Enrichment of neural-related genes in human mesenchymal stem cells from neuroblastoma patients

  • Authors:
    • Miguel Ángel Rodriguez-Milla
    • Isabel Mirones
    • Luis Mariñas-Pardo
    • Gustavo J. Melen
    • Isabel Cubillo
    • Manuel Ramírez
    • Javier García-Castro
  • View Affiliations

  • Published online on: May 22, 2012     https://doi.org/10.3892/ijmm.2012.1008
  • Pages: 365-373
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Abstract

Neuroblastoma (NB) is one of the most common pediatric solid tumors and, like most human cancers, is characterized by a broad variety of genomic alterations. Although mesenchymal stem cells (MSCs) are known to interact with cancer cells, the relationship between MSCs and metastatic NB cancer cells in bone marrow (BM) is unknown. To obtain genetic evidence about this interaction, we isolated ΒΜ-derived MSCs from children with NB and compared their global expression patterns with MSCs obtained from normal pediatric donors, using the Agilent 44K microarrays. Significance analysis of microarray results with a false discovery rate (FDR) <5% identified 496 differentially expressed genes showing either a 2-fold upregulation or downregulation between both groups of samples. Comparison of gene ontology categories of differentially expressed genes revealed the upregulation of genes categorized as ‘neurological system process’, ‘cell adhesion’, ‘apoptosis’, ‘cell surface receptor linked signal transduction’, ‘intrinsic to membrane’ and ‘extracellular region’. Among the downregulated genes, several immunology-related terms were the most abundant. These findings provide preliminary genetic evidence of the interaction between MSCs and NB cancer cells in ΒΜ as well as identify relevant biological processes potentially altered in MSCs in response to NB.

Introduction

Neuroblastoma (NB), a poorly differentiated tumor derived from neural crest cells that affects mainly children, is the most common extracranial pediatric solid tumor. The origin of stroma in primary NB tumors, formed by Schwann cells, and whether the stroma is the cause or consequence of the maturation potential of tumor cells remain controversial. It has been hypothesized that crosstalk between Schwann cells and neuroblasts influences the biology and clinical behavior of NB tumors. However, little is known about the role of the NB microenvironment in metastasis localizations, especially in bone marrow (BM). Recent articles suggest that mesenchymal stem cells (MSCs) have a major role in maintaining stem cells niches in the BM (1), as well as in creating the tumor microenvironment (2). In this study we focused in BM-derived MSC from NB patients as a key factor in the development of metastasis.

During the past years several studies have used microarray-based high-throughput technologies to identify biological processes altered in NB cells. Hiyama et al (3) surveyed the differences in gene expression between unfavorable and maturing/regressing NB. Interestingly, in favorable NB, neuronal differentiation signals such as CD44, IGF2, NTRK1 and ANK1 were overexpressed in maturing tumors. Similarly, Kamei et al (4) identified genes that exhibited altered gene expression in NB tumors associated with a favorable outcome. More recently, Chen et al (5) performed parallel global protein and mRNA expression profiling on NB tumors and identified that cell adhesion, nervous system development and cell differentiation processes were downregulated in stage 4 MYCN-amplified NB tumors, suggesting a less mature neural and a more invasive phenotype of this type of cells. Finally, Abel et al (6), based on expression profiling, identified 4 molecular subgroups of NB that can be distinguished by a 6-gene signature. These studies show that microarray techniques are useful tools for gene expression profiling in NB tumors.

It is well known that the BM is a common site for metastasis in patients with high-risk NB. Although little is known about the control of NB tumor growth by the BM microenvironment, there has been an increasing interest in the role of MSCs and their BM niches in cancer (7,8). Some reports implicate MSCs having tumor-promoting effects whereas others show inhibition of tumor growth. Regarding the relationship between MSCs and NB, Ma et al (9) reported that MSCs in BM may enhance metastasis of NB via SDF-1/CXCR4 and SDF-1/CXCR7 signaling. Moreover, CXCR5 may be involved in the attraction of human metastatic NB cells to the BM (10). Despite these findings, the relationship between MSCs and NB cancer cells is still unknown, and research in this area would add new scientific knowledge and provides new therapeutic ideas and targets. Thus, we isolated MSCs from BM of NB patients and control donors and compared their global expression patterns using microarrays. Our findings provide preliminary genetic evidence of the interaction between MSCs and NB cancer cells in BM as well as identify relevant biological processes potentially altered in MSCs in response to NB.

