segmental chromosome aberrations converge on overexpression of mitotic spindle regulatory genes in...

GENES, CHROMOSOMES & CANCER 51:545–556 (2012)

Segmental Chromosome Aberrations Converge onOverexpression of Mitotic Spindle Regulatory Genesin High-Risk Neuroblastoma

Wen Fong Ooi,11,† Angela Re,11,† Viktoryia Sidarovich,1 Valentina Canella,1 Natalia Arseni,1 Valentina Adami,2

Giulia Guarguaglini,3 Maria Giubettini,3 Paola Scaruffi,4,5 Sara Stigliani,4 Patrizia Lavia,3 Gian Paolo Tonini,4 and

Alessandro Quattrone1*

1Laboratoryof Translational Genomics,Centre for Integrative Biology and Departmentof Information Engineering and ComputerScience,Universityof Trento, 38122 Trento,Italy2HighThroughput Screening Facility,Centre for Integrative Biology,Universityof Trento, 38122 Trento,Italy3Institute of Molecular Biology and Pathology,CNR, c/o Sapienza Universityof Rome,00185 Rome,Italy4Translational Oncopathology,National Cancer Research Institute (IST),16132 Genoa,Italy5Centerof Physiopathologyof Human Reproduction,Departmentof Obstetrics and Gynecology,‘‘San Martino’’Hospital,16132 Genoa,Italy

Integration of genome-wide profiles of DNA copy number alterations (CNAs) and gene expression variations (GEVs) could

provide combined power to the identification of driver genes and gene networks in tumors. Here we merge matched ge-

nome and transcriptome microarray analyses from neuroblastoma samples to derive correlation patterns of CNAs and

GEVs, irrespective of their genomic location. Neuroblastoma correlation patterns are strongly asymmetrical, being on average

10 CNAs linked to 1 GEV, and show the widespread prevalence of long range covariance. Functional enrichment and net-

work analysis of the genes covarying with CNAs consistently point to a major cell function, the regulation of mitotic spindle

assembly. Moreover, elevated expression of 14 key genes promoting this function is strongly associated to high-risk neuro-

blastomas with 1p loss and MYCN amplification in a set of 410 tumor samples (P < 0.00001). Independent CNA/GEV profil-

ing on neuroblastoma cell lines shows that increased levels of expression of these genes are linked to 1p loss. By this

approach, we reveal a convergence of clustered neuroblastoma CNAs toward increased expression of a group of prognostic

and functionally cooperating genes. We therefore propose gain of function of the spindle assembly machinery as a lesion

potentially offering new targets for therapy of high-risk neuroblastoma. VVC 2012 Wiley Periodicals, Inc.

INTRODUCTION

Neuroblastoma (NB) is an important clinical

problem, accounting for 15% of the pediatric can-

cers. It is characterized by remarkable phenotypic

variability (Cohn et al., 2009), ranging from an

exceptionally favorable outcome, with spontane-

ous regression, to an aggressive malignant course,

which qualifies the so-called high-risk NBs. In the

past years the introduction of whole-genome

profiling (Michels et al., 2007; Mosse et al., 2007;

Scaruffi et al., 2007; Tomioka et al., 2008) enabled

the identification of chromosome hyperdiploidy or

aneuploidy (numerical aberrations) and of several

nonrandom copy number aberrations (CNAs)

below the chromosome size (segmental altera-

tions). While numerical aberrations almost always

qualify for a benign course (Ambros et al., 2009;

Cohn et al., 2009), various structural CNAs are pre-

dictive of an adverse disease course (Ambros et al.,

2009; Cohn et al., 2009). Amplification of the

MYCN (MNA) oncogene and losses of the 11q

chromosomal arm have been shown to be signifi-

cantly associated with poor outcome (Cohn et al.,

2009). Since MNA and 11q loss are almost mutu-

ally exclusive (Caren et al., 2008; Ambros et al.,

2009), these two aberrations were suggested to

molecularly characterize two distinct tumor sub-

groups (Caren et al., 2010). Loss of the 1p chromo-

somal arm is another negative prognosis marker,

occurring at significantly higher frequency in the

MNA cases; gain of chromosome arm 17q is the

Additional Supporting Information may be found in the onlineversion of this article.

yThese authors contributed equally to this work.

Supported by: The Autonomous Province of Trento (http://www.provincia.tn.it); the Associazione Italiana per la Lotta alNeuroblastoma (http://www.neuroblastoma.org).

*Correspondence to: Centre Alessandro Quattrone, Laboratoryof Translational Genomics, for Integrative Biology, University ofTrento, 38122 Trento, Italy. E-mail: [email protected]

Received 28 August 2011; Accepted 7 January 2012

DOI 10.1002/gcc.21940

Published online 15 February 2012 inWiley Online Library (wileyonlinelibrary.com).

VVC 2012 Wiley Periodicals, Inc.

