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The RASSF1A tumor suppressor

The RASSF1A tumor suppressor Commentary 3163 1 2 1, Howard Donninger , Michele D. Vos and Geoffrey J. Clark * Molecular Targets Group, Department of Medicine, J. G. Brown Cancer Center, University of Louisville, 119C Baxter Boulevard, 580 S. Preston Street, Louisville, KY 40202, USA Research Analysis and Evaluation Branch, NCI, Rockville, MD, USA *Author for correspondence (e-mail: gjclar01@louisville.edu) Accepted 23 July 2007 Journal of Cell Science 120, 3163-3172 Published by The Company of Biologists 2007 doi:10.1242/jcs.010389 Summary RASSF1A (Ras association domain family 1 isoform A) is RASSF1A lacks apparent enzymatic activity but a recently discovered tumor suppressor whose inactivation contains a Ras association (RA) domain and is potentially is implicated in the development of many human cancers. an effector of the Ras oncoprotein. RASSF1A modulates Although it can be inactivated by gene deletion or point multiple apoptotic and cell cycle checkpoint pathways. mutations, the most common contributor to loss or Current evidence supports the hypothesis that it serves as reduction of RASSF1A function is transcriptional silencing a scaffold for the assembly of multiple tumor suppressor of the gene by inappropriate promoter methylation. This complexes and may relay pro-apoptotic signaling by K- epigenetic mechanism can inactivate numerous tumor Ras. suppressors and is now recognized as a major contributor to the development of cancer. Key words: Epigenetic, RASSF1A, Ras, Tumor suppressor Introduction RASSF1A is a tumor suppressor For many years it was suspected that one or more tumor Tumor suppressor genes are classically defined by Knudson’s suppressors lurk in the 3p21.3 region of the human genome, ‘two-hit’ hypothesis (Knudson, 1971), which states that because this area frequently suffers loss of heterozygosity inactivation of both alleles of a tumor suppressor gene is (LOH) in lung cancer (Lerman and Minna, 2000). In 2000, required for tumorigenesis. Loss of a RASSF1A allele is a Damman et al. serendipitously cloned a gene located in this frequent phenomenon in primary human cancer (Burbee et al., region that they termed RASSF1 (Ras association domain 2001; Pfeifer and Dammann, 2005). In a study of sporadic lung family 1), because the protein contains a putative Ras cancers, 76% of the tumors showing allelic imbalance at association (RA) domain. One of the isoforms produced by the 3p21.3 (the RASSF1A locus) also showed RASSF1A promoter gene, RASSF1A, has properties compatible with a tumor hypermethylation (Agathanggelou et al., 2001). Similar suppressor function. Moreover, the gene appears to suffer findings in non-small-cell lung cancer (NSCLC) (Tomizawa et frequent transcriptional inactivation in tumor cells due to al., 2002), bladder transitional carcinoma (Chan et al., 2003) aberrant promoter methylation (Burbee et al., 2001; Dammann and cervical squamous cell carcinoma (Yu et al., 2003) have et al., 2000). Simultaneously, a bioinformatics-based approach been reported. Thus RASSF1A alleles can be inactivated by a revealed a proapoptotic novel Ras-binding protein that inhibits combination of genetic and epigenetic mechanisms, and tumor cell growth and is encoded by a gene localizing to RASSF1A conforms to the Knudson two-hit model. 3p21.3. This protein turned out to be RASSF1C, a smaller Hypermethylation of both alleles of the RASSF1A promoter has isoform produced by the RASSF1 gene (Vos et al., 2000). Thus, been shown to cause loss of expression of the gene (Lusher et RASSF1 appeared to be one of the elusive tumor suppressors al., 2002). Moreover, although early studies reported located at 3p21.3. infrequent mutation of RASSF1A, other studies have Subsequent work showed that specific point mutations suggested that up to 15% of tumors may contain inactivating compromise the ability of RASSF1A to inhibit tumor cell point mutations (Pan et al., 2005). Thus, the evidence that growth (Dreijerink et al., 2001; Kuzmin et al., 2002; RASSF1A is inactivated in a high percentage of human tumors Shivakumar et al., 2002). A small deluge of papers began, is strong (Table 1). demonstrating frequent epigenetic inactivation of RASSF1A in If the inactivation of RASSF1A contributes to the a wide variety of tumors. Bearing in mind that RASSF1A can development of the transformed phenotype, then one might also suffer point mutations in up to 15% of primary tumors expect that re-introduction of RASSF1A into RASSF1A- (Pan et al., 2005), RASSF1A is one of the most frequently negative cells would impair tumorigenicity. Indeed, this is the inactivated proteins ever identified in human cancer. case. Re-expression of the gene in RASSF1A-negative cancer RASSF1A lacks any obvious enzymatic activity but may cells results in reduced colony formation in soft agar and serve as a scaffold for signaling complexes, key components of reduced tumorigenicity in nude mice (Burbee et al., 2001; which have recently been identified (Fig. 1). Here, we discuss Dammann et al., 2000; Dreijerink et al., 2001; Kuzmin et al., work that has implicated RASSF1A in the regulation of the cell 2002). Two groups have independently knocked out the cycle, apoptosis and genetic instability (Agathanggelou et al., RASSF1A gene in mice (Tommasi et al., 2005; van der 2005), and the molecular mechanisms involved. Weyden et al., 2005). In each case the animals exhibit an Journal of Cell Science 3164 Journal of Cell Science 120 (18) Mitogenic stimuli Death receptor Fig. 1. Summary of some of the known partners and pathways of RASSF1A. RASSF1A binds to at least Ras three microtubule-binding proteins (MAPs), complexes PMCa with microtubules and RASSF1A regulates mitosis, the cell JNK cycle and apoptosis in E4F p120 MOAP-1 response to mitogenic or MST1 SAV apoptotic stimuli. Direct Cdc20 MAP1B Cyclin A2 C19ORF5 LATs1/2 interaction between Cyclin D1 CNK1 BAX RASSF1A and microtubule- APC associated proteins localizes RASSF1A to the G1 S microtubules, stabilizing them and, thereby, regulating Cell cycle mitosis. Repression of cyclins Microtubule dynamics A and D1 by RASSF1A M G2 results in cell cycle arrest and interactions with CNK1, MST1, Salvador and MOAP1 may allow RASSF1A to Cell cycle arrest Mitotic arrest Migration Apoptosis modulate apoptosis. enhanced tendency to develop spontaneous tumors. Thus, 2005; Endoh et al., 2005; Hesson et al., 2005; Lambros et al., RASSF1A is a tumor suppressor that is frequently impaired in 2005; Vos et al., 2003b). Inactivation of RASSF2 correlates human tumors. with activation of Ras in tumor cells (Hesson et al., 2005). Reintroduction of RASSF2 into tumor cells impairs RASSF1A is part of a family of potential tumor tumorigenesis and knocking down RASSF2 enhances suppressors tumorigenesis (Akino et al., 2005). Thus, RASSF2 also RASSF1A is a member of a family of six related proteins, each appears to be an epigenetically inactivated tumor suppressor. of which exhibits multiple splice variants. With the exception The effector pathways controlled by it remain unknown. of some minor splice variants, each protein contains an RA RASSF3 can also bind to Ras and inhibit cell growth domain and a C-terminal SARAH protein-protein interaction (unpublished observations). However, it is the only family motif. Each family member, with the exception of RASSF3, member whose RNA is not downregulated in tumors (Tommasi has now been implicated as a human tumor suppressor. et al., 2002). Whether it is involved in tumor suppression thus RASSF5 (Nore1a) is the best characterized member of the remains unknown. family after RASSF1A. RASSF5 was the first member of the RASSF4 is frequently downregulated by promoter family cloned and it was originally designated Nore1a for methylation in human tumor cells, binds to Ras and induces novel Ras effector 1 (Vavvas et al., 1998). RASSF5 binds apoptosis. It localizes mostly to the cytosol but can be recruited activated Ras directly and is present in an endogenous complex to the plasma membrane by activated Ras (Eckfeld et al., with Ras in cells. RASSF5 is pro-apoptotic and kills cells in a 2004). Ras-dependent manner (Khokhlatchev et al., 2002; Vos et al., RASSF6 exhibits similar biological properties to its brethren 2003a). It is frequently inactivated in human tumors by and is often downregulated in primary human tumors (Allen et promoter methylation (Table 2). Moreover, it is linked to the al., 2007). Intriguingly, the RASSF6 locus is implicated in development of a rare familial form of cancer (Chen et al., susceptibility to bronchiolitis induced by respiratory syncytial 2003), which confirms its role as a tumor suppressor in vivo. virus (Hull et al., 2004). RASSF6 might therefore have a role Its mechanisms of action remain largely unknown, although it in inflammation; indeed it can suppress the NFB pathway may regulate the pro-apoptotic kinase MST1 (Praskova et al., (Allen et al., 2007). 