Abstract 2052: Regulation of p16Ink4a - Rb pathway by Cyclin D1- Dmp1 interaction

Cyclin D1 is a crucial regulator in mammalian cell cycle that binds to and activates CDK4/6, which is an kinase that drives cells to enter S phase. It has an important oncogenic role in breast cancer because the CCND1 gene is amplified in ∼15% of cases and its protein expression is elevated in up to 50% of cases. Previous studies demonstrate that cyclin D1 overexpression can cause pRb hyper-phosphorylation and therefore induce cell hyperproliferation, which can result in the development of mammary adenocarcinoma. In addition, cyclin D1 is found to contribute to tumorigenesis by affecting the activity of transcriptional factors via physical interaction. One of the affected transcriptional factors is called Dmp1, which acts as a tumor suppressor when receiving oncogenic signals. It activates Arf by binding to its promoter region, therefore causing Arf, p53-dependent cell cycle arrest in normal cells. Our recent study in MMTV-neu mice showed that loss of Dmp1 significantly accelerates mammary carcinogenesis. Although both Dmp1 and cyclin D1 play vital roles in breast cancer prevention and development respectively, the biological functions and significance of Dmp1-cyclin D1 interaction remains to be explored. To identify the impact of their interaction on the activation of p16 Ink4a / p19 Arf promoters induced by cyclin D1, MEFs were overexpressed cyclin D1 and we found the endogenous p16 Ink4a and p19 Arf transcripts were increased up to 7 fold. However, their transcription level was not affected in Dmp1-null MEFs. Moreover, cyclin D1 mutant α142-253 that cannot bind to Dmp1could not fully activate the p16 Ink4a /p19 Arf promoters, indicating the activation of p16 Ink4a /p19 Arf promoters by cyclin D1 is dependent on Dmp1, and loss of Dmp1-cyclin D1 interaction will attenuate this effect. To identify the potential regulation of p16 Ink4a by Dmp1, we performed EMSA and luciferase reporter assay, and we found that Dmp1 activates the p16 Ink4a promoter by directly binding to its promoter. Moreover, the p16 Ink4a mRNA and protein level were increased upon Dmp1 overexpression. The above results suggest that Dmp1 is a physiological regulator of p16 Ink4a . To explore the effect of Dmp1 on Cdk4 kinase activity, 293T cells were overexpressed cyclin D1 alone or together with Dmp1, and we found there was less cyclin D1 associated with CDK4 after CDK4 immunoprecipitation. As a consequence, pRb phosphorylation was decreased in CDK4 kinase assay. These results suggest that Dmp1 decreases CDK4 kinase activity by impairing cyclin D1-CDK4 interaction. Taken together, our data suggest that Dmp1 decreases pRb phosphorylation in two ways: one is to directly bind to and activate p16 INK4a , and the other one is to block the CDK4 kinase activity by binding to cyclin D1. In addition, cyclin D1-Dmp1 interaction is indispensible in activation of p16 Ink4a /p19 Arf promoters induced by cyclin D1. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2052. doi:1538-7445.AM2012-2052