RESEARCH ARTICLES

last major rearrangement predicting poor outcome,

and equally associates with either 11q or 1p losses

(Cohn et al., 2009). In addition to genomic altera-

tions, gene expression variations (GEVs) have pro-

ven to be predictive of NB outcome (Schramm

et al., 2005; Katleen et al., 2006; Oberthuer et al.,

2006, 2010; Wang et al., 2006).

Besides their prognostic potential, integration

of genomic and transcriptomic profiles can reveal

important features of the biology of tumors. A tra-

ditional approach to this integration has been

based on the assumption that genomic aberrations

contribute to oncogenesis through local gene dos-

age effects (Mosse et al., 2007; Lin et al., 2008;

Turner et al., 2010). There is no ground, how-

ever, to exclude long range effects of genomic

instability on gene expression.

Cell cycle alterations are a hallmark of all tumors,

so that cell cycle kinase-targeted compounds have

been proposed as new potential antitumor agents,

by which targeting of altered cell cycle checkpoints

could selectively kill cancer cells (Shapiro, 2006).

Among the cell-cycle related molecular aberrations

occuring in NB, amplification of Cyclin D1, respon-

sible for the G1 checkpoint, is present at low fre-

quency, but the gene is overexpressed in more

than 70% of the tumors (Molenaar et al., 2003).

Two other cell-cycle genes recently entered in the

NB field are CHK1, coding for another kinase

involved in the G1 and G2/M checkpoints, which

has been recently identified as more expressed in

MYCN-amplified tumors and selected as a thera-

peutic target (Cole et al., 2011), and CDK2, whoseproduct regulates the S-phase checkpoint and

whose inhibition induces apoptosis only in MYCNoverexpressing cells (Molenaar et al., 2009). An

integrated analysis of transcriptome profile and

MYCN binding sites using a cell model of MYCNoverexpression identified also an enrichment of

cell cycle related genes among the transcriptional

targets ofMYCN (Murphy et al., 2011).

We address here the genome-wide effects of

CNAs on GEVs in NB, by integrating aCGH

analyses and transcriptome profiles of tumors and

cell lines. An unbiased correlation analysis of NB

tumors through a bicluster-based approach of

matched genome-wide DNA and transcript pro-

files (Tanay et al., 2002; Lee et al., 2008) identi-

fies genes active in a specific cell cycle event,

spindle formation at mitotic metaphase, as targets

of recurrent genomic alterations, all associated to

increased expression of these genes. We also

demonstrate that such behavior is confined to NB

high-risk tumors with MNA and 1p loss.

MATERIALS AND METHODS

Bicluster Identification and Selection

The data for biclustering came from matched

array-based CNA measurements and Affymetrix

microarray total mRNA measurements obtained for

82 NB samples (Wang et al., 2006), and deposited

in the GEO database as GSE7230 and GSE3960.

We modeled the relationship between CNAs and

GEVs in a form of correlation matrix. Firstly, Pear-

son’s correlation coefficients (PCCs) between the

log2-transformed intensities of each array-CGH

probe and gene expression probe set were calcu-

lated (Supporting Information Fig. S1). Second, we

applied the SAMBA algorithm (Tanay et al., 2002)

to the correlation matrix in order to look for biclus-

ters, where a bicluster is a group of CNAs and

GEVs such that the GEV patterns show high corre-

lation with the CNA patterns across the samples.

Notice that the same GEV and CNA are allowed

to occur in more than one bicluster. Each bicluster

was scored by summing PCCs in absolute values.

The statistical confidence corresponding to a

bicluster score was defined by a bootstrapping pro-

cedure where 10,000 shufflings of the genes in a

bicluster provided the null empirical distribution of

scores to compute the bicluster P-value. The P-val-

ues were then adjusted by the Benjamini & Hoch-

berg false discovery rate (FDR) controlling

procedure (0.01 confidence threshold).

Annotation of the High Correlation Biclusters

We used the Gene Ontology (GO) Biological

Process (BP) and Cellular Component (CC) gene

sets (version 2.5) recorded in the Molecular Sig-

natures Database (http://www.broadinstitute.org/

gsea/msigdb/) to perform the functional enrich-

ment analysis of the genes in each bicluster. The

Fisher’s exact test P-values were adjusted by the

Benjamini and Hochberg FDR controlling proce-

dure (0.05 confidence threshold).

Array-CGH and Transcriptome Profiling of NB

Cell Lines

The characteristics and growth conditions of

the 14 NB cell lines are described in Supporting

Information. Total DNA and RNA were isolated

by using the DNA Blood and Tissue Extraction

Kit (Qiagen) and the RNeasy Mini Kit (Qiagen,

Hilde, Germany), respectively, according to man-

ufacturer’s protocols. Total RNA was quantified

and quality control assessed by RNA 6000 NanoVR

assay on the 2100 Bioanalyzer (Agilent

546 OOI ET AL.

Genes, Chromosomes & Cancer DOI 10.1002/gcc

Technologies). Only samples with a RNA Integ-

rity Number >7 were included in the study.

