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Selection of a highly invasive neuroblastoma cell population through long-term human cytomegalovirus infection

The human cytomegalovirus (HCMV) is suspected to increase tumour malignancy by infection of cancer and/or stroma cells (oncomodulation). So far, oncomodulatory mechanisms have been attributed to the presence of HCMV and direct action of its gene products on cancer cells. Here, we investigated whether the prolonged presence of HCMV can result in the irreversible selection of a cancer cell population with increased malignancy. The neuroblastoma cell line UKF-NB-4 was long-term (200 passages) infected with the HCMV strain Hi91 (UKF-NB-4Hi) before virus eradication using ganciclovir (UKF-NB-4HiGCV). Global gene expression profiling of UKF-NB-4, UKF-NB-4Hi and UKF-NB-4HiGCV cells and subsequent bioinformatic signal transduction pathway analysis revealed clear differences between UKF-NB-4 and UKF-NB-4Hi, as well as between UKF-NB-4 and UKF-NB-4HiGCV cells, but only minor differences between UKF-NB-4Hi and UKF-NB-4HiGCV cells. Investigation of the expression of a subset of five genes in different chronically HCMV-infected cell lines before and after virus eradication suggested that long-term HCMV infection reproducibly causes specific changes. Array comparative genomic hybridisation showed virtually the same genomic differences for the comparisons UKF-NB-4Hi/UKF-NB-4 and UKF-NB-4HiGCV/UKF-NB-4. UKF-NB-4Hi cells are characterised by an increased invasive potential compared with UKF-NB-4 cells. This phenotype was completely retained in UKF-NB-4HiGCV cells. Moreover, there was a substantial overlap in the signal transduction pathways that differed significantly between UKF-NB-4Hi/UKF-NB-4HiGCV and UKF-NB-4 cells and those differentially regulated between tumour tissues from neuroblastoma patients with favourable or poor outcome. In conclusion, we present the first experimental evidence that long-term HCMV infection can result in the selection of tumour cell populations with enhanced malignancy.


INTRODUCTION
Human cytomegalovirus (HCMV), a ubiquitous herpes virus, infection leads (in immunocompetent individuals usually symptomless) to a life-long persistence after primary infection. About 50 to 100% of the general adult population is infected. HCMV routinely reactivates, but this is usually controlled by the host immune response in healthy people. In immuno-compromised individuals such as recipients of organ transplants or AIDS patients, HCMV is a major pathogen. 1,2 HCMV has been suspected to have a role in cancer diseases for decades, based on seroepidemiological evidence and on the detection of viral DNA, messenger RNA (mRNA) and/or antigens in tumour tissues. 2,3 However, in contrast to viruses from different RNA virus (retrovirus, flavivirus) and DNA virus (hepadna virus, herpes virus, papova virus) families that are known to be oncogenic in humans, 4 HCMV is not considered a tumour virus because of a lack of proven transformation potential in human cells. 2 With our studies about the influence of HCMV on cancer cells, [5][6][7] we introduced the concept of oncomodulation, meaning that HCMV may infect cancer cells and/or stromal cells in established tumours and increase tumour malignancy also in the absence of transformation potential. [1][2][3]6,8,9 In the meantime, oncomodulatory effects exerted by HCMV or single cytomegalovirus proteins have been reported in vitro and in vivo by numerous groups. 2,3,[8][9][10][11] Moreover, application of sensitive (although not yet indisputably accepted) pathological methods applied by numerous independent research groups indicated the presence of HCMV and/or virus constituents in cancers from different cancer entities. [1][2][3][12][13][14][15][16][17] In glioblastomas, the presence of HCMV was correlated with higher disease stage and worse outcome. [1][2][3]12,18,19 In addition, expression of HCMV proteins appeared to promote oncogenic signalling events. 