Oncogene (2008) 27, 3329–3338 & 2008 Nature Publishing Group All rights reserved 0950-9232/08 $30.00 www.nature.com/onc ORIGINAL ARTICLE Expression of the tumour suppressor gene CADM1 is associated with favourable outcome and inhibits cell survival in neuroblastoma S Nowacki1, M Skowron1, A Oberthuer1, A Fagin2, H Voth1, B Brors3, F Westermann4, A Eggert2, B Hero1, F Berthold1 and M Fischer1 1Department of Pediatric Oncology and Hematology, Children’s Hospital, University of Cologne, Cologne, Germany; 2Department of Pediatric Oncology and Hematology, University Hospital of Essen, Germany; 3Department of Theoretical Bioinformatics (B080), German Cancer Research Center, Heidelberg, Germany and 4Department of Tumour Genetics (B030), German Cancer Research Center, Heidelberg, Germany Cell adhesion molecule 1 (CADM1) is a putative tumour Introduction suppressor gene, which is downregulated in many solid tumours. In neuroblastoma, loss of CADM1 expression Neuroblastoma is a paediatric tumour arising from the has recently been found in disseminated tumours with developing sympathetic nervous system. The biological adverse outcome, prompting us to investigate its role in and clinical behaviour of this malignancy is remarkably neuroblastoma tumour progression. Oligonucleotide- heterogeneous ranging from aggressive progression to microarray analysis of 251 neuroblastoma specimens spontaneous regression, and several clinical and mole- demonstrated that CADM1 downregulation is associated cular parameters have been identified to be associated with unfavourable prognostic markers like disseminated with either phenotype. Although patients with meta- stage 4, age >18 months, MYCN amplification and static disease (stage 4) and an age above 2 years in most chromosome 11q alterations (Po0.001 each). Further- cases have a poor outcome, spontaneous tumour more, low CADM1 expression was significantly correlated involution is regularly observed in infants o1-year old with unfavourable gene expression-based classification with localized stages or with the special stage 4S, which (Po0.001) and adverse patient outcome (Po0.001). is defined by the age of the patient (o1 year) and Bisulphite sequencing and genetic analysis of 18 primary dissemination limited to bone marrow, liver and/or skin. neuroblastomas suggested that neither haploinsufficiency It has been shown that characteristic cytogenetic nor hypermethylation is regularly involved in CADM1 aberrations are associated with these contrasting sub- gene silencing in neuroblastoma, which is in contrast to types of neuroblastoma. Aggressive tumours of patients results obtained in other malignancies. In addition, no with poor survival often show either an amplification of mutations disrupting the CADM1 reading frame were the oncogene MYCN together with a 1p deletion, or a found in 25 primary neuroblastomas. Over-expression of deletion of chromosomes 11q and 3p, whereas favour- CADM1 in neuroblastoma cells resulted in significant able tumours usually lack gross structural chromosomal reduction of proliferation, viability and colony formation aberrations. Moreover, the expression of various in soft agar. Collectively, our results suggest that down- genes such as NTRK1 and FYN have been described regulation of CADM1 tumour suppressor gene expression to be indicative of neuroblastoma tumour behaviour is a critical event in neuroblastoma pathogenesis resulting (Nakagawara et al., 1993; Berwanger et al., 2002). in tumour progression and unfavourable patient outcome. Recently, prognostic gene expression signatures were Oncogene (2008) 27, 3329–3338; doi:10.1038/sj.onc.1210996; shown to even more accurately predict the natural published online 17 December 2007 courses of this malignancy (Ohira et al., 2005; Oberthuer et al., 2006). Together, these data strongly suggest that Keywords: CADM1; neuroblastoma; tumor suppressor; spontaneously regressing and aggressive neuroblastoma outcome assessment (health care); cell proliferation; represent two distinct subtypes of the disease. However, disease progression the exact molecular mechanisms underlying these contrasting phenotypes and the genes involved in spontaneous regression and tumour progression remain to be determined. To identify genes involved in spontaneous regression of neuroblastoma, we have previously generated gene expression profiles from spontaneously regressing stage Correspondence: Dr M Fischer, Department of Pediatric Oncology 4S and fatal stage 4 disease using serial analysis of gene and Hematology, Children’s Hospital, University of Cologne, expression (SAGE). Differential expression of a number Kerpener Street 62, Cologne 50924, Germany. E-mail: matthias.fi[email protected] of transcripts was confirmed by quantitative real-time Received 6 September 