Expression of the Tumour Suppressor Gene CADM1 Is Associated with Favourable Outcome and Inhibits Cell Survival in Neuroblastoma

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 demonstrate that CADM1 tran- suppressor gene CADM1 are substantially decreased in script levels discriminate neuroblastoma patients with stage 4 tumours, which was confirmed in a cohort of 76 beneficial and adverse outcome. neuroblastoma samples (stage 4S, n ¼ 27; stage 4, n ¼ 49) using quantitative real-time RT-PCR (Fischer et al., 2006). To address the question whether downregulation No evidence for hypermethylation of the CADM1 of CADM1 is a marker of adverse outcome in general in promoter region in neuroblastoma this tumour, its expression levels were evaluated in a To investigate whether hypermethylation of the cohort of 251 neuroblastoma samples (stage 1, n ¼ 69; CADM1 promoter is involved in regulating CADM1 stage 2, n ¼ 44; stage 3, n ¼ 40; stage 4, n ¼ 67; stage 4S, expression levels in neuroblastoma, eight CpG sites n ¼ 31) reflecting the whole spectrum of the disease using located in the promoter region that have been demon- the microarray technology (Oberthuer et al., 2006), and strated to be methylated in several tumours (Fukami associations of CADM1 transcript levels with five et al., 2003; Steenbergen et al., 2004; Kikuchi et al., prognostic markers were examined. Similar ranges of 2006) were tested for methylation by bisulphite sequen- CADM1 transcript levels were observed for stage 1–3 cing. Hypermethylation was defined as methylation of a

Oncogene CADM1 is a tumour suppressor gene in neuroblastoma S Nowacki et al 3331

Figure 1 Correlation of cell adhesion molecule 1 (CADM1) expression levels with prognostic markers. Association of (a) tumour stage, (b) age at diagnosis, (c) MYCN amplification status, (d) chromosome 11q status and (e) gene expression-based classification (PAM classifier) with relative CADM1 transcript levels in 251 neuroblastoma samples as determined by oligonucleotide microarray experiments. Boxes, median expression values (horizontal line) and 25th and 75th percentiles; whiskers, distances from the end of the box to the largest and smallest observed values that are o1.5 box lengths from either end of the box; open circles, outlying values; N, patient numbers.

specific CpG site in >50% of the clones sequenced. No evidence for inactivating mutations in the coding Tumour samples from 18 patients with various CADM1 region of the CADM1 gene in neuroblastoma expression levels were investigated (high IGFS4 expres- To investigate whether inactivating mutations can be sion as defined above, n ¼ 5; intermediate CADM1 found in the coding sequence of CADM1 in neuroblas- expression, n ¼ 7; low CADM1 expression, n ¼ 6). In toma, cDNA sequence analysis was performed in 20 this cohort, the same methylation pattern was found primary tumours with low expression and five primary throughout the whole group of patients (Figure 3; tumours with high expression levels. Within the group of Supplementary data). In addition, the finding of low neuroblastoma with low CADM1 expression, a 6 bp in- CADM1 expression levels in five samples without 11q frame deletion in exon 1 was found in a single tumour, alterations on the one hand, and intermediate resulting in loss of Leu26 and Arg27, and a point expression levels in two samples with 11q deletions on mutation in exon 7 was observed in three tumours, the other (Figure 3) suggest that CADM1 expression resulting in an Asp285Glu replacement. All mutations is not directly affected by chromosome 11q aberrations. were confirmed on the genomic level. As none of these Together, these data suggest that neither global mutations disrupts the reading frame of the transcript, promoter hypermethylation nor biallelic inactivation and as aspartate and glutamate represent similarly by loss of heterozygosity and methylation nor haploin- structured hydrophilic acidic amino acids, it appears sufficiency is essential for repression of CADM1 in questionable whether these sequence alterations repre- neuroblastoma. sent loss-of-function mutations.

Oncogene CADM1 is a tumour suppressor gene in neuroblastoma S Nowacki et al 3332

Figure 2 Survival curves of patients with neuroblastoma according to expression of cell adhesion molecule 1 (CADM1) mRNA. The Kaplan–Meier curves show the probability of (a) event-free (EFS) and (b) overall survival (OS), respectively, in terms of the level of CADM1 expression as determined by microarray analysis. ‘CADM1 high’, ‘CADM1 intermediate’ or ‘CADM1 low’ indicate patients with CADM1 mRNA levels deﬁned as high (>75th percentile), intermediate (>25th and o75th percentile) or low (o25th percentile), respectively.