Materials and methods

Mesenchymal stem cell isolation, culture and characterization

MSCs were isolated from 4 NB pediatric patients (with no amplification of the N-Myc gene) and 4 healthy donors. The study protocol was approved by the Ethics Committee of the Hospital. All patients and volunteers were informed about the purpose of the study and provided written consent, by the parents or legal guardians, regarding their participation in the study. BM-derived MSCs were obtained by adherence to plastic. Mononucleated cells were obtained after centrifugation using a Ficoll-Paque gradient. Cells were cultured at 37°C with 5% CO2 in DMEM (Lonza) supplemented with 10% fetal bovine serum (PAN-Biotech GmbH). MSCs cultures were characterized according the International Society for Cellular Therapy criteria (11).

RNA isolation and cRNA labeling

MSCs were stabilized in PrepProtect™ (Miltenyi Biotec) and total-RNA was isolated using standard RNA extraction protocols (NucleoSpin® RNA II, Macherey-Nagel). RNA integrity and overall quality was checked via the Agilent 2100 Bioanalyzer expert software (Agilent Technologies). All RNA samples revealed an RNA Integrity Number (RIN) between 7.3 and 10. For the linear T7-based amplification step, 1 μg of each total-RNA sample was used. To produce Cy3-labeled cRNA, the RNA samples were amplified and labeled using the Agilent Low RNA Input Linear Amp kit (Agilent Technologies) following the manufacturer’s protocol. Yields of cRNA and the dye-incorporation rate were measured with the ND-1000 Spectrophotometer (NanoDrop Technologies).

Microarray hybridization

The hybridization procedure was performed according to the Agilent 60-mer oligo microarray processing protocol using the Agilent Gene Expression Hybridization kit (Agilent Technologies). Briefly, 1.65 μg Cy3-labeled fragmented cRNA in hybridization buffer was hybridized overnight (17 h, 65°C) to Agilent Whole Human Genome Oligo Microarrays 4×44K using Agilent’s recommended hybridization chamber and oven. Finally, the microarrays were washed once with 6X SSPE buffer containing 0.005% N-lauroylsarcosine for 1 min at room temperature followed by a second wash with preheated 0.06X SSPE buffer (37°C) containing 0.005% N-lauroylsarcosine for 1 min. The last washing step was performed with acetonitrile for 30 sec. Fluorescence signals of the hybridized Agilent microarrays were detected using Agilent’s Microarray Scanner System (Agilent Technologies). The Agilent Feature Extraction Software (FES) was used to read out and process the microarray image files.

Microarray data analysis

The microarray raw data have been deposited at the NCBI Gene Expression Omnibus under the accession number GSE35133 (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?token=rdszfaigqkmgqdw&acc=GSE35133). Intensity values of flagged spots below background were filtered out, data were normalized using the array median and the mean values of 3 replicates for each biological sample were calculated. Statistical analysis of microarray significance (SAM) was performed to identify genes with significant changes in expression, and permutations were used to estimate the false discovery rate (FDR) (12). Genes were selected if they showed a 2-fold regulation and a FDR <0.05%. These statistical analyses were performed using TMEV and default parameters were used unless specified (13).

Gene ontology (GO) analysis

To better characterize the functionally-related genes which showed at least a 2-fold regulation in the microarray analysis, the genes were assigned to Gene Ontologies using a database for annotation, visualization and integrated discovery (DAVID) (14). We used the 3 ontologies produced by the GO consortium, namely ‘biological process’, ‘cellular component’ and ‘molecular function’. GO terms were collected, redundant terms were excluded, and P-values were used to evaluate the significance of the terms.

Results

We isolated MSCs from NB patients (NB-MSCs) as well as from normal donors. All MSC cultures were characterized according to the International Society for Cellular Therapy criteria: morphology, positive/negative markers and differentiation properties (data not shown) (11). We analyzed the expression profiles of RNA from NB-MSCs compared to those from normal donors, using the Agilent Whole Human Genome Oligo Microarrays. The results from the SAM analysis (≥2-fold regulation and FDR <0.05%) revealed that 454 genes had transcript levels significantly higher in NB-MSCs, whereas 42 genes had transcript levels significantly lower in NB-MSCs (Fig. 1). A list of the genes showing the strongest upregulation in NB-MSCs is shown in Table I whereas the downregulated genes are shown in Table II.

Table I

List of upregulated genes in hMSCs from NB patients.

Table I

List of upregulated genes in hMSCs from NB patients.

