della resistenza ormonale Giuseppe Naso, Roberta Ferraldeschi Oncologia Medica B Università di Roma Sapienza Novel therapeutic.

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Presentazione sul tema: "Meccanismo di azione e superamento della resistenza ormonale Giuseppe Naso, Roberta Ferraldeschi Oncologia Medica B Università di Roma Sapienza Novel therapeutic."— Transcript della presentazione:

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

2 American Cancer Society. Breast Cancer Facts and Figures ; Jemal et al. CA Cancer J Clin ; 54 : 8-29 ; Rhodes et al. J Clin Pathol ; 53 : ; Johnston et al. Nat Rev Cancer ; 3 : Breast cancer tumors can be divided into two basic types :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 therapyAbout 75 % of breast cancers are estrogen receptor (ER) and/or progesterone receptor (PgR) positive and therefore are suitable for endocrine therapy Endocrine Dependency in Breast Cancer

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

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

5 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

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

7 Struttura dei ER e loro parti funzionali Review Breast Cancer Res (2004), 6: 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

8 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 lattività 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 Receptors

9 (adipe, ovaie, mammelle…) ER + Estrone AndrostenedioneTestosterone Estradiolo AROMATASI RECETTORI ESTROGENICI: ERα/ERβ ER- ER- ER- Cellula mammaria tumorale ?

10 Attività genomica classicaAttività genomica classica (nucleare) – attività NISS (Nuclear Initiated Steroid Signaling) Attività genomica non classica Attività 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

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

12 ER Genomic Function - non Classical ER E E E E E E E E E E E E Tumor Growth Tumor Growth mRNA Target Gene AP-1 FosFos JunJun

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

14 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

15 BRCA1 and HSP 90 exert a strong control on Er BRCA1 and HSP 90 exert a strong control on Er GATA-3 turn on the Er transcription

16 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).

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

19 Ligand-induced estrogen receptor α degradation by the proteasome

20 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

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

22 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

23 17 ESTRADIOLO AF1 AF2 TAMOXIFENE ERE ATTIVAZIONE PARZIALE DELLA TRASCRIZIONE (solo AF1) ANTAGONISTA AGONISTA INIBITORI DELLE AROMATASI BLOCCO COMPLETO DELLA TRASCRIZIONE, PERSISTENZA DELLA VIA RECETTORIALE ESTROGENICA FULVESTRANT BLOCCO COMPLETO DELLA TRASCRIZIONE, ABOLIZIONE COMPLETA DEL SEGNALE ESTROGENICO RE AF1 AF2 ERE RE AF1 AF2 RE ERE DIFFERENTE MECCANISMO DAZIONE: SERM, IA, SERD 17 ESTRADIOLO

24 What are the mechanisms of resistance to hormonal therapies?

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

26 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 RESISTENZA ORMONALE: IPOTESI

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

28 Pathway Activation-Other Receptors

29 Pancholi S, SABCS 2007

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

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

32 Chan CM et al. J Ster Biochem & Mol Biol 2002 ER, oestrogen receptor PgR, progesterone receptor No. of weeks in oestrogen-deprived medium Number of cells/mL (x10 5 ) Wild type (FBS) 11–20>20–50 MCF-7 cells – oestradiol Increased basal proliferation (>20 weeks) Quiescent (<15 weeks) ER PgR In vitro model of long-term oestrogen deprived (LTED) cells

33 C Oestradiol (M) Cells (x10 6 ) Wt LTED Martin et al, JBC 2003 ER IS ACTIVATED IN MCF-7 CELLS RESISTANT TO LTED

34 C Oestradiol (M) Cells (x10 6 ) Wt LTED Martin et al, JBC 2003 ER IS ACTIVATED IN MCF-7 CELLS RESISTANT TO LTED Hypersensitivity to E2 induced proliferation

35 C Oestradiol (M) WtLTED ERa Ser 118 ERa perbB2 pEGFR EGFR erbB Wt-MCF7LTED Cell line Fold activity Basal transcription Cells (x10 6 ) Wt LTED Martin et al, JBC 2003 ER IS ACTIVATED IN MCF-7 CELLS RESISTANT TO LTED

36 RECETTORI ESTROGENICI: ER RECETTORI ESTROGENICI: ER RECETTORE ESTROGENICO a Cdk 2 p38RSK PKA AKTc-src family MAPK ER AF-1DBD LBD/AF-2

37 NEW STRATEGIES TO OVERCOME ENDOCRINE RESISTANCE 1)Integrating targeted therapies with hormonal therapies 2)SERD: Fulvestrant

38 NEW STRATEGIES TO OVERCOME ENDOCRINE RESISTANCE 1)Integrating targeted therapies with hormonal therapies 2)SERD: Fulvestrant

39 12 KK SHC PI3K GRB2 SOS AKT RAS PTEN mTOR FKHRGSK-3BAD 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 , R115777) RAF inhibithors mTOR inhibitors (CCI 779) Mek inhibitors (CI 1040)

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

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

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

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

44 NEW STRATEGIES TO OVERCOME ENDOCRINE RESISTANCE 1)Integrating targeted therapies with hormonal therapies 2)SERD: Fulvestrant

45 Concentrazione (nM) Percentuae di inibizione E2 Faslodex Tam SELETTIVITA DEL LEGAME 3H-ESTRADIOLO/RE Estradiolo vs Faslodex vs Tam

46 Mean ± 1SEM 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 2001 DeFriend DJ, et al. Cancer Res. 1994;54: ng/ml 2.5ng/ml 5ng/ml 23ng/ml 6mg SA s/c (*n=6) 7ng/ml 500mg LA = 14ng/ml ? Post-treatment Mean ER H-scores

47 Robertson JFR et al. Cancer Res 2001; 61: 6739–6746. Post-treatment Mean PgR H-scores NS = not significant NS p=0.003 p= p= Placebo (n=28) 50mg Fulvestrant (n=29) 125mg Fulvestrant (n=29) 250mg Fulvestrant (n=29) Tamoxifen (n=21) Mean ± 1SEM Overall treatment effect p=0.0001

48 Wild type M oestradiol LTED Cont Fulvestrant (M) Fold change in cell growth compared with 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 long- term oestrogen-deprived (LTED) MCF-7 cells

49 C Cells (x10 6 ) Drug concentration (M) E2 Tamoxifen Fulvestrant ER TamFulvestrant LTED-R cells are hypersensitive to E2, refractory to tamoxifen, but sensitive to fulvestrant Martin et al, JBC 2003

50 Fulvestrant: Summary of Preclinical Data Downregulates estrogen receptors in breast cancer cellsDownregulates estrogen receptors in breast cancer cells No estrogenic activityNo estrogenic activity Completely blocks estrogen actionCompletely blocks estrogen action

51 MCF 7 MCF7-T = TAM RESISTENT MCF7-F = FULVESTRANT RESISTENT Fan M, Clin Cancer Res 2006

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55 p85 p110 AKT raptor-mTOR EGFR p85 p110 AKT raptor-mTOR EGFR ERBB3 P P HIF-activationcell-growth

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

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

58 Grazie per lattenzione!!!!!


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