NORE1A is a Ras senescence effector that controls the apoptotic/senescent balance of p53 via HIPK2.

The Ras oncoprotein is a key driver of cancer. However, Ras also provokes senescence, which serves as a major barrier to Ras-driven transformation. Ras senescence pathways remain poorly characterized. NORE1A is a novel Ras effector that serves as a tumor suppressor. It is frequently inactivated in tumors. We show that NORE1A is a powerful Ras senescence effector and that down-regulation of NORE1A suppresses senescence induction by Ras and enhances Ras transformation. We show that Ras induces the formation of a complex between NORE1A and the kinase HIPK2, enhancing HIPK2 association with p53. HIPK2 is a tumor suppressor that can induce either proapoptotic or prosenescent posttranslational modifications of p53. NORE1A acts to suppress its proapoptotic phosphorylation of p53 but enhance its prosenescent acetylation of p53. Thus, we identify a major new Ras signaling pathway that links Ras to the control of specific protein acetylation and show how NORE1A allows Ras to qualitatively modify p53 function to promote senescence.

The RASSF1A tumor suppressor regulates XPA-mediated DNA repair.

RASSF1A may be the most frequently inactivated tumor suppressor identified in human cancer so far. It is a proapoptotic Ras effector and plays an important role in the apoptotic DNA damage response (DDR). We now show that in addition to DDR regulation, RASSF1A also plays a key role in the DNA repair process itself. We show that RASSF1A forms a DNA damage-regulated complex with the key DNA repair protein xeroderma pigmentosum A (XPA). XPA requires RASSF1A to exert full repair activity, and RASSF1A-deficient cells exhibit an impaired ability to repair DNA. Moreover, a cancer-associated RASSF1A single-nucleotide polymorphism (SNP) variant exhibits differential XPA binding and inhibits DNA repair. The interaction of XPA with other components of the repair complex, such as replication protein A (RPA), is controlled in part by a dynamic acetylation/deacetylation cycle. We found that RASSF1A and its SNP variant differentially regulate XPA protein acetylation, and the SNP variant hyperstabilizes the XPA-RPA70 complex. Thus, we identify two novel functions for RASSF1A in the control of DNA repair and protein acetylation. As RASSF1A modulates both apoptotic DDR and DNA repair, it may play an important and unanticipated role in coordinating the balance between repair and death after DNA damage.

NORE1A tumor suppressor candidate modulates p21CIP1 via p53.

NORE1A (RASSF5) is a proapoptotic Ras effector that is frequently inactivated by promoter methylation in human tumors. It is structurally related to the RASSF1A tumor suppressor and is itself implicated as a tumor suppressor. In the presence of activated Ras, NORE1A is a potent inducer of apoptosis. However, when expressed at lower levels in the absence of activated Ras, NORE1A seems to promote cell cycle arrest rather than apoptosis. The mechanisms underlying NORE1A action are poorly understood. We have used microarray analysis of an inducible NORE1A system to screen for physiologic signaling targets of NORE1A action. Using this approach, we have identified several potential signaling pathways modulated by NORE1A. In particular, we identify the cyclin-dependent kinase inhibitor p21(CIP1) as a target for NORE1A activation and show that it is a vital component of NORE1A-mediated growth inhibition. In primary human hepatocellular carcinomas (HCC), loss of NORE1A expression is frequent and correlates tightly with loss of p21(CIP1) expression. NORE1A down-regulation in HCC also correlates with poor prognosis, enhanced proliferation, survival, and angiogenic tumor characteristics. Experimental inactivation of NORE1A results in the loss of p21(CIP1) expression and promotes proliferation. The best characterized activator of p21(CIP1) is the p53 master tumor suppressor. Further experiments showed that NORE1A activates p21(CIP1) via promoting p53 nuclear localization. Thus, we define the molecular basis of NORE1A-mediated growth inhibition and implicate NORE1A as a potential component of the ill-defined connection between Ras and p53.

The RASSF1A tumor suppressor.

