PPM1D Is a Potential Target for 17Q Gain in Neuroblastoma1

[CANCER RESEARCH 63, 1876–1883, April 15, 2003] PPM1D Is a Potential Target for 17q Gain in Neuroblastoma1

Fumiko Saito-Ohara,2 Issei Imoto,2 Jun Inoue, Hajime Hosoi, Akira Nakagawara, Tohru Sugimoto, and Johji Inazawa3 Department of Molecular Cytogenetics, Medical Research Institute, Tokyo Medical and Dental University, Tokyo 113-8510 [F. S-O., I. I., J. Ino., J. Ina.]; Core Research for Evolutional Science and Technology of Japan Science and Technology Corporation, Saitama 332-0012 [F. S-O., I. I., J. Ina.]; Theranostics Research Center, Otsuka Pharmaceutical Co. Ltd., Tokushima 771-0192 [J. Ino.]; Department of Pediatrics, Kyoto Prefectural University of Medicine, Kyoto 602-8566 [H. H., T. S.]; and Division of Biochemistry, Chiba Cancer Center Research Institute, Chiba 260-8717 [A. N.], Japan

ABSTRACT treatment known as the INSS (3). However, clinical criteria are not likely to predict disease outcome in a reliable way. Neuroblastomas (NBs) show complex patterns of genetic abnormalities, The remarkable variability in clinical course of NB reflects diverse which may include amplification of the MYCN gene, deletion of 1p, or a genetic changes acquired by the tumor cells. Among the genomic gain of DNA at 17q, the last being the most frequent observation in NB tumors. However, the specific genes and the molecular mechanisms re- abnormalities identified in NB tumors to date, some, including am- sponsible for development and progression of NB remain poorly under- plification of MYCN (4), deletion of 1p (5), gain of 17q (6), and stood. We investigated aberrations of DNA copy number in 25 NB cell diploidy or tetraploidy (7), are associated with poor prognosis. LOH lines using comparative genomic hybridization and identified a minimal and FISH analyses have revealed other alterations of subchromosomal common region of gain at 17q23. Although gain of distal 17q is the most regions, e.g., losses at 3p, 4p, 9p, 11q, 14q and gains at 5q and 18q (8, powerful genetic predictor of adverse outcome currently available for 9). Recent CGH analyses have confirmed cytogenetic abnormalities patients with NB, thus far, no potential target genes have been reported that were previously reported and also revealed additional genetic for that region. Therefore, we defined the 17q23 amplicon in detail and aberrations in NB cells (10–12). Despite detection of numerous non- determined expression levels of 15 genes located within the smallest region random alterations, to date MYCN is the only gene corresponding to of overlap observed among our NB cell lines to identify the most likely any of the affected chromosomal regions that has been identified. To target gene(s). Among them, seven (CLTC, VMP1, delta-tubulin, RPS6KB1, FLJ22087, APPBP2, and PPM1D) were consistently overexpressed gain new insights into the pathogenesis of NB and to establish through increases in regional copy number. Analysis of expression levels molecular targets for diagnosis and therapy, candidate genes in the of those seven genes in 32 primary NB tumors revealed a significant altered regions, 1p and 17q in particular, must be identified and correlation between higher expression and poorer clinical outcome only investigated. with respect to PPM1D. Moreover, down-regulation of PPM1D by trans- Tumor cell lines provide valuable resources for gene discovery and fection of an antisense oligonucleotide suppressed the growth of NB cell functional studies because their molecular and cytogenetic aberrations lines to a remarkable degree, at least partly by participating in a process and biological properties may reflect at least a subset of primary leading to apoptotic cell death. Taken together, our results indicate that tumors. Many cell lines have been established from NB over the past PPM1D is the most likely target of the 17q23 gain/amplification in NB several years, and detailed knowledge of specific chromosomal aber- tumors and may have an important role in the pathogenesis of this disease. rations leading to losses, gains, or amplification of particular chromosomal regions in these tumor-derived cells will be instrumental in identifying target genes. We have already carried out extensive CGH INTRODUCTION studies in various other types of tumor cell lines and have identified NB,4 the most common extra-cranial solid tumor in children, is genes present in detected amplicons that may be involved in tumor- characterized by diverse clinical behavior ranging from spontaneous igenesis (13–15). regression to rapid malignant progression. These differences reflect In the work reported here, we examined 25 NB cell lines by CGH biological heterogeneity among individual tumors, with the conse- to explore genomic alterations that might affect the development quence that both prognosis and response to therapy can vary widely and/or progression of this disease. As in previous studies, we found from one patient to another (1, 2). In view of this heterogeneity, the most frequent gains at distal 17q. Among the cell lines showing understanding the characteristics of a given NB tumor is crucial for 17q gains, we identified one line in which amplification at 17q23 was choosing appropriate therapy. Extensive efforts in the past led to detected as a remarkably HLG by CGH and as a HSR by FISH. Gains standardization of criteria for diagnosis, staging, and response to of distal 17q are observed mainly in advanced stages of NB; this change is considered to be the most powerful genetic predictor of Received 10/28/02; accepted 2/18/03. adverse outcome