Modulation of the Mdm2 signaling axis sensitizes triple-negative breast cancer cells to carboplatin

Abstract

Triple-negative breast cancers (TNBCs) are highly refractive to current treatment strategies, and new multi-targeted treatments need to be elucidated. Combination therapy that includes targeting the murine double minute 2 (Mdm2) signaling axis offers a promising approach. Protein-protein interaction inhibitors such as Nutlin-3a block the binding of key signaling molecules such as p53, p73α, and E2F1 to the hydrophobic pocket of Mdm2 and can lead to activation of cell-death signaling pathways. Since clinical trials for TNBC are evaluating the DNA damaging agent carboplatin, the objective of this thesis was to evaluate the therapeutic potential and mechanism of action of combination carboplatin and Nutlin-3a to treat TNBC. In TNBC cell lines with a mutant p53 background, we determined if modulation of Mdm2 function in the context of carboplatin-mediated DNA damage resulted in a synergistic inhibition of cell growth. Several ratios of carboplatin:Nutlin-3a were strongly synergistic in increasing cell death, with combination indices of 0.5 and lower. Mechanistic studies indicated that drug sensitivity and Mdm2 expression were dependent on p73. Mdm2 localized to a larger degree in the chromatin fraction isolated from cells treated with the combination treatment consistent with observations by others that Mdm2 binds to the Mre11/Rad50/Nbs1 complex, inhibits the DNA damage response, and increases drug sensitivity. In vivo efficacy experiments were conducted in the TMD231 orthotopic mammary fat pad model in NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) mice. For assessment of baseline tumor burden and randomization, fluorescent imaging of E2-Crimson expressing TMD231 cells was performed. Following Nutlin-3a and carboplatin combination treatment, there was a statistically significant reduction in primary tumor volume as well as lung metastases with significantly increased probability of survival compared to Vehicle and single drug treatments (p<0.001). While there was a decrease in bone-marrow cellularity, this did not lead to bone-marrow aplasia, and body weights recovered to normal levels within 7 days post-treatment. The present studies demonstrate the promise of Mdm2 as a therapeutic target in combination with conventional therapy, increase our understanding of how to potentiate DNA damage in cancers, and may lead to new clinical therapies for triple-negative primary and metastatic breast cancer.