Role of the Ras-Association Domain Family 1 Tumor Suppressor Gene in Human Cancers

Review

Angelo Agathanggelou, Wendy N. Cooper, and Farida Latif

Section of Medical and Molecular Genetics, Division of Reproductive and Child Health, The Institute of Biomedical Research, University of Birmingham, Edgbaston, Birmingham, United Kingdom

Abstract renal cell carcinomas was identified from region 3p25 confirming In recent years, the list of tumor suppressor genes (or this hypothesis (3). This prompted the search for TSGs within the candidate TSG) that are inactivated frequently by epigenetic other regions of 3p. An important TSG was suspected to reside in events rather than classic mutation/deletion events has been 3p21.3 because instability of this region is the earliest and most growing. Unlike mutational inactivation, methylation is frequently detected deficiency in lung cancer. Overlapping homo- reversible and demethylating agents and inhibitors of histone zygous deletions in lung and breast tumor cell lines reduced the deacetylases are being used in clinical trails. Highly sensitive critical region in 3p21.3 to 120 kb and this region was found to be and quantitative assays have been developed to assess exceptionally gene rich. From this critical region, eight genes were methylation in tumor samples, early lesions, and bodily fluids. identified, including CACNA2D2, PL6/placental protein 6, CYB561D2/101F6, TUSC4/NPRL2/G21, ZMYND10/BLU, RASSF1/ Hence, gene silencing by promoter hypermethylation has potential clinical benefits in early cancer diagnosis, prognosis, 123F2, TUSC2/FUS1, and HYAL2/LUCA2. However, despite extensive treatment, and prevention. The hunt for a TSG located at genetic analysis in lung and breast tumors, none of these candidate 3p21.3 resulted in the identification of the RAS-association genes were frequently mutated. Meanwhile, Dammann et al. (2) domain family 1, isoform A gene (RASSF1A). RASSF1A falls identified RASSF1 in a yeast two-hybrid screen baited with XPA. into the category of genes frequently inactivated by methyl- Although the interaction was not formally shown, this novel gene ation rather than mutational events. This gene is silenced and was of interest because it mapped to the 3p21.3 entry in Genbank, it frequently inactivated by promoter region hypermethylation shared high-sequence homology with a known RAS effector in in many adult and childhood cancers, including lung, breast, mouse (Nore1), and expression of one of the isoforms, RASSF1A, but kidney, gastric, bladder, neuroblastoma, medulloblastoma, not RASSF1C, was lost in most lung tumor cell lines. Subsequently, hypermethylation of the RASSF1A promoter region CpG island was and gliomas. It has homology to a mammalian Ras effector (i.e., Nore1). RASSF1A inhibits tumor growth in both in vitro identified as the major cause for loss of expression. Cells treated with and in vivo systems, further supporting its role as a TSG. We demethylating agents reexpressed RASSF1A, which confirmed the and others identified the gene in 2000, but already there are role of DNA methylation in the inactivation of RASSF1A in tumor cell over a 150 publications demonstrating RASSF1A methylation lines. Finally, overexpression of RASSF1A but not RASSF1C in non– in a large number of human cancers. Many laboratories small cell lung cancer (NSCLC) A549 cells reduced colony formation including ours are actively investigating the biology of this efficiency and suppressed growth of tumor cells in nude mice in vitro novel protein family. Thus far, it has been shown to play and in vivo growth assays, respectively. important roles in cell cycle regulation, apoptosis, and Since the discovery of the first tumor suppressor gene, RB, microtubule stability. This review summarizes our current Knudson’s ‘‘two-hit’’ hypothesis (4) has been used to define this knowledge on genetic, epigenetic, and functional analysis of class of genes. The hypothesis states that inactivation of both RASSF1A tumor suppressor gene and its homologues. (Cancer alleles of a TSG, classically by genetic insult, are required for Res 2005; 65(9): 3497-508) tumorigenesis. Recently, this model was extended to include epigenetically inactivated genes. Hypermethylated in cancer-1, HIC1, a gene on 17p13.3, is hypermethylated in a number of Gene Identification +/À sporadic human tumors. In a study of Hic1 mice by Chen et al. The RAS-association domain family 1, isoform A (RASSF1A) (5), development was normal but the mice were predisposed to tumor suppressor gene (TSG), is a member of a new group of RAS malignant tumors at 70 weeks. Examination of the tumors effectors thought to regulate cell proliferation and apoptosis. revealed loss of Hic1 expression in the context of an intact allele. Formally known as 123F2, two laboratories independently cloned Methylation analysis then showed that the remaining allele was and sequenced RASSF1 (1, 2). Loss of heterozygosity (LOH) studies in inactivated by hypermethylation. Hence, inactivation of Hic1 in lung, breast, and kidney tumors identified several loci in chromo- this mouse model satisfied Knudson’s criteria for a TSG. The first some 3p likely to harbor one or more tumor suppressor genes, ‘‘hit’’ being the genetic knockout of one allele and the second including 3p12, 3p14, 3p21.3, and 3p25-26. In 1993, the von Hippel- ‘‘hit’’ being hypermethylation of Hic1 promoters. Using a Lindau (VHL) TSG inactivated in familial and sporadic clear cell combination of combined bisulfite and restriction analysis and LOH analysis inactivation of RASSF1A in sporadic lung tumors was shown to obey Knudson’s model for a TSG. In SCLC, Note: Due to space limitation, it has not been possible to include all references relating to RASSF1A methylation. hypermethylation was detected in 76% tumors with allelic Requests for reprints: Farida Latif, Section of Medical and Molecular Genetics, imbalance at 3p21.3 indicating that genetic and epigenetic Division of Reproductive and Child Health, The Institute of Biomedical Research, mechanisms provide the two ‘‘hits’’ to inactivate RASSF1A (6). University of Birmingham, Edgbaston, Birmingham, B15 2TT, United Kingdom. Phone: 44-121-627-2741; Fax: 44-121-627-2618; E-mail: [email protected]. Similarly, RASSF1A methylation was detected in 52% of NSCLC, I2005 American Association for Cancer Research. 72% of bladder transitional cell carcinoma, and 70% of cervical www.aacrjournals.org 3497 Cancer Res 2005; 65: (9). May 1, 2005

Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 2005 American Association for Cancer Research. Cancer Research squamous cell carcinoma (SCC) with allelic imbalance at 3p21.3 RASSF1B and RASSF1C. The entire first exon of each RASSF1 (7–9). In primary medulloblastoma, however, in the absence of transcript is contained within the CpG islands. allelic imbalance at 3p21.3, there is evidence to suggest biallelic A Ras association or RalGDS/AF-6 domain encoded by exons 4 methylation is the major mechanism for RASSF1A inactivation in and 5 defines the RASSF1 gene and is located at the COOH this tumor type (10). terminus of isoforms A-E. This domain mediates interactions with Ras and other small GTPases. Despite the absence of any sequence homology, it has a similar structure to the RasGTP binding domain RASSF1 Gene Locus and Protein Structure of Raf1 (kinase), the most studied RasGTP effector. The Ras The RASSF1 gene locus spans about 11,151 bp of the human association domain of RalGDS is comprised of a five-stranded genome and is comprised of eight exons. Differential promoter mixed h-sheet and three a helices. Ras association domains usage and alternative splicing generates seven transcripts homodimerize via intermolecular disulfide bonds, formed using (RASSF1A-G; Fig. 1). Isoforms A and C are ubiquitously two cysteine residues from each monomer. The interaction expressed, whereas isoform B is mainly expressed in cells of between RalGDS homodimers and Ras is mediated mostly by two the hemopoietic system. Isoforms D and E are specifically antiparallel h-strands within the Ras association domain and Ras. expressed in cardiac and pancreatic cells, respectively. They are RASSF1A and RASSF1D-G isoforms have a NH2-terminal protein very similar to RASSF1A except for slight differences in the kinase C (PKC) conserved region 1 (C1) domain encoded by exons splice sites used in exons 2ah (RASSF1D) and 3 (RASSF1E) 1a and 1h. In PKC, this is a diacylglycerol (DAG)/phorbol ester providing each protein with four additional amino acids. Two binding domain that regulates kinase activity. Overlapping the C1 CpG islands are associated with the promoter regions of RASSF1. domain is a putative zinc-binding domain, ZnF_NFX. This domain The smaller of the two (737 bp, 85 CpGs, 71.5% GC and OE:0.89) is present in a transcriptional repressor of HLA-DRA, NK-X1, and in spans the promoter region of RASSF1A (and RASSF1D, RASSF1E, Drosophila shuttle craft protein, vital for the late stages of embryonic RASSF1F, and RASSF1G). The second CpG island (1365 bp, 139CpG, neurogenesis. A SARAH domain, (Sav/RASSF/Hpo) is present at the 67.9% GC and OE:0.88) encompasses the promoter regions for COOH terminus of RASSF1A-E. SARAH domains mediate heterotypic

Figure 1. Transcription map of the RASSF1 gene locus in 3p21.3. RASSF1A to RASSF1G are generated by differential promoter usage (arrows) and alternative splicing. The positions of promoter associated CpG islands (black lines) as predicted by the UCSC Genome Browser May 2004. The domain structure of the protein products (predicted using Prosite): C1, DAG-binding domain; RA, RalGDS/AF6 Ras association domain; and SARAH, Sav/RASSF/Hpo interaction domain. ATM is the ATM-kinase phosphorylation consensus sequence (11). Position of the extra 4aa in RASSF1D and RASSF1E (white).

