Identification of RASSF8 As a Candidate Lung Tumor Suppressor

Oncogene (2006) 25, 3934–3938 & 2006 Nature Publishing Group All rights reserved 0950-9232/06 $30.00 www.nature.com/onc ORIGINAL ARTICLE Identiﬁcation of RASSF8 as a candidate lung tumor suppressor gene

FS Falvella1, G Manenti1, M Spinola1, C Pignatiello1, B Conti2, U Pastorino2, TA Dragani1

1Department of Experimental Oncology and Laboratories, Istituto Nazionale Tumori, Milan, Italy and 2Unit of Thoracic Surgery, Istituto Nazionale Tumori, Milan, Italy

The RASSF8 gene,which maps close to the KRAS2 In mammals, the RAS signaling pathway is mediated gene,contains a RAS-associated domain and encodes by numerous effectors, including proteins with a RAS- a protein that is evolutionarily conserved from fish associated (RalGDS/AF-6) (RA) domain (Malumbres to humans. Analysis of the RASSF8 transcript revealed and Barbacid, 2003; Wohlgemuth et al., 2005). Interest- a complex expression pattern of 50-UTR mRNA isoforms ingly, the genes C11ORF13 at 25.9kb from the HRAS1 in normal lung and in lung adenocarcinomas (ADCAs), gene on Chromosome 11 and RASSF8 (Ras association with no apparent differences. However,RASSF8 (RalGDS/AF-6) domain family 8) at 70.8 kb from the gene transcript levels were Bseven-fold-lower in lung KRAS2 gene on Chromosome 12 (http://www.ensembl. ADCAs as compared to normal lung tissue. Expression of org/Homo_sapiens/), contain a RAS (RalGDS/AF-6) RASSF8 protein by transfected lung cancer cells led to domain mapping near the two RAS genes. The presence inhibition of anchorage-independent growth in soft agar in of such domains close to RAS genes suggests the A549 cells and reduction of clonogenic activity in NCI- conservation of functional genomic fragments (Wall H520 cells. These results raise the possibility protein and Pritchard, 2003) and a possible involvement of these encoded by RASSF8 is a novel tumor suppressor for lung proteins in the RAS signaling pathway, although the cancer. presence of the RA domain does not necessarily signify Oncogene (2006) 25, 3934–3938. doi:10.1038/sj.onc.1209422; RAS binding affinity of a protein (Kiel et al., 2005; published online 6 February 2006 Wohlgemuth et al., 2005). In the light of the important role of RAS genes in Keywords: lung cancer; KRAS; RAS genes; cancer cancer, it seems likely that proteins involved in the RAS progression signaling pathway are also involved in modulation of cancer development. Indeed, several proteins containing an RA domain are inactivated in human cancers and act as tumor suppressor genes (Dammann et al., 2000; Introduction Hesson et al., 2003). Here, we analysed mRNA expression of the RASSF8 RAS genes, such as KRAS2, frequently undergo gene in human normal and tumor lung tissue, and activating mutations in several human and other animal studied functional activities of the full-length transcript cancers, including lung adenocarcinomas (ADCAs) through overexpression in A549or NCI-H520 lung (Ellis and Clark, 2000; Mascaux et al., 2005). KRAS2 carcinoma cells. mutations play a causative role in lung cancer development, since transgenic mice carrying an activated KRAS2 gene develop lung cancer early in life and at Results high frequency (Johnson et al., 2001). In human lung cancer, the presence of KRAS2 mutations is associated RASSF8 gene is downregulated in lung ADCAs with poor prognosis (De Gregorio et al., 1998; Screening of the full-length human lung cDNA library Huncharek et al., 1999). revealed a RASSF8 clone (B4-E11) of 5472 bp (Acc. Ras proteins are pivotal in many signal transduction #AY665468) encoding a 419-amino acids putative pathways that transfer information from the extracel- protein (Acc. #AAV54603), which showed 99% identity lular environment to the internal cellular compartments with the annotated protein BAC98838. Since analysis (Malumbres and Barbacid, 2003). Thus, mutation- of EST clones reported in public databases suggested induced deregulation of Ras activity may result in the presence of several 50-UTR variants for the RASSF8 important alterations of cell growth control and transcript, we used primers mapping in common differentiation. regions to analyse 50-UTR expression in human lung mRNAs. Four 50-UTR variants originating from alternative splicing of exons 1–3 were identified, whose Correspondence: Dr TA Dragani, Istituto Nazionale Tumori, Via G. relative abundance was similar in normal and tumor Venezian 1, 20133 Milan, Italy. E-mail: [email protected] samples (Figure 1), suggesting no tumor-specific expres- Received 13 July 2005; revised 9December 2005; accepted 29December sion of these variants. However, kRT–PCR analysis 2005; published online 6 February 2006 of RASSF8 mRNA levels carried out in paired normal RASSF8 gene in lung cancer FS Falvella et al 3935 1234567 M NTNTNTT N N T N T N 123A 3B A B C D

