The p53 tumor suppressor acts as the major sensor for a regulatory circuit that monitors signaling pathways from diverse sources, including DNA damage, oncogenic events, ribosomal stress and others abnormal cellular processes (Vousedn and Prives, 2009;Qian and Chen, 2010; Xiong et al., 2011; Zhang and Lu, 2009). It is well accepted that Mdm2 and its related protein Mdmx (also called Mdm4) play a major part in the scope of specifically inhibiting p53 activities in cancer cells. Despite its high sequence homology with Mdm2 and the presence of a RING domain, Mdmx does not have intrinsic E3-ligase activity for p53 but was instead shown to inhibit p53- induced transcription via their interactions; nevertheless, Mdmx knockout mice die even in the presence of Mdm2 and this lethality is also rescued by inactivation of p53 (Maine et al., 2007). Thus, the role of Mdmx in repressing p53 function is as critical as that of Mdm2 and physiological levels of Mdmx are required in a non-redundant manner to regulate p53 activity in vivo. Mdmx over expression is found in several types of human tumors retaining wild type p53 (Maine et al., 2007; Danovi et al., 2004; Li et al., 2010; Gilkes and Chen, 2007). Notably, Mdmx is a very stable protein in most human cancer cells and it is predominantly localized in the cytoplasm of cancer cells under normal conditions. The molecular mechanism by which Mdmx is regulated in vivo is an extremely important issue but needs further elucidation. Both p53 and Mdmx can be ubiquitinated and degraded by the same E3 ligase (Mdm2); interestingly, however, while p53 is rapidly degraded by Mdm2, Mdmx is a very stable protein in unstressed cancer cells, raising a critical issue regarding how Mdmx degradation by Mdm2 is specifically protected without affecting the degradation of p53. In our preliminary studies, we have identified the noncoding 5S rRNA as a major component of Mdmx-associated complexes from human cells. We show that 5S rRNA acts as a natural inhibitor of Mdmx degradation in cancer cells. 5S rRNA expression inhibits Mdm2- mediated degradation of Mdmx but has no obvious effects on p53 degradation by Mdm2 in human cells. This part of the work reveals an unexpected role of noncoding 5S rRNA in modulating the stability of Mdmx. Moreover, to understand the mechanism by which Mdmx sub cellular localization is regulated in cells, we have indentified Pellino 1 (Peli1), as a novel Mdmx regulator through protein purification. We found Peli1 interacts with Mdmx both in vitro and in vivo. Peli1 expression does not affect the Mdmx levels but induces cytoplasmic localization of Mdmx. Thus, we have indentified two critical cofactors (the noncoding 5S rRNA and Peli1) from the Mdmx protein complex that are critically involved in regulating Mdmx function. The central hypothesis to be tested is that Mdmx requires multiple layers (the stability and sub cellular localization) of regulatory networks to ensure temporal and spatial controls of p53 activity in tumor cells. It includes the following two specific aims and we have obtained substantial preliminary data to support the feasibility of each aim.
In Aim1, we will elucidate the stability control of Mdmx by th noncoding 5S rRNA in human cancer cells.
In Aim 2, we will investigate the regulatory role of Peli1, a newly identified Mdmx-associated protein, in modulating the sub cellular localization of Mdmx as well as subsequent effects on p53 functions. To further investigate the role of the Peli1-Mdmx interplays in vivo, we will use Peli1 mutant mice to dissect the role of Peli1 in Mdmx-mediated pathogenesis.
Restoration of p53 activity remains an important goal in the quest for more effective cancer therapeutics. Because the p53 gene often remains wild-type in Mdm2- or Mdmx- over expressing cancers, it has long been conceived that targeting these p53 repressors could restore p53 activity. Interestingly, over expression of MDM2 and MDMX are mutually exclusive in cancer cells, suggesting that dysregulation of a major repressor is sufficient to inactivate p53 leading to tumor development. This study will elucidate novel molecular mechanisms for controlling the stability and sub cellular localization of Mdmx in regulating p53-mediated tumor suppression and yield crucial insights regarding how to target this pathway in cancer therapy.