2004). A smaller splice version of RASSF5 has been identified Two further RA-domain-containing proteins have been and designated Nore1b or RAPL (Katagiri et al., 2003; identified and are now being described as RASSF8 (Falvella Tommasi et al., 2002). This protein demonstrates more et al., 2006) and RASSF7. These were previously known as restricted expression than RASSF5 and can form an HOJ-1 and HRC1. Although these proteins can bind to Ras and endogenous complex with the Ras-related protein Rap. It inhibit cell growth (our unpublished observations), they do not regulates lymphocyte adhesion and has also been implicated as display great homology with RASSF1-RASSF6 and do not a tumor suppressor (Katagiri et al., 2003; Macheiner et al., contain SARAH motifs. This raises the issue of how to define 2006). a RASSF protein. RASSF proteins can heterodimerize with RASSF2 is a pro-apoptotic Ras effector that is frequently each other (Ortiz-Vega et al., 2002) and this might serve as a downregulated in human tumors by promoter methylation, functional definition. Although we have found that RASSF2- histone deacetylation and sometimes deletion (Akino et al., RASSF6 readily heterodimerize with RASSF1A, the RASSF7 Journal of Cell Science RASSF1A 3165 Table 1. Primary tumors containing RASSF1A promoter methylation Tumor type Frequency* References Lung: SCLC 88% Grote et al., 2006 Lung: NSCLC 39% 28% 15% Chen et al., 2006; Grote et al., 2006; Safar et al., 2005 Breast 95% 81% Yeo et al., 2005; Shinozaki et al., 2005 Colorectal 20% 52% Miranda et al., 2006; Oliveira et al., 2005 Prostate 99% Jeronimo et al., 2004 Cervical Adenocarcinoma 45% Cohen et al., 2003 Esophageal 34% Wong et al., 2006 Gastric 44% Oliveira et al., 2005 Renal 56-91% Yoon et al., 2001; Dreijerink et al., 2001 Hepatocellular 75% Katoh et al., 2006 Bladder 30-50% Marsit et al., 2006 Pancreatic 63% Liu et al., 2005a Ovarian 26% 30% Teodoridis et al., 2005; Makarla et al., 2005 Nasopharyngeal 68% Tan et al., 2006 Leukemia 0% 15% Johan et al., 2005; Harada et al., 2002 Neuroblastoma 83% Lazcoz et al., 2006 Thyroid 71% 35% Schagdarsurengin et al., 2006; Nakamura et al., 2005 Cholangiocarcinoma 67% Tischoff et al., 2005 Ependymoma 36% 86% Michalowski et al., 2006; Hamilton et al., 2005 Glioma 57% 54% Hesson et al., 2004 Horiguchi et al., 2003 Hodgkin Lymphoma 65% Murray et al., 2004 Medulloblastoma 79% Lusher et al., 2002 Retinoblastoma 59% Harada et al., 2002 Testicular Seminoma 40% Honorio et al., 2003 Testicular Nonseminoma 83% Honorio et al., 2003 Wilms tumor 54% Wagner et al., 2002 Rhabdomyosarcoma 61% Harada et al., 2002 Pheochromocytomas 22% Astuti et al., 2001 Head and neck 15% 17% Dong et al., 2003; Hogg et al., 2002 Melanoma 41% Spugnardi et al., 2003 *Frequency of RASSF1A promoter hypermethylation in tumor type. and RASSF8 proteins do not (our unpublished observation). contain an RA domain located towards the C-terminus of the Thus, RASSF7 and RASSF8 may be a separate sub-family protein and a SARAH (Sav-RASSF-Hpo) protein-protein distinct from the ‘true’ RASSF proteins. interaction motif at the very C-terminus. A putative ATM phosphorylation site for the DNA repair checkpoint kinase RASSF1 produces multiple isoforms ATM is found in isoforms A, C, D, E and H (Fig. 2B). The RASSF1 locus at 3p21.3 spans approximately 11,000 bp. Only isoforms A and C have been subjected to extensive It contains eight exons, and alternative splicing and usage of biological analysis. Little information is available regarding the two different promoters (Fig. 2A) give rise to eight different functions of splice variants B, D, E, F, G and H. RASSF1C transcripts, RASSF1A-RASSF1H. Epigenetic inactivation of appears to share many of the biological characteristics of genes often involves the methylation of CpG islands in their RASSF1A and has been implicated as a tumor suppressor in promoters (Hesson et al., 2007). There are two CpG islands both in vitro and in vivo studies (Li et al., 2004; Vos et al., associated with the RASSF1 promoters. A smaller, 737 bp 2000). However, it has unique functions not shared by island contains 85 CpGs and spans the promoter for RASSF1A, RASSF1A, such as coupling DNA damage to the activation of RASSF1D, RASSF1E, RASSF1F and RASSF1G. A larger 1365 the SAPK-JNK signaling pathway (Kitagawa et al., 2006). bp island, containing 139 CpGs, spans the promoter region for RASSF1C and RASSF1A use different promoters, and Latif RASSF1B and RASSF1C (Agathanggelou et al., 2005). and co-workers report that RASSF1C is not subject to RASSF1A is a 340-residue protein that migrates at 39 kDa. epigenetic inactivation (Agathanggelou et al., 2005). However, It contains a cysteine-rich domain (CRD) reminiscent of the we have observed differential loss of RASSF1C protein in diacylglycerol-binding–CRD domain of Raf-1 towards the N- some tumor lines (our unpublished observations). Perhaps the terminus (residues 50-101), which is not present in the other regulation of RASSF1C involves more significant post- ubiquitously expressed isoform RASSF1C. Isoforms A-E also transcriptional mechanisms than regulation of RASSF1A. Table 2. Primary tumors containing NORE1A promoter methylation Tumor type Frequency* References Lung, SCLC 0% Hesson et al., 2003 Lung, NSCLC 24% 28% Hesson et al., 2003; Irimia et al., 2004 Hepatocellular Carcinoma 37.5% Calvisi et al., 2006 Clear cell renal Carcinoma 32% Chen et al., 2003 Neuroblastoma 3% Lazcoz et al., 2006 Wilms tumor 15% Morris et al., 2003 *Frequency of NORE1A promoter hypermethylation in tumor type. Journal of Cell Science 3166 Journal of Cell Science 120 (18) 5 3 1α 1β 1γ 2 36 4 5 51-101 125-138 194-289 291-337 RASSF1A C1/DAG ATM RA SARAH 340aa 43-138 140-186 RASSF1B RA SARAH 189aa 55-68 121-219 221-267 RASSF1C ATM RA SARAH 270aa 51-105 129-142 198-292 294-341 RASSF1D C1/DAG ATM RA SARAH 344aa 51-101 129-142 198-293 295-341 RASSF1E C1/DAG ATM RA SARAH 344aa 51-85 RASSF1F C1/DAG 92aa 51-103 RASSF1G C1/DAG 152aa 55-68 RASSF1H ATM 75aa Fig. 2. RASSF1 gene locus and domain structure of the different RASSF1 isoforms. (A) The RASSF1 gene locus is characterized by eight exons (boxed regions) and two different promoters (arrows) with two associated CpG islands (black bars). Black boxes represent coding regions and white boxes are non-coding regions. (B) Schematic representation of the different RASSF1 isoforms. C1/DAG, conserved region 1 diacylglycerol-binding domain; ATM, ATM-kinase consensus phosphorylation sequence; RA, RalGDS/AF6 Ras association domain; SARAH, Sav/RASSF/Hpo interaction domain. The position of each domain (as outlined in the Swiss-Prot/TrEMBL database) is indicated above each isoform and the number of amino acids in each isoform is shown on the right. RASSF1A as a Ras effector between Ras and RASSF1A. They suggest that the interaction Activated forms of K-Ras, although being transforming is indirect and due to heterodimerization of RASSF1A with oncoproteins, also have growth inhibitory effects, including the RASSF5 (Ortiz-Vega et al., 2002). The use of unfarnesylated induction of apoptosis (Cox and Der, 2003; Downward, 1998). Ras in their studies may have led them to