for patients (6, 16). Copy-number gain/amplification The costs of publication of this article were defrayed in part by the payment of page of this region, and possible target genes present there, have been charges. This article must therefore be hereby marked advertisement in accordance with reported in breast and gastric cancers (17–21). Taken together, these 18 U.S.C. Section 1734 solely to indicate this fact. 1 Supported by grants-in-aid for Scientific Research (B) and Scientific Research on lines of evidence strongly suggested that 17q23 might harbor one or Priority Areas (C) from the Japanese Ministry of Education, Culture, Sports, Science, and more genes whose amplification renders them oncogenic, although no Technology, from Core Research for Evolutional Science and Technology of Japan Science and Technology Corporation, and the Special Program of Integrated Genomics potentially significant candidate for NB had yet been identified within and Advanced Medical Frontier Research. this region. Therefore, we carried out further molecular definition of 2 These authors contributed equally to this work. the 17q23 amplicon in NB cell lines by examining expression levels 3 To whom requests for reprints should be addressed, at Department of Molecular Cytogenetics, Medical Research Institute, Tokyo Medical and Dental University, 1-5-45 of candidate genes located within the amplicon in cell lines and Yushima, Bunkyo-ku, Tokyo 113-8510, Japan. Phone: 03-5803-5820; Fax: 03-5803- primary tumors of NB, to identify genes whose products might play 0244; E-mail: [email protected]. important roles in the tumorigenesis of NB. 4 The abbreviations used are: NB, neuroblastoma; INSS, International Neuroblastoma Staging System; LOH, loss of heterozygosity; FISH, fluorescence in situ hybridization; CGH, comparative genomic hybridization; HLG, high-level gain; HSR, homogeneously MATERIALS AND METHODS staining region; BAC, bacterial artificial chromosome; PAC, P1-derived artificial chromosome; UCSC, University of California at Santa Cruz; NCBI, National Center for NB Cell Lines and Tumors. All human NB cell lines we examined (Table Biotechnology Information; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; SRO, smallest region of overlap; OPT, oligonucleotide phosphorothioate; NMA, MYCN ampli- 1) had been established from surgically resected tumors and maintained in fication. RPMI 1640 supplemented with 10% fetal bovine serum. 1876

Table 1 Summary of 25 NB cell lines used in this study generated from total RNAs of cell lines and primary NB tumors using the No. Namea NMAb HLG sitesc SuperScript First-Strand Synthesis System (Invitrogen, Carlsbad, CA) according to the manufacturer’s directions. Real-time quantitative PCR was per- 1 KP-N-RT (ϩ) DM 2p22-p24 2 KP-N-SIFA (Ϫ) formed using LightCycler (Roche Diagnostics) with CYBR Green according to 3 KP-N-SILA (Ϫ) 1q43-qter the manufacturer’s protocol. Primer sequences for each gene are available on 4 KP-N-YN (ϩ) DM 2p23-p24 request. The GAPDH gene (Roche Diagnostics) served as an endogenous ϩ 5 KP-N-YS ( ) HSR 2p23-p24, 5p15.1-pter, 19q12-q13.1 control, and each sample was normalized on the basis of its GAPDH content. 6 KP-N-AYR (ϩ) DM 2p22-p24, 17q22-qter 7 KP-N-TK (ϩ) HSR 17q23-qter PCR amplification was performed in duplicate for each sample. 8 KP-N-AY (ϩ) DM 2p21-pter Analysis for TP53 Mutations. Exons 5–8oftheTP53 gene were analyzed -MP-N-TS c-MYC (؉) HSR 8q24.1-q24.3, 17q23 by direct sequencing of their genomic PCR- or reverse transcriptase-PCR 9 Ϫ 10 KP-N-NY ( ) 12q13-q15 amplified products as described by Smith et al. (27) and Fleckenstein et al. 11 KP-N-NH (Ϫ) 17q22-qter 12 MP-N-MS (ϩ) DM 2p22-p25, 17q22-qter (28), respectively, using an ABI377 sequencer (PE Biosystems, Foster 13 SMS-KCN (ϩ) DM 2p22-p25 City, CA). 14 SJ-N-KP (Ϫ) Transfection with Antisense OPT. Antisense experiments were per- Ϫ 15 SK-N-SH ( ) formed as previously described (15), with minor modifications. Briefly, we 16 SK-N-DZ (ϩ) HSR 2p23-p24 17 IMR32 (ϩ) HSR 2p22-p24, 2p13-p14, 17q22-q24 synthesized the following oligonucleotides containing phosphorothioate back- 18 SMS-KAN (ϩ) DM 2p21-p24 bones (OPT; Espec Oligo Service Co., Tsukuba, Japan): PPM1D-AS, nucle- 19 NB-1 (ϩ) HSR 1q21-q32, 2p21-p24 otides 223–240 of PPM1D cDNA (GenBank accession no. NM_003620) in the Ϫ 20 SK-N-AS ( ) 17q22-q24 antisense direction; PPM1D-IV, the inverse control for PPM1D-AS; PPM1D- 21 CHP134 (ϩ) HSR 2p22-p24, 17q22-qter 22 SMS-KCNR (ϩ) DM 2p22-p25 SC, a scrambled control for PPM1D-AS; c-MYC-AS, nucleotides 554–573 of 23 GOTO (ϩ) HSR 20q13.2-qter c-MYC cDNA (GenBank accession no. NM_002467; Ref. 29) in the antisense 24 SJ-N-CG (ϩ) HSR 17q23-q24 direction; c-MYC-SC, a scrambled control for c-MYC-AS (29); MYCN-AS, ϩ 25 RT-BMV-C6 ( ) DM 2p22-p24 nucleotides 175–189 of MYCN cDNA (GenBank accession no. NM_005378; a None of cell lines carried mutations in exons 5–8oftheTP53 gene. Ref. 30) in the antisense direction; and MYCN-SC, a scrambled control for b The status of MYCN amplification (NMA) was determined by FISH using N-myc- specific probe. Note that MP-N-TS cells have c-MYC amplification (bold) instead of MYCN-AS (30). The OPT used in this study was delivered into cells using NMA. DM, double minute chromosomes. Oligofectamine (Invitrogen) according to the manufacturer’s instructions. c Bold indicates HLG on part of 17q. For evaluation of gene expression, 2 ϫ 105 cells were plated on a 10-cm dish, transfected, and harvested 24 h later. RNA levels were determined by real-time quantitative RT-PCR. For measurements of viable cells (cell