RASSF1A (Ras association domain family 1 isoform A) is a recently discovered tumor suppressor whose inactivation is implicated in the development of many human cancers. Although it can be inactivated by gene deletion or point mutations, the most common contributor to loss or reduction of RASSF1A function is transcriptional silencing of the gene by inappropriate promoter methylation. This epigenetic mechanism can inactivate numerous tumor suppressors and is now recognized as a major contributor to the development of cancer. RASSF1A lacks apparent enzymatic activity but contains a Ras association (RA) domain and is potentially an effector of the Ras oncoprotein. RASSF1A modulates multiple apoptotic and cell cycle checkpoint pathways. Current evidence supports the hypothesis that it serves as a scaffold for the assembly of multiple tumor suppressor complexes and may relay pro-apoptotic signaling by K-Ras.

RASSF family proteins and Ras transformation.

There are six members of the RASSF gene family, with RASSF1 being the best characterized. All six genes produce proteins that contain Ras Association (RA) domains that can interact directly with activated Ras in overexpression studies. Their role in mediating the biological effects of Ras remains under investigation. However, they seem to modulate some of the growth inhibitory responses mediated by Ras. Moreover, evidence is accumulating that RASSF family members may serve as tumor suppressors that succumb to inactivation during the evolution of the transformed phenotype. Thus, RASSF proteins may be described as effector/tumor suppressors, in contrast to traditional Ras effectors such as Raf and PI-3 kinase, which may be considered to be effector/oncoproteins.

The RASSF1A tumor suppressor activates Bax via MOAP-1.

The novel tumor suppressor RASSF1A is frequently inactivated during human tumorigenesis by promoter methylation. RASSF1A may serve as a node in the integration of signaling pathways controlling a range of critical cellular functions including cell cycle, genomic instability, and apoptosis. The mechanism of action of RASSF1A remains under investigation. We now identify a novel pathway connecting RASSF1A to Bax via the Bax binding protein MOAP-1. RASSF1A and MOAP-1 interact directly, and this interaction is enhanced by the presence of activated K-Ras. RASSF1A can activate Bax via MOAP-1. Moreover, activated K-Ras, RASSF1A, and MOAP-1 synergize to induce Bax activation and cell death. Analysis of a tumor-derived point mutant of RASSF1A showed that the mutant was defective for the MOAP-1 interaction and for Bax activation. Moreover, inhibition of RASSF1A by shRNA impaired the ability of K-Ras to activate Bax. Thus, we identify a novel pro-apoptotic pathway linking K-Ras, RASSF1A and Bax that is specifically impaired in some human tumors.

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.

RASSF4/AD037 is a potential ras effector/tumor suppressor of the RASSF family.

Activated Ras proteins interact with a broad range of effector proteins to induce a diverse series of biological consequences. Although typically associated with enhanced growth and transformation, activated Ras may also induce growth antagonistic effects such as senescence or apoptosis. It is now apparent that some of the growth-inhibitory properties of Ras are mediated via the RASSF family of Ras effector/tumor suppressors. To date, four members of this family have been identified (Nore1, RASSF1, RASSF2, and RASSF3). We now identify a fifth member of this group, RASSF4 (AD037). RASSF4 shows approximately 25% identity with RASSF1A and 60% identity with RASSF2. RASSF4 binds directly to activated K-Ras in a GTP-dependent manner via the effector domain, thus exhibiting the basic properties of a Ras effector. Overexpression of RASSF4 induces Ras-dependent apoptosis in 293-T cells and inhibits the growth of human tumor cell lines. Although broadly expressed in normal tissue, RASSF4 is frequently down-regulated by promoter methylation in human tumor cells. Thus, RASSF4 appears to be a new member of the RASSF family of potential Ras effector/tumor suppressors.

RASSF1A interacts with microtubule-associated proteins and modulates microtubule dynamics.

