Abstract A40: Investigating the functional response of the ARF tumor suppressor to acute p53 loss

The tumor suppressor, p19 ARF , is classically considered to function in response to hyerproliferative signals by binding and sequestering MDM2 in the nucleolus, thereby activating p53. Steady-state levels of ARF are kept low to prevent activation of p53 and are thought to monitor nucleolar function. Interestingly, p53 itself inhibits the Arf locus by recruiting proteins involved in heterochromatin formation. Cell lines that lack functional p53, therefore, accumulate enormous amounts of ARF protein but proliferate relatively well. Given that ARF is known to have functions independent of p53 both in vitro and in vivo, we wanted to investigate the function of ARF in the setting of acute p53 loss using conditional deletion of p53 from p53 Flox/Flox mouse embryonic fibroblasts (MEFs). Cre -mediated excision of p53 leads to an induction of ARF by 4 days post infection, but levels do not peak until 10 days. Adeno-Cre infected MEFs have a higher proliferation rate than Adeno-LacZ controls, and do not undergo replicative senescence. However, knockdown of Arf in p53 Δ/Δ MEFs using lentiviral shRNAs results in an increase in proliferation and enhanced growth in semisolid medium following Ras I/12 transformation. These findings suggest that the induced ARF levels are acting to inhibit p53-null cells in some capacity. Loss of p53 also leads to a dramatic downregulation of MDM2 protein levels. Immunoprecipitation experiments indicate that ARF predominantly interacts with MDM2 in LacZ -infected cells, but binds the ribosomal chaperone nucleophosmin (NPM) more readily in p53 Δ/Δ MEFs. Since inhibition of NPM by ARF has been shown to negatively affect overall protein synthesis, we hypothesize that in the absence of p53, ARF functions to negatively regulate overall protein synthesis through NPM in an effort to slow proliferation. Indeed, p53 Δ/Δ MEFs contain less total protein per cell, copies of 47S rRNA, and have reduced active translation compared to their LacZ counterparts. We are currently testing whether knockdown Arf in p53 Δ/Δ MEFs can rescue protein synthesis, thus allowing these cells to proliferate at an even faster rate. These findings could explain why certain cancers (i.e., pancreatic cancer) preferentially inactivate both ARF and p53. We are also attempting to understand why the high levels of endogenous ARF seen in the absence of p53 are not able to induce a cell cycle arrest when ARF is clearly capable of eliciting such a response independently of p53. As p53 mutation occurs in over 50% of all cancers it would be of interest to know how we could activate this function of ARF in those patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the Second AACR International Conference on Frontiers in Basic Cancer Research; 2011 Sep 14-18; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2011;71(18 Suppl):Abstract nr A40.