Cancer is characterized by uncontrolled cell growth and division. Alterations in the regulatory mechanism of tumor suppressor genes result in the proliferation of these abnormal cells. The Murine Double Minute 2 (MDM2) protein negatively regulates the tumor suppressor protein p53 and its over expression results in transformation of cells. Previous reports suggest that MDM2 also has a p53 independent role in tumorigenesis. MDM2 transgenic mice lacking p53 have an increased incidence of malignancies as compared with mice deficient in p53 alone. Also, alternative spliced forms of MDM2 that lack their p53 binding domain can be detected in mouse cells after induction of genotoxic stress. MDM2-ALT1 is the most common alternative spliced form observed in human cancers, and it alters p53 activity through its interaction with MDM2. Even though these findings suggest a role for the alternative splicing of MDM2 in tumorigenesis, the regulation of these spliced forms is not well understood. Furthermore, the p53 independent target interactions of these alternative spliced forms with regulators of cell cycle control and DNA damage response pathway are not well known. We hypothesize that changes in the splicing regulation of MDM2 have a p53 independent role to impair MDM2 interactions with proteins involved in cell cycle control and DNA damage pathways resulting in transformation. To understand the regulation of these spliced forms in cancer, we generated an MDM2 splicing competent minigene system to identify and characterize negative and positive splicing regulators. Mutations affecting sequences specific for the binding of splicing factors SC35 and SF2/ASF in exon 11 result in changes in the MDM2 alternative spliced isoform, both in normal and in stress conditions. This suggests a role for these splicing factors in the alternative splicing of MDM2. In order to determine the function of the alternative MDM2 isoforms, our lab has generated a double transgenic CD19-Cre inducible MDM2-ALT1 mouse model for the expression of this isoform in B cells. This tool will be used to query MDM2-ALT1 interactions with full length MDM2 and with proteins involved in cell cycle control and DNA damage response pathways. Our goal is to identify full length and alternative spliced MDM2 interactions with p53 independent proteins involved in cell cycle control and DNA damage response pathways. Even more we aim to determine the dependence of MDM2-ALT1's activity on MDM2 and p53. Overall this project will result in a better understanding of the mechanism behind the alternative splicing of MDM2, its role in tumorigenesis and in the identification of protein interactions that can be targeted as potential intervention points for the treatment of cancer.
MDM2 is the most important regulator of the tumor suppressor protein p53. p53 function is critical to prevent inappropriate cell growth and proliferation. This research proposal will elucidate the role of alternative splicing of MDM2 in transformation and regulation of the p53 pathway. Since regulation of the p53 tumor suppressor pathway is under control of MDM2, understanding the mechanism behind its alternative splicing could lead to the discovery of novel potential intervention points that can be targeted for the development of anti cancer therapies.
