Estrogen, mTOR, and Metastasis

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Dr. Lisa Henske is a Senior Member of the Medical Science Division at Fox Chase Cancer Center. Research in her lab focuses on both Tuberous Sclerosis Complex (TSC) and Lymphangioleiomyomatosis (LAM). This is her presentation from the December Boston LAM/TSC Research Seminar

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  • Estrogen, mTOR, and Metastasis

    1. 1. Estrogen, mTOR, and Metastasis Elizabeth (Lisa) Henske, MD Fox Chase Cancer Center Philadelphia
    2. 2. Estrogen, mTOR, and Metastasis <ul><li>Biochemical mechanisms </li></ul><ul><ul><li>Rheb inhibits C-Raf kinase (J Biol Chem 2006) </li></ul></ul><ul><ul><li>Estrogen activates Rheb and mTOR in MCF-7 cells </li></ul></ul><ul><ul><ul><li>(Cancer Research 2006) </li></ul></ul></ul><ul><li>Models (unpublished) </li></ul><ul><ul><li>Estrogen promotes metastasis of TSC2-null cells </li></ul></ul><ul><ul><li>Birt-Hogg-Dube: a yeast model for lung cysts </li></ul></ul>
    3. 3. What is Lymphangiomyomatosis (LAM)? Normal Lung LAM
    4. 4. Crino, Nathanson, and Henske. N Engl J Med 2006 Angiomyolipomas and LAM: a Working Model B-Raf C-Raf? Estrogen? Mechanism?
    5. 5. Tsc2 -/- MEFs: Tsc2 +/+ MEFs: + + + + serum starved 10 min FBS Phospho-p42/44MAPK TSC2 Activates B-Raf and p42/44 MAPK 32 P-MEK1 B-Raf IP: B-Raf Active Rheb B-Raf Im et al. Oncogene 2002 directly binds
    6. 6. Rheb Inhibits B-Raf: Rapamycin Resistant Phospho-S6 Myc-Rheb Myc-Wt-Rheb: Wt-B-Raf: + - + + HEK293 cells Karbowniczek et al., J Biol Chem 2004 Phospho- p42/44 MAPK IP: B-Raf B-Raf Activity Rapamycin: + - + + + + Rheb mTOR B-Raf Rapamycin
    7. 7. B-Raf MEK1/2 p42/44 MAPK Proliferation Differentiation Does Rheb also inhibit C-Raf? C-Raf/Raf-1
    8. 8. Whole cell lysate C-Raf: Rheb: 10 min, 20% FBS: + + + + + + + + 24 h, 20 nM Rapamycin + + + + + + + + Phospho-p42/44 MAPK Myc-Rheb IP: Flag Flag-C-Raf Phospho-Ser 338 Rheb inhibits C-Raf (as well as B-Raf) Magda Karbowniczek but could not detect interaction 32 P-MEK1 Rheb mTOR B-Raf Rapamycin
    9. 9. Whole cell lysate B-Raf: Rheb: 10 min, 20% FBS: + + + + + + + + 24 h, 20 nM Rapamycin + + + + + + + + HA-B-Raf Rheb Inhibits the Association of B-Raf/C-Raf IP: Magda Karbowniczek Endogenous C-Raf HA-B-Raf IP: C-Raf B-Raf C-Raf Rheb mTOR B-Raf Rapamycin
    10. 10. Rheb Inhibits B-Raf/Ras Binding Magda Karbowniczek HA-B-Raf IP: H-Ras H-Ras B-Raf: Q64L-Rheb: + + + + + + IP: IgG IP Whole cell lysate 10 min, 20% FBS: HA-B-Raf Myc-Rheb Tsc2 -/- MEFs + - - + Control siRNA: Rheb siRNA: Rheb Whole cell lysate IgG B-Raf H-Ras IP: B-Raf
    11. 11. Rheb inhibits B-Raf and C-Raf Kinases: Model Rheb B-Raf Im et al. Oncogene 2002 Karbowniczek JBC 2004 Karbowniczek JBC 2006 What role, if any, in TSC pathogenesis? B-Raf Ras B-Raf C-Raf
    12. 12. Crino, Nathanson, and Henske. N Engl J Med 2006 Angiomyolipomas and LAM: a Working Model Estrogen? MCF-7 cells
    13. 13. Estradiol-induced activation of mTOR in MCF-7 cells 0 15 30 0 15 30 0 15 30 Rapamycin Wortmannin Phospho-p70S6K (Thr389) Phospho-S6 (Ser235/236) Beta-actin E2 (min) Fold: 1 5 Fold: 1 4 p85S6K Jane Yu “ Non-genomic” or Cytoplasmic Estrogen Signaling
    14. 14. Estrogen Signaling Pathways (James D. Yager, and Nancy E. Davidson, NEJM 2006) Genomic pathway: Gene Transcription E2 activates mTOR: Rheb dependent? PI3K/Akt and MAPK activation

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