Array-CGH profiling was performed using Agilent

Human Genome CGH 244K oligonucleotide

microarrays (Agilent Technologies). RNA expres-

sion profiling was performed starting from 500 ng

of total RNA. Each sample was hybridized on

Human GE 4x44K v2 Microarray Kit (Agilent

Technologies). Slides were scanned by G2565BA

scanner (Agilent Technologies). Normalization

and statistical analyses are described in Support-

ing Information. All data is MIAME compliant.

Array-CGH and gene expression data have been

deposited in the MIAME-compliant Gene

Expression Omnibus (GEO) data repository, as

detailed on the MGED Society website http://

www.mged.org/Workgroups/MIAME/miame.html

(GEO series accession number GSE22785).

Mapping of the Genome-Wide Associations

between CNAs and Prognostic Genes in NB Cell

Lines

Absolute PCCs between CNAs and GEVs

were computed for each prognostic gene across

the NB cell lines. The Rank Product (Hong

et al., 2006) method (RankProd, R package) was

used to identify the CNAs that most consistently

ranked within the top 1% of the CNAs associat-

ing with GEVs of prognostic genes.

Cell Immunofluorescence

Cells were grown on sterile polylysine-coated

coverslips and fixed either in cold methanol for 6

min (preferred method for high resolution of the

spindle microtubules), or in 3.7% PFA/0.2% Tri-

tonX-100 in PHEM (45 mM PIPES pH 6.9; 45

mM HEPES pH 6.9; 10 mM EGTA; 5 mM

MgCl2 and 1 mM PMSF) for 10 min at room

temperature (RT). Blocking steps and incubation

with antibodies were performed at room tempera-

ture in 0.05% Tween 20, 3% BSA in PBS. Pri-

mary antibodies were: mouse anti alpha-tubulin,

either unconjugated (1:2000, B-5-1-2, Sigma, St

Louis, MO) or FITC (fluorescein isothiocynate)-

conjugated (1:150, DM-1A, Sigma), rabbit anti-

pericentrin (2 mg/ml, ab4448, Abcam, Cambridge,

MA), mouse anti-gamma-tubulin (1:1000, GTU-

88, Sigma). Secondary antibodies were conjugated

to FITC (Jackson Immunoresearch Laboratories,

West Grove, PA) or Texas Red (Vector Laborato-

ries). Cells were counterstained with 4,6-diami-

dino-2-phenylindole (DAPI, 0.05 lg/ml) and

mounted using Vectashield (Vector Laboratories,

Peterborough, UK). Samples were analyzed under

a 90i microscope (Nikon, Langen, Germany)

equipped with a Qimaging CCD camera. Color

encoded images were acquired using the Nis-Ele-

ments AR 3.1 software (Nikon) and processed

with Adobe Photoshop CS 8.0.

RESULTS

A Large Number of Genomic Alterations Have

Strong, Long Range Effects on Gene Expression in

Neuroblastoma

In our integrative analysis of cancer genome

and transcriptome variations, we initially

employed a public dataset with matched array-

CGH and expression array profiles of 82 NB

tumors (Wang et al., 2006). CNAs were defined

by applying the pipeline presented in Fig. S1. Af-

ter filtering CNAs from recurrent somatic varia-

tions with reference to the HapMap data (Frazer

et al., 2007), we obtained 134 NB-specific CNAs

across the samples. Normalized transcriptome

data resulted in 7970 GEVs for the same samples

(Supporting Information). CNA and GEV pat-

terns were next integrated into a pairwise correla-

tion matrix via the mapping of 1942 array-CGH

probes and 7970 probe sets, respectively. The

application of the SAMBA algorithm (Tanay

et al., 2002) to the correlation matrix gave 409

statistically significant biclusters (P-value <0.0001), each composed by a number of directly

or inversely covariant CNAs and GEVs, irrespec-

tive of their genomic location. The median num-

ber of CNAs and genes defining a bicluster were

14 and 135, respectively, with 97.32% of CNAs

and 55.44% of genes belonging to more than 10

biclusters, while 14.14% of CNAs and only 0.63%

of genes were shared by more than 50 biclusters.

This structure clearly defines the relationship

between highly covariant GEVs and CNAs as

‘‘one-gene-to-many-CNAs,’’ with an average cor-

relation of 1 gene to 9.24 CNAs (Fig. 1A). Note-

worthy, a prevalence of long range effects of

genomic imbalances on transcript level variations

in NB clearly emerged. Indeed, we found that

almost all the covariations were not colocalized

(Fig. 1B), with only 0.23% of all them being such

that the probed mRNA was from a gene locus

intersecting the CNA. These loci are reported in

Table S1. Therefore, considering the colocalized

effects of genomic lesions on transcriptome

changes and ignoring long-range effects, as in

CNAS IDENTIFY ALTERED SPINDLE ASSEMBLY GENES IN NB 547


previous studies (Heidenblad et al., 2005; Mat-

suda et al., 2011), might be simplistic. Moreover,

our analysis identified most negative associations

among noncolocalized CNAs and genes (Fig. 1B),

while confirming the expected positive correla-

tion among colocalized CNAs and genes (Sup-

porting Information Table S1).

