Inhibition of a New Differentiation Pathway in Neuroblastoma by Copy Number Defects of N-Myc, Cdc42, and Nm23 Genes

Research Article

Inhibition of a New Differentiation Pathway in Neuroblastoma by Copy Number Defects of N-myc, Cdc42, and nm23 Genes

Linda J. Valentijn, Arjen Koppen, Ronald van Asperen, Heather A. Root, Franciska Haneveld, and Rogier Versteeg

Department of Human Genetics, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands

Abstract are of variable length as well, but can be smaller with a more The best studied oncogenic mechanisms are inactivating telomeric SRO (3, 4). The different SROs in N-myc-amplified defects in both alleles of tumor suppressor genes and neuroblastomas have raised the idea that more than one tumor suppressor gene maps on distal chromosome 1p (5). activating mutations in oncogenes. Chromosomal gains and losses are frequent in human tumors, but for many regions, N-myc-amplified neuroblastomas follow a very aggressive course like 1p36 and 17q in neuroblastoma, no mutated tumor (6). Overexpression of transfected N-myc genes in neuroblastoma suppressor genes or oncogenes were identified. Amplification cell lines strongly increased proliferation rates (7, 8). Recent mRNA of N-myc in neuroblastoma is strongly correlated with loss of expression profiling experiments, serial analysis of gene expression 1p36 and gain of 17q. Here we report that N-myc down- (SAGE) and microarray analysis, identified many myc-regulated regulates the mRNA expression of many genes with a role in genes (9–13). We used SAGE to identify genes regulated by N-myc cell architecture. One of them is the 1p36 gene Cdc42. in neuroblastoma. Genes up-regulated by N-myc were involved in Restoring the Cdc42 expression in neuroblastoma cells rRNA processing and protein synthesis (9). Two recently identified strongly induced differentiation. N-myc also inhibited Cdc42 N-myc targets, nm23-H1 and nm23-H2, map to chromosomal band functioning at the protein level. This was mediated by nm23- 17q21, in the chromosome 17q region overrepresented in H1 and nm23-H2, which are located in the amplified 17q neuroblastoma (14). region. Nm23-H1 and nm23-H2 are strongly up-regulated Additional copies of the distal chromosome 17q arm are present in about 70% of neuroblastoma tumors, including all N-myc downstream targets of N-myc. Nm23-H1 was shown to bind Cdc42 and prevented the induction of differentiation. Over- amplified cases. The copy number ranges between two and eight expression of Nm23 due to gain of 17q and induction by N-myc extra alleles and the minimal region of gain is 17q21-17qter. The combined with weak expression of Cdc42 due to loss of 1p36 map position of the nm23-H1 and nm23-H2 genes at 17q21 makes and down-regulation by N-myc can thus block differentiation. them interesting candidates for a role in neuroblastoma patho- Although this marks Cdc42 as a candidate tumor suppressor genesis. The nm23 proteins encode nucleoside diphosphate gene, no mutations were found. Further silencing of Cdc42 by kinases, the suppliers of nucleoside triphosphates. In several small interfering RNA induced massive apoptosis, indicating human tumors, like breast cancer and melanoma, decreased that tumor cell survival requires a minimal Cdc42 activity. expression was correlated with poor prognosis (15, 16). Up- Three regions of chromosomal gain and loss thus affect genes regulation of nm23-H1 reverted melanoma cells from metastatic to nonmetastatic (17). In contrast, in several other tumors including functioning in one pathway in neuroblastoma. They converge to bring the pathway out of balance and prevent Cdc42 neuroblastoma and osteosarcoma, elevated expression of nm23-H1 mediated differentiation. (Cancer Res 2005; 65(8): 3136-45) and nm23-H2 was associated with a poor prognosis (18, 19). The extra copies of 17q and the induction by N-myc result in a strongly increased expression of the nm23 genes in neuroblastoma cell lines Introduction and tumors (14). Besides the function of the nm23 proteins as Neuroblastoma is a childhood tumor originating from neural crest– dinucleoside kinases, growing evidence exists for additional derived cells. Approximately 20% of neuroblastomas have N-myc functions in signal transduction (20–22). amplification, which is usually accompanied by loss of heterozygosity In this article, we identified genes down-regulated by N-myc in of distal chromosome 1p and additional copies of chromosome 17q neuroblastoma. One of the key genes is Cdc42. The Cdc42 gene (1). Deletions of the short arm of chromosome 1 in N-myc maps on 1p36, and one copy is consistently deleted in N-myc amplified neuroblastomas are typically very large. The shortest 1p amplified neuroblastoma. In addition, we provide evidence that the deletion we observed in a series of N-myc amplified neuroblasto- activity of the Cdc42 protein is down-regulated by nm23-H1 and mas was 24.4 Mb, from the telomere till the RUNX3 gene (ref. 2; nm23-H2, which are induced by N-myc. http://genome.cse.ucsc.edu). The region is deleted in 90% to 95% of all N-myc amplified tumors, whereas about 5% of N-myc amplified Materials and Methods neuroblastomas have no apparent 1p deletions. In addition, N-myc Cell culture. The SHEP-2 and SHEP-21N cell lines were cultured in single copy neuroblastomas can have 1p deletions. These deletions RPMI, the SKNAS-NmycER cells in DMEM. Both media were supplemented with 10% FCS, 2 mmol/L glutamine, 50 units/mL penicillin, and 50 units/ mL streptomycin. N-myc expression in SHEP-21N was repressed by tetracycline (final concentration, 50 ng/mL; Sigma, St. Louis, MO). The Requests for reprints: Linda J. Valentijn, Department of Human Genetics M1-134, NmycER protein in the SKNAS-NmycER cells was activated upon addition Academic Medical Center, University of Amsterdam, P.O. Box 22700, 1100 DE of 4-hydroxytamoxifen (final concentration, 50 nmol/L; Sigma). The ER Amsterdam, the Netherlands. Phone: 31-20-566-2386; Fax: 31-20-691-8626; E-mail: [email protected]. domain has a point mutation, which enables transactivation of the I2005 American Association for Cancer Research. chimearic protein by 4-hydroxytamoxifen and not estrogens (23).

