The PTEN/Phosphatidylinositol 3'-kinase (PI3-kinase) growth factor signaling pathway plays a critical role in epithelial tumor development in a multitude of tissue types. Deletion of the Pten tumor suppressor gene in murine urothelial cells in vivo results in upregulation of cyclin-dependent kinase inhibitor p21. We have previously shown in mice that p21 expression blocks an increase in urothelial cell proliferation due to Pten deletion. In this study, we utilized human urothelial carcinoma cells UMUC-3 and UMUC-14 to identify the signaling pathways downstream of PI3-kinase that regulate p21.
Cells were treated with a combination of PI3-kinase stimulating growth factors and kinase inhibitors, or transfected with exogenous genes in order to identify the signaling events that are necessary for p21 induction. Mice with conditional deletion of Pten in bladder urothelium were also examined for evidence of PI3-kinase pathway signaling events that affect p21 expression.
When cells were treated with PI3-kinase activating growth factors EGF or PDGF, we found that p21 levels increased, in a manner similar to that observed in mice. We used the inhibitors LY294002, Akti-1/2, and rapamycin, to show that p21 induction is dependent upon PI3-kinase and AKT activity, and partially dependent on mTOR. We treated the cells with proteasome inhibitor MG-132 and found that p21 may be degraded in the proteasome to regulate protein levels. Importantly, our findings show that GSK-3β plays a role in diminishing p21 levels in cells. Treatment of cells with the GSK-3β inhibitor SB-216763 increased p21 levels, while exogenous expression of GSK-3β caused a decrease in p21, indicating that GSK-3β actively reduces p21 levels. We found that a combined treatment of LY294002 and SB-216763 improved the cytotoxic effect against UMUC-3 and UMUC-14 carcinoma cells over LY294002 alone, suggesting potential therapeutic uses for GSK-3β inhibitors. Immunohistochemical staining in bladders from wild-type and Pten-deleted mice indicated that GSK-3β inhibitory phosphorylation increases when Pten is deleted.
PI3-kinase and AKT cause an upregulation of p21 by suppressing GSK-3β activity and activating mTOR in both cultured human urothelial carcinoma cells and mouse urothelial cells in vivo.