growth) Primary tumor samples from 32 NB patients were provided by the Chiba or cell numbers, the day before transfection 2 ϫ 103 or 3 ϫ 104 cells were Cancer Center Hospital, with written consent from each patient in formal style seeded respectively in 96- or 6- well plates. Viable cells were assessed 48, 72 after approval by the local ethics committee. The stage of each tumor was or 96 h after transfection by the microtiter-plate colorimetric WST assay (Cell classified according to INSS criteria (stage 1, 12 tumors; stage 2, 5 tumors; counting kit-8; Dojindo Laboratories, Kumamoto, Japan). Numbers of living stage 3, 2 tumors; and stage 4, 13 tumors; Ref. 3). All 32 patients were and dead cells were assessed 48 or 72 h after transfection by direct counting diagnosed and treated between 1996 and 1998, according to previously de- using the trypan-blue exclusion method. For assessing nuclear morphology, scribed protocols (22). The status of MYCN amplification, Trk-A expression, cells that had been treated with OPT as the same manner in the analysis of cell LOH on 1p36, and DNA ploidy in each tumor had been determined before the numbers were fixed with 4% paraformaldehyde, and then stained with 4Ј,6- present study began (23, 24). diamidino-2-phenylindole. Experiments were repeated three times, and per- CGH Analysis. CGH experiments were performed using directly fluoro- formed in triplicate each time. chrome-conjugated DNA as described by Kallioniemi et al. (25), with minor Statistical Analysis. The Mann-Whitney U test was used to compare the modifications (13). Briefly, tumor and normal DNAs were labeled, respec- mRNA expression level of each gene among subgroups of primary tumors. tively, with Spectrum Green-dUTP and Spectrum Orange-dUTP (Vysis, Chi- Possible correlations between variables of the analyzed tumor samples and cago, IL) by nick translation, denatured, and hybridized to normal male gene expression status were tested by the ␹2 or Fisher’s exact test. Kaplan- metaphase chromosome spreads together with Cot-1 DNA. The slides were Meier survival plots were constructed, and log-rank tests were used for washed and counterstained with 4Ј,6-diamidino-2-phenylindole. Shifts in CGH comparisons between groups. Survival data were also subjected to Cox profiles were rated as gains or losses if they reached at least the 1.2 or 0.8 proportional-hazards regression analysis. One-way analyses of variance (one- thresholds, respectively, and overrepresentations were considered to be HLGs way ANOVA) with subsequent Scheffe´’s tests determined the significance of when fluorescence ratios exceeded 1.5, as described elsewhere (13). Hetero- differences in multiple comparisons. P Ͻ 0.05 was required for significance in chromatic regions near the centromeres and the entire Y chromosome were each case. excluded from the analysis. FISH. Metaphase chromosome slides were prepared for FISH experiments in the manner described previously (13). The location of each BAC (RPCI-11 RESULTS and CTC library) or PAC within the region of interest was compiled from information described by Wu et al. (10) and archived by the UCSC5 or the DNA Copy Number Aberrations Detected by CGH in NB Cell NCBI.6 We confirmed or modified the mapping data according to the results Lines. An overview of the chromosomal aberrations we detected of FISH using normal metaphase chromosomes. A MYCN-specific BAC among 25 NB cell lines is shown in Fig. 1. All lines we tested showed (355H10) and a c-MYC-specific PAC (80K22) were also used as probes. chromosomal imbalances. On average, 9.5 genetic changes were Probes were labeled with biotin-16-dUTP or digoxigenin-11-dUTP by nick- found/cell line, including 5.9 (range, 2–11) gains and 3.4 (range, 1–6) translation (Roche Diagnostics, Tokyo, Japan). Chromosomal in situ suppres- losses. Minimal common regions for the most frequent copy number sion hybridization and fluorescent detection of hybridization signals were gains were at 17q23 (23 of 25, 92%), 2p23-p24 (19 of 25, 76%), carried out as described elsewhere (13). The copy number and molecular 1q31-q41 (14 of 25, 56%), 7p11.2-p21 (9 of 25, 36%), 7q22-q36 (9 of organization of the region of interest were assessed according to the hybrid- 25, 36%), and 8q24.3 (8 of 25, 32%). The most common losses were ization patterns observed on both metaphase and interphase chromosomes. Real-Time Quantitative Reverse Transcriptase-PCR. The quantification observed at 1p36.2-pter (16 of 25, 64%) and 11q21-q22 (7 of 25, of mRNA of genes present within the 17q23 amplicon was carried out using a 28%). Most of the cell lines (22 of 25, 88%) displayed prominent, real-time fluorescence detection method (26). Single-stranded cDNAs were localized regions of HLG indicative of gene amplification (Table 1, Fig. 1). The chromosomal region at 2p23-p24 showing HLG (14 of 5 Internet address: genome.ucsc.edu. 25, 64%) contains MYCN, a gene that is amplified in many NB tumors 6 Internet address: www.ncbi.nlm.nih.gov. (4). Another frequent HLG was detected at 17q23 in nine NB cell 1877

Fig. 1. Summary of genetic imbalances detected by CGH in 25 NB cell lines. The 22 autosomes and X chromosome are represented by ideograms showing G-banding patterns. As judged by the computerized green-to-red profiles, vertical lines on the left of each chromosome ideogram show losses of genomic materials in cell lines, whereas those on the right correspond to copy number gains. HLGs are represented as open rectangles. The number at the top of each vertical line corresponds to the cell line in which each change was recorded (see Table 1).