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Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 2005 American Association for Cancer Research. RASSF1 Tumor Suppressor Gene interactions between proteins as shown for Hpo/Sav and homotypic region (15, 25). Hence, reexpression of RASSF1A in 5-aza-2V interactions as shown with Mst1. In vitro studies have identified a deoxycytidine–treated neuroblastoma, rhabdomyosarcoma and peptide sequence within exon 3 that is a substrate for ataxia retinoblastoma cell lines is coincident with RASSF1F reexpression telagectasia mutant (ATM) kinase. ATM is vital for the activation of (12) Although not reported, expression of RASSF1E, RASSF1D, and p53 following exposure to ionizing radiation. Phosphorylation levels RASSF1G are also likely to be linked with expression of RASSF1A of the RASSF1 peptide sequence WETPDLSQAEIEQK were compa- because they also share the same promoter region. Expression of rable with levels of the ATM consensus sequence in p53 suggesting RASSF1A has been investigated in primary tumors and matched that RASSF1 might also be a substrate for ATM (11). The RASSF1 normal tissue. Whereas contaminating normal cells can be ATM site is present in isoforms A, C, D, and E. problematic, down-regulation of RASSF1A was shown to correlate with promoter methylation in breast, renal, thyroid, and bladder RASSF1 Homologues and Orthologues in Model tumors (13, 21, 24, 35, 41). Organisms RASSF1A methylation has been reported in at least 37 tumor types. Normal tissue controls are used in the majority of studies to RASSF1A homologues with 38% to 85% identity are present in show methylation is tumor specific and therefore involved in rodents, fish, and nematodes (Fig. 2A). Each has a COOH-terminal tumorigenesis. Methylation of RASSF1A occurs rarely in normal Ras association domain and SARAH domain and a NH -terminal 2 tissues. Combined with the high frequency reported in cancer, this C1 domain. In Xenopus,aRASSF1C homologue (MGC82975) has makes RASSF1A a candidate molecular marker for tumor been identified but the genomic sequence is not available to verify diagnosis. Detection of methylation in premalignant breast cancer the presence of RASSF1A.InDrosophila, an 89.6-kDa protein lesions and in sputum samples from patients who later developed (LD40758p) has the closest homology with RASSF1A. However, lung cancer (45, 46) have highlighted the potential importance of although it has a Ras association domain at the COOH terminus, a RASSF1A in early diagnosis. Several studies have investigated the LIM domain not a C1 domain is predicted at the NH terminus. 2 prospect of using DNA methylation as a tumor cell marker in Homology searches within the human genome have identified samples obtained by noninvasive techniques such as in plasma, other Ras association domain containing genes called RASSF2 sputum, urine, throat washings, and nipple aspirates (47–50, 53, (20pter-p12.1), RASSF3 (12q14.1), AD037 (10q11.21), NORE1 56). When comparing RASSF1A methylation in nasopharyngeal (1q32.1), and RASSF6 (4q21.21; Fig. 2B). In common with RASSF1, carcinoma with corresponding nasopharyngeal swab and mouth/ these genes code for multiple transcripts and CpG islands are throat washings, Chang et al. showed 100% concordance and 98% associated with their promoter regions. The Ras association and concordance with plasma and buffy coat samples (47). In ovarian SARAH domains of these genes are located at the COOH terminus cancer, detection of RASSF1A methylation in corresponding indicating that they share similar structural organization with peritoneal fluid and serum from patients with all histologic types, RASSF1. NORE1 which has the most sequence identity with RASSF1 grades, and stages of disease even in samples, where the CA-125 (49%) also has a putative NH -terminal C1 domain. 2 (serum marker) levels were low (<35), underscored the importance of having a reliable tumor marker when early detection is crucial to Tumor-Associated Methylation of RASSF1A patient outcome (48). In addition, RASSF1A methylation in urine Since the discovery of RASSF1A inactivation in lung tumors, a from kidney cancer patients corresponded with methylation of the wealth of literature has been published implicating RASSF1A in tumors in 73% of cases (53). Compared with other methods of the pathogenesis of a wide spectrum of tumors (Table 1 and tumor surveillance, detection of methylated DNA by methylation- references within). specific PCR is very sensitive and relatively cheap. Therefore, A direct correlation between promoter methylation and loss RASSF1A methylation has the potential to be used (along with a of RASSF1A expression has been shown in many tumor cell lines, panel of other TSGs to ensure 100% coverage) as a marker for early including SCLC, NSCLC, breast, kidney, nasopharyngeal carci- detection and monitoring. noma, prostate, ovarian, hepatocellular, Hodgkin’s lymphoma, mela- The value of RASSF1A methylation as a prognostic marker has noma, thyroid, bladder, neuroblastoma, medulloblastoma, been investigated most in NSCLC. Tomizawa et al. suggest that rhabdomyosarcoma, and retinoblastoma (2, 10, 12, 13, 15, 16, 18, RASSF1A methylation correlated with poor survival rate in patients 21, 22, 24, 25, 27, 31–35, 38). In most studies, reverse transcription- with stage I lung adenocarcinoma disease (7). RASSF1A methyla- PCR for the expression of RASSF1C was used as a control for RNA tion was associated with poorly differentiated tumors, predomi- integrity and loading. This served a duel purpose because it nantly with vascular invasion and pleural involvement. A similar emphasized that methylation of the RASSF1A promoter region correlation with poor survival was also reported by Burbee et al. does not affect expression of the C-isoform. Expression of RASSF1B (15). Conversely, three separate studies did not find any correlation and RASSF1F have also been examined (2, 12, 13, 25). Northern with survival and RASSF1A methylation (57, 59, 60). Instead, Wang blotting showed RASSF1B is expressed most strongly in hemo- et al. suggested RASSF1A methylation was predictive of early poeitic cells and expression was lost in two of four tumor cell lines relapse (60). of lymphoid origin (2). Loss of RASSF1B expression coincided with loss of RASSF1A expression in this study. Similarly, loss of RASSF1B expression occurred in seven of eight bladder cancer cell lines with Correlation between RASSF1A Methylation and loss of RASSF1A expression and was recovered along with RASSF1A Other Oncogenic Events following treatment with demethylating agent (13). Whereas the Due to the evidence linking RASSF1A to Ras signaling pathways, reasons for the loss of RASSF1B expression are not yet understood, several studies have looked for correlation between mutation of none of the samples analyzed exclusively down-regulated RASSF1B. K-Ras and inactivation of RASSF1A by methylation. An inverse Conversely, expression of RASSF1F is intimately connected with correlation was observed in colorectal cancers (61), pancreatic RASSF1A expression because they share a common promoter adenocarcinomas (62), and NSCLC (63). However, a different www.aacrjournals.org 3499 Cancer Res 2005; 65: (9). May 1, 2005

Figure 2. A, comparison of the amino acid (aa) sequence of RASSF1A homologues in Homosapiens (H), Mus musculus (M), Dario rerio (D), and Caenorhabditis elegans (C). Identical aa (black), conserved aa (dark gray), semiconserved aa (pale gray). RA domain (‘‘heavy’’ box); C1 domain (‘‘light’’ box); SARAH domain (hatched box); putative ATM phosphorylation consensus sequence (bold text and underlined). Position of missense changes (*). B, schematic illustration of isoform A of RASSF1A (AAD44174), RASSF2A (AAN59975), RASSF3A (AAO61687), AD037A (AAH32593), NORE1A (NP_872604), and RASSF6 (NP_803876) showing putative functional domains: RA, RalGDS/AF6 Ras association domain; C1, DAG-binding domain; and SARAH, Sav/RASSF/Hpo interaction domain (predicted using Prosite). study of NSCLC (64) found no correlation between methylation at infection of the tumor cells. The HPV can only replicate in dividing RASSF1A and activating mutations in K-Ras; however, individuals cells; therefore, it encodes viral proteins (E6 and E7) to subvert with both defects had a poorer outcome suggesting a synergistic control of the cell cycle by inactivating p53 and Rb, respectively. mechanism of action. Synergy was also proposed in melanomas DNA from HPV types 16 and 18 are particularly associated with (65) where in addition to mutation screening of N-Ras, K-Ras, and transformation. HPV DNA was never found in cervical carcinomas H-Ras, B-Raf was also analyzed and whereas no tumors with B- with RASSF1A methylation (68) suggesting that the presence of Raf mutations had additional mutations in N- or K-Ras, the viral proteins abrogated any requirement for RASSF1A inactivation majority of tumors with RASSF1A methylation had mutations in implicating them both in the same pathway. A similar correlation either B-Raf or N-Ras. However, in thyroid cancer the reverse was observed in a study of HNSCC (69). However, other studies situation was observed such that no tumors in which RASSF1A (9, 70) have not found an association between RASSF1A was methylated had mutations in B-Raf (66). These inconstancies methylation and absence of viral DNA. may reflect other alterations in Ras signaling (67). The ubiquitous human herpes virus, EBV, can transform B cells Cervical cancer and head and neck squamous cell carcinoma into continually growing lymphoblastoid cell lines in vitro. Viral (HNSCC) are associated with human papillomavirus (HPV) DNA and transcripts are associated with a number of neoplasia,

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Table 1. Summary of RASSF1A methylation data in Table 1. Summary of RASSF1A methylation data in primary tumors primary tumors (Cont’d)

Tumor type Percentage References Tumor type Percentage References RASSF1A RASSF1A methylation methylation in tumors in tumors

