Role of the Ras-Association Domain Family 1 Tumor ... · some 3p likely to harbor one or more tumor suppressor genes, including 3p12, 3p14, 3p21.3, and 3p25-26. In 1993, the von Hippel-Lindau

Role of the Ras-Association Domain Family 1 Tumor

Suppressor Gene in Human Cancers

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

In recent years, the list of tumor suppressor genes (orcandidate TSG) that are inactivated frequently by epigeneticevents rather than classic mutation/deletion events has beengrowing. Unlike mutational inactivation, methylation isreversible and demethylating agents and inhibitors of histonedeacetylases are being used in clinical trails. Highly sensitiveand quantitative assays have been developed to assessmethylation in tumor samples, early lesions, and bodily fluids.Hence, gene silencing by promoter hypermethylation haspotential clinical benefits in early cancer diagnosis, prognosis,treatment, and prevention. The hunt for a TSG located at3p21.3 resulted in the identification of the RAS-associationdomain family 1, isoform A gene (RASSF1A). RASSF1A fallsinto the category of genes frequently inactivated by methyl-ation rather than mutational events. This gene is silenced andfrequently inactivated by promoter region hypermethylationin many adult and childhood cancers, including lung, breast,kidney, gastric, bladder, neuroblastoma, medulloblastoma,and gliomas. It has homology to a mammalian Ras effector(i.e., Nore1). RASSF1A inhibits tumor growth in both in vitroand in vivo systems, further supporting its role as a TSG. Weand others identified the gene in 2000, but already there areover a 150 publications demonstrating RASSF1A methylationin a large number of human cancers. Many laboratoriesincluding ours are actively investigating the biology of thisnovel protein family. Thus far, it has been shown to playimportant roles in cell cycle regulation, apoptosis, andmicrotubule stability. This review summarizes our currentknowledge on genetic, epigenetic, and functional analysis ofRASSF1A tumor suppressor gene and its homologues. (CancerRes 2005; 65(9): 3497-508)

Gene Identification

The RAS-association domain family 1, isoform A (RASSF1A)tumor suppressor gene (TSG), is a member of a new group of RASeffectors thought to regulate cell proliferation and apoptosis.Formally known as 123F2 , two laboratories independently clonedand sequenced RASSF1 (1, 2). Loss of heterozygosity (LOH) studies inlung, breast, and kidney tumors identified several loci in chromo-some 3p likely to harbor one or more tumor suppressor genes,including 3p12 , 3p14 , 3p21.3 , and 3p25-26 . In 1993, the von Hippel-Lindau (VHL) TSG inactivated in familial and sporadic clear cell

renal cell carcinomas was identified from region 3p25 confirmingthis hypothesis (3). This prompted the search for TSGs within theother regions of 3p. An important TSG was suspected to reside in3p21.3 because instability of this region is the earliest and mostfrequently detected deficiency in lung cancer. Overlapping homo-zygous deletions in lung and breast tumor cell lines reduced thecritical region in 3p21.3 to 120 kb and this region was found to beexceptionally gene rich. From this critical region, eight genes wereidentified, including CACNA2D2 , PL6 /placental protein 6 ,CYB561D2/101F6 , TUSC4/NPRL2/G21 , ZMYND10/BLU, RASSF1/123F2 , TUSC2/FUS1 , andHYAL2/LUCA2 . However, despite extensivegenetic analysis in lung and breast tumors, none of these candidategenes were frequently mutated. Meanwhile, Dammann et al. (2)identified RASSF1 in a yeast two-hybrid screen baited with XPA.Although the interaction was not formally shown, this novel genewas of interest because it mapped to the 3p21.3 entry in Genbank, itshared high-sequence homology with a known RAS effector inmouse (Nore1), and expression of one of the isoforms, RASSF1A , butnot RASSF1C , was lost in most lung tumor cell lines. Subsequently,hypermethylation of the RASSF1A promoter region CpG island wasidentified as themajor cause for loss of expression. Cells treatedwithdemethylating agents reexpressed RASSF1A , which confirmed therole of DNAmethylation in the inactivation of RASSF1A in tumor celllines. Finally, overexpression of RASSF1A but not RASSF1C in non–small cell lung cancer (NSCLC) A549 cells reduced colony formationefficiency and suppressed growth of tumor cells in nudemice in vitroand in vivo growth assays, respectively.Since the discovery of the first tumor suppressor gene, RB ,

Knudson’s ‘‘two-hit’’ hypothesis (4) has been used to define thisclass of genes. The hypothesis states that inactivation of bothalleles of a TSG, classically by genetic insult, are required fortumorigenesis. Recently, this model was extended to includeepigenetically inactivated genes. Hypermethylated in cancer-1,HIC1 , a gene on 17p13.3, is hypermethylated in a number ofsporadic human tumors. In a study of Hic1+/� mice by Chen et al.(5), development was normal but the mice were predisposed tomalignant tumors at 70 weeks. Examination of the tumorsrevealed loss of Hic1 expression in the context of an intact allele.Methylation analysis then showed that the remaining allele wasinactivated by hypermethylation. Hence, inactivation of Hic1 inthis mouse model satisfied Knudson’s criteria for a TSG. The first‘‘hit’’ being the genetic knockout of one allele and the second‘‘hit’’ being hypermethylation of Hic1 promoters. Using acombination of combined bisulfite and restriction analysis andLOH analysis inactivation of RASSF1A in sporadic lung tumorswas shown to obey Knudson’s model for a TSG. In SCLC,hypermethylation was detected in 76% tumors with allelicimbalance at 3p21.3 indicating that genetic and epigeneticmechanisms provide the two ‘‘hits’’ to inactivate RASSF1A (6).Similarly, RASSF1A methylation was detected in 52% of NSCLC,72% of bladder transitional cell carcinoma, and 70% of cervical

Note: Due to space limitation, it has not been possible to include all referencesrelating to RASSF1A methylation.

Requests for reprints: Farida Latif, Section of Medical and Molecular Genetics,Division of Reproductive and Child Health, The Institute of Biomedical Research,University of Birmingham, Edgbaston, Birmingham, B15 2TT, United Kingdom. Phone:44-121-627-2741; Fax: 44-121-627-2618; E-mail: [email protected].

I2005 American Association for Cancer Research.

