Antagonizing MDM2 Overexpression Induced by MDM4 Inhibitor CEP-1347 Effectively Reactivates Wild-Type p53 in Malignant Brain Tumor Cells

SIMPLE SUMMARY: In human cancer, the major tumor suppressor p53 is often functionally inactivated through the deregulation of its negative regulators MDM2 and MDM4. The inhibitors of MDM4 have recently been attracting increasing attention as a promising approach to the reactivation of wild-type p53 in cancer cells. However, due to the multiple, complex feedback loops that involve p53, MDM2, and MDM4, the mechanisms by which the inhibition of MDM4 affects MDM2 and whether the additional inhibition of MDM2 enhances the p53-activating effects of MDM4 inhibitors remain unclear. Here, we demonstrated that CEP-1347, an inhibitor of MDM4 protein expression, induced the overexpression of MDM2 and that the concomitant inhibition of MDM2, particularly the selective disruption of the MDM2–p53 interaction, promoted the CEP-1347-mediated activation of p53 and the inhibition of cell growth. The present results suggest the potential of combining the inhibition of MDM4 with the disruption of the MDM2–p53 interaction to maximally activate p53 in cancer cells. ABSTRACT: The development of MDM4 inhibitors as an approach to reactivating p53 in human cancer is attracting increasing attention; however, whether they affect the function of MDM2 and how they interact with MDM2 inhibitors remain unknown. We addressed this question in the present study using CEP-1347, an inhibitor of MDM4 protein expression. The effects of CEP-1347, the genetic and/or pharmacological inhibition of MDM2, and their combination on the p53 pathway in malignant brain tumor cell lines expressing wild-type p53 were investigated by RT-PCR and Western blot analyses. The growth inhibitory effects of CEP-1347 alone or in combination with MDM2 on inhibition were examined by dye exclusion and/or colony formation assays. The treatment of malignant brain tumor cell lines with CEP-1347 markedly increased MDM2 protein expression, while blocking CEP-1347-induced MDM2 overexpression by genetic knockdown augmented the effects of CEP-1347 on the p53 pathway and cell growth. Blocking the MDM2–p53 interaction using the small molecule MDM2 inhibitor RG7112, but not MDM2 knockdown, reduced MDM4 expression. Consequently, RG7112 effectively cooperated with CEP-1347 to reduce MDM4 expression, activate the p53 pathway, and inhibit cell growth. The present results suggest the combination of CEP-1347-induced MDM2 overexpression with the selective inhibition of MDM2′s interaction with p53, while preserving its ability to inhibit MDM4 expression, as a novel and rational strategy to effectively reactivate p53 in wild-type p53 cancer cells.