Acute leukemia 15 (3/20) (12) 71 (37/52) (52) Bladder 62 (34/55) (13) 63 (7/11) (34) 35 (34/98) (14) Rhabdomyosarcoma 61 (11/18) (12) Breast 62 (28/45) (41) Retinoblastoma 59 (10/17) (12) 49 (19/39) (15) Schwannoma 10 (1/10) (26) Biliary tract carcinoma 27 (10/37) (17) Testicular nonseminoma 83 (15/18) (54) Cervical SCC 9.5 (4/42) (68) 21 (9/44) (55) Cervical adenosquamous 21 (4/19) (68) Testicular seminoma 40 (4/10) (54) carcinoma Thyroid 71 (27/38) (35) Cervical adenocarcinoma 24 (8/34) (68) 37 (19/51) (66) Cervical 24 (12/50) (9) Wilms’ tumor 71 (22/31) (58) Cholangiosarcoma 69 (9/13) (19) 54 (21/39) (16) 65 (47/72) (20) Colon 45 (13/29) (16) 20 (45/222) (61) NOTE: Data discussed in text. Esophageal SCC 51 (24/47) (23) Ewing’s sarcoma 0 (0/8) (12) Gastric EBV+ 67 (14/21) (71) Gastric EBVÀ 4 (2/56) (71) Gastric 43 (39/90) (72) including nasopharyngeal carcinoma, endemic Burkitt’s lymphoma, Glioma 54 (25/46) (26) Hodgkin’s disease, and gastric carcinomas. Whereas RASSF1A 57 (36/63) (78) methylation is detected in the majority of nasopharyngeal Hepatoblastoma 19 (5/27) (12) carcinomas (67%), it is difficult to draw any correlations regarding Hepatocellular 85 (70/83) (28) virus infection of the tumor cells because virtually all cases are EBV 100 (29/29) (29) positive. However, compared with other head and neck tumors, Head and neck 17 (4/24) (30) RASSF1A methylation occurs at a much higher frequency in 15 (7/46) (69) Hodgkin’s lymphoma 65 (34/52) (18) nasopharyngeal carcinoma. Comparisons between virus infection Kidney RCC 91 (39/43) (21) and RASSF1A methylation have been possible with Hodgkin’s 26 (44/165) (22) lymphoma and gastric carcinomas because only a subset of these Lung: SCLC 72 (21/29) (6) are EBV associated. In a cohort of 52 Hodgkin’s lymphomas, 27 79 (22/28) (24) were EBV positive and 34 showed RASSF1A methylation (18). Lung: NSCLC 43 (32/75) (36) However, RASSF1A methylation did not correlate with EBV 30 (32/107) (15) infection. In gastric carcinoma, a correlation between EBV 32 (65/204) (37) infection and RASSF1A methylation was detected (71). RASSF1A 42 (42/100) (25) was methylated in 14 of 21 (66.7%) of EBV-positive gastric 37 (190/514) (39) carcinomas, whereas only 2 of 56 (3.6%) of EBV-negative gastric 34 (14/41) (6) 38 (22/58) (2) carcinomas were methylated. In this study, EBV-associated 32 (35/110) (7) methylation of several other TSGs, including PTEN, p16, THBS1, Medulloblastoma 79 (27/34) (10) and MINT12 was also examined and in general the frequency of Malignant mesothelioma 32 (21/66) (40) CpG island methylation was higher in virus infected gastric Melanoma 41 (14/44) (27) carcinomas. Methylation of the viral genome occurs shortly after 15 (3/20) (42) infection to restrict expression of viral transcripts which enable Meningioma 17 (2/12) (26) EBV to evade immune surveillance. This data suggest that EBV is Multiple myeloma 28 (9/32) (43) associated with a methylator phenotype in gastric carcinoma and 0 (0/56) (44) the virus may be involved in disrupting normal DNA methylation Nasopharyngeal carcinoma 67 (20/30) (47) mechanisms. Coincidentally, another study on gastric carcinomas 67 (14/21) (31) Neuroblastoma 55 (37/67) (32) found RASSF1A methylation was associated with poorly differen- 52 (14/27) (12) tiated gastric carcinomas (72), which is also the histologic Osteosarcoma 0 (0/11) (12) subgroup most associated with EBV. Ovarian 40 (8/20) (33) Malignant mesothelioma arises from mesothelial cells lining the 50 (25/50) (48) peritoneum, pleura, and pericardium. About 50% of malignant Pancreatic 62 (47/75) (62) mesotheliomas are associated with SV40. In a study comparing the Phaeochromocytoma 22 (5/23) (32) methylation of profile of 66 malignant mesotheliomas and 40 lung Prostate 53 (53/101) (51) adenocarcinomas, Toyooka et al. showed methylation of RARb, CDH13, MGMT, p16, and APC was significantly greater in lung www.aacrjournals.org 3501 Cancer Res 2005; 65: (9). May 1, 2005

Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 2005 American Association for Cancer Research. Cancer Research adenocarcinomas (22-52%) than in malignant mesotheliomas (0- nance, whereas DNMT3b is involved in de novo DNA methylation. 10%; ref. 73). However, methylation of RASSF1A was not However, in tumor cells, a paradox exists with global hypomethy- significantly different between the two tumor types, 32% malignant lation of the genome, hypermethylation of CpG islands, and mesothelioma versus 43% lung adenocarcinoma. SV40 Tag was overexpression of DNMTs. Hypomethylation contributes to tumor- present in 48% (32 of 66) of malignant mesotheliomas and igenesis through loss of imprinting, genomic instability, and was shown to correlate with a higher methylation index than in activation of transposable elements. Whereas the mechanisms SV40-negative malignant mesotheliomas. Furthermore, RASSF1A that lead to aberrant CpG island methylation are not fully methylation was significantly higher in SV40-positive malignant understood, recent data by Kawasaki et al. may provide clues to mesotheliomas (48%) than SV40-negative cases (16%). The unraveling this mystery (80). Many organisms use RNA interference authors investigated the connection between aberrant methyla- (RNAi) to regulate gene expression post-transcriptionally. Micro- tion and SV40 by infecting mesothelial cells with virus and RNAs (miRNAs), 21 to 23-nucleotide ds-RNAs, are the effector examining promoter methylation in early (8-30) and late (15-86) molecules for RNAi. Exploitation of this mechanism using passaged cells. No promoter methylation was detected in early chemically synthesized small inhibitory RNAs (siRNAs) is proving passaged cells but in two of six foci of late-passage cells. RASSF1A very fruitful for functional genomics, especially for the study of promoter methylation was detected with concomitant down- tumor suppressor gene inactivation in cell lines. However, if siRNA regulation of RASSF1A. These cells also displayed morphologic is designed to target CpG islands within gene promoters, sequence- signs of transformation (74). Expression was recovered using specific methylation of DNA is induced with concomitant down- 5-aza-2V deoxycytidine but not with the histone deacetylase inhib- regulation of transcription. siRNA-directed methylation of the itor trichostatin A indicating that SV40 induces methylation rather E-cadherin promoter has been shown in breast a tumor cell line (80). than histone deacetylation of the RASSF1A promoter. How this is achieved is not yet understood, but recent microarray data from 27 prostate cancers suggests that endogenous antisense transcripts required to generate siRNAs are synthesized in vivo from Epigenetic Inactivation of Other Ras Association intragenic promoters including RASSF1 (81). It is tempting to Domain Family Members speculate, therefore, that RNAi may lie at the heart of epigenetic NORE1A but not NORE1B was shown down-regulated in lung inactivation of RASSF1A and other commonly inactivated TSGs. tumor cell lines. Down-regulation of NORE1A expression correlated with promoter CpG island hypermethylation (75) and was found Mutation Analysis of RASSF1A Gene methylated in 24% of NSCLC tumors. Two missense changes were As already mentioned, mutation of RASSF1A occurs rarely in identified in a mutational screen of 20 lung tumor cell lines (75). human cancers. As far as we can ascertain, only one frame-shift Hypermethylation of the NORE1A CpG island in NSCLC has an mutation at codon 277 (within the Ras association domain) has been inverse correlation with K-RAS mutations suggesting that they have reported derived from a nasopharyngeal carcinoma (31). A number a cooperative role in carcinogenesis (76). Chen et al. (77) identified of missense changes and polymorphisms have been found in two breakpoint-spanning genes, LSAMP and NORE1 in a familial nasopharyngeal carcinoma, lung, breast, and kidney carcinomas clear cell renal cell carcinoma (CCRCC)–associated translocation (6, 15, 21, 31). Many of these localize to the functional domains of t(1;3)(q32.1;q13.3). NORE1A was found methylated in 32% of RASSF1A, three in the DAG binding domain, four in the ATM sporadic CCRCC. RASSF2, like NORE1, is widely expressed (with phosphorylation domain, and five in the Ras association domain particularly high levels in the brain and blood), isoform A of (Figs. 2B and 3). The functional significance of these changes has not RASSF2 (RASSF2A) is frequently methylated and silenced in been fully investigated, but data suggests that they are defective. For colorectal and lung tumors and tumor cell lines.1 ADO37 seems instance, unlike wild-type RASSF1A, a C65R/V211A mutant does not expressed in most tissues and is methylated in lung and breast suppress growth of LNCaP prostate carcinoma cells or KRC/Y renal tumors but rarely in nasopharyngeal carcinomas or glioma tumors cell carcinoma cells, in vitro (34, 82). Phosphorylation of RASSF1A (78, 79). RASSF3 is unmethylated in human cancers (78), whereas is reduced in the S131F mutation of the putative ATM site, which RASSF6 methylation status in tumors has not been reported. also results in less efficient inhibition of cell proliferation (83). Two missense changes, C65R and R257Q, relocate RASSF1A from the Methylation and RNA Interference cytoplasm to the nucleus and have diminished ability to halt the cell cycle (84). This strongly suggests that cellular localization of During embryogenesis, tissue-specific DNA methylation patterns RASSF1A is also vital to its function. Hence, study of naturally are established for the correct regulation of imprinted genes such occurring tumor associated missense changes is proving fruitful for as insulin-like growth factor II and X chromosome inactivation. dissecting out the relative importance of the different putative The majority of the rest of the genome is methylated with the functional domains of RASSF1A. exception of CpG islands, most of which are associated with the promoter regions of housekeeping genes. As well as transcriptional regulation, methylation of the genome is thought to suppress the RASSF1 Function activity of transposable elements (e.g., LINE-1 and Alu) that can Frequent inactivation of RASSF1A gene in human cancers disrupt gene function. Some of the DNA methyltransferases suggests that it must have a pivotal role in tumor prevention. This (DNMT1, DNMT2, DNMT3a, DNMT3b, and DNMTL) involved in notion is supported by the phenotype of tumor cell lines with these processes have been identified. DNMT1 ensures mainte- constitutive overexpression of RASSF1A. The observations in NSCLC, prostate, kidney, nasopharyngeal carcinoma, and glioma cell lines (2, 15, 21, 34, 78, 85) indicate that RASSF1A expressing cells are less viable, growth suppressed, less invasive, and have reduced 1 Hesson and Latif, unpublished data. anchorage/substrate independence. Using a tetracycline regulation

