The Molecular Mechanism Regulation Of P21^WAF1/CIP1 Mediated By LKB1

Background and object:Cancer is a major public health problem all over the world.Based on GLOBOCAN estimates,the incidence and mortality of tumor is increasing rapidly.The mutation of tumor suppressor gene is known to play a key role in the progression of the cancer.A new tumor suppressor gene,LKB1 attracts more an more attention.LKB1,which is the the causative gene for Peutz-Jeghers syndrome,is a predisposition to certain cancers.The inactivation of LKB1 is rare in most sporadic cancers.While the percentage of LKB1 loss-of-function somatic mutations in NSCLC is up to 30%.Previous studies showed that LKB1 leads to induction of p21^WAF1/CIP1 expression,arresting cell cycle and inhibiting cell growth in He La S3（cervical cancer cell line）and G361 cells（melanoma cell line）.While the mechanism of the regulation p21^WAF1/CIP1 mediated by LKB1 in NSCLC is still not clear.Besides,one of the downstream kinase of LKB1,AMPK can increase p21^WAF1/CIP1 protein through the directly up-regulation of p53 Ser15 under low energy levels and in vitro.No published work has shown the direct evidence for LKB1 is through its downstream kinase AMPK to induce p21^WAF1/CIP1 expression.Based on this,firstly we established the LKB1 stable knockdown cell line;secondly we explored the regulation p21^WAF1/CIP1 mediated by LKB1 in NSCLC;thirdly we attempted to explore whether LKB1 is through its downstream kinase AMPK to induce p21^WAF1/CIP1 expression.Methods:（1）A549,a NSCLC cell line with LKB1 mutant-type and p53 wide-type,was transfected with the wild-type and K78M mutant LKB1 plasmids.Cell colony formation assay was performed to detect the cell growth.Next,A549 and H460（LKB1 mutated-type and p53 wide-type）cells were transfected with the wild-type,K78M mutant LKB1 plasmids and the control plasmids.The expression of p21^WAF1/CIP1 is measured by Western Blot.Cell cycle phase distribution were analyzed by flow cytometry.Then,H1299 and Calu-1（LKB1 wide-type and p53mutant-type）were transfected with the wild-type LKB1 plasmids and the control plasmids.p21^WAF1/CIP1 expression was assessed by quantitative real-time PCR and Western Blot.Cell cycle phase distribution were analyzed by flow cytometry.At last,the isogenic cell lines H1299-LKB1sh RNA and H1299-PLKO.1 were assessed the expression of p21^WAF1/CIP1 by Western Blot.（2）LKB1 stable knockdown HCT116 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.（3）The isogenic cell lines HCT116 408（LKB1 knock down）and HCT 116407（PLKO.1）were assessed the expression of p21^WAF1/CIP1.And the isogenic cell lines HCT116 p53^-/-and HCT116 were transfected with LKB1 Si RNA.The expression of p21^WAF1/CIP1 was measured by Western Blot.Next,HCT116 was transfected with the wild-type,K78M mutant LKB1 plasmids and the control plasmids.The expression of p-AMPK,p-p53-Ser 15 and p21^WAF1/CIP1 were assessed by Western Blot.Then we treated the HCT116 cell with 2-deoxyglucose（2-DG）to determine the expression of p21^WAF1/CIP1.Lastly,The isogenic cell lines HCT116408/HCT 116 407 and HCT 116p53^-/-/HCT 116 were treated with 2-DG to determine the expression of p-AMPK,p-p53-Ser 15 and p21^WAF1/CIP1 by Western Blot.Results:（1）A549 cells transfected with the wild-type LKB1 plasmid exhibited fewer cell colony formation compared with transfected with the K78M mutant LKB1plasmids.After transfected with the wild-type LKB1 plasmid,the cells（A549 and H460）were significantly increased the expression of p21^WAF1/CIP1 and arrested the cell cycle in G1 phase.While the cells（H1299 and Calu-1）had little impact on the expression of p21^WAF1/CIP1 after the transfection with the wild-type LKB1 plasmid and cell cycle showed no delayed in G1/S transition.The expression of p21^WAF1/CIP1showed no suppression in in LKB1 stable knockdown H1299 cell lines.（2）LKB1 m RNA and protein expression were significantly suppressed in LKB1 stable knockdown HCT116 cell line.（3）p21^WAF1/CIP1 protein expression were significantly suppressed in LKB1 stable knockdown H1CT116 cell line.As for the isogenic cell lines HCT116 p53^-/-and HCT116,the HCT 116 was significantly suppressed p21^WAF1/CIP1 erpression after the transfected with LKB1 Si RNA,while HCT 116 p53^-/-showed no significantly suppression.And the expression of p-AMPK,p-p53-Ser 15 and p21^WAF1/CIP1 were significantly increased in HCT116 cells after transfected with LKB1 plasmid.HCT116 407 were more sensitive to the 2-DG treatment than HCT 116 408 as for the expression of p-AMPK,p-p53-Ser 15 and p21^WAF1/CIP1.However,the expression of p21 was no significantly increased in HCT 116 p53^-/-after the treatment of 2-DG.Conclusion:（1）In NSCLC cell lines,LKB1 leads to induction of p21^WAF1/CIP1 expression,arresting cell cycle and inhibiting cell growth,which requires its kinase activity.（2）LKB1 induces p21^WAF1/CIP1 through a p53-dependent manner.（3）The mechanism is LKB1 is through its downstream kinase AMPK to phosphorylate p53-Ser15 and induce the expression of p21^WAF1/CIP1.