Adenovirus-mediated expression of CADM1 in reduction of proliferation to merely 20% at day 4 post- neuroblastoma cell lines infection and to about 10% from day 7 on. Steadily To examine whether CADM1 functions as a tumour decreasing cell proliferation was observed in IMR-32, suppressor in neuroblastoma, CADM1 expression was Kelly and SH-SY5Y cells as well, even though slightly reconstituted in neuroblastoma cell lines. For this delayed as compared to SH-EP cells. Similar results purpose, we selected two human neuroblastoma cell were obtained for each cell line in two independent lines with MYCN amplification (IMR-32 and Kelly) and experiments, each of which was performed in triplicate. two with normal MYCN status (SH-SY5Y and SH-EP). These findings indicate a critical role of CADM1 in Cells were infected with either CADM1 or LacZ regulating growth properties in human neuroblastoma transducing adenovirus. Two days post-infection, sa- cells in general, as not only a single cell line with certain turation of infection was monitored using b-Gal clonal characteristics, but a panel of four different staining, while no cytopathic effect was observed using neuroblastoma cell lines showed a similar decline in Trypan blue staining (data not shown). Three days post- proliferation following CADM1 over-expression. infection, northern and western blot analysis demonstrated that CADM1 expression was effectively restored in all of the cell lines, resulting in recombinant protein Decrease in cell viability in response to CADM1 levels in range of physiological levels observed in over-expression patients (Figure 4). Peak expression of CADM1 mRNA Given the established role of CADM1 expression in was evident between days 2 and 3, with detectable levels inducing apoptosis when expressed transiently in A549 by northern blot until day 10 post-infection (data not cells (Mao et al., 2004), we tested whether the anti- shown). proliferative effect of CADM1 observed in the XTT assay could be due to decreased cell viability by examining transgene-mediated changes in lactate dehy- CADM1 expression inhibits proliferation of drogenase (LDH) release. Leakage of LDH into the neuroblastoma cells culture supernatant inversely correlates with the number To investigate whether CADM1 is involved in the of viable cells (Racher et al., 1990). Starting 5 h post- regulation of proliferation in human neuroblastoma cell infection, LDH released into the culture medium was lines, IMR-32, Kelly, SH-SY5Y and SH-EP cells were measured for the following three days. Under these infected with adenovirus transducing either CADM1 or experimental conditions, a significantly increased LDH LacZ as a negative control, and cell proliferation was release in all four neuroblastoma cell lines was observed examined for up to 10 days using the XTT colorimetric when comparing CADM1-transduced to LacZ-infected assay. At day 10, cell proliferation was significantly control cells (Figure 5b). CADM1 increased LDH decreased in all cell lines expressing CADM1 as release rates to about 20–60% above negative control compared to LacZ-infected controls (Figure 5a). In levels throughout the measurement interval. These SH-EP cells, the most severe effect was detected with a findings are consistent with results of the proliferation

Oncogene CADM1 is a tumour suppressor gene in neuroblastoma S Nowacki et al 3333

Figure 4 Physiological cell adhesion molecule 1 (CADM1) expression levels and re-expression of CADM1 in neuroblastoma cell lines IMR-32, Kelly, SH-SY5Y and SH-EP as determined by (a) northern and (b) western blot analysis. Cells were transduced with either adLacZ or adCADM1, and harvested 3 days post- infection. In northern blot analysis, only mRNA levels of the recombinant CADM1 transcript are shown, which is shorter than the endogenous transcript. Representative samples of patients with high (patient A) and low (patient B) CADM1 expression levels demonstrate that re-expression of CADM1 resulted in protein levels similar to physiological levels observed in patients with high CADM1 expression. In CADM1-transduced SH-EP cells, the b- actin control was undetectable, which was consistent with northern blot analysis results (data not shown).

