Ras^V12-Mediated Down-regulation of CCAAT/Enhancer Binding Protein in Immortalized Fibroblasts Requires Loss of p19^Arf and Facilitates Bypass of Oncogene-Induced Senescence

Basic Research Laboratory, Center for Cancer Research, National Cancer Institute-Frederick, Frederick, Maryland

^* To whom correspondence should be addressed. E-mail: johnsopf{at}ncifcrf.gov.

	Abstract

The transcription factor CCAAT/enhancer binding protein (C/EBP) is involved in cellular responses to oncogenic and physiologic Ras signals. C/EBP is required for premature senescence of primary mouse fibroblasts induced by expression of H-Ras^V12, demonstrating its role in oncogene-induced senescence. Here, we have investigated the mechanisms by which Ras inhibits proliferation of normal cells but transforms immortalized cells. We show that oncogenic Ras down-regulates C/EBP expression in NIH 3T3 cells, which are immortalized by a deletion of the CDKN2A locus and, therefore, lack the p16^Ink4a and p19^Arf tumor suppressors. Ras^V12-induced silencing of C/EBP occurred at the mRNA level and involved both the Raf–mitogen-activated protein/extracellular signal-regulated kinase (ERK) kinase–ERK and phosphatidylinositol 3-kinase signaling pathways. Oncogenic Ras decreased C/EBP expression in Ink4a/Arf^-/- mouse embryo fibroblasts (MEF) but increased C/EBP levels in wild-type MEFs. C/EBP down-regulation in NIH 3T3 cells was reversed by expression of p19^Arf, but not of p53 or p16^Ink4a, highlighting a critical role for p19^Arf in sustaining C/EBP levels. Ectopic expression of p34 C/EBP (LAP) inhibited Ras^V12-mediated transformation of NIH 3T3 cells, suppressed their tumorigenicity in nude mice, and reactivated expression of the proapoptotic Fas receptor, which is also down-regulated by Ras. Our findings indicate that Cebpb gene silencing eliminates a growth inhibitory transcription factor that would otherwise restrain oncogenesis. We propose that C/EBP is part of a p53-independent, p19^Arf-mediated network that enforces Ras-induced cell cycle arrest and tumor suppression in primary fibroblasts. [Cancer Res 2009;69(6):2588–98]

Key Words: Ras, C/EBP, p19^Arf, oncogenesis, tumor suppression