The LKB1 Downregulates The Expression Of GLUT1 In Lung Cancer Cells Through Inhibiting The Activation Of AKT Signaling Pathway Due To Promotion Of PTEN Phosphorylation

Liver kinase B1(LKB1)also known as serine/threonine kinase 11(STK11),is a tumor suppressor gene found for the first time in 1998 in patients with Peutz Jeghers syndrome(PJs).Its germ line mutation is the main pathogenic factor of PJs patients,and is also an important genetic basis for cancer susceptibility.LKB1 has serine / threonine protein kinase activity.Through phosphorylation of substrate protein or binding with target protein,LKB1 protein regulates gene expression and participates in the regulation of cell cycle,apoptosis and other life activities.Our previous studies have found that LKB1 is inversely proportional to the expression of h TERT and h TERC,the main components of telomerase,and LKB1 can negatively regulate their expression levels.It has been reported that the expression level of GLUT1 is significantly increased in PJs patients.In lung cancer,the molecular mechanism by which LKB1 affects the expression of GLUT1 remains to be elucidated.Phosphatase and Tensin Homolog deleted on Chromosome 10(PTEN)is a dual phosphatase with both protein and lipid phosphatase activities.PTEN has been identified as a tumor suppressor gene with high frequency mutations in many cancers.The downregulation of PTEN promoted the proliferation of lung adenocarcinoma cells.LKB1 interacts with and promotes the phosphorylation of PTEN.The loss of this interaction may be one of the causes of tumorigenesis.It has been reported that activated PTEN plays an anti-cancer role by negatively regulating protein kinase B(AKT)activity,inhibits cells proliferation and migration and promotes apoptosis by promoting AKT phosphorylation.Experiments of co-immunoprecipitation and in vitro de-phosphorylation assay demonstrated that PTEN physically interacts with AKT and drives its dephosphorylation,and so limiting the expression of GLUT1 at the plasma membrane.In the present study,we revealed for the first time that LKB1 protein in lung cancer cells activated its anticancer activity by inducing PTEN phosphorylation,then lost its carcinogenic activity by inducing AKT dephosphorylation,and finally down regulated GLUT1 expression at protein and mRNA levels Objective:Therefore,the purpose of this study is to elucidate the mutual regulatory mechanism of LKB1,PTEN,AKT,and GLUT1 in lung cancer cells,and provide new strategies for the treatment of lung cancer..Materials and Methods : First of all,we used cell transfection and cell interference technology to bibitively regulate LKB1.Western blotting was used to detect the protein expression changes of LKB1,PTEN,AKT,GLUT1,p-PTEN and p-AKT,and real-time quantitative polymerase chain reaction(q RT-PCR)was used to detect the mRNA expression changes of LKB1,PTEN,AKT and GLUT1.Then,after the regulation of PTEN by cell interference technology,the expression changes of PTEN,AKT,GLUT1 and p-AKT protein were detected by Western blotting,and the mRNA expression changes of PTEN,AKT and GLUT1 were detected by q RT-PCR.and by adding pathway inhibitors to inhibit the effect of the signal pathway to clarify its effect on the expression and localization of GLUT1.Then we applied nucleoplasmic separation technology to clarify the regulatory mechanism of LKB1 on PTEN;immunofluorescence technology was used to further confirm the effect of various indicators on GLUT1.SPSS22.0statistical analysis software was used to conduct t-test analysis on the experimental data,and P<0.05 was considered statistically significant.Results : 1.The overexpression of LKB1 upregulated the p-PTEN expression but downregulated the p-AKT and GLUT1 expression To investigate the effects of LKB1 on p-PTEN,p-AKT,and GLUT1,pc DNA3-LKB1-His vectors were transiently transfected into the A549 low-expressing cell line.Results showed that the LKB1 overexpression significantly upregulated the PTEN and p-PTEN expression but downregulated the p-AKT expression and GLUT1 expression at both the protein and mRNA levels.However,the increase of PTEN expression was significantly lower than that of p-PTEN,and AKT expression had little or no change.On the other hand,the inhibition of LKB1 received the opposite results in NCI-H460 cells.2.The knockdown of PTEN upregulates the p-AKT and GLUT1 expression,In order to further verify the regulatory effects of p-PTEN on p-AKT and GLUT1,PTEN-specific Si RNA was used to knockdown the PTEN expression of in H460 cells.Results showed that the PTEN inhibition significantly upregulated the p-AKT expression and GLUT1 expression at both the protein and mRNA levels,while the AKT expression had little or no change.3.The knockdown of PTEN upregulates the GLUT1 expression by activating AKT signaling pathway.We first detected the expression level of PTEN after Si RNA interference and then conducted the subsequent experiments.After PTEN Si RNA interference,the expression level of total AKT,a key protein of the AKT signaling pathway was not altered,but the expression level of p-AKT was significantly increased.Along with the expression level of p-AKT increased,the expression level of GLUT1 was also upregulated.However,we added the AKT signaling pathway inhibiter Miltefosine to NCI-H460 cells,the expression levels of p-AKT and GLUT1 evidently reversed.4.Overexpression of LKB1 up-regulates the expression of p-PTEN in the cytoplasm,up-regulates the expression of PTEN in the cytoplasm,and down-regulates the expression of PTEN in the nucleus.5.Down-regulate LKB1 down-regulate the expression of p-PTEN in the cytoplasm,down-regulate the expression of PTEN in the cytoplasm,and up-regulate the expression of PTEN in the nucleus.6.Transfect LKB1 Si RNA into NCI-H460 cells,and observe the expression of GLUT1 fluorescence signal in the cytoplasm and cell membrane and the signal enhancement by immunofluorescence technology.7.By transfecting PTEN Si RNA into NCI-H460 cells,it was observed that the fluorescence signal of GLUT1 was localized in the cytoplasm and cell membrane and the signal was enhanced.After adding the AKT pathway inhibitor,the fluorescence signal of GLUT1 was almost in the cytoplasm and the fluorescence signal was Weaker.Conclusion: 1.This project verified that LKB1 can inhibit the expression of GLUT1 protein and mRNA in lung cancer cell models.2.LKB1 promotes the transport of PTEN from the nucleus to the cytoplasm by regulating the phosphorylation of PTEN in the cytoplasm at Ser380.3.LKB1 regulates the activation of PTEN,thereby inhibiting the expression of GLUT1 by mediating the activation of the AKT signaling pathway.4.Our research results provide new evidence for the inhibitory effects of LKB1 and PTEN on lung cancer and their regulatory mechanism on GLUT1,and may suggest new therapeutic targets.