The candidate tumor suppressor gene RASSF1A is inactivated in many types of adult and childhood cancers. However, the mechanisms by which RASSF1A exerts its tumor suppressive functions have yet to be elucidated. To this end, we performed a yeast two-hybrid screen to identify novel RASSF1A-interacting proteins in a human brain cDNA library. Seventy percent of interacting clones had homology to microtubule-associated proteins, including MAP1B and VCY2IP1/C19ORF5. RASSF1A association with MAP1B and VCY2IP1/C19ORF5 was subsequently confirmed in mammalian cell lines. This suggested that RASSF1A may exert its tumor-suppressive functions through interaction with the microtubules. We demonstrate that RASSF1A associates with the microtubules, causing them to exist as hyperstabilized circular bundles. We found that two naturally occurring tumor-associated missense substitutions in the RASSF1A coding region, C65R and R257Q, perturb the association of RASSF1A with the microtubules. The C65R and R257Q in addition to VCY2IP1/C19ORF5 showed reduced ability to induce microtubule acetylation and were unable to protect the microtubules against the depolymerizing action of nocodazole. In addition, wild-type RASSF1A but not the C65R or the R257Q is able to block DNA synthesis. Our data identify a role for RASSF1A in the regulation of microtubules and cell cycle dynamics that could be part of the mechanism(s) by which RASSF1A exerts its growth inhibition on cancer cells.

A role for the RASSF1A tumor suppressor in the regulation of tubulin polymerization and genomic stability.

The high frequency with which the novel tumor suppressor RASSF1A is inactivated by promoter methylation suggests that it plays a key role in the development of many primary human tumors. Yet the mechanism of RASSF1A action remains unknown. We now show that RASSF1A associates with microtubules and that this association is essential for RASSF1A to mediate its growth inhibitory effects. Overexpression of RASSF1A promotes the formation of stable microtubules, whereas a dominant-negative fragment of RASSF1A destabilizes microtubule networks. The RASSF1 protein is expressed as two main isoforms, 1A and 1C. The smaller 1C isoform also associates with microtubules but is less effective at stabilizing them. Because RASSF1A and RASSF1C localize to the mitotic spindle, we examined their effects upon genomic instability. RASSF1A and RASSF1C block activated Ras-induced genomic instability. However, a point mutant of RASSF1C, identified in human tumors, was severely defective for stabilizing tubulin and was unable to block the genomic destabilizing effects of Ras. Thus, we identify a role for RASSF1A/C in the control of microtubule polymerization and potentially in the maintenance of genomic stability.

Novel functional interaction between the plasma membrane Ca2+ pump 4b and the proapoptotic tumor suppressor Ras-associated factor 1 (RASSF1).

Plasma membrane calmodulin-dependent calcium ATPases (PMCAs) are enzymatic systems implicated in the extrusion of calcium from the cell. We and others have previously identified molecular interactions between the cytoplasmic COOH-terminal end of PMCA and PDZ domain-containing proteins. These interactions suggested a new role for PMCA as a modulator of signal transduction pathways. The existence of other intracellular regions in the PMCA molecule prompted us to investigate the possible participation of other domains in interactions with different partner proteins. A two-hybrid screen of a human fetal heart cDNA library, using the region 652-840 of human PMCA4b (located in the catalytic, second intracellular loop) as bait, revealed a novel interaction between PMCA4b and the tumor suppressor RASSF1, a Ras effector protein involved in H-Ras-mediated apoptosis. Immunofluorescence co-localization, immunoprecipitation, and glutathione S-transferase pull-down experiments performed in mammalian cells provided further confirmation of the physical interaction between the two proteins. The interaction domain has been narrowed down to region 74-123 of RASSF1C (144-193 in RASSF1A) and 652-748 of human PMCA4b. The functionality of this interaction was demonstrated by the inhibition of the epidermal growth factor-dependent activation of the Erk pathway when PMCA4b and RASSF1 were co-expressed. This inhibition was abolished by blocking PMCA/RASSSF1 association with an excess of a green fluorescent protein fusion protein containing the region 50-123 of RASSF1C. This work describes a novel protein-protein interaction involving a domain of PMCA other than the COOH terminus. It suggests a function for PMCA4b as an organizer of macromolecular protein complexes, where PMCA4b could recruit diverse proteins through interaction with different domains. Furthermore, the functional association with RASSF1 indicates a role for PMCA4b in the modulation of Ras-mediated signaling.

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.

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.