Abstract B24: A p19Arf-Egr-C/EBPβ axis underlying oncogene-induced senescence and tumor suppression
Authors: Salotti Jacqueline , Sakchaisri Krisada , Johnson Peter 
Published: January 2014

Abstract
Cellular senescence is an intrinsic tumor suppression mechanism that protects cells from neoplastic transformation by activated oncogenes. As such, strategies to enhance senescence could be a promising avenue for cancer prevention. The transcription factor (TF) CCAAT/enhancer binding protein β (C/EBPβ) is induced and post-translationally activated by oncogenic Ras signaling in primary mouse embryonic fibroblasts (MEFs) and cooperates with RB:E2F to implement RasV12-induced senescence. In contrast, immortalized cells lacking the tumor suppressor p19Arf, such as NIH 3T3 murine fibroblasts, bypass senescence and are transformed by RasV12. Our lab previously showed that C/EBPβ expression is down-regulated in association with RasV12 transformation of NIH 3T3 cells. Furthermore, p19Arf acts in a p53-independent manner to maintain C/EBPβ levels in RasV12-expressing fibroblasts. Thus, C/EBPβ functions as part of an Arf-dependent tumor suppressor network activated by oncogenic stress. The opposing effects of oncogenic Ras on Cebpb expression in primary MEFs and immortalized NIH 3T3 cells prompted us to investigate the mechanisms that regulate Cebpb gene transcription. The early growth response (Egr) family of zinc-finger TFs is composed of 4 members, Egr-1-4. Here we show that Egrs are also down-regulated in RasV12-transformed NIH 3T3 cells (3T3Ras) and their ectopic expression restores C/EBPβ levels. Over-expression of any of the Egr proteins causes growth arrest and senescence in 3T3Ras cells in a manner that is partially dependent on reactivation of C/EBPβ. Egr proteins stimulate transcription from a promoter-reporter construct containing ∼3 kb of the Cebpb 5' flanking region. The proximal Cebpb promoter contains several putative Egr binding motifs, and mutating three of these sites disrupts Egr-mediated transactivation of the Cebpb reporter. Chromatin immunoprecipitation assays showed that ectopically expressed and endogenous Egrs bind to the Cebpb proximal promoter. In addition, serum stimulation of normal NIH 3T3 cells induces C/EBPβ mRNA and protein levels, which peak between 2-5 hours and coincide with transient induction of Egr proteins. Moreover, Egrs are only weakly induced by serum in 3T3Ras cells compared to their robust induction in the parental cells, with the exception of Egr-4 which was not detected in either cell line. Thus, decreased Egr levels can account for RasV12-induced down-regulation of C/EBPβ in transformed fibroblasts. Furthermore, microarray data from Oncomine show a strong correlation between CEBPB and EGR expression in human tumor samples. Collectively, our findings identify the Egr-C/EBPβ axis as a critical pathway in determining the cellular response (transformation or senescence) to oncogenic Ras-p19Arf signaling.
Citation Format: Jacqueline Salotti, Krisada Sakchaisri, Peter Johnson. A p19Arf-Egr-C/EBPβ axis underlying oncogene-induced senescence and tumor suppression. [abstract]. In: Proceedings of the Eleventh Annual AACR International Conference on Frontiers in Cancer Prevention Research; 2012 Oct 16-19; Anaheim, CA. Philadelphia (PA): AACR; Cancer Prev Res 2012;5(11 Suppl):Abstract nr B24.