Gene symbolGene descriptionGenBank accession no.Fold-changeQ-value
GPR128G protein-coupled receptor 128NM_03278744.2694.5173
TMEFF2Transmembrane protein with EGF-like and two follistatin-like domains 2NM_01619218.6550.7810
ANXA10Annexin A10NM_00719313.7001.2968
ITGA2Integrin, α 2 (CD49B, α 2 subunit of VLA-2 receptor)NM_00220311.4350.0000
A_32_P208076UnknownA_32_P20807610.7070.0000
A_24_P365349UnknownA_24_P3653499.6641.6333
THC2402993UnknownTHC24029939.0110.0000
NEF3Neurofilament 3 (150 kDa medium)NM_0053828.4681.0034
IGFBP1Insulin-like growth factor binding protein 1, transcript variant 1NM_0005968.2554.6632
WDR69WD repeat domain 69NM_1788218.1323.9950
C1orf110Chromosome 1 open reading frame 110BC0400187.9583.6213
TREM1Triggering receptor expressed on myeloid cells 1NM_0186437.9314.5173
FLJ37228cDNA FLJ37228 fis, clone BRAMY2000411AK0945477.1770.0000
KCTD4Potassium channel tetramerisation domain containing 4NM_1984047.1624.2453
CNIH3Cornichon homolog 3NM_1524956.9843.9107
ENST00000379108Unknown ENST000003791086.8823.3557
CALB2Calbindin 2, 29 kDa (calretinin)NM_0017406.5034.3242
COL4A5Collagen, type IV, α 5, transcript variant 2NM_0333806.4382.1503
SLC6A15Solute carrier family 6, member 15, transcript variant 1NM_1827676.2340.9502
TSPAN8Tetraspanin 8NM_0046165.9551.4171
SLC7A14mRNA for KIAA1613 proteinAB0468335.8890.0000
THC2438492UnknownTHC24384925.5880.0000
ENST00000222543Similar to tissue factor pathway inhibitor 2 precursor (TFPI-2) ENST000002225435.5773.2311
PSG7Pregnancy specific β-1-glycoprotein 7NM_0027835.5532.6056
DCBLD2Discoidin, CUB and LCCL domain containing 2NM_0809275.5470.7810
HTR1F5-hydroxytryptamine (serotonin) receptor 1FNM_0008665.5391.6929
SLC7A14Solute carrier family 7, member 14NM_0209495.4241.2573
RNF128Ring finger protein 128, transcript variant 1NM_1944635.3874.2453
SHC3SHC (Src homology 2 domain containing) transforming protein 3NM_0168485.3292.0866
HLA-DR BHLA class II DR-βX125445.3133.5143
SAMD3Sterile α motif domain containing 3, transcript variant 2NM_1525525.2540.0000
SEMA3ESemaphorin 3ENM_0124315.2272.0866
LOC284344Similar to biliary glycoprotein 1 precursorAK0976725.1974.8517
PSG4Pregnancy specific β-1-glycoprotein 4, transcript variant 2NM_2136335.1641.2496
RGS4Regulator of G-protein signaling 4NM_0056135.1511.4010
AREGAmphiregulin (schwannoma-derived growth factor)NM_0016575.0434.0968
DNERDelta-notch-like EGF repeat-containing transmembraneNM_1390724.9432.4280
AK094786cDNA FLJ37467 fis, clone BRAWH2011920AK0947864.7871.2384
HGDHomogentisate 1,2-dioxygenase (homogentisate oxidase)NM_0001874.7841.8692
RGS18Regulator of G-protein signaling 18NM_1307824.7524.6632
SAMD3Sterile α motif domain containing 3, transcript variant 1NM_0010173734.7491.1850
SLC24A3Solute carrier family 24, member 3NM_0206894.7363.4520
F2RL1Coagulation factor II (thrombin) receptor-like 1NM_0052424.6832.6953
CST1Cystatin SNNM_0018984.6251.6333
TMEM158Transmembrane protein 158NM_0154444.6183.4313
THC2455389ORF2280 gene homologTHC24553894.6083.1260
AK127194cDNA FLJ45259 fis, clone BRHIP2020695AK1271944.5883.1367
PSCDBPPleckstrin, Sec7 and coiled-coil domains, binding proteinNM_0042884.5392.4280
CLGNCalmeginNM_0043624.5333.7978
RP11-138L21.1Similar to contactin associated protein (Caspr)AK0546454.4362.3612
BCANBrevicanBC0050814.3700.0000
NPTX1Neuronal pentraxin INM_0025224.2902.8954
HLA-DRB5Major histocompatibility complex, class II, DR β5NM_0021254.2843.3557
SULT4A1Sulfotransferase family 4A, member 1NM_0143514.2462.5183
THC2335868ALU5_HUMAN (P39192) Alu subfamily SCTHC23358684.2310.0000
FATE1Fetal and adult testis expressed 1NM_0330854.1964.3477

Table II

List of downregulated genes in hMSCs from NB patients.

