mycn-mediated overexpression of mitotic spindle regulatory genes

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Original Articles MYCN-mediated overexpression of mitotic spindle regulatory genes and loss of p53-p21 function jointly support the survival of tetraploid neuroblastoma cells Sina Gogolin a , Richa Batra b,1 , Nathalie Harder b,1 , Volker Ehemann c,1 , Tobias Paffhausen a,1 , Nicolle Diessl b,2 , Vitaliya Sagulenko a , Axel Benner d , Stephan Gade e , Ingo Nolte f , Karl Rohr b , Rainer König b , Frank Westermann a,a Division of Tumor Genetics, German Cancer Research Center (DKFZ), Im Neuenheimer Feld 280, 69120 Heidelberg, Germany b Department of Bioinformatics and Functional Genomics, University of Heidelberg, BIOQUANT, IPMB and German Cancer Research Center (DKFZ), Im Neuenheimer Feld 267, 69120 Heidelberg, Germany c Department of Pathology, University of Heidelberg, Im Neuenheimer Feld 224, 69120 Heidelberg, Germany d Division of Biostatistics, German Cancer Research Center (DKFZ), Im Neuenheimer Feld 580/581, 69120 Heidelberg, Germany e Division of Molecular Genetics, Cancer Genome Research, German Cancer Research Center (DKFZ), Im Neuenheimer Feld 280, 69120 Heidelberg, Germany f Small Animal Clinic, University of Veterinary Medicine Hannover, Bünteweg 9, 30559 Hannover, Germany article info Article history: Received 25 July 2012 Received in revised form 5 November 2012 Accepted 8 November 2012 Keywords: p53 pRB Genomic instability Mitotic catastrophe Aneuploidy specific lethality genes abstract High-risk neuroblastomas often harbor structural chromosomal alterations, including amplified MYCN, and usually have a near-di/tetraploid DNA index, but the mechanisms creating tetraploidy remain unclear. Gene-expression analyses revealed that certain MYCN/MYC and p53/pRB-E2F target genes, espe- cially regulating mitotic processes, are strongly expressed in near-di/tetraploid neuroblastomas. Using a functional RNAi screening approach and live-cell imaging, we identified a group of genes, including MAD2L1, which after knockdown induced mitotic-linked cell death in MYCN-amplified and TP53-mutated neuroblastoma cells. We found that MYCN/MYC-mediated overactivation of the metaphase–anaphase checkpoint synergizes with loss of p53-p21 function to prevent arrest or apoptosis of tetraploid neuro- blastoma cells. Ó 2012 Elsevier Ireland Ltd. All rights reserved. 1. Introduction Aneuploidy and chromosomal instability are hallmarks of most if not all cancers and play an essential role in tumor formation and progression [1]. Neuroblastoma, the most common solid extracra- nial tumor in early childhood, is characterized by contrasting clin- ical courses, ranging from low-risk to high-risk disease. To adjust therapy and improve prognosis, markers have been identified, such as loss of chromosome arm 1p/11q or MYCN amplification, that are associated with an aggressive disease and poor overall survival [2– 5]. Several studies have further shown an association of tumor ploidy and outcome in neuroblastoma [6–9]. Thus, near-diploid and near-tetraploid neuroblastomas are associated with poor out- come, whereas near-triploid/near-pentaploid tumors are associ- ated with low-risk disease and may even undergo spontaneous maturation or regression [10,11]. Near-triploid/near-pentaploid neuroblastomas lack structural chromosomal alterations, whereas near-diploid and near-tetraploid neuroblastomas are frequently associated with structural chromosomal alterations, suggesting that numerical, whole-chromosome aneuploidy and ploidy changes associated with chromosomal instability may arise from at least two different mechanisms. To date, several models have been proposed about how aneu- ploidy might arise in cancer. One extensively investigated mecha- nism in recent years is the development of aneuploidy through a tetraploid genetically unstable intermediate [12,13]. An associa- tion between unscheduled tetraploidy, cell transformation and tu- mor formation has been shown at least in mice [14]. The appearance of tetraploid cells is characteristic especially for the pre-malignant condition Barrett’s esophagus [15] and early stages of cervical carcinogenesis [16] but can also be detected in several 0304-3835/$ - see front matter Ó 2012 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.canlet.2012.11.028 Corresponding author. Tel.: +49 (0) 6221 423275; fax: +49 (0) 6221 423277. E-mail address: [email protected] (F. Westermann). 1 These authors are contributed equally. 2 Current address: Department of Genomics and Proteomics Core Facility, High Throughput Sequencing, German Cancer Research Center (DKFZ), Im Neuenheimer Feld 580, 69120 Heidelberg, Germany. Cancer Letters 331 (2013) 35–45 Contents lists available at SciVerse ScienceDirect Cancer Letters journal homepage: www.elsevier.com/locate/canlet

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Page 1: MYCN-mediated overexpression of mitotic spindle regulatory genes

Cancer Letters 331 (2013) 35–45

Contents lists available at SciVerse ScienceDirect

Cancer Letters

journal homepage: www.elsevier .com/locate /canlet

Original Articles

MYCN-mediated overexpression of mitotic spindle regulatory genesand loss of p53-p21 function jointly support the survival of tetraploidneuroblastoma cells

0304-3835/$ - see front matter � 2012 Elsevier Ireland Ltd. All rights reserved.http://dx.doi.org/10.1016/j.canlet.2012.11.028

⇑ Corresponding author. Tel.: +49 (0) 6221 423275; fax: +49 (0) 6221 423277.E-mail address: [email protected] (F. Westermann).

1 These authors are contributed equally.2 Current address: Department of Genomics and Proteomics Core Facility, High

Throughput Sequencing, German Cancer Research Center (DKFZ), Im NeuenheimerFeld 580, 69120 Heidelberg, Germany.

Sina Gogolin a, Richa Batra b,1, Nathalie Harder b,1, Volker Ehemann c,1, Tobias Paffhausen a,1,Nicolle Diessl b,2, Vitaliya Sagulenko a, Axel Benner d, Stephan Gade e, Ingo Nolte f, Karl Rohr b,Rainer König b, Frank Westermann a,⇑a Division of Tumor Genetics, German Cancer Research Center (DKFZ), Im Neuenheimer Feld 280, 69120 Heidelberg, Germanyb Department of Bioinformatics and Functional Genomics, University of Heidelberg, BIOQUANT, IPMB and German Cancer Research Center (DKFZ), Im Neuenheimer Feld 267,69120 Heidelberg, Germanyc Department of Pathology, University of Heidelberg, Im Neuenheimer Feld 224, 69120 Heidelberg, Germanyd Division of Biostatistics, German Cancer Research Center (DKFZ), Im Neuenheimer Feld 580/581, 69120 Heidelberg, Germanye Division of Molecular Genetics, Cancer Genome Research, German Cancer Research Center (DKFZ), Im Neuenheimer Feld 280, 69120 Heidelberg, Germanyf Small Animal Clinic, University of Veterinary Medicine Hannover, Bünteweg 9, 30559 Hannover, Germany

a r t i c l e i n f o a b s t r a c t

Article history:Received 25 July 2012Received in revised form 5 November 2012Accepted 8 November 2012

Keywords:p53pRBGenomic instabilityMitotic catastropheAneuploidy specific lethality genes

High-risk neuroblastomas often harbor structural chromosomal alterations, including amplified MYCN,and usually have a near-di/tetraploid DNA index, but the mechanisms creating tetraploidy remainunclear. Gene-expression analyses revealed that certain MYCN/MYC and p53/pRB-E2F target genes, espe-cially regulating mitotic processes, are strongly expressed in near-di/tetraploid neuroblastomas. Using afunctional RNAi screening approach and live-cell imaging, we identified a group of genes, includingMAD2L1, which after knockdown induced mitotic-linked cell death in MYCN-amplified and TP53-mutatedneuroblastoma cells. We found that MYCN/MYC-mediated overactivation of the metaphase–anaphasecheckpoint synergizes with loss of p53-p21 function to prevent arrest or apoptosis of tetraploid neuro-blastoma cells.