underestimate the K-Ras must thus have pro-apoptotic effector proteins, which binding affinity. Confirmation of RASSF1A as a bona fide Ras are likely to be downregulated during the development of Ras- effector awaits the demonstration that the endogenous proteins dependent tumors. RASSF1A is a pro-apoptotic protein that form a complex in vivo. has a potential RA domain, and so it could mediate some of If RASSF1A serves as a pro-apoptotic Ras effector, then one the pro-apoptoptic effects of K-Ras. This hypothesis is might expect Ras activation to correlate with RASSF1A supported by the observation that the related RASSF5 protein inactivation in tumors. Several studies have failed to detect can be detected in an endogenous complex with Ras (Vavvas such a relationship (Dammann et al., 2003; Li et al., 2003; van et al., 1998). Engeland et al., 2002). However, these experiments used the The RA domain of RASSF1A can bind to Ras directly in presence or absence of an activating Ras mutation to identify vitro (Vos et al., 2000), and RASSF1A forms a complex with Ras-dependent tumors. In fact, there is a surprisingly poor activated K-Ras when overexpressed in cells (Rodriguez- correlation between the presence of a mutation in the Ras gene Viciana et al., 2004). Formation of the complex depends on an and the abundance of activated Ras protein in tumor cells intact effector domain for Ras and farnesylation of K-Ras (Fig. (Eckert et al., 2004). Thus, resolving this issue will require 3). We have found that K-Ras binds better than H-Ras, even direct measurements of Ras-GTP levels in the cells. though both share an identical effector domain and both are farnesylated. Other Ras-related proteins also demonstrate the Biological functions of RASSF1A potential to bind RASSF1A, including M-Ras and R-Ras but Numerous studies have shown that overexpression of not Rap (our unpublished observations). M-Ras and R-Ras are RASSF1A promotes apoptosis, cell cycle arrest and reduces post translationally modified by geranylgeranyl, not farnesyl, the tumorigenicity of cancer cell lines (for a review, see and this may contribute to the weaker interaction with Agathangelou et al., 2005). RNAi experiments have implicated RASSF1A. RASSF1A downregulation in loss of cell cycle control, Ortiz-Vega et al., however, have failed to see direct binding enhanced genetic instability, enhanced cell motility and Journal of Cell Science RASSF1A 3167 Microtubules are polymers that continually switch between phases of elongation and shortening; this is known as dynamic instability. Microtubule dynamics can be modulated by a series of microtubule-associated proteins (MAPs) that bind directly to tubulin (Halpain and Dehmelt, 2006). Two-hybrid analysis has identified three such proteins – MAP1b (Dallol et al), C19ORF5 (also known VCY2IP1 or RABP1) (Liu et al., 2002; Song et al., 2005) and MAP4 (G.J.C., unpublished observation) – as direct binding partners of RASSF1A. Thus, RASSF1A could associate with microtubules via MAPs. MAP1b has been shown to promote tubulin polymerization (Togel et al., 1998) and MAP4 has been shown to impede microtubule depolymerization (Nguyen et al., 1998). C19ORF5 has also been shown to enhance microtubule polymerization (Liu et al., Fig. 3. RASSF1A binds Ras. (A) HEK-293-T cells were transfected 2005; Orbán-Németh et al., 2005). Thus, RASSF1A has the with FLAG-tagged RASSF1A and HA-tagged forms of K-Ras12v. potential to scaffold proteins that we might expect would have The cells were lysed and immunoprecipitated (IP) before being immunoblotted (IB) with HA and FLAG. Upper panel shows a synergistic effect on microtubule polymerization. immunoprecipitation, lower panel shows protein levels in the cell We have identified a minimum domain in RASSF1A that is lysate. Wild-type K-Ras, a Y40C effector mutant of K-Ras12v and a required for the microtubule-stabilizing effects. When this farnesylation-defective mutant of K-Ras12v (K-RasCX) were isolated domain is itself overexpressed, it causes a catastrophic defective for binding RASSF1A. collapse of the microtubule network (Vos et al., 2004). The underlying mechanism and whether it involves the direct resistance to K-Ras and tumor necrosis factor  (TNF)- interaction of RASSF1A with tubulin remains under induced apoptosis (Baksh et al., 2005; Dallol et al., 2005; Song investigation, but it appears that RASSF1A has the capacity to et al., 2004; Vos et al., 2004; Vos et al., 2006). Thus, RASSF1A profoundly influence the dynamic balance of microtubules appears to regulate multiple biological processes. The both positively and negatively. mechanisms behind these activities are multifold and remain under investigation but the emerging evidence suggests a role Maintenance of genomic stability for RASSF1A as a scaffolding protein that can assemble and Genomic instability is one of the hallmarks of transformed modulate multiple effector protein complexes. cells (Saavedra et al., 2000) and defects in spindle regulation can lead to genomic instability (Wassmann and Benezra, RASSF1A regulates microtubules 2001). Since RASSF1A localizes to the centrosome and RASSF1A localizes to microtubules and promotes their mitotic spindle, and can modulate tubulin dynamics (Song et stabilization (Liu et al., 2003; Dallol et al., 2004; Song et al., al., 2005; Vos et al., 2004; Dallol et al., 2004), it is not 2004; Vos et al., 2004). During interphase, it is localized to surprising that RASSF1A has been implicated in the cytoplasmic microtubules; during prophase it localizes to maintenance of genomic stability (Song et al., 2005; Vos et al., centrosomes; during metaphase and anaphase, it localizes to 2004). both spindle microtubules and the spindle poles; and it is found C19ORF5 may play a key role in recruiting RASSF1A to at the midzone and midbody during early and late telophase, the centrosome and spindle. Moreover, inhibition of C19ORF5 respectively (Fig. 4). expression by RNAi can promote genetic instability similar to Fig. 4. RASSF1A associates with microtubules and localizes to centrosome and spindles during mitosis. COS cells were transfected with GFP-RASSF1A and the nuclei stained blue with DAPI. Journal of Cell Science 3168 Journal of Cell Science 120 (18) that observed when RASSF1A is downregulated (Song et al., 2004). An elegant explanation for the M-phase arrest mediated 2005; Dallol et al., 2007). Song et al. suggest that the by RASSF1A has been put forward by Song et al., who mechanism of C19ORF5 action is to enhance the ability of suggested that it is brought about by the direct interaction of RASSF1A to stabilize mitotic cyclins. Thus, loss of function RASSF1A with Cdc20 (Song et al., 2004; Song et al., 2005). of C19ORF5 leads to premature destruction of mitotic cyclins Cdc20 is an essential cell cycle regulator required for the and accelerated, aberrant mitosis (Song et al., 2004). However, completion of mitosis (Yu, 2007). Cdc20 binds and activates in similar experiments, Dallol et al. observed delayed rather the ubiquitin ligase activity of a large molecular machine than accelerated mitotic progression and showed a role for designated the anaphase-promoting complex (APC). This C19ORF5 in anchoring  and  tubulin to the centrosomes promotes the ubiquitylation and degradation of cyclins A and (Dallol et al., 2007). This suggests that the abnormalities in B, leading to anaphase and mitotic exit. Song et al. suggest that sister chromatid separation observed when C19ORF5 is the interaction with RASSF1A blocks the ability of Cdc20 to downregulated is due to aberrations in spindle dynamics. This activate the APC and that the resultant stabilization of cyclins is clearly a complicated issue that may require further A and B blocks the mitotic progression that usually follows experimentation to resolve. their degradation (Mathe, 2004; Peters, 2002; Zachariae and Both RASSF1A and RASSF1C contain a potential ATM Nasmyth, 1999). Liu et al., however, have been unable to kinase (mutated in ataxia telangiectasia) phosphorylation site confirm the interaction of RASSF1A with