Table II

List of downregulated genes in hMSCs from NB patients.

Gene symbolGene descriptionGenBank accession no.Fold-changeQ-value
IFI27Interferon, α-inducible protein 27NM_0055320.04874.052
CRIP1Cysteine-rich protein 1 (intestinal)NM_0013110.09944.324
CCL8Chemokine (C-C motif) ligand 8NM_0056230.10213.438
LSP1Lymphocyte-specific protein 1, transcript variant 3NM_0010132540.11881.462
CDCA7Cell division cycle associated 7, transcript variant 1NM_0319420.15090.000
ENST00000372045cDNA clone CS0DI016YJ18 (CR623913) ENST000003720450.17572.385
IL21RInterleukin 21 receptor, transcript variant 2NM_1810780.19754.052
C2Complement component 2NM_0000630.20054.448
MT1JPMTBAF3489940.20144.517
IFITM1Interferon induced transmembrane protein 1 (927)NM_0036410.20493.995
ENST00000313624cDNA clone DKFZp667P0410 (AL831953) ENST000003136240.21183.438
FAM70AFamily with sequence similarity 70, member ANM_0179380.22312.385
ISG20Interferon stimulated exonuclease gene 20 kDaNM_0022010.24192.385
DKFZP761M1511cDNA FLJ39342 fis, clone OCBBF2018873AK0966610.24593.995
MBOAT1cDNA FLJ16207 fis, clone CTONG2019822AK1312690.24750.000
JPH2Junctophilin 2, transcript variant 1NM_0204330.25241.448
C1RComplement component 1, r subcomponentNM_0017330.26394.724
ENPP2Ectonucleotide pyrophosphatase/phosphodiesterase 2 (autotaxin), transcript variant 1NM_0062090.27891.462
TMEM119Transmembrane protein 119NM_1817240.28262.385
E2F2E2F transcription factor 2NM_0040910.28632.385
OLFML2BOlfactomedin-like 2BNM_0154410.28792.854
EXO1Exonuclease 1, transcript variant 3NM_0036860.28812.487
CCNE2Cyclin E2, transcript variant 1NM_0577490.29511.448
C12orf46Chromosome 12 open reading frame 46NM_1523210.30282.385
ECGF1Endothelial cell growth factor 1 (platelet-derived)NM_0019530.30671.462
SLC2A12Solute carrier family 2 (facilitated glucose transporter), member 12NM_1451760.33214.724
NAV2Steerin3 protein, alternative exon 1bAJ4882020.33873.145
A_24_P927205UnknownA_24_P9272050.34804.052
IL7Interleukin 7NM_0008800.38114.463
ENST00000270031Unknown ENST000002700310.39654.448
GBP1Guanylate binding protein 1, interferon-inducible, 67 kDaNM_0020530.40152.385
RAB42RAS oncogene familyNM_1523040.42144.517
HELLSHelicase, lymphoid-specificNM_0180630.42404.448
BARD1BRCA1 associated RING domain 1NM_0004650.42443.699
RAD51AP1RAD51 associated protein 1NM_0064790.42562.392
FLJ39660cDNA clone DKFZp434P055AL8345370.42922.576
HIRAHistone cell cycle regulation defective homolog ANM_0033250.43274.448
THC2376015UnknownTHC23760150.43854.168
POLE2Polymerase (DNA directed), ɛ 2 (p59 subunit)NM_0026920.46232.854
GBP2Guanylate binding protein 2, interferon-inducibleNM_0041200.47084.183
ATAD2ATPase family, AAA domain containing 2NM_0141090.48384.069

To illustrate the differences between NB and normal samples the genes whose expression was induced or repressed by at least 2.5-fold in NB samples are shown as heat map in the Fig. 2, where NB and normal samples are clearly differentiated. Interestingly, several of the genes are known to play roles in NB (ANXA10, ITGA2, COL4A5 and SHC3) or other types of cancer (TMEFF2, TSPAN8, DCBLD2, PSCDBP and BCAN) (Table I). Table V lists the genes involved in neuronal processes that were >2.0-fold upregulated.