� 2012 Elsevier Ireland Ltd. All rights reserved.

1. Introduction

Aneuploidy and chromosomal instability are hallmarks of mostif not all cancers and play an essential role in tumor formation andprogression [1]. Neuroblastoma, the most common solid extracra-nial tumor in early childhood, is characterized by contrasting clin-ical courses, ranging from low-risk to high-risk disease. To adjusttherapy and improve prognosis, markers have been identified, suchas loss of chromosome arm 1p/11q or MYCN amplification, that areassociated with an aggressive disease and poor overall survival [2–5]. Several studies have further shown an association of tumorploidy and outcome in neuroblastoma [6–9]. Thus, near-diploid

and near-tetraploid neuroblastomas are associated with poor out-come, whereas near-triploid/near-pentaploid tumors are associ-ated with low-risk disease and may even undergo spontaneousmaturation or regression [10,11]. Near-triploid/near-pentaploidneuroblastomas lack structural chromosomal alterations, whereasnear-diploid and near-tetraploid neuroblastomas are frequentlyassociated with structural chromosomal alterations, suggestingthat numerical, whole-chromosome aneuploidy and ploidychanges associated with chromosomal instability may arise fromat least two different mechanisms.

To date, several models have been proposed about how aneu-ploidy might arise in cancer. One extensively investigated mecha-nism in recent years is the development of aneuploidy through atetraploid genetically unstable intermediate [12,13]. An associa-tion between unscheduled tetraploidy, cell transformation and tu-mor formation has been shown at least in mice [14]. Theappearance of tetraploid cells is characteristic especially for thepre-malignant condition Barrett’s esophagus [15] and early stagesof cervical carcinogenesis [16] but can also be detected in several

Page 2: MYCN-mediated overexpression of mitotic spindle regulatory genes

36 S. Gogolin et al. / Cancer Letters 331 (2013) 35–45

other cancers independent of the tumor stage [17]. Tetraploidy canarise via cell fusion, cytokinesis failure, endoreduplication or mito-tic slippage. Prolonged activation of the metaphase–anaphasecheckpoint resulting from overexpressed metaphase–anaphasecheckpoint genes, such as Mad2 [18], has been shown to provokemitotic slippage associated with incomplete cytokinesis resultingin tetraploidization [19]. The main function of the metaphase–ana-phase checkpoint is to inhibit the anaphase-promoting complex orcyclosome (APC/C) until the spindles have properly attached to allkinetochores, thereby preventing chromosome mis-segregation.Only one unattached kinetochore is sufficient to stop anaphaseby Mad2 activation, which subsequently forms complexes withCdc20, BubR1 and Bub3. These complexes prevent APC/C-mediatedubiquitylation of securin and, consequentially, separase-mediatedcohesin degradation [20]. Deregulation of mitotic checkpointgenes, including MAD2L1, either by overexpression, reducedexpression or mutation has been reported for many cancers,including neuroblastoma [1,21,22].

Recently, it has been suggested that overactivation of the meta-phase–anaphase checkpoint might trigger aneuploidy induction incells with a defective G1-S arrest as a consequence of non-func-tional pRB and p53-p21 [23]. Both the p53-p21 and pRB axes arederegulated especially in relapse neuroblastomas and tumor-de-rived cell lines through genetic aberrations that include TP53mutation and/or amplification of MDM2, CDK4 or CCND1 [24–26].Furthermore, MYCN impairs the p53-p21 and pRB pathwaythrough transcriptional inhibition of p21 and upregulation ofMDM2 and CDK4, which are direct p53 and pRB inhibitors, respec-tively [27–30]. An association between tetraploidy and loss of p53function has been described for Barrett’s esophagus [31] and, morerecently, for medulloblastoma [32]. Whether impaired p53-p21/pRB-mediated checkpoints might further contribute to tetraploidyin neuroblastoma cells and whether deregulation of the meta-phase–anaphase checkpoint contributes to the development ofaneuploidy in neuroblastomas has not been investigated to date.

To address both points, we analyzed the expression of MYCN/MYC, p53 and pRB-E2F target genes, which are primarily involvedin cell cycle regulation or neuronal differentiation, in associationwith tumor DNA index and structural chromosomal aberrationsin a large cohort of primary neuroblastomas. We then used a func-tional siRNA screening approach combined with a live-cell imagingmicroscopy-based readout in two neuroblastoma cell lines witheither low MYCN expression and functional p53 or amplified MYCNand mutated TP53 to determine the consequences of metaphase–anaphase checkpoint gene repression.

2. Material and methods

2.1. Tumor samples

Clinical data and tumor samples from 483 patients enrolled in the German Neu-roblastoma Trial and diagnosed between 1998 and 2010 were used in this study.Informed consent was collected within the trial protocol.

2.2. Cell culture

Cell lines were maintained at 37 �C and 5% CO2 DMEM (SK-N-BE(2)-C) or RPMI(SH-EP and WAC2 [33,34]) supplemented with 10% FCS. SH-EP and SK-N-BE(2)-Cwere stably transfected with the H2B-GFP expression vector using Lipofectamine2000 (Invitrogen Ltd., Paisley, UK) as previously described [35], and were main-tained in DMEM supplemented with 10% FCS and 1.5 mg/ml G418. SH-EP-MYCN(TET21 N) cells stably expressing a tetracycline-regulatable MYCN transgene and ap21CIP1 shRNA were cultured and induced as previously described [29,36].

2.3. Ploidy and cell cycle analysis

Native cryo-conserved tumor samples were minced with scissors in 2.1% citricacid/0.5% Tween-20 [37,38]. Phosphate buffer (7.2 g Na2HPO4 � 2H2O in 100 ml dis-tilled water, pH 8.0) containing 50 lg/ml 2,4 diamino-2-phenylindole (DAPI) was

used to stain DNA. High resolution flow cytometric analyses were performed onthe Galaxy pro flow cytometer (Partec, Münster, Germany) equipped with a mer-cury vapor lamp 100 W and DAPI filter. Data was acquired in the FCS-mode and his-togram analyses were generated using the Multicycle program (Phoenix FlowSystems, San Diego, CA). Each histogram included 30,000–100,000 cells to calculatethe DNA index and for cell cycle analysis. Human lymphocytes from healthy donorswere used as an internal standard to calibrate the diploid DNA index. The meancoefficient of variation for diploid lymphocytes was 0.9. For cell cycle analysis, cellswere plated in 75 cm2 flasks, and 24 h later induced with doxycycline and/or trea-ted with vincristine or doxorubicin.