2,3,12,20,21 Neuroblastoma, a paediatric cancer entity, has been associated with increased HCMV antibody titres and HCMV immediate-early antigen (IEA) expression in a fraction of tumours. 2,3,22,23 After primary HCMV infection of different neuroblastoma cell lines, a balance is established between virus production and cell division. 6,7,24,25 Chronically HCMV-infected neuroblastoma cells show a more malignant phenotype indicated by properties such as increased invasive potential, metastasis formation in nude mice and resistance to chemotherapy. 2,6,7,24 1 So far, HCMV-induced oncomodulatory effects were attributed to the presence of HCMV and direct action of its gene products, 2,3,10,24 and therefore suspected to be reversible after virus eradication. Here, we investigated the effects of long-term HCMV strain Hi91 infection on UKF-NB-4 neuroblastoma cells. Long-term HCMVinfected (UKF-NB-4 Hi ) cells showed a very close relationship with ganciclovir-cured UKF-NB-4 Hi (UKF-NB-4 HiGCV ) cells at the level of gene expression and genomic copy number changes, whereas substantial differences were detected between UKF-NB-4 Hi /UKF-NB-4 HiGCV cells and parental UKF-NB-4 cells. Moreover, UKF-NB-4 HiGCV showed the same increased invasive potential as UKF-NB-4 Hi cells compared with UKF-NB-4. Bioinformatics signal transduction pathway analysis suggested a substantial overlap in pathways differentially regulated between UKF-NB-4 Hi /UKF-NB-4 HiGCV cells and UKF-NB-4 cells, as well as between tumour tissues from neuroblastoma patients with poor or favourable outcome. These data indicate that the long-term presence of HCMV can result in the irreversible selection of a cancer cell population with increased malignancy. Investigation of the expression of a subset of five genes in additional long-term HCMV-infected neuroblastoma cells and their cidofovir-or ganciclovir-cured sub-lines suggested that long-term HCMV infection of different neuroblastoma cells reproducibly results in characteristic changes.
Thereafter, UKF-NB-4 Hi cells were treated with 20 mM ganciclovir (a concentration that did not affect the viability of UKF-NB-4 or UKF-NB-4 Hi cells) for six passages until no HCMV gene expression was detected anymore (UKF-NB-4 HiGCV ; Figure 1). Further cultivation of UKF-NB-4 HiGCV cells for 10 passages did not result in recurrence of HCMV gene expression ( Figure 1). The absence of HCMV from UKF-NB-4 HiGCV cells was confirmed by virus yield assay and quantitative PCR detecting HCMV genome (Supplementary Bioinformatic signal transduction pathway analysis of global gene expression data Bioinformatic signal transduction pathway analysis, performed using the PANTHER database (www.pantherdb.org), showed that the top five differentially regulated pathways for the comparisons UKF-NB-4 Hi vs UKF-NB-4 and UKF-NB-4 HiGCV vs UKF-NB-4 were the same, although the order differed (Table 1). In total, 13 pathways were significantly differentially regulated in both comparisons (P-valueo0.05, corrected for multiple testing; Table 1), whereas only three pathways were significantly differentially regulated between UKF-NB-4 Hi and UKF-NB-4 cells but not between UKF-NB-4 HiGCV and UKF-NB-4 cells (Supplementary Table 3). Four pathways were significantly differentially regulated between UKF-NB-4 HiGCV and UKF-NB-4 cells but not between UKF-NB-4 Hi and UKF-NB-4 cells (Supplementary Table 4). No significant differentially regulated pathways were detected between UKF-NB-4 Hi and UKF-NB-4 HiGCV , confirming the high similarity of these cells at the level of gene expression.