Cdc20 (Liu et al., (Kim et al., 1999). ATM functions as part of the DNA damage 2007). Thus the role of Cdc20 and APC in RASSF1A- checkpoint and has been implicated in regulation of genomic mediated cell cycle control requires further investigation. stability (Levitt and Hickson, 2002; Shiloh, 2003). Point mutations that destroy the RASSF1A or RASSF1C ATM Modulation of apoptosis phosphorylation site have been found in human tumors RASSF family proteins are pro-apoptotic (Vos et al., 2000; (Burbee et al., 2001; Shivakumar et al., 2002). We have been Khokhlatchev et al., 2002; Eckfeld et al., 2004; Vos et al., unable to detect any obvious difference in the microtubule- 2003a; Vos et al., 2003b) and several pathways by which stabilizing activities of wild-type RASSF1A and RASSF1A RASSF1A may modulate apoptosis have now been identified. mutated at the ATM site. However, the equivalent mutant of MST1 and MST2 are pro-apoptotic serine/threonine kinases RASSF1C (S61F) is clearly impaired (Vos et al., 2004). that activate the SAPK-JNK signaling pathway and Indeed, this RASSF1C mutant can induce genomic instability phosphorylate histone H2B (Cheung et al., 2003; Ura et al., at frequencies comparable to those evident in RASSF1A- 2007). They bind directly to RASSF1A and other RASSF knockdown studies (our unpublished observation). Thus, family members via their SARAH motifs (Avruch et al., 2005; RASSF1A and RASSF1C may be mediators through which Hwang et al., 2007; Khokhlatchev et al., 2002; Oh et al., 2006; ATM maintains genomic stability. Moreover, mutant Praskova et al., 2004). Consequently, they are obvious pro- RASSF1C has the potential to serve as an oncogene. apoptotic effectors for RASSF1A. However, the role of RASSF1A in the regulation of MST1 appears complex. In RASSF1A modulates the cell cycle mammalian cells, contradictory effects of RASSF1A on MST1 Initial studies examining the role of RASSF1A in the cell cycle kinase activity have been reported. Praskova et al. found that demonstrated a role for RASSF1A at the G1-S checkpoint and MST1 kinase activity is inhibited by RASSF1A whereas Oh et showed that RASSF1A modulates the levels of cyclin D1 al. and Guo et al. have found that it is activated (Oh et al., 2006; (Shivakumar et al., 2002). Subsequent work confirmed this and Praskova et al., 2004; Guo et al., 2007). Our own studies implicated inhibition of the JNK pathway as a mechanism support the results of Oh and Guo. Thus, the effects of (Whang et al., 2005). RASSF1A could connect to JNK by RASSF1A on MST1 may be context dependent. direct interactions with the kinase MST1 (Khokhlatchev et al., RASSF1A also forms a complex with the pro-apoptotic 2002), which can modulate JNK activity (Ura et al., 2007). adapter protein CNK1 through interaction with the CRIC and RASSF1A could also impact the G1 transition via its direct PDZ domains of CNK1 (Rabizadeh et al., 2004). The ability E4F interaction with the transcription factor p120 (Fenton et al., of CNK1 to induce apoptosis appears to require interaction E4F 2004). p120 can negatively regulate the transcription of with a RASSF1A-MST1 complex and an as-yet-unidentified cyclin A2, leading to cell cycle arrest in G1 phase (Fajas et al., effector. Thus, RASSF1A may function as a scaffold for E4F 2001). RASSF1A enhances the ability of p120 to suppress assembly of an apoptotic complex containing CNK1. E4F cyclin A2 and synergizes with p120 to induce cell cycle Studies in Drosophila have recently led to the identification arrest (Fenton et al., 2004; Ahmed-Choudury et al., 2005). of a pro-apoptotic tumor suppressor kinase cascade. This However, the role of RASSF1A in cyclin A2 regulation may pathway involves the coupling of MST kinases to the LATs be complex because it appears to be able to increase cyclin A2 kinases via an adaptor protein called Salvador that acts as a protein levels under some circumstances (Song et al., 2004). tumor suppressor in Drosophila (Harvey and Tapon, 2007). Variations in experimental procedures could be responsible for LATs kinases are pro-apoptotic and transgenic mice lacking E4F this observed discrepancy. Intriguingly, p120 has also been LATs1 develop tumors (St John et al., 1999; Tao et al., 1999; detected at the mitotic spindle and has been implicated in Yabuta et al., 2000). One target of the LATs kinases that has genomic instability (Le Cam et al., 2004). This localization is been identified is the key transcriptional repressor YAP (Huang likely to be mediated by RASSF1A and may indicate a et al., 2005). E4F biological role of the RASSF1A-p120 interaction that is Structural modeling led to the prediction that RASSF1A independent of the latter’s transcription factor function. might bind Salvador through heterodimerization of their RASSF1A has been implicated in control of mitotic arrest SARAH motifs (Scheel and Hofmann, 2003). Although studies in prometaphase (Liu et al., 2003; Vos et al., 2004; Rong et al., in Drosophila appeared to show that RASSF1A does not Journal of Cell Science RASSF1A 3169 interact with Salvador (Polesello et al., 2006), recent studies different cell types used. Further studies will clearly be have confirmed that human RASSF1A does bind human required to reveal the exact nature of the involvement of Salvador (Guo et al., 2007). Analysis of the protein sequence RASSF1A in these pathways. of the ‘RASSF1A’ described in Drosophila suggests that it is closer to human RASSF5 than to RASSF1A. In our hands, Conclusion human RASSF5 does not appear to bind Salvador, and this may The RASSF1A protein modulates a broad range of cellular explain the apparent contradiction. Nevertheless, RASSF1A is functions that are essential for normal growth control. therefore connected to the LATs kinase tumor suppressor RASSF1A expression is lost in a wide variety of human system via Salvador (Guo et al., 2007; O’Neill et al., 2005). tumors by silencing resulting primarily from promoter Because RASSF1A can bind Salvador and MST kinases, this hypermethylation. The high frequency with which RASSF1A is is an obvious example of a scaffolding function for RASSF1A. silenced in tumors suggests that it plays a pivotal role in the Bax is a member of the Bcl2 family and an important development of human cancer. component of the apoptotic machinery (Sharpe et al., 2004; Lacking enzymatic activity, the RASSF1A protein appears Tan et al., 2001). Recent work has shown that RASSF1A can to serve as a node that can scaffold multiple tumor suppressor also regulate Bax activity (Baksh et al., 2005; Vos et al., 2006). pathways. Those pathways known to contain potential effectors This is accomplished by the direct binding of RASSF1A to of RASSF1A function are shown in Fig. 1. There are almost modulator of apoptosis-1 (MOAP1), a Bax-binding protein certainly others, and the role of the Salvador-LATs pathway in (Tan et al., 2001). The interaction between RASSF1A and RASSF1A is of particular current interest. All of these MOAP1 is enhanced by activated K-Ras (Vos et al., 2006), and pathways have the potential to be modified by Ras, although knocking down RASSF1A impairs the ability of oncogenic K- the physiological interaction between Ras and RASSF1A has Ras to activate Bax. RASSF1A mutants found in human yet to be confirmed. RASSF1A represents an important tumors exhibit impaired interaction with MOAP1, which potential diagnostic and therapeutic target. Because the gene suggests that subversion of this pathway is important for the remains intact but dormant in most tumors, reactivation by development of a tumor. Thus a Ras-RASSF1A-MOAP1 promoter demethylation would present a novel approach to complex appears to be essential for Ras-induced apoptosis. therapy. Baksh et al. have observed that RASSF1A expression also References enhances TNF--induced apoptosis in transformed and non- Agathanggelou, A., Honorio, S., Macartney, D. 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H., Law, F. B., Tin, P. C., Cheung, anaphase-promoting complex. Genes Dev. 13, 2039-2058. Journal of Cell Science http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Cell Science The Company of Biologists