tumour suppressor gene in neuroblastoma. To further investigate the effect of CADM1 expression on tumorigenicity, we compared anchorage-independent growth rates of CADM1-transduced to LacZ-infected control Figure 3 Cell adhesion molecule 1 (CADM1) expression, methylation status of the CADM1 promoter in a CpG island and 11q cells. Cells were grown in soft agar for 7 days starting 2 status in 18 neuroblastoma patients. (a) Long vertical bars indicate hours post-infection. In three of four cell lines analysed the eight CpG sites examined upstream of the predicted TATA box (SH-EP, SH-SY5Y, Kelly), a significantly reduced sequence. The open box and asterisk indicate exon 1 and the colony-forming ability in soft agar (20–50%) was predicted TATA box sequence, respectively. ATG indicates the initial methionine codon. White and black circles represent observed after 7 days, whereas the decline of 20% unmethylated and methylated CpGs, respectively (detailed infor- observed in IMR-32 cells was not significant (Figure 5c). mation of each sequenced clone are given in Supplementary Figure Together, these results demonstrate that reconstitution 1 and in the Supplementary sequencing files). Normalized log ratios of CADM1 expression attenuates the malignant pheno- of CADM1 expression levels were determined by oligonucleotide- type of neuroblastoma cells in vitro. microarray analysis and defined as high (>75th percentile; n ¼ 62), intermediate (>25th and o75th percentiles; n ¼ 127) and low (o25th percentile, n ¼ 62). Status of chromosome 11q was defined as normal (n), imbalance (im) or deleted (del). Representative results of interphase fluorescence in situ hybridization (FISH) of (b) Discussion deletion and (c) retention of 11q23 alleles. The putative tumour suppressor gene CADM1 has been described to be inactivated in numerous solid human tumours including liver, pancreatic, breast, brain and assay and demonstrate that a reduced number of viable prostate cancers (Kuramochi et al., 2001; Fukuhara cells might account for CADM1-induced decline in et al., 2002; Jansen et al., 2002; Houshmandi et al., 2006; proliferation. Heller et al., 2007), and there has been considerable interest in understanding the biological significance of CADM1 loss in tumourigenesis. Previous investigations CADM1 expression reduces the ability of colony forming have supported the hypothesis of CADM1 as a general in soft agar tumour suppressor-mediating apoptosis (Mao et al., The results obtained from the analysis of proliferation 2004), differentiation (Wakayama et al., 2003) and cell and viability support the hypothesis of CADM1 as a cycle regulation (Ito et al., 2003b). We recently found

Oncogene CADM1 is a tumour suppressor gene in neuroblastoma S Nowacki et al 3334 neuroblastoma tumour behaviour. First, CADM1 expression was correlated with established prognostic variables and patient outcome in a cohort of 251 neuroblastomas. In general, a strong association of low CADM1 mRNA levels with unfavourable prognostic markers like stage 4, age >18 months, MYCN amplification and chromosome 11q alterations was found. Furthermore, low CADM1 transcript levels significantly correlated with unfavourable gene expression-based classification according to the PAM classifier we have defined previously (Oberthuer et al., 2006). In line with these results, low and intermediate/high CADM1 expression levels discriminated patients with beneficial and adverse 5-year survival, respectively, thereby supporting the assumption that CADM1 may serve as a tumour suppressor in neuroblastoma. Similarly, an inverse correlation of CADM1 expression with increased malignancy grade and reduced patient survival has been reported for meningioma (Surace et al., 2004). One might therefore suggest that this tumour suppressor requires a critical transcript level to prevent aggressive behaviour of malignant cells in solid tumours resulting in progressive and advanced cancer. Promoter hypermethylation of CADM1 has been observed in many malignancies (Fukami et al., 2003; Li et al., 2005; Ehrlich et al., 2006; Kikuchi et al., 2006; Worsham et al., 2006) and has thus been suggested to represent the most common molecular mechanism responsible for gene silencing of CADM1 in human cancers. In contrast to these reports, we could not detect any associations of CADM1 expression level with the methylation status of the CADM1 promoter region in a set of 18 primary neuroblastomas. Therefore, the molecular mechanism underlying transcriptional repression in these cases is unlikely to be hypermethylation. Figure 5 Cell adhesion molecule 1 (CADM1)-induced changes in Although we cannot rule out the possibility that (a) cell proliferation (XTT assay) and (b) cell viability (LDH hypermethylation of a distinct set of CpG sites in the (lactate dehydrogenase)-release assay) and (c) soft agar colony promoter region might have occurred as described for formation in neuroblastoma cell lines IMR-32, Kelly, SH-SY5Y and SH-EP. Cells were transduced with either adLacZ or cervical cancer (Li et al., 2005), the eight CpG sites adCADM1, and adLacZ-transduced controls were set as 100%. chosen for our analysis have been considered to be The result of a representative experiment from two independent representative for global hypermethylation in several experiments with similar results is shown. Each value represents the studies (Fukami et al., 2003; Steenbergen et al., 2004; mean of triplicate determinations; error bars are SD for triplicate analyses. Most of the error bars are too small to be visible. P-values Kikuchi et al., 2006). Nevertheless, hypermethylation for end-point measurements were calculated using the unpaired, can be indicative, but is not necessarily involved in two-sided Student’s t-test. downregulation of CADM1 expression as suggested from previous analysis of two prostate cancer cell lines (Fukuhara et al., 2002). Alternatively, loss of CADM1 expression could be a result of haploinsufficiency, as CADM1 to be differentially expressed with strong deletion of the chromosomal region 11q23 is frequently statistical significance between stage 4S and 4 neuro- observed in unfavourable neuroblastoma. However, in blastoma that usually correspond to regressing and our analysis, low CADM1 expression levels were progressing phenotypes, respectively (Fischer et al., observed in five samples despite normal status of 2006). High CADM1 transcript levels in stage 4S chromosome 11q (Figure 3). On the other hand, neuroblastoma samples may suggest that CADM1 intermediate CADM1 expression levels were detected expression is related to spontaneous regression of in two tumours with chromosome 11q deletions disseminated neuroblastoma. Furthermore, this gene (Figure 3). These results indicate that neither hyper- has been highlighted as a candidate tumour suppressor methylation nor haploinsufficiency nor biallelic inacti- gene in human neuroblastoma, because it was found to vation by a combination of loss of heterozygosity be downregulated in neuroblastoma cells in comparison and methylation is regularly involved in CADM1 with foetal neuroblasts (De Preter et al., 2006). These downregulation in neuroblastoma. Furthermore, se- data prompted us to investigate the role of CADM1 in quence analysis of 20 tumours with low and five

