Abstract P6-06-05: Down-regulation of the circadian factor Period 2 by the oncogenic E3 ligase Mdm2: Relevance of circadian components for cell cycle progression

Circadian rhythms are mechanisms that measure time on a scale of about 24 h and that adjusts our body to external environmental signals. Core circadian clock genes are defined as genes whose protein products are necessary components for the generation and regulation of circadian rhythms. Circadian proteins also regulate genes involved in either cell division or death; and a perturbation of the balance among these processes leads to cancer development and progression. A key aspect of cancer research is identifying new regulatory pathways involved in proliferation and differentiation of cell. Disruption of circadian rhythm has recently emerged as a new potential risk factor in the development of cancer, pointing to the core gene period 2 (per2) as a tumor suppressor. However, it remains unclear how the circadian network regulates tumor suppression, nor which, if any, of its components is either the ultimate effector that influences the fate of the cell. Initial experiments were devoted to identifying new interacting partners for Per2 using a two-hybrid system. Interestingly, among the positive clones analyzed was the oncogenic protein Mdm2. This result was validated by immunoprecipitation of recombinant and endogenous Per2/Mdm2 complexes from unstressed cells. Pull-down assays using tagged-expressed proteins fragments and labeled proteins were later used to map the interacting regions between Per2 and Mdm2. Our results show Mdm2 binds to the central flexible region of Per2 known to interact with various protein partners. Thus, we hypothesized that binding of Mdm2 to Per2 might act by mediating its ubiquitination and therefore altering Per2 stability. We next examined the formation of the Mdm2/p53/Per2 complex by immunoprecipitation. Our data show anti-p53 antibody is able to co-immunoprecipitate Per2 and Mdm2. Moreover, in vitro and in vivo ubiquitination assays show that binding of Per2 to p53 prevented ubiquitination of p53 by Mdm2 without altering their binding. Immunofluorescence studies using H1299 cells (p53-) confirmed Per2 role in p53 stabilization and for localization. Overall our results suggest that Mdm2 modulates the stability of Per2 and p53 in unstressed cells, and might be responsible for the oscillatory levels of these proteins observed in a 24 h cycle. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P6-06-05.