The RASSF1A tumor suppressor

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Abstract

Commentary 3163 1 2 1, Howard Donninger , Michele D. Vos and Geoffrey J. Clark * Molecular Targets Group, Department of Medicine, J. G. Brown Cancer Center, University of Louisville, 119C Baxter Boulevard, 580 S. Preston Street, Louisville, KY 40202, USA Research Analysis and Evaluation Branch, NCI, Rockville, MD, USA *Author for correspondence (e-mail: gjclar01@louisville.edu) Accepted 23 July 2007 Journal of Cell Science 120, 3163-3172 Published by The Company of Biologists 2007 doi:10.1242/jcs.010389 Summary RASSF1A (Ras association domain family 1 isoform A) is RASSF1A lacks apparent enzymatic activity but a recently discovered tumor suppressor whose inactivation contains a Ras association (RA) domain and is potentially is implicated in the development of many human cancers. an effector of the Ras oncoprotein. RASSF1A modulates Although it can be inactivated by gene deletion or point multiple apoptotic and cell cycle checkpoint pathways. mutations, the most common contributor to loss or Current evidence supports the hypothesis that it serves as reduction of RASSF1A function is transcriptional silencing a scaffold for the assembly of multiple tumor suppressor of the gene by inappropriate promoter methylation. This complexes and may relay pro-apoptotic signaling by K- epigenetic mechanism can inactivate numerous tumor Ras. suppressors and is now recognized as a major contributor to the development of cancer. Key words: Epigenetic, RASSF1A, Ras, Tumor suppressor Introduction RASSF1A is a tumor suppressor For many years it was suspected that one or more tumor Tumor suppressor genes are classically defined by Knudson’s suppressors lurk in the 3p21.3 region of the human genome, ‘two-hit’ hypothesis (Knudson, 1971), which states that because this area frequently suffers loss of heterozygosity inactivation of both alleles of a tumor suppressor gene is (LOH) in lung cancer (Lerman and Minna, 2000). In 2000, required for tumorigenesis. Loss of a RASSF1A allele is a Damman et al. serendipitously cloned a gene located in this frequent phenomenon in primary human cancer (Burbee et al., region that they termed RASSF1 (Ras association domain 2001; Pfeifer and Dammann, 2005). In a study of sporadic lung family 1), because the protein contains a putative Ras cancers, 76% of the tumors showing allelic imbalance at association (RA) domain. One of the isoforms produced by the 3p21.3 (the RASSF1A locus) also showed RASSF1A promoter gene, RASSF1A, has properties compatible with a tumor hypermethylation (Agathanggelou et al., 2001). Similar suppressor function. Moreover, the gene appears to suffer findings in non-small-cell lung cancer (NSCLC) (Tomizawa et frequent transcriptional inactivation in tumor cells due to al., 2002), bladder transitional carcinoma (Chan et al., 2003) aberrant promoter methylation (Burbee et al., 2001; Dammann and cervical squamous cell carcinoma (Yu et al., 2003) have et al., 2000). Simultaneously, a bioinformatics-based approach been reported. Thus RASSF1A alleles can be inactivated by a revealed a proapoptotic novel Ras-binding protein that inhibits combination of genetic and epigenetic mechanisms, and tumor cell growth and is encoded by a gene localizing to RASSF1A conforms to the Knudson two-hit model. 3p21.3. This protein turned out to be RASSF1C, a smaller Hypermethylation of both alleles of the RASSF1A promoter has isoform produced by the RASSF1 gene (Vos et al., 2000). Thus, been shown to cause loss of expression of the gene (Lusher et RASSF1 appeared to be one of the elusive tumor suppressors al., 2002). Moreover, although early studies reported located at 3p21.3. infrequent mutation of RASSF1A, other studies have Subsequent work showed that specific point mutations suggested that up to 15% of tumors may contain inactivating compromise the ability of RASSF1A to inhibit tumor cell point mutations (Pan et al., 2005). Thus, the evidence that growth (Dreijerink et al., 2001; Kuzmin et al., 2002; RASSF1A is inactivated in a high percentage of human tumors Shivakumar et al., 2002). A small deluge of papers began, is strong (Table 1). demonstrating frequent epigenetic inactivation of RASSF1A in If the inactivation of RASSF1A contributes to the a wide variety of tumors. Bearing in mind that RASSF1A can development of the transformed phenotype, then one might also suffer point mutations in up to 15% of primary tumors expect that re-introduction of RASSF1A into RASSF1A- (Pan et al., 2005), RASSF1A is one of the most frequently negative cells would impair tumorigenicity. Indeed, this is the inactivated proteins ever identified in human cancer. case. Re-expression of the gene in RASSF1A-negative cancer RASSF1A lacks any obvious enzymatic activity but may cells results in reduced colony formation in soft agar and serve as a scaffold for signaling complexes, key components of reduced tumorigenicity in nude mice (Burbee et al., 2001; which have recently been identified (Fig. 1). Here, we discuss Dammann et al., 2000; Dreijerink et al., 2001; Kuzmin et al., work that has implicated RASSF1A in the regulation of the cell 2002). Two groups have independently knocked out the cycle, apoptosis and genetic instability (Agathanggelou et al., RASSF1A gene in mice (Tommasi et al., 2005; van der 2005), and the molecular mechanisms involved. Weyden et al., 2005). In each case the animals exhibit an Journal of Cell Science 3164 Journal of Cell Science 120 (18) Mitogenic stimuli Death receptor Fig. 1. Summary of some of the known partners and pathways of RASSF1A. RASSF1A binds to at least Ras three microtubule-binding proteins (MAPs), complexes PMCa with microtubules and RASSF1A regulates mitosis, the cell JNK cycle and apoptosis in E4F p120 MOAP-1 response to mitogenic or MST1 SAV apoptotic stimuli. Direct Cdc20 MAP1B Cyclin A2 C19ORF5 LATs1/2 interaction between Cyclin D1 CNK1 BAX RASSF1A and microtubule- APC associated proteins localizes RASSF1A to the G1 S microtubules, stabilizing them and, thereby, regulating Cell cycle mitosis. Repression of cyclins Microtubule dynamics A and D1 by RASSF1A M G2 results in cell cycle arrest and interactions with CNK1, MST1, Salvador and MOAP1 may allow RASSF1A to Cell cycle arrest Mitotic arrest Migration Apoptosis modulate apoptosis. enhanced tendency to develop spontaneous tumors. Thus, 2005; Endoh et al., 2005; Hesson et al., 2005; Lambros et al., RASSF1A is a tumor suppressor that is frequently impaired in 2005; Vos et al., 2003b). Inactivation of RASSF2 correlates human tumors. with activation of Ras in tumor cells (Hesson et al., 2005). Reintroduction of RASSF2 into tumor cells impairs RASSF1A is part of a family of potential tumor tumorigenesis and knocking down RASSF2 enhances suppressors tumorigenesis (Akino et al., 2005). Thus, RASSF2 also RASSF1A is a member of a family of six related proteins, each appears to be an epigenetically inactivated tumor suppressor. of which exhibits multiple splice variants. With the exception The effector pathways controlled by it remain unknown. of some minor splice variants, each protein contains an RA RASSF3 can also bind to Ras and inhibit cell growth domain and a C-terminal SARAH protein-protein interaction (unpublished observations). However, it is the only family motif. Each family member, with the exception of RASSF3, member whose RNA is not downregulated in tumors (Tommasi has now been implicated as a human tumor suppressor. et al., 2002). Whether it is involved in tumor suppression thus RASSF5 (Nore1a) is the best characterized member of the remains unknown. family after RASSF1A. RASSF5 was the first member of the RASSF4 is frequently downregulated by promoter family cloned and it was originally designated Nore1a for methylation in human tumor cells, binds to Ras and induces novel Ras effector 1 (Vavvas et al., 1998). RASSF5 binds apoptosis. It localizes mostly to the cytosol but can be recruited activated Ras directly and is present in an endogenous complex to the plasma membrane by activated Ras (Eckfeld et al., with Ras in cells. RASSF5 is pro-apoptotic and kills cells in a 2004). Ras-dependent manner (Khokhlatchev et al., 2002; Vos et al., RASSF6 exhibits similar biological properties to its brethren 2003a). It is frequently inactivated in human tumors by and is often downregulated in primary human tumors (Allen et promoter methylation (Table 2). Moreover, it is linked to the al., 2007). Intriguingly, the RASSF6 locus is implicated in development of a rare familial form of cancer (Chen et al., susceptibility to bronchiolitis induced by respiratory syncytial 2003), which confirms its role as a tumor suppressor in vivo. virus (Hull et al., 2004). RASSF6 might therefore have a role Its mechanisms of action remain largely unknown, although it in inflammation; indeed it can suppress the NFB pathway may regulate the pro-apoptotic kinase MST1 (Praskova et al., (Allen et al., 2007). 