Oncogene CADM1 is a tumour suppressor gene in neuroblastoma S Nowacki et al 3335 tumours with high CADM1 expression revealed no investigated, which underlines its attenuating effect on evidence of inactivating mutations of the CADM1 in vitro tumorigenicity. coding region. Although a total of four tumours with In conclusion, this study demonstrates that expression low CADM1 mRNA levels were found to harbour of the putative tumour suppressor gene CADM1 is mutations within the coding region, none of these downregulated in aggressive neuroblastoma in general. resulted in disruption or termination of the CADM1 The mechanisms that regulate CADM1 expression in reading frame, which may indicate that they probably neuroblastoma remain elusive, however, mutations in represent polymorphisms of the gene. Together, we the coding region, promoter hypermethylation and therefore speculate that different mechanisms of gene haploinsufficiency apparently do not contribute to a silencing (for example, downregulation of essential significant extent for this event. The finding of impaired activators or enhancers, changes in chromatin structure proliferation, reduced cell viability and decreased or upregulation of specific repressors) are responsible colony-forming ability after reconstitution of CADM1 for decreased CADM1 expression in neuroblastoma, expression in neuroblastoma cell lines supports its role which needs to be examined by further investigations. as a molecule with critical regulatory functions in solid To determine whether CADM1 directly contributes to tumour progression. Further analysis of this gene is the suppression of an aggressive phenotype in neuro- expected to contribute to the understanding of the blastoma, we examined its effect on growth properties of molecular processes underlying the biological and neuroblastoma cells after in vitro re-expression. Low clinical differences observed in neuroblastoma. endogenous CADM1 protein expression was found in three of four neuroblastoma cell lines, which corre- sponds well to the range of CADM1 transcript levels in Materials and methods patients with poor outcome (Figures 1 and 4). Different levels of endogenous CADM1 expression have been Microarray data and patients’ characteristics reported for several human cancer cell lines (Kuramochi Gene expression profiles from 251 patients (stage 1, n ¼ 69; et al., 2001; Ito et al., 2003b), and were considered to be stage 2, n ¼ 44; stage 3, n ¼ 40; stage 4, n ¼ 67; stage 4S, n ¼ 31) a result of a complex regulatory network of important of the German Neuroblastoma Trials NB90-NB2004 were molecules, such as APC (Naishiro et al., 2005), ErbB4 generated as dye-flipped dual-colour replicates using (Ojeda et al., 2006) and MITF (Ito et al., 2003a). The customized 11 K oligonucleotide-microarrays as previously described (Oberthuer et al., 2006). Patients’ age at diagnosis CADM1 significant protein level of endogenous in Kelly ranged from 0 to 296 months (median age, 15 months). cells might thus result from individual molecular Median follow-up for patients without fatal events was 4.5 characteristics of this cell line such as high MYCN years (range, 0.8–15.6 years). Stage was classified according to expression. In comparison to LacZ-transduced controls, the International Neuroblastoma Staging System (Brodeur proliferation was significantly reduced in XTT turnover et al., 1993). All raw and normalized microarray data are assays in all four CADM1-transduced cell lines irrespec- available at the ArrayExpress database (http://www.ebi.ac.uk/ tive of endogenous CADM1 expression. These results arrayexpress; accession: E-TABM-38). were in line with previous reports in several cancers (Surace et al., 2004; Houshmandi et al., 2006). As the Data analysis and statistics cell lines used in this study differ in several biological Statistical analysis was performed using SPSS software version and molecular characteristics like doubling time, mor- 11.0.1 for Windows (SPSS GmbH Software, Munich, Germany). phology, MYCN status or tissue (metastatic or primary Levels of CADM1 mRNA determined by microarray analysis tumour tissue) they were derived from (Tumilowicz were compared in patient groups defined by MYCN status et al., 1970; Schwab et al., 1983; Ciccarone et al., 1989), (normal vs amplified), age (o18 months vs >18 months), tumour stage (stage 1–3 and 4S vs stage 4), status of our results suggest a strong antiproliferative effect of chromosome 11q (normal vs deletion/imbalance) and gene CADM1 expression on neuroblastoma tumour beha- expression-based classification (favourable vs unfavourable) viour in general. In line with these results, a substantial according to the published PAM classifier (Oberthuer et al., reduction of cell viability was found in an LDH release 2006). Two-tailed nonparametric tests (Wilcoxon, Mann– assay, suggesting that over-expression of CADM1 in Whitney U, and Kruskal–Wallis test) were used where human neuroblastoma cell lines does not only reduce appropriate to test whether CADM1 expression levels differed proliferation, but might also function as a proapoptotic significantly in these groups. Kaplan–Meier estimates for or pronecrotic regulator. CADM1 over-expression is OS and EFS were calculated and compared by log-rank known to induce apoptosis in A549 non–small cell lung test. Recurrence, progression and death from disease were cancer cells (Mao et al., 2004), but many human considered as events. neuroblastoma cell lines show considerable resistance to apoptosis when exposed to common apoptosis Bisulphite sequencing inducing triggers (Teitz et al., 2000; Lombet et al., CADM1 promoter methylation was analysed by bisulphite 2001). Therefore, further experiments are necessary to sequencing as described elsewhere (Waha et al., 2003). Sodium bisulphite-modified genomic DNA isolated from primary more precisely delineate the mechanisms by which neuroblastoma specimens (Puregene DNA isolation Kit; CADM1 triggers changes in growth properties and loss Gentra Systems, Minneapolis, MN, USA) was tested for of cell viability. Finally, CADM1 significantly decreased methylation at eight CpG sites located in the CADM1 anchorage-independent clonal growth in short-term soft promoter region that have been demonstrated to be differen- agar assays in three of four neuroblastoma cell lines tially methylated in several tumours (Fukami et al., 2003;

