Elevated MDM2 contributes to antitumor activity of p53-MDM2 binding inhibitors

C280 The p53 tumor suppressor is a powerful growth suppressive and pro-apoptotic molecule frequently inactivated in human cancer. MDM2, a negative p53 regulator, inhibits the transcriptional activity of p53 and facilitates its ubiquitin-dependent degradation. MDM2 is overproduced in many human tumors and effectively disables p53 function. Recently developed small-molecule MDM2 antagonists, the nutlins, can restore p53 function in cancer cells leading to induction of cell cycle arrest and apoptosis. By activating p53, nutlins also elevate the cellular level of its transcriptional target MDM2. They selectively block the physical interaction between p53 and MDM2 but may not interfere with other p53-independent functions of MDM2. Here, we present evidence that nutlin-induced MDM2 contribute to the overall anti-tumor activity of MDM2 antagonists by facilitating the degradation of p53 inhibitor MDMX. MDM2 and MDMX levels were analyzed in a panel of 12 randomly selected solid tumor lines. In the presence of nutlins-3a, MDM2 increased in all and MDMX decreased in 9/10 cell lines with normal MDM2 expression, except MCF7. MDMX protein levels were virtually undetectable in the MDM2 overexpressing osteosarcoma cell lines, SJSA1 and MHM. Kinetically, cellular MDMX decreased with the increase of MDM2 and was partially restored by treatment with the proteasome inhibitor MG132 or MDM2 knockdown. In vitro experiments indicated that nutlin-3a does not affect MDM2 self-ubiquitination. Taken together these data suggest that MDM2 contributes to antitumor activity of nutlins at least in part by facilitating MDMX degradation. Although there was overall reverse correlation between MDMX level and nutlin-induced apoptosis, MDMX appear to be a major determinant of the apoptotic response only in one of 12 cancer cell lines, MCF7. Knockdown of MDMX expression by siRNA in the presence of nutlins-3a increased the apoptotic fraction in cancer cells with high MDMX level (e.g. MCF7) but did not affect the response of cells with low MDMX.