2004). A smaller splice version of RASSF5 has been identified Two further RA-domain-containing proteins have been and designated Nore1b or RAPL (Katagiri et al., 2003; identified and are now being described as RASSF8 (Falvella Tommasi et al., 2002). This protein demonstrates more et al., 2006) and RASSF7. These were previously known as restricted expression than RASSF5 and can form an HOJ-1 and HRC1. Although these proteins can bind to Ras and endogenous complex with the Ras-related protein Rap. It inhibit cell growth (our unpublished observations), they do not regulates lymphocyte adhesion and has also been implicated as display great homology with RASSF1-RASSF6 and do not a tumor suppressor (Katagiri et al., 2003; Macheiner et al., contain SARAH motifs. This raises the issue of how to define 2006). a RASSF protein. RASSF proteins can heterodimerize with RASSF2 is a pro-apoptotic Ras effector that is frequently each other (Ortiz-Vega et al., 2002) and this might serve as a downregulated in human tumors by promoter methylation, functional definition. Although we have found that RASSF2- histone deacetylation and sometimes deletion (Akino et al., RASSF6 readily heterodimerize with RASSF1A, the RASSF7 Journal of Cell Science RASSF1A 3165 Table 1. Primary tumors containing RASSF1A promoter methylation Tumor type Frequency* References Lung: SCLC 88% Grote et al., 2006 Lung: NSCLC 39% 28% 15% Chen et al., 2006; Grote et al., 2006; Safar et al., 2005 Breast 95% 81% Yeo et al., 2005; Shinozaki et al., 2005 Colorectal 20% 52% Miranda et al., 2006; Oliveira et al., 2005 Prostate 99% Jeronimo et al., 2004 Cervical Adenocarcinoma 45% Cohen et al., 2003 Esophageal 34% Wong et al., 2006 Gastric 44% Oliveira et al., 2005 Renal 56-91% Yoon et al., 2001; Dreijerink et al., 2001 Hepatocellular 75% Katoh et al., 2006 Bladder 30-50% Marsit et al., 2006 Pancreatic 63% Liu et al., 2005a Ovarian 26% 30% Teodoridis et al., 2005; Makarla et al., 2005 Nasopharyngeal 68% Tan et al., 2006 Leukemia 0% 15% Johan et al., 2005; Harada et al., 2002 Neuroblastoma 83% Lazcoz et al., 2006 Thyroid 71% 35% Schagdarsurengin et al., 2006; Nakamura et al., 2005 Cholangiocarcinoma 67% Tischoff et al., 2005 Ependymoma 36% 86% Michalowski et al., 2006; Hamilton et al., 2005 Glioma 57% 54% Hesson et al., 2004 Horiguchi et al., 2003 Hodgkin Lymphoma 65% Murray et al., 2004 Medulloblastoma 79% Lusher et al., 2002 Retinoblastoma 59% Harada et al., 2002 Testicular Seminoma 40% Honorio et al., 2003 Testicular Nonseminoma 83% Honorio et al., 2003 Wilms tumor 54% Wagner et al., 2002 Rhabdomyosarcoma 61% Harada et al., 2002 Pheochromocytomas 22% Astuti et al., 2001 Head and neck 15% 17% Dong et al., 2003; Hogg et al., 2002 Melanoma 41% Spugnardi et al., 2003 *Frequency of RASSF1A promoter hypermethylation in tumor type. and RASSF8 proteins do not (our unpublished observation). contain an RA domain located towards the C-terminus of the Thus, RASSF7 and RASSF8 may be a separate sub-family protein and a SARAH (Sav-RASSF-Hpo) protein-protein distinct from the ‘true’ RASSF proteins. interaction motif at the very C-terminus. A putative ATM phosphorylation site for the DNA repair checkpoint kinase RASSF1 produces multiple isoforms ATM is found in isoforms A, C, D, E and H (Fig. 2B). The RASSF1 locus at 3p21.3 spans approximately 11,000 bp. Only isoforms A and C have been subjected to extensive It contains eight exons, and alternative splicing and usage of biological analysis. Little information is available regarding the two different promoters (Fig. 2A) give rise to eight different functions of splice variants B, D, E, F, G and H. RASSF1C transcripts, RASSF1A-RASSF1H. Epigenetic inactivation of appears to share many of the biological characteristics of genes often involves the methylation of CpG islands in their RASSF1A and has been implicated as a tumor suppressor in promoters (Hesson et al., 2007). There are two CpG islands both in vitro and in vivo studies (Li et al., 2004; Vos et al., associated with the RASSF1 promoters. A smaller, 737 bp 2000). However, it has unique functions not shared by island contains 85 CpGs and spans the promoter for RASSF1A, RASSF1A, such as coupling DNA damage to the activation of RASSF1D, RASSF1E, RASSF1F and RASSF1G. A larger 1365 the SAPK-JNK signaling pathway (Kitagawa et al., 2006). bp island, containing 139 CpGs, spans the promoter region for RASSF1C and RASSF1A use different promoters, and Latif RASSF1B and RASSF1C (Agathanggelou et al., 2005). and co-workers report that RASSF1C is not subject to RASSF1A is a 340-residue protein that migrates at 39 kDa. epigenetic inactivation (Agathanggelou et al., 2005). However, It contains a cysteine-rich domain (CRD) reminiscent of the we have observed differential loss of RASSF1C protein in diacylglycerol-binding–CRD domain of Raf-1 towards the N- some tumor lines (our unpublished observations). Perhaps the terminus (residues 50-101), which is not present in the other regulation of RASSF1C involves more significant post- ubiquitously expressed isoform RASSF1C. Isoforms A-E also transcriptional mechanisms than regulation of RASSF1A. Table 2. Primary tumors containing NORE1A promoter methylation Tumor type Frequency* References Lung, SCLC 0% Hesson et al., 2003 Lung, NSCLC 24% 28% Hesson et al., 2003; Irimia et al., 2004 Hepatocellular Carcinoma 37.5% Calvisi et al., 2006 Clear cell renal Carcinoma 32% Chen et al., 2003 Neuroblastoma 3% Lazcoz et al., 2006 Wilms tumor 15% Morris et al., 2003 *Frequency of NORE1A promoter hypermethylation in tumor type. Journal of Cell Science 3166 Journal of Cell Science 120 (18) 5 3 1α 1β 1γ 2 36 4 5 51-101 125-138 194-289 291-337 RASSF1A C1/DAG ATM RA SARAH 340aa 43-138 140-186 RASSF1B RA SARAH 189aa 55-68 121-219 221-267 RASSF1C ATM RA SARAH 270aa 51-105 129-142 198-292 294-341 RASSF1D C1/DAG ATM RA SARAH 344aa 51-101 129-142 198-293 295-341 RASSF1E C1/DAG ATM RA SARAH 344aa 51-85 RASSF1F C1/DAG 92aa 51-103 RASSF1G C1/DAG 152aa 55-68 RASSF1H ATM 75aa Fig. 2. RASSF1 gene locus and domain structure of the different RASSF1 isoforms. (A) The RASSF1 gene locus is characterized by eight exons (boxed regions) and two different promoters (arrows) with two associated CpG islands (black bars). Black boxes represent coding regions and white boxes are non-coding regions. (B) Schematic representation of the different RASSF1 isoforms. C1/DAG, conserved region 1 diacylglycerol-binding domain; ATM, ATM-kinase consensus phosphorylation sequence; RA, RalGDS/AF6 Ras association domain; SARAH, Sav/RASSF/Hpo interaction domain. The position of each domain (as outlined in the Swiss-Prot/TrEMBL database) is indicated above each isoform and the number of amino acids in each isoform is shown on the right. RASSF1A as a Ras effector between Ras and RASSF1A. They suggest that the interaction Activated forms of K-Ras, although being transforming is indirect and due to heterodimerization of RASSF1A with oncoproteins, also have growth inhibitory effects, including the RASSF5 (Ortiz-Vega et al., 2002). The use of unfarnesylated induction of apoptosis (Cox and Der, 2003; Downward, 