lines (36%). MYCN amplifications were confirmed by FISH using a defined between BAC758H09 and 215P18, except for BAC264B14 probe specific for that gene (Table 1). Notably, the MP-N-TS cell line and 136H19 (SRO I), or else between BAC758H09 and 106H22 (SRO showed amplification of c-MYC rather than MYCN (Table 1), and II) if we consider one copy number difference to be significant. The none of the 25 lines we tested carried mutations in exons 5–8ofthe respective sizes of SROs I and II are ϳ2.2 and 1.8 Mb, according to TP53 gene. information in the UCSC database. To avoid the oversight of impor- Definition of the 17q23 Amplicon by FISH. We focused on the tant target gene(s) for the 17q23 amplification in additional analyses, 17q23 amplification detected in NB for the following reasons: (a) the SRO I was adopted as a critical region harboring targets. distal 17q is the region most frequently involved in NB tumors, as Expression of Candidate Target Genes within the 17q23 Am- reported elsewhere (10, 11, 16, 31); (b) we were able to narrow the plicon in NB Cell Lines. Genes activated by copy number increases common region of gain to 17q23 by CGH analysis (Fig. 1); (c) gain and involved in the progression of tumors are likely to be located in of distal 17q is the most powerful known genetic predictor of adverse the SROs of amplicons (14, 19). To determine whether genes ampli- outcome for NB patients (6, 16, 32); and (d) no potential target gene fied at 17q23 were overexpressed in association with amplification, had yet been identified within this region in NB tumors. To generate we assessed expression status of transcripts located within SRO I in a defined map of the 17q23 amplicon, we performed FISH analyses in NB cell lines. On the basis of our map constructed on the basis of the four of our NB cell lines (MP-N-TS, SJ-N-CG, KP-N-NH, and SMS- FISH results (Fig. 2B), 15 transcripts, consisting of 10 known genes KAN) that had exhibited remarkable copy number gains (data not and five uncharacterized transcripts, were selected from the genome shown), using 20 BACs spanning the amplified region as probes. databases archived by UCSC and NCBI (Fig. 2A, Table 2). The Relative positions of BACs on a map of the 17q23 region are indi- expression level in NB cell lines of each transcript, normalized with cated in Fig. 2A. Copy numbers, as well as molecular organization of GAPDH, was divided by the average value found in NB cell lines the amplicon, were assessed by analysis of the hybridization patterns having normal copy numbers at 17q23 (KP-N-DZ and KP-N-NY, data on metaphase and interphase chromosomes. not shown) and recorded as a fold increase in relative expression level In one cell line (MP-N-TS), 11 BACs (758H09, 619I22, 700K10, (19). Although the NCBI database predicted some additional tran- 178C03, 767P09, 371B04, 15E18, 106H22, 332H18, 15K02, and scribed sequences within our SRO, we excluded those from expres- 215P18) produced the highest number of signals as HSRs on marker sion screening because their genomic structures and/or expression chromosomes (Fig. 2, B and C). Fewer signals were detected with the status suggested that they were unlikely to be real transcripts. Indeed, remaining 9 BACs (142B17, 1081P03, 329E11, 1096F01, 264B14, our preliminary reverse transcriptase-PCR experiments failed to am- 136H19, 320E04, 342K02, and 42F20), suggesting that they were plify a single product of such predicted sequences (data not shown). located outside the amplicon. The other three cell lines examined by As summarized in Table 2, 7 (CLTC, VMP1, delta-tubulin, FISH each yielded more than five signals; however, the number of RPS6KB1, FLJ22087, APPBP2, PPM1D) of 15 transcripts were con- signals in each line was not different among all 20 BACs, except for sistently overexpressed in cell lines exhibiting increased copy num- one increase observed between BAC1096F01 and 106H22 in the bers at 17q23 (Fig. 2, A and B) compared with lines exhibiting normal SJ-N-CG cell line (Fig. 2, B and C). Therefore, the SRO could be copy numbers, strongly suggesting that these transcripts might be 1878

Fig. 2. A, map of 17q23 covering the region amplified in NB cell lines. Selected sequence-tagged site (STS) markers are mapped on the left of the vertical chromosome. BACs and PACs are indicated as vertical bars; those used for FISH are shown in black. The 15 genes and uncharacterized transcripts we analyzed are boxed (see Table 2). All markers and transcripts are positioned according to the information provided by Wu et al. (24) and archived by UCSC and NCBI. B, summarized results of DNA copy-number analysis by FISH in four different cell lines. The horizontal axis (top) shows the number of FISH signals detected with the BAC/PAC probes described above. The number of signals was truncated at 15 because it was difficult to enumerate them above that level. Lines connect the measurements made for each cell line. The SRO with the maximal amplification was defined between BAC758H09 and 215P18, except for BAC264B14 and 136H19 (SRO I) or between BAC758H09 and 106H22 (SRO II) if the change of one copy number is considered significant. C, representative images of FISH on metaphase chromosomes from