This document discusses the relationships between estrogen, mTOR, and metastasis. It summarizes that estrogen activates Rheb and mTOR in MCF-7 cells, promoting metastasis of TSC2-null cells. It also discusses models showing that Rheb inhibits B-Raf and C-Raf kinases, and that Rheb inhibits the association of B-Raf and C-Raf with Ras. Finally, it shows that estradiol activates mTOR in MCF-7 cells in a rapamycin-sensitive manner, indicating a non-genomic estrogen signaling pathway involving mTOR.

1. Estrogen, mTOR, and Metastasis Elizabeth (Lisa) Henske, MD Fox Chase Cancer Center Philadelphia

3. What is Lymphangiomyomatosis (LAM)? Normal Lung LAM

4. Crino, Nathanson, and Henske. N Engl J Med 2006 Angiomyolipomas and LAM: a Working Model B-Raf C-Raf? Estrogen? Mechanism?

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. 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. B-Raf MEK1/2 p42/44 MAPK Proliferation Differentiation Does Rheb also inhibit C-Raf? C-Raf/Raf-1

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. 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. 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. 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. Crino, Nathanson, and Henske. N Engl J Med 2006 Angiomyolipomas and LAM: a Working Model Estrogen? MCF-7 cells

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. 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