Cancer Res 2005; 65: (8). April 15, 2005 3136 www.aacrjournals.org

Plasmids. The pCdc42flag plasmid was constructed by amplification of interfering RNA (siRNA) mixture was transfected serum-free with 30 AL cDNA with primers Cdc42Efor (5V-TATATAGAATTCATTTCAGCAATGCA- Lipofectamine (Invitrogen, Carlsbad, CA) as described by the manufacturer. GACAATTAAG-3V)andCdc42Xrev(5V-TATATACTCGAGTAGCAGCACA- After 4 hours, the medium was supplemented with FCS to a final con- CACCTGCG-3V). The amplified product was digested with EcoRI and XhoI centration of 10%. and cloned into the EcoRI and XhoI sites of pCMV-Tag4 vector (Stratagene, For immunofluorescence analysis cells were grown on glass slides in La Jolla, CA). The pCdc42G12Vflag and pCdc42T17Nflag constructs were 6-well plates and transfected with 15 AL siRNA and 15 AL Lipofectamine. produced by amplification of Cdc42 from vector pCdc42flag using primer After 72 hours, the cells were fixed with formaldehyde and stained with T3 (5V-AATTAACCCTCACTAAAGGG-3V) in combination with mutant TRITC-phalloidin (Sigma) and 4V,6-diamidino-2-phenylindole. primers CdcG12Vrev (5V-GTGTAGGATATCAGGAGACATGTTTTACCAA- CAGCAACATCGC-3V) or CdcT17Nrev (5V-GTGTAGGATATCAGGAGA- CAGTTTTTACC-3V). The PCR products were digested with BamHI and Results EcoRV and ligated in the BamHI and EcoRV sites of pCdc42flag vector. Genes down-regulated by N-myc. We have applied the SAGE Northern blot analysis. Total RNA (20 Ag per lane) was separated on a technique to identify downstream target genes of N-myc. The 1% agarose gel in the presence of 6.7% formaldehyde and blotted on neuroblastoma cell line SHEP has no N-myc amplification and Hybond-N membranes (Amersham, Piscataway, NJ) in 10Â SSC. Hybridiza- expression. A tetracycline-regulated N-myc expression vector has tion was carried out overnight in 0.5 mol/L NaHPO4 (pH 7.0), 7% SDS, j been introduced into these cells, resulting in the SHEP-21N clone 1 mmol/L EDTA at 65 C. Filters were washed in 40 mmol/L NaHPO4,1% SDS at 65jC. Probes were labeled by random priming of sequence-verified (8). SAGE libraries were constructed from N-myc expressing PCR products. SHEP-21N cells and from SHEP-2 (empty vector control) cells (9). Western blotting. Primary antibodies: anti-N-myc (PharMingen, San We identified 34,443 tags for SHEP-21N and 44,450 tags for SHEP- Diego, CA), anti-Cdc42-B8 (Santa Cruz Biotechnology, Santa Cruz, CA), 2, each representing one mRNA expressed in these cells. 112 Tags anti--tubulin (Roche, Nutley, NJ), anti-Caspase-3 (Cell Signaling Technology, were significantly (P < 0.01) down-regulated in the N-myc Beverly, MA), and anti-poly(ADP-ribose) polymerase (Biomol Research expressing cells. We could reliably assign 78 of these tags to a Labs, Plymouth Meeting, PA). Western blotting was done according to total of 64 genes (http://bioinfo.amc.uva.nl/HTMseq/controller; standard procedures, secondary antibody anti-mouse linked to horseradish Table 1). We analyzed 14 genes by Northern blot, which peroxidase (Amersham), detected with Enhanced Chemiluminescence KIT confirmed the SAGE data (Fig. 1A). The overall expression (Amersham). pattern showed that N-myc down-regulates many genes involved Transient transfection and immunofluoresence microscopy. For in the architecture of the cell and the extracellular matrix. transient expression, the cells were grown for 48 hours on glass slides in h medium supplemented with 1% FCS. Four micrograms of plasmid DNA and Cytoskeletal components like -actin and vimentin are down- 4 AL DAC-30 (Eurogentec, Seraing, Belgium) in 1 mL medium, without FCS regulated, but also genes which regulate the structures, like and antibiotics, was incubated for 30 minutes at room temperature. For transgelin (TAGLN). The adhesion properties of cells depend on double transfections, 4 Ag of each plasmid were incubated with 8 Ag DAC- the extracellular matrix and membrane molecules. Genes of both 30. The DNA/DAC-30 mix was added dropwise to the cell cultures. After groups are repressed by N-myc. Several collagens are regulated by 16 hours, the medium was replaced by fresh medium with 1% FCS. Forty N-myc, but also connective tissue growth factor (CTGF) and hours after transfection, the slides were fixed with 4% paraformaldehyde in SPARC, which regulate the build-up of the extracellular matrix. In PBS for 25 minutes. The cells were permeabilized with PBS/0.05% Triton X- addition, cadherin 11 (CDH11) and other genes encoding 100. The slides were washed thrice with PBS/TX (PBS, 0.01% Triton X-100). membrane proteins are down-regulated. The slides were blocked for 30 minutes in Abdil (PBS, 0.01% Triton X-100, To investigate whether the genes were indeed down-regulated by 1% bovine serum albumin). The antibodies were diluted in Abdil each incubated for 30 minutes each incubation followed by four washes in PBS/ N-myc, we did Northern blot analysis of a time course experiment TX. Antibodies: 400 times diluted mouse-anti-flag M2 (Stratagene), 100 in which N-myc was switched off and on in the SHEP-21N cell line. times diluted TRITC- or FITC-conjugated anti-mouse (Sigma), 200 times The N-myc expression was switched off by tetracycline for 7 days, diluted goat-anti-NFL (Santa Cruz Biotechnology), and 200 times diluted after which N-myc was switched on for another 7 days (Fig. 1B).