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squamous cell carcinoma (SCC) with allelic imbalance at 3p21.3(7–9). In primary medulloblastoma, however, in the absence ofallelic imbalance at 3p21.3, there is evidence to suggest biallelicmethylation is the major mechanism for RASSF1A inactivation inthis tumor type (10).

RASSF1 Gene Locus and Protein Structure

The RASSF1 gene locus spans about 11,151 bp of the humangenome and is comprised of eight exons. Differential promoterusage and alternative splicing generates seven transcripts(RASSF1A-G ; Fig. 1). Isoforms A and C are ubiquitouslyexpressed, whereas isoform B is mainly expressed in cells ofthe hemopoietic system. Isoforms D and E are specificallyexpressed in cardiac and pancreatic cells, respectively. They arevery similar to RASSF1A except for slight differences in thesplice sites used in exons 2ah (RASSF1D) and 3 (RASSF1E)providing each protein with four additional amino acids. TwoCpG islands are associated with the promoter regions of RASSF1.The smaller of the two (737 bp, 85 CpGs, 71.5% GC and OE:0.89)spans the promoter region of RASSF1A (and RASSF1D , RASSF1E ,RASSF1F, and RASSF1G). The second CpG island (1365 bp, 139CpG,67.9% GC and OE:0.88) encompasses the promoter regions for

RASSF1B and RASSF1C . The entire first exon of each RASSF1transcript is contained within the CpG islands.A Ras association or RalGDS/AF-6 domain encoded by exons 4

and 5 defines the RASSF1 gene and is located at the COOHterminus of isoforms A-E . This domain mediates interactions withRas and other small GTPases. Despite the absence of any sequencehomology, it has a similar structure to the RasGTP binding domainof Raf1 (kinase), the most studied RasGTP effector. The Rasassociation domain of RalGDS is comprised of a five-strandedmixed h-sheet and three a helices. Ras association domainshomodimerize via intermolecular disulfide bonds, formed usingtwo cysteine residues from each monomer. The interactionbetween RalGDS homodimers and Ras is mediated mostly by twoantiparallel h-strands within the Ras association domain and Ras.RASSF1A and RASSF1D-G isoforms have a NH2-terminal proteinkinase C (PKC) conserved region 1 (C1) domain encoded by exons1a and 1h. In PKC, this is a diacylglycerol (DAG)/phorbol esterbinding domain that regulates kinase activity. Overlapping the C1domain is a putative zinc-binding domain, ZnF_NFX. This domainis present in a transcriptional repressor of HLA-DRA, NK-X1, and inDrosophila shuttle craft protein, vital for the late stages of embryonicneurogenesis. A SARAH domain, (Sav/RASSF/Hpo) is present at theCOOH 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 alternativesplicing. The positions of promoter associated CpG islands (black lines ) as predicted by the UCSC Genome Browser May 2004. The domain structure of the proteinproducts (predicted using Prosite): C1, DAG-binding domain; RA, RalGDS/AF6 Ras association domain; and SARAH, Sav/RASSF/Hpo interaction domain. ATM is theATM-kinase phosphorylation consensus sequence (11). Position of the extra 4aa in RASSF1D and RASSF1E (white ).

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interactions between proteins as shown for Hpo/Sav and homotypicinteractions as shown with Mst1. In vitro studies have identified apeptide sequence within exon 3 that is a substrate for ataxiatelagectasia mutant (ATM) kinase. ATM is vital for the activation ofp53 following exposure to ionizing radiation. Phosphorylation levelsof the RASSF1 peptide sequence WETPDLSQAEIEQK were compa-rable with levels of the ATM consensus sequence in p53 suggestingthat RASSF1 might also be a substrate for ATM (11). The RASSF1ATM site is present in isoforms A, C, D, and E.

RASSF1 Homologues and Orthologues in ModelOrganisms

RASSF1A homologues with 38% to 85% identity are present inrodents, fish, and nematodes (Fig. 2A). Each has a COOH-terminalRas association domain and SARAH domain and a NH2-terminalC1 domain. In Xenopus , a RASSF1C homologue (MGC82975) hasbeen identified but the genomic sequence is not available to verifythe presence of RASSF1A . In Drosophila , an 89.6-kDa protein(LD40758p) has the closest homology with RASSF1A . However,although it has a Ras association domain at the COOH terminus, aLIM domain not a C1 domain is predicted at the NH2 terminus.Homology searches within the human genome have identifiedother Ras association domain containing genes called RASSF2(20pter-p12.1), RASSF3 (12q14.1), AD037 (10q11.21), NORE1(1q32.1), and RASSF6 (4q21.21; Fig. 2B). In common with RASSF1 ,these genes code for multiple transcripts and CpG islands areassociated with their promoter regions. The Ras association andSARAH domains of these genes are located at the COOH terminusindicating that they share similar structural organization withRASSF1 . NORE1 which has the most sequence identity with RASSF1(49%) also has a putative NH2-terminal C1 domain.

Tumor-Associated Methylation of RASSF1A

Since the discovery of RASSF1A inactivation in lung tumors, awealth of literature has been published implicating RASSF1A inthe pathogenesis of a wide spectrum of tumors (Table 1 andreferences within).A direct correlation between promoter methylation and loss

of RASSF1A expression has been shown in many tumor cell lines,including SCLC, NSCLC, breast, kidney, nasopharyngeal carci-noma, prostate, ovarian, hepatocellular, Hodgkin’s lymphoma, mela-noma, thyroid, bladder, neuroblastoma, medulloblastoma,rhabdomyosarcoma, and retinoblastoma (2, 10, 12, 13, 15, 16, 18,21, 22, 24, 25, 27, 31–35, 38). In most studies, reverse transcription-PCR for the expression of RASSF1C was used as a control for RNAintegrity and loading. This served a duel purpose because itemphasized that methylation of the RASSF1A promoter regiondoes not affect expression of the C-isoform. Expression of RASSF1Band RASSF1F have also been examined (2, 12, 13, 25). Northernblotting showed RASSF1B is expressed most strongly in hemo-poeitic cells and expression was lost in two of four tumor cell linesof lymphoid origin (2). Loss of RASSF1B expression coincided withloss of RASSF1A expression in this study. Similarly, loss of RASSF1Bexpression occurred in seven of eight bladder cancer cell lines withloss of RASSF1A expression and was recovered along with RASSF1Afollowing treatment with demethylating agent (13). Whereas thereasons for the loss of RASSF1B expression are not yet understood,none of the samples analyzed exclusively down-regulated RASSF1B .Conversely, expression of RASSF1F is intimately connected withRASSF1A expression because they share a common promoter