NB cell lines. In MP-N-TS cells, BAC142B17, located outside the amplified region, shows only two signals (top), whereas BAC15E18, containing the PPM1D gene, shows an HSR pattern on the marker chromosome (middle, arrowhead). BAC15E18 shows six signals in SJ-N-CG cells as well (bottom). targets for 17q23 gain/amplification in NB (14, 19). On the other expression group or a high expression group, respectively, and the hand, none of the other eight transcripts within SRO I was consistently clinicopathological implications of each gene’s expression level were overexpressed in conjunction with copy number gains in the region. evaluated by comparisons between the two groups. The clinicopath- Notably, none of the three transcripts located outside SRO II showed ological factors we analyzed are shown in Table 3. Among the 32 consistent overexpression as a consequence of amplification, although primary NB tumors, only two carried mutations in exons 5–8ofthe one or more of them are positional and functional targets for 17q TP53 gene. In accord with the results of our nonparametric analysis, amplification in breast cancers (33). only PPM1D mRNA expression status showed a statistically signifi- Expression of Potential Targets for the 17q23 Amplification in cant correlation with patient outcome. Moreover, the PPM1D high Primary NB Tumors. To identify the most likely target gene(s) expression group showed a significantly poorer outcome compared among the seven candidates, we additionally determined their expres- with the PPM1D low expression group in all patients, as well as in sion levels in each of 32 primary NB tumors and compared the patients who were positive for MYCN amplification (NMA-positive expression patterns with clinicopathological data, especially the prog- patients; Table 3). In contrast, none of the other six transcripts showed nosis of patients. As a screening procedure, we compared living, significant correlation between expression level and any clinicopath- disease-free patients with those who had died of their tumors, by a ological factor (data not shown). nonparametric Mann-Whitney U test. Only PPM1D mRNA expres- Fig. 3B shows Kaplan-Meier survival curves for all 32 NB patients sion revealed a significant difference between the two groups and for the 16 NMA-positive patients. Patients whose tumors showed (P ϭ 0.0160, Fig. 3A); the other six genes showed none (Fig. 3A). To high PPM1D expression had significantly shorter overall survival confirm this result, we considered cases with values of less than or times than did patients with low PPM1D expression in their tumors more than the mean expression level of each gene to belong to a low (P ϭ 0.0034, log-rank test; Fig. 3B, left). In addition, a Cox propor- 1879

Table 2 Relative expression levels of 15 positional candidate target transcripts for on NB cell growth/survival by down-regulating this gene with an 17q-gain/amplification in NB cell lines antisense oligonucleotide (15). MP-N-TS cells, which had exhibited amplification and consequent overexpression of PPM1D, were transfected with an antisense OPT for the PPM1D gene (PPM1D-AS). PPM1D-AS, but not the control OPT (IV or SC) or the transfecting reagent Oligofectamine alone (Mock), induced a decrease in PPM1D mRNA (Fig. 4A). Growth of PPM1D-AS-treated cells was remarkably inhibited compared with control OPT-treated cells (PPM1D-IV

Table 3 Correlation between patient profiles and PPM1D mRNA expression in 32 cases with NB PPM1D expressiona

low high a Genes and uncharacterized transcripts were selected from the SRO of the 17q23 Variables n (n ϭ 23) (n ϭ 9) Pb amplicon in NB cell lines (Fig. 2). b http://www.ncbi.nlm.nih.gov/LocusLink/. Age Ͻ c Expression level of each gene in each cell line was divided by the average value of 1 yr 19 15 4 0.4269 м1yr 13 8 5 that in SK-N-DZ and KP-N-NY cell lines (reference value), which have normal copy c number of 17q, after normalization with GAPDH, and recorded as a fold increase in Stage relative expression level. Fold increase in relative expression levels Ͼ 2.0 were considered 1, 2 17 14 3 0.2433 significant and shown in bold type. Gray background indicates genes/transcript that was 3, 4 15 9 6 ϭ ϭ ϭ overexpressed as a consequence of 17q23 gain/amplification. Ploidy (n 30) (n 23) (n 7) d NBL cell lines having remarkable gains in the copy number (м5) within the SRO of Diploid 13 11 2 0.4268 the 17q23 amplicon (Fig. 2). Others 17 12 5 e 0.00 indicates that expression level is below the lower limit of quantification. MYCN amplification (NMA) Negative 16 13 3 0.4331 Positive 16 10 6 TrkA expression tional hazard regression analysis also demonstrated that high PPM1D Non-low 16 13 3 0.2733 High 16 10 6 expression correlated with poorer overall survival in all cases (hazard 1p36 LOH (n ϭ 23) (n ϭ 15) (n ϭ 8) ratio ϭ 6.182, 95% confidence interval ϭ 1.533–24.294, P ϭ 0.0104). No LOH 13 9 4 0.6850 Among the 16 NMA-positive NB patients, the high PPM1D expres- LOH 10 6 4 TP53 mutation sion group showed the same tendency toward poorer prognosis, Point mutation 2 1 1 Ͼ0.9999 although the difference did not reach statistical significance Wild-type 30 22 8 (P ϭ 0.0730, log-rank test, Fig. 3B, right). Outcome All cases