Genes Highly Correlated to Neuroblastoma CNAs

Point Primarily to Functions Related to Mitotic

Spindle Assembly

The mRNAs in the 409 high correlation biclus-

ters are extensively associated to multiple CNAs,

therefore their coding genes are sites of conver-

gence of different tumor DNA structural varia-

tions influencing their expression. We speculate

that these genes could be responsible for cell

activities critical for NB development. Ontologi-

cal overrepresentation analysis of Gene Ontology

(GO) Biological Process (BP) and Cellular Com-

ponent (CC) annotations performed on CNA/

GEV covariation biclusters immediately conveyed

a main biological message: the emergence of

genes (consistently present in two third of the

biclusters) involved in spindle formation and

attachment to the kinetochore of chromosomes in

the M phase of the cell cycle (Fig. 2).

We also reasoned that our network of covari-

ance of CNAs and genes, in which the CNA con-

nectivity is much higher than the gene

connectivity, offered another way to prioritize

Figure 1. One-to-many structure and low colocalization of CNA/GEV associations in neuroblastoma tumors. A: Box-whisker plotsshowing the distribution of the average number of CNAs associatedwith a GEV in all biclusters. B: Piechart showing the fractions of colo-calized and noncolocalized associations in all biclusters. Positive andnegative associations are highlighted within each fraction. The fractionof colocalized associations, which is extremely small, is magnified forbetter representation.

Figure 2. Neuroblastoma segmental alterations converge on mitotic spindle assembly genes. Heatmapof negative log10 transformed enrichment P-values for all pairs of overrepresented GO (BP and CC)annotations and biclusters. GO annotations are denoted by their official names or identifiers. Biclustersare denoted by their numerical identifiers.

548 OOI ET AL.


genes, by focusing on the genes associated with

the maximally dispersed CNAs. This was based

on the assumption that the more a specific gene

expression change is targeted by multiple CNAs

across tumors, the more it is expected to be rele-

vant for tumor pathogenesis. By applying this

selection approach, we obtained the same indica-

tion of large prevalence of genes involved in the

mitotic spindle assembly process (Supporting In-

formation Table S2, Fig. S2).

Therefore, genes that strongly covary in

expression with the maximally dispersed, recur-

rent DNA lesions in NB tumors show a clear

functional focus toward regulatory pathways of

spindle formation in mitotic metaphase.

Overexpression of Spindle Assembly Genes Highly

Targeted by CNAs is Predictive of Poor Outcome

in Neuroblastomas Bearing 1p Loss and MYCN

Amplification

We then evaluated the relationship of the mi-

totic spindle assembly genes in NB with disease

characteristics through the association between

transcriptome variations and NB prognostic

markers. To this end, we increased the number

of studied tumors from the initial 82 profiled NB

samples (Wang et al., 2006) by including three

additional gene expression datasets (McArdle

et al., 2004; Oberthuer et al., 2006; Łastowska

et al., 2007; Fischer et al., 2010) to reach a total

number of 410 samples (Supporting Information).

These additional samples lacked genome-wide

DNA profiles, but were carefully stratified accord-

ing to stage (INSS), disease outcome, and the

presence of the known major prognostic segmen-

tal alterations (MNA, 1p loss, 11q loss and 17q

gain, whose relative distribution in the samples is

reported in Supporting Information Fig. S3A).

Crossing this stratification with a genome-wide

cell cycle related phenotypic annotation obtained

by gene silencing (Neumann et al., 2010) showed

that only genes annotated with phenotypes indic-

ative of mitotic impairment were predictive of

poor outcome. We also noted that extending the

analysis to genes the silencing of which more

generally compromises proliferation failed to

reach statistical significance (P > 0.05, Supporting

Information Fig. S3B).

Next, we came back to the high correlation

biclusters, wherein we shortlisted 33 informative

genes, based on their assignment to the GO BP

and CC annotations that are stably overrepre-

sented across biclusters (Pearson’s chi-squared

test, P-value < 0.01). The network formed by

these genes is illustrated in Supporting Informa-

tion Fig. S4A, and associations are detailed in

Supporting Information Table S3. We then

sought to determine if the 33 GO informative

genes could be related to poor outcome in NB,

considering the same clinically annotated datasets

used for the phenotype-based investigation. As

reported in Figure 3A, a clear higher expression

(Wilcoxon rank sum test, P-value � 5E-6) with

respect to all the other samples emerged in 23

out of the 33 selected genes, and only for tumors

bearing MNA, 1p loss or both. A much smaller

effect was seen for the same genes in tumors

with 17q gain and combinations of this lesion

with MNA and 1p loss, while absolutely no sig-

nificant association emerged for 11q loss in any

combination. The same high expression signature

was also observed in correlation with unfavorable

Figure 3. Overexpression of spindle assembly genes is associatedwith high-risk neuroblastoma tumors bearing MNA and 1p loss. P-val-ues for significance of overexpression and underexpression of the 33GO informative genes in sample groups are reported in A and B,respectively, compared with all the other samples each time. Thegroups are formed cumulatively starting from four different neuro-blastoma expression profiling datasets (410 samples in total) accord-ing to the major prognostic cytogenetic marker profiles (MNA, 1pdel, 11q del, 17q gain), histology and disease endpoint.