Gross genomic differences of UKF-NB-4 Hi and UKF-NB-4 HiGCV cells compared with UKF-NB-4 cells by array comparative genomic hybridization (CGH)
Array CGH is a technology developed to detect genomic copy number variations. UKF-NB-4 cells were compared with UKF-NB-4 Hi cells or UKF-NB-4 HiGCV cells by array CGH in order to investigate whether genomic differences underlie the observed changes in gene expression. Results revealed virtually identical array CGH profiles in DNA copy number for both the comparisons UKF-NB-4 Hi vs UKF-NB-4 and UKF-NB-4 HiGCV vs UKF-NB-4 ( Figure 3). There is evidence of aneuploidy, chromosome segment gain and loss and individual loci loss/gain. Specifically, we detected aneuploidy (gain) of chromosomes 6, 13 and 20, with loss of 14, 15 and 18. Structural gains were observed at terminal 2p, the whole of 9q (with a small portion of 9p), 12q, terminal 17p and 20q. Losses are detected of chromosome 1q (and about a third of proximal 1p), parts of chromosome 3, terminal 5q, a central portion of 8q, most of terminal 9p, terminal 15q (in addition to the aforementioned aneuploidy) and 20p. Reciprocal gains and losses of p and q arms on chromosomes 9 and 20 (and possibly chromosome 12) may indicate the presence of isochromosomes. Single locus gains (gene amplifications) were apparent on chromosomes 4q and 15q, with losses on 7p, 7q, 9p, 10 (near the centromere), 15q and 16q.
UKF-NB-4, UKF-NB-4 Hi or UKF-NB-4 HiGCV adhesion to and transmigration through endothelial monolayers UKF-NB-4, UKF-NB-4 Hi and UKF-NB-4 HiGCV were investigated for cancer cell adhesion to and migration through human umbilical vein endothelial cell (HUVEC) monolayers. These events are considered to be important during metastasis formation. HCMVinfected neuroblastoma cells had been shown earlier to exhibit a stronger invasive potential than non-infected cells. 28  Previous results had shown that interaction of UKF-NB-4 cells with endothelial cells depended on a5b1 integrin binding. 28 Here, adhesion of UKF-NB-4, UKF-NB-4 Hi and UKF-NB-4 HiGCV cells to and transmigration through endothelial cell monolayers could be inhibited by an antibody blocking a5b1 integrin (Figures 4a and  b). Moreover, antibody-mediated inhibition of a5b1 integrin binding also interfered with endothelial cell monolayer disruption by UKF-NB-4 Hi or UKF-NB-4 HiGCV cells.
These findings suggest that the enhanced malignant properties of UKF-NB-4 Hi cells are at least partly retained in UKF-NB-4 HiGCV cells after and despite virus eradication.
Next, signal transduction pathways differentially regulated between UKF-NB-4 and UKF-NB-4 Hi /UKF-NB-4 HiGCV cells were analysed in the context of two gene expression data sets derived from neuroblastoma patients. The first data set (Oberthuer et al.) was derived from a study comparing gene expression signatures between N-myc-amplified neuroblastoma tissues from patients with favourable outcome (event-free survival) or poor outcome (death or relapse of disease) (www.ebi.ac.uk/arrayexpress; accession E-TABM-38; Oberfhuer et al. 29 (Table 2). In the Asgharzadeh et al. data set, 32 out of the 153 pathways were significantly differentially regulated, and 12 of the 13 pathways differentially regulated between UKF-NB-4 and UKF-NB-4 Hi /UKF-NB-4 HiGCV were also significantly differentially regulated between favourable and poor outcome patients (Table 2). Moreover, 7 of the 13 pathways (Angiogenesis, platelet-derived growth factor signalling pathway, Ras pathway, p53 pathway, p53 pathway feedback loops 2, Parkinson disease, Integrin signalling pathway) belonged to the 10 most strongly differentially Abbreviations: EGF, epidermal growth factor; PDGF, platelet-derived growth factor. a Pathways were ranked by their P-values with the lowest P-value being rank 1.

