caro meeting 2012
TRANSCRIPT
Sestrin2 sensitizes breast and lung cancer cells to ionizing radiation through
modulation of AMP-activated protein kinase (AMPK).
Toran Sanli, Katja Linher-Melville, Yaryna Storozhuk, Gurmit Singh, & Theodoros Tsakiridis
Presented by: Toran Sanli, M.Sc., Ph.D.
Department of Oncology,
McMaster University
Radiation TherapyRadiation Therapy
• Radiation Therapy (RT) is a common treatment modality for breast and lung cancer.
• However, the overall response and survival of patients undergoing RT remains poor, indicating the need to enhance the efficiency of radiation.
Pathways Effected by Ionizing Radiation
• Recently, we observed that therapeutic doses of IR rapidly activates AMP-activated protein kinase (AMPK).
Sanli et al, 2010
• Ionizing Radiation (IR) causes DNA damage, leading to cell cycle arrest & apoptosis in cancer cells.
• DNA repair and survival following IR are regulated through complex molecular pathways involving oncogenes and tumour suppressors.
P Thr-172
LKB1 CaMKK TAK1
αα1, α2
ββ1, β2
γγ1, γ2, γ3
AMPK
AMPK acts as a master regulator of cellular energy homeostasis
AMPK: Structure and FunctionAMPK: Structure and Function
Energy Production ↑Enhanced fatty acid oxidiationand glucose uptake
Energy Consumption ↓Decreased fat, glycogen, and protein synthesis
AMP ATP
Energy Homeostasis
AMP : ADP : ATP
Low Energy
The Sestrins
• Sestrins (SESN) are p53-target genes that function as antioxidants that accumulate in cells exposed to genotoxic stresses.
• Mammals express 3 SESN family members (SESN1-3).
Budanov et al, 2010 Science
• SESN1/2 have been shown to interact with and regulate AMPK activity in response to pathological stress stimuli.
Hypothesis & Objectives
• Sestrin2 (SESN2) sensitizes cancer cells to IR and is required for radiation-induced AMPK expression and activity.
Objectives:• Determine which subunits of AMPK interact
with SESN2.• Evaluate the effect of SESN2 overexpression
on AMPK.• Determine the role of sestrins in mediating
cancer cell sensitivity to IR.
SESN2 associates with AMPKα1β1γ1 and increases its expression/activity
- + IP-Sesn2F
AMPK β1
AMPK α1
AMPK γ1
Immunoprecipitation Assay
0
50
100
150
200
250
300
350
400
450
500
0 ug 0.05 ug 0.125 ug 0.25 ug 0.5 ug 1 ugS
ES
N2
leve
ls (%
of c
on
tro
l)
B.
*
**
*
**
SESN2
Western Blotting
A.
AMPKα1
AMPKβ1
AMPKγ1
C.
Actin
P-AMPKα
0 0.05 0.125 0.25 0.5 1 .0 Sesn2F (μg)
Actin
AMPKα1
Actin
AMPKβ1
A.
SESN2
AMPKγ1
P-ACC
- + - + SESN2 siRNA
- - + + 8Gy IR
P-AMPKα
**
*
*
**
#
##
#
#
B.
**
MCF7
Western Blotting
- + - + SESN2 siRNA
- - + + 8Gy IR
AMPKα1
Actin
AMPKβ1
SESN2
AMPKγ1
C.
P-ACC
P-AMPKα
D.
**
*
*
*
*
##
##
*
A549
IR Increases AMPK Expression Through SESN2
Control 10Gy IRE.
Western Blotting
SESN2 Overexpression Inhibits mTOR and Sensitizes Cancer Cells to IR
A.
B.
P-Akt (Thr308)
P-Akt (Ser473)
+ - + - Dox
- - + + 8Gy IR
SESN2
P-p70 S6K
P-mTOR
** **
**
*
##
##
*
**
0 2 4 6 80.01
0.1
1
Basal
SESN2
Dose (Gy)
Su
rviv
ing
Fra
cti
on
C.
*
D.
**
*
#
1 1
1
Ionizing Radiation
Activity/Expression
Sestrin2 P
Survival
mTOR
Akt
AMPK
Model of IR-mediate SESN2-AMPK Model of IR-mediate SESN2-AMPK SignallingSignalling
Conclusions
• SESN2 directly interacts with AMPK subunits.
• Overexpression of SESN2 is sufficient to increase AMPK activity and expression, and is required for IR-mediated regulation of this enzyme.
• Enhanced SESN2 levels can act as a radiation sensitizer in cancer cells.
• Future work will be focused on understanding the direct mechanism in which sestrins regulate AMPK, and the potential for sestrins to act as biomarker for cancer.
Acknowledgments:
Juravinski Cancer Centre• Dr. Gurmit Singh • Dr. Theos Tsakiridis• Dr. Katja Linher
• CIHR
• Dr. Andrei Budanov