Pumping the brakes on RAS - negative regulators and death effectors of RAS.

Mutations that activate the RAS oncoproteins are common in cancer. However, aberrant upregulation of RAS activity often occurs in the absence of activating mutations in the RAS genes due to defects in RAS regulators. It is now clear that loss of function of Ras GTPase-activating proteins (RasGAPs) is common in tumors, and germline mutations in certain RasGAP genes are responsible for some clinical syndromes. Although regulation of RAS is central to their activity, RasGAPs exhibit great diversity in their binding partners and therefore affect signaling by multiple mechanisms that are independent of RAS. The RASSF family of tumor suppressors are essential to RAS-induced apoptosis and senescence, and constitute a barrier to RAS-mediated transformation. Suppression of RASSF protein expression can also promote the development of excessive RAS signaling by uncoupling RAS from growth inhibitory pathways. Here, we will examine how these effectors of RAS contribute to tumor suppression, through both RAS-dependent and RAS-independent mechanisms.

Ras signaling through RASSF proteins.

There are six core RASSF family proteins that contain conserved Ras Association domains and may serve as Ras effectors. They lack intrinsic enzymatic activity and appear to function as scaffolding and localization molecules. While initially being associated with pro-apoptotic signaling pathways such as Bax and Hippo, it is now clear that they can also connect Ras to a surprisingly broad range of signaling pathways that control senescence, inflammation, autophagy, DNA repair, ubiquitination and protein acetylation. Moreover, they may be able to impact the activation status of pro-mitogenic Ras effector pathways, such as the Raf pathway. The frequent epigenetic inactivation of RASSF genes in human tumors disconnects Ras from pro-death signaling systems, enhancing Ras driven transformation and metastasis. The best characterized members are RASSF1A and RASSF5 (NORE1A).

Nonstructural protein 5B promotes degradation of the NORE1A tumor suppressor to facilitate hepatitis C virus replication.

Hepatitis C virus (HCV) infection is a common risk factor for the development of liver cancer. The molecular mechanisms underlying this effect are only partially understood. Here, we show that the HCV protein, nonstructural protein (NS) 5B, directly binds to the tumor suppressor, NORE1A (RASSF5), and promotes its proteosomal degradation. In addition, we show that NORE1A colocalizes to sites of HCV viral replication and suppresses the replication process. Thus, NORE1A has antiviral activity, which is specifically antagonized by NS5B. Moreover, the suppression of NORE1A protein levels correlated almost perfectly with elevation of Ras activity in primary human samples. Therefore, NORE1A inactivation by NS5B may be essential for maximal HCV replication and may make a major contribution to HCV-induced liver cancer by shifting Ras signaling away from prosenescent/proapoptotic signaling pathways. CONCLUSION: HCV uses NS5B to specifically suppress NORE1A, facilitating viral replication and elevated Ras signaling. (Hepatology 2017;65:1462-1477).

Micro-RNA-186-5p inhibition attenuates proliferation, anchorage independent growth and invasion in metastatic prostate cancer cells.

BACKGROUND: Dysregulation of microRNA (miRNA) expression is associated with hallmarks of aggressive tumor phenotypes, e.g., enhanced cell growth, proliferation, invasion, and anchorage independent growth in prostate cancer (PCa). METHODS: Serum-based miRNA profiling involved 15 men diagnosed with non-metastatic (stage I, III) and metastatic (stage IV) PCa and five age-matched disease-free men using miRNA arrays with select targets confirmed by quantitative real-time PCR (qRT-PCR). The effect of miR-186-5p inhibition or ectopic expression on cellular behavior of PCa cells (i.e., PC-3, MDA-PCa-2b, and LNCaP) involved the use bromodeoxyuridine (BrdU) incorporation, invasion, and colony formation assays. Assessment of the impact of miR-186-5p inhibition or overexpression on selected targets entailed microarray analysis, qRT-PCR, and/or western blots. Statistical evaluation used the modified t-test and ANOVA analysis. RESULTS: MiR-186-5p was upregulated in serum from PCa patients and metastatic PCa cell lines (i.e., PC-3, MDA-PCa-2b, LNCaP) compared to serum from disease-free individuals or a normal prostate epithelial cell line (RWPE1), respectively. Inhibition of miR-186-5p reduced cell proliferation, invasion, and anchorage-independent growth of PC-3 and/or MDA-PCa-2b PCa cells. AKAP12, a tumor suppressor target of miR-186-5p, was upregulated in PC-3 and MDA-PCa-2b cells transfected with a miR-186-5p inhibitor. Conversely, ectopic miR-186-5p expression in HEK 293 T cells decreased AKAP12 expression by 30%. Both pAKT and ß-catenin levels were down-regulated in miR-186-5p inhibited PCa cells. CONCLUSIONS: Our findings suggest miR-186-5p plays an oncogenic role in PCa. Inhibition of miR-186-5p reduced PCa cell proliferation and invasion as well as increased AKAP12 expression. Future studies should explore whether miR-186-5p may serve as a candidate prognostic indicator and a therapeutic target for the treatment of aggressive prostate cancer.

