Abstract A19: A Ras-Arf-Egr-C/EBPβ axis underlying oncogene-induced senescence and cancer

In normal cells, persistent oncogenic Ras activation triggers senescence, an intrinsic tumor suppression mechanism that protects cells from malignant transformation. Oncogene-induced senescence (OIS) is dependent on tumor suppressors such as p19/p14Arf, and loss of those genes facilitates transformation by oncogenic Ras. We previously reported that C/EBPβ, a transcription factor that has both pro- and anti-oncogenic functions, contributes to OIS in primary fibroblasts. Notably, C/EBPβ is down-regulated in H- or K-RasV12-transformed NIH 3T3 cells in a manner that requires loss of p19Arf. The potential role of C/EBPβ down-regulation in RasV12-induced transformation prompted us to investigate the mechanisms of Cebpb gene transcription. Here we show that Cebpb is an early growth factor-induced gene whose induction is suppressed by H-RasV12 in NIH 3T3 cells and p19Arf-/- MEFs. The early growth response (Egr) transcription factors are also down-regulated by H-RasV12 in these cells. Over-expression of any of Egr proteins (Egr1-4) restored C/EBPβ levels in 3T3Ras cells. Interestingly, Egrs induce growth arrest and senescence in 3T3Ras cells in a manner that is only partially dependent on reactivation of C/EBPβ. Egr1-4 proteins stimulated transcription from a promoter-reporter construct containing ∼3 kb of the Cebpb 59 flanking region. The proximal Cebpb promoter contains several putative Egr binding motifs, and mutating three of these sites disrupted Egr-mediated transactivation of the Cebpb promoter. Chromatin immunoprecipitation assays showed that ectopically expressed and endogenous Egrs bind to the Cebpb proximal promoter. Furthermore, microarray data from Oncomine show a strong correlation between CEBPB and EGR expression in human tumor samples, regardless of whether C/EBPβ levels increase or decrease in tumors compared to normal tissue. Since CEBPB and EGRs are down-regulated in lung adenocarcinomas and their elevated expression is associated with a better prognosis in lung cancer patients (Prognoscan), we over-expressed Egrs in A549 lung adenocarcinoma cells, which carry a KRAS mutation. Ectopic Egr2-4 induced CEBPB mRNA expression and dramatically suppressed cell proliferation. Collectively, our data indicate that decreased Egr levels account for oncogenic Ras-induced down-regulation of C/EBPβ in certain transformed cells, and restoration of Egrs induces growth arrest and C/EBPβ expression. Our findings identify the Egr-C/EBPβ axis as an important pathway in determining the cellular response (transformation or senescence) to oncogenic Ras-Arf signaling. Citation Format: Jacqueline Salotti, Krisada Sakchaisri, Peter F. Johnson. A Ras-Arf-Egr-C/EBPβ axis underlying oncogene-induced senescence and cancer. [abstract]. In: Proceedings of the AACR Special Conference on RAS Oncogenes: From Biology to Therapy; Feb 24-27, 2014; Lake Buena Vista, FL. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(12 Suppl):Abstract nr A19. doi: 10.1158/1557-3125.RASONC14-A19