Oncogenic ras activates the ARF-p53 pathway to suppress epithelial cell transformation

Abstract

Chemically induced skin carcinomas in mice are a paradigm for epithelial neoplasia, where oncogenic ras mutations precede p53 and INK4a͞ARF mutations during the progression toward malignancy. To explore the biological basis for these genetic interactions, we studied cellular responses to oncogenic ras in primary murine keratinocytes. In wild-type keratinocytes, ras induced a cell-cycle arrest that displayed some features of terminal differentiation and was accompanied by increased expression of the p19 ARF , p16 INK4a , and p53 tumor suppressors. In ARF-null keratinocytes, ras was unable to promote cell-cycle arrest, induce differentiation markers, or properly activate p53. Although oncogenic ras produced a substantial increase in both nucleolar and nucleoplasmic p19 ARF , Mdm2 did not relocalize to the nucleolus or to nuclear bodies but remained distributed throughout the nucleoplasm. This result suggests that p19 ARF can activate p53 without overtly affecting Mdm2 subcellular localization. Nevertheless, like p53-null keratinocytes, ARF-null keratinocytes were transformed by oncogenic ras and rapidly formed carcinomas in vivo. Thus, oncogenic ras can activate the ARF-p53 program to suppress epithelial cell transformation. Disruption of this program may be important during skin carcinogenesis and the development of other carcinomas. N ormal cells possess natural defense mechanisms that counter uncontrolled mitogenic signaling, and these safeguards may be eliminated by mutation during multistage carcinogenesis (1). One such safeguard is illustrated by the transforming interactions between oncogenic ras and various tumor suppressor genes in primary fibroblasts. The ras family of proto-oncogenes are small GTPases that, when activated by mutation, constitutively transmit growth promoting signals via the mitogen-activated protein kinase (MAPK) cascade and other pathways (2). Oncogenic ras readily transforms immortal rodent cell lines; however, it is unable to transform primary cells because it induces a premature senescence program involving tumor suppressors such as p53, p16 INK4a , p19 ARF , p15 INK4b , and the promyelocytic leukemia gene product (PML;. Importantly, mutations that disable the arrest program cooperate with ras in transformation (5,(8)(9)(10). Given the high frequency of ras, p53, and INK4a͞ARF mutations in human cancers (11), this safeguard may be an important tumor suppressor mechanism.The INK4a͞ARF locus is a critical sensor of hyperproliferative signals produced by ras and other oncogenes (12). This locus encodes the two structurally unrelated tumor suppressors p16 INK4a and p19 ARF (13,14). p16 INK4a , an inhibitor to cyclin D-dependent kinase, regulates the retinoblastoma (Rb) protein to block the G 1 -S transition (15). In contrast, p19 ARF can activate p53 by interfering with the p53 antagonist Mdm2 (16-19), leading to cell-cycle arrest or apoptosis depending on context (12). Both p16 INK4a and p19 ARF accumulate during the serial passaging of cells in culture (20)(21)(22), and can be indu...

Human BRE1 Is an E3 Ubiquitin Ligase for Ebp1 Tumor Suppressor

“…Nucleolar ARF stabilizes nucleoplasmic p53 through binding to MDM2 and sequestering it in the nucleolus (Weber et al, 1999). Nonetheless, nucleoplasmic ARF activates p53 function by directly inhibiting the ubiquitin ligase activity of MDM2 (Midgley et al, 2000;Lin and Lowe, 2001;Llanos et al, 2001). The extent of p53 ubiquitination under different cellular conditions is determined not only by changes in MDM2 and ARF-BP1 activities but also by phosphorylation of the substrate p53 (Gao and Karin, 2005).…”

Regulation of cellular response to oncogenic and oxidative stress by Seladin-1

“…Microtubules rapidly splayed outward from the spindle centre, after which the two half spindles appeared to become disconnected and the degree of anti-parallel microtubule overlap decreased markedly. This effect was specific to the hydrolytic activity of PARG, as simultaneous addition of 100 mM of the PARG inhibitor ADP-HPD 13 blocked the phenotypic effect of PARG (Fig. 2b).…”

“…4,13). Intriguingly, while Ras-mediated p16 INK4a induction was unaffected in Seladin-1-ablated WI38-TERT cells, silencing of Seladin-1 prevented Ras-induced p53 accumulation (Fig.…”

Ras proteins: paradigms for compartmentalised and isoform-specific signalling

“…In normal human fibroblasts, the consequence of hyperactive Ras signalling is activation of the ARF/p53 tumour suppressor pathway and induction of replicative senescence [23,24]. Deletion of components of the tumour suppressor pathway such as ARF are needed to generate the hyperproliferative state and induce carcinogenesis [25]. However, in contrast to these in vitro studies where supraphysiological levels of Ras are expressed, mouse embryonic fibroblasts (MEFs) derived from mice engineered to express endogenous levels of oncogenic K-Ras are immortalised and do not become senescent [19,26].…”