1998). Ras in their studies may have led them to underestimate the K-Ras must thus have pro-apoptotic effector proteins, which binding affinity. Confirmation of RASSF1A as a bona fide Ras are likely to be downregulated during the development of Ras- effector awaits the demonstration that the endogenous proteins dependent tumors. RASSF1A is a pro-apoptotic protein that form a complex in vivo. has a potential RA domain, and so it could mediate some of If RASSF1A serves as a pro-apoptotic Ras effector, then one the pro-apoptoptic effects of K-Ras. This hypothesis is might expect Ras activation to correlate with RASSF1A supported by the observation that the related RASSF5 protein inactivation in tumors. Several studies have failed to detect can be detected in an endogenous complex with Ras (Vavvas such a relationship (Dammann et al., 2003; Li et al., 2003; van et al., 1998). Engeland et al., 2002). However, these experiments used the The RA domain of RASSF1A can bind to Ras directly in presence or absence of an activating Ras mutation to identify vitro (Vos et al., 2000), and RASSF1A forms a complex with Ras-dependent tumors. In fact, there is a surprisingly poor activated K-Ras when overexpressed in cells (Rodriguez- correlation between the presence of a mutation in the Ras gene Viciana et al., 2004). Formation of the complex depends on an and the abundance of activated Ras protein in tumor cells intact effector domain for Ras and farnesylation of K-Ras (Fig. (Eckert et al., 2004). Thus, resolving this issue will require 3). We have found that K-Ras binds better than H-Ras, even direct measurements of Ras-GTP levels in the cells. though both share an identical effector domain and both are farnesylated. Other Ras-related proteins also demonstrate the Biological functions of RASSF1A potential to bind RASSF1A, including M-Ras and R-Ras but Numerous studies have shown that overexpression of not Rap (our unpublished observations). M-Ras and R-Ras are RASSF1A promotes apoptosis, cell cycle arrest and reduces post translationally modified by geranylgeranyl, not farnesyl, the tumorigenicity of cancer cell lines (for a review, see and this may contribute to the weaker interaction with Agathangelou et al., 2005). RNAi experiments have implicated RASSF1A. RASSF1A downregulation in loss of cell cycle control, Ortiz-Vega et al., however, have failed to see direct binding enhanced genetic instability, enhanced cell motility and Journal of Cell Science RASSF1A 3167 Microtubules are polymers that continually switch between phases of elongation and shortening; this is known as dynamic instability. Microtubule dynamics can be modulated by a series of microtubule-associated proteins (MAPs) that bind directly to tubulin (Halpain and Dehmelt, 2006). Two-hybrid analysis has identified three such proteins – MAP1b (Dallol et al), C19ORF5 (also known VCY2IP1 or RABP1) (Liu et al., 2002; Song et al., 2005) and MAP4 (G.J.C., unpublished observation) – as direct binding partners of RASSF1A. Thus, RASSF1A could associate with microtubules via MAPs. MAP1b has been shown to promote tubulin polymerization (Togel et al., 1998) and MAP4 has been shown to impede microtubule depolymerization (Nguyen et al., 1998). C19ORF5 has also been shown to enhance microtubule polymerization (Liu et al., Fig. 3. RASSF1A binds Ras. (A) HEK-293-T cells were transfected 2005; Orbán-Németh et al., 2005). Thus, RASSF1A has the with FLAG-tagged RASSF1A and HA-tagged forms of K-Ras12v. potential to scaffold proteins that we might expect would have The cells were lysed and immunoprecipitated (IP) before being immunoblotted (IB) with HA and FLAG. Upper panel shows a synergistic effect on microtubule polymerization. immunoprecipitation, lower panel shows protein levels in the cell We have identified a minimum domain in RASSF1A that is lysate. Wild-type K-Ras, a Y40C effector mutant of K-Ras12v and a required for the microtubule-stabilizing effects. When this farnesylation-defective mutant of K-Ras12v (K-RasCX) were isolated domain is itself overexpressed, it causes a catastrophic defective for binding RASSF1A. collapse of the microtubule network (Vos et al., 2004). The underlying mechanism and whether it involves the direct resistance to K-Ras and tumor necrosis factor  (TNF)- interaction of RASSF1A with tubulin remains under induced apoptosis (Baksh et al., 2005; Dallol et al., 2005; Song investigation, but it appears that RASSF1A has the capacity to et al., 2004; Vos et al., 2004; Vos et al., 2006). Thus, RASSF1A profoundly influence the dynamic balance of microtubules appears to regulate multiple biological processes. The both positively and negatively. mechanisms behind these activities are multifold and remain under investigation but the emerging evidence suggests a role Maintenance of genomic stability for RASSF1A as a scaffolding protein that can assemble and Genomic instability is one of the hallmarks of transformed modulate multiple effector protein complexes. cells (Saavedra et al., 2000) and defects in spindle regulation can lead to genomic instability (Wassmann and Benezra, RASSF1A regulates microtubules 2001). Since RASSF1A localizes to the centrosome and RASSF1A localizes to microtubules and promotes their mitotic spindle, and can modulate tubulin dynamics (Song et stabilization (Liu et al., 2003; Dallol et al., 2004; Song et al., al., 2005; Vos et al., 2004; Dallol et al., 2004), it is not 2004; Vos et al., 2004). During interphase, it is localized to surprising that RASSF1A has been implicated in the cytoplasmic microtubules; during prophase it localizes to maintenance of genomic stability (Song et al., 2005; Vos et al., centrosomes; during metaphase and anaphase, it localizes to 2004). both spindle microtubules and the spindle poles; and it is found C19ORF5 may play a key role in recruiting RASSF1A to at the midzone and midbody during early and late telophase, the centrosome and spindle. Moreover, inhibition of C19ORF5 respectively (Fig. 4). expression by RNAi can promote genetic instability similar to Fig. 4. RASSF1A associates with microtubules and localizes to centrosome and spindles during mitosis. COS cells were transfected with GFP-RASSF1A and the nuclei stained blue with DAPI. Journal of Cell Science 3168 Journal of Cell Science 120 (18) that observed when RASSF1A is downregulated (Song et al., 2004). An elegant explanation for the M-phase arrest mediated 2005; Dallol et al., 2007). Song et al. suggest that the by RASSF1A has been put forward by Song et al., who mechanism of C19ORF5 action is to enhance the ability of suggested that it is brought about by the direct interaction of RASSF1A to stabilize mitotic cyclins. Thus, loss of function RASSF1A with Cdc20 (Song et al., 2004; Song et al., 2005). of C19ORF5 leads to premature destruction of mitotic cyclins Cdc20 is an essential cell cycle regulator required for the and accelerated, aberrant mitosis (Song et al., 2004). However, completion of mitosis (Yu, 2007). Cdc20 binds and activates in similar experiments, Dallol et al. observed delayed rather the ubiquitin ligase activity of a large molecular machine than accelerated mitotic progression and showed a role for designated the anaphase-promoting complex (APC). This C19ORF5 in anchoring  and  tubulin to the centrosomes promotes the ubiquitylation and degradation of cyclins A and (Dallol et al., 2007). This suggests that the abnormalities in B, leading to anaphase and mitotic exit. Song et al. suggest that sister chromatid separation observed when C19ORF5 is the interaction with RASSF1A blocks the ability of Cdc20 to downregulated is due to aberrations in spindle dynamics. This activate the APC and that the resultant stabilization of cyclins is clearly a complicated issue that may require further A and B blocks the mitotic progression that usually follows experimentation to resolve. their degradation (Mathe, 2004; Peters, 2002; Zachariae and Both RASSF1A and RASSF1C contain a potential ATM Nasmyth, 1999). Liu et al., however, have been unable to kinase (mutated in ataxia telangiectasia) phosphorylation