Radiation Oncology research talk, Sept 2012

1. 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

2. 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.

3. 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.

4. P Thr-172
LKB1 CaMKK TAK1
α
α1, α2
β
β1, β2
γ
γ1, 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 oxidiation
and glucose uptake
Energy Consumption ↓
Decreased fat, glycogen, and
protein synthesis
AMP ATP
Energy Homeostasis
AMP : ADP : ATP
Low Energy

5. 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.

6. 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.

7. 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 ug
SESN2levels(%ofcontrol)
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

8. AMPKα1
Actin
AMPKβ1
A.
SESN2
AMPKγ1
P-ACC
- + - + SESN2 siRNA
- - + + 8Gy IR
P-AMPKα
**
*
*
**
#
#
#
#
#
B.
**
MCF7
Western Blotting

9. - + - + SESN2 siRNA
- - + + 8Gy IR
AMPKα1
Actin
AMPKβ1
SESN2
AMPKγ1
C.
P-ACC
P-AMPKα
D.
**
*
*
*
*
#
#
#
#
*
A549
IR Increases AMPK Expression Through SESN2
Control 10Gy IR
E.
Western Blotting

10. 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 8
0.01
0.1
1
Basal
SESN2
Dose (Gy)
SurvivingFraction
C.
*
D.
*
*
*
#

11. 1
1
1
Ionizing Radiation
Activity/Expression
Sestrin2 P
Survival
mTOR
Akt
AMPK
Model of IR-mediate SESN2-AMPKModel of IR-mediate SESN2-AMPK
SignallingSignalling

12. 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.

13. Acknowledgments:
Juravinski Cancer Centre
• Dr. Gurmit Singh
• Dr. Theos Tsakiridis
• Dr. Katja Linher
• CIHR
• Dr. Andrei Budanov

14. Thank You
Questions?

15. Supplemental Data
AMPK α1
IP: α1 α2 β1 β2 γ1 γ2 γ3
A.