Ras Regulates Rb via NORE1A.

Mutations in the Ras oncogene are one of the most frequent events in human cancer. Although Ras regulates numerous growth-promoting pathways to drive transformation, it can paradoxically promote an irreversible cell cycle arrest known as oncogene-induced senescence. Although senescence has clearly been implicated as a major defense mechanism against tumorigenesis, the mechanisms by which Ras can promote such a senescent phenotype remain poorly defined. We have shown recently that the Ras death effector NORE1A plays a critical role in promoting Ras-induced senescence and connects Ras to the regulation of the p53 tumor suppressor. We now show that NORE1A also connects Ras to the regulation of a second major prosenescent tumor suppressor, the retinoblastoma (Rb) protein. We show that Ras induces the formation of a complex between NORE1A and the phosphatase PP1A, promoting the activation of the Rb tumor suppressor by dephosphorylation. Furthermore, suppression of Rb reduces NORE1A senescence activity. These results, together with our previous findings, suggest that NORE1A acts as a critical tumor suppressor node, linking Ras to both the p53 and the Rb pathways to drive senescence.

Ras regulates SCF(ß-TrCP) protein activity and specificity via its effector protein NORE1A.

Ras is the most frequently activated oncogene found in human cancer, but its mechanisms of action remain only partially understood. Ras activates multiple signaling pathways to promote transformation. However, Ras can also exhibit a potent ability to induce growth arrest and death. NORE1A (RASSF5) is a direct Ras effector that acts as a tumor suppressor by promoting apoptosis and cell cycle arrest. Expression of NORE1A is frequently lost in human tumors, and its mechanism of action remains unclear. Here we show that NORE1A forms a direct, Ras-regulated complex with ß-TrCP, the substrate recognition component of the SCF(ß-TrCP) ubiquitin ligase complex. This interaction allows Ras to stimulate the ubiquitin ligase activity of SCF(ß-TrCP) toward its target ß-catenin, resulting in degradation of ß-catenin by the 26 S proteasome. However, the action of Ras/NORE1A/ß-TrCP is substrate-specific because IκB, another substrate of SCF(ß-TrCP), is not sensitive to NORE1A-promoted degradation. We identify a completely new signaling mechanism for Ras that allows for the specific regulation of SCF(ß-TrCP) targets. We show that the NORE1A levels in a cell may dictate the effects of Ras on the Wnt/ß-catenin pathway. Moreover, because NORE1A expression is frequently impaired in tumors, we provide an explanation for the observation that ß-TrCP can act as a tumor suppressor or an oncogene in different cell systems.

Cell cycle restriction is more important than apoptosis induction for RASSF1A protein tumor suppression.

The Ras association domain family protein 1A (RASSF1A) is arguably one of the most frequently inactivated tumor suppressors in human cancer. RASSF1A modulates apoptosis via the Hippo and Bax pathways but also modulates the cell cycle. In part, cell cycle regulation appears to be dependent upon the ability of RASSF1A to complex with microtubules and regulate their dynamics. Which property of RASSF1A, apoptosis induction or microtubule regulation, is responsible for its tumor suppressor function is not known. We have identified a short conserved motif that is essential for the binding of RASSF family proteins with microtubule-associated proteins. By making a single point mutation in the motif, we were able to generate a RASSF1A variant that retains wild-type apoptotic properties but completely loses the ability to bind microtubule-associated proteins and complex with microtubules. Comparison of this mutant to wild-type RASSF1A showed that, despite retaining its proapoptotic properties, the mutant was completely unable to induce cell cycle arrest or suppress the tumorigenic phenotype. Therefore, it appears that the cell cycle/microtubule effects of RASSF1A are key to its tumor suppressor function rather than its apoptotic effects.

Magnetic nanoparticle-supported lipid bilayers for drug delivery.

