Abstract A23: Mdm2 phosphorylation by Akt is critical for modulating cellular responses to oxidative stress

Abstract

Abstract The serine/threonine kinase Akt is activated by a myriad of growth signals and promotes cell proliferation, survival, and metabolism. Akt is frequently activated in a wide assortment of human hematologic malignancies and solid tumors. In vitro studies demonstrated that Akt phosphorylates the Mdm2 oncoprotein at ser166 and ser186 (163 and 183 in mice) to promote Mdm2 translocation into the nucleus. This enhances Mdm2-mediated p53 ubiquitination and the subsequent proteasomal-mediated degradation of p53. Wild-type p53 protein is a tumor suppressor that is activated in response to stress signals such as DNA damage, oxidative stress, and aberrant oncogene activation. Activated p53 induces the expression of genes that trigger cell cycle arrest, senescence, and apoptosis. This inhibits the growth of damaged and/or potentially oncogenic cells, underscoring the role of p53 in tumor suppression. However, the role of Akt-mediated phosphorylation of Mdm2 on p53 functions in vivo remains unknown. We hypothesize that Mdm2 phosphorylation by Akt at ser183 promotes cell growth and tumorigenesis by reducing p53 levels and function in vivo. Using CRISPR/Cas9-mediated gene editing, we generated mice with mutant Mdm2 protein that cannot be selectively phosphorylated at ser183, by substituting this serine with an alanine. Unlike Mdm2-/- mice that are embryonic lethal, Mdm2S183A mice are viable, fertile, and obtained at Mendelian ratios. There are no apparent growth/weight differences between unchallenged Mdm2WT and Mdm2S183A mice. Other stress-induced kinases like ATM and c-Abl phosphorylate Mdm2 to regulate the levels and functional activity of p53 in response to stress such as DNA damage. In contrast, Akt-mediated phosphorylation of Mdm2 does not alter p53 levels and function in response to DNA damage both in vitro and in vivo. However, under low-density stress plating, the ability of Mdm2S183A MEFs to form colonies is completely abrogated. In addition, Mdm2S183A MEFs fail to proliferate under normal cell culture conditions and exhibit salient features of senescence including positive staining for SA-β Gal. The expression of p53-target genes that mediate senescence is elevated in Mdm2S183A MEFs than in Mdm2WT MEFs. The severe proliferation defect and premature senescent phenotypes observed in Mdm2S183A MEFs are rescued by culturing cells at low oxygen tension with media supplemented with N-acetyl cysteine, a reactive oxygen species (ROS) scavenger. Our results from various assays examining the levels and effects of ROS suggest that under normal cell culture conditions, Mdm2S183A MEFs are more sensitive to oxidative stress compared to Mdm2WT MEFs. So far, our data suggest that Mdm2 phosphorylation by Akt at ser183 is required for proper regulation of the oxidative stress response and is necessary to promote cell growth under normal plating stress conditions. We are currently analyzing RNA-seq data to determine whether there are certain gene expression changes that could aid our quest to decipher the underlying mechanism responsible for the senescent phenotype of Mdm2S183A MEFs. In addition, we have crossed Mdm2S183A to lung and liver cancer models in which ROS plays a pivotal role to examine whether blocking Mdm2 phosphorylation by Akt alters the incidence and progression of tumors. Citation Format: Loretah Chibaya, Hong Zhang, Stephen N. Jones. Mdm2 phosphorylation by Akt is critical for modulating cellular responses to oxidative stress [abstract]. In: Proceedings of the AACR Special Conference: Advances in Modeling Cancer in Mice: Technology, Biology, and Beyond; 2017 Sep 24-27; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(10 Suppl):Abstract nr A23.