Inhibition of Cell Growth and Induction of Cell Death by Alive 23 20 3 0.0056 Down-Regulation of PPM1D Expression with Antisense OPT. Dead 9 3 6 ␦ NMA-positive cases (n ϭ 16) (n ϭ 9) (n ϭ 7) PPM1D encodes a protein phosphatase 1D magnesium-dependent, Alive 6 6 0 0.0114 isoform, the amplification of which has been reported recently to Dead 10 3 7 contribute to the development of human breast cancers by suppressing a Low and high expression groups were determined by a mean value of PPM1D p53 activation (34, 35). Because our molecular cytogenetic analyses mRNA of all cases. b P are from Fisher’s exact test and were statistically significant when Ͻ0.05. Statis- indicated that PPM1D was likely to be a target for gain/amplification tically significant values are in boldface type. at 17q23 in NB, we investigated the effect of overexpressed PPM1D c Tumor stage was classified according to the INSS.

Fig. 3. A, expression of PPM1D mRNA in 32 primary NB tumors, compared with patient outcomes. We compared living, disease-free patients with those who had died of their tumors, by a nonparametric Mann-Whitney U test. Only PPM1D mRNA expression revealed a significant difference between the two groups (P ϭ 0.0160). Median values are indicated with horizontal bars in the boxes. The vertical bars indicate the range and the horizontal boundaries of the boxes represent the first and third quartiles. B, Kaplan-Meier curves for overall survival rates of all 32 patients (left)or MNA-positive patients (right), categorized according to levels of PPM1D expression (see Table 3). In all patients, the overall survival rate for tumors with high PPM1D expression was significantly lower than that for patients with low PPM1D expression (P ϭ 0.0034, log-rank test). Among the 16 NMA- positive patients, the high-PPM1D-expression group showed a tendency for poorer prognosis, although the difference did not reach statistical significance (P ϭ 0.0730, log-rank test).

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Fig. 4. Targeting of PPM1D with antisense OPT to inhibit the growth of NB cells that exhibited amplification and overexpression of the gene. A, expression levels of PPM1D mRNA determined by real-time quantitative reverse transcriptase-PCR experiments. MP-N-TS cells were treated with 300 nM of each OPT or Oligofectamine alone (mock control) and harvested 24 h after transfection. PPM1D-AS, antisense OPT targeting the PPM1D gene; PPM1D-IV, the inverse control OPT for PPM1D-AS; PPM1D-SC, a scrambled control OPT for PPM1D-AS. B, effect of PPM1D-AS on the viability of MP-N-TS cells treated with the indicated concentrations of OPT. Cell viability was determined by WST assay 72 h after transfection (left) or at the indicated times after transfection with 300 nM OPT (right). Percentages were calculated against the absorbance of control cells treated with Oligofectamine alone (mock control). The data presented are the means Ϯ SE of three separate experiments. Statistical analysis used the Mann-Whitney U test: a, PPM1D-AS versus PPM1D-IV; b, PPM1D-AS versus PPM1D-SC. All, P Ͻ 0.05. C, effect of PPM1D-AS on the numbers of living MP-N-TS cells at the indicated times after transfection with 300 nM OPT or controls, as determined by direct counting using the trypan-blue exclusion method. The data presented are the means Ϯ SE of three separate experiments. Statistical analysis used the Mann-Whitney U test: c, PPM1D-AS versus mock; d, PPM1D-AS versus PPM1D-IV. All, P Ͻ 0.05. D, effect of PPM1D-AS on the numbers of dead MP-N-TS cells at the indicated times after transfection with 300 nM OPT or Oligofectamine alone (mock), as determined by direct counting using the trypan-blue exclusion method. The data presented are the means Ϯ SE of three separate experiments. Statistical analysis used the Mann-Whitney U test: e, PPM1D-AS versus mock; f, PPM1D-AS versus PPM1D-IV. All, P Ͻ 0.05. E, typical changes in nuclear morphology observed 72 h after transfection of MP-N-TS cells with 300 nM PPM1D-AS. F, additive effects of PPM1D-AS and c-MYC-AS on the viability of MP-N-TS cells. Cells were treated with combinations of 300 nM of each OPT, and cell viability was determined 48 or 72 h after transfection. The same results were observed in CHIP134 cells treated with the combination of PPM1D-AS and MYCN-AS (data not shown). Differences among multiple comparisons were analyzed by one-way ANOVA with subsequent Scheffe´’s tests: g, c-MYC-SCϩPPM1D-AS versus c-MYC-SCϩPPM1D-IV; h, c-MYC-SCϩPPM1D-AS versus c-MYC-SCϩPPM1D-IV; i, c-MYC-ASϩPPM1D-AS versus c-MYC- SCϩPPM1D-IV; j, c-MYC-ASϩPPM1D-AS versus c-MYC-ASϩPPM1D-IV; k, c-MYC-ASϩPPM1D-AS versus c-MYC-SCϩPPM1D-AS. All, P Ͻ 0.05. or SC; Fig. 4B) in a time and a dose-dependent manner. Correlations tion of MYCN and 17q gain. However, MP-N-TS cells, which exhib- between inhibitory effects on PPM1D expression level and cell ited remarkably strong amplification of PPM1D, showed amplifica- growth were observed during our preliminary experiments using tion of c-MYC instead of MYCN. Therefore, we investigated the effect several different antisense OPTs targeting different regions of the of cotransfection of antisense OPTs for PPM1D and c-MYC (c-MYC- PPM1D gene, indicating