Mel-18, a polycomb group protein, inhibits Akt-mediated cytoplasmic localization of p27Kip1

Abstract

Polycomb group (PcG) proteins are chromatin modifiers that can act as transcription repressors of their target genes and also are involved in the control of cell proliferation. Recently, we reported that Mel-18, a polycomb group protein, has been shown to have anti-proliferative activity in breast cancer cells. However, its function of anti-tumor effect has not been fully established. Here we report that Mel-18 inhibits cytoplasmic localization of p27Kip1 mediated by Akt pathway. First, to explore the role of Mel-18 in cell cycle regulation, we infected human breast cancer cells with retrovirus encoding either Mel-18 or Mel-18 antisense construct and then examined cell growth and cell cycle. In SK-BR-3 cells, which express Mel-18 at the low level, Mel-18 overexpression suppressed cell proliferation and led to G1 arrest. Also Cyclin D1 expression and phosphorylation of p27Kip1 at Thr157 were downregulated. Consistent with these observations, knockdown of Mel-18 in T-47D cells, which moderately express Mel-18, stimulated cell proliferation and increased the proportion of cells in S phase. In T-47D cells, repression of Mel-18 expression enhanced protein levels of Cyclin D1, Cyclin E and phosphorylated p27Kip1 at Thr157 as well as binding of Cyclin D1/CDK4, Cyclin E/CDK2. Especially, cyclin D1 expression was changed at the transcriptional level. Both Cyclin D1 expression and phosphorylation of p27Kip1 at Thr157 can be regulated by Akt signaling. In SK-BR-3 cells, phosphorylated Akt at Ser473 was significantly reduced by Mel-18 overexpression. Phosphorylation of GSK-3β at Ser9 was also downregulated. In T-47D cells, blockade of Mel-18 caused to upregulate phosphorylation of Akt at Ser473 and GSK-3β at Ser9. As a result of LY294002 inhibition of PI3K but not MEK inhibitor PD98059, the increase in Cyclin D1 expression and phosphorylation of p27Kip1 by Mel-18 blockade in T-47D cells were abolished. Akt can regulate p27Kip1 localization through phosphorylation of p27Kip1 at Thr157. In Mel-18 infected SK-BR-3 cells, immunocytochemistry with a p27Kip1 antibody showed reduction of cytoplasmic localization of p27Kip1. This result was confirmed by immunoblot analysis with cytoplasmic and nuclear fractions. Furthermore, seven of 10 human breast primary tumors revealed significantly decreased Mel-18 expression compared with normal breast tissues. Mel-18 expression was inversely related with phosphorylation of Akt and p27Kip1. Taken together, Mel-18 has a critical role in the development of human breast cancer as a novel negative Akt regulator, which resulted in decreased cytoplasmic localization of p27Kip1. These findings also indicated that Mel-18 is a possible candidate for a tumor suppressor gene.