Figure 1 mRNA expression of the four 50-UTR alternative transcripts of the RASSF8 gene, originating from different combinations of exons 1–3, in pairs of normal (N) lung and lung adenocarcinoma (T) tissues. Lane M shows a DNA size marker. The RT–PCR results are shown as an inverted color image of an ethidium bromide-stained agarose gel. Transcripts A to D consist of sequences AY665469, AY665470, AY665471, and AY665468, respectively.

transfected cells (not shown). Comparison of growth curves of A549cells transfected with control vector and 0.0 of RASSF8 stable transfected cells showed no apparent differences (not shown). Anchorage-independence assays in soft agar revealed similar-sized large colonies formed from nontransfected or vector-transfected A549 -2.0 cells, whereas the stable RASSF8 transfectant and the

Ct two independent RASSF8 bulk transfectants formed ∆ - smaller, similarly sized colonies (Figure 3c). Thus, the -4.0 two control groups and the three RASSF8-transfected clones were grouped together, respectively, and used for comparison. Mean colony area was B1.7-fold higher in control than in RASSF8-transfected A549cells -6.0 (Figure 3b, Po0.001), and the number of colonies >0.12 mm in diameter was 2.6-fold higher in control than in RASSF8-transfected A549cells (not shown). Transfection of RASSF8 in NCI-H520 cells produced Normal ADCA a B12-fold reduction in clonogenic activity: the number Figure 2 Higher RASSF8 mRNA levels in normal lung tissue as of colonies with diameter >0.5 mm was 22.273.1 compared to lung ADCAs. kRT–PCR was carried out in 20 paired (mean7s.e.) and 280.676.8 in transfected and control normal and tumor samples; for each sample, the RASSF8 mRNA cells, respectively (Po0.0001; Figure 4). level was normalized against that of the housekeeping gene HMBS. The line within each box represents the median ÀDCt value; the upper and lower edges of each box represent the 75th and 25th percentile, respectively; the upper and lower bars indicate the Discussion highest and lowest values determined, respectively. RASSF8 protein is evolutionarily very well-conserved with 66% identity and 83% positives between the Danio rerio homologous NP_956657 protein sequence or ADCA tissue of 20 lung ADCA patients revealed a and the human AAV54603. The RASSF8 cDNA clear expression of transcript AY665468 in normal that we have isolated (AY665468) consists of ﬁve exons lungs, a B6.7-fold decreased abundance in lung ADCA and it covers 113.8 kb. RASSF8 has been implicated in (Figure 2, Po0.0001), and >50-fold decreased abun- a complex type of synpolydactyly by the reciprocal dance in both A549and NCI-H520 cancer cells (not chromosomal translocation t(12;22)(p11.2;q13.3), which shown). involves genes RASSF8 and FBLN1 (Debeer et al., 2002). The biochemical activity of the RASSF8 protein RASSF8-transfected clones show reduced remains unclear, although the presence of an RA anchorage-independent growth domain in the N-terminal region suggests involvement A549cells stably transfected with RASSF8 were of the protein in the RAS signaling pathway. Alterations screened for RASSF8 protein expression. A single clone in such a pathway are involved in a variety of and two bulk transfectants (containing approximately malignancies, including lung cancer, and affect both 100–200 individual clones) stably expressing RASSF8 tumor initiation and tumor progression (Campbell and protein (Figure 3a and not shown) were assayed for Der, 2004). In silico prediction of the RAS binding clonogenicity and anchorage-independent growth. afﬁnity using the computer algorithm FoldX (http:// Clonogenic assays of A549transfectants carried out in foldx.embl.de) (Guerois et al., 2002; Wohlgemuth et al., complete serum (10%) or in reduced serum (0.5%) 2005) showed that the predicted complex stabilities revealed similar colony numbers in vector- or RASSF8- are in the twighlight zone (based on the À7 kcal/mol