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Figure 3. Schematic representation of RASSF1A summarizing sites of protein interactions (horizontal lines). Position of missense changes (black circles). Bottom, scale of amino acid positions. system, RASSF1C has also been shown to inhibit colony formation mouse embryonic fibroblasts (MEFs) generated from rassf1 null in vitro (82). In addition, mutations detected in a gene inactivation mice are smaller than wild-type MEFs and show increased test suggest that RASSF1C may also function as a TSG (82). sensitivity to the tubulin depolymerizing agent nocodazole (89). Subsequent studies have indicated RASSF1A functions as a We and others have shown that transfection of RASSF1A into modulator of two pathways commonly deregulated in cancer, RASSF1A-negative cell lines induced the microtubular structures apoptosis, and cell cycle (83, 86). Microarray analysis of tumor cell radiating from the centrosome to become hyperstabilized circular lines (NSCLC and neuroblastoma) stably expressing RASSF1A structures and also protected the cells from the action of identified expression changes consistent with the observed pheno- nocodazole (84, 89–91). Microtubular association of tumor typic effects such as reduced cell cycle effectors (Cyclins D1 and D3), suppressor proteins is not without precedent, as both the increased cell adhesion molecules (N-cadherin) and alterations in adenomatous polyposis coli (APC) and von Hippel-Lindau (pVHL) the levels of a number of extracellular matrix modifiers (SPARC and proteins have been shown to bind to and stabilize microtubules ANPEP; ref. 87). Several of 66 RASSF1A-regulated genes identified by suggesting that microtubule destabilization may be an important this study are also affected by activated Ras during cellular facet of tumorigenesis. The RASSF1A mutants C65R and R257Q, transformation. Interestingly, our data found that RASSF1A and which fail to localize to the microtubules, were unable to protect Ras had the reciprocal effect on their expression, which might be against this nocodazole-induced depolymerization (84). Deletion further evidence to suggest that these molecules do indeed function analysis (89) showed that a region between amino acids 120 and in the same pathway. By identifying RASSF1A-interacting proteins 288 of RASSF1A was required for microtubule association, and a (Figs. 3 and 4), we are beginning to understanding how RASSF1A subsequent deletion analysis defined a smaller region amino acids achieves growth suppression or induces apoptosis and may provide 120 to 185 as the microtubule association domain (90). The new therapeutic targets for the treatment of human cancers. RASSF1A peptide comprised of amino acids 120 to 185 destabilized microtubules and was termed dominant-negative RASSF1A for its ability to inhibit RASSF1A-induced cell death. Acetylation Microtubules, Adhesion, and Migration of a-tubulin is associated with microtubule stability, and transfec- The first mice lacking RASSF1 were made by Smith et al. (88) tion of NCI-H1299 cells with RASSF1A induced high levels of who generated a targeting vector to the mouse chromosome acetylated a-tubulin; however, the RASSF1A mutants C65R and syntenic to the minimal region of human chromosome 3p21.3 R257Q were less competent at induction of microtubule acetylation deleted in lung tumors, this 370-kb deletion knocked out 12 genes (84). The microtubule stability induced by RASSF1A could have including rassf1. Heterozygous mice with this contiguous gene implications for cell adhesion and motility, especially in the light of deletion were viable and fertile; however, the homozygous null data showing that genes for cell adhesion and motility such as embryos died before 13.5d.p.c. Subsequently, mice were generated tropomyosin I and CDH2 (N-CAD) were up-regulated in A549 cells with a targeted deletion of the rassf1-coding region (89). These mice stably expressing RASSF1A (87). have been bred to homozygosity and shown to have no obvious Wild-type RASSF1A was shown to associate with microtubules in phenotype; however, they have not been grown to old age nor have immunofluorescence (84, 89) and cosedimentation assays, whereas they been treated with tumor promoting agents. Interestingly mutant versions showed varying abilities to colocalize with www.aacrjournals.org 3503 Cancer Res 2005; 65: (9). May 1, 2005

Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 2005 American Association for Cancer Research. Cancer Research microtubules. Fewer than 30% of cells transfected with C65R or effects of nocadozole. RASSF1C can also associate with micro- R257Q showed RASSF1A localizing to microtubules, whereas tubules (90); however, it is less effective at stabilizing them. f90% of wild type and 80% of mutants K21Q, S131F, A133S, RASSF1C has also been shown to interacts with C19ORF5 (93), R201H, V211A, Y325C, A336T localized to the microtubules (84). which in turn can interact with the SEC-1 domain of leucine-rich Bromodeoxyuridine incorporation assays showed that the mutants pentapeptide repeat motif–containing protein (LRPPRC, also called deficient in their ability to bind microtubules (C65R or R257Q) were gp130 and LRP130). LRPPRC also interacts with UXT (93), which in also deficient in their ability to stop DNA synthesis in NCI-H1299 turn interacts with BUB3 that is involved in the kinetochore cells (84) suggesting a potential connection between competency to checkpoint that ensures cells containing misaligned chromosomes bind microtubules and ability to induce cell cycle arrest. do not exit from mitosis prematurely. C19ORF5 and LRPPRC The localization of RASSF1A can be seen to alter during mitosis colocalize with h-tubulin in the cytoplasm; however, in apoptotic (92); cytoplasmic microtubular association was confirmed during cells, they are found to colocalize in the nucleus (93). An interphase. However, as the cells progressed into prophase, interaction between C19ORF5 and the mitochondrial proteins RASSF1A relocated to the separated centrosomes, then to the (NADH-dehydrogenase subunit 1, cyclooxygenase-1) and LRPPRC spindle fibers and poles during metaphase and anaphase and finally associates RASSF1 with mitochondria, an organelle with pivotal to the midbody during cytokinesis in HeLa cells. functioning in control of apoptosis. Microtubule association is likely to play an important role in the function of RASSF1. A yeast two hybrid screen (84) for RASSF1- interacting proteins showed binding to MAP1B and C19ORF5 (also Apoptosis called BPY2IP or VCY2IP1); in fact, 70% of interacting clones had RASSF1 was predicted to form a soluble cytoplasmic protein homology to microtubule-associated proteins. The RASSF1A with a Ras association domain (1). Ras is a small inner membrane– interacting protein C19ORF5 colocalized with RASSF1A to the embedded GTPase that relays proliferative signals from cell surface microtubules, but overexpression of C19ORF5 unlike overexpres- receptors such as receptor tyrosine kinases. Ras becomes activated sion of RASSF1A could not protect against the depolymerizing by binding to GTP causing a shift in conformation revealing

Figure 4. A summary of RASSF members pathways and interactions. RASSF proteins can regulate the microtubule network, cell cycle progression, or apoptosis by recruiting common and unique effectors. Initial stimuli are likely to originate from mitogen receptors, ion channels, etc. RASSF members are also linked to several pivotal proteins such as Ras proteins (including Ran0, pRb, p53, p14ARF, and Cdc20). Homodimerization and heterodimerization between the RASSF members and between MSTs may be a central regulatory feature of the dynamics of these pathways.