region (15, 25). Hence, reexpression of RASSF1A in 5-aza-2Vdeoxycytidine–treated neuroblastoma, rhabdomyosarcoma andretinoblastoma cell lines is coincident with RASSF1F reexpression(12) Although not reported, expression of RASSF1E , RASSF1D , andRASSF1G are also likely to be linked with expression of RASSF1Abecause they also share the same promoter region. Expression ofRASSF1A has been investigated in primary tumors and matchednormal tissue. Whereas contaminating normal cells can beproblematic, down-regulation of RASSF1A was shown to correlatewith promoter methylation in breast, renal, thyroid, and bladdertumors (13, 21, 24, 35, 41).RASSF1A methylation has been reported in at least 37 tumor

types. Normal tissue controls are used in the majority of studies toshow methylation is tumor specific and therefore involved intumorigenesis. Methylation of RASSF1A occurs rarely in normaltissues. Combined with the high frequency reported in cancer, thismakes RASSF1A a candidate molecular marker for tumordiagnosis. Detection of methylation in premalignant breast cancerlesions and in sputum samples from patients who later developedlung cancer (45, 46) have highlighted the potential importance ofRASSF1A in early diagnosis. Several studies have investigated theprospect of using DNA methylation as a tumor cell marker insamples obtained by noninvasive techniques such as in plasma,sputum, urine, throat washings, and nipple aspirates (47–50, 53,56). When comparing RASSF1A methylation in nasopharyngealcarcinoma with corresponding nasopharyngeal swab and mouth/throat washings, Chang et al. showed 100% concordance and 98%concordance with plasma and buffy coat samples (47). In ovariancancer, detection of RASSF1A methylation in correspondingperitoneal fluid and serum from patients with all histologic types,grades, and stages of disease even in samples, where the CA-125(serum marker) levels were low (<35), underscored the importanceof having a reliable tumor marker when early detection is crucial topatient outcome (48). In addition, RASSF1A methylation in urinefrom kidney cancer patients corresponded with methylation of thetumors in 73% of cases (53). Compared with other methods oftumor surveillance, detection of methylated DNA by methylation-specific PCR is very sensitive and relatively cheap. Therefore,RASSF1A methylation has the potential to be used (along with apanel of other TSGs to ensure 100% coverage) as a marker for earlydetection and monitoring.The value of RASSF1A methylation as a prognostic marker has

been investigated most in NSCLC. Tomizawa et al. suggest thatRASSF1A methylation correlated with poor survival rate in patientswith stage I lung adenocarcinoma disease (7). RASSF1A methyla-tion was associated with poorly differentiated tumors, predomi-nantly with vascular invasion and pleural involvement. A similarcorrelation with poor survival was also reported by Burbee et al.(15). Conversely, three separate studies did not find any correlationwith survival and RASSF1A methylation (57, 59, 60). Instead, Wanget al. suggested RASSF1A methylation was predictive of earlyrelapse (60).

Correlation between RASSF1A Methylation andOther Oncogenic Events

Due to the evidence linking RASSF1A to Ras signaling pathways,several studies have looked for correlation between mutation ofK-Ras and inactivation of RASSF1A by methylation. An inversecorrelation was observed in colorectal cancers (61), pancreaticadenocarcinomas (62), and NSCLC (63). However, a different

RASSF1 Tumor Suppressor Gene




study of NSCLC (64) found no correlation between methylation atRASSF1A and activating mutations in K-Ras; however, individualswith both defects had a poorer outcome suggesting a synergisticmechanism of action. Synergy was also proposed in melanomas(65) where in addition to mutation screening of N-Ras, K-Ras, andH-Ras, B-Raf was also analyzed and whereas no tumors with B-Raf mutations had additional mutations in N- or K-Ras, themajority of tumors with RASSF1A methylation had mutations ineither B-Raf or N-Ras. However, in thyroid cancer the reversesituation was observed such that no tumors in which RASSF1Awas methylated had mutations in B-Raf (66). These inconstanciesmay reflect other alterations in Ras signaling (67).Cervical cancer and head and neck squamous cell carcinoma

(HNSCC) are associated with human papillomavirus (HPV)

infection of the tumor cells. The HPV can only replicate in dividingcells; therefore, it encodes viral proteins (E6 and E7) to subvertcontrol of the cell cycle by inactivating p53 and Rb, respectively.DNA from HPV types 16 and 18 are particularly associated withtransformation. HPV DNA was never found in cervical carcinomaswith RASSF1A methylation (68) suggesting that the presence ofviral proteins abrogated any requirement for RASSF1A inactivationimplicating them both in the same pathway. A similar correlationwas observed in a study of HNSCC (69). However, other studies(9, 70) have not found an association between RASSF1Amethylation and absence of viral DNA.The ubiquitous human herpes virus, EBV, can transform B cells

into continually growing lymphoblastoid cell lines in vitro . ViralDNA and transcripts are associated with a number of neoplasia,

Figure 2. A, comparison of the amino acid (aa) sequence of RASSF1A homologues in Homosapiens (H), Mus musculus (M), Dario rerio (D), and Caenorhabditiselegans (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 Aof 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 interactiondomain (predicted using Prosite).

