RRP22 is a farnesylated, nucleolar, Ras-related protein with tumor suppressor potential.

Ras proteins are members of a superfamily of related small GTPases. Some members, such as Ras, are oncogenic. However, other members seem to serve as tumor suppressors, such as Rig and Noey2. We now identify and characterize a novel member of the Ras superfamily, RRP22. Like Ras, RRP22 can be posttranslationally modified by farnesyl. Unlike Ras, RRP22 inhibits cell growth and promotes caspase-independent cell death. Examination of human tumor cells shows that RRP22 is frequently down-regulated due to promoter methylation. Moreover, reexpression of RRP22 in an RRP22-negative neural tumor cell line impairs its growth in soft agar. Unusually for a Ras-related protein, RRP22 localizes to the nucleolus in a GTP-dependent manner, suggesting a novel mechanism of action. Thus, we identify a new member of the Ras superfamily that can serve as a potential tumor suppressor.

p75-Ras-GRF1 is a c-Jun/AP-1 target protein: its up regulation results in increased Ras activity and is necessary for c-Jun-induced nonadherent growth of Rat1a cells.

The c-Jun/AP-1 transcription complex is associated with diverse cellular processes such as differentiation, proliferation, transformation, and apoptosis. These different biological endpoints are likely achieved by the regulation of specific target gene expression. We describe the identification of Ras guanine nucleotide exchange factor 1, Ras-GRF1, by microarray analysis as a c-Jun/AP-1 regulated gene essential for anchorage-independent growth of immortalized rat fibroblasts. Increased Ras-GRF1 expression, in response to inducible c-Jun expression in Rat1a fibroblasts, was confirmed by both real-time PCR and Northern blot analysis. We show that c-Jun/AP-1 can bind and activate the Ras-GRF1 promoter in vivo. A 75-kDa c-Jun/AP-1-inducible protein, p75-Ras-GRF1, was detected, and the inhibition of its expression with antisense oligomers significantly blocked c-Jun-regulated anchorage-independent cell growth. p75-Ras-GRF1 expression occurred with a concomitant increase in activated Ras (GTP bound), and the activation of Ras was significantly inhibited by antisense Ras-GRF1 oligomers. Moreover, p75-Ras-GRF1 could be coprecipitated with a Ras dominant-negative glutathione S-transferase (GST) construct, GST-Ras15A, demonstrating an interaction between p75-Ras-GRF1 and Ras. A downstream target of Ras activation, Elk-1, had increased transcriptional activity in c-Jun-expressing cells, and this activation was inhibited by dominant-negative Ras. In addition, c-Jun overexpression resulted in an increase in phospho-AKT while phosphorylation of ERK1/2 remained largely unaffected. The inhibition of phosphatidylinositol 3-kinase (PI3K)-AKT signal transduction by Ly294002 and wortmannin significantly blocked c-Jun-regulated morphological transformation, while inhibition of basal MEK-ERK activity with PD98059 and U0126 had little effect. We conclude that c-Jun/AP-1 regulates endogenous p75-Ras-GRF1 expression and that c-Jun/AP-1-regulated anchorage-independent cell growth requires activation of Ras-PI3K-AKT signal transduction.

RASSF2 is a novel K-Ras-specific effector and potential tumor suppressor.

Ras proteins regulate a wide range of biological processes by interacting with a broad assortment of effector proteins. Although activated forms of Ras are frequently associated with oncogenesis, they may also provoke growth-antagonistic effects. These include senescence, cell cycle arrest, differentiation, and apoptosis. The mechanisms that underlie these growth-inhibitory activities are relatively poorly understood. Recently, two related novel Ras effectors, NORE1 and RASSF1, have been identified as mediators of apoptosis and cell cycle arrest. Both of these proteins exhibit many of the properties normally associated with tumor suppressors. We now identify a novel third member of this family, designated RASSF2. RASSF2 binds directly to K-Ras in a GTP-dependent manner via the Ras effector domain. However, RASSF2 only weakly interacts with H-Ras. Moreover, RASSF2 promotes apoptosis and cell cycle arrest and is frequently down-regulated in lung tumor cell lines. Thus, we identify RASSF2 as a new member of the RASSF1 family of Ras effectors/tumor suppressors that exhibits a specificity for interacting with K-Ras.