Oncogene CADM1 is a tumour suppressor gene in neuroblastoma S Nowacki et al 3336 Steenbergen et al., 2004; Kikuchi et al., 2006). Briefly, 1 mg Gateway LR Clonase-mediated recombination using pAd/ DNA was diluted in 50 mlH2O, denatured with 5.5 ml2M CMV/V5-DEST (Invitrogen) as acceptor and the pDONR201- NaOH for 10 min at 37 1C and incubated with 30 ml10mM CADM1 plasmid as donor. The resulting plasmid was purified hydroquinone and 520 ml3M sodium bisulphite at 50 1C for (NucleoBond Kit PC 500; Macherey-Nagel, Du¨ren, Germany) 16 h. After denaturation with 5.5 ml3M NaOH, the DNA was and sequenced. Cell supernatants containing recombinant precipitated with 10 M ammonium acetate and 100% ethanol, adenovirus transducing either LacZ (adLacZ) or CADM1 washed with 70% ethanol and resuspended in 30 ml HPLC- (adCADM1) were produced in HEK-293A cells according to H2O. Bisulphite-treated DNA was then amplified using the manufacturer’s protocol (Virapower Adenovirus Expres- internal primers for the promoter region of CADM1 as sion System; Invitrogen). Viral titres were determined using the previously described (Steenbergen et al., 2004). The PCR Adeno-X Rapid Titer Kit (Clontech, Heidelberg, Germany). mixtures contained 20 ng of modified genomic DNA, primers Neuroblastoma cells were infected with either adLacZ at 0.5 mM, each of the four deoxynucleotide triphosphates at (control) or adCADM1 using equal amounts of virus particles 200 mM, 1.5 mM MgCl2 and 1.25 U of AmpliTaq Gold DNA per cell. Transduction efficiencies were monitored by b-Gal polymerase (Applied Biosystems, Weiterstadt, Germany) in a staining by using a b-Gal Staining Kit (Invitrogen). total volume of 50 ml. Amplification conditions were 95 1C for 1 1 7 min, followed by 30 cycles of 94 C for 30 s, 59 C for 30 s and Northern blot analysis 1 1 72 C for 45 s, and a final elongation step at 72 C for 10 min. CADM1 mRNA levels were analysed 72 h after adenoviral Finally, the resulting PCR products were subcloned for infection of neuroblastoma cell lines with either adLacZ or sequencing (Seqlab, Go¨ttingen, Germany) and sequencing adCADM1. Total RNA was isolated using TRIzol reagent data were compiled and analysed using BiQ Analyzer software (Invitrogen), and northern blotting with 3–10 mg of total RNA (Bock et al., 2005; see Supplementary data for detailed was performed as previously described (Oberthuer et al., 2004). information). A sequence verified PCR product of 624 bp generated with CADM1-specific primers (forward: 50-GTGAACTCAAAG Sequencing of the coding region of CADM1 TATCATTGACAA-30; reverse: 50-CAGCGGTATGTAC After extraction of total RNA from primary neuroblastoma CATTATCTG-30) served as template for the CADM1 probe. specimens, cDNA was generated using Superscript II reverse transcriptase (Invitrogen, Carlsbad, CA, USA) as previously Western blot analysis described (Oberthuer et al., 2006). The complete coding region CADM1 protein levels were analysed 72 h after adenoviral was then amplified by PCR, and amplificates were directly infection of neuroblastoma cell lines with either adLacZ or sequenced using the BigDye Terminator sequencing Kit adCADM1. The NuPAGE electrophoresis and blotting system (Version 3.1, Applied Biosystems). Mutations that were found (Invitrogen) was used for protein analysis. About 10 mgof in the cDNA were verified on the DNA level by amplification protein obtained from transduced cell lines or primary tumour and direct