2.4. Reverse transfection on cell arrays

Two different siRNAs (Ambion, Austin, Texas, USA) were used for knockdown ofG2/M regulatory genes chosen from the microarray analysis [28]. Two control siR-NAs (Ambion) and mock-transfection were used as negative controls. Transfectionmixtures were prepared and one-chamber LabTeks (#155361, Thermo scientificnunc, Langenselbold, Germany) were spotted using an automated system in dupli-cates for each siRNA and dried as previously described [39]. To create cell array,60,000 SH-EP/H2B-GFP or 100,000 SK-N-BE(2)-C/H2B-GFP were seeded per cham-ber and incubated with 1.5 ml growth medium at 37 �C and 5% CO2. The experimentwas repeated four times for each cell line.

2.5. Image acquisition, image analysis

Live-cell imaging was performed 16 h after transfection for 5 days at an acqui-sition rate of 35–40 min using an automated wide-field fluorescence microscopewith 10� magnification as previously described [39]. Automatic segmentationand tracking was performed for all images, and mitotic events were detected usingmorphological features [40]. Images of cell nuclei were classified into interphase(round/elliptical nuclei with smooth boundaries), apoptotic (small bright nuclearfragments), mitotic (prometaphase, metaphase or anaphase nuclei) or clusters(two or more nuclei grouped too closely to be identified as separate objects) usingSupport Vector Machines (SVMs) in R with package e1071. Nuclei classified as arti-facts (very low contrast) were discarded. The classification protocol was optimizedfor both neuroblastoma cell lines, where the performance of the classifier was man-ually evaluated. Classification errors were automatically corrected by checking thecomputed class sequences along the respective trajectories regarding their biolog-ical validity using a state transition model. The average classification accuracy forSH-EP and SK-N-BE(2)-C nuclei were 90% and 79%, respectively. Automatic errorcorrection was only applied for SH-EP nuclei, since the tracking result for SK-N-BE(2)-C nuclei was less reliable due to frequent unresolvable cell clusters.

2.6. Quantitative analysis of classified images

The classified time series were further processed to identify the consequencesof gene knockdown and to cluster genes with similar phenotype profiles. Our quan-titative analysis included the following steps: (1) An adapted Z-Score normalizationwas performed as described elsewhere [41] for each cell array, time point and phe-notype class to account for edge effects and other spatial errors on the cell arrays.(2) The time series for each gene was integrated in overlapping time windows foreach well and phenotype class, yielding 13 time windows per 120 h of imaging witha single time window spanning 24 h including an 8 h overlap with the previoustime window. The integrated time series of each time window was designatedthe ‘‘phenotype signal’’ for that time window. (3) The phenotype signals for eachgene (four replicates using two siRNA constructs per gene) were compared to thephenotype signals to the phenotype signals of all other genes assayed using theWilcoxon rank-sum test to determine if the gene phenotype was higher or lowerthan the overall population (significant if p-value 60.05). (4) For each gene, a ‘‘phe-notype signature’’ was defined by the time window with the lowest p-value as com-puted above, and the first time window with a significant p-value. The ‘‘phenotypesignature’’ included all phenotype classes (interphase, apoptosis, mitosis). (5) Forfurther analysis, we only considered genes, which exhibited higher cell death, high-er mitotic index and lower interphase counts than the overall population. (6) Geneswere clustered into ‘‘phenoclusters’’ based on their phenotype signatures. Euclideandistances were computed, as similarity measure, for phenotype signatures of allpairs of genes. Using this similarity measure, hierarchical clustering of genes wasperformed using the R software package pvclust.

2.7. RNA interference

WAC2 (SH-EPMYCN) or parental SH-EP cells were stably transfected with thedoxycycline-inducible pcDNA6TR repressor following the manufacturer’s protocol(Invitrogen). Specific shRNA fragments targeting MAD2L1 (AATACGGACT-CACCTTGCTTG, Gen BankTM accession number NM_002358) or, as control, SCRAM-BLE (AACAGTCGCGTTTGCGACTGG, Ambion) were cloned into the pTER+vector [42].WAC2pcDNA6TR or SH-EPpcDNA6TR cells were stably transfected with the pTER+

vector harboring shMAD2L1 or shSCRAMBLE using Effectene (QIAGEN, Hilden, Ger-many). Zeocin-resistant clones were cultured in RPMI supplemented with 10%

Page 3: MYCN-mediated overexpression of mitotic spindle regulatory genes

1.0 1.5 2.0 2.5 3.0

010

2030

40

01

23

45

6

DNA index

Standardized log-rank statistic

Freq

uenc

y

1.11 1.77 2.11

p=0.05

Fig. 1. Tumor near-di/tetraploidy correlates with poor outcome in neuroblastomapatients. Overlay of a maximally selected log-rank statistic of overall survival and ahistogram of the DNA indices of 483 neuroblastomas. The DNA index cut-off points,1.11 (⁄P = 1.155e�07) and 1.77, separate neuroblastoma patient subgroups withsignificantly different outcome. Tumors with DNA indices between 1.0 and 1.11 andbetween 1.77 and 2.11 (near-di/tetraploidy) were correlated with unfavorablepatient outcome. Tumors with DNA indices between 1.11 and 1.77 or >2.11 (near-tri/pentaploidy) were correlated with favorable patient outcome.

S. Gogolin et al. / Cancer Letters 331 (2013) 35–45 37

FCS, and selected for with 7.5 lg/ml blasticidine and 50 lg/ml zeocin. WAC2-shMAD2L1 or SH-EP-shMAD2L1 clones were assayed for efficient MAD2L1 downreg-ulation (western blotting upon doxycycline addition (100 nM)).

2.8. Protein expression

Whole cell lysates were prepared as previously described [43]. 50 lg of proteinlysate was separated per lane on 12% SDS–PAGE. Blots were probed with antibodiesdirected against hMAD2 (1:2000; #610679, BD Biosciences, Franklin Lakes, NJ,USA), MYCN (1:1000; #sc-53993, Santa Cruz, CA, USA) and b-actin (1:5000;#A5441, Sigma–Aldrich, St. Louis, Missouri, USA). HSR–peroxidase labeled anti-mouse antibody (1:1000; #115-035-003, Dianova, Hamburg, Germany) was usedas secondary antibody. Proteins were visualized using the ECL detection system(Amersham/GE Healthcare, Freiburg, Germany) and a chemiluminescence reader(VILBER Eberhardzell, Germany).

2.9. Fluorescence in situ hybridization (FISH)

Centromeric regions of chromosomes 3, 6, 8 and 18 were localized with fluores-cently labeled plasmids (chromosome 3: pAE0.68 – Cy3 (GE Healthcare), chromo-some 6: pEDZ6 – Cy3.5, chromosome 8: pZ8.4 – FITC (Molecular Probes, Eugene,Oregon, USA), and chromosome 18: 2Xba – DEAC (Molecular Probes)) as previouslydescribed [44], and 4-color FISH analysis was performed as previously described[45]. Images were analyzed using a Zeiss axiophot microscope and IPLap 10software.

