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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
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Stress, Survival and Cancer Metastasis: A working hypothesis
- REGULATION OF ENERGY 1. Tumor cell growth - EZRIN PLAYS KEY ROLE 2. Angiogenesis 3. Invasion 5. Survival in circulation 4. Intravasation - Early metastasis or survival of cancer cells - Protection from death in a foreign environment requires new protein synthesis to survive. 6. Arrest in capillary bed (new organ) DEATH Proliferation SINGLE CELL MICROMETASTASIS CLINICALLY DETECTABLE METASTASIS 7. Extravasation
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Thiazolidinediones Metformin
Upstream regulators of AMPK: LKB1 and CaMKKs phosphorylate the same residue (Thr-172) on the α subunit of AMPK Upstream regulators of AMPK LKB1 and CaMKKs phosphorylate the same residue (Thr-172) on the α subunit of AMPK. Phosphorylation by LKB1, but not by CaMKKs, is stimulated by binding of AMP to AMPK. Thiazolidinediones Metformin 3 Hardie D G et al. J Physiol 2006;574:7-15 ©2006 by The Physiological Society
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Fatty acid/cholesterol
Protein synthesis AMPK EF2/mTOR/p70S6K GS pTORC2 ACC1/HMGR Fatty acid oxidation ACC2 PFK2 Glycolysis Mitochondrial biogenesis ? Glucose uptake Glycogen synthesis Gluconeogenesis Fatty acid/cholesterol synthesis
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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 -Metformin/glucophage is a widely used antidiabetic agent, which regulates glucose homeostasis through inhibition of gluconeogenesis and stimulates uptake of glucose in skeletal muscle -French lilac plant that is known for centuries to reduce diabetes symptoms
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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
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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 State which cell lines and why these cell lines (K7M2 metastasis in vivo, HOS-MNNG/143B clonally related yet differ in phenotype) HOS is non-tumorogenic in nude mice, when MNNG drug added forms tumors in nude mice Cell Counting Kit-8 (CCK-8) allows convenient assays using Dojindo's tetrazolium salt, WST-8 CCK-8 is a sensitive nonradioactive colorimetric assay for determining the number of viable cells in cytotoxicity assays WST-8 is bioreduced by cellular dehydrogenases to an orange formazan product that is soluble in tissue culture medium. The amount of formazan produced is directly proportional to the number of living cells Wound healing assays are a classic and commonly used method for studying cell migration and the biology underlying it [ 4]. They have been used with multiple cell types and, as the monolayers heal the wound in a characteristic manner, they have been used to study cell polarization, matrix remodeling, cell migration, and numerous other processes
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Similar results observed in other sarcoma cell lines
Metformin modestly inhibits cell growth in a time & dose dependent manner Similar results observed in other sarcoma cell lines
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Similar results seen in other sarcoma cells
Metformin Significantly decreases anchorage independent growth Control 50M 500M 5mM 143B/GFP cells Similar results seen in other sarcoma cells
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Metformin Decreases cell motility in a wound healing scratch assay
Hour 0 Hour 24 control 50uM 500uM 5mM 143B/GFP cells Similar results in other sarcoma cell lines
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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) For in vivo studies we will utilize an established system of osteosarcoma models with metastatic phenotype in our laboratory (K7M2) Assay reliably predicts metastatic phenotype in both carcinomas and sarcomas in HOS & K7M2 Experimental metastasis model-briefly describe-tail vein injection of which tumor cells. When will mice be treated with metformin? Treat tumor is palpable, 3 days before amputation & after amputation Do you want to look at primary tumor growth? If so, you should probably reword the aim. If you do primary tumor stuff, state how (i.e. I.M., subcut., etc.). How will you quantify response? How many mice, lung culture repeats, etc. What will your controls be? At the end of experiments proposed in Aim 2 what do you expect to be able to say?
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Correlation b/w in vitro & vivo dose for metformin difficult to ascertain
Physiological exposure in plasma 40M/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:
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Whole Organ Lung Metastasis Culture (ex vivo)
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Percent Relative Fluorescence
Metformin Significantly decreased metastatic progression of HOS-MNNG/GFP cells Day 14 Percent Relative Fluorescence Lung Culture Untreated 1mM Metformin 5mM Metformin
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Started metformin 200ug/ml (1.2mM) in drinking water n=10 mice
EXPERIMENTAL METASTASIS MODEL Started metformin ug/ml (1.2mM) in drinking water n=10 mice DAY 7 Mice euthanized once symptomatic from lung metastasis & Confirm metastasis Tail vein injection 1 x 106 tumor cells n=20 SCID mice DAY 0
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METFORMIN MODESTLY IMPROVES SURVIVAL OF MICE IN
EXPERIMENTAL METASTASIS p value .009
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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 State which material you will use (i.e. fresh tumor samples from mice treated with drug in Aim 2, in vitro cell lysates from Aim 1, etc.). State what you'll be isolating (protein) How you'll isolate it (tumor tissue processing by dissection/homogenization/lysis using something like a modified RIPA buffer with complete protease and phosphatase inhibitors) In vitro cell lysate harvesting using a buffer like above followed by protein quantitation to ensure equal protein loading for Western blot analysis
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EXPOSURE TO METFORMIN PHOSPHORYLATES AMPK
HOS-MNNG Metformin 50M 500M 5mM 143B 50M 500M 5mM p-AMPK Total AMPK β-Actin
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METFORMIN DECREASES PHOSPHORYLATION OF DOWNSTREAM TARGET OF MTORC1
HOS-MNNG Metformin 50M 500M 5mM 143B 50M 500M 5mM p-S6K Total S6K β-Actin
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EXPOSURE TO METFORMIN DECREASES PHOSPHORYLATION OF AKT
HOS-MNNG Metformin 50M 500M 5mM 143B 50M 500M 5mM p-Akt Ser (450) Total Akt β-Actin
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METFORMIN DECREASE S PHOSPHORYLATION
OF ERM AND PKC 143B 50M 500M 5mM Metformin p-PKC p-ERM Total ERM β-Actin
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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
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Metformin inhibits features of metastatic phenotype
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
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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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