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Role of Metformin in Cancer Metastasis

Role of Metformin in Cancer Metastasis. Manpreet Ahluwalia Hematology Oncology Fellow Children’s National Medical Center Pediatric Oncology Branch National Cancer Institute 11/06/09. Stress, Survival and Cancer Metastasis: A working hypothesis. - REGULATION OF ENERGY.

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Role of Metformin in Cancer Metastasis

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  1. Role of Metformin in Cancer Metastasis Manpreet Ahluwalia Hematology Oncology Fellow Children’s National Medical Center Pediatric Oncology Branch National Cancer Institute 11/06/09

  2. Stress, Survival and Cancer Metastasis: A working hypothesis - REGULATION OF ENERGY 1. Tumor cell growth -EZRIN PLAYS KEY ROLE 2. Angiogenesis 5. Survival in circulation 3. Invasion 4. Intravasation 6. Arrest in capillary bed (new organ) 7. Extravasation DEATH Proliferation Proliferation MICROMETASTASIS CLINICALLY DETECTABLE METASTASIS SINGLE CELL

  3. Upstream regulators of AMPK: LKB1 and CaMKKs phosphorylate the same residue (Thr-172) on the α subunit of AMPK Thiazolidinediones Metformin 3 Hardie D G et al. J Physiol 2006;574:7-15 ©2006 by The Physiological Society

  4. Protein synthesis Glycolysis Glycogen synthesis EF2/mTOR/p70S6K PFK2 GS AMPK Fatty acid oxidation Gluconeogenesis ACC2 pTORC2 ? ACC1/HMGR Fatty acid/cholesterol synthesis Glucose uptake Mitochondrial biogenesis

  5. Background • Metformin widely used anti-diabetic • Biguanide originates from French lilac • Recent studies have suggested an anticancer property to metformin • Epidemiological studies suggested metformin exposure was protective • from cancer in diabetics • Evans JM, Donnelly LA, et al. BMJ 2005 • Metformin selectively impaired p53 deficient tumor cell growth • Buzzai M, Joines RS, et al. Cancer Res 2007 • Metformin inhibits mTOR dependent translation initiation in breast cancer • cells through activation of AMPK • Dowling RJ, Sonenberg N, et al. Cancer Res 2007

  6. Rationale • An over-riding hypothesis in our lab is that sarcoma cells are uniquely programmed to endure the stress of metastasis. • One such stress may be related to energy availability and utilization. • Modulating these stress responses in sarcoma cells may be a means to improve patient outcomes. • Metformin may be a mean to modulate this stress response Question… The extent to which metformin inhibits mesenchymal tumor cell growth metastasis specifically in sarcomas, is currently unknown

  7. Question 1 • Determine the extent to which metformin inhibits in vitro biology of sarcoma cells • Cell lines: • Human  HOS-MNNG,143B, LM7 (SaOS2), MG63.2, RD Methods: • Proliferation assay • Cell viability assay • Migration assay • Colony formation in soft agar

  8. Metforminmodestly inhibits cell growth in a time & dose dependent manner Similar results observed in other sarcoma cell lines

  9. Metformin Significantly decreases anchorage independent growth 50M 143B/GFP cells Control 500M 5mM Similar results seen in other sarcoma cells

  10. Metformin Decreases cell motility in a wound healing scratch assay Hour 0 143B/GFP cells Hour 24 50uM Hour 24 control Hour 24 500uM Hour 24 5mM Similar results in other sarcoma cell lines

  11. Question 2 • Determine the extent to which metformin inhibits tumor growth & metastasis in sarcoma cells ex vivo and in vivo • Whole Organ Lung Metastasis Culture (ex vivo) • Primary tumor growth (in vivo) • Experimental/Spontaneous metastasis (in vivo)

  12. Correlation b/w in vitro & vivo dose for metformin difficult to ascertain • Physiological exposure in plasma 40M/L • Higher concentrations achieved in the tissues Wilcock C, Bailey CJ. Xenobiotica. 1994;24:49 -57 Wilcock C, Wyre ND, Bailey CJ. J Pharm Pharmacoll.1991;43:442- 444

  13. Whole Organ Lung Metastasis Culture (ex vivo)

  14. Metformin Significantly decreased metastatic progression of HOS-MNNG/GFP cells Day 14 Untreated Percent Relative Fluorescence Lung Culture 1mM Metformin 5mM Metformin

  15. EXPERIMENTAL METASTASIS MODEL Started metformin 200ug/ml (1.2mM) in drinking water n=10 mice Mice euthanized once symptomatic from lung metastasis & Confirm metastasis Tail vein injection 1 x 106 tumor cells n=20 SCID mice DAY 7 DAY 0

  16. METFORMIN MODESTLY IMPROVES SURVIVAL OF MICE IN EXPERIMENTAL METASTASIS p value .009

  17. Question 3 • Characterize the intracellular signaling pathways activated in sarcoma cells treated with metformin which correlate with changes in cell phenotype • Western blot analysis (AMPK/LKB1/mTOR/S6K) • Protein from in vitro cell lysates obtained from cell lines inhibited by metformin • Protein from fresh tumor samples from mice treated with metformin

  18. EXPOSURE TO METFORMIN PHOSPHORYLATES AMPK HOS-MNNG 143B Metformin 50M 500M 5mM 0 50M 500M 5mM 0 p-AMPK  Total AMPK β-Actin

  19. METFORMIN DECREASES PHOSPHORYLATION OF DOWNSTREAM TARGET OF MTORC1 HOS-MNNG 143B Metformin 5mM 500M 50M 0 50M 500M 5mM 0 p-S6K Total S6K β-Actin

  20. EXPOSURE TO METFORMIN DECREASES PHOSPHORYLATION OF AKT HOS-MNNG 143B Metformin 5mM 500M 50M 0 50M 500M 5mM 0 p-Akt Ser (450) Total Akt β-Actin

  21. METFORMIN DECREASE S PHOSPHORYLATION OF ERM AND PKC Metformin 143B p-PKC  50M 500M 5mM 0 p-ERM Total ERM β-Actin

  22. WORKING MODEL METFORMIN LKB1 CaMKK Ca2+ Ezrin AMPK PKC TSC2/TSC1 mTORC2 mTORC1 p70 S6K 4E-BP1 • Ezrin mediated metastasis linked to Akt/mTOR/p70S6K1/4E-BP1 pathway • Rapamycin / Rapalogue reduced metastasis in murine model of sarcoma PROTEIN SYNTHESIS

  23. SUMMARY • Metformin inhibits features of metastatic phenotype - in vitro - ex vivo - in vivo • Metformin has a wide therapeutic index in mice • Mechanism of action of metformin unclear - activates AMPK - inhibits mTORC1 - inhibits activation of Akt/PKC/ERM

  24. Acknowledgements • TMBS, Pediatric Oncology Branch • Chand Khanna • Sung-Hyeok Hong • Arnulfo Mendoza • Ling Ren • Joseph Briggs • Lauren Buquo • Martin Mendoza • Tanasa Osborne • Kaylee Nuckolls • Comparative Oncology Program • Melissa Paoloni • Christina Mazcko

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