Abstract 4639: Blockade of MDM2-mediated signaling in context of DNA damage increases E2F1 expression and enhances cell death in triple-negative breast cancer cells.
Authors: Tonsing-Carter Eva , Shannon Harlan E. , Bailey Barbara J. , Mayo Lindsey D. , Pollok Karen E. 
Published: August 2014

Metastatic breast cancer is highly refractive to current treatment strategies and new multi-targeted treatments need to be elucidated. In metastatic disease, blocking key protein-protein interactions with the murine double minute 2 (MDM2) could be beneficial for developing new treatment modalities since this signaling pathway is a critical regulatory point in cancer progression. Inhibition of protein binding to the hydrophobic pocket of MDM2 by Nutlin-3a can result in increases in pro-apoptotic proteins such as p53, p73, and E2F1. Since the DNA damaging agent carboplatin is being studied in clinical trials of triple-negative breast cancers (TNBCs), our objective was to evaluate the effects of carboplatin and Nutlin-3a in combination in TNBC cancer in a mutant p53 background. We utilized TMD231 breast cancer cells derived from the MDA-MB-231 human TNBC cancer line. TMD231 cells are highly metastatic in vivo and readily metastasize from the primary tumor to the lung. Combination studies were performed using different ratios of carboplatin to Nutlin-3a to evaluate the range of carboplatin-mediated DNA damage required to obtain synergism with inhibition of MDM2 function. A fixed ratio of 1:1 carboplatin:Nutlin-3a was strongly synergistic with a combination index of &lt; 0.5. In cell proliferation assays there was increased sensitivity to the drugs when given together and a significant reduction in number of colonies in colony formation assays. In order to understand how DNA damage and inhibition of MDM2 signaling leads to enhanced cell death, dose-response and time-course studies were performed. Western analyses demonstrated increases in E2F1 when treated with carboplatin and Nutlin-3a alone, and a time-dependent increase in E2F1 following combination treatment at a fixed 1:1 ratio. Following DNA damage, E2F1 has been shown to promote the transcription of pro-apoptotic genes and repression of survival genes once E2F1 is released from interaction with Rb. Here, E2F1 levels correlated with Rb phosphorylation at serine 780, a phosphorylation site which inhibits binding of E2F1 to Rb. The E3 ubiquitin ligase Itch, which ubiquitinates p73, increased following treatment with carboplatin alone or in combination with Nutlin-3a in a dose- and time-dependent manner but appeared not to regulate p73 since p73 levels remained unchanged following combination treatment. Studies are ongoing to further understand how modulation of DNA damage by blockade of MDM2 results in a robust synergistic response and how upregulation of E2F1 affects downstream targets. The impact of the combination of DNA damaging carboplatin and MDM2 blockade by Nutlin-3a on DNA repair kinetics is currently being evaluated in an orthotopic TMD231 model. These studies will lead to a better understanding of how to potentiate DNA damage and may lead to new clinical therapies in the future.

Citation Format: Eva Tonsing-Carter, Harlan E. Shannon, Barbara J. Bailey, Lindsey D. Mayo, Karen E. Pollok. Blockade of MDM2-mediated signaling in context of DNA damage increases E2F1 expression and enhances cell death in triple-negative breast cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4639. doi:10.1158/1538-7445.AM2013-4639