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Meccanismo di azione e superamento della resistenza ormonale

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  1. 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

  2. Endocrine Dependency in Breast Cancer • 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 American Cancer Society. Breast Cancer Facts and Figures2003-2004 ; 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.

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

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

  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. 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 ReviewBreast Cancer Res (2004), 6: 39-52

  8. 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.

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

  10. 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)

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

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

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

  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. GATA-3 turn on the Era transcription BRCA1 and HSP 90 exert a strong control on Era

  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).

  17. Irminger-Finger et al.Nature Reviews Cancer6, 382–391 (May 2006) | doi:10.1038/nrc1878

  18. Ligand-induced estrogen receptor α degradation by the proteasome

  19. 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

  20. 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

  21. 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

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

  23. What are the mechanisms of resistance to hormonal therapies?

  24. 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

  25. 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

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

  27. Pathway Activation-Other Receptors

  28. Pancholi S, SABCS 2007

  29. 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)

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

  31. ER Number of cells/mL (x105) PgR PgR Increased basal proliferation (>20 weeks) Quiescent (<15 weeks) Wild type (FBS) 1 1–20 >20–50 MCF-7 cells – oestradiol No. of weeks in oestrogen-deprived medium In vitro model of long-term oestrogen deprived (LTED) cells ER, oestrogen receptor PgR, progesterone receptor Chan CM et al. J Ster Biochem & Mol Biol 2002

  32. 2.5 2.0 1.5 1.0 0.5 0 10-15 10-13 10-11 10-9 10-8 C Oestradiol (M) ER IS ACTIVATED IN MCF-7 CELLS RESISTANT TO LTED Wt Cells (x106) LTED Martin et al, JBC 2003

  33. 2.5 2.0 1.5 1.0 0.5 0 10-15 10-13 10-11 10-9 10-8 C Oestradiol (M) ER IS ACTIVATED IN MCF-7 CELLS RESISTANT TO LTED Wt Hypersensitivity to E2 induced proliferation Cells (x106) LTED Martin et al, JBC 2003

  34. 2.5 2.0 1.5 1.0 0.5 0 10-15 10-13 10-11 10-9 10-8 C Oestradiol (M) pEGFR Basal transcription EGFR 10 perbB2 7.5 Fold activity 5.0 erbB2 2.5 0 Wt-MCF7 LTED Cell line ER IS ACTIVATED IN MCF-7 CELLS RESISTANT TO LTED Wt Wt LTED Cells (x106) ERa Ser118 LTED ERa Martin et al, JBC 2003

  35. RSK AKT p38 c-src family Cdk 2 MAPK PKA AF-1 DBD LBD/AF-2 ER a RECETTORI ESTROGENICI: ERa RECETTORE ESTROGENICO a

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

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

  38. 1 2 Anti ErbB1-2 receptors Mabs (Trastuzumab, 2C4, C225) HER1,HER2,HER4, Tyrosine KinaseInhibitors (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

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

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

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

  42. TANDEM:Clinical Benefit 50 Anastazole (n =104) 40 P=0.026 42,7 Anastazole + herceptin (n= 103) 30 27,9 20 Patients (%) 10 0 Clinical Benefit Machej JR, SABCS 2006

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

  44. SELETTIVITA’ DEL LEGAME 3H-ESTRADIOLO/REEstradiolo vs Faslodex vs Tam 100 90 80 70 E2 60 Faslodex Percentuae di inibizione 50 Tam 40 30 20 10 0 1 5 10 50 100 300 1000 3000 10000 Concentrazione (nM)

  45. 7ng/ml 23ng/ml ? 500mg LA = 14ng/ml Post-treatment Mean ER H-scores 120 100 1ng/ml 80 2.5ng/ml 5ng/ml Mean ± 1SEM 60 40 20 0 6mg SA s/c (*n=6) 50mg 'Faslodex' (n=31) 125mg 'Faslodex' (n=32) 250mg 'Faslodex' (n=32) 18mg SA s/c (*n=12) Placebo(n=29) J Robertson et al, Cancer Research 2001DeFriend DJ, et al. Cancer Res. 1994;54:408-414.

  46. p=0.0001 100 p=0.0001 p=0.0001 80 p=0.0002 p=0.003 NS 60 Mean ± 1SEM 40 20 Overall treatment effect p=0.0001 0 50mg Fulvestrant (n=29) Placebo(n=28) 125mg Fulvestrant (n=29) 250mg Fulvestrant (n=29) Tamoxifen (n=21) Post-treatment Mean PgR H-scores NS = not significant Robertson JFR et al. Cancer Res 2001; 61: 6739–6746.

  47. Fulvestrant inhibits the growth of long-term oestrogen-deprived (LTED) MCF-7 cells Fold changein cell growth comparedwith control 1.4 LTED Wild type + 10-9M oestradiol 1.2 • Fulvestrant is an effective inhibitor of LTED cell growth • Consistent with ER signalling mechanism and supersensitisation of cells to oestradiol 1.0 0.8 0.6 0.4 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

  48. LTED-R cells are hypersensitive to E2, refractory to tamoxifen, but sensitive tofulvestrant 3.0 E2 2.0 Cells (x106) Tamoxifen 1.0 Fulvestrant 0 10-12 10-13 10-11 10-10 10-9 10-8 C Drug concentration (M) ER ER Tam Fulvestrant Martin et al, JBC 2003

  49. Downregulates estrogen receptors in breast cancer cells No estrogenic activity Completely blocks estrogen action Fulvestrant: Summary of Preclinical Data