Table V

List of neuronal-related genes upregulated in hMSCs from NB patients.

Table V

List of neuronal-related genes upregulated in hMSCs from NB patients.

Gene symbolGene descriptionGenBank accession no.Fold changeQ-value
ITGA2Integrin, α 2 (CD49B, α 2 subunit of VLA-2 receptor)NM_00220311.4350.0000
HTR1F5-hydroxytryptamine (serotonin) receptor 1FNM_0008665.5391.6929
SHC3SHC (Src homology 2 domain containing) transforming protein 3NM_0168485.3292.0866
NPTX1Neuronal pentraxin INM_0025224.2902.8954
DFNB31Autosomal recessive deafness type 31 protein 2AK0561903.4870.0000
SYT1Synaptotagmin INM_0056393.4524.6886
RTP3Receptor transporter protein 3NM_0314403.2822.3923
OXTROxytocin receptorNM_0009163.2743.5862
SNCASynuclein, α (non A4 component of amyloid precursor)NM_0073082.9802.8954
ESPNEspinNM_0314752.5181.5910
CRHCorticotropin releasing hormoneNM_0007562.4963.3557
CLN8 Ceroid-lipofuscinosis, neuronal 8 (epilepsy, progressive with mental retardation)NM_0189412.4791.0034
COCHCoagulation factor C homolog, cochlinNM_0040862.4203.6986
DMDDystrophin (muscular dystrophy, Duchenne and Becker types)NM_0040102.4084.0521
DTNADystrobrevin, α, transcript variant 1NM_0013902.3302.4868
HAP1 Huntingtin-associated protein 1 (neuroan 1), transcript variant 1NM_0039492.3112.1529
OR12D3Olfactory receptor, family 12, subfamily D, member 3NM_0309592.3011.0207
OR5AK2Olfactory receptor, family 5, subfamily AK, member 2NM_0010053232.2930.7810
TCF15Transcription factor 15 (basic helix-loop-helix)NM_0046092.1512.5183
PRKCGProtein kinase C, γNM_0027392.1383.4520
PRKCAProtein kinase C, αNM_0027372.1321.3963
OR4F4Olfactory receptor, family 4, subfamily F, member 4NM_0010041952.1041.6012
OPN1SWOpsin 1 (cone pigments), short-wave-sensitive (color blindness, tritan)NM_0017082.0691.1850
UTS2Urotensin 2, transcript variant 1NM_0219952.0671.3372
OR10J5Olfactory receptor, family 10, subfamily J, member 5NM_0010044692.0521.0034
USH1CcDNA: FLJ21290 fis, clone COL01954AK0249432.0111.0034
SLC6A4Solute carrier family 6 (neurotransmitter transporter, serotonin), member 4NM_0010452.0014.5173

To further examine the differences in the expression profiles between NB-MSCs and normal donors, the 496 significantly up or downregulated genes were analyzed with the DAVID software and classified into the 3 main GO domains. For the upregulated genes, in the gene ontology ‘biological process’ we identified 138 terms, in ‘cellular component’ 23 terms, and in ‘molecular function’ 17 terms. The highest ranked terms are shown in Table III. For the downregulated genes, in the gene ontology ‘biological process’ we identified 24 terms, in ‘cellular component’ 1 term, and in ‘molecular function’ 9 terms. The highest ranked terms are shown in Table IV.

Table III

Main GO terms of the different GO categories enriched in upregulated genes in hMSCs from NB patients.

Table III

Main GO terms of the different GO categories enriched in upregulated genes in hMSCs from NB patients.