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including nasopharyngeal carcinoma, endemic Burkitt’s lymphoma,Hodgkin’s disease, and gastric carcinomas. Whereas RASSF1Amethylation is detected in the majority of nasopharyngealcarcinomas (67%), it is difficult to draw any correlations regardingvirus infection of the tumor cells because virtually all cases are EBVpositive. However, compared with other head and neck tumors,RASSF1A methylation occurs at a much higher frequency innasopharyngeal carcinoma. Comparisons between virus infectionand RASSF1A methylation have been possible with Hodgkin’slymphoma and gastric carcinomas because only a subset of theseare EBV associated. In a cohort of 52 Hodgkin’s lymphomas, 27were EBV positive and 34 showed RASSF1A methylation (18).However, RASSF1A methylation did not correlate with EBVinfection. In gastric carcinoma, a correlation between EBVinfection and RASSF1A methylation was detected (71). RASSF1Awas methylated in 14 of 21 (66.7%) of EBV-positive gastriccarcinomas, whereas only 2 of 56 (3.6%) of EBV-negative gastriccarcinomas were methylated. In this study, EBV-associatedmethylation of several other TSGs, including PTEN , p16 , THBS1 ,and MINT12 was also examined and in general the frequency ofCpG island methylation was higher in virus infected gastriccarcinomas. Methylation of the viral genome occurs shortly afterinfection to restrict expression of viral transcripts which enableEBV to evade immune surveillance. This data suggest that EBV isassociated with a methylator phenotype in gastric carcinoma andthe virus may be involved in disrupting normal DNA methylationmechanisms. Coincidentally, another study on gastric carcinomasfound RASSF1A methylation was associated with poorly differen-tiated gastric carcinomas (72), which is also the histologicsubgroup most associated with EBV.Malignant mesothelioma arises from mesothelial cells lining the

peritoneum, pleura, and pericardium. About 50% of malignantmesotheliomas are associated with SV40. In a study comparing themethylation of profile of 66 malignant mesotheliomas and 40 lungadenocarcinomas, Toyooka et al. showed methylation of RARb ,CDH13 , MGMT, p16 , and APC was significantly greater in lung

Table 1. Summary of RASSF1A methylation data inprimary tumors

Tumor type Percentage

RASSF1Amethylation

in tumors

References

Acute leukemia 15 (3/20) (12)

Bladder 62 (34/55) (13)

35 (34/98) (14)

Breast 62 (28/45) (41)49 (19/39) (15)

Biliary tract carcinoma 27 (10/37) (17)

Cervical SCC 9.5 (4/42) (68)Cervical adenosquamous

carcinoma

21 (4/19) (68)

Cervical adenocarcinoma 24 (8/34) (68)

Cervical 24 (12/50) (9)Cholangiosarcoma 69 (9/13) (19)

65 (47/72) (20)

Colon 45 (13/29) (16)

20 (45/222) (61)Esophageal SCC 51 (24/47) (23)

Ewing’s sarcoma 0 (0/8) (12)

Gastric EBV+ 67 (14/21) (71)Gastric EBV� 4 (2/56) (71)

Gastric 43 (39/90) (72)

Glioma 54 (25/46) (26)

57 (36/63) (78)Hepatoblastoma 19 (5/27) (12)

Hepatocellular 85 (70/83) (28)

100 (29/29) (29)

Head and neck 17 (4/24) (30)15 (7/46) (69)

Hodgkin’s lymphoma 65 (34/52) (18)

Kidney RCC 91 (39/43) (21)

26 (44/165) (22)Lung: SCLC 72 (21/29) (6)

79 (22/28) (24)

Lung: NSCLC 43 (32/75) (36)30 (32/107) (15)

32 (65/204) (37)

42 (42/100) (25)

37 (190/514) (39)34 (14/41) (6)

38 (22/58) (2)

32 (35/110) (7)

Medulloblastoma 79 (27/34) (10)Malignant mesothelioma 32 (21/66) (40)

Melanoma 41 (14/44) (27)

15 (3/20) (42)Meningioma 17 (2/12) (26)

Multiple myeloma 28 (9/32) (43)

0 (0/56) (44)

Nasopharyngeal carcinoma 67 (20/30) (47)67 (14/21) (31)

Neuroblastoma 55 (37/67) (32)

52 (14/27) (12)

Osteosarcoma 0 (0/11) (12)Ovarian 40 (8/20) (33)

50 (25/50) (48)

Pancreatic 62 (47/75) (62)Phaeochromocytoma 22 (5/23) (32)

Prostate 53 (53/101) (51)

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

Tumor type Percentage

RASSF1Amethylation

in tumors

References

71 (37/52) (52)

63 (7/11) (34)

Rhabdomyosarcoma 61 (11/18) (12)Retinoblastoma 59 (10/17) (12)

Schwannoma 10 (1/10) (26)

Testicular nonseminoma 83 (15/18) (54)

21 (9/44) (55)Testicular seminoma 40 (4/10) (54)

Thyroid 71 (27/38) (35)

37 (19/51) (66)

Wilms’ tumor 71 (22/31) (58)54 (21/39) (16)

NOTE: Data discussed in text.





adenocarcinomas (22-52%) than in malignant mesotheliomas (0-10%; ref. 73). However, methylation of RASSF1A was notsignificantly different between the two tumor types, 32% malignantmesothelioma versus 43% lung adenocarcinoma. SV40 Tag waspresent in 48% (32 of 66) of malignant mesotheliomas andwas shown to correlate with a higher methylation index than inSV40-negative malignant mesotheliomas. Furthermore, RASSF1Amethylation was significantly higher in SV40-positive malignantmesotheliomas (48%) than SV40-negative cases (16%). Theauthors investigated the connection between aberrant methyla-tion and SV40 by infecting mesothelial cells with virus andexamining promoter methylation in early (8-30) and late (15-86)passaged cells. No promoter methylation was detected in earlypassaged cells but in two of six foci of late-passage cells. RASSF1Apromoter methylation was detected with concomitant down-regulation of RASSF1A . These cells also displayed morphologicsigns of transformation (74). Expression was recovered using5-aza-2V deoxycytidine but not with the histone deacetylase inhib-itor trichostatin A indicating that SV40 induces methylation ratherthan histone deacetylation of the RASSF1A promoter.

Epigenetic Inactivation of Other Ras AssociationDomain Family Members

NORE1A but not NORE1B was shown down-regulated in lungtumor cell lines. Down-regulation of NORE1A expression correlatedwith promoter CpG island hypermethylation (75) and was foundmethylated in 24% of NSCLC tumors. Two missense changes wereidentified in a mutational screen of 20 lung tumor cell lines (75).Hypermethylation of the NORE1A CpG island in NSCLC has aninverse correlation with K-RAS mutations suggesting that they havea cooperative role in carcinogenesis (76). Chen et al. (77) identifiedtwo breakpoint-spanning genes, LSAMP and NORE1 in a familialclear cell renal cell carcinoma (CCRCC)–associated translocationt(1;3)(q32.1;q13.3). NORE1A was found methylated in 32% ofsporadic CCRCC. RASSF2 , like NORE1 , is widely expressed (withparticularly high levels in the brain and blood), isoform A ofRASSF2 (RASSF2A) is frequently methylated and silenced incolorectal and lung tumors and tumor cell lines.1 ADO37 seemsexpressed in most tissues and is methylated in lung and breasttumors but rarely in nasopharyngeal carcinomas or glioma tumors(78, 79). RASSF3 is unmethylated in human cancers (78), whereasRASSF6 methylation status in tumors has not been reported.

