Role Of LKB1 In Akt Mediated Phosphorylation Of FOXO3a In NSCLC

Background and object: Lung cancer,with increasing morbidity and mortality in recent years,is one of the most common malignant tumors.Non-small cell lung cancer(NSCLC)accounts for about 80% ～ 85% of all types of lung cancer.Although the treatment of lung cancer has made considerable progress in recent years,the treatment effect is poor because non-small cell lung cancer tends to metastasis in early phase.Further research on the molecular mechanism of the development of non-small cell lung cancer will help to provide new diagnosis and treatment ideas for these patients,thus improving the quality of life of patients.The development of lung cancer is related to environment,genetics,the activation of oncogene and inactivation of tumor suppressor gene.The recent research has shown that a tumor suppressor gene LKB1(liver kinase B1)mutations occur in a variety of tumor cells,which is rare in sporadic tumors but occur in up to 20-30% of non-small cell lung cancer.LKB1 plays an important role in regulating cell energy metabolism,cell growth and cell polarity through downstream kinases.The downstream effluent molecule AMPK(AMP-regulated protein kinase)is one of the important target proteins of LKB1 and plays an important role in regulating the energy metabolism of cells.LKB1/AMPK signal can inhibit m TOR signaling pathway and inhibit tumor cell growth.When LKB1 is inactivated,it can promote the development of lung cancer through multiple pathways.PI3K/Akt signal pathway is one of the most common abnormal pathways in tumor cells.It is involved in regulating cell proliferation and apoptosis,angiogenesis and invasion and metastasis of tumors.In the downstream of Akt,FOXO3 a tumor suppressor transcription factors are widely found in various tissues of the human body and it play an important role in regulating cell proliferation,differentiation,apoptosis and regulation of immunity.The loss of FOXO3 a function caused by different mechanisms may promote the regulation of cell proliferation and the accumulation of cell damage,leading to the occurrence of tumor and other diseases.Our previous work has found that activation of Akt could increase the phosphorylation level of FOXO3 a in Thr32 in LKB1 wild-type cell line,but not in LKB1 wild-type cell line,which suggested that LKB1 may be involved in the regulation of phosphorylation of FOXO3 a which is a downstream target molecule of Akt.Based on the previous studies,this study further explored the regulation of phosphorylation of FOXO3 a downstream of Akt by LKB1 and its related mechanisms through cytological experiments in vitro and molecular biology techniques.Methods:(1)we transfect both LKB1 mutant and wild NSCLC cell lines with p EGFP-C2 plasmid,LKB1 wild type plasmid and LKB1-K78M(KD,kinase-deficent)plasmid.By detecting the expression of p-FOXO3 a,verifying the role of LKB1 in the regulation of phosphorylation of FOXO3 a,which is related to its kinase activity.(2)LKB1 stable knockdown H1299 cell line was established using a lentiviral short hairpin RNA(sh RNA).To identify the knockdown effect,LKB1 m RNA and protein expression level were evaluated with quantitative real-time PCR and Western Blot.And then the expression of p-FOXO3 a and Bim were measured by Western Blot.(3)We treated the NSCLC cell with A-769662 to determine the expression of P-FOXO3 by Western Blot.Results:(1)Compared with K78 M mutant LKB1 plasmids and control,after transfected with the wild-type LKB1 plasmid,the expression of P-FOXO3 a were significantly increased in cells with both LKB1 mutant and wild type.(2)LKB1 m RNA and protein expression were significantly suppressed in LKB1 stable knockdown H1299 cell line,suggesting that we established isogenic LKB1 stable knockdown cell lines using LKB1 wild type H1299 cells.Detecting the expression of p-FOXO3 a and its downstream target protein Bim verified that LKB1 positively regulates the phosphorylation of FOXO3 a.(3)Compared with control,in H1299 cell line treated with AMPK activating agent A-769662,higher expression level of P-FOXO3 was deteced.Conclusion:(1)In both LKB1 mutant and wild NSCLC cell lines,the over-expression of LKB1 leads to higher expression level of p-FOXO3 a,which is related to its kinase activity.(2)The knockdown of LKB1 in NSCLC leads to less expression of p-FOXO3 a and higher expression of the downstream target protein Bim.(3)LKB1 may participate in the regulation of P-FOXO3 a through its downstream kinase activity molecular AMPK.