TRITC-conjugated anti-goat (Sigma). The slides were rinsed with H2O, dried Two distinct patterns of N-myc regulation emerged. Expression of a and mounted in Vectashield (Vector Laboratories, Burlingame, CA) with limited number of genes increased within 24 hours after N-myc was 4V,6-diamidino-2-phenylindole (nuclear staining). turned off. This group includes galectin-1 (LGALS), S100A10, and Glutathione S-transferase pulldown. Nm23H1 was amplfied with caveolin-1 (CAV1; Fig. 1B). Reexpression of N-myc decreased the V adaptor oligonucleotides (5-TATATAGGATCCATGGCCAACTGTGAGCG- expression of these genes (Fig. 1B). The majority of genes follow a TAC-3V and 5V-TATATGAATTCTCTGCCCTCCTGTCATTCAT-3V). The prod- slow N-myc dependent regulation. The expression of COL1A1, uct was digested with BamHI/EcoRI and cloned in the pGEX2TK vector (Pharmacia, Piscataway, NJ) to yield GST-nm23H1. TAGLN, SPARC, FN1, and SDC2 increased 4 days after N-myc was GST-nm23H1 transformed bacteria were lysed in PBS-0.01%Triton X-100. switched off (Fig. 1B). These analyses show that genes involved in Cell lysis and pulldown were done as described by Sander et al. (24). cell architecture and adhesion are important targets of down- RNA interference. The sequences of the single stranded RNA mole- regulation by N-myc. Most of these genes are slowly regulated and cules (Isogen, Maarsen, The Netherlands) are for Cdc42-a 5V-CUAUGCAGU- thus most likely represent secondary targets. CACAGUUAUGTT-3Vand 5V-CAUACCUGUGACUGCAUAGTT-3V, for Cdc42-b Cdc42 expression is down-regulated by N-myc. One of the 5V-CUCACCACUGUCCAAAGACTT-3V and 5V-GUCUUUGGACAGUGGUGAG- genes identified as an N-myc downstream target is Cdc42 (Table 1). TT-3V, for N-myc-a 5V-CACCAAGGCUGUCACCACATT-3Vand 5V-UGUGGU- The SAGE-tag frequencies show that Cdc42 is expressed at a level V V GACAGCCUUGGUGTT-3, for N-myc-b 5-CCCAGACCUCGAGUUUGACTT- of 12.6 mRNAs per 20,000 mRNAs in SHEP-2 and 2.9 per 20,000 in 3Vand 5V- GUCAAACUCGAGGUCUGGGTT-3V, and for GFP 5V-GACCCGCGC- SHEP-21N. Cdc42 is a member of the Rho GTPase subfamily and CGAGGUGAAGTT-3V and 3V-CUUCACCUCGGCGCGGGUCTT-3V. Cells were grown to 30% confluency in 6-cm plates. RNA interference functions in a series of signaling pathways, which are particularly was done as described by Elbashir et al. (25). In short, 20 Amol/L per important in cytoskeletal remodeling (26, 27). As many genes with single stranded RNA molecule in 30 AL annealing buffer [0.1 mol/L KAc, a function in these processes are down-regulated by N-myc, Cdc42 30 mmol/L HEPES/KOH (pH 7.4), and 2 mmol/L MgAc] was heated at 95jC might be a key target of N-myc to control cytoskeletal remodeling. for 5 minutes, followed by incubation at 37jC for 1 hour. The small Northern blot analysis confirmed the decreased expression of www.aacrjournals.org 3137 Cancer Res 2005; 65: (8). April 15, 2005