site confirm the interaction of RASSF1A with Cdc20 (Liu et al., (Kim et al., 1999). ATM functions as part of the DNA damage 2007). Thus the role of Cdc20 and APC in RASSF1A- checkpoint and has been implicated in regulation of genomic mediated cell cycle control requires further investigation. stability (Levitt and Hickson, 2002; Shiloh, 2003). Point mutations that destroy the RASSF1A or RASSF1C ATM Modulation of apoptosis phosphorylation site have been found in human tumors RASSF family proteins are pro-apoptotic (Vos et al., 2000; (Burbee et al., 2001; Shivakumar et al., 2002). We have been Khokhlatchev et al., 2002; Eckfeld et al., 2004; Vos et al., unable to detect any obvious difference in the microtubule- 2003a; Vos et al., 2003b) and several pathways by which stabilizing activities of wild-type RASSF1A and RASSF1A RASSF1A may modulate apoptosis have now been identified. mutated at the ATM site. However, the equivalent mutant of MST1 and MST2 are pro-apoptotic serine/threonine kinases RASSF1C (S61F) is clearly impaired (Vos et al., 2004). that activate the SAPK-JNK signaling pathway and Indeed, this RASSF1C mutant can induce genomic instability phosphorylate histone H2B (Cheung et al., 2003; Ura et al., at frequencies comparable to those evident in RASSF1A- 2007). They bind directly to RASSF1A and other RASSF knockdown studies (our unpublished observation). Thus, family members via their SARAH motifs (Avruch et al., 2005; RASSF1A and RASSF1C may be mediators through which Hwang et al., 2007; Khokhlatchev et al., 2002; Oh et al., 2006; ATM maintains genomic stability. Moreover, mutant Praskova et al., 2004). Consequently, they are obvious pro- RASSF1C has the potential to serve as an oncogene. apoptotic effectors for RASSF1A. However, the role of RASSF1A in the regulation of MST1 appears complex. In RASSF1A modulates the cell cycle mammalian cells, contradictory effects of RASSF1A on MST1 Initial studies examining the role of RASSF1A in the cell cycle kinase activity have been reported. Praskova et al. found that demonstrated a role for RASSF1A at the G1-S checkpoint and MST1 kinase activity is inhibited by RASSF1A whereas Oh et showed that RASSF1A modulates the levels of cyclin D1 al. and Guo et al. have found that it is activated (Oh et al., 2006; (Shivakumar et al., 2002). Subsequent work confirmed this and Praskova et al., 2004; Guo et al., 2007). Our own studies implicated inhibition of the JNK pathway as a mechanism support the results of Oh and Guo. Thus, the effects of (Whang et al., 2005). RASSF1A could connect to JNK by RASSF1A on MST1 may be context dependent. direct interactions with the kinase MST1 (Khokhlatchev et al., RASSF1A also forms a complex with the pro-apoptotic 2002), which can modulate JNK activity (Ura et al., 2007). adapter protein CNK1 through interaction with the CRIC and RASSF1A could also impact the G1 transition via its direct PDZ domains of CNK1 (Rabizadeh et al., 2004). The ability E4F interaction with the transcription factor p120 (Fenton et al., of CNK1 to induce apoptosis appears to require interaction E4F 2004). p120 can negatively regulate the transcription of with a RASSF1A-MST1 complex and an as-yet-unidentified cyclin A2, leading to cell cycle arrest in G1 phase (Fajas et al., effector. Thus, RASSF1A may function as a scaffold for E4F 2001). RASSF1A enhances the ability of p120 to suppress assembly of an apoptotic complex containing CNK1. E4F cyclin A2 and synergizes with p120 to induce cell cycle Studies in Drosophila have recently led to the identification arrest (Fenton et al., 2004; Ahmed-Choudury et al., 2005). of a pro-apoptotic tumor suppressor kinase cascade. This However, the role of RASSF1A in cyclin A2 regulation may pathway involves the coupling of MST kinases to the LATs be complex because it appears to be able to increase cyclin A2 kinases via an adaptor protein called Salvador that acts as a protein levels under some circumstances (Song et al., 2004). tumor suppressor in Drosophila (Harvey and Tapon, 2007). Variations in experimental procedures could be responsible for LATs kinases are pro-apoptotic and transgenic mice lacking E4F this observed discrepancy. Intriguingly, p120 has also been LATs1 develop tumors (St John et al., 1999; Tao et al., 1999; detected at the mitotic spindle and has been implicated in Yabuta et al., 2000). One target of the LATs kinases that has genomic instability (Le Cam et al., 2004). This localization is been identified is the key transcriptional repressor YAP (Huang likely to be mediated by RASSF1A and may indicate a et al., 2005). E4F biological role of the RASSF1A-p120 interaction that is Structural modeling led to the prediction that RASSF1A independent of the latter’s transcription factor function. might bind Salvador through heterodimerization of their RASSF1A has been implicated in control of mitotic arrest SARAH motifs (Scheel and Hofmann, 2003). Although studies in prometaphase (Liu et al., 2003; Vos et al., 2004; Rong et al., in Drosophila appeared to show that RASSF1A does not Journal of Cell Science RASSF1A 3169 interact with Salvador (Polesello et al., 2006), recent studies different cell types used. Further studies will clearly be have confirmed that human RASSF1A does bind human required to reveal the exact nature of the involvement of Salvador (Guo et al., 2007). Analysis of the protein sequence RASSF1A in these pathways. of the ‘RASSF1A’ described in Drosophila suggests that it is closer to human RASSF5 than to RASSF1A. In our hands, Conclusion human RASSF5 does not appear to bind Salvador, and this may The RASSF1A protein modulates a broad range of cellular explain the apparent contradiction. Nevertheless, RASSF1A is functions that are essential for normal growth control. therefore connected to the LATs kinase tumor suppressor RASSF1A expression is lost in a wide variety of human system via Salvador (Guo et al., 2007; O’Neill et al., 2005). tumors by silencing resulting primarily from promoter Because RASSF1A can bind Salvador and MST kinases, this hypermethylation. The high frequency with which RASSF1A is is an obvious example of a scaffolding function for RASSF1A. silenced in tumors suggests that it plays a pivotal role in the Bax is a member of the Bcl2 family and an important development of human cancer. component of the apoptotic machinery (Sharpe et al., 2004; Lacking enzymatic activity, the RASSF1A protein appears Tan et al., 2001). Recent work has shown that RASSF1A can to serve as a node that can scaffold multiple tumor suppressor also regulate Bax activity (Baksh et al., 2005; Vos et al., 2006). pathways. Those pathways known to contain potential effectors This is accomplished by the direct binding of RASSF1A to of RASSF1A function are shown in Fig. 1. There are almost modulator of apoptosis-1 (MOAP1), a Bax-binding protein certainly others, and the role of the Salvador-LATs pathway in (Tan et al., 2001). The interaction between RASSF1A and RASSF1A is of particular current interest. All of these MOAP1 is enhanced by activated K-Ras (Vos et al., 2006), and pathways have the potential to be modified by Ras, although knocking down RASSF1A impairs the ability of oncogenic K- the physiological interaction between Ras and RASSF1A has Ras to activate Bax. RASSF1A mutants found in human yet to be confirmed. RASSF1A represents an important tumors exhibit impaired interaction with MOAP1, which potential diagnostic and therapeutic target. Because the gene suggests that subversion of this pathway is important for the remains intact but dormant in most tumors, reactivation by development of a tumor. Thus a Ras-RASSF1A-MOAP1 promoter demethylation would present a novel approach to complex appears to be essential for Ras-induced apoptosis. therapy. Baksh et al. have observed that RASSF1A expression also References enhances TNF--induced apoptosis in transformed and non- Agathanggelou, A., Honorio, S., Macartney, D. 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Journal of Cell ScienceThe Company of Biologists

Published: Sep 15, 2007

References