The pro-apoptotic Ras effector Nore1 may serve as a Ras-regulated tumor suppressor in the lung.

Ras oncoproteins mediate multiple biological effects by activating multiple effectors. Classically, Ras activation has been associated with enhanced cellular growth and transformation. However, activated forms of Ras may also inhibit growth by inducing senescence, apoptosis, and differentiation. Induction of apoptosis by Ras may be mediated by its effector RASSF1, which appears to function as a tumor suppressor. We now show that the Ras effector Nore1, which is structurally related to RASSF1, can also mediate a Ras-dependent apoptosis. Moreover, an analysis of Nore1 protein expression showed that it is frequently down-regulated in lung tumor cell lines and primary lung tumors. Like RASSF1, this correlates with methylation of the Nore1 promoter rather than gene deletion. Finally, re-introduction of Nore1, driven by its own promoter, impairs the growth in soft agar of a human lung tumor cell line. Consequently, we propose that the Ras effector Nore1 is a member of a family of Ras effector/tumor suppressors that includes RASSF1.

Tamoxifen and the farnesyl transferase inhibitor FTI-277 synergize to inhibit growth in estrogen receptor-positive breast tumor cell lines.

Farnesyl transferase inhibitors (FTIs) serve to specifically inhibit farnesyl isoprenoid lipid modification of proteins. Although originally developed as anti-Ras oncoprotein drugs, it now appears that these compounds function independently of Ras. FTIs have been shown to inhibit transformation by a variety of mechanisms, including apoptosis involving cytochrome c release from mitochondria. Tamoxifen exhibits both anti-estrogenic and estrogenic properties and is widely used as an estrogen antagonist for the treatment of estrogen receptor (ER) positive human breast tumors. Tamoxifen can induce ER-dependent apoptosis in human breast tumor cells by a mechanism involving the Bcl2/mitochondrial arm of the apoptotic machinery. Since tamoxifen and FTIs may stimulate distinct components of the mitochondrial-based apoptotic machinery, we reasoned that their effects might be synergistic. Here we show that anti-estrogens and an FTI (FTI-277) synergize to inhibit cell growth and enhance cell death in ER positive, human breast tumor cell lines. However, the drugs exhibited only additive effects on an ER negative cell line. Analysis of treated ER positive T-47D cells demonstrated that a synergistic increase in apoptosis was induced, as measured by increased caspase 3 activity. Thus, tamoxifen and FTIs may synergize to promote apoptotic cell death in ER positive human breast tumor cells.

Rig is a novel Ras-related protein and potential neural tumor suppressor.

The Ras superfamily consists of a large group of monomeric GTPases demonstrating homology to Ras oncoproteins. Although structurally similar, Ras-superfamily proteins are functionally diverse. Whereas some members exhibit oncogenic properties, others may serve as tumor suppressors. We have identified a novel Ras-related protein that suppresses cell growth and have designated it Rig (Ras-related inhibitor of cell growth). Overexpression of Rig inhibited Ras-mediated cellular transformation and activation of downstream signaling in NIH 3T3 cells. rig mRNA is expressed at high levels in normal cardiac and neural tissue. However, Rig protein expression is frequently lost or down-regulated in neural tumor-derived cell lines and primary human neural tumors. Moreover, expression of exogenous Rig in human astrocytoma cells suppressed growth. Rig has a C-terminal CAAX motif that codes for posttranslational modification by both farnesyl and geranylgeranyl isoprenoid lipids. Consequently, Rig may play a role in the cellular response to farnesyl transferase inhibitors. Rig bears 63% overall sequence homology to a recently described Ras-family member Noey2, a tumor suppressor in breast and ovarian tissue. Therefore, Rig and Noey2 may represent a new subfamily of Ras-like tumor suppressors.