Table 1. Down-regulation of gene expression by N-myc

c c b Tag sequence Ratio* SHEP2 SHEP21N P Unigene Gene Description

Cytoskeleton GTTTCTAATA 10 5.8 0.6 0.006 31095 MAP4 Microtubule-associated protein 4 TGCTACGAAA 10 5.8 0.6 0.006 146550 MYH9 Myosin, heavy polypeptide 9, nonmuscle TGCTAAAAAA 5 35.1 7.5 0.000 146550 MYH9 Myosin, heavy polypeptide 9, nonmuscle TTAAAGATTT 9 26.5 2.9 0.000 133892 TPM1 Tropomyosin 1 (a) ACAGGCTACG 9 11.2 1.2 0.000 410977 TAGLN Transgelin TGTAGAAAAA 7 8.5 1.2 0.001 512712 TUBB Tubulin, h polypeptide TCCAAATCGA 6 6.7 1.2 0.008 435800 VIM Vimentin AAGAGGTTTG 5 8.5 1.7 0.004 74368 CKAP4 Cytoskeleton-associated protein 4 GGTTAATTGA >4 4 0 0.007 14376 ACTG1 Actin, g 1 TAATATTTTT 4 11.7 2.9 0.002 443619 ACTN4 Actinin, a 4 CCCTTAGCTT 4 13.5 3.5 0.001 233936 MRCL3 Myosin regulatory light chain MRCL3 GCTTTATTTG 3 31.9 9.9 0.000 426930 ACTB Actin, h AACTGCTTCA 2 25.6 10.5 0.001 433506 ARPC1B Actin-related protein 2/3 complex, subunit 1B, 41 kDa TGTACCTGTA 2 28.3 11.6 0.001 446608 K-ALPHA-1 Tubulin, a, ubiquitous CTGCCAAGTT 2 20.7 8.7 0.004 75873 ZYX Zyxin ECM structural ATCTTGTTAC 22 13.5 0.6 0.000 418138 FN1 Fibronectin 1 TGCAATATGC >12 11.7 0 0.000 750 FBN1 Fibrillin 1 (Marfan syndrome) TTAAATAGCA 11 6.7 0.6 0.002 172928 COL1A1 Collagen, type I, a 1 TGGAAATGCC 8 13.9 1.7 0.000 172928 COL1A1 Collagen, type I, a 1 TTCGGTTGGT 5 20.2 4.1 0.000 172928 COL1A1 Collagen, type I, a 1 GGAGGAGAGC >5 4.9 0 0.003 172928 COL1A1 Collagen, type I, a 1 TGGAAATGAC 5 85.9 15.7 0.000 172928 COL1A1 Collagen, type I, a 1 TGAAATTGTC >4 4.5 0 0.004 172928 COL1A1 Collagen, type I, a 1 ACCAAAAACC 2 42.7 23.2 0.002 172928 COL1A1 Collagen, type I, a 1 GACCGCAGGA 11 6.7 0.6 0.002 437173 COL4A1 Collagen, type IV, a 1 ACAGATTTGA >9 9.4 0 0.000 114599 COL8A1 Collagen, type VIII, a 1 TAAAAACAAA 9 5.4 0.6 0.010 114599 COL8A1 Collagen, type VIII, a 1 TAATCCTCAA 7 15.3 2.3 0.000 413175 COL18A Collagen, type XVIII, a 1 TCTTGATATT >5 5.4 0 0.004 283393 COL5A2 Collagen, type V, a 2 ECM regulator TTTGCACCTT 58 35.1 0.6 0.000 410037 CTGF Connective tissue growth factor ATGTGAAGAG 22 539 25 0.000 111779 SPARC Secreted protein, acidic, cysteine-rich (osteonectin) AAAAAGCTGC >15 14.8 0 0.000 111779 SPARC Secreted protein, acidic, cysteine-rich (osteonectin) GGTGGACACG >12 12.1 0 0.000 111779 SPARC Secreted protein, acidic, cysteine-rich (osteonectin) CTAAAAACCT >9 9.4 0 0.000 111779 SPARC Secreted protein, acidic, cysteine-rich (osteonectin) TTAGTGTCGT 8 9.9 1.2 0.000 111779 SPARC Secreted protein, acidic, cysteine-rich (osteonectin) TAAAAATGTT 8 35.1 4.6 0.000 119000 PAI1 Plasminogen activator inhibitor type 1 TCTCTGATGC 3 25.2 8.1 0.000 6441 TIMP2 Tissue inhibitor of metalloproteinase 2 Extracellular CTTTCTTTGA >8 7.6 0 0.000 130865 DKK3 DKK3 dickkopf homologue 3 (Xenopus laevis) GTTTTTTTTA 6 10.3 1.7 0.001 437656 LEPRE1 Leucine proline-enriched proteoglycan (leprecan) 1 CATTATAACT 13 7.6 0.6 0.001 84359 DULLARD Dullard homologue (Xenopus laevis) AAGGAAATGA >4 4.5 0 0.004 484063 SLIT3 Slit homologue 3 (Drosophila) CATATCATTA >15 14.8 0 0.000 435795 IGFBP7 Insulin-like growth factor binding protein 7 GCCCCAATAA >10 10.3 0 0.000 407909 LGALS1 Lectin, galactoside-binding, soluble, 1 (galectin 1) Signal transduction AAGCCTTGCT 9 5.4 0.6 0.010 411366 GRB2 Growth factor receptor-bound protein 2 TCCGTGGTTG >8 7.6 0 0.000 511745 BASP1 Brain abundant, membrane attached signal protein 1 TCTCAATTCT 4 12.6 2.9 0.001 146409 CDC42 Cell division cycle 42 (GTP binding protein, 25 kDa) TCCTGTAAAG 4 14.4 4.1 0.001 74034 CAV1 Caveolin 1, caveolae protein, 22 kDa AGCAGATCAG 2 26.5 11 0.001 143873 S100A10 S100 calcium binding protein A10 (annexin II ligand, calpactin I, light polypeptide (p11)) Integral to membrane TGGCCTAATA 43 25.6 0.6 0.000 1501 SDC2 Syndecan 2 (heparan sulfate proteoglycan 1) TGGCCTAAAA >4 4.5 0 0.004 1501 SDC2 Syndecan 2 (heparan sulfate proteoglycan 1) TACAATAAAC 11 13 1.2 0.000 9071 PGRMC2 Progesterone receptor membrane component 2 AGTTTCCCAA 10 12.1 1.2 0.000 279929 HSGP25L2G gp25L2 protein

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Table 1. Down-regulation of gene expression by N-myc (Cont’d)