tumor phenotype (Stages 3 or 4 versus Stages 1,

2, and 4S) or tumor lethality. Of the 23 highly

expressed genes, spindle assembly genes were

14, DNA replication genes 6 and splicing genes 3

(Supporting Information Fig. S4A). When we

repeated the same analysis checking for

decreased expression among the initial 33 inform-

ative genes, none of the clinically clustered sub-

groups of samples reached statistical significance

(Fig. 3B). Based on their established function in

mitosis, 11 out of the 14 spindle assembly genes

participate in two functional machineries, a com-

plex involved in spindle microtubule assembly

regulation [RAN, TPX2, KIF11 (EG5), DLGAP5(HURP), AURKA] and the so-called mitotic or

spindle assembly checkpoint [TTK (MPS1),BUB1, BUB1B (BUBR1), CENPE (CENP-E),MAD2L1, ZWINT].Taken together, these results identify genes

necessary for productive mitosis, but not for the

more general activity of proliferation, as signifi-

cantly more expressed in subsets of NB samples

characterized by MNA and 1p loss, and by

aggressive course of the disease. The same asso-

ciation profile becomes statistically much stronger

when we consider genes prioritized for being co-

variant with recurrent CNAs in NB, among which

the majority are mitotic genes related to spindle

assembly.

Profiling of Genome and Transcriptome

Covariations from a Panel of Neuroblastoma Cell

Lines Reveals a Major Association of 1p Loss with

Increased Expression of the Spindle Assembly

Genes

A simple mechanistic explanation for the

increased expression of these 23 genes could be

that they are direct transcriptional targets of

MYCN. Nevertheless, when we attempted to ver-

ify this hypothesis, our analysis excluded a direct,

common influence on these genes by MYCN (Sup-

porting Information Table S4, Fig. S5). Moreover,

in our dataset of 410 NB tumors almost all sam-

ples with MNA had also 1p loss (59 of 63, 94%)

while a smaller fraction of 1p loss tumors (59 of

97, 61%) were also MNA, suggesting a role of 1p

loss in the coherent higher expression.

To gain further information about the recurrent

CNAs associated to this increase in expression of

the 23 genes, we moved from tumor samples to a

panel of parental (not subcloned in vitro) NB cell

lines. The use of NB cell lines allows a much

higher sensitivity for CNA profiling, due to the

absence of nontumor contaminating cells (Vol-

chenboum et al., 2009). We profiled these cells

using high density array-CGH and gene expres-

sion microarrays from the same platform, with the

aim to minimize technical variability. The stable

top 1% CNAs associated to 18 out of the 23 prog-

nostic genes were found to distribute, in order of

CNA frequency, on the 7q, 1p, 17q, 18q, 14q, 2p,

15q, 11p, 1q, and 10q chromosome arms (Sup-

porting Information Table S5). The complex net-

work of top scoring CNA and GEV covariance is

represented in Figure 4. Two general features are

immediately evident from this scheme: (a) no

colocalized CNA/GEV association is present in

the network, confirming no role of this type of

imbalance for the informative genes; (b) when

multiple informative genes are associated to the

same CNA region they are homogeneously

affected, positively or negatively, by these CNAs.

The two main CNA clusters correlating with the

expression of informative genes result by far to

be 7q gains (10 different CNAs, from 7q21.3 to

7q36.3), negatively correlated with the informa-

tive genes and therefore associated to decreased

gene expression, and 1p losses (9 different CNAs,

from 1p31.1 to 1p36.31), again negatively corre-

lated, and therefore associated to increased gene

expression. This last association confirms the tu-

mor-derived higher expression clustering reported

in Figure 3A, whereas no strong correlation is

identified between MNA (MYCN is located in

2p24.3) and any of the informative genes, despite

the fact that 11 out of the 14 profiled NB cell

lines are MNA. Interestingly, 17q gain (4 differ-

ent CNAs, from 17q12 to 17q23.2) is positively

correlated almost exclusively with spindle assem-

bly genes. The robust negative association

between gains of 7q and the informative genes

detected in cell lines is instead of more difficult

interpretation in the framework of this study.