Methylation and RNA Interference

During embryogenesis, tissue-specific DNA methylation patternsare established for the correct regulation of imprinted genes suchas insulin-like growth factor II and X chromosome inactivation.The majority of the rest of the genome is methylated with theexception of CpG islands, most of which are associated with thepromoter regions of housekeeping genes. As well as transcriptionalregulation, methylation of the genome is thought to suppress theactivity of transposable elements (e.g., LINE-1 and Alu) that candisrupt gene function. Some of the DNA methyltransferases(DNMT1, DNMT2, DNMT3a, DNMT3b, and DNMTL) involved inthese processes have been identified. DNMT1 ensures mainte-

nance, whereas DNMT3b is involved in de novo DNA methylation.However, in tumor cells, a paradox exists with global hypomethy-lation of the genome, hypermethylation of CpG islands, andoverexpression of DNMTs. Hypomethylation contributes to tumor-igenesis through loss of imprinting, genomic instability, andactivation of transposable elements. Whereas the mechanismsthat lead to aberrant CpG island methylation are not fullyunderstood, recent data by Kawasaki et al. may provide clues tounraveling this mystery (80). Many organisms use RNA interference(RNAi) to regulate gene expression post-transcriptionally. Micro-RNAs (miRNAs), 21 to 23-nucleotide ds-RNAs, are the effectormolecules for RNAi. Exploitation of this mechanism usingchemically synthesized small inhibitory RNAs (siRNAs) is provingvery fruitful for functional genomics, especially for the study oftumor suppressor gene inactivation in cell lines. However, if siRNAis designed to target CpG islands within gene promoters, sequence-specific methylation of DNA is induced with concomitant down-regulation of transcription. siRNA-directed methylation of theE-cadherin promoter has been shown in breast a tumor cell line (80).How this is achieved is not yet understood, but recent microarraydata from 27 prostate cancers suggests that endogenous antisensetranscripts required to generate siRNAs are synthesized in vivo fromintragenic promoters including RASSF1 (81). It is tempting tospeculate, therefore, that RNAi may lie at the heart of epigeneticinactivation of RASSF1A and other commonly inactivated TSGs.

Mutation Analysis of RASSF1A Gene

As already mentioned, mutation of RASSF1A occurs rarely inhuman cancers. As far as we can ascertain, only one frame-shiftmutation at codon 277 (within the Ras association domain) has beenreported derived from a nasopharyngeal carcinoma (31). A numberof missense changes and polymorphisms have been found innasopharyngeal carcinoma, lung, breast, and kidney carcinomas(6, 15, 21, 31). Many of these localize to the functional domains ofRASSF1A , three in the DAG binding domain, four in the ATMphosphorylation domain, and five in the Ras association domain(Figs. 2B and 3). The functional significance of these changes has notbeen fully investigated, but data suggests that they are defective. Forinstance, unlike wild-type RASSF1A, a C65R/V211A mutant does notsuppress growth of LNCaP prostate carcinoma cells or KRC/Y renalcell carcinoma cells, in vitro (34, 82). Phosphorylation of RASSF1Ais reduced in the S131F mutation of the putative ATM site, whichalso results in less efficient inhibition of cell proliferation (83). Twomissense changes, C65R and R257Q, relocate RASSF1A from thecytoplasm to the nucleus and have diminished ability to halt the cellcycle (84). This strongly suggests that cellular localization ofRASSF1A is also vital to its function. Hence, study of naturallyoccurring tumor associated missense changes is proving fruitful fordissecting out the relative importance of the different putativefunctional domains of RASSF1A.

RASSF1 Function

Frequent inactivation of RASSF1A gene in human cancerssuggests that it must have a pivotal role in tumor prevention. Thisnotion is supported by the phenotype of tumor cell lines withconstitutive overexpression of RASSF1A. The observations inNSCLC, prostate, kidney, nasopharyngeal carcinoma, and gliomacell lines (2, 15, 21, 34, 78, 85) indicate that RASSF1A expressing cellsare less viable, growth suppressed, less invasive, and have reducedanchorage/substrate independence. Using a tetracycline regulation1 Hesson and Latif, unpublished data.

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system, RASSF1C has also been shown to inhibit colony formationin vitro (82). In addition, mutations detected in a gene inactivationtest suggest that RASSF1C may also function as a TSG (82).Subsequent studies have indicated RASSF1A functions as amodulator of two pathways commonly deregulated in cancer,apoptosis, and cell cycle (83, 86). Microarray analysis of tumor celllines (NSCLC and neuroblastoma) stably expressing RASSF1Aidentified expression changes consistent with the observed pheno-typic effects such as reduced cell cycle effectors (Cyclins D1 and D3),increased cell adhesion molecules (N-cadherin) and alterations inthe levels of a number of extracellular matrix modifiers (SPARC andANPEP; ref. 87). Several of 66 RASSF1A-regulated genes identified bythis study are also affected by activated Ras during cellulartransformation. Interestingly, our data found that RASSF1A andRas had the reciprocal effect on their expression, which might befurther evidence to suggest that these molecules do indeed functionin the same pathway. By identifying RASSF1A-interacting proteins(Figs. 3 and 4), we are beginning to understanding how RASSF1Aachieves growth suppression or induces apoptosis and may providenew therapeutic targets for the treatment of human cancers.