DISCUSSION
Oncomodulation is a concept postulating that HCMV may infect cancer cells and/or stromal cells in established tumours and increase tumour malignancy also in the absence or independently of transformation potential. 2,3,6 To study long-term effects of HCMV on cancer cells, chronically HCMV-infected neuroblastoma cell cultures were established. 6 To enhance the confidence that the observed effects are associated with the presence of HCMV and to receive some more information about the specificity of the HCMV-induced selection process, five genes were selected that had been shown earlier to be significantly differentially expressed in various chronically HCMV-infected neuroblastoma cell lines relative to the corresponding non-infected cell lines: DHFR (encoding for dihydrofolate reductase), ENPP2 (encoding for autotaxin), KIAA0101 (encoding for a protein that is also known as PCNA-associated factor/p15(PAF)), NDRG1 (encoding for N-myc downstream regulated 1) and TMPRSS15 (also known as PRSS7, encoding for a protein known as enterokinase or transmembrane protease, serine 15). 25 These genes were also significantly differentially regulated between UKF-NB-4 and both UKF-NB-4 Hi and UKF-NB-4 HiGCV cells (as indicated by gene expression microarray data and confirmed by real-time PCR, Supplementary Table 5  Various processes might be involved in the observed establishment of a novel, more malignant neuroblastoma cell population due to long-term HCMV infection. In fully permissive normal cells such as human fibroblasts, HCMV results in cell cycle arrest and subsequent cell lysis. 2,31 However, (at least a fraction of) chronically HCMV-infected cell cultures must escape from HCMVinduced cell cycle block and death. In this context, cell cycle deregulation is regarded as 'hallmark' of cancer, 32 and HCMV may not be able to induce an effective cell cycle block in certain cancer cells. 2 HCMV antigen-positive glioblastoma cells were shown to divide, resulting in two HCMV antigen-expressing progeny cells. 33 In sarcoma or glioblastoma cells, virus variants arose that replicated more slowly and produced lower virus yields than the original strain used for primary infection. 2,34,35 Therefore, changes of virus properties may contribute to the selection of a more malignant cell fraction in our model, although HCMV strain Hi91 reisolated from UKF-NB-4 Hi cells showed virtually the same growth kinetics after acute infection in UKF-NB-4 cells and human foreskin fibroblasts as the original virus used for primary infection.
Stress conditions activate cellular survival signalling pathways, resulting in a condition thought to favour adaptation processes in malignant cells. 36 In this context, HCMV is known to exert proinflammatory effects and to activate crucial events in survival signalling, such as NFkB, PI3K-Akt, MAPKs and/or JNK, and inhibit apoptosis by effects on p53 and p73, as well as by enhancing expression of anti-apoptotic proteins such as bcl-2. 1,2,37 Moreover, HCMV is suspected to promote chromosomal instability in infected cells. 2,38 Stable expression of the HCMV UL76 protein was sufficient to induce chromosomal aberrations in glioblastoma cells. 39 Indeed, the long-term presence of HCMV had resulted in chromosomal imbalances in UKF-NB-4 Hi cells in our experiments. As HCMV is known to also modify epigenetic features such as histone acetylation of infected cells, 19,[40][41][42] epigenetic mechanisms are also likely to contribute.
Finally, cancer cells are characterised by chromosomal instability, resulting in clonal evolution, that is, the permanent appearance and disappearance of novel (sub-)clones (characterised by various mutations and/or genomic aberrations) that compete for space and resources. 32,43 Therefore, HCMV may also exert a selection pressure on clones initially present in a cancer cell population or on clones that develop in the presence, but without the involvement, of HCMV. Five metaphases from UKF-NB-4, UKF-NB-4 Hi or UKF-NB-4 HiGCV cells were investigated by spectral karyotyping. Apparently, six abnormalities are consistently found in cells from all three cell lines, suggesting the cells to be of a common origin (Supplementary Table 6). Nevertheless, the longterm presence of HCMV may exert a selection pressure that favours the growth of (a) specific cell clone(s).
