Negative regulation of mTOR activity by LKB1-AMPK signaling in non-small cell lung cancer cells
Abstract
Li-xia DONG1, #, Lin-lin SUN2, #, Xia ZHANG1, Li PAN1, Lin-juan LIAN1, Zhe CHEN1, Dian-sheng ZHONG1, 2, *
1Department of Respiratory Medicine, Tianjin Medical University General Hospital, Tianjin 300052, China; 2Tianjin Lung Cancer Institute, Tianjin Medical University General Hospital, Tianjin 300052, China
Aim: To investigate the role of LKB1 in regulation of mTOR signaling in non-small cell lung cancer (NSCLC) cells.
Methods: LKB1 protein expression and phosphorylation of AMPK, 4E-BP1, and S6K in the cells were assessed using Western blotting in various NSCLC cell lines (A549, H460, H1792, Calu-1, and H1299). Energy stress was mimicked by treating the cells with 2-deoxyglucose (2-DG). Compound C was used to inhibit AMPK activity. Cell growth was measured using the MTS assay.
Results: LKB1 protein was expressed in LKB1 wild-type Calu-1, H1299, and H1792 cells, but it was undetected in LKB1 mutant A549 and H460 cells. Treatment of the LKB1 wild-type cells with 2-DG (5, 10, and 25 mmol/L) augmented the phosphorylation of AMPK in dose- and time-dependent manners. In the LKB1 wild-type cells, 2-DG dramatically suppressed the phosphorylation of two mTOR targets, 4E-BP1 and S6K, whereas the LKB1 mutant A549 and H460 cells were highly resistant to 2-DG-induced inhibition on mTOR activity. In addition, stable knockdown of LKB1 in H1299 cells impaired 2-DG-induced inhibition on mTOR activity. Pretreatment of H1299 and H1792 cells with the AMPK inhibitor compound C (10 µmol/L) blocked 2-DG-induced inhibition on mTOR activity. 2-DG inhibited the growth of H1299 cells more effectively than that of H460 cells; stable knockdown of LKB1 in H1299 cells attenuated the growth inhibition caused by 2-DG.
Conclusion: In non-small cell lung cancer cells, LKB1/AMPK signaling negatively regulates mTOR activity and contributes to cell growth inhibition in response to energy stress.
Keywords: non-small cell lung cancer; LKB1/AMPK/mTOR; 2-deoxyglucose
The project was supported by grants from the National Natural Science Foundation of China (No 30971307 and No 81071915) and the Tianjin Natural Science Foundation (No 10JCYBJC13700).
# These authors contributed equally to this work.
* To whom correspondence should be addressed.
E-mail zhongdsh@hotmail.com
Received 2012-07-21 Accepted 2012-09-17
Keywords:
1Department of Respiratory Medicine, Tianjin Medical University General Hospital, Tianjin 300052, China; 2Tianjin Lung Cancer Institute, Tianjin Medical University General Hospital, Tianjin 300052, China
Aim: To investigate the role of LKB1 in regulation of mTOR signaling in non-small cell lung cancer (NSCLC) cells.
Methods: LKB1 protein expression and phosphorylation of AMPK, 4E-BP1, and S6K in the cells were assessed using Western blotting in various NSCLC cell lines (A549, H460, H1792, Calu-1, and H1299). Energy stress was mimicked by treating the cells with 2-deoxyglucose (2-DG). Compound C was used to inhibit AMPK activity. Cell growth was measured using the MTS assay.
Results: LKB1 protein was expressed in LKB1 wild-type Calu-1, H1299, and H1792 cells, but it was undetected in LKB1 mutant A549 and H460 cells. Treatment of the LKB1 wild-type cells with 2-DG (5, 10, and 25 mmol/L) augmented the phosphorylation of AMPK in dose- and time-dependent manners. In the LKB1 wild-type cells, 2-DG dramatically suppressed the phosphorylation of two mTOR targets, 4E-BP1 and S6K, whereas the LKB1 mutant A549 and H460 cells were highly resistant to 2-DG-induced inhibition on mTOR activity. In addition, stable knockdown of LKB1 in H1299 cells impaired 2-DG-induced inhibition on mTOR activity. Pretreatment of H1299 and H1792 cells with the AMPK inhibitor compound C (10 µmol/L) blocked 2-DG-induced inhibition on mTOR activity. 2-DG inhibited the growth of H1299 cells more effectively than that of H460 cells; stable knockdown of LKB1 in H1299 cells attenuated the growth inhibition caused by 2-DG.
Conclusion: In non-small cell lung cancer cells, LKB1/AMPK signaling negatively regulates mTOR activity and contributes to cell growth inhibition in response to energy stress.
Keywords: non-small cell lung cancer; LKB1/AMPK/mTOR; 2-deoxyglucose
The project was supported by grants from the National Natural Science Foundation of China (No 30971307 and No 81071915) and the Tianjin Natural Science Foundation (No 10JCYBJC13700).
# These authors contributed equally to this work.
* To whom correspondence should be addressed.
E-mail zhongdsh@hotmail.com
Received 2012-07-21 Accepted 2012-09-17