Microtubules, Adhesion, and Migration

The first mice lacking RASSF1 were made by Smith et al. (88)who generated a targeting vector to the mouse chromosomesyntenic to the minimal region of human chromosome 3p21.3deleted in lung tumors, this 370-kb deletion knocked out 12 genesincluding rassf1 . Heterozygous mice with this contiguous genedeletion were viable and fertile; however, the homozygous nullembryos died before 13.5d.p.c. Subsequently, mice were generatedwith a targeted deletion of the rassf1-coding region (89). These micehave been bred to homozygosity and shown to have no obviousphenotype; however, they have not been grown to old age nor havethey been treated with tumor promoting agents. Interestingly

mouse embryonic fibroblasts (MEFs) generated from rassf1 nullmice are smaller than wild-type MEFs and show increasedsensitivity to the tubulin depolymerizing agent nocodazole (89).We and others have shown that transfection of RASSF1A intoRASSF1A-negative cell lines induced the microtubular structuresradiating from the centrosome to become hyperstabilized circularstructures and also protected the cells from the action ofnocodazole (84, 89–91). Microtubular association of tumorsuppressor proteins is not without precedent, as both theadenomatous polyposis coli (APC) and von Hippel-Lindau (pVHL)proteins have been shown to bind to and stabilize microtubulessuggesting that microtubule destabilization may be an importantfacet of tumorigenesis. The RASSF1A mutants C65R and R257Q,which fail to localize to the microtubules, were unable to protectagainst this nocodazole-induced depolymerization (84). Deletionanalysis (89) showed that a region between amino acids 120 and288 of RASSF1A was required for microtubule association, and asubsequent deletion analysis defined a smaller region amino acids120 to 185 as the microtubule association domain (90). TheRASSF1A peptide comprised of amino acids 120 to 185 destabilizedmicrotubules and was termed dominant-negative RASSF1Afor its ability to inhibit RASSF1A-induced cell death. Acetylationof a-tubulin is associated with microtubule stability, and transfec-tion of NCI-H1299 cells with RASSF1A induced high levels ofacetylated a-tubulin; however, the RASSF1A mutants C65R andR257Q were less competent at induction of microtubule acetylation(84). The microtubule stability induced by RASSF1A could haveimplications for cell adhesion and motility, especially in the light ofdata showing that genes for cell adhesion and motility such astropomyosin I and CDH2 (N-CAD) were up-regulated in A549 cellsstably expressing RASSF1A (87).Wild-type RASSF1A was shown to associate with microtubules in

immunofluorescence (84, 89) and cosedimentation assays, whereasmutant versions showed varying abilities to colocalize with

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.





microtubules. Fewer than 30% of cells transfected with C65R orR257Q showed RASSF1A localizing to microtubules, whereasf90% of wild type and 80% of mutants K21Q, S131F, A133S,R201H, V211A, Y325C, A336T localized to the microtubules (84).Bromodeoxyuridine incorporation assays showed that the mutantsdeficient in their ability to bind microtubules (C65R or R257Q) werealso deficient in their ability to stop DNA synthesis in NCI-H1299cells (84) suggesting a potential connection between competency tobind microtubules and ability to induce cell cycle arrest.The localization of RASSF1A can be seen to alter during mitosis

(92); cytoplasmic microtubular association was confirmed duringinterphase. However, as the cells progressed into prophase,RASSF1A relocated to the separated centrosomes, then to thespindle fibers and poles during metaphase and anaphase and finallyto the midbody during cytokinesis in HeLa cells.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 (alsocalled BPY2IP or VCY2IP1); in fact, 70% of interacting clones hadhomology to microtubule-associated proteins. The RASSF1Ainteracting protein C19ORF5 colocalized with RASSF1A to themicrotubules, but overexpression of C19ORF5 unlike overexpres-sion of RASSF1A could not protect against the depolymerizing

effects of nocadozole. RASSF1C can also associate with micro-tubules (90); however, it is less effective at stabilizing them.RASSF1C has also been shown to interacts with C19ORF5 (93),which in turn can interact with the SEC-1 domain of leucine-richpentapeptide repeat motif–containing protein (LRPPRC, also calledgp130 and LRP130). LRPPRC also interacts with UXT (93), which inturn interacts with BUB3 that is involved in the kinetochorecheckpoint that ensures cells containing misaligned chromosomesdo not exit from mitosis prematurely. C19ORF5 and LRPPRCcolocalize with h-tubulin in the cytoplasm; however, in apoptoticcells, they are found to colocalize in the nucleus (93). Aninteraction between C19ORF5 and the mitochondrial proteins(NADH-dehydrogenase subunit 1, cyclooxygenase-1) and LRPPRCassociates RASSF1 with mitochondria, an organelle with pivotalfunctioning in control of apoptosis.

Apoptosis

RASSF1 was predicted to form a soluble cytoplasmic proteinwith a Ras association domain (1). Ras is a small inner membrane–embedded GTPase that relays proliferative signals from cell surfacereceptors such as receptor tyrosine kinases. Ras becomes activatedby 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 byrecruiting common and unique effectors. Initial stimuli are likely to originate from mitogen receptors, ion channels, etc. RASSF members are also linked to severalpivotal proteins such as Ras proteins (including Ran0, pRb, p53, p14ARF, and Cdc20). Homodimerization and heterodimerization between the RASSF members andbetween MSTs may be a central regulatory feature of the dynamics of these pathways.

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cytoplasmic epitopes that mediate interactions with effectorproteins such as Raf kinases; Ras signaling may be an importantchemotherapeutic target. There are three Ras family members, H-Ras, K-Ras, and N-Ras, which are located at 11p15.5, 12p12.1, and1p13.2, respectively. K-Ras has been shown to most stronglyinteract with RASSF1. RASSF1 has been shown to directly bind toRas in a GTP-dependent fashion (86) and also to interact viaheterodimerization with NORE1 (94). Ras is an importantmodulator of the apoptotic response and is thought to mediatecell survival in response to hyperproliferative signals in trans-formed cells through PI3-K– and Tiam-1–mediated pathways andto induce apoptosis in untransformed cells through as yetincompletely understood pathways that may be mediated byRASSF1 and NORE1.The difficulty of generating NIH 3T3 cells stably expressing

