Molecular Mechanisms Of Cell Cycle Regulation Mediated By Tumor Suppressor LKB1

BACKGROUND AND OBJECTIVES: Cancer is fundamentally a disease of abnormal cell proliferation: Cancer cells multiply when and where they should not. Some of the key oncogenic events in cancer directly perturb proteins that regulate progression through the cell division cycle, others alter cell cycle progression indirectly, through effects on signaling pathway that impinge on the cell cycle. This biology is fundamentally important in cancer therapy. Because of the large network of proteins involved in cell cycle progression and regulation, we are still far from understanding the details of its functioning. However, intense cancer research has uncovered many genes and the roles they play in this cycle. Hopefully, through a gradual understanding of the mechanisms underlying this complex system, we can find the long-awaited cure for this disease.Germline mutations of the LKB1 gene are the leading cause of Peutz–Jeghers syndrome and were detected in around 90% of patients. In addition, recent studies revealed that somatic inactivation of LKB1 is associated with a variety of sporadic cancers, particularly non-small cell lung carcinoma, in which 30-40% of cases have this lesion. LKB1 was also found to be somatically mutated in 20% of cervical carcinomas, 19% of squamous cell carcinomas, 13% of invasive ductal carcinomas of breast, and other cancers. Therefore, LKB1 is highlighted as a tumor suppressor. Previous studies had found that expression LKB1 in LKB1 deficient cancer cells resulted in a G1 cell cycle arrest, but the underlying mechanisms are still elusive. Besides, most of these cell culture experiments involved overexpression assays under LKB1 null conditions in cancer cells, and the physiological regulation and activity of LKB1 in vivo therefore remains unclear.Herein, we validated the roles of LKB1 gene on cell cycle progression and investigated the underlying mechanisms involved, in two normal cell lines: human embryonic kidney 293T cells (HEK293T cells) and human umbilical vein endothelial cells (HUVECs), and in LKB1-deficient Hela cells.METHODS: Three shRNA sequences directed against LKB1 mRNA were generated. DNA constructs (pshRNA-1, pshRNA-2, and pshRNA-3) were transfected into HEK293T cells and HUVECs. The scrambled shRNA vector (pshRNA-N), which has no match with any mRNA of the homo sapiens database, was also used as a negative control to examine inhibitory specificity. Ablation of endogenous LKB1 expression was examined by western blot and immunohistochemistry. Cell cycle profile and phase distribution were analyzed by flow cytometry. Protein levels of total Rb, phosphorylated Rb, cyclin D1, cyclin E, p53, p21 and p16 were analyzed by western blot.A stable Hela cell line constitutively expressing wild-type LKB1 protein was established by using G418 antibiotic selection pressure. The recombinant LKB1 protein was characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and western blot. Cell cycle profile and phase distribution were analyzed by flow cytometry. Protein levels of phosphorylated Rb, PCNA, AMPKαand phosphorylated AMPKαwere analyzed by western blot. AMPK inhibitor Compound C was also used to examine the effect of AMPK inhibition on cell proliferation.A LKB1 mutant plasmid deficient in kinase activity was prepared by site-directed mutagenesis of invariant nucleotide binding site in LKB1 kinase domain. Wild-type and mutant LKB1 plasmids were transfected into HEK293T cells. The recombinant LKB1 protein was characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and western blot. Cell cycle profile and phase distribution were analyzed by flow cytometry. Protein levels of phosphorylated Rb, PCNA, AMPKαand phosphorylated AMPKαwere analyzed by western blot.RESULTS: Transfection of LKB1-specific shRNAs (particularly pshRNA-1 and pshRNA-3) significantly reduced LKB1 expression in HEK293T cells and HUVECs, compared with that in cells transfected with pshRNA-N. Downregulation of endogenous LKB1 led to a facilitated G1/S transition, accompanied by a concomitant increase in Rb phosphorylation. Furthermore, reduced expression of p53 and p16 was observed in LKB1 ablated cells, while no differences were detected for cyclin D1 and cyclin E.Constitutive expression of LKB1 in Hela stable cells led to a delayed G1/S transition, accompanied by a concomitant decrease in Rb phosphorylation. Reduced expression of PCNA and elevated phosphorylation of AMPKαwere also observed in Hela stable cells, while no differences were detected for total AMPKαprotein. Besides, the expression of PCNA was greatly increased in Hela stable cells treated with AMPK inhibitor Compound C.Compared with mock plasmid transfected HEK293T cells, HEK293T cells transfected with wild-type LKB1 plasmid showed a delayed G1/S transition, accompanied by decreased Rb phosphorylation, reduced PCNA expression and elevated AMPKαphosphorylation, while no differences were detected for mutant LKB1 plasmid transfected cells.CONCLUSIONS: Endogenous LKB1 knockdown accelerates cell cycle progression through G1/S checkpoint in HEK293T cells and HUVECs, which is at least in part, mediated by decline of p53 and p16 pathways. Overexpression of LKB1 protein in Hela cells and HEK293T cells inhibited cell proliferation, which are mediated by LKB1 induced AMPK activation. Our findings provide a plausible mechanism by which loss of LKB1 expression in normal cells contributes to the formation of malignancies and further confirms the negative role of LKB1 in cell proliferation.