Mel-18, a novel interacting partner of Cyclin D2 negatively regulates cell cycle progression through regulating G1 Cyclins in human breast cancer

Abstract

Cyclin D2 is a member of the D-type cyclins. D-type cyclins play a crucial role in progression through the G1 phase as a regulatory subunit of cyclin-dependent kinase and have been reported to be able to interact with several transcription regulators. In this work, we performed a yeast two hybrid screen using cyclin D2 as bait. Here we report that Mel-18, one of the Polycomb group gene products is a novel interacting partner of cyclin D2. Further, we found that the proline/serine-rich domain of Mel-18 and N-terminal region of cyclin D2 are required for interaction. Confocal microscopy analysis showed that cyclin D2 colocalized with Mel-18 in the nucleus. According to several studies, Mel-18 is a transcriptional repressor and negatively regulates cell cycle progression. The human homolog of Mel-18 is located at 17q, on which candidate tumor suppressor genes for breast cancer have been suggested for a long time. To investigate the role of Mel-18 on cell cycle status and cyclin D2 activity, we blocked Mel-18 expression using an antisense strand system and/or induced cyclin D2 using a retroviral infection in T47D breast cancer cell, which moderately expresses Mel-18 but no detectable cyclin D2. Repression of Mel-18 increased cell proliferation and caused upregulation of cyclin D1 and cyclin E. Induced expression of cyclin D2 caused increase in cyclin E expression. In contrast, repression of Mel-18 in cyclin D2 overexpressing cell resulted in downregulation of cyclin E. These results indicate that Mel-18 may modulate cyclin D2 activity through inhibition of binding of cyclin D2 and cdk2 which mainly binds cyclin E. We found that Mel-18 mRNA and protein levels were lower in more aggressive estrogen receptor (ER)-negative breast cancer cell lines compared with ER-positive breast cancer cell lines. Also, the expression level of Mel-18 protein was significantly reduced in invasive ductal carcinomas compared with normal tissue. Taken together, Mel-18 is involved in cell cycle through interacting with cyclin D2 and regulating cyclin D1 and cyclin E and may have a role in anti-tumor therapy.