2.10. Indirect immunofluorescence microscopy

Cells were washed in PBS, then fixed in ice-cold methanol/acetone (1:1) ontomicroscope slides for 7 min. Fixed cells were then directly used for indirect immu-nofluorescence microscopy or stored at �20 �C. Following fixation cells wereblocked in PBS-GSA (136.9 mM NaCL, 2.68 mM KCL, 0.01 M Na2HPO4, 1.76 mM KH2-

PO4, pH 7.4, 1% GSA) for 30 min, then incubated with primary human anti-centro-mere antibodies (CREST, 1:20) [46] in PBS-GSA for 60 min at ambient temperaturein a humidified chamber. Slides were washed several times with PBS, incubatedwith a species-specific fluorescent secondary antibody (Alexa Fluor 488 mouseanti-human, 1:1000; Molecular Probes Invitrogen) for 30 min at ambient tempera-ture, then washed several more times with PBS, once with distilled H2O and oncewith 100% ETOH. Slides were counterstained for 2 min with DAPI (0.25 lg/ml ddH2-

O; Sigma–Aldrich) and mounted in VECTASHIELD (Vector Laboratories, Inc., Burlin-game, CA, USA). Immunofluorescence images were collected using a Zeiss ImagerZ.1microscope and the Isis Metasystems, Version 5.0 software (MetaSystems, Altluss-heim, Germany).

2.11. Pharmacological inhibition

Cell cultures were treated with 0.1 lg/ml doxorubicin (TOC-2252-M010, BiozolEching, Germany) and 0.05 lM vincristine (BML-T117-0005, Alexis Biochemicals,Lörrach, Germany), where indicated.

2.12. GO term enrichment/cluster analysis

Oligo sequences of 144 probes from the custom Agilent gene expression array[47] were mapped to the current version of the human genome (Genome ReferenceConsortium GRCh37) using BLAST (http://blast.ncbi.nlm.nih.gov/Blast.cgi). A totalof 123 probes had mapped perfect matches to confirmed transcripts. Gene ontology(GO) term enrichment and annotation cluster analysis was performed using the DA-VID Bioinformatics Database 6.7 [48]. The transcript IDs mapped to 115 unique DA-VID IDs. GO terms were enriched with an ease score <0.001. The ‘‘highest’’-stringency settings were chosen to generate functional groups with tightly associ-ating genes for annotation cluster analysis.

2.13. Statistics

To identify a model describing the relationship between survival and tumor cellDNA index (tumor cell ploidy), the functional form of this relationship was testedby maximally selected log-rank statistics as previously described [49]. Statisticalanalyses were conducted using the R software package, version 2.12.1.

We also applied classification and regression trees to evaluate the functionaldependency between the tumor cell DNA index (tumor cell ploidy) and age, stage,MYCN amplification and overall survival. Conditional inference trees were used toestimate the regression relationship by binary recursive partitioning in a condi-tional inference framework. At each node of the tree multiplicity-adjustedMonte-Carlo tests were applied using 10,000 random permutations. In order to splita node the p-value of the corresponding Monte-Carlo test had to be smaller thanalpha = 5%. The algorithm works as follows: (1) Test the global null hypothesis ofindependence between the DNA index and the response. Stop if this hypothesis can-

not be rejected. (2) Implement a binary split in the DNA index. (3) Recursively rep-eate steps (1) and (2). For a general description of the methodology see Hothornet al. [50].

2.14. Gene expression analysis

Differential expression of the 144 oligonucleotide probes from a previouslypublished gene expression-based classifier [47] was assessed for primary neurobl-astomas from 133 patients out of the whole patient cohort and used for two-wayhierarchical cluster analysis as previously described [28].

3. Results

3.1. Tumor DNA index cut-off points 1.11 and 1.77 separateneuroblastoma patients with different outcomes

To test the association of tumor ploidy with clinical and geneticmarkers, we analyzed the DNA indices of 483 primary tumors frompatients enrolled in the German Neuroblastoma Trial (Supplemen-tary Table 1) using flow cytometry. The frequency of tumor DNAindices peaked primarily at 1.0 and 1.5, and to a lesser extent at2.0 and 2.5 (Fig. 1), which each defined tumor subtypes in thenear-diploid, near-triploid, near-tetraploid and near-pentaploidrange, respectively. We further analyzed the biological relevanceof these subtypes by assessing their correlation with clinical out-come. A maximally selected log-rank statistic was used to analyzethe relationship between overall survival and tumor DNA index.Using all 483 cases, we identified a DNA index cut-off value of1.11 (maximally selected log-rank statistics, p < 0.001, Fig. 1) thatseparates two patient subgroups with significantly different out-come. Intriguingly, a second DNA index cut-off value at 1.77 wasidentified, when all near-diploid tumors with a DNA index 6 1.11were excluded from the analysis (maximally selected log-rank sta-tistics, p < 0.001, Supplementary Fig. 1), indicating that the groupof aneuploid tumors (DNA index > 1.11) consists of at least two dis-tinct subtypes associated with significantly different patient out-comes. Using maximally selected log-rank statistics, we did notfind a third DNA index cut-off value that further separates the tu-mors in the higher aneuploid range (>1.77). We also applied clas-sification and regression trees to evaluate the functionaldependency between the tumor cell DNA index and overall sur-vival (Supplementary Fig. 2A). This confirmed the two DNA index

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38 S. Gogolin et al. / Cancer Letters 331 (2013) 35–45

cut-off values at 1.11 and 1.77 (p = 0.039 and p = 0.004, respec-tively) and revealed a third DNA index cut-off value at 2.11(p = 0.032). Together, these analyses defined patients with favor-able outcomes as having near-triploid tumors (57%) with DNAindices between 1.11 and 1.77 or near-pentaploid (>2.11) tumors(4%) and patients with poor outcomes as having near-diploid tu-mors (28%) with DNA indices between 1.0 and 1.11 or near-tetra-ploid tumors with DNA indices between 1.77 and 2.11 tumors(11%) (Fig. 1). Furthermore, we evaluated a functional dependencybetween the DNA index cut-off values and the clinical or geneticmarkers, MYCN status, age at diagnosis >1.5 years, and stage 4,showing a significant association of these markers of unfavorablebiology with near-di/tetraploidy subtypes (SupplementaryFig. 2B–D, Table 1). This supports not only that tumor DNA indexis a prognostic marker for neuroblastoma patients, but also sug-gests that at least two distinct modes of mitotic failure result ineither near-di/tetraploid or near-tri/pentaploid neuroblastomas.

3.2. Mitotic regulatory genes are overexpressed in neuroblastomaswith unfavorable biology

Oncogenic mutations in pathways (e.g. MYC, pRB, p53) that reg-ulate genomic stability may trigger aneuploidy and chromosomalinstability through the activation of transcriptional programs[51]. However, aneuploidy itself may induce transcriptional re-sponses because aneuploid cells need to develop specific adapta-tions in order to proliferate with their altered genomes. To getinsights into transcriptional changes associated with near-di/tetra-ploid and/or near-tri/pentaploid neuroblastomas, we analyzedgene expression profiles from our primary neuroblastoma cohortfor expression of the 144 genes in a previously published geneexpression-based classifier that distinguishes high- from low-riskneuroblastomas [47] and is enriched with MYCN/MYC target genesand p53/pRB-E2F-regulatory genes [28,52]. Signature expressionprofiles from 133 primary neuroblastomas from our patient cohortwere analyzed using a two-way hierarchical cluster analysis.Expression of the 144-gene signature classified these 133 patientsinto four distinct subgroups that were enriched with either near-di/tetraploid or near-tri/pentaploid tumors: Group I patients were<1.5 years of age at diagnosis, had tumors with favorable prognos-tic markers, including tumor stage 1 or 2, near-tri/pentaploidy,normal MYCN status and normal chromosome 1p/11q status, anda favorable outcome. Patients in group II had tumors that werecharacterized by near-di/tetraploidy, amplified MYCN, deletion orimbalance of chromosome arm 1p and poor outcome. Group III pa-tients had near-di/tetraploid, single-copy MYCN tumors with dele-tion or imbalance of chromosome arm 11q. Tumors from groups IIand III patients had similar expression profiles of the 144-gene

Table 1Correlation of tumor ploidy and prognostic markers.