Magnetic nanoparticle-supported lipid bilayers (SLBs) constructed around core-shell Fe3O4-SiO2 nanoparticles (SNPs) were prepared and evaluated as potential drug carriers. We describe how an oxime ether lipid can be mixed with SNPs to produce lipid-particle assemblies with highly positive ζ potential. To demonstrate the potential of the resultant cationic SLBs, the particles were loaded with either the anticancer drug doxorubicin or an amphiphilic analogue, prepared to facilitate integration into the supported lipid bilayer, and then examined in studies against MCF-7 breast cancer cells. The assemblies were rapidly internalized and exhibited higher toxicity than treatments with doxorubicin alone. The magnetic SLBs were also shown to increase the efficacy of unmodified doxorubicin.

K-RAS Is Complicated.

There is little argument that the K-RAS onco-protein is the most important single oncoprotein in human cancer [...].

BRCA1 and NORE1A Form a Her2/Ras Regulated Tumor Suppressor Complex Modulating Senescence.

BRCA1 is a tumor suppressor with a complex mode of action. Hereditary mutations in BRCA1 predispose carriers to breast cancer, and spontaneous breast cancers often exhibit defects in BRCA1 expression. However, haploinsufficiency or suppression of BRCA1 expression leads to defects in DNA repair, which can induce DNA damage responses, leading to senescence. Activating mutation or overexpression of the Her2 oncoprotein are also frequent drivers of breast cancer. Yet, over-activation of Her2, working through the RAS oncoprotein, can also induce senescence. It is thought that additional defects in the p53 and Rb tumor suppressor machinery must occur in such tumors to allow an escape from senescence, thus permitting tumor development. Although BRCA1 mutant breast cancers are usually Her2 negative, a significant percentage of Her2 positive tumors also lose their expression of BRCA1. Such Her2+/BRCA1- tumors might be expected to have a particularly high senescence barrier to overcome. An important RAS senescence effector is the protein NORE1A, which can modulate both p53 and Rb. It is an essential senescence effector of the RAS oncoprotein, and it is often downregulated in breast tumors by promotor methylation. Here we show that NORE1A forms a Her2/RAS regulated, endogenous complex with BRCA1 at sites of replication fork arrest. Suppression of NORE1A blocks senescence induction caused by BRCA1 inactivation and Her2 activation. Thus, NORE1A forms a tumor suppressor complex with BRCA1. Its frequent epigenetic inactivation may facilitate the transformation of Her2+/BRCA1- mediated breast cancer by suppressing senescence.

Salvador protein is a tumor suppressor effector of RASSF1A with hippo pathway-independent functions.

The RASSF1A tumor suppressor binds and activates proapoptotic MST kinases. The Salvador adaptor protein couples MST kinases to the LATS kinases to form the hippo pathway. Upon activation by RASSF1A, LATS1 phosphorylates the transcriptional regulator YAP, which binds to p73 and activates its proapoptotic effects. However, although serving as an adaptor for MST and LATS, Salvador can also bind RASSF1A. The functional role of the RASSF1A/Salvador interaction is unclear. Although Salvador is a novel tumor suppressor in Drosophila and mice, its role in human systems remains largely unknown. Here we show that Salvador promotes apoptosis in human cells and that Salvador inactivation deregulates the cell cycle and enhances the transformed phenotype. Moreover, we show that although the salvador gene is seldom mutated or epigenetically inactivated in human cancers, it is frequently down-regulated posttranscriptionally. Surprisingly, we also find that although RASSF1A requires the presence of Salvador for full apoptotic activity and to activate p73, this effect does not require a direct interaction of RASSF1A with MST kinases or the activation of the hippo pathway. Thus, we confirm a role for Salvador as a human tumor suppressor and RASSF1A effector and show that Salvador allows RASSF1A to modulate p73 independently of the hippo pathway.

The t(1;3) breakpoint-spanning genes LSAMP and NORE1 are involved in clear cell renal cell carcinomas.

By positional cloning, we identified two breakpoint-spanning genes in a familial clear cell renal cell carcinoma (CCRCC)-associated t(1;3)(q32.1;q13.3): LSAMP and NORE1 (RASSF1 homolog). Both genes are downregulated in 9 of 9 RCC cell lines. While the NORE1A promoter predominantly presents partial methylation in 6 of the cell lines and 17/53 (32%) primary tumors, the LSAMP promoter is completely methylated in 5 of 9 cell lines and in 14/53 (26%) sporadic and 4 familial CCRCCs. Expression of LSAMP and NORE1A proteins in CCRCC cell lines inhibited cell proliferation. These characteristics indicate that LSAMP and NORE1A may represent new candidate tumor suppressors for CCRCC.

Detection of Endogenous RASSF1A Interacting Proteins.