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Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 2005 American Association for Cancer Research. RASSF1 Tumor Suppressor Gene cytoplasmic epitopes that mediate interactions with effector the two proteins; this was shown by deletion of the C-terminal proteins such as Raf kinases; Ras signaling may be an important (RASSF1 interacting) region of MST1 that prevented MST1 from chemotherapeutic target. There are three Ras family members, H- interacting with CNK1. Despite the fact that both RASSF1A and Ras, K-Ras, and N-Ras, which are located at 11p15.5, 12p12.1, and RASSF1C can interact with CNK1, only RASSF1A could augment 1p13.2, respectively. K-Ras has been shown to most strongly CNK-induced apoptosis (98). interact with RASSF1. RASSF1 has been shown to directly bind to Ras in a GTP-dependent fashion (86) and also to interact via heterodimerization with NORE1 (94). Ras is an important Cell Cycle modulator of the apoptotic response and is thought to mediate Deregulation of cell cycling is an essential prerequisite for cell survival in response to hyperproliferative signals in trans- tumorigenesis. In normal cells, cycling is exquisitely controlled by a formed cells through PI3-K– and Tiam-1–mediated pathways and number of protein complexes whose activity is required for the cell to induce apoptosis in untransformed cells through as yet to pass through specific checkpoints. For example, mitotis- incompletely understood pathways that may be mediated by promoting factor, a cyclinB/Cdc2 complex that phosphorylates RASSF1 and NORE1. mitotic regulators allowing the cell to progress from G2 to M. The difficulty of generating NIH 3T3 cells stably expressing Anaphase promoting complex/cyclosome (APC/C) is a protein RASSF1C gave an initial clue that RASSF1 may play a role in complex that interacts with ubiquitin-conjugating and activating apoptosis (86). Activated Ras-GTP can directly bind RASSF1C and enzymes to catalyze the poly-ubiquitylation of proteins destined for can enhance RASSF1-induced apoptosis in 293 cells (86). Transient degradation (99) to allow the cell cycle to progress. APC/C is transfections were used to show that RASSF1C could induce cell activated by complexing with Cdc20 or cdh1 (proteins that contain death in 293 cells, and that activated Ras could augment this cell WD40 repeats). These WD40 repeat proteins are required for APC/C death, whereas dominant-negative Ras inhibited the cell death. The to interact with target proteins. During S, G2, and prophases, APC/C caspase inhibitor z-VAD-fmk reduced RASSF1C-induced cell death is inhibited by sequestration of Cdc20 by Emi1; later during implicating an apoptotic mechanism. This information was prometaphase, RASSF1A takes on the role of regulator as it in turn augmented by demonstration in a yeast two hybrid assay that sequesters Cdc20, and coimmunoprecipitation of RASSF1A with human RASSF1, murine NORE1, and Caenorhabditis elegans Cdc20 was shown by Song et al. (92). To allow progression this T24F1.3 all specifically bound to the Ste20-related proapoptotic repression by RASSF1A must be released allowing APC/C to become kinase mammalian Sterile20-like (MST1) through their common active inducing the polyubiquitylation and degradation of cyclin A. conserved COOH-terminal tails (95). MST1 and MST2 (also called Coexpression of Cdc20 with RASSF1A suggests that it is the relative STK4 and STK3, respectively) are serine/threonine kinases that levels of these proteins that is important. During prometaphase, initiate apoptosis when overexpressed (96). The Drosophila loss of RASSF1A and Cdc20 are colocalized, but during mitosis, only a small MST function mutant (hippo) fails to undergo accurate develop- proportion coprecipitates (92). Overexpression of RASSF1A leads to mental apoptosis. MST becomes autoactivated by autophospho- accumulation in prometaphase with raised levels of cyclins A and B rylation of threonine (Thr183 of MST1 and Thr180 of MST2), and this (92); this arrest is before the metaphase to anaphase transition. The autoactivation of MST1 is inhibited by cotransfection with spindle checkpoint keeps the cells in metaphase until chromosomes RASSF1A or RASSF1C or NORE1A or NORE1C but is augmented are correctly aligned; the kinetochores induce assembly of the by cotransfection of membrane-localized NORE1A-CAAX (97). inhibitory proteins Mad2 and Mad3 on incorrectly tensioned MST1 contains caspase cleavage sites, cleavage with caspase 3 spindles. These inhibit the APC/C until alignment is correct when generates a 36-kDa fragment seen during apoptosis, and cleavage the repression is relieved allowing the APC/C-Cdc20 to ubiquitinate with caspase 6 or 7 generates a 41-kDa product. securin (a separase inhibitor) targeting it for degradation and Activated Ras can bind NORE1 and RASSF1. RASSF1 and NORE1 initiating sister chromatid separation and anaphase. can form both homodimers and heterodimers (94), which complex During telophase, an APC/C-cdh1 complex targets mitotic with Mst-1 regulating its seriene/threonine kinase activity. The cyclins for destruction resulting in exit from mitosis. The APC/C MST1 binding site is at the COOH-terminal end of RASSF1A (amino complex is kept in its inactive form by phosphorylation of cdh1 acids 358-413 of NORE1; ref. 95). Whereas overexpression of MST1 inhibiting its ability to bind APC/C. cdk1 maintains cdh1 in its could induce apoptosis in NIH 3T3 cells, and coexpression with phosphorylated state, whereas Cdc14 induces dephosphorylation of NORE1 had a small effect on increasing apoptosis, the addition of a cdh1 when the spindle becomes correctly orientated. CAAX motif to NORE1 to induce membrane targeting enabled The cyclins D1 and D3 were also shown RASSF1A regulable, NORE1 alone to induce apoptosis, and NORE1 together with MST1 because they were down-regulated in A549 cells stably transfected had a greater proapoptotic effect than either protein alone (95). with RASSF1A (87). Another cyclin, cyclin A (that regulates CDK2 This cell death was inhibitable by the caspase inhibitor z-VAD-fmk thereby controlling progression through S phase) is regulated by the (95). A model was proposed (97) whereby NORE1 and MST1 transcription factor p120E4F (100). We have shown that p120E4F can constitutively form a complex maintaining MST in an inactive bind to RASSF1A (101) and this interaction was shown mediated by reservoir until activation by serum stimulation is able to induce amino acids 1 to 119 of RASSF1A. Cotransfection of RASSF1A with E4F association with Ras. p120 -induced G1 arrest of a greater magnitude than that induced CNK1 (CNKSR1) is a scaffold protein that in Drosophila is by transfection with either construct alone. p120E4F provides a required for Ras to activate Raf kinase. Transfection of CNK1 into mechanistic link to other known tumor suppressor genes such as 293 cells can induce apoptosis (98), and this can be abrogated by p14ARF, Rb, and p53 that are known to interact with p120E4F. This dominant-negative inhibitors of MST1/2. CNK1 has been shown to suggests that RASSF1A maybe able to affect cyclin A expression. bind to RASSF1A or RASSF1C but not to NORE1 (98). RASSF1 has Systematic studies were undertaken by two hybrid screens to previously been shown to interact with MST1/2 (95); therefore, it determine possible RASSF1-interacting partners. Mst-1, C19ORF5, was thought that RASSF1 may be providing the mechanism linking MAP1B, p120E4F, and CNK1 were identified as interacting partners www.aacrjournals.org 3505 Cancer Res 2005; 65: (9). May 1, 2005