Tumor ploidy

Near-di/tetraploidy (unfavorable)

Age in years<1.5 76P1.5 113

Stagea

1, 2, 3, 4s 864 103

Amplified MYCNNo 126Yes 63

1p Deletion/imbalanceNo 88Yes 86

a INSS = International Neuroblastoma Staging System.

classifier with strong expression of direct MYCN/MYC and p53/pRB-E2F target genes and low expression of neuronal differentia-tion genes. Gene expression profiles from group I tumors were re-versed, with low expression of MYCN/MYC and p53/pRB-E2F targetgenes and strong neuronal differentiation gene expressions. Pa-tients in group IV had tumors that were characterized by favorableclinical markers, near-tri/pentaploidy, normal 1p or 11q and nor-mal MYCN status as well as favorable outcome, but presented anintermediate phenotype between groups I and II/III regarding theexpression of the 144 genes of the gene expression-based classifier.Expression levels of neuronal differentiation genes were similar totumors in group I, whereas expression of p53/pRB-E2F target geneswere similar to group II/III tumors, and MYCN/MYC target geneexpression was lower than in group II/III tumors (Fig. 2 and Supple-mentary Table 2).

We classified the genes making up the 144-gene expressionclassifier according to their biological function using Gene Ontol-ogy (GO) term enrichment, then used annotation cluster analysisto create larger functionally descriptive groups using the DAVIDBioinformatics Database. Most genes in the 144-gene classifierare involved in chromosome segregation, spindle organization,microtubule-based processes, nuclear division or DNA replication.These could be clustered together into one main group regulatingmitosis. A small subset of genes was involved in processes neces-sary for DNA repair and to respond to DNA damaging stimuli.Annotation cluster analysis of significantly enriched cellular com-ponents showed that genes in the classifier were assigned func-tions associated with the kinetochore or centromeric region(Supplementary Table 3). Both, results of the gene expressionand GO term enrichment analyses revealed that near-di/tetraploidneuroblastomas possess functional expression signatures associ-ated with mitotic checkpoint activation, which argues in favor ofa MYCN/MYC-induced mitotic stress response and checkpoint acti-vation via suppressed p53 and pRB functionality in these tumors.In contrast, near-tri/pentaploid neuroblastomas showed lowMYCN/MYC target gene activation and retained neuronal differen-tiation signatures. However, mitotic checkpoint activation con-trolled by p53 and pRB was variable (low in group I versus highin group IV), which could argue for a weakening of p53 and pRBfunctionality at least in a subset of favorable near-tri/pentaploidtumors.

3.3. Loss of p53-p21 function is associated with tetraploidization inneuroblastoma cells

Similar to the metaphase–anaphase checkpoint, the p53-p21axis has an important role in limiting the expansion of aneuploidhuman cells. Loss of p53-p21 has been shown to result in cycling

P

Near-tri/pentaploidy (favorable) Fisher’s exact test

20490 <0.001

24054 <0.001

27618 <0.001

22052 <0.001

Page 5: MYCN-mediated overexpression of mitotic spindle regulatory genes

OutcomeMYCN1p Status11q StatusPloidyAgeStage

133 patients

p53/

pRB-

E2F

MYC

N/

MYC

Array probe Gene sym bolA _23_P 415443 B RRN1A _23_P 88331 D LG7A _32_P 62997 P B KA _24_P 323598 E S C O2A _23_P 138507 C D C 2A _23_P 51085 S pc25A _23_P 115872 C 10orf3A _23_P 65757 C C NB 2A _24_P 297539 UB E 2CA _23_P 74349 C D C A 1A _23_P 7636 P TTG1A _23_P 48669 C D K N3A _23_P 104651 C D C A 5A _23_P 107421 TK 1A _23_P 49972 C D C 6A _23_P 50096 TYMSA _23_P 28886 P C NAA _24_P 234196 RRM2A _23_P 401 C E NP FA _24_P 96780 C E NP FA _23_P 23303 E X O1A _23_P 10385 RA MPA _24_P 53519 C HAF1AA _23_P 254733 M LF1IPHs87507.1 B RIP 1A _23_P 155765 HMGB 2A _23_P 131866 S TK 6A _23_P 96325 FLJ20105A _24_P 413884 C E NP AA _23_P 323751 C 20orf129A _23_P 122197 C C NB 1Hs23960.1 C C NB 1A _32_P 151800 MGC 57827A _23_P 71727 C K S 2Hs79078.10 MA D 2L1A _23_P 92441 MA D 2L1A _23_P 133123 GA JA _23_P 252740 D C C 1A _23_P 87351 RRM1A _23_P 18196 RFC 4Hs422789.1 V RK 1A _23_P 373119 HMG4LA _23_P 90612 MC M6Hs155462.1 MC M6A _23_P 88740 B M039Hs24763.1 RA NB P 1A _23_P 36076 S S RP 1A _23_P 35219 NE K 2A_23_P122 443 HIS T1H1C

A _23_P 41280 P A IC SA_23_P 21033 GMP SA _23_P 143958 LOC 200916A_23_P17575 A HC YA _24_P 57367 AHC YA _23_P 114232 PRD X4A _24_P 182182 S LC 25A 5A _32_P 98348 ZNF525A _23_P 217609 RP L36AA _23_P 126291 S NRP EA _23_P 18422 MRP L3A _23_P 78888 FB LA _23_P 200507 HS P C 163Hs55424.1 FLJ10151A _23_P 58280 NOLA 1A _32_P 73903 B X 119435

Array probe Gene sym bolp53/pRB-E2F target genes MYCN/MYC target genes

group IIIgroup I group II group IV

Neu

rona

l diff

eren

tiatio

n (e

.g. N

TRK1

, CAM

TA1)

Fig. 2. Mitosis regulatory genes are strongly expressed targets of MYCN/MYC and p53/pRB-E2F signaling in unfavorable neuroblastomas. Two-way hierarchical clusteranalysis of the 144 genes in the previously published gene expression-based classifier in 133 primary neuroblastomas (blue = high expression, green = low expression). Theclinical covariates, patient outcome (black = death from disease, grey = progression/relapse, white = no event), MYCN status (black = amplified, white = non-amplified),chromosome arm 1p or 11q status (black = deletion/imbalance, grey = not defined, white = normal), ploidy (black = near-di/tetraploidy, white = near-tri/pentaploidy), age atdiagnosis (white <1.5 years, black P1.5 years) and tumor stage using the International Neuroblastoma Staging System (black = 4, grey = 3, white = 1 or 2, yellow = 4s), wereadded to the gene expression heatmap. Patients were classified into four groups (I-IV). The gene expression cluster of direct MYCN/MYC and p53/pRB-E2F target genes ishighlighted. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