RASSF1A is a Ras effector that promotes the anti-proliferative properties of Ras. It acts as a scaffold protein that regulates several pro-apoptotic signaling pathways, thereby linking Ras to their regulation. However, accumulating evidence suggests that RASSF1A functions as a regulator of other additional biological processes, such as DNA repair and transcription, thereby implicating Ras in the modulation of these biological processes. The mechanisms by which RASSF1A modulates these processes is not fully understood but likely involves interacting with other effectors associated with these functions and coordinating their activity. Thus, to fully understand how RASSF1A manifests its activity, it is critical to identify RASSF1A interacting partners.Unfortunately, the reagents available for the detection of RASSF1A are of poor quality and also exhibit low sensitivity. Here we describe an immunoprecipitation protocol, taking into consideration the limitations of currently available reagents, that can reliably detect the endogenous interaction between RASSF1A and its binding partners.

The RASSF1A Tumor Suppressor Binds the RasGAP DAB2IP and Modulates RAS Activation in Lung Cancer.

Lung cancer is the leading cause of cancer-related death worldwide. Lung cancer is commonly driven by mutations in the RAS oncogenes, the most frequently activated oncogene family in human disease. RAS-induced tumorigenesis is inhibited by the tumor suppressor RASSF1A, which induces apoptosis in response to hyperactivation of RAS. RASSF1A expression is suppressed in cancer at high rates, primarily owing to promoter hypermethylation. Recent reports have shown that loss of RASSF1A expression uncouples RAS from apoptotic signaling in vivo, thereby enhancing tumor aggressiveness. Moreover, a concomitant upregulation of RAS mitogenic signaling upon RASSF1A loss has been observed, suggesting RASSF1A may directly regulate RAS activation. Here, we present the first mechanistic evidence for control of RAS activation by RASSF1A. We present a novel interaction between RASSF1A and the Ras GTPase Activating Protein (RasGAP) DAB2IP, an important negative regulator of RAS. Using shRNA-mediated knockdown and stable overexpression approaches, we demonstrate that RASSF1A upregulates DAB2IP protein levels in NSCLC cells. Suppression of RASSF1A and subsequent downregulation of DAB2IP enhances GTP loading onto RAS, thus increasing RAS mitogenic signaling in both mutant- and wildtype-RAS cells. Moreover, co-suppression of RASSF1A and DAB2IP significantly enhances in vitro and in vivo growth of wildtype-RAS cells. Tumors expressing wildtype RAS, therefore, may still suffer from hyperactive RAS signaling when RASSF1A is downregulated. This may render them susceptible to the targeted RAS inhibitors currently in development.

Assessment of NORE1A as a putative tumor suppressor in human neuroblastoma.

The putative tumor suppressor NORE1A (RASSF5) is a member of the Ras association domain family and is commonly inactivated in human cancer. The closely related gene family member and functional collaborator RASSF1A is a bona fide tumor suppressor and is frequently involved in neuroblastoma. In the present study, we sought to investigate the role of NORE1A in human neuroblastoma. A panel of tumors (36 neuroblastomas and 4 ganglioneuromas) and neuroblastoma cell lines was assessed for NORE1A gene expression by Taqman quantitative RT-PCR. Promoter methylation was quantitatively determined by methylation sensitive pyrosequencing. The antitumourigenic role was functionally investigated in Nore1a transfected SK-N-BE (2) cells by fluorescent inhibition of caspase activity and BrdU incorporation assays. Neuroblastoma cells showed very low or absent NORE1A mRNA expression, which could not be reversed by trichostatin A or 5-aza-cytidine treatments. Neuroblastoma tumors showed suppressed NORE1A gene expression that was particularly pronounced in cases without MYCN amplification or 1p loss. Methylation of the NORE1A promoter was not observed in primary tumors and only one out of seven neuroblastoma cell lines displayed weak partial methylation. Transient expression of Nore1a resulted in enhanced apoptosis and delayed cell cycle progression. In conclusion NORE1A appears to be strongly suppressed in neuroblastic tumors and reconstitution of its expression diminishes the tumorigenic phenotype. Promotor methylation is not a common mechanism responsible for NORE1A transcriptional suppression in this tumor type.

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.

RASSF1A Deficiency Enhances RAS-Driven Lung Tumorigenesis.