Oncogene RASSF8 gene in lung cancer FS Falvella et al 3936 threshold for binding/nonbinding domains) (Wohlge- muth et al., 2005), leaving it uncertain whether the RA domain of RASSF8 indeed binds to Ras proteins. Nevertheless, sequence alignment of the RA domain family shows that RASSF8 contains the positively charged amino-acid residues (Lys, Arg, and Arg) in b1, b1 and the first helix, respectively, important for binding to Ras proteins (Kiel et al., 2005; Wohlgemuth et al., 2005). Since the prediction method is heavily based on the use of different template structures in order to account for small structural changes in the interface, it is possible that the position of secondary structure elements differs slightly, so that small details in the interface cannot be modelled correctly (Kiel et al., 2005). The RASSF8 gene revealed a complex pattern of expression of its 50-UTR mRNA isoforms in normal lung and in lung ADCAs, with no apparent differences between normal and tumor tissue in the relative abundance of these isoforms (Figure 1). However, quantitative real-time RT–PCR showed a B6.7-fold decrease in RASSF8 transcript abundance in lung ADCA as compared to normal lung tissue (Figure 2), raising the possibility that this gene plays a role in the negative control of tumor development. A549cells, derived from a human lung cancer, expressed the RASSF8 protein when transfected with a recombinant mammalian expression vector containing the RASSF8 protein (Figure 3). Clonogenic assay of RASSF8- transfected and control A549cells revealed no signifi- cant effect of RASSF8 protein overexpression on cancer Figure 3 RASSF8 inhibits anchorage-independent growth of cell viability, since colony numbers were similar. A549lung cancer cells. ( a) Expression of RASSF8 protein in However, anchorage-independent growth in soft agar, transfected A549cells. Results of immunoprecipitation followed by which is correlated with tumor progression and meta- Western blotting using an anti-V5 monoclonal antibody are shown. stasis formation (Nikiforov et al., 1996; Clezardin, 1998; Arrow indicates the RASSF8-V5His fusion protein overexpressed in the single clone transfectant as compared to control cells Jiang et al., 2001; Malaney and Daly, 2001; Nakanishi (molecular weights markers are shown on the left. (b) After et al., 2002), was significantly reduced in the RASSF8- anchorage-independent growth in soft agar, the mean (7s.e.) area transfected A549cells (Figure 3b and c). Moreover, of A549control colonies was B1.7-fold higher than that of RASSF8 transfection inhibited clonogenic activity in RASSF8-transfected A549colonies ( Po0.0001). (c) Microscopic analysis reveals large colonies formed in soft agar by nontrans- NCI-H520 cancer cell line. Thus, the RASSF8 gene may fected or vector-transfected A549cells but only small colonies represent a novel molecular target involved in lung formed by RASSF8-transfected A549cells. cancer development.

Figure 4 Analysis of colony formation in human NCI-H520 lung cancer cell transfectants. Giemsa-stained plates reveal inhibition of in vitro colony formation activity in NCI-H520 cells transfected with RASSF8 gene (right), as compared to vector-transfected cells (left).