S. Gogolin et al. / Cancer Letters 331 (2013) 35–45 39

4 N cells that underwent endoreduplication [53,54]. To investigatewhether loss of p53-p21 function favors the propagation of viabletetraploid neuroblastoma cells, we analyzed the DNA index of SK-

N-BE(2)-C cells, which harbor both amplified MYCN and mutatedTP53. These cells are characterized by an impaired p53-p21/pRB-mediated G1 arrest and low levels of apoptosis upon irradation-in-

Page 6: MYCN-mediated overexpression of mitotic spindle regulatory genes

40 S. Gogolin et al. / Cancer Letters 331 (2013) 35–45

duced DNA damage [55] and resistance to various chemotherapeu-tic drugs [56]. Flow cytometric analysis identified both diploid andtetraploid cell clones in SK-N-BE(2)-C cell cultures. The tetraploidcell clone expanded from �16% to �90% within nine rounds of pas-saging. DNA damage prior mitosis may increase tetraploidy in cellswith impaired cell cycle checkpoints as a consequence of non-func-tional p53-p21 signaling [57]. Treatment of SK-N-BE(2)-C cultureswith the DNA damage-inducing drug, doxorubicin, expanded thetetraploid fraction to �80% in only four rounds of passaging, andcreated an entirely tetraploid cell culture by passage 18. The dip-loid fraction was reduced accordingly in each passage after doxo-rubicin treatment (Table 2). To test the consequence of p21inhibition at varying levels of MYCN on tumor cell ploidy, we usedthe SH-EP-MYCN cell model, which stably express a tetracycline-regulatable MYCN transgene allowing MYCN induction by removalof tetracycline from the culture medium [36]. Targeted MYCNexpression in these cells caused a transient down-regulation ofp21, which was compensated for over time (24 h) through p53induction by MYCN [29]. To further abrogate p21 levels, we stablytransfected SH-EP-MYCN cells with a shRNA targeting p21CIP1, orwith scrambled shRNA as a control. Expression of the p21 proteinwas consistently reduced to <10% whether MYCN was induced ornot in SH-EP-MYCN cells stably expressing p21CIP1 shRNA com-pared to the control cells (Supplementary Fig. 3). Repression ofp21CIP1 by shRNA resulted in tetraploidization of about 30% ofSH-EP-MYCN cells. Provoking DNA damage by using doxorubicintreatment increased the tetraploid fraction to �76%, similar toSK-N-BE(2)-C cells (Table 2), while targeted MYCN induction inSH-EP-MYCN cells expressing scrambled shRNA control did not in-duce tetraploidization (Supplementary Fig. 4). Furthermore, en-hanced MYCN expression did not significantly further increasetetraploidization after shRNA-mediated p21 repression. These re-sults indicate that an impaired p53-p21 axis is involved in tetra-ploidization of neuroblastoma cells, whereas transcriptionalrepression of p21CIP1 by targeted MYCN induction is insufficientto induce tetraploidization at least in MYCN-single-copy neuroblas-toma cells.

3.4. Selective inhibition of mitotic checkpoint genes causes mitotic-linked cell death in MYCN-amplified and TP53-mutatedneuroblastoma cells

Mitotic regulatory genes are overexpressed in unfavorable neu-roblastomas with suppressed p53 and pRB functionality. To testwhether an overactivated metaphase–anaphase checkpoint sup-ports the survival of cells lacking functional p53-p21 signaling,we performed a functional siRNA screen targeting mitotic regula-tory genes controlled by MYCN/MYC and p53/pRB-E2F. We focusedon a set of 240 genes with significantly higher expression in neu-

Table 2Loss of p53–p21 functionality is associated with tetraploidization in neuroblastoma cells.

Untreated

% Diploid % Aneuploid

SK-N-BE(2)-Ca

passage 7 83.4 ± 1.0 16.6 ± 1.0passage 9 69.8 ± 2.0 30.2 ± 2.0passage 13 36.9 ± 1.8 63.1 ± 1.8passage 18 9.2 ± 0.5 90.8 ± 0.5SH-EP-MYCNb

p21off + MYCNon 66.1 ± 3.7 33.9 ± 3.7p21off + MYCNoff 66.8 ± 3.3 33.2 ± 3.3

* DI = DNA index (SD < 0.02).a Mean ± SD of triplicates from one representative experiment.b Mean ± SD of triplicates from two independent experiments.

roblastomas with unfavorable than favorable biology, which in-cluded those in the 144 gene-expression classifier(Supplementary Table 4). Each candidate gene was silenced usingtwo different small interfering siRNAs. Two unrelated scrambledsiRNAs were used as negative controls. Screening was performedin two neuroblastoma cell lines with different MYCN and TP53 ge-netic backgrounds stably expressing the H2B-GFP fluorescent chro-matin marker to allow live-cell imaging microscopy-basedreadout. SH-EP were used as the control since they harbor single-copy MYCN and wild-type TP53. SK-N-BE(2)-C harbor both ampli-fied MYCN and mutated TP53. Cells were classified as being ininterphase, mitosis or apoptosis during a 5-day culture periodusing 24-h time windows with an 8-h overlap to the previous timewindow. Genes were then clustered depending on the specificRNAi-mediated knockdown phenotype, which were defined as fol-lows: (1) interphase arrest, (2) mitotic arrest, (3) primary apopto-sis, (4) mitotic-linked cell death (secondary apoptosis out ofmitotic arrest) and (5) mitotic slippage followed by interphase ar-rest and/or cell death (Fig. 3). Knockdown of 108 out of 240 genessignificantly induced a phenotype in both cell lines, while knock-down of 56 and 39 genes induced a phenotype in only SK-N-BE(2)-C or SH-EP cells, respectively. Mitotic-linked cell death wasinduced in SK-N-BE(2)-C cells via knockdown of a group of sevengenes that included MAD2L1, ANLN, NCD80, RAD51, DPH5, ECSITand SLC1A5, which are associated with metaphase–anaphasecheckpoint regulation, mitotic exit or DNA repair [58–61]. Knock-down of any of these genes except RAD51 induced mitotic or inter-phase arrest in SH-EP cells instead of mitotic-linked cell death(Fig. 3). These results indicate that functional p53 compensatesfor a non-functional metaphase–anaphase checkpoint. In line withthis, TP53 knockdown in SH-EP cells resulted in mitotic-linked celldeath (Fig. 3, blue box). These results demonstrate that deregulat-ing distinct genes downstream of p53/pRB-E2F at the mitoticcheckpoint, and probably downstream of high MYCN activity, sup-port the survival of MYCN-amplified neuroblastoma cells duringmitotic arrest and checkpoint activation.