GO termCountP-valueGenes
Biological process
  GO:0007166. Cell surface receptor linked signal transduction330.09GPR128, CDK5R1, TACR3, EDN1, F2RL1, PTPN22, OXTR, OPN1SW, LGR6, OR4F4, DNER, TGM2, HHIP, SHC3, HTR1F, PTPRC, RET, VAV3, GPR135, ITGA2, RGS18, GPR132, OR10J5, CCND1, GPR34, OR12D3, OR5AK2, HIPK2, GPR56, CRH, STC1, IGFBP1, AREG
  GO:0050877. Neurological system process270.01RTP3, SYT1, UTS2, SNCA, SLC6A4, OXTR, OPN1SW, OR4F4, ESPN, DFNB31, NPTX1, DMD, SHC3, HAP1, HTR1F, DTNA, PRKCA, COCH, ITGA2, OR10J5, PRKCG, OR12D3, OR5AK2, CRH, USH1C, CLN8, TCF15
  GO:0007242. Intracellular signaling cascade250.05DCBLD2, EDN1, OXTR, TLR7, TGM2, SHC3, KNDC1, HTR1F, PRKCA, PTPRC, RET, VAV3, PRKCG, RGNEF, CCND1, UACA, CNIH3, RGS4, C7ORF16, HIPK2, ASB1, TREM1, ABL1, GRB14, DUSP6
  GO:0042981. Regulation of apoptosis210.01PRKCA, PTPRC, IER3, CDK5R1, VAV3, SNCA, SOX9, GDNF, ACVR1C, CDKN2A, UACA, TNFRSF10D, HIPK2, CRH, TGM2, BIK, CTSB, ABL1, CLN8, PHLDA1, ANGPTL4
  GO:0007155. Cell adhesion170.03DCBLD2, PTPRC, OLFM4, CDK5R1, RET, PCDH10, BCAN, ITGA2, MGP, CDH2, PCDH7, SOX9, BTBD9, GPR56, CNTNAP3, ABL1, CDH10
  GO:0010605. Negative regulation of macromolecule metabolic process170.04PRKCA, PTPRC, SNCA, EDN1, PRKCG, SOX9, NR0B1, PKIA, PROX1, CDKN2A, HIPK2, RNF128, HES2, ASB1, PRDM1, CLN8, C1D
  GO:0007610. Behavior130.02PRKCA, SNCA, OXTR, PRKCG, GDNF, ESPN, C7ORF16, HIPK2, CRH, SERPIND1, SHC3, CLN8, TCF15
  GO:0006928. Cell motion120.06PRKCA, KLF7, CDK5R1, RET, VAV3, ANK3, DNER, PRSS3, ITGA2, CDH2, GDNF, TNP2
  GO:0060341. Regulation of cellular localization110.01PRKCA, SYT1, CDKN2A, UACA, SNCA, EDN1, CRH, OXTR, PKIA, GDNF, ACVR1C
  GO:0048584. Positive regulation of response to stimulus100.01PRKCA, PTPRC, HIPK2, F2RL1, CRH, TGM2, PTPN22, ITGA2, PRKCG, TLR7
Cellular component
  GO:0031224. Intrinsic to membrane980.04GPR128, SYT1, SLC9A7, KLRC2, SLC6A4, F2RL1, TSPAN8, TLR7, PAPPA, CREB3L3, HTR1F, TMEFF2, LRRC3, RET, GPR135, PLXNB3, TMEM132B, GPR132, CYP2E1, PTPRO, HLA-DQA1, SLITRK1, GPR56, NEU3, RTP3, DCBLD2, OPN1SW, RIC3, HLA-DRB5, MFAP3L, CACNA2D1, MLC1, PCDH10, ITGA2, SLC6A15, NCKAP1L, SLAMF8, REEP2, KCTD4, P2RX5, GPR34, OR12D3, CLGN, OR5AK2, CNIH3, AREG, CD200, SLC45A3, KCNJ16, SLC16A14, IER3, SLC5A4, MSR1, TACR3, SLC20A1, DPP10, BCAN, CDCP1, LGR6, ACVR1C, ST6GALNAC5, SLC24A3, CNTNAP3, HHIP, CEACAM3, PPAP2C, MMP16, OR10J5, PCDH7, SLC7A14, PSG8, PSG7, TNFRSF10D, PSG4, TREM1, CLN8, GPAM, KCNH5, TMCO2, FATE1, MFSD4, TMEM158, OXTR, CDH2, C12ORF53, OR4F4, LOC151162, DNER, TMEM35, RNF128, HS6ST3, PTPRC, KIAA1244, TMPRSS9, TMEM51, GDPD1, CYBB, SLC17A3, BIK, CDH10
  GO:0005576. Extracellular region54 6×10−5UTS2, MSR1, EDN1, BCAN, CDCP1, GDNF, PSG11, SERPINA9, PAPPA, SEMA3E, SERPINE1, CNTNAP3, HHIP, ANGPT2, TFPI2, PRSS35, COCH, TMEFF2, SCUBE3, CA11, CRISP1, CST2, MGP, PSG3, CST1, MMP16, PSG1, C17ORF69, MMP12, PSG9, PSG8, PSG7, PSG4, TFPI, STC1, CTSB, TREM1, GPHA2, CA2, OLFM4, DEFB126, MIA2, DEFB114, PRSS2, PRSS3, ANGPTL4, COL4A5, TSLP, IGSF21, UACA, MCFD2, CRH, AREG, IGFBP1, SERPIND1, TLL2
  GO:0042995. Cell projection180.04SYT1, CDK5R1, FBXO2, SNCA, OXTR, ITGA2, PRKCG, CDH2, OPN1SW, ESPN, DFNB31, PPP1R9A, GPR34, ANK3, DNER, DRP2, USH1C, CA2
Molecular function
  GO:0005509. Calcium ion binding32 6×10−7SYT1, CDK5R1, SNCA, CDH2, CALB2, NPTX1, SLC24A3, PRSS2, DNER, DMD, PRSS3, TGM2, DTNA, PRKCA, CACNA2D1, RET, SCUBE3, PCDH10, MGP, ITGA2, PRKCG, MMP16, PCDH7, PADI1, MMP12, CLGN, ANXA10, EFHB, DRP2, MCFD2, TLL2, CDH10
  GO:0004857. Enzyme inhibitor activity14 5×10−5SNCA, CST2, CST1, PKIA, SERPINA9, CDKN2A, PPP1R1C, C7ORF16, SERPINE1, TFPI, SERPINB4, SERPIND1, TFPI2, ANGPTL4

