Ras-Raf-Arf signaling critically depends on the Dmp1 transcription factor.

The INK4a-ARF locus on human chromosome 9p21 is disrupted in approximately 40% of human cancers (42). This unusual locus encodes two distinct tumor suppressor proteins, p16INK4a and p14ARF (p19Arf in the mouse), encoded in part via alternative reading frames. p16Ink4a binds to cyclin-dependent kinase 4 (Cdk4) to inhibit Rb phosphorylation, whereas p19Arf binds to the p53 negative regulator, Mdm2, thereby stabilizing and activating p53 (26, 47). Arf is induced by potentially harmful growth-promoting signals stemming from overexpression of a variety of oncoproteins, including c-Myc, E2F-1, mutated Ras, v-Abl, and β-catenin (47). This forces incipient cancer cells to undergo p53-dependent proliferative arrest or apoptosis, providing a powerful mode of tumor suppression. In turn, Arf-null mice are highly prone to spontaneous tumor development and die of various forms of cancer by 15 months of age (21, 22). Recently, the creation of Arf-GFP knock-in mice has provided direct experimental evidence that the Arf promoter monitors latent oncogenic signals in vivo (56). How Arf responds to oncogenic Ras signaling remains unclear. Ras family proteins play crucial roles in the control of cell growth and differentiation (29). Overexpression of activated Ras initiates DNA synthesis independent of growth factor stimulation. In immortal rodent cell lines, transformation by oncogenic Ras involves its ability to bind and activate a series of effector proteins, including Raf-1, phosphoinositide 3-OH kinase [PI(3)K], and Ral-GDS (23). Each of these molecules, in turn, activates distinct downstream targets, thereby producing different aspects of the transformed phenotype. The Ras-Raf interaction initiates the mitogen-activated protein kinase (MAPK) cascade, which involves the sequential activation of a series of protein kinases that transmit mitogenic signals to nuclear transcription factors. These kinases include Raf-1, the MEKs (MEK1 and MEK2), and the ERKs (ERK1 and ERK2). On the other hand, the ability of Ras to activate PI(3)K promotes membrane ruffling (20), and the Ral-GDS proteins act as exchange factors that can activate the Ral family of small GTPases (50). Although each of these effector pathways contributes to the transforming activity of Ras in established rodent fibroblast cell lines, activation of the Raf-MEK-ERK pathway is sufficient for transformation (10). Paradoxically, sustained overexpression of oncogenic Ras and its various effectors in nonimmortalized cells has the capacity to elicit irreversible cell cycle arrest by upregulating the levels of p16Ink4a, p19Arf, and p53 in mice and p16INK4a and p53 in humans (29, 33, 44, 48, 57). The ability of oncogenic Ras to induce premature senescence depends on the activity of the Raf-MEK-ERK pathway that mediates proliferation (25) but is nullified in primary mouse embryo fibroblasts (MEFs) lacking either Arf or p53 (21, 33, 48). Among known Arf activators, the Dmp1 transcription factor (cyclin D-interacting Myb-like protein 1) is a bona fide tumor suppressor (18, 19). Dmp1 was originally isolated in a yeast two-hybrid screen of a murine T-lymphocyte library with cyclin D2 as bait (14). The protein binds to nonameric CCCG(G/T)ATG(T/C) DNA consensus sequences, a subset of which is also bound by proteins of the Ets family. Dmp1 can physically interact with any of the three D-type cyclins, each of which can interfere, in a Cdk4-independent manner, with Dmp1's ability to bind to DNA (15). Overexpression of Dmp1 in mouse fibroblasts arrests cell cycle progression, an effect that can be overridden by coexpression of D-type cyclins (15). Importantly, Dmp1 directly binds to the Arf promoter to activate its expression, thereby inducing p53-dependent cell cycle arrest (17). Several lines of evidence have implicated Dmp1 in the process by which Ras induces Arf and p53. When primary Dmp1-null MEFs were explanted into culture and continuously passaged, p19Arf and p53 levels remained uncharacteristically low and the cells exhibited a prolonged proliferative capacity, readily yielding established cell lines that retained wild-type Arf and p53. Such cells were susceptible to transformation by oncogenic Ras alone without any requirement for an immortalizing oncogene, such as Myc or adenovirus E1A. Thus, the activity of the Arf-p53 pathway is strikingly impaired in Dmp1-null cells (18). Dmp1-null mice are prone to spontaneous tumor development in their second year of life, and tumor formation was accelerated when the animals were neonatally treated with ionizing radiation or dimethylbenzanthracene (18, 19), a carcinogen that induces Ras mutations in vivo (37). When crossed onto a Dmp1+/− or Dmp1−/− background, lymphomas induced by an Eμ-Myc transgene were greatly accelerated with no differences between cohorts lacking one or two Dmp1 alleles. The retention and expression of the wild-type Dmp1 allele in tumors arising in heterozygotes indicated that Dmp1 is haplo-insufficient for tumor suppression (19, 38). Interestingly, the combined frequencies of p53 mutation and Arf deletion in the Dmp1−/− and Dmp1+/− lymphomas were significantly lower than those in Dmp1+/+ tumors (∼14% versus ∼50%). Thus, Dmp1 is a physiological regulator of the Arf-p53 pathway in vivo (19). The present studies were undertaken in an attempt to define the mechanism(s) by which Ras induces Arf. Here we show that Dmp1 is a key mediator of this process.