3.5. MAD2L1 repression in the presence of vincristine inducestetraploidization in neuroblastoma cells with functional p53-p21

Tetraplodization, however, was not only observed in neuroblas-tomas harboring mutated TP53 but also in primary tumors withfunctional p53 but deregulated MYCN. We further investigated thisapparent interplay between deregulated MYCN and the meta-phase–anaphase checkpoint in cells with functional p53-p21 inthe WAC2-neuroblastoma cell model, which stably expresses aMYCN transgene driven by a CMV promoter in a wild-type TP53 ge-netic background. WAC2 cells are characterized by a near-diploidDNA index and express high levels of MYCN protein [33,34], which

Doxorubicin-treated

DI* % Diploid % Aneuploid DI*

1.92 12.1 ± 3.1 87.9 ± 3.1 2.001.91 24.4 ± 2.1 75.6 ± 2.1 1.991.89 19.5 ± 13.7 80.5 ± 13.7 1.971.87 - 100.0 ± 0.0 2.00

1.93 21.1 ± 6.4 78.9 ± 6.4 1.981.92 27.4 ± 8.0 72.6 ± 8.0 1.97

Page 7: MYCN-mediated overexpression of mitotic spindle regulatory genes

mitotic-linked cell death:

gene symbol full gene name function MAD2L1 mitotic arrest deficient-like 1 mitotic cell cycle checkpoint ANLN anillin, actin binding protein septin ring assembly (regulation of exit from mitosis) DPH5 DPH5 homolog (S. cerevisiae) diphthine synthase activity ECSIT evolutionary conserved signaling intermediate

in Toll pathways cytoplasmic signaling in Toll-like and BMP signal transduction pathways

RAD51 RAD51 homolog (S. cerevisiae) mitotic recombination/DNA repair NDC80 NDC80 homolog, kinetochore complex

component (S. cerevisiae) mitotic checkpoint signaling

SLC1A5 solute carrier family 1 (neutral amino acid transporter), member 5

neutral amino acid transmembrane transporter activity

SK-N-BE(2)-C

interphase arrestm

itotic arrestprim

ary apoptosism

itotic-linked cell death*

**

SH-EP

interphase arrestm

itotic arrestprim

ary apoptosism

itotic-linked cell death

***

***

#

Height Height0 5 10 15 0 5 10 15

cluster dendrogramm

eith AU/BP values (%

)

distance: eucliceancluster m

ethod: centroid

Fig. 3. Mitotic-linked cell death after selective inhibition of metaphase–anaphase checkpoint genes in MYCN-amplified neuroblastoma cells. Genes were grouped according tothe phenotype after specific gene knockdown in SK-N-BE(2)-C/H2B-GFP (MYCN-amplified and TP53 inactivating mutation) and SH-EP/H2B-GFP (MYCN-single-copy and wild-type TP53) cells using hierarchical cluster analysis. ⁄Gene knockdown in this cluster revealed interphase arrest escape into mitotic arrest or vice versa. ⁄⁄Gene knockdown inthis cluster caused secondary cell death after interphase arrest. ⁄⁄⁄Gene knockdown in this cluster caused a mixture of mitotic-linked cell death, secondary apoptosis afterinterphase arrest and mitotic slippage into interphase arrest followed by secondary apoptosis. # Knockdown of KIF11 caused primary apoptosis and knockdown of SIAH2caused mitotic-linked cell death. (1) MYCN/MYC target gene and (2) E2F target gene, (NA) = neither MYCN/MYC nor E2F target gene. Phenoclusters were generated usingEuclidean distance as a dissimilarity measure. Values on branches are in percentages. P-values are shown for the approximate unbiased test (red) and bootstrap probabilities(green). The group of seven genes that induced ‘‘mitotic-linked cell death’’ in SK-N-BE(2)-C but not in SH-EP cells is highlighted (red boxes). (For interpretation of thereferences to color in this figure legend, the reader is referred to the web version of this article.)

S. Gogolin et al. / Cancer Letters 331 (2013) 35–45 41

Page 8: MYCN-mediated overexpression of mitotic spindle regulatory genes

80

60

40

20

0

cells

[%]

8N

WAC2-shMAD2L1

MAD2L1

MYCN

β-actin

doxycycline + -

A

B

C

control MAD2L1 expressed+ vincristine

MAD2L1 repressed+ vincristine

8N 8NG0/1 S G2/M2N 4N

G0/1 S G2/M2N 4N

G0/1 S G2/M2N 4N

80

60

40

20

0

80

60

40

20

0

N8N4N2

D

SH-EP-shMAD2L1

+ -

8N4N2N4N2N4N2N

G0/G1

S

G2/M

96% 58%

Fig. 4. MAD2L1 silencing after vincristine treatment induces tetraploidization in neuroblastoma cells with functional p53-p21. (A) Western blot showing MAD2L1 and MYCNexpression in whole-cell lysates from WAC2 and SH-EP cells stably transfected with shRNA targeting MAD2L1. (B) Flow cytometric analysis of cell cycle and ploidy in WAC2-shMAD2L1 cell cultures 36 h after treatment. Curves are paired with bar-graph quantifications (below) for each treatment group. (C) 2-color FISH of WAC2-shMAD2L1 after36 h of vincristine treatment and MAD2L1 shRNA induction using centromeric probes for chromosome 6 and 8 (pink and green, respectively) and counterstained with DAPI(blue). Representative images from 250 interphases are shown. (D) Merged immunofluorescence images of WAC2-shMAD2L1 stained for centromers with CREST antibodies(green) and DNA (blue) to visualize altered nuclear size after combined MAD2L1 silencing and vincristine treatment. (For interpretation of the references to color in this figurelegend, the reader is referred to the web version of this article.)

42 S. Gogolin et al. / Cancer Letters 331 (2013) 35–45

Page 9: MYCN-mediated overexpression of mitotic spindle regulatory genes

high MYCN

metaphase-anaphase checkpoint activation

e.g. MAD2L1

near-diploid neuroblastomaselevated cellular fitness

pRB

**MAD2L1 inhibition reduces cellular fitness

*inactivating mutation

p53

p21

Activated aneuploid checkpointsImpaired p53-p21-dependent

aneuploid checkpoint

high MYCN

metaphase-anaphase checkpoint activation

e.g. MAD2L1

tetraploid neuroblastomaselevated cellular fitness

pRB p53

p21

*

**

Fig. 5. Schematic model describing the role of MYCN and aneuploidy checkpoints in the development of tetraploid neuroblastomas.

S. Gogolin et al. / Cancer Letters 331 (2013) 35–45 43

results in metaphase–anaphase checkpoint overactivation. Theparental SH-EP cell line, which is characterized by single-copyMYCN, barely detectable MYCN levels and a lower metaphase–ana-phase checkpoint activation status, was used as a control. To testthe effect of metaphase–anaphase checkpoint deregulation in afunctional p53-p21 background, we stably transfected the WAC2and SH-EP cell lines with a doxycycline-inducible shRNA targetingthe metaphase–anaphase checkpoint regulator, MAD2L1. The SH-EP-MYCN neuroblastoma cell model was not suitable for theseexperiments because these cells already express a tetra-/doxycy-cline inducible system. MAD2L1 was effectively repressed uponshMAD2L1 induction in both SH-EP and WAC2 cells, as demon-strated by western blotting (Fig. 4A). The cell proportions in differ-ent phases of the cell cycle were similar for SH-EP-shMAD2L1 andWAC2-shMAD2L1 cells with or without MAD2L1 knockdown and tocontrol cells expressing scrambled shRNA. To mimic a weakenedp53-p21 checkpoint, we treated both cell lines with vincristine,which disrupts microtubule formation and reduces nuclear accu-mulation of p53, consequently preventing the transcriptional acti-vation of p21 [62]. Vincristine treatment resulted in G2/M arrest of4 N cells in both WAC2-shMAD2L1 and SH-EP-shMAD2L1 cellsexpressing MAD2L1 (Fig. 4B and Supplementary Fig. 5). Combinedinhibition of the p53-p21 axis (vincristine treatment) and themetaphase–anaphase checkpoint (MAD2L1 silencing) resulted inapproximately 40% of the WAC2-shMAD2L1 cells being 8 N, indic-ative of cycling tetraploid cells (Fig. 4B and SupplementaryFig. 5A). Only about 5% of SH-EP-shMAD2L1 cells were 8 N follow-ing combined vincristine treatment and MAD2L1 silencing (Supple-mentary Fig. 5B). These results strengthen our hypothesis thatderegulated MYCN is associated with tetraploidization whenp53-p21 signaling is weakened and the metaphase–anaphasecheckpoint unbalanced.