c c b Tag sequence Ratio* SHEP2 SHEP21N P Unigene Gene Description

TCTTCTGCCA 6 9.4 1.7 0.002 86392 KIAA1402 CSGlcA-T KIAA1402 protein TTCCTCCTTT >7 7.2 0 0.000 151155 TM6SF1 Transmembrane 6 superfamily member 1 TTACTTATAC >6 6.3 0 0.000 159 TNFRSF1A Tumor necrosis factor receptor superfamily, member 1A TAAGAAAATG >6 5.8 0 0.001 443435 CDH11 Cadherin 11, type 2, OB-cadherin (osteoblast) AGAACCTTAA >5 4.9 0 0.003 181244 HLA-A Major histocompatibility complex, class I, A GCCCTTTCTC 3 25.2 7.5 0.000 7835 MRC2 Mannose receptor, C type 2 GTTGTGGTTA 2 60.3 28.5 0.000 75415 B2M h-2-microglobulin Transcription TACAGAGGGA 8 9.4 1.2 0.000 406096 ZNF216 Zinc finger protein 216 AAAAGATACT 6 7.6 1.2 0.003 82071 CITED2 Cbp/p300-interacting transactivator, with Glu/Asp-rich COOH-terminal domain, 2 GCCATATTAT >5 5.4 0 0.002 170752 CRIM1 Cysteine-rich motor neuron 1 CTCGAATAAA >4 4.5 0 0.004 34871 ZFHX1B Zinc finger homeobox 1b Others AATTTTCATT 10 5.8 0.6 0.006 339024 LOC253827 Hypothetical protein LOC253827 TTTTGTATTT 10 5.8 0.6 0.006 439190 C14orf31 Chromosome 14 open reading frame 31 GGTGAAGACA 7 8.5 1.2 0.001 26951 RUSC2 RUN and SH3 domain containing 2 CCCTGATTTT 6 19.8 3.5 0.000 183684 EIF4G2 Eukaryotic translation initiation factor 4 g,2 ATGTGAAGAA 6 6.7 1.2 0.008 13662 MGC5508 Hypothetical protein MGC5508 GAATAAATGT 6 7.2 1.2 0.005 497972 FKBP9 FK506 binding protein 9, 63 kDa GCTGTTTTGT >6 6.3 0 0.000 426312 AMOTL2 Angiomotin like 2 TTCCGGTTCC 5 9 1.7 0.003 172609 NUCB1 Nucleobindin 1 GTGGCTGAAA >5 4.9 0 0.003 401929 rpL10 ribosomal protein L10 CTTAATCCTG 4 30.6 8.1 0.000 298275 SLC38A2 Solute carrier family 38, member 2 GAAATAATGG 4 18.4 4.6 0.000 343879 SPANXC SPANX family, member C AAGGCAATTT 4 12.6 2.9 0.001 301626 FLJ11739 Homo sapiens cDNA FLJ11739 fis AAGTGAAACA 4 10.3 2.9 0.006 93659 ERP70 Protein disulfide isomerase related protein (calcium-binding protein, intestinal-related) TTAAAACAAA >4 4 0 0.01 370610 WAC WW domain containing adaptor with coiled coil AAAACATTCT 2 67.5 32.5 0.00 323562 DKFZp564K142 DKFZp564K142 implantation-associated protein

*Fold down-regulation of SHEP-2/SHEP-21N. cThe expression in SHEP-2 and SHEP-21N is given in number of tags normalized per 20,000 tags. bPs were calculated with the Monte Carlo simulation of the SAGE software (40).

Cdc42 mRNA in SHEP-21N compared with SHEP-2 (Fig. 2A). We therefore analyzed whether Cdc42 could function as a tumor When N-myc expression in SHEP-21N was down-regulated by suppressor gene in neuroblastoma. tetracycline, Cdc42 mRNA expression increased within 24 hours Activated Cdc42 induces neuronal differentiation in SHEP- (Fig. 2A). Western blot analysis showed that also the Cdc42 21N cells. Cdc42 is a G-protein that is active in the GTP-bound protein level increased after N-myc was switched off (Fig. 2B). The state and inactive in the GDP-bound state. We investigated the role regulation of Cdc42 was also investigated in cell line SKNAS- of Cdc42 in neuroblastoma by transient expression of the Cdc42 NmycER, which expresses a hybrid N-myc-estrogen receptor protein coupled to a COOH-terminal FLAG tag (Cdc42flag). We protein that can be activated by 4-hydroxytamoxifen. The first transfected a mutant Cdc42 form that is permanently active SKNAS-NmycER cells were treated with 4-hydroxytamoxifen and (Cdc42-G12V). After 40 hours, 42% of the SHEP-21N cells the Cdc42 mRNA expression decreased within 24 hours after N-myc expressing the Cdc42-G12Vflag protein showed neuronal differen- activation (Fig. 2C). The Cdc42 reduction was moderate (3-fold). In tiation, as evident by long neurite extensions of at least twice the addition, we analyzed the expression of Cdc42 mRNA in a panel of length of the cell body (Fig. 3A and B). The extensions were neuroblastoma tumors by Northern blot. Cdc42 expression was analyzed for expression of neurofilament, a marker for neuronal reduced in 10 tumors with N-myc amplification compared with 10 differentiation. The extensions in cells transfected with Cdc42- N-myc single copy tumors (Fig. 2D). The tumors show no G12V show formation of neural fibers with strong expression of the expression of c-myc (Fig. 2D). We conclude that Cdc42 mRNA neurofilament light chain (Fig. 3C). The differentiation was not expression in neuroblastoma cells is down-regulated by N-myc, observed in untransfected cells. In addition, we transfected SHEP- resulting in decreased Cdc42 protein levels. 21N with a wild-type Cdc42flag construct and with a dominant- The Cdc42 gene maps at 21.5 Mb from the 1p telomere, within negative Cdc42 mutant (Cdc42-T17N). Wild-type Cdc42flag and the 24.4 Mb region consistently deleted in N-myc amplified the dominant-negative Cdc42-T17Nflag-transfected cells showed neuroblastomas (2). The net result is that Cdc42 is 50% reduced only 3% and 6% differentiation, respectively (Fig. 3A and B), by deletion of one allele in N-myc amplified neuroblastoma suggesting that the wild-type Cdc42 protein in SHEP-21N cells is in tumors, and the expression is further down-regulated by N-myc. the inactive GDP-bound state. However, when N-myc was switched www.aacrjournals.org 3139 Cancer Res 2005; 65: (8). April 15, 2005

Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 2005 American Association for Cancer Research. Cancer Research off with tetracycline, also wild-type Cdc42flag-transfected cells We also analyzed the functional interaction of nm23-H1 and displayed neuronal differentiation (28%), whereas the dominant- nm23-H2 with Cdc42. Transient transfection of a wild-type negative Cdc42 construct remained inactive (Fig. 3B). This strongly Cdc42-flag construct into SHEP-21N cells resulted in 8% suggests that N-myc not only down-regulates the Cdc42 mRNA differentiation of the transfected cells (Fig. 3E). When N-myc expression level but also keeps the Cdc42 protein in its inactive was switched off in this experiment, the Cdc42flag-induced GDP-bound form. differentiation increased to 20% of the transfected cells. As N- Nm23 suppresses the activity of Cdc42. Cycling between the myc down-regulation resulted in a decrease of nm23-H1 and inactive GDP-bound and active GTP-bound state of Cdc42 is nm23-H2, we reconstituted these proteins by cotransfections. regulated by GTPase-activating proteins (GAP) and guanine SHEP-21N cells in which N-myc expression was switched off by nucleotide exchange factors. The inhibition of Cdc42-induced tetracycline were cotransfected with wild-type Cdc42flag and differentiation by N-myc indicated that N-myc controls a GAP or nm23-H1 and/or nm23-H2 expression vectors. Cotransfection of guanine nucleotide exchange factor of Cdc42. Previous experiments either nm23-H1 or nm23-H2 could completely replace the effect have shown that N-myc strongly up-regulates the expression of the of N-myc, as the differentiation reduced from 20% to 6% nm23-H1 and nm23-H2 genes in SHEP-21N (14). We confirmed the (Fig. 3E). The percentage of differentiated cells is not further up-regulation of the nm23-H1 gene in the SKNAS-NmycER cell line reduced by transfection of both nm23-H1 and nm23-H2. treated with 4-hydroxytamoxifen (Fig. 2C). In addition, in tumors Therefore, nm23-H1 and nm23-H2 prevent Cdc42-induced with N-myc amplification, nm23-H1 is also highly expressed differentiation. As N-myc strongly up-regulates nm23-H1 and (Fig. 2D). nm23-H2, they are likely to mediate the effect of N-myc. Nm23 proteins are primarily known to function as dinucleo- Cdc42 RNA interference: Cdc42 is essential for cell division. side kinases, but a possible role for nm23-H1 in GDP and GTP Neuroblastomas with N-myc amplification still have one Cdc42 exchange of Rad1 has been reported (20). We investigated allele. The classic paradigm for tumor suppressor gene whether the nm23-H1 and Cdc42 proteins can physically interact inactivation is deletion of one copy and mutation of the second with each other. For this purpose, we constructed the allele. We screened the coding region of Cdc42 for mutations in glutathione S-transferase (GST) fusion protein GST-nm23-H1. over 50 neuroblastoma tumors and cell lines but no mutations The GST-nm23-H1 protein was immobilized on glutatione- were found. The Cdc42 expression in N-myc amplified tumors sepharose beads and incubated with a lysate of SHEP-21N cells suggests haplo-insufficiency as a potential mechanism for transfected with Cdc42. The beads were analyzed for bound tumorigenesis. We further decreased the expression of Cdc42 proteins by Western blot. Cdc42 was pulled down by the GST- by RNA interference. The effect of two Cdc42-specific siRNAs was nm23-H1 protein and not by the GST protein alone (Fig. 3D). As compared with two N-myc-siRNAs, and siRNA for green fluores- a control for unspecific protein binding in the pull-down assay, cent protein, which has no target in the SHEP-21N cells. Western the Western blots were incubated with an N-myc antibody. N- blot and Northern blot analysis showed that 48 hours after myc did not bind to the GST or GST-nm23-H1 protein (Fig. 3D). transfection the expression of N-myc or Cdc42 was strongly We concluded that nm23-H1 and Cdc42 can physically interact. reduced by N-myc-siRNA and Cdc42-siRNA, respectively (Fig. 4A

Figure 1. Gene repression by N-myc. A, confirmation of the SAGE data. Northern blot analysis of 14 genes in SHEP-2 and SHEP-21N cells. Blot was also hybridized with N-myc (confirmation N-myc expression) and cofilin-1 (CFL1, an equally expressed gene). The 28S ribosomal band as loading control. B, Northern blot analysis of a time course experiment of N-myc. SHEP-21N cells were treated for 0 to 7 days with tetracycline, which switches off the ectopic N-myc expression. After 7 days, the cells were grown for a week in the absence of tetracycline (7+/7À). Right, analyzed genes.

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Figure 2. Cdc42 is down-regulated by N-myc in cell lines and tumors. A, Northern blot analysis of a time course experiment of N-myc. SHEP-21N cells were treated for 0 to 6 days with tetracycline, which switches off the ectopic N-myc expression. Lane 1, total RNA of SHEP-2 cells. The 28S ribosomal band as loading control. B, Western blot analysis of N-myc and Cdc42 proteins in a similar time course experiment in SHEP-21N. Part of the Coomassie-stained SDS-PAGE as loading control. C, Northern blot of SKNAS-N-mycER cells treated for 0 to 3 days with 4-hydroxytamoxifen (4OHT), which activates the N-mycER protein. Expression of Cdc42 and nm23-H1 mRNA. D, Northern blot analysis of N-myc, nm23-H1, Cdc42, and c-myc expression of 10 N-myc-amplified and 10 N-myc single-copy tumors.