Table IV

Main GO terms of the different GO categories enriched in downregulated genes in hMSCs from NB patients.

Table IV

Main GO terms of the different GO categories enriched in downregulated genes in hMSCs from NB patients.

GO termCountP-valueGenes
Biological process
GO:0006955. Immune response90.00003EXO1, IL7, ENPP2, CCL8, RSAD2, C1R, C2, GBP2, GBP1
GO:0006259. DNA metabolic process70.003EXO1, CCNE2, RAD51AP1, POLE2, HELLS, ISG20, BARD1
GO:0006952. Defense response50.02LSP1, CCL8, RSAD2, C1R, C2
GO:0007049. Cell cycle50.06EXO1, CCNE2, E2F2, HELLS, BARD1
GO:0006959. Humoral immune response40.0004EXO1, IL7, C1R, C2
GO:0002252. Immune effector process40.002EXO1, RSAD2, C1R, C2
GO:0046649. Lymphocyte activation40.01EXO1, IL7, IL21R, HELLS
GO:0006281. DNA repair40.01EXO1, RAD51AP1, POLE2, BARD1

Discussion

The role of MSCs in tumor progression is unclear as it has been suggested that MSCs may promote or suppress tumor growth (8). Therefore, identifying potential genes regulated in BM-derived MSCs by NB cancer cells would be of great importance to assess their role in determining disease outcome. Previously, Hahn et al (15) studied the effect of conditioned medium of BM cultures in NB cell growth in vitro (15). They showed that BM cultures may stimulate the proliferation and differentiation suppression of NB cells. In this model, monocytes seem to be the mediators of these effects. However, there is no data on how NB cells modify the characteristics of BM-resident cell populations. In this study, we analyzed for the first time the expression profiles of BM-derived MSCs from NB patients and report the identification of 496 genes with more than a 2-fold increase or decrease transcript levels. Our findings suggest that NB cancer cells may have an impact on several processes of MSCs localized in BM.

Interestingly, our microarray analysis revealed that some of the top ranked upregulated genes in NB-MSCs (ANXA10, ITGA2, COL4A5 and SHC3) have been previously reported to have a potential role in NB (Table I). For instance, Annexin A10 (ANXA10) has been identified in a microarray analysis of human NB stem cells as a gene associated with malignancy (16). Similarly, integrin upregulation has been reported as a marker of NB cell differentiation (17). The same study identified the overexpression of COL4A5 in unfavorable NB. Finally, a distinct role of ShcC (SHC3) docking protein in the differentiation of NB has been proposed (18). In addition, we identified several genes reported in other studies to be regulated in different types of cancer: TMEFF2 (19,20), TSPAN8 (21,22), DCBLD2 (23,24). Taken together, these observations suggest that the interaction between MSCs and NB cancer cells in the BM microenvironment induces changes in the expression of cancer-related genes in the MSCs. An unlikely explanation would be the cellular fusion of NB cells and MSCs, in a similar manner to Rizvanov et al (25) who observed rare in vitro cell fusion in co-cultures of NB tumor cells and MSCs.

