Background Mouse double minute 2 (MDM2) is an E3 ubiquitin ligase that is over-expressed in many cancers and regulates target proteins through ubiquitination. Full-length MDM2 (MDM2-FL) is best known for targeting wild-type p53 for degradation by the proteasome, but the functions of the many splice variants of MDM2 are under-explored. The three well-studied alternative MDM2 isoforms are MDM2-A/ALT2, MDM2-B/ALT1, and MDM2-C/ALT3. MDM2-A and MDM2-B are capable of down-regulating MDM2-FL activity and have transforming activity in cancers with mutant p53. The MDM2 isoform MDM2-C is over-expressed in breast cancer and correlates with decreased survival in the context of mutant p53 expression. Therefore, MDM2-C requires further study to determine if it has biochemical activities similar to MDM2-FL. Hypothesis: We hypothesized that like MDM2-FL, the MDM2-C isoform (lacking exons 5–9 and containing a full C-terminal RING finger sequence) would maintain E3 ubiquitin ligase activity. Materials and Methods In order to explore the biochemical function of MDM2-C, we used an in vitro ubiquitination assay and a glutaraldehyde cross-linking assay. Results Here we report, for the first time, that MDM2-C has E3 auto-ubiquitin ligase activity, which can promote ubiquitination of wild-type p53 and mutant p53 R273H, and also can form a protein–protein interaction with p53 proteins. Conclusion This information strongly positions MDM2-C as a protein with biochemical activities that may explain the varied outcomes observed in patients with high-level expression of MDM2-C in the presence of wild-type p53 versus mutant p53.

Human breast cancers often overexpress MDM family proteins MDM2 and MDMX(MDM4). MDM2 is an E3 ubiquitin ligase and MDMX is an E4 that promotes ubiquitination of MDM2 targets (however this is an understudied area). The Cancer Genome Atlas (TCGA) shows high MDMX/MDM2 expression in all breast cancer subtypes. We identified that MDMX depletion reduces circulating tumor cells (CTCs) and metastasis of breast cancers with mtp53. Our ongoing objective is to determine the mechanisms of how MDMX promotes CTCs and metastasis, and the potential MDMX regulated ubiquitinated targets. In TNBC primary tumors, knockdown of mdmx correlates with reduced expression of the G protein coupled receptor, CXCR4. We used mtp53 expressing T47D ER+, and MDA-MB-468, and MDA-MB-231 metastatic TNBC cells and their derived CTC lines, and shRNA-mediated knockdown of MDM2 and MDMX derived lines, to address the potential MDMX targets. We assessed metastatic properties of migration and invasion changed by the knockdown of MDM2/MDMX in parental and CTC cell lines using multi-faceted cell biological and biochemical approaches. Depletion of MDM2 in T47D and MDA-MB-231 cells increased the level of MDMX, demonstrating a critical function of MDM2 for regulating MDMX. MDMX depletion surprisingly did not change MDM2 levels, but did reduce the migration of both MDA-MB-231 cells and their derived CTC lines. Interestingly, the parental MDA-MB-231 cells migrated more rapidly in scratch assays, compared to both their derived CTCs and MDMX-depleted CTCs, suggesting migration to be critical for intravasation, but not for latter cancer cell survival. Experiments are in progress to address if loss of MDMX perturbs CXCL12 ligand communication with the CXCR4 receptor in parental and CTC breast cancer lines. Preliminary data suggests activated MDMX expression correlates with the activation of these metastasis signal transduction pathways. The ubiquitination targets of MDMX/MDM2 in the parental and CTC cell lines are being defined by affinity purification in the presence, and absence, of MDMX/MDM2, and add back of purified MDMX/MDM2 to validate targets. As expected, purified MDMX increases MDM2 E3 ubiquitin ligase activity, and on its own MDMX is inert. Stable isotope labeling in cell culture suggested protein targets involved in DNA replication and repair pathways. This preliminary data informed our interest in studying DNA damage signaling by pharmacologically inhibiting the DNA repair protein PARP. We examined how MDMX/MDM2 pathways respond to DNA damage caused by PARP inhibition. Our preliminary data affirms regulation of the MDMX/MDM2 stress-response circuit by replication stress response pathways, and reveals the requirement of MDMX/MDM2 for their function in the studied cell lines. Disrupting MDMX function has potential to be beneficial for inhibition of breast cancer progression and metastasis. Supported by BCRF-20-011. Citation Format: Rusia Lee, Viola Ellison, Gu Xiao, Dominique Forbes, Pam Leybengrub, Alexandra Kern, Falande Alexandre, George Annor, Jill Bargonetti. Identification of MDMX/MDM2 metastasis signaling pathways in breast cancer cells with mutant p53 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2437.