Even if 7q gains are a common chromosomal

imbalance in NB tumors, no clear prognostic

value has been demonstrated for them (Mora

et al., 2002; Stallings et al., 2003). Of interest, 7q

gains have been recently shown to be enriched,

using a large tumor set, in 11q loss tumors (Buck-

ley et al., 2010). These tumors in our analysis

(Fig. 3) are not associated with enhanced expres-

sion or decreased expression of any of the inform-

ative genes, Therefore the biological significance

of the link between 7q gains and decreased gene

expression needs further analysis.

To better understand if our signature of

increased transcriptome levels of the informative

550 OOI ET AL.


genes in cells with 1p loss was reflected in the

proteome, we finally profiled the panel of NB

cell lines for polysomal RNA after velocity sed-

imentation of the cell lysates in sucrose gra-

dients (Supporting Information). Polysomal

RNA is a better proxy of protein changes than

total RNA (Zong et al., 1999), since translation

is controlled at its initiation, and therefore the

quantity of mRNAs loaded on polysomes is a

likely direct indicator of translated proteins. A

comparison of the polysomal signals in the 11

cell lines with 1p loss of our panel with those

in the SK-N-SH cell line, not bearing 1p loss

or MNA, clearly shows for the 23 informative

genes a general increase in levels (Table S6

and Supporting Information).

Mitotic Analysis of Neuroblastoma Cell Lines

Suggests Resolution of Spindle Abnormalities

We finally selected 3 cell lines tumorigenic in

nude mice bearing different cytogenetic prognos-

tic markers, in which we analyzed the mitotic ap-

paratus by fluorescence microscopy-based

immunolocalization of microtubule and spindle-

related proteins. The cell lines were: CHP-134,

diploid, with MNA and 1p loss (Schlesinger

et al., 1976); SK-N-BE(2), near-diploid, with

Figure 4. Overexpression of prognostic spindle assembly genes isassociated with 1p loss and 17q gain in neuroblastoma cell lines. Thegraph shows the top scoring PCCs between genome-wide CNAs andthe prognostic genes derived from our analysis by using an independ-ent panel of 14 neuroblastoma parental cell lines. The two base barsof the graph display the strongly associated copy number losses (leftside, green bar) and the strongly associated copy number gains (rightside, red bar), while the two vertical bars display the prognosticgenes in strong direct (top bar) and strong inverse (bottom bar)associations along with their locus cytoband. The lines connecting the

base bars with the vertical bars represent, with a different color foreach chromosome location, the top ranking CNA/GEV covariancesfor the prognostic genes. The figure illustrates the origin of thescored PCCs at four selected pairs of CNA and genes, each corre-sponding to a type of CNA/GEV association shown in the figure. Ascatterplot displays the relationship observed in the NB cell linesbetween the DNA copy numbers and the gene expression levels fora selected pair of CNA and gene; each scatterplot shows the linearregression line along with the corresponding PCC.



MNA and 1p loss (Biedler and Spengler, 1976);

SK-N-MC, near-diploid, with 11q loss. The first

two cell lines were representative of high-risk

tumors strongly associated with the spindle as-

sembly gene signature found, while the last one

was indicative of high-risk tumors with no rela-

tion to these genes. The SK-N-BE(2) cell line

displayed mitotic figures with grossly normal

spindle structures in prometaphase and meta-

phase (an example is shown in Fig. 5A), except

for a fraction (6.18%) that displayed monopolar

spindles. Scoring of anaphase and telophase fig-

ures revealed a low fraction (2.98%) harboring

chromosome segregation defects (lagging chromo-

somes and chromatin bridges). Noteworthy, this

cell line showed a high accumulation of cells in

metaphase (19.80%), which was statistically sig-

nificant compared with the others (Pearson’s chi-

Figure 5. MNA and 1p loss neuroblastoma cell lines display a phe-notype compatible with protection from mitotic abnormalities. Immu-nofluorescence panels of representative mitotic phenotypes in theindicated cell lines. Mitotic spindles are visualized by staining alpha-tubulin, the pericentriolar material (PCM) is depicted by either peri-centrin or gamma-tubulin staining and chromosomes are stained withDAPI. A: SK-N-BE(2) metaphase with a bipolar spindle (n ¼ 320

counted mitoses, four independent analyses). B: CHP-134 prometa-phase diplaying a monopolar spindle (n ¼ 352 counted mitoses in fiveanalyses). C: S-KN-MC prometaphase with a multipolar spindle andfragmented PCM. D: S-KN-MC telophase with abnormally segregatingchromosomes (n ¼ 301 counted S-KN-MC mitoses, three analyses).Bars: 10 lm.