Oncogene RASSF8 gene in lung cancer FS Falvella et al 3937 Materials and methods cDNA library (OriGene Technologies) using primers 50-ggtgcaccatggaacttaaa-30 and 50-gacatatatgccttcaggatta-30. Human tissue samples PCR aliquots were subcloned in the pEF6/V5-His TOPO Lung adenocarcinoma (ADCA) surgical specimens and the vector to be in-frame with the V5 epitope and polyhistidine corresponding normal lung tissue were obtained from patho- tag. Clones were screened and sequenced using vector-specific logically documented patients enrolled at Istituto Nazionale primers. Tumori, Milan, Italy. Clinical data were available with Human cell lines A549and NCI-H520, derived from lung informed consent and approval of the Institute Ethics carcinoma and lung squamous cell carcinoma, respectively, Committee. Total RNA was extracted from normal and tumor were transfected with 2.5 mg DNA of recombinant or control lung tissue with RNeasy Midi kit (Qiagen, Valencia, CA, vector (pEF6/V5-His-TOPO) using SuperFect Transfection USA). Reagent (Qiagen) with or Nupherint-neuron (Biomol). Transfected clones were selected with blasticidin 5 mg/ml cDNA cloning (Invitrogen) for 3 (A549) or 4 (NCI-H520) weeks, methanol- RASSF8 transcripts were cloned by screening a full-length fixed and stained with 10% Giemsa. The clonogenic assay was human lung cDNA library (OriGene Technologies, Rockville, carried out in selective medium containing 10% serum; MD, USA) according to the manufacturer’s instructions using additionally, A549cells were tested in low-serum (0.5% PCR primers designed for the coding region (50-gggaacc FBS) medium. gaaaaggaaatca-30,50-gcaccttttgcttaaactc-30). PCR was carried Cell proliferation was measured using Alamar Blue (Bio- out in 1X buffer II (10 mM Tris-HCl pH 8.3, 50 mM KCl) Source International) diluted to 10% in complete cell culture (Applied Biosystems, Foster City, CA, USA) and 1.5 mM medium. Thousand cells were plated in 48-well plate and MgCl2, 200 mM dNTPs, 7 pmoles primers, and 0.6 U AmpliTaq fluorescence was measured daily for 1 week using Ultra Gold (Applied Biosystems). fluorometer (Tecan). Cell proliferation was measured as The 50-ends of the RASSF8 gene were determined by RT– Alamar Blue reduction. PCR of a cDNA sample prepared from human normal lung Protein extracts from confluent A549control and trans- tissue, using the primers 50-ctgcgggggcggcgcagtt-30 (exon 1) fected cells were mixed with 1 mg of anti-V5 monoclonal and 50-ctgaactccatccacccatact-30 (exon 3), and subcloned into a antibody (Invitrogen) and Protein-G Sepharose (Sigma) and plasmid vector. Nucleotide sequences of the isolated plasmid incubated overnight at 41C. The mixture was washed, eluted, clones were obtained using an ABI PRISM 377 automatic separated electrophoretically on a polyacrylamide gel, and sequencer or a 3100 Genetic Analyser (Applied Biosystems). transfrerred onto Hybond-C nitrocellulose membranes (Amer- sham) using the Trans-Blot Semi-Dry system (Biorad). 1 mRNA expression and quantitative real-time RT–PCR analysis Membranes were incubated overnight at 4 C with hybridiza- cDNAs were prepared by reverse–transcription using oli- tion solution containing anti-V5 antibody (1:5000 dilution). go(dT) primers and ThermoScript RT (Invitrogen, Carlsbad, The ECL system (Amersham) was used for detection. CA, USA). Kinetically monitored reverse transcriptase PCR Anchorage-independent growth was assayed based on (kRT–PCR) amplification mixtures contained 0.5 ml template growth in soft agar prepared in medium containing serum cDNA, 12.5 ml2Â QuantiTect SYBRGreen PCR Master Mix and 0.5% agarose (Sigma) in 24-multi-well plates and over- layered with 3000 cells resuspended in medium containing (Qiagen), and 0.3 mM intron-spanning gene-specific primers (50-agcagttcatccagcagaca-30;50-gagatgaaccaggtcgcttc-30) de- serum and 0.33% agarose (Sigma). Cells were incubated at 1 signed using Primer3 software (http://www.broad.mit.edu/ 37 C for 4 weeks. Resulting colonies were stained overnight cgi-bin/primer/primer3_www.cgi). Final volume was adjusted with the dye contained in the Cell Transformation Detection to 25 ml with water. The human hydroxymethylbilane synthase kit (Chemicon). (HMBS) gene (GenBank Acc. No. NM_000190) served as a housekeeping control for possible differences in the amount of Data analysis cDNA used (primers: 50-aggatgggcaactgtacctg-30;50-gcctac Nucleotide and protein sequences were aligned and compared caactgtgggtcat-30). Relative expression was calculated with using Genetics Computer Group (GCG) software, Genomatix ÀDCt the 2 method, where DCt ¼ Ct (target mRNA) – Ct Dialign software (http://www.genomatix.de), or BLAST soft- (control gene). All real-time assays were run in duplicate on ware (http://www.ncbi.nlm.nih.gov/BLAST). Differences be- an ABI GeneAmp 5700 sequence detection system (Applied tween groups in cell growth, clonogenic activity, or mRNA Biosystems). levels were determined using univariate analysis of variance mRNA expression of the 50-UTR of the RASSF8 gene was (SPSS 10.1, SPSS Inc., Chicago, IL, USA). determined in six pairs of human normal lung and lung adenocarcinoma tissues, using the previously indicated primers Acknowledgements for exons 1 and 3. PCR products were run on 5% agarose gels and stained with ethidium bromide. We thank Christina Kiel for the in silico analysis of the RAS binding domain of the RASSF8 protein. This work was funded Transfection and in vitro assays in part by grants from Associazione and Fondazione Italiana The RASSF8 gene coding region was amplified from the Ricerca Cancro (AIRC and FIRC) and from FIRB. GenBank cDNA clone isolated from a full-length human lung plasmid Acc. Numbers: AY665468 to AY665471.

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