The GO functional classification analysis through DAVID showed a number of mainly affected categories further suggesting that NB-MSCs are altered (Table III). Overall, we noted that in the ‘cellular component’ domain, NB upregulated a large number of genes encoding proteins ‘intrinsic to membrane’ (n=98) and localized to the ‘extracellular region’ (n=54), suggesting that NB cancer cells may exert a large repertoire of changes in these MSCs compartments. It is likely that functional relationships between NB and MSCs are mostly mediated through these proteins. Analysis of the category ‘biological process’ revealed effects on MSCs in terms previously described in NB cancer cells in the literature. Most importantly, we remark on the upregulation of genes in the term ‘neurological system processes’. Despite of the wide variety of proteins encoded by these genes, they may provide insights into potential neurological functions altered in NB-MSCs. Interestingly, regulation of neural-related genes has also been shown in previous microarray analysis of NB tumors. Thus, Hiyama et al (3) reported that in favorable NB neuronal differentiation signals were overexpressed in maturing tumors whereas Chen et al (5) found that in NB tumors of stage 4+, proteins with functions in nervous system development were downregulated, suggesting a less mature neural and a more invasive phenotype of these tumors.

The absence of NB markers in our MSC cultures pants to the absence of a tumor cell contamination. Then, it is tempting to speculate whether our findings reflect that MSCs in the BM microenvironment of NB patients redirect toward neuronal lineage. Therefore, MSCs have been proposed to adopt neural cell phenotypes, although this occurs at a very low frequency (26). In this sense, it has been proposed that NB cells would induce MSCs differentiation into Schwann cells (27). However in our data we did not observe an increase of classical Schwann-markers such as S100, Egr-1 or Egr-2 in NB-MSCs. On the other hand, MSCs would suffer a dedifferentiation process since a neuroectodernal origin of fetal MSCs localised in BM has been proposed (28).

The term ‘cell surface receptor linked signal transduction’ included several members of the G protein-coupled receptor family (GPR128, GPR135, GPR132, GPR56 and GPR34). Indeed, GPR128 was the strongest upregulated gene in our microarray analysis (Table I). The G protein-coupled receptor (GPCR) superfamily has long been proposed to have vital dual roles in cellular adhesion and signaling (29). One of the best described GPCRs is GPR56. In addition to its role in neural progenitor cell migration (30), a role in suppression of tumor growth by the microenvironment have been investigated (31). By interacting with an extracellular matrix ligand, TG2 (transglutaminase 2), GPR56 seems to suppress tumor growth and metastasis in vivo; conversely, reduced expression is associated with tumor progression. In addition, it is overexpressed in many human glioblastomas and functions in tumor cell adhesion (32). SHC3, another component of this GO term, has been described in the literature. Miyake et al (18) observed a significantly higher level of ShcC protein in NBs with poor prognostic factors and indicated that the expression of ShcC potentially has a function in inhibiting the differentiation of NB cells (18).

Upregulation of ‘cell adhesion’ genes supports the hypothesis that NB-MSCs may undergo changes in their extracellular matrix and cell adhesion properties. Previous research has shown the modulation of NB cell differentiation by the extra-cellular matrix (33). In this study, the authors showed how extracellular matrix rigidity potentiates NB cell differentiation and decreases cell proliferation; and, as we mentioned above, the receptors of extracellular matrix molecules have been reported as markers of NB cell differentiation (17). Similarly, Chen et al (5) reported that suppression of cell adhesion proteins in NB tumors of stage 4+ indicates the metastatic nature of this kind of NB tumors.

In addition, increased transcript levels of a number of relevant genes involved in cell-cycle regulation or apoptosis (e.g., CCND1, CDKN2A (p14/p16), RET and GDNF) suggest that there may be alterations in these processes in NB-MSCs. Similarly, the number of upregulated genes in the ‘calcium ion binding’ category may indicate that intracellular calcium is likely involved in the response of MSCs to NB. ANXA10 belongs to the annexin super-family of closely related calcium and membrane-binding proteins, and many studies have shown their potential role in tumor development and progression (34). An alternative explanation would be based in the evidence that NB cells stimulate osteoclasts to generate osteolytic lesions and set free calcium, in which interactions of NB cells with BM-derived MSCs play a critical role (35).

Finally, most of the downregulated GO terms contained genes encoding immune-related proteins. However, our results also showed upregulation of HLA-DRB5 and HLA-DOA1, which encode MHC class II molecules. In this sense, Johann et al (36) showed that NK cell cytotoxicity was significantly impaired after co-culturing NB cells with NB-MSCs, compared with MSCs of