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 develop- ment, 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 alter- native 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.