Manpreet AhluwaliaHematology Oncology Fellow

Children’s National Medical Center

Pediatric Oncology BranchNational Cancer Institute

11/06/091

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

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

3

4

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

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

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

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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Hour 0

Hour 24 control

Hour 24 50uM

Hour 24 500uM

Hour 24 5mM

Similar results in other sarcoma cell lines

143B/GFP cells

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)

14

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

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

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

Total AMPK

p-AMPK

β-Actin

EXPOSURE TO METFORMIN PHOSPHORYLATES AMPK

HOS-MNNG

Metformin 0 50M 500M 5mM

143B

0 50M 500M 5mM

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

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

21β-Actin

Total ERM

p-ERM

p-PKC

METFORMIN DECREASE S PHOSPHORYLATION OF ERM AND PKC

143B

0 50M 500M 5mMMetformin

• 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

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