Role of glycogen synthase kinase 3β in rapamycin-mediated cell cycle regulation and chemosensitivity

The mammalian target of rapamycin is a serine-threonine kinase that regulates cell cycle progression. Rapamycin and its analogues inhibit the mammalian target of rapamycin and are being actively investigated in clinical trials as novel targeted anticancer agents. Although cyclin Dl is down-regulated by rapamycin, the role of this down-regulation in rapamycin-mediated growth inhibition and the mechanism of cyclin Dl down-regulation are not well understood. Here, we show that overexpression of cyclin Dl partially overcomes rapamycin-induced cell cycle arrest and inhibition of anchorage-dependent growth in breast cancer cells. Rapamycin not only decreases endogenous cyclin D1 levels but also decreases the expression of transfected cyclin Dl, suggesting that this is at least in part caused by accelerated proteolysis. Indeed, rapamycin decreases the half-life of cyclin Dl protein, and the rapamycin-induced decrease in cyclin D1 levels is partially abrogated by proteasome inhibitor N-acetyl-leucyl-leucyl-norleucinal. Rapamycin treatment leads to an increase in the kinase activity of glycogen synthase kinase 3(3 (GSK3(beta), a known regulator of cyclin Dl proteolysis. Rapamycin-induced down-regulation of cyclin Dl is inhibited by the GSK3beta inhibitors lithium chloride, SB216763, and SB415286. Rapamycin-induced G(1) arrest is abrogated by nonspecific GSK3 inhibitor lithium chloride but not by selective inhibitor SB216763, suggesting that GSK3beta is not essential for rapamycin-mediated Gt arrest. However, rapamycin inhibits cell growth significantly more in GSK3beta wild-type cells than in GSK3beta-null cells, suggesting that GSK3beta enhances rapamycin-mediated growth inhibition. In addition, rapamycin enhances paclitaxel-induced apoptosis through the mitochondrial death pathway; this is inhibited by selective GSK3beta inhibitors SB216763 and SB415286. Furthermore, rapamycin significantly enhances paclitaxel-induced cytotoxicity in GSK30 wild-type but not in GSK3beta-null cells, suggesting a critical role for GSK30 in rapamycin-mediated paclitaxel-sensitization. Taken together, these results show that GSK3beta plays an important role in rapamycin-mediated cell cycle regulation and chemosensitivity and thus significantly potentiates the antitumor effects of rapamycin.