2007; revised 24 October 2007; accepted 21 RT-PCR in these stages, including the putative tumour November 2007; published online 17 December 2007 suppressor gene cell adhesion molecule 1 (CADM1), CADM1 is a tumour suppressor gene in neuroblastoma S Nowacki et al 3330 which is also known as TSLC1, IGSF4, SynCAM and tumours and stage 4S tumours, but both subgroups Necl-2 (Fischer et al., 2006). This gene maps to exhibited significantly higher expression levels in com- chromosome 11q23.2, a region frequently deleted in parison to stage 4 (Po0.001; Figure 1a). CADM1 levels tumours such as malignant melanoma (Katoh, 2003, were substantially decreased in patients older than 18 2004), lung cancer (Gomyo et al., 1999) and neuroblas- months at diagnosis (Po0.001; Figure 1b) as well as toma (Mertens et al., 1997), and encodes an immuno- in tumours with MYCN amplification (Po0.001; globulin superfamily transmembrane molecule primarily Figure 1c) and chromosome 11q aberrations (defined involved in epithelial cell adhesion (Murakami, 2005). as deletion or imbalance; Po0.001; Figure 1d). Further- Loss of CADM1 expression has been described in many more, low CADM1 transcript levels significantly corre- malignancies, including liver, pancreatic, breast, brain lated with unfavourable gene expression-based and prostate cancers, particularly in those showing classification (Po0.001; Figure 1e) according to a invasion or metastasis (Kuramochi et al., 2001; classifier that we have previously defined using the Fukuhara et al., 2002; Jansen et al., 2002; Houshmandi prediction analysis for microarrays algorithm (PAM; et al., 2006; Heller et al., 2007). Promoter hypermethyla- Oberthuer et al., 2006). This predictive signature tion was reported to account for downregulation of consists of 144 genes, but does not include CADM1. CADM1 in several human cancers (Murakami, 2005; Taken together, these results clearly demonstrate that Ehrlich et al., 2006; Kikuchi et al., 2006; Worsham et al., downregulation of CADM1 is significantly associated 2006). Moreover, high expression levels of CADM1 with unfavourable markers in neuroblastoma. from a recombinant adenovirus vector were shown to inhibit cell proliferation and to induce apoptosis in the Low CADM1 expression is associated with adverse non–small cell lung cancer cell line A549 (Mao et al., outcome of neuroblastoma patients 2004). To determine whether CADM1 expression levels are Together, these data suggested that loss of CADM1 related to differences in patients’ outcome, Kaplan– expression might represent a critical event in tumour Meier estimates for event-free survival (EFS) and overall progression of unfavourable neuroblastoma. We there- survival (OS) were calculated and compared by log-rank fore examined the role of CADM1 expression in test. As expected from the strong correlation of CADM1 neuroblastoma pathogenesis by analysing mRNA levels transcript levels with established prognostic markers, it of CADM1 in a cohort of 251 primary tumours and turned out that low CADM1 expression was correlated their correlation with prognostic markers and patient with adverse clinical outcome (Figure 2). When using survival. To address whether promoter hypermethyla- the 25th percentile of CADM1 mRNA levels as a cut-off tion might account for CADM1 gene silencing, the for low expression (n ¼ 62), patients with intermediate methylation status of this genomic region was deter- (defined as >25th and 75th percentile, n ¼ 127) and mined in 18 primary neuroblastomas, and the CADM1 o high (defined as >75th percentile, n ¼ 62) CADM1 coding region was sequenced in 25 tumours to identify transcript levels were found to have a significantly better inactivating mutations,. Finally, CADM1 expression EFS as compared to patients with low expression (high was restored in four neuroblastoma cell lines by and intermediate CADM1 levels, 5-year EFS 86±5 and adenoviral gene transfer to assess its functional role in 786±4%, respectively vs low CADM1 levels, 5-year regulating tumour growth of neuroblastoma. EFS 366±7%; Po0.001; Figure 2a). Similar results with high statistical significance were observed for OS of these patients (high and intermediate CADM1 levels, Results 5-year OS 986±2 and 896±3%, respectively vs low CADM1 levels, 5-year OS 526±8%; P 0.001; Low CADM1 transcript levels are correlated with o Figure 2b). In addition, a trend towards a significantly markers of poor outcome in neuroblastoma differing EFS and a significant difference in OS was Comparison of expression profiles of five stage 4S and detected between patients with high and intermediate three stage 4 neuroblastoma generated by SAGE CADM1 expression levels (P ¼ 0.130 and 0.047, respec- revealed that transcript levels of the putative tumour tively). These results