CARO meeting 2012

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

Thank You

Questions?

Supplemental Data

AMPK α1

IP: α1 α2 β1 β2 γ1 γ2 γ3

A.