that the effects of our antisense OPTs were AS) on the growth of MP-N-TS cells. As shown in Fig. 4F, the exerted specifically through down-regulation of PPM1D expression combination of PPM1D-AS with c-MYC-AS induced more effective (data not shown). PPM1D-AS also inhibited growth of CHP134 cells, reduction of cell survival than either treatment alone. The same results which have four copies of the PPM1D gene (data not shown). were observed in CHP134 cells that have amplification of MYCN To investigate additionally the mechanism of PPM1D-AS-induced when they are treated with the combination of PPM1D-AS with growth inhibition, we assessed the number of living and dead MP- MYCN-AS (data not shown). N-TS cells after treatment with PPM1D-AS. As expected, the rate of increase in numbers of living cells was slower in PPM1D-AS-treated DISCUSSION cells than in PPM1D-IV- or mock-treated cells (Fig. 4C). PPM1D-AS treatment induced a significant increase in dead cells (Fig. 4D); In this study, we have demonstrated that (a) gain of genomic DNA moreover, cells with typical apoptotic changes such as condensation in part of 17q was the most frequent aberration detected by CGH in of nuclear chromatin were observed more frequently in the PPM1D- our panel of 25 NB cell lines; (b) the SRO of 17q gain/amplification AS-treated group than in the controls (Fig. 4E), indicating that apo- was confined to a small portion of 17q23 in NB cells; (c) among the ptotic cell death had been induced by PPM1D-AS treatment in transcripts located within the SRO, PPM1D was overexpressed MP-N-TS cells. through its copy number increase and appeared to be involved in Overexpression of PPM1D can confer oncogenic phenotypes on poorer outcomes among patients with NB tumors; and (d) down- cells by complementing other oncogenes such as RAS, MYC,orNEU1 regulation of PPM1D expression by antisense OPT inhibited growth and by inactivating wild-type p53 during transformation of primary and induced cell death in a cell line overexpressing PPM1D. The 17q fibroblasts (34, 35). All of the NB cell lines we examined contained arm may harbor one or more genes that are rendered oncogenic by wild-type TP53 (Table 1), whereas most of them showed amplifica- copy number gains in NB tumors (31). However, until now no 1881

Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 2003 American Association for Cancer Research. PPM1D IN NEUROBLASTOMA potential target genes had ever been identified in NB, although several because the common criterion for a putative target gene is that its candidates in that region had been identified positionally and/or amplification leads to consistent overexpression (14, 19). Even after functionally in other types of tumors (17–21). Our findings indicate this step, however, we still had seven genes as candidates for NB, as that PPM1D is the most significant candidate as a potential target of in other tumors (17–21). Therefore, we hypothesized that overexpres- distal 17q gain/amplification in NB tumors. Because amplified re- sion of those candidate genes might correlate with poor prognosis gions often contain more than two target genes (17–21), we adopted and/or some other prognostic factor(s) and chose to compare their the strategy outlined here for determining the most likely target(s) in expression levels in primary NB tumors with clinicopathological data. this type of neural tumor. Of the seven genes in question, only PPM1D expression showed a In our CGH analysis of 25 NB cell lines, minimal common regions significant correlation with the prognosis of primary NB (Table 4, Fig. for the most frequent chromosomal gains were observed at 17q23, 3). The other six genes might be amplified merely as a consequence 2p23-p24, 1q31-q41, 7p11-p21, 7q22-q36, and 8q24.3, whereas losses of their proximity to PPM1D. However, additional analysis of a larger were most common at 1p36.2-pter and 11q21-q22. On the whole, our series of primary NB tumors will be necessary to clarify the roles, if results are consistent with findings of previous studies that examined any, of these six genes in the pathogenesis of NB. primary NB tumors, except that we found gains at 1q and 2p and PPM1D is rapidly and transiently induced in response to radiation losses at 1p more often in the cell lines (10–12). Such alterations in a wild-type p53-dependent manner and encodes a serine/threonine occur more frequently in advanced cases of NB, indicating that genes protein phosphatase (43). Li et al. (35) have demonstrated that over- in those regions may be involved in malignant progression (4, 5, 8, 9). expression of PPM1D confers two oncogenic phenotypes on cells in Moreover, the gains at 5p15.3,11q24 and 20q13.2-q13.3 and the culture: attenuation of apoptosis induced by serum starvation and losses at 3p21-pter, 6q24-qter, 9p23-pter, 10q24-qter, 15q26, 16q24, transformation of primary cells in cooperation with RAS. Bulavin et 17p12, and 18q22-qter that were detected by our CGH analysis (see al. (34) also demonstrated that overexpressed PPM1D reduces p53 Fig. 1) also have been observed in advanced NB tumors (12, 36–38). phosphorylation at Ser33 and Ser46 through inactivation of the p38 Therefore, our NB cell lines appear to