To further verify induction of tetraploidization in WAC2-shMAD2L1 cells, centromeric regions of chromosome 6 and 8 werelabeled with fluorescent dyes (Cy3.5 and FITC, respectively) and 2-color FISH was performed. In addition to cells with two signals foreach centromer, indicative of normal diploid cells, interphase nu-clei with four or eight centromeric signals, indicative of cycling tet-raploid cells, were also observed after MAD2L1 repression andvincristine treatment in WAC2-shMAD2L1 cells (Fig. 4C). For com-plete confirmation, 4-color FISH for chromosome 3, 6, 8 and 18

centromeres was also performed, and at least 250 interphase nu-clei were manually counted from WAC2-shMAD2L1 cell culturestreated with vincristine and expressing MAD2L1 or not. Thenumerical index of centromeric signals was used as a direct indica-tor for nuclear DNA content. The fraction of 8 N cells reached 8.7%18 h after shMAD2L1 induction and vincristine treatment. After36 h, the 8 N fraction had increased from 8.7% to 30.1% (Supple-mentary Table 5). Interphase nuclei of 8 N cells appeared stronglyenlarged with an asymmetrical shape in contrast to small, rounddiploid cells (Fig. 4C and D). We also detected 3.5% 16 N cells36 h after induction and treatment. Vincristine treatment alone re-sulted in 5.1% 8 N cells after 36 h of treatment (Supplementary Ta-ble 5). Immunofluorescence imaging using a CREST antibody thatunspecifically binds to centromeric regions further supported theobservation of polyploidization after MAD2L1 knockdown and vin-cristine treatment (Fig. 4D).

4. Discussion

In this study, we show that overactivation of the metaphase–anaphase checkpoint acts as a pro-survival mechanism in thedevelopment of tetraploid neuroblastoma cells lacking functionalp53-p21 signaling. Elevated expression of mitotic spindle regula-tory genes has been shown to be associated with MYCN amplifica-tion and 1p loss in neuroblastomas [52,63,64]. This is consistentwith our gene expression analyses in primary neuroblastoma tu-mors, which further showed that overexpression of MYCN/MYCand p53/pRB-E2F target genes, especially those involved in regulat-ing mitotic processes, such as sister chromatid segregation, micro-tubule organization or metaphase–anaphase checkpointregulation, is associated with near-di/tetraploidy and poor out-come in neuroblastoma patients. One gene we identified in this re-gard was MAD2L1, which is a direct MYC and E2F-1 target [22,65].This suggests that MYCN/MYC-mediated overactivation of themetaphase–anaphase checkpoint might be causally involved inthe development of near-di/tetraploidy by initially provoking sus-tained mitotic arrest, as shown for Mad2 overexpression [66]. Cellsmight escape this sustained arrest by mitotic slippage – an adapta-tion that consequently results in the failure of cytokinesis and tet-raploidy [1]. Our observation that neuroblastoma cells lacking p53-

Page 10: MYCN-mediated overexpression of mitotic spindle regulatory genes

44 S. Gogolin et al. / Cancer Letters 331 (2013) 35–45

p21 function, either through TP53 mutation or mediated byp21CIP1 knockdown, consist of diploid and tetraploid cell fractionsindicates that functional p53-p21-mediated checkpoints are re-quired to arrest these cells [53,67] and to subsequently initiate celldeath or senescence programs [57]. Evidence that p53 and p21inactivation is mainly involved in the origin of tetraploidy existsin the mouse model p53-R172P equivalent to R175P in human,which harbor an Arg-to-Pro TP53 mutation. The p53 in cells fromthese mice is incapable of inducing apoptosis, but still activatedp21-mediated G1-S arrest [68]. Accordingly, these mice developedtumors with a diploid DNA index. Crossing the TP53-mutant miceinto a p21�/� background resulted in formation of aneuploid tu-mors. Some evidence exists for a potential association between tet-raploidy and loss of p53-p21 functionality in neuroblastoma.Additionally to SK-N-BE(2)-C cells, other neuroblastoma cell linesharboring mutant TP53 are characterized by a near-tetraploidDNA index, including LA-N-1, NMB and NB-6 [69–72]. Whole gen-ome sequencing of primary neuroblastomas revealed that at leastsome tetraploid tumors harbored TP53 mutations (unpublisheddata). This genetic alteration, although frequent in many other can-cers, occurs mainly in relapse neuroblastomas and is associatedwith therapy resistance [55], suggesting a direct connection ofp53 functionality to neuroblastoma biology.

A direct connection between the p53-p21 axis, mitotic spindleregulatory genes and tetraplodization was presented by Schvartz-man, et al. using the same TP53-mutant and p21-deficient mice. Heshowed that p21 is a direct negative Mad2 regulator and that nor-malization of Mad2 expression reduced the aneuploid cell fractionin these murine tumors [73]. In cells lacking p53 and/or p21 function,overactivation of metaphase–anaphase checkpoint members might,therefore, facilitate the development and survival of tetraploid cells(Fig. 5). We observed that tetraploid cell fractions increased afterseveral passages or drug-induced DNA damage in both the TP53-mu-tated neuroblastoma cell line and in neuroblastoma cells silenced forp21CIP1 expression. Our siRNA screening approach also demon-strated that knockdown of genes involved in metaphase–anaphasecheckpoint regulation, including MAD2L1, induced mitotic-linkedcell death only in neuroblastoma cells lacking p53-p21 function asa consequence of TP53 mutation. These results from various in vivoand in vitro studies show that functional p53-p21 signaling is crucialto control the expression of metaphase–anaphase checkpoint genesand to inhibit the survival of tetraploid cells.

In summary, the results we present here reveal novel insightsinto how genetic aberrations of the p53-p21 axis contribute to tet-raploidy in neuroblastoma cells. These data enhance our under-standing of how MYCN/MYC mediates aggressive behavior inneuroblastomas. Since overactivation of the metaphase–anaphasecheckpoint supports the survival of tetraploid cells lacking p53-p21 function, targeted inhibition of certain metaphase–anaphasecheckpoint members, such as MAD2L1, may provide a therapeuticoption for neuroblastomas harboring genomic alterations reducingp53-p21 function.

Acknowledgments

We thank Geoffrey M. Wahl for providing the H2B-GFP expres-sion vector, Frank Berthold, Barbara Hero and the German Neuro-blastoma Study Group for providing clinical data, and KathyAstrahantseff for manuscript editing.

Appendix A. Supplementary data

Supplementary data associated with this article can be found, inthe online version, at http://dx.doi.org/10.1016/j.canlet.2012.11.028.

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