and B). Cell growth of the N-myc-siRNA treated cells was which are particularly important in cytoskeletal remodeling (26, strongly reduced compared with the gfp-siRNA control (Fig. 4C). 27). It has a major role in actin reorganization, as Cdc42-GTP Silencing of Cdc42 had an even more dramatic effect on cell growth. binds to WASP, which in turn binds and activates the Arp2/3 The majority of SHEP-21N cells treated with Cdc42-siRNA died complex (28). This induces the branching of actin filaments and within 72 hours after transfection (Fig. 4C). In contrast, cells treated the formation of the cytoskeleton. Several genes functioning in with gfp-orN-myc-siRNA survived the treatment. The experiments this process are regulated by N-myc.N-myc not only down- were repeated on glass slides and the actin cytoskeleton and nuclei regulates Cdc42, but also actin (ACTG1 and ACTB) and ARPC1B were stained 72 hours after transfection. In the absence of Cdc42, (Table 1). Furthermore, Cdc42 has been shown to mediate the actin was organized in a peripheral membrane-associated ring, laminin-dependent differentiation in PC12 cells and murine indicating that these cells were unable to rearrange the cytoskeleton neuroblastoma cells (29–31). Here, we have shown that after cell division (Fig. 4D). The cells transfected with Cdc42-siRNA reconstitution of active Cdc42 allowed differentiation of human showed 16% apoptotic nuclei, compared with 2% and 5% in the neuroblastoma cells. The Cdc42 expression and activity is gfp-siRNA and N-myc-siRNA–treated cells, respectively (Fig. 4E). down-regulated at three different levels in N-myc amplified The apoptosis was further analyzed on Western blot. The cells neuroblastoma cells (Fig. 5). First, the expression is 50% were treated for 48 hours with siRNAs and the lysates were reduced by loss of heterozygosity of 1p36, as it maps in the screened for cleaved proteins. In cells treated with Cdc42-siRNAs, 1p region consistently deleted in N-myc amplified neuroblasto- both caspase3 and poly(ADP-ribose) polymerase showed the mas. Second, a further 4- to 5-fold down-regulation of Cdc42 apoptosis-induced cleavage products (Fig. 4F). No cleaved mRNA is caused by the overexpression of N-myc. Together, this products were visible after treatment with gfp-orN-myc-siRNA. results in an estimated 8- to 10-fold down-regulation at the Therefore, Cdc42 is an essential gene for the growth and survival of mRNA level. Third, N-myc inhibits the differentiation induced neuroblastoma cells. This provides an explanation for the absence by the Cdc42 protein. This effect of N-myc is fully reconstituted of Cdc42 mutations: a minimal amount of Cdc42 is required for by transfection of nm23-H1 and nm23-H2 constructs, which cell survival. strongly suggests that the N-myc effect is mediated by the nm23 proteins. Regulation of the nm23-H1 and nm23-H2 genes in neuroblas- Discussion toma is a mirror image of the Cdc42 regulation. The nm23 genes Cellular transformation involves increased proliferation, alter- map in the minimal region of 17q gain and are present with ations in cell shape and adhesion. The N-myc protein regulates two to eight extra copies. The expression of these nm23 copies genes involved in these processes. Here, we have shown that N- is 6- to 10-fold up-regulated by N-myc, resulting in a 12- to 80- myc represses genes determining the cell structure. The N-myc fold overexpression of nm23-H1 and -H2 (14). Therefore, the targets include structural genes but also one of the key ratio between Cdc42 and nm23 protein levels is probably at least regulators Cdc42. Cdc42 is a member of the Rho GTPase 100-fold out of balance. The differentiation-blocking effect of N- subfamily and functions in a series of signaling pathways, myc is thus amplified by chromosomal imbalances of the www.aacrjournals.org 3141 Cancer Res 2005; 65: (8). April 15, 2005

Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 2005 American Association for Cancer Research. Cancer Research downstream mediators, Cdc42 and nm23-H1 and nm23-H2. (21). In this study, we showed that nm23-H1 can bind to the Moreover, neuroblastomas frequently have an unbalanced Cdc42 protein and nm23 can inhibit the Cdc42-induced differen- translocation between chromosomes 1p and 17q, causing loss tiation. These data suggest that nm23-H1 and nm23-H2 also exerts of heterozygosity of 1p and gain of 17q (32, 33). This would, by a GAP-activity for Cdc42. single hit, disturb the balance between Cdc42 and the nm23 Is Cdc42 the elusive neuroblastoma suppressor gene at distal genes. The translocation event is often followed by duplication chromosome 1p? If Cdc42 indeed functions as a tumor of the derivative chromosome 1 with the extra 17q material, suppressor gene, it would be unlikely to be the only 1p suppressor further increasing the imbalance. Amplification of N-myc would gene in neuroblastoma. Deletions of 1p in N-myc amplified boost the effect of the imbalance, by down-regulation of the neuroblastomas are at least 24.4 Mb. If Cdc42, which maps at 21.5 remaining Cdc42 copy and up-regulation of the overrepresented Mb from the telomere, would be the one and only suppressor nm23 genes. The down-regulation of Cdc42 and up-regulation of gene, a certain frequency of interstitial deletions encompassing the nm23 genes by N-myc is very consistent in neuroblastoma Cdc42 would be expected. The length of the deletions has cell lines and tumors. In the two cell lines in which we can previously raised the idea that more than one suppressor gene is manipulate N-myc activity, Cdc42 is down-regulated and the involved (5). Thus far, only one homozygous 1p36 deletion was nm23 genes are up-regulated. Furthermore, comparison of a identified in a neuroblastoma cell line (35). The scarcity of panel of 10 N-myc amplified tumors and 10 N-myc single copy homozygous 1p36 deletions in N-myc amplified neuroblastomas neuroblastomas strongly supports an identical effect of N-myc has suggested that the postulated tumor suppressor gene(s) may on Cdc42 and the nm23 genes in vivo (this article and ref. 14). function by haploinsufficiency. Our data suggest that Cdc42 may The nm23 proteins could function as a GAP for Cdc42, keeping represent the first example of such a gene. The chromosome 17q Cdc42 in the inactive, GDP-bound state. Several lines of evidence region gained in neuroblastoma tumors is also very large. support this role for nm23-H1. Direct GAP activity of nm23-H1 Therefore, in addition to the nm23 genes, other 17q genes may was shown for the Rad1 GTPase in melanoma cells (20). contribute to neuroblastoma pathogenesis as well. Involvement in the regulation of Cdc42-family member Rac1 Neuroblastomas are embryonal tumors that originate from was established as well. Nm23-H1 is recruited toward adhesive neural crest–derived cells. During normal embryogenesis N-myc is junctions by ARF6-GTP (34). At these junctions, nm23-H1 expressed in migrating neuroblasts (36) and probably prevents negatively regulates TIAM1 and subsequently Rac1 is inactivated premature differentiation. When neuroblasts arrive at their

