The role of Akt1 in G1/S cell cycle checkpoint bypass and cell migration after genotoxin stress

A growing body of evidence underscores the central role of the Akt pathway in the pathogenesis of lung cancer. Akt has been documented to play a role not only in cell survival but also in cell migration and invasion. Certain forms of hexavalent chromium [Cr(VI)] are human respiratory carcinogens and genotoxins. Our studies identified Akt as crucial to the death/survival balance after Cr(VI) exposure in both lung fibroblasts and epithelial cells. The objective of this dissertation was to elucidate the role of Akt1 in G1/S checkpoint bypass and migration after genotoxin exposure. Our studies indicate that a broad range protein tyrosine phosphatase (PTP) inhibitor, sodium orthovanadate (SOV) partially abrogated Cr(VI)-induced growth arrest and induced G1/S cell cycle checkpoint override in human lung fibroblasts (HLF). This checkpoint override was dependent on Akt activation, resulting in the modulation of subcellular localization and expression of the G1/S transition effectors, pRb and p27, following genotoxin exposure. Furthermore, biological upregulation of Akt1 indicated that Akt1 was sufficient to bypass a Cr(VI)-induced G1/S checkpoint arrest via the regulation of G1/S checkpoint effector expression and localization. Recent evidence suggests that p27 localization and phosphorylation status is responsible for cytoskeletal reorganization via its interaction with RhoA. Our data indicates that Akt upregulation, either biologically or in the presence of SOV, causes an increase in the interaction between p27 and RhoA. Furthermore, PTP inhibition significantly decreased the GTPase activity of RhoA after Cr(VI) treatment, whereas Akt1 activation significantly increased GPTase activity of RhoA after Cr(VI) treatment. Moreover, PTP inhibition and Akt1 upregulation both significantly increased HLF migration after Cr(VI) exposure, as assessed by a wound healing assay. Similarly, Cr(VI)-induced decrease in cell adhesion was abrogated by PTP inhibition and Akt1 upregulation. Consistent with its effect on migration, the Cr(VI)-induced disruption of F-actin stress fibers was abrogated by PTP inhibition. Notably, Akt1 activation was sufficient to overcome Cr(VI)-induced cytoskeletal alterations in our preliminary studies, as transient MyrAkt1 transfection abrogated the Cr(VI)-induced disruption in F-actin stress fibers. Given the evidence for the purported role of fibroblasts in epithelial mesenchymal transition (EMT), we investigated the effect of PTP inhibition and Akt1 upregulation in the presence of Cr(VI) treatment in fibroblasts, on the migratory capacity of epithelial cells. Our data indicate that PTP inhibition in fibroblasts, in the context of genotoxic stress, abrogates the Cr(VI)-induced decrease in migration of virally transformed BEAS2B lung epithelial cells. Furthermore, PTP inhibition in the presence of genotoxic stress in fibroblasts further enhances the Cr(VI)-induced increase in migration of metastatic Calu3 cells. Akt1 upregulation in the context of genotoxic stress in fibroblasts increases the migration of BEAS2B cells, while having no effect on the migration of Calu3 lung adenocarcinoma cells. In conclusion, the results of our studies provide new insights to the understanding of Cr(VI)-induced lung carcinogenesis. The ability of Akt activation to enhance cell migration in conjunction with checkpoint override in the face of genotoxic exposure could play a role in neoplastic progression. Since fibroblasts are involved in accelerating the EMT, our data also highlight a potential role by which survival pathway activation may affect the cellular microenvironment after initial exposure to a genotoxin.