552 OOI ET AL.


squared test, P-value < 0.01). These observations

suggest a possible scenario wherein spindle

microtubules established defective attachments

to the kinetochores of chromosomes. Cells possi-

bly attempted to correct this defect during pro-

longed metaphase, eventually yielding proper

chromosome segregation in the vast majority of

cells. CHP-134 showed a higher frequency of mi-

totic figures with abnormal spindle structure

(13.35%), mostly represented by monopolar spin-

dles (7.38%). These monopolar figures seemed to

be typically characterized by defective separation

of the microtubule asters, as well as hampered

microtubule growth (exemplified in Fig. 5B). In

late mitotic stages, however, most mitotic cells

segregated chromosomes normally (only 3.30% of

late mitotic cells showed chromosome segregation

defects). This pattern suggests that the spindle

assembly abnormalities observed in earlier mitotic

sub stages were resolved as mitosis progressed,

with little consequence on chromosome segrega-

tion. The SK-N-MC cell line showed the highest

frequency of abnormal mitotic figures: almost

20% of cells assembled a mitotic apparatus with

abnormal structure; of those, 11.20% were multi-

polar spindles and showed fragmented pericen-

triolar material (examples are shown in Fig. 5C).

An analysis of later mitotic stages showed segre-

gation defects in 12.16% of the cells (examples in

Fig. 5D). In this cell line, therefore, the occur-

rence of spindle abnormalities, particularly at the

level of spindle poles, is paralleled by a similar

frequency of chromosome missegregation, sug-

gesting a proneness to develop genetic abnormal-

ities at each mitotic round.

With the strong limitation of a minimal sam-

pling of NB cell lines, this analysis could suggest

protection from spindle abnormalities in cell

models of tumors associated with the spindle as-

sembly gene signature.

DISCUSSION

Integration of matching genome and transcrip-

tome data from tumor samples is expected to pro-

vide useful information on the nature of gene

expression alterations driving tumor onset and

progression, and therefore to guide the search for

tailored therapies. In our integrative analysis of

CNAs and GEVs in NB we applied a modular

strategy (Tanay et al., 2002; Lee et al., 2008),

which initially enabled us to prioritize strictly

covarying CNAs and GEVs. Our results pointed

to a picture of colocalized and noncolocalized

covariations between genome regions and tran-

script abundances, providing a firm ground for

the use of genome-spanning approaches in func-

tionally integrating DNA and mRNA tumor pro-

files. We also realized that the connectivity

among CNAs and GEVs is strongly asymmetrical,

with a GEV being associated on average with

almost 10 different CNAs. Cell activities related

to spindle formation were identified in the CNA/

GEV covariation biclusters by both the GO over-

representation analysis and the measure of CNA

dispersion. The result was also robust with regard

to the unbiased selection of prioritized genes in

the biclusters (more than 50% of the 33 priori-

tized genes related to spindle functions) and to

the evaluation of the association of these genes

with poor outcome using a large NB sample data-

set obtained from four different studies (60% of

the 23 informative genes). The higher expression

of spindle assembly genes observed in these

tumors with respect to the other tumors might

simply reflect a higher proliferative index. None-

theless, extending the gene annotation from mi-

totic spindle assembly to more general terms

such as ‘‘proliferation’’ did not produce any statis-

tical significance (Fig. S3). From recent low and

high resolution profiling studies (Michels et al.,

2006; Ambros et al., 2009) MNA and 1p loss

define a specific combination of chromosome seg-

mental alteration among NB tumors, character-

ized by few other genomic lesions (median ¼ 4),

poor prognosis and the shortest survival among

high-risk patients. This specific and very aggres-

sive NB disease is undoubtedly the main source

of our mitotic spindle gene signature, with a

lower signal associated to 17q gain (Fig. 3A).

Owing to the strong co-occurrence of MNA and

1p loss (Cohn et al., 2009), identifying the lesion

from which the coherently increased expression is

likely to have originated is not obvious. Our

approaches allowed us to robustly associate the

gene expression signature with 1p loss, but not

with MNA. When we employed, in an independ-

ent and unbiased manner, DNA and transcript

profiles of parental NB cell lines to measure the

association of prioritized spindle assembly genes

with CNAs, CNAs spanning from 1p31.1 to

1p36.31 strongly associated with all genes except

two (Fig. 4), whereas MNA did not. Moreover,

we showed the lack of indication of direct MYCNregulation on the prioritized genes (Supporting

Information Fig. S5). Therefore, not overlooking

the inherently correlative origin of our outcomes,

we expect 1p loss to be more likely responsible



for the characteristic gene expression signature

than MNA. Another hint emerging from the cell

line network is that 17q gains are associated to

increased expression of over 8 of the 14 spindle

genes (Fig. 4), albeit with lower multiplicity than

for 1p. This would be consistent with the weaker

association of 17q gain in the tumors with the

higher expression signature (Fig. 3A). We notice

that the 14 more highly expressed genes in MNA

and 1p loss NB tumors define two functional

machineries. The first one includes TPX2, KIF11,DLGAP5, and AURKA, all of which contribute to

regulate mitotic microtubule growth and organiza-

tion of a normal bipolar spindle under the control

of the other signature gene RAN. The proteins

encoded by these genes are actually found to be

part of a single protein complex in reconstitution

systems (Koffa et al., 2006) and also show func-

tional interactions in living cells (Ciciarello et al.,

2007; Clarke and Zhang, 2008). The second ma-

chinery identifies the spindle assembly check-

point (SAC) network, preventing metaphase-to-

anaphase transition until all chromosomes are cor-

rectly attached to spindle fibers. TTK, BUB1,BUB1B, CENPE, ZWINT and MAD2L1 are SAC

components (Musacchio and Salmon, 2007). The

remaining three genes are involved in related

processes: CENPF regulates microtubule dynam-

ics as well as the stability of microtubule attach-

ments to kinetochores, and can therefore be

viewed as a gene bridging the two machineries;