Mutant K-RAS has been shown to have both tumor-promoting and -suppressing functions, and growing evidence suggests that the RASSF family of tumor suppressors can act as RAS apoptosis and senescence effectors. It has been hypothesized that inactivation of the RASSF1A tumor suppressor facilitates K-RAS-mediated transformation by uncoupling it from apoptotic pathways such as the Hippo pathway. In human lung tumors, combined activation of K-RAS and inactivation of RASSF1A is closely associated with the development of the most aggressive and worst prognosis tumors. Here, we describe the first transgenic mouse model for activation of K-RAS in the lung in a RASSF1A-defective background. RASSF1A deficiency profoundly enhanced the development of K-RAS-driven lung tumors in vivo Analysis of these tumors showed loss of RASSF1A-uncoupled RAS from the proapoptotic Hippo pathway as expected. We also observed an upregulation of AKT and RALGEF signaling in the RASSF1A- tumors. Heterozygosity of RASSF1A alone mimicked many of the effects of RAS activation on mitogenic signaling in lung tissue, yet no tumors developed, indicating that nonstandard Ras signaling pathways may be playing a key role in tumor formation in vivo In addition, we observed a marked increase in inflammation and IL6 production in RASSF1A-deficient tumors. Thus, RASSF1A loss profoundly affects RAS-driven lung tumorigenesis and mitogenic signaling in vivo Deregulation of inflammatory pathways due to loss of RASSF1A may be essential for RAS-mediated tumorigenesis. These results may have considerable ramifications for future targeted therapy against RAS+/RASSF1A- tumors.Significance: A transgenic mouse model shows that suppression of RASSF1A dramatically enhances Ras-driven tumorigenesis and alters Ras signaling pathway activity.Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/78/10/2614/F1.large.jpg Cancer Res; 78(10); 2614-23. ©2018 AACR.

The Ras effectors NORE1A and RASSF1A are frequently inactivated in pheochromocytoma and abdominal paraganglioma.

NORE1A (RASSF5) and RASSF1A are newly described Ras effectors with tumour suppressor functions. Both molecules are frequently inactivated in various cancers. In this study, we aimed to explore the potential involvement of NORE1A and RASSF1A in pheochromocytoma and abdominal paraganglioma tumorigenesis. A panel of 54 primary tumours was analysed for NORE1A and RASSF1A mRNA expression by TaqMan quantitative RT-PCR. Furthermore, NORE1A and RASSF1A promoter methylation was assessed by combined bisulphite restriction endonuclease assay and methylation-sensitive Pyrosequencing respectively. The anti-tumorigenic role of NORE1A was functionally investigated in Nore1A-transfected PC12 rat pheochromocytoma cells by fluorescent inhibition of caspase activity and soft agar assays. Significantly suppressed NORE1A and RASSF1A mRNA levels were detected in primary tumours compared with normal adrenal medulla (P<0.001). Methylation of the NORE1A promoter was not observed in primary tumours. On the other hand, 9% (5/54) of the primary tumours examined showed RASSF1A promoter methylation greater than 20% as detected by Pyrosequencing. Methylation of the RASSF1A promoter was significantly associated with malignant behaviour (P<0.05). Transient expression of Nore1a resulted in enhanced apoptosis and impaired colony formation in soft agar. Our study provides evidence that NORE1A and RASSF1A are frequently suppressed in pheochromocytoma and abdominal paraganglioma. Silencing of NORE1A contributes to the transformed phenotype in these tumours.

Transcriptional regulation of cyclin A2 by RASSF1A through the enhanced binding of p120E4F to the cyclin A2 promoter.

Recent advances in the study of RASSF1A, the candidate tumor suppressor gene, indicate a possible role of RASSF1A in cell cycle regulation; however, very little is known regarding molecular mechanisms underlying this control. Using small interfering RNA to knockdown endogenous RASSF1A in the breast tumor cell line HB2 and in the cervical cancer cell line HeLa, we identify that a key player in cell cycle progression, cyclin A2, is concomitantly increased at both protein and mRNA levels. In A549 clones stably expressing RASSF1A, cyclin A2 levels were diminished compared with vector control. A known transcriptional regulator of cyclin A2, p120(E4F) (a repressor of cyclin A2), has been shown previously by our group to interact with RASSF1A. We show that levels of p120(E4F) are not affected by RASSF1A small interfering RNA in HB2 and HeLa cells. However, electrophoretic mobility shift assays indicate that knockdown of endogenous RASSF1A in HB2 and HeLa cells leads to a reduction in the binding capacity of p120(E4F) to the cyclin A2 promoter, whereas in the A549 clone stably expressing RASSF1A the binding capacity is increased. These data are further corroborated in vitro by the luciferase assay and in vivo by chromatin immunoprecipitation experiments. Together, these data identify the cyclin A2 gene as a cellular target for RASSF1A through p120(E4F) and for the first time suggest a transcriptional mechanism for RASSF1A-dependent cell cycle regulation.

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.