(refs. 84, 95, 98, 101; Figs. 3 and 4). Additionally, the catalytic mechanism; cell cycle analysis also showed a decreased proportion domain of plasma membrane calmodulin-dependent calcium of cells in G2-M implicating G0-G1 arrest. ATPase (PMCA4b, also called ATP2B4) was found via a bacterial AD037 (also called RASSF4) has been shown to bind activated, two hybrid screen to interact with RASSF1 through amino acids 74 but not wild-type K-Ras and for the two proteins to act to 123 of RASSF1C or amino acids 144 to 193 of RASSF1A (102). synergistically to induce apoptosis in 293T cells. AD037 can also PMCA4b is involved in expelling calcium from cells and may also inhibit the growth of human tumor cell lines, and this inhibition play a role in signaling because it interacts with proteins such as can be enhanced by the addition of a tag (CAAX) to induce nitric oxide synthase I and calcium/calmodulin–dependent serine membrane localization thus mimicking the effect of activation (79). protein kinase. The interaction between RASSF1 and PMCA4b No functional studies on RASSF3 or RASSF6 have been reported. reduced epidermal growth factor (EGF)–dependent activation of Erk, a downstream target of the Raf, Ras, and mitogen-activated protein kinase cascade (102). Concluding Remarks RASSF1A tumor suppressor gene undergoes frequent tumor- specific epigenetic inactivation in a wide range of tumors. RASSF1A Functional Analysis of Other RASSF Family methylation has also been shown in preneoplastic lesions and in Members patient’s DNA obtained using noninvasive procedures such as urine NORE1 (novel Ras effector, also called RASSF5) was originally from kidney cancer patients and sputum from lung cancer patients. identified (103) from a mouse T-cell library by its ability to bind to Hence, RASSF1A methylation can potentially be developed as a Ras-GTP in a yeast two hybrid screen. Confirmation of this molecular biomarker for screening cancer patients and populations interaction by immunoprecipitaion showed that NORE1 only bound at risk. At least three other RASSF1 homologues (NORE1, AD037, to Ras after stimulation with EGF or 12-O-tetradecanoylphorbol-13- and RASSF2) are also inactivated in tumors by promoter hyper- acetate. NORE1 expression inhibited cell growth and this inhibition methylation. Whereas somatic mutational inactivation is a rare was augmented by expression of H-Ras, whereas cotransfection event for all RASSF family members analyzed thus far. Although, with the antiapoptotic agent Bcl2 blocked this growth inhibition several of the missense changes reported in RASSF1A have been implicating an apoptotic mechanism (104). Transfection of NORE1A shown functionally relevant. The inverse correlation reported for or NORE1B into cell lines with low endogenous NORE expression RASSF1A and NORE1A inactivation and K-ras alterations in some suppressed colony formation in A549 and G361 lines (which have tumor types may provide alternative pathways for affecting Ras disrupted Ras signaling due to a Ras activating mutation or signaling. constitutively active B-Raf kinase respectively). However, when Studies are in progress to elucidate the function of the protein transfected into other lines with similar disruptions to Ras signaling products of the RASSF1 family of TSGs. Reintroduction of RASSF1A (NCI-H460 and M14), there was no effect on colony formation (105). in tumor cell lines lacking endogenous expression decreased In the A549 cell line, NORE1A inhibited anchorage-independent in vitro colony formation and in vivo tumorigenicity. It can induce growth and induced G1 arrest but failed to induce apoptosis (105). apoptosis likely through interactions with the MST proteins. Deletion of the COOH-terminal MST-interacting domain and the RASSF1A can also bind to and stabilize microtubules, a property Ras binding domain and/or mutation of the zinc finger domain had that seems central to its function, and through modulation of APC/C minimal effect on the growth suppressive activities of NORE1, activity can affect cell cycle regulation. Rassf1À/À mice are viable leading Aoyama et al. (105) to conclude that the growth suppression and fertile; it remains to be seen if they are prone to increased of NORE1 was due to amino acids 188 to 250. However, binding to spontaneous and or induced tumor formation. There has been an MST1/2 or Ras-like GTPases had previously been shown required explosion of reports on RASSF1A methylation in cancer; hopefully, for growth suppression (95, 104). NORE1 had been shown to induce the next few years will yield further insights into the biology of this apoptosis in 293T cells (104) rather than the cell cycle delay seen in important family of tumor suppressor genes. A549 cells (105). RASSF2 was first identified by Comincini et al. (106) and it seems up-regulated in radiation workers (107). RASSF2 (originally called Addendum Rasfadin or KIAA0168) can bind to K-Ras in a GTP-dependent After this article went to press, RASSF1A isoform-specific knockout mice were manner through its Ras effector domain (108); however, it only reported to demonstrate increased susceptibility to spontaneous and chemically induced tumors (Tomassi S, Denissenko MF, Pfeifer GP. Cancer Res 2005;65:92–8). weekly binds to H-Ras. Overexpression of RASSF2 inhibited growth of lung tumor cell lines, and Vos et al. (108) were unable to generate cell lines stably overexpressing RASSF2. In transient assays, RASSF2 Acknowledgments inhibited cell growth, and this inhibition was augmented when cotransfected with K-Ras, whereas H-Ras had little additional effect. Received 11/15/2004; revised 1/19/2005; accepted 2/1/2005. Grant support: Sport Aiding Medical Research for Kids, the Wellcome Trust, Cell death induced by RASSF2 coexpressed with activated K-Ras Breast Cancer Campaign, Association for International Cancer Research, and was shown mediated by caspase 3 and hence via an apoptotic Birmingham Children’s Hospital Research Fund.

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Cancer Res 2005; 65: (9). May 1, 2005 3508 www.aacrjournals.org

RASSF1 Tumor Suppressor Gene In the review on RASSF1 tumor suppressor gene in the May 1, 2005 issue of Cancer Research (1), there was an error in Fig. 1. The correct Fig. 1 appears below. Also, in the Addendum, the reference should have read as follows: Tommasi S, Dammann R, Zhang Z, et al. Cancer Res 2005;65:92–8.

1. Agathanggelou A, Cooper WN, Latif F. Role of the Ras-association domain family 1 tumor suppressor gene in human cancers. Cancer Res 2005;65:3497–508.

Cancer Res 2005; 65: (12). June 15, 2005 5480 www.aacrjournals.org Role of the Ras-Association Domain Family 1 Tumor Suppressor Gene in Human Cancers

Angelo Agathanggelou, Wendy N. Cooper and Farida Latif

Cancer Res 2005;65:3497-3508.

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