Negative regulation of mTOR activity by LKB1-AMPK signaling in non-small cell lung cancer cells

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-deoxyglu- cose （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 tar- gets, 4E-BP1 and S6K, whereas the LKB1 mutant A549 and H460 cells were highly resistant to 2-DG-induced inhibition on mTOR activ- ity. 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 pmol/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.