In conclusion, we present evidence that long-term HCMV infection of neuroblastoma cells can lead to the selection of a novel cell population with increased malignant properties. It is the first report demonstrating that HCMV infection can irreversibly modulate the malignant properties of cancer cells in such a way that the HCMV-induced enhanced cancer cell malignancy is retained (at least in part) even when the virus is eradicated from the cancer cells. This indicates that HCMV can act as a selection agent in cancer cells. Therefore, our findings represent a profound extension to the concept of oncomodulation 2,3,6 and a novel way by which viruses may influence cancer diseases.

Immunostaining for HCMV antigen expression
As described previously, 44 cells producing HCMV-specific antigens were detected by immunoperoxidase staining using monoclonal antibodies directed against the UL123-coded 72-kDa IEA1 (DuPont, Bad Homburg, Germany) or late antigen gB (kindly provided by K Radsak, Institut fü r Virologie, Marburg, Germany).
Virus propagation HCMV Strain Hi91 was isolated from the urine of an AIDS patient with HCMV retinitis. 27 Virus stocks were prepared in human foreskin fibroblasts maintained in minimal essential medium with 4% foetal calf serum. The titres were determined by plaque titration as described previously. 44 Virus yield assay The amount of infectious virus was determined by virus yield assay in a single-cycle assay format using human foreskin fibroblasts as described before. 44 Virus titres were expressed as 50% of tissue culture infectious dose (TCID 50 ) 120 h post infection.  26 Cells were grown at 37 1C in Iscove's modified Dulbecco's medium supplemented with 10% heat-inactivated foetal calf serum and containing 100 IU/ml of penicillin and 100 mg/ml streptomycin. HUVECs were cultivated as described before. 44 Global gene expression analysis Triplicates of UKF-NB-4, UKF-NB-4 Hi and UKF-NB-4 HiGCV cells were analysed for global cellular gene expression at the mRNA level using GeneChip HGU133 Plus 2.0 (Affymetrix) by the Fraunhofer Institut fü r Zelltherapie und Immunologie (Leipzig, Germany). mRNA was isolated using the RNeasy kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. Expression data were processed using the R/bioconductor packages 'gcrma' and 'limma' (www.r-project.org/; www.bioconductor.org/) in order to detect fold changes and FDRs. 45 FDRs were corrected for multiple testing after Benjamini and Hochberg. 46 Hierarchical clustering analysis was performed and heatmaps were visualised using R (www.rproject.org).

Signal transduction pathway bioinformatics
Statistical analysis to identify significant expression changes was focusing on a pathway analysis using the PANTHER database, which identifies global patterns in expression. 47 For each expert-curated pathway in the database, potential differential expression was determined by a binomial test 48 with subsequent Bonferroni correction for multiple testing, using the PANTHER human gene reference list matching our microarrays and lists of differentially expressed genes that passed a FDR threshold of 0.05.

Array CGH
DNA was isolated from UKF-NB-4, UKF-NB-4 Hi and UKF-NB-4 HiGCV cell lines using the DNAeasy Blood & Tissue kit (Qiagen) following the manufacturer's instructions. Using DNA from cell line UKF-NB-4 as reference cell lines, UKF-NB-4 Hi and UKF-NB-4 HiGCV were profiled for DNA copy number changes by applying the Nimblegen oligonucleotide microarray platform. Hybridisations on Nimblegen HG18_WG_CGH_v2D_HX1 microarrays and microarray data processing were carried out at Source BioScience imaGenes (Berlin, Germany). The chips used contain 2.5 mio oligonucleotides tiling the human genome at an average probe spacing of 1.2 kb. Microarray images were processed in the NimbleScan 2 software (Roche NimbleGen, Madison, WI, USA).
Quantitative real-time PCR Total RNA was isolated from cell cultures using TRI reagent (Sigma-Aldrich). Quantitative real-time reverse transcriptase PCR (qRT-PCR) for viral mRNA was performed as described previously. 44 Cellular mRNA was detected using TaqMan Gene Expression Assays (Applied Biosystems, Darmstadt, Germany).
DNA was purified using the DNeasy Blood & Tissue Kit (Qiagen). Viral DNA quantification was carried out by real-time PCR as described before. 44