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The Preliminary Study of LKB1-AMPK-mTOR Signaling Pathway in None Small Cell Lung Cancer Cell Lines

Author: ZhangXia
Tutor: ChenZhe
School: Tianjin Medical University
Course: Internal Medicine
Keywords: Lung cacer LKB1 AMPK mTOR signaling pathway
CLC: R734.2
Type: Master's thesis
Year: 2011
Downloads: 299
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Abstract


Objective:Lung cancer is one of the most harmful tumors for human health and lives nowdays. It is generally considered to be a genetic disease with complex mechanisms, in which activation of oncogenes, inactivation of the tumor suppressor genes as well as dysregulation of multiple signaling pathaways play significant roles in tumorigenesis and development. LKB1, also known as Serine/Threonine Kinase 11-STK11,is a new suppressor gene. Studies have shown that somatic mutations of LKB1 are rare in the majority of sporadic tumors, however, its mutation rate in none-small cell lung cancer (NSCLC) is up to 15%-35%. Mouse model Studies suggest that LKB1 inhibits lung cancer incidence and progress, but the signaling pathway underlying LKB1 function is not well understood currently. Reuben J.Shaw study in MEF(mouse embryonic fibroblasts) demonstrated that LKB1 can phosphorylate and activate AMP-activated kinase(AMPK) which negatively regulates mammalian target of rapamycin (mTOR) activity, taken together, there is an LKB1-AMPK-mTOR signling pathway and hyper-activation of the mTOR is related to tumorigenesis and development.In this study, we use western blot to investigate the LKB1-AMPK-mTOR signaling pathway in various NSCLC cell lines including A549、H460、H1792、H1299、Calul and Homogeneous H1299-LKB1shRNA, H1299-pLK0.1 (with similar genetic background), thus providing rational for molecular targeted therapy of lung cancer.Methods:1、Harvest the cell lysate of A549、H460、H1792、H1299、Calu1、1299-PLK0.1, H1299-LKB1shRNA, and detect LKB1 expression by Western- Blot analysis.2、Treat H1792、H1299、Calul with different concentration of 2-Deoxyglucose (2-DG)(0mM、5mM、10mM、25mM) for 2 hours; Or treat them with 25mM 2-DG for different time(0min、30mn、1 hours、2 hours、4hours、8 hours、24 hours); Treat A549、H460、H1299-PLK0.1 and H1299-LKB1shRNA with 25mM 2-DG for 2 hours; compare the levels of AMPK-alpha phosphorylation (P-AMPK-a).3、Treat A549、H460、H1792、H1299、Calu1、H1299-PLK0.1 and H1299-LKB1shRNA with 25mM 2-DG for 2 hours, then measure the level of S6K and 4EBP1 phosphorylation.4、Pretreat H1792 cell with 10μM Compound C for 30 min before 2-DG treatment for 2 hours, then compare the levels of S6K and 4EBP1 phosphorylation.Results:1、LKB1 expression can be detected in H1299、H1792 and Calul(LKB1 wide-type cell lines),but not in A549 and H460 cells(LKB1 mutane cell lines); LKB1 expression is dramatically suppressed in H1299-LKB1shRNA compared with the control H1299-PLK0.1 cells.2、AMPK phosphorylation is increased in LKB1 wild-type cells (H1299、H1792 and Calul) upon 2-DG treatment, but not in LKB1 mutant cells(A549 and H460). H1299-LKB1shRNA is deficient in 2-DG-induced AMPK phosphorylation; 2-DG led to AMPK phosphorylation in a dose and time-dependent manner, with 5mM or 30minite- treatment of 2-DG starting to function.3、2-DG treatment led to inhibition of S6K and 4EBP1 phosphorylation in H1299, H1792 and Calu1 cell lines,but not in A549、H460、H1299-LKB1shRNA.4、The negative regulation of S6K and 4EBP1 phosphorylation in H1792 is blocked by AMPK inhibitor (Compound C).Conclusion:1、2-DG treatment led to AMPK phosphorylaiton in LKB1wide-type cell lines H1299、H1792 and Calu1, but not in A549 and H460 cells. H1299-LKB1shRNA impairs AMPK phosphorylation.2、LKB1 negatively regulatates mTOR activity(S6K and 4EBP1 phosphorylation) through activating AMPK; Loss of LKB1 and AMPK activity can block the negative regulation of mTOR signaling induced by 2-DG.3、LKB1-AMPK-mTOR signaling pathway is confirmed in NSCLC cell lines.

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CLC: > Medicine, health > Oncology > Respiratory system tumors > Lung tumors
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