Down-regulation of E-cadherin is considered the initial and critical step of the EMT process. In this study, we found that cyclin G1 delivery by adenovirus led to significant repression of E-cadherin promoter activity in hepatoma cells. Biological functional

studies demonstrated that cyclin G1 enhanced the metastatic potential of HCC cells and induced more metastatic lesions in the livers and lungs of nude mice. Therefore, XL184 in vivo these data indicated that cyclin G1 might promote HCC metastasis via, at least partially, induction of EMT in hepatoma cells. To date, multiple oncogenic pathways have been reported to participate in EMT regulation, such as MAPKs, NF-κB, Hedgehog signaling, and so forth. To determine whether cyclin G1 affects these pathways, we analyzed the activity of AP-1, NF-κB, and Gli-1 by luciferase reporter

assay in cells with cyclin G1 overexpression. The results excluded the involvement of these cascades in cyclin G1 function. Increasing evidence has demonstrated that the activated PI3K/Akt pathway plays a central role in the EMT process and correlates with an aggressive phenotype in several kinds of malignancies.22, 26-30 It is therefore of interest to investigate whether Akt participates in cyclin G1–meditated EMT. We found that overexpression of cyclin G1 remarkably intensified the phosphorylation of Akt, which was further enhanced RXDX-106 upon the stimulation of mitogen or carcinogen. Meanwhile, we also found that enhanced cyclin G1 expression intensified the kinase activity of PI3K. These results suggest that cyclin G1 may activate Akt by modulating PI3K activity. Activation of PI3K is usually triggered by the ligand-activated receptor tyrosine kinase. However,

cyclin G1 overexpression did not show significant influence on the autophosphorylation of several critical RTKs. Considering PI3K activity is principally regulated by the p85 subunit, we hypothesized that cyclin G1 might interact with p85 and eventually increase the Fenbendazole catalytic activity of PI3K. As expected, cyclin G1 and p85α were coprecipitated in an immunoprecipitation assay, indicating that cyclin G1–induced Akt activation may depend on the interaction of cyclin G1 and p85. This notion was further supported by the result that overexpression of Δp85 remarkably abolished cyclin G1–induced Akt activation. Snail acts as the most important transcription factor in the negative regulation of E-cadherin expression and EMT process of epithelial cells.33, 34 Phosphorylation and subsequent degradation of Snail is controlled by GSK-3β, which is predominantly regulated by PI3K/Akt.35 Through the activation of PI3K/Akt signaling, cyclin G1 suppressed GSK-3β activity, and the level of Snail was correspondingly increased, which led to the down-regulation of E-cadherin and the subsequent EMT of hepatoma cells.