Mel-18, a polycomb group protein, negatively regulated cell cycle progression via inhibition of Akt-mediated cytoplasmic localization of p27Kip1

초록

Background We have previously identified that Mel-18, a polycomb group protein, is a novel binding partner of Cyclin D2 and blockade of Mel-18 expression enhanced cell proliferation of T-47D breast cancer cells (Chun et al., FEBS Lett. 2005 Oct 10;579(24):5275-80). However, its functional mechanism has not been fully elucidated. Methods Using a retroviral vector system, SK-BR-3 cells, which expression Mel-18 at the low level, were infected with Mel-18 cdNA, while T-47D cells, which exhibit moderate expression level of Mel-18, were infected with Mel-18 antisense construct. The effect of cell growth was estimated by MTT assay and cell cycle was analyzed by flow cytometry. The expression levels of cell cycle regulators and Akt signaling molecules were measured by immunoblotting. The cellular localization of p27Kip1 was observed using immunocytochemistry and cytoplasmic/nuclear fractionations. Results Upon Mel-18 overexpression in SK-BR-3, attenuation of cell growth and G1 arrest were observed and Cyclin D1 expression and p27Kip1 phosphorylation at Thr157 were reduced. Likewise, suppression of Mel-18 in T-47D cells lead to enhanced cell growth and acceleration of G1/S phase transition, upregulation of Cyclin D1, Cyclin E expressions and phosphorylated p27Kip1 at Thr157. Moreover, phosphorylation of Akt at Ser473 and GSK-3 at Ser9 were reduced by Mel-18 overexpression in SK-BR-3 cells and enhanced by Mel-18 antisense expression in T-47D cells. As a result of LY294002 inhibition of PI3K, the increase in Cyclin D1 expression and phosphorylation of p27Kip1 by Mel-18 blockade in T-47D cells were abolished. Particularly, Mel-18 inhibited Akt-mediated cytoplasmic localization of p27Kip1. In Mel-18 infected SK-BR-3 cells, immunocytochemistry with a p27Kip1 antibody showed reduction of cytoplasmic localization of p27Kip1. Conclusion Mel-18 has a critical role in the cell cycle regulation as a novel negative Akt regulator, which resulted in decreased cytoplasmic localization of p27Kip1 in human breast cancer.