exhibit a pattern of chromo- mitogen-activated protein kinase; abrogates RAS-induced apoptosis; somal alterations that mimics patterns of more advanced primary NB and can (a) partially rescue RAS-overexpressing cells from cell cycle tumors. Because the affected chromosomal segments may include arrest and promote transformation in vitro and (b) expedite tumor oncogenes or tumor suppressor genes associated with the pathogen- formation in vivo after injection of mouse embryonic fibroblasts esis of NB tumors and because a few target genes already have been expressing E1AϩRAS into nude mice. Moreover, cells established identified from the regions in question, cell lines we used in this study from Ppm1dϪ/Ϫ mice show decreased proliferation rates (44). Those can serve as a resource for exploring those targets further. findings suggest that PPM1D is likely to be a proto-oncogene that can Bown et al. (6) showed that partial gain of 17q is strongly predic- be involved in tumorigenesis in concert with other activated onco- tive of poor prognosis in NB, although gain of the whole chromosome genes and wild-type p53. In keeping with the requirements for as- is not, indicating that the position of translocation breakpoints on 17q suming an oncogenic function of PPM1D, the cell lines and primary may be important for differential biological/clinical behaviors of NB tumors of NB we analyzed in this study showed frequent amplifica- tumors. Because breakpoints tend to be clustered within the proximal tion of MYCN and infrequent mutation of TP53 (Tables 1 and 3). half of 17q, albeit in a variety of positions (31, 32), the dosage of Overexpression of PPM1D through an increase in its copy number specific gene(s) localized distal to the breakpoints may be critical for may be a good explanation for both the strong link between 17q gain progression of NB. Thus, the precise definition of common regions of and MYCN amplification and a low frequency of TP53 mutation in additional chromosomal material in tumors is an important step to- NB tumors. ward localizing candidate genes. 17q23.1-qter appeared to be the To clarify the functional role of PPM1D in NB, we down-regulated smallest region of gain on 17q in NB tumors (32, 39). However, the its expression by transfecting antisense OPT into NB cell lines that physical size of this region (Ͼ26 Mb), as well as relatively high amplified and overexpressed this gene (Fig. 4A). Down-regulation of density of genes on 17q, have made identification of target genes PPM1D suppressed cell growth in those experiments, indicating that difficult. Indeed, only a few genes such as NME1 (17q22; Ref. 40) and PPM1D plays an important role in the growth of MP-N-TS (Fig. 4B) survivin (17q25; Ref. 24) have been proposed as possible targets in and CHIP134 cells (data not shown). The growth inhibitory activity of NB, and none of them has proved to be a true target in this disease. PPM1D-AS was at least partly correlated with induction of apoptotic Our CGH and FISH analyses identified the smallest, and remarkably cell death (Fig. 4, C–E). Analysis of the phosphorylation status of p53 amplified, region on 17q23.2 in NB cell line MP-N-TS. Because (a) and of p38 mitogen-activated protein kinase activity in NB tumors proximal and distal sides of the amplified region in MP-N-TS showed will be necessary to clarify the mechanism by which PPM1D con- normal copy numbers, (b) other cell lines also showed maximal copy tributes to the pathogenesis of NB. Our experiments using combina- numbers in this region, and (c) 17q gain is a highly frequent event in tions of antisense OPTs for PPM1D and c-MYC or MYCN indicated NB cell lines, this amplicon seems to harbor gene(s) that are critical additive inhibitory effects on cell growth (c-MYC; Fig. 4F, MYCN; for exerting dosage effects in the progression of this disease. Notably, data not shown), supporting a hypothesis that overexpressed PPM1D 17q23 overlaps areas that are frequently gained/amplified in breast confers oncogenic phenotypes on cells by complementing other on- (17–20) and gastric (21) cancers in anaplastic meningiomas (41) and cogenes (34, 35). Because some NB tumors having 17q gains show in malignant tumors of the peripheral nerve sheath (42). Those ob- normal copy numbers for MYCN (6), oncogenes other than MYCN servations suggest that some gene(s) located on 17q23 may be re- may contribute to the development and/or progression of NB. sponsible for the pathogenesis of those tumors as well as NB. Gene expression profiling of the 17q23 amplicon has been done most precisely in breast cancer, and some of the putative target genes for that disease such as CLTC, RPS6KB1, APPBP2, and TBX2 are located ACKNOWLEDGMENTS within our SRO (17–20, 33). We thank Professor Yusuke Nakamura (Human Genome Center, The In- After narrowing the amplicon to a relatively small chromosomal stitute of Medical Science, University of Tokyo) for his continuous encour- region, we compared the expression level of each positional candidate agement. We also thank Ai Watanabe and Yuri Yoshinaga for their technical transcript within the SRO with its copy number in NB cell lines assistance. 1882

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