RASSF1C gave an initial clue that RASSF1 may play a role inapoptosis (86). Activated Ras-GTP can directly bind RASSF1C andcan enhance RASSF1-induced apoptosis in 293 cells (86). Transienttransfections were used to show that RASSF1C could induce celldeath in 293 cells, and that activated Ras could augment this celldeath, whereas dominant-negative Ras inhibited the cell death. Thecaspase inhibitor z-VAD-fmk reduced RASSF1C-induced cell deathimplicating an apoptotic mechanism. This information wasaugmented by demonstration in a yeast two hybrid assay thathuman RASSF1, murine NORE1, and Caenorhabditis elegansT24F1.3 all specifically bound to the Ste20-related proapoptotickinase mammalian Sterile20-like (MST1) through their commonconserved COOH-terminal tails (95). MST1 and MST2 (also calledSTK4 and STK3, respectively) are serine/threonine kinases thatinitiate apoptosis when overexpressed (96). The Drosophila loss ofMST function mutant (hippo) fails to undergo accurate develop-mental apoptosis. MST becomes autoactivated by autophospho-rylation of threonine (Thr183 of MST1 and Thr180 of MST2), and thisautoactivation of MST1 is inhibited by cotransfection withRASSF1A or RASSF1C or NORE1A or NORE1C but is augmentedby cotransfection of membrane-localized NORE1A-CAAX (97).MST1 contains caspase cleavage sites, cleavage with caspase 3generates a 36-kDa fragment seen during apoptosis, and cleavagewith caspase 6 or 7 generates a 41-kDa product.Activated Ras can bind NORE1 and RASSF1. RASSF1 and NORE1

can form both homodimers and heterodimers (94), which complexwith Mst-1 regulating its seriene/threonine kinase activity. TheMST1 binding site is at the COOH-terminal end of RASSF1A (aminoacids 358-413 of NORE1; ref. 95). Whereas overexpression of MST1could induce apoptosis in NIH 3T3 cells, and coexpression withNORE1 had a small effect on increasing apoptosis, the addition of aCAAX motif to NORE1 to induce membrane targeting enabledNORE1 alone to induce apoptosis, and NORE1 together with MST1had a greater proapoptotic effect than either protein alone (95).This cell death was inhibitable by the caspase inhibitor z-VAD-fmk(95). A model was proposed (97) whereby NORE1 and MST1constitutively form a complex maintaining MST in an inactivereservoir until activation by serum stimulation is able to induceassociation with Ras.CNK1 (CNKSR1) is a scaffold protein that in Drosophila is

required for Ras to activate Raf kinase. Transfection of CNK1 into293 cells can induce apoptosis (98), and this can be abrogated bydominant-negative inhibitors of MST1/2. CNK1 has been shown tobind to RASSF1A or RASSF1C but not to NORE1 (98). RASSF1 haspreviously been shown to interact with MST1/2 (95); therefore, itwas thought that RASSF1 may be providing the mechanism linking

the two proteins; this was shown by deletion of the C-terminal(RASSF1 interacting) region of MST1 that prevented MST1 frominteracting with CNK1. Despite the fact that both RASSF1A andRASSF1C can interact with CNK1, only RASSF1A could augmentCNK-induced apoptosis (98).

Cell Cycle

Deregulation of cell cycling is an essential prerequisite fortumorigenesis. In normal cells, cycling is exquisitely controlled by anumber of protein complexes whose activity is required for the cellto pass through specific checkpoints. For example, mitotis-promoting factor, a cyclinB/Cdc2 complex that phosphorylatesmitotic regulators allowing the cell to progress from G2 to M.Anaphase promoting complex/cyclosome (APC/C) is a protein

complex that interacts with ubiquitin-conjugating and activatingenzymes to catalyze the poly-ubiquitylation of proteins destined fordegradation (99) to allow the cell cycle to progress. APC/C isactivated by complexing with Cdc20 or cdh1 (proteins that containWD40 repeats). These WD40 repeat proteins are required for APC/Cto interact with target proteins. During S, G2, and prophases, APC/Cis inhibited by sequestration of Cdc20 by Emi1; later duringprometaphase, RASSF1A takes on the role of regulator as it in turnsequesters Cdc20, and coimmunoprecipitation of RASSF1A withCdc20 was shown by Song et al. (92). To allow progression thisrepression by RASSF1Amust be released allowing APC/C to becomeactive inducing the polyubiquitylation and degradation of cyclin A.Coexpression of Cdc20 with RASSF1A suggests that it is the relativelevels of these proteins that is important. During prometaphase,RASSF1A and Cdc20 are colocalized, but during mitosis, only a smallproportion coprecipitates (92). Overexpression of RASSF1A leads toaccumulation in prometaphase with raised levels of cyclins A and B(92); this arrest is before the metaphase to anaphase transition. Thespindle checkpoint keeps the cells in metaphase until chromosomesare correctly aligned; the kinetochores induce assembly of theinhibitory proteins Mad2 and Mad3 on incorrectly tensionedspindles. These inhibit the APC/C until alignment is correct whenthe repression is relieved allowing the APC/C-Cdc20 to ubiquitinatesecurin (a separase inhibitor) targeting it for degradation andinitiating sister chromatid separation and anaphase.During telophase, an APC/C-cdh1 complex targets mitotic

cyclins for destruction resulting in exit from mitosis. The APC/Ccomplex is kept in its inactive form by phosphorylation of cdh1inhibiting its ability to bind APC/C. cdk1 maintains cdh1 in itsphosphorylated state, whereas Cdc14 induces dephosphorylation ofcdh1 when the spindle becomes correctly orientated.The cyclins D1 and D3 were also shown RASSF1A regulable,

because they were down-regulated in A549 cells stably transfectedwith RASSF1A (87). Another cyclin, cyclin A (that regulates CDK2thereby controlling progression through S phase) is regulated by thetranscription factor p120E4F (100). We have shown that p120E4F canbind to RASSF1A (101) and this interaction was shown mediated byamino acids 1 to 119 of RASSF1A. Cotransfection of RASSF1A withp120E4F-induced G1 arrest of a greater magnitude than that inducedby transfection with either construct alone. p120E4F provides amechanistic link to other known tumor suppressor genes such asp14ARF, Rb , and p53 that are known to interact with p120E4F. Thissuggests that RASSF1A maybe able to affect cyclin A expression.Systematic studies were undertaken by two hybrid screens to

determine possible RASSF1-interacting partners. Mst-1, C19ORF5,MAP1B, p120E4F, and CNK1 were identified as interacting partners





(refs. 84, 95, 98, 101; Figs. 3 and 4). Additionally, the catalyticdomain of plasma membrane calmodulin-dependent calciumATPase (PMCA4b, also called ATP2B4) was found via a bacterialtwo hybrid screen to interact with RASSF1 through amino acids 74to 123 of RASSF1C or amino acids 144 to 193 of RASSF1A (102).PMCA4b is involved in expelling calcium from cells and may alsoplay a role in signaling because it interacts with proteins such asnitric oxide synthase I and calcium/calmodulin–dependent serineprotein kinase. The interaction between RASSF1 and PMCA4breduced epidermal growth factor (EGF)–dependent activation ofErk, a downstream target of the Raf, Ras, and mitogen-activatedprotein kinase cascade (102).