CAPC is a condensin subunit involved in control-

ling chromosome condensation; KIF23 regulates

cytokinesis. In many instances, downregulation of

these genes [e.g. AURKA (Hata et al., 2005; De

Luca et al., 2008), DLGAP5 (Sillje et al., 2006;

Wong and Fang, 2006), MAD2L1 (Michel et al.,

2001), CENPF (Holt et al., 2005; Yang et al.,

2005)] resulted in aberrant mitosis and chromo-

some missegregation in model cell lines. Accord-

ingly, cell phenotypes associated with silencing of

either TPX2/AURKA or BUB1/BUB1B/CENPE/MAD2L1 in a genome-wide phenotyping screen-

ing (Neumann et al., 2010) produced a strikingly

common profile of mitotic failure, consistent with

their coordinated activity (Supporting Information

Fig. S4B). In the CNA/GEV network we built

from NB cell lines (Fig. 4) the strongest associa-

tion with the signature genes in terms of cover-

age and multiplicity, 7q gain, is linked to lower

expression of the spindle assembly genes. Gain

of 7q is not an identified predictor of prognosis in

NB, but it is a recurring NB chromosomal imbal-

ance (Stallings et al., 2003; George et al., 2007),

and prior studies (Buckley et al., 2010) have indi-

cated a positive correlation between 7q gain and

11q loss. Nevertheless, when our informative

genes were tested in the available expression NB

datasets for lower expression in clinical annota-

tion subgroups with respect to all the other sam-

ples, no gene reached even low significance in

the 11 q loss subgroup (Fig. 3B). Therefore, from

these data we infer that coherent high expression

of spindle assembly genes is associated to high-

risk tumors and to loss of 1p in NBs.

A crucial point is then to surmise how this high

production of functionally related proteins in a

specific high-risk NB class may impact the phe-

notype. The best studied proteins of the two

identified machineries, AURKA and MAD2L1,were already demonstrated to be important NB

prognostic genes when at high expression levels.

AURKA overexpression in NBs is actually associ-

ated with high-risk and high-stage tumors, unfav-

orable histology, MNA, disease relapse and

decreased progression-free survival (Shang et al.,

2009) and offers a promising target for NB ther-

apy (Shang et al., 2009; Maris et al., 2010).

MAD2L1 overexpression has also been associated

to NB poor prognosis (Hernando et al., 2004).

This provides a clear validation of our procedures

for prioritizing genes involved in NB

pathogenesis.

From the above data it is tempting to speculate

that in NB a long range CNA-induced increased

expression of genes regulating the formation of

spindle at metaphase could produce a ‘‘gain-of-

function’’ phenotype through an unknown mech-

anism, that enables NB cells to cope with produc-

tive mitosis in an otherwise challenging genomic

landscape. Our small sampling of mitotic pheno-

types in NB-derived cell lines showed aneu-

ploidy-producing missegregation only in a cell

line with 11q loss, whereas mitoses with low fre-

quency of missegregation were consistently char-

acterized in two independent 1p loss and MNA

cell lines (Fig. 5). We speculate that in a specific

high-risk NB type coordinated enhanced expres-

sion of genes converging on mitotic spindle orga-

nization and function can evoke an ‘‘adaptation’’

mechanism ensuring mitotic progression, while at

the same time generating a small fraction of

defects which elude correction at each mitotic

round. In this light, the NB spindle assembly

gene signature identified here should not neces-

sarily be expected to be coupled to the induction

of heavy mitotic aberrations, but rather of subtle

defects that, in cooperation with other lesions,

554 OOI ET AL.


could confer increased malignancy to the surviv-

ing transformed cells.

The findings reported here advance our current

understanding of the elusive pathogenic mecha-

nisms underlying the aggressiveness of this

deadly disease. We propose a model in which

individually prognostic CNAs converge on the

long range high expression of a number of spin-

dle assembly genes, which could thus become a

mitotic adaptive, low aneuploidy generating ma-

chinery able to constantly promote disease pro-

gression. If verified, this model might have

profound therapeutic implications for NB.

ACKNOWLEDGMENTS

The authors are grateful to Gabriella Viero,

Toma Tebaldi, Erik Dassi and Francesca De

Michelis for comments on the manuscript.

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