Figure 3. Neuronal differentiation is mediated by active Cdc42 and counteracted by nm23-H1. SHEP-21N cells were grown without and with tetracycline (tet) and transfected with flag-tagged Cdc42, Cdc42-G12V, or Cdc42-T17N. Cells were fixed 40 hours after transfection and stained with an anti-FLAG antibody to identify transfected cells. A, examples of an undifferentiated (Cdc42-T17N transfected) and differentiated (Cdc42-G12V transfected) cell. B, differentiation frequencies in over 500 flag-positive cells per transfected construct. Bars, SE. *, P < 0.01 (v 2 test). C, expression of neurofilament (NFL) upon differentiation in SHEP-21N cells positive for Cdc42-G12V-flag expression. Note the nodules of the neural structures are negative for neurofilament. D, Cdc42 and nm23-H1 physically interact. SHEP-21N cells were transfected with Cdc42. The lysates were incubated with fusion protein GST-nm23H1 (lanes 1 and 2) or GST (lanes 3 and 4) linked to gluthatione-sepharose beads and analyzed for bound Cdc42 protein (pull-down) on Western blot. Part of the lysate was analyzed for Cdc42 expression. Blots were stripped and incubated with anti-N-myc. E, analysis of neuronal differentiation by Cdc42 in the presence or absence of nm23. The SHEP-21N cells were grown in the absence or presence of tetracycline and transfected with flag-tagged Cdc42 in combination with empty vector, nm23-H1 or nm23-H2. Cells were fixed 40 hours after transfection and stained with an anti-flag antibody to identify transfected cells. Approximately 1,000 flag-positive cells were scored for neuronal differentiation. Bars, SE. *, P < 0.01 (v2 test).

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Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 2005 American Association for Cancer Research. N-myc, Cdc42, and nm23 Cooperate in Neuroblastoma destination in the trunk, N-myc expression ceases and differenti- observed during cell division and should be followed by actin ation proceeds, in which the Cdc42/nm23 pathway may play an rearrangent and the appearance of new stress fibers. Absence of important role. The strong differentiation-inducing potential of Cdc42 causes a collapse of the cytoskeleton and might thus Cdc42 and the suppression of this effect by N-myc and nm23 inhibit cell cycle progression. Alternatively, the observed cell suggest that this is a major mechanism in neuroblast differenti- death may result from the involvement of Cdc42 in spindle ation. We therefore consider Cdc42 as a strong candidate tumor formation and cell cycle progression by the Cdc42-PAR6-PKC3 suppressor gene for neuroblastoma. pathway, as observed in Caenorhabditis elegans and rat astrocytes The classic proof of principle for a tumor suppressor gene is (37, 38). Surprisingly, SHEP-21N cells treated with siRNA still show inactivation of both gene copies. One copy of Cdc42 is deleted, many metaphases after 72 hours. This phenotype resembles the but we detected no mutations in the remaining copy. Our siRNA yeast Cdc42-V44A mutant, which cannot interact with multiple experiments showed that a minimal Cdc42 activity is required for effector proteins (39). Half of these mutant cells have two or more viability of neuroblastoma cells, explaining why Cdc42 cannot be nuclei, indicating a G2-M-phase delay and a defect in cytokinesis homozygously inactivated. Down-regulation of Cdc42 by siRNA and cell separation. caused a major reduction in cell number and dislocation of actin The mechanism of inactivation of Cdc42 might represent a new to the inner surface of rounded cells. Normally, this is only paradigm for tumor suppressor gene inactivation. Chromosomal

Figure 4. RNA interference of Cdc42 inhibits cell division and actin remodeling. Equal numbers of SHEP-21N cells were treated with siRNA for gfp (negative control), N-myc, and Cdc42. For Cdc42 and N-myc, two siRNAs were used (a and b), directed against different regions. A, Western blot of cell lysates isolated 48 hours post-transfection. Right, incubated anti-bodies. Controls: anti-h-tubulin (tub); Coomassie-stained SDS-PAGE gel (loading control). B, Northern blot of siRNA-treated SHEP-21N cells. Right, hybridized probes. Controls: cofilin (CFL1), 18S (loading control). C, light microscopy picture of cells (40Â magnification), 72 hours post-transfection. D, cytoskeleton staining with phalloidine-TRITC of the cells 72 hours post-transfection (1,000Â magnification). The nuclei were stained with 4V,6-diamidino-2-phenylindole and scored for apoptosis. Arrowhead, apoptotic cell with apoptotic bodies. E, quantification of apoptosis. Bars, SE. F, Western blot of cell lysates 48 hours post-transfection. Blots were incubated with anti-caspase3 and anti-poly(ADP-ribose) polymerase (PARP). Fraction of cleaved protein (bottom of lanes).

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Figure 5. Model for the down-regulation of Cdc42 activity by N-myc, loss of heterozygosity of chromosomal band 1p36, and gain of the chromosome 17q arm.

regions that consistently show hemizygosity or moderate copy in the pathway and restore the function of the remaining Cdc42 number gains are frequent in human tumors. For many of them, allele. no tumor suppressor genes or oncogenes have been identified as yet. Here we show that these relatively limited changes in copy Acknowledgments numbers can dramatically amplify the oncogenic effect of an Received 7/10/2004; revised 1/4/2005; accepted 1/31/2005. activated pathway, in this case the N-myc pathway. Importantly, Grant support: Dutch Cancer Society (L.J. Valentijn and A. Koppen). in this model the actual tumor suppressor gene does not have The costs of publication of this article were defrayed in part by the payment of page to be completely inactivated. Cdc42 is not fully destroyed like charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. classic tumor suppressor genes but can be reactivated. This We thank K. Berns for providing the pBSK-ER plasmid and J. Collard for the GST- offers a clinical perspective, as innovative drugs may interfere plasmid.

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Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 2005 American Association for Cancer Research. Inhibition of a New Differentiation Pathway in Neuroblastoma by Copy Number Defects of N-myc, Cdc42, and nm23 Genes

Linda J. Valentijn, Arjen Koppen, Ronald van Asperen, et al.

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