Functional Analysis of Other RASSF FamilyMembers

NORE1 (novel Ras effector, also called RASSF5) was originallyidentified (103) from a mouse T-cell library by its ability to bind toRas-GTP in a yeast two hybrid screen. Confirmation of thisinteraction by immunoprecipitaion showed that NORE1 only boundto Ras after stimulation with EGF or 12-O-tetradecanoylphorbol-13-acetate. NORE1 expression inhibited cell growth and this inhibitionwas augmented by expression of H-Ras, whereas cotransfectionwith the antiapoptotic agent Bcl2 blocked this growth inhibitionimplicating an apoptotic mechanism (104). Transfection of NORE1Aor NORE1B into cell lines with low endogenous NORE expressionsuppressed colony formation in A549 and G361 lines (which havedisrupted Ras signaling due to a Ras activating mutation orconstitutively active B-Raf kinase respectively). However, whentransfected into other lines with similar disruptions to Ras signaling(NCI-H460 and M14), there was no effect on colony formation (105).In the A549 cell line, NORE1A inhibited anchorage-independentgrowth and induced G1 arrest but failed to induce apoptosis (105).Deletion of the COOH-terminal MST-interacting domain and theRas binding domain and/or mutation of the zinc finger domain hadminimal effect on the growth suppressive activities of NORE1,leading Aoyama et al. (105) to conclude that the growth suppressionof NORE1 was due to amino acids 188 to 250. However, binding toMST1/2 or Ras-like GTPases had previously been shown requiredfor growth suppression (95, 104). NORE1 had been shown to induceapoptosis in 293T cells (104) rather than the cell cycle delay seen inA549 cells (105).RASSF2 was first identified by Comincini et al. (106) and it seems

up-regulated in radiation workers (107). RASSF2 (originally calledRasfadin or KIAA0168) can bind to K-Ras in a GTP-dependentmanner through its Ras effector domain (108); however, it onlyweekly binds to H-Ras. Overexpression of RASSF2 inhibited growthof lung tumor cell lines, and Vos et al. (108) were unable to generatecell lines stably overexpressing RASSF2. In transient assays, RASSF2inhibited cell growth, and this inhibition was augmented whencotransfected with K-Ras, whereas H-Ras had little additional effect.Cell death induced by RASSF2 coexpressed with activated K-Raswas shown mediated by caspase 3 and hence via an apoptotic

mechanism; cell cycle analysis also showed a decreased proportionof cells in G2-M implicating G0-G1 arrest.AD037 (also called RASSF4) has been shown to bind activated,

but not wild-type K-Ras and for the two proteins to actsynergistically to induce apoptosis in 293T cells. AD037 can alsoinhibit the growth of human tumor cell lines, and this inhibitioncan be enhanced by the addition of a tag (CAAX) to inducemembrane localization thus mimicking the effect of activation (79).No functional studies on RASSF3 or RASSF6 have been reported.

Concluding Remarks

RASSF1A tumor suppressor gene undergoes frequent tumor-specific epigenetic inactivation in a wide range of tumors. RASSF1Amethylation has also been shown in preneoplastic lesions and inpatient’s DNA obtained using noninvasive procedures such as urinefrom kidney cancer patients and sputum from lung cancer patients.Hence, RASSF1A methylation can potentially be developed as amolecular biomarker for screening cancer patients and populationsat risk. At least three other RASSF1 homologues (NORE1 , AD037 ,and RASSF2) are also inactivated in tumors by promoter hyper-methylation. Whereas somatic mutational inactivation is a rareevent for all RASSF family members analyzed thus far. Although,several of the missense changes reported in RASSF1A have beenshown functionally relevant. The inverse correlation reported forRASSF1A and NORE1A inactivation and K-ras alterations in sometumor types may provide alternative pathways for affecting Rassignaling.Studies are in progress to elucidate the function of the protein

products of the RASSF1 family of TSGs. Reintroduction of RASSF1Ain tumor cell lines lacking endogenous expression decreasedin vitro colony formation and in vivo tumorigenicity. It can induceapoptosis likely through interactions with the MST proteins.RASSF1A can also bind to and stabilize microtubules, a propertythat seems central to its function, and through modulation of APC/Cactivity can affect cell cycle regulation. Rassf1�/� mice are viableand fertile; it remains to be seen if they are prone to increasedspontaneous and or induced tumor formation. There has been anexplosion of reports on RASSF1A methylation in cancer; hopefully,the next few years will yield further insights into the biology of thisimportant family of tumor suppressor genes.

Addendum

After this article went to press, RASSF1A isoform-specific knockout mice werereported to demonstrate increased susceptibility to spontaneous and chemicallyinduced tumors (Tomassi S, Denissenko MF, Pfeifer GP. Cancer Res 2005;65:92–8).

Acknowledgments

Received 11/15/2004; revised 1/19/2005; accepted 2/1/2005.Grant support: Sport Aiding Medical Research for Kids, the Wellcome Trust,

Breast Cancer Campaign, Association for International Cancer Research, andBirmingham Children’s Hospital Research Fund.

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Cancer Research





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. Thecorrect Fig. 1 appears below. Also, in the Addendum, the referenceshould have read as follows: Tommasi S, Dammann R, Zhang Z,et al. Cancer Res 2005;65:92–8.

Correction

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

1. Agathanggelou A, Cooper WN, Latif F. Role of the Ras-association domainfamily 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

2005;65:3497-3508. Cancer Res Angelo Agathanggelou, Wendy N. Cooper and Farida Latif Suppressor Gene in Human CancersRole of the Ras-Association Domain Family 1 Tumor

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Role of the Ras-Association Domain Family 1 Tumor ... · some 3p likely to harbor one or more tumor suppressor genes, including 3p12, 3p14, 3p21.3, and 3p25-26. In 1993, the von Hippel-Lindau