sequencing of the respective genomic regions. samples were separated by sodium dodecyl sulphate–poly- Primer sequences for amplification and sequencing are acrylamide gel electrophoresis with 4–12% bis-Tris gels and available from the authors upon request. transferred to nitrocellulose membranes by tank blotting. The membranes were blocked with 5% dry milk powder in 0.05% Cell culture Tween 20/phosphate-buffered saline before incubation with The neuroblastoma cell lines SH-SY5Y, Kelly and SHEP were primary antibody (SynCAM antibody ab3910, dilution 1:1000; obtained from American Tissue Culture Collection (ATCC, Abcam, Cambridge, UK) and horseradish peroxidase-labelled Rockville, MD, USA), and the neuroblastoma cell line IMR- secondary goat anti-rabbit antibody (dilution 1:2000; Dako, 32 was purchased from DSMZ-German Collection of Micro- Glostrup Denmark). The antigen–antibody complex was organisms and Cell Cultures (Braunschweig, Germany). All detected with Visualizer Spray & Glow (Upstate, Schwalbach, neuroblastoma cell lines were maintained in RPMI-1640 Germany). (PAA, Co¨lbe, Germany) supplemented with 10% fetal À1 calf serum (Invitrogen) and 5 mgml ciprofloxacin (Bayer, In vitro growth properties assays Leverkusen, Germany), which is referred to as RPMI-1640 The effects of CADM1 expression on cell proliferation were throughout the article. Human embryonic kidney cells HEK- assessed using the Cell Proliferation Kit II (XTT; Roche 293A (Invitrogen) were grown in Dulbecco’s modified Eagle’s Diagnostics GmbH, Mannheim, Germany) as described by the medium (PAA) supplemented with 10% fetal calf serum manufacturer. Neuroblastoma cells were seeded in 24-well (Invitrogen). All cell lines were maintained in a humidified plates at a density of 30 000 cells per well in 800 ml RPMI-1640 1 incubator at 37 C with 5% CO2 and passaged at 90% and transduced the next day with either adLacZ or confluence using Accutase (PAA). adCADM1. To assure continuous supply of nutrients throughout the measurement interval, 400 ml fresh RPMI- Plasmids and adenoviruses 1640 per well were added on day 5. XTT turnover was Adenovirus transducing CADM1 cDNA was prepared with measured at days 4, 7 and 10 post-infection by adding XTT the ViraPower Adenovirus Expression System (Invitrogen) labelling mixture and incubation for 7 h at 37 1C to allow according to the manufacturers instructions. A pAd/CMV/V5- colour development. The formation of formazan was quanti- GW/lacZ vector supplied with the kit served as a positive fied in 96-well plates measuring the absorbance at 450 nm control for infection efficiency. A human CADM1 full open- against a reference wavelength of 690 nm with an enzyme- reading frame Gateway Shuttle Clone (pDONR201-CADM1, linked immunosorbent assay reader (Multiscan Ascent plate RZPDo834A0712D2) was obtained from the RZPD reader; Thermo Labsystems, Helsinki, Finland). (Deutsches Ressourcenzentrum fu¨r Genomforschung GmbH, Cell viability was determined by measuring cell membrane Berlin-Charlottenburg, Germany). The nucleotide sequence integrity, as indicated by the leakage of LDH into the was confirmed by direct sequencing using the BigDye culture supernatant (Decker and Lohmann-Matthes, 1988). Terminator sequencing Kit (Applied Biosystems). The adeno- The LDH activity in the extracellular medium was quantified viral plasmid pAd/CMV/V5-CADM1 was generated by using the colorimetric Cytotoxicity Detection Kit Plus (Roche

Oncogene CADM1 is a tumour suppressor gene in neuroblastoma S Nowacki et al 3337 Diagnostics GmbH) according to the manufacturer’s instruc- the manufacturer’s instructions. In this short-term assay, tions. Neuroblastoma cells were seeded in 96-well plates at a CADM1- and LacZ-transduced cells were grown in soft agar density of 4500 cells per well in 100 ml RPMI-1640. On the next for 7 days starting 2 hours post-infection. For end-point day, the medium was changed to contain 1% bovine serum quantiﬁcation of soft agar growth, the soft agar matrix was albumin (Roth, Karlsruhe, Germany) instead of 10% fetal calf solubilized, cells were stained using MTT, lysed and measured serum and cells were transduced with either adLacZ or at 570 nm against a reference wavelength of 690 nm. adCADM1. LacZ-transduced cells were used as a control to All assays were conducted in triplicate and reproduced in determine background release of LDH caused by viral two independent experiments. The statistical signiﬁcance of infection. At 5, 24, 48 and 72 h post-infection, LDH substrate differences in end-point measurements was evaluated by the mixture was added according to the manufacturer’s instruc- unpaired, two-sided Student’s t-test. tions, followed by incubation in the dark for 30 min at room temperature. The reaction was terminated by the addition of Acknowledgements stop solution, and absorbance was measured at 450 nm against a reference wavelength of 690 nm with a Multiscan Ascent We thank Yvonne Kahlert for excellent technical assistance. plate reader (Thermo Labsystems). This work was supported by the Deutsche Krebshilfe (grants The ability of CADM1 to inhibit soft agar colony forming of 50-2719-Fi1 and 106847), the Bundesministerium fu¨r Bildung neuroblastoma cells was measured using the CytoSelect 96- und Forschung through the National Genome Research well Cell Transformation Assay (Colorimetric, Cell Recovery Network 2 (NGFN2 grants 01GS0456 and 01GR0450) and a Compatible; Cell Biolabs, San Diego, CA, USA) according to grant of the Kind-Philipp Stiftung.

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Supplementary Information accompanies the paper on the Oncogene website (http://www.nature.com/onc).

Oncogene