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Manpreet AhluwaliaHematology Oncology Fellow
Children’s National Medical Center
Pediatric Oncology BranchNational Cancer Institute
11/06/091
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1. Tumor cell growth
2. Angiogenesis
3. Invasion
4. Intravasation
5. Survival in circulation
6. Arrest in capillary bed (new organ)
7. Extravasation
DEATH Proliferation Proliferation
SINGLE CELL MICROMETASTASIS CLINICALLY DETECTABLE METASTASIS
- REGULATION OF ENERGY
- EZRIN PLAYS KEY ROLE
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Upstream regulators of AMPK: LKB1 and CaMKKs phosphorylate the same residue (Thr-172) on the α subunit of AMPK
Hardie D G et al. J Physiol 2006;574:7-15
©2006 by The Physiological Society
MetforminThiazolidinediones
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Protein synthesis
Glycogen synthesis
Gluconeogenesis
Fatty acid/cholesterol synthesis
AMPK
EF2/mTOR/p70S6K
GS
pTORC2
ACC1/HMGR
Fatty acid oxidation
ACC2
PFK2
Glycolysis
Mitochondrialbiogenesis
?
Glucoseuptake
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Metformin widely used anti-diabetic
Biguanide originates from French lilac
Recent studies have suggested an anticancer property to metformin
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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
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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
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The extent to which metformin inhibits mesenchymal tumor cell growth metastasis specifically in sarcomas, is currently unknown
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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
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METFORMIN MODESTLY INHIBITS CELL GROWTH IN A TIME & DOSE DEPENDENT MANNER
143B GFP cells treated with metformin at 100hrs Calcein AM stain
0
20
40
60
80
100
120
Control 50uM 500uM 5000uM
concentration
% g
row
th o
f n
orm
al
% survival
Similar results observed in other sarcoma cell lines
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Control 50M
500M 5mM
METFORMIN SIGNIFICANTLY DECREASES ANCHORAGE INDEPENDENT GROWTH
Similar results seen in other sarcoma cells
143B/GFP cells
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10
Hour 0
Hour 24 control
Hour 24 50uM
Hour 24 500uM
Hour 24 5mM
Similar results in other sarcoma cell lines
143B/GFP cells
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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)
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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;43:442- 444
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WHOLE ORGAN LUNG METASTASIS CULTURE (EX VIVO)
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Untreated
1mM Metformin
5mM Metformin
Day 14P
erce
nt
Rel
ativ
e F
luo
resc
ence
Lu
ng
Cu
ltu
re
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EXPERIMENTAL METASTASIS MODEL
Tail vein injection1 x 106 tumor cells
& Confirm metastasis
Mice euthanizedonce symptomaticfrom lung metastasis
DAY 0
Started metformin 200ug/ml (1.2mM) in
drinking water n=10 mice
DAY 7
n=20 SCID mice
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METFORMIN MODESTLY IMPROVES SURVIVAL OF MICE IN EXPERIMENTAL METASTASIS
Survival of HOS MNNG Experimental Metastasis
0 200
25
50
75
100Placebo
Metformin
30 40 50 60 70 80
Days
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 metformin17
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Total AMPK
p-AMPK
β-Actin
EXPOSURE TO METFORMIN PHOSPHORYLATES AMPK
HOS-MNNG
Metformin 0 50M 500M 5mM
143B
0 50M 500M 5mM
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p-S6K
Total S6K
β-Actin
METFORMIN DECREASES PHOSPHORYLATION OF DOWNSTREAM TARGET OF MTORC1
HOS-MNNG
Metformin 0 50M 500M 5mM
143B
0 50M 500M 5mM
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20β-Actin
Total Akt
p-Akt Ser (450)
EXPOSURE TO METFORMIN DECREASES PHOSPHORYLATION OF AKT
143B
0 50M 500M 5mM
HOS-MNNG
Metformin 0 50M 500M 5mM
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21β-Actin
Total ERM
p-ERM
p-PKC
METFORMIN DECREASE S PHOSPHORYLATION OF ERM AND PKC
143B
0 50M 500M 5mMMetformin
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• Ezrin mediated metastasis linked to Akt/mTOR/p70S6K1/4E-BP1 pathway• Rapamycin / Rapalogue reduced metastasis in murine model of sarcoma
LKB1
AMPK
TSC2/TSC1
mTORC1
p70S6K
4E-BP1
PROTEIN SYNTHESIS
METFORMINMETFORMIN
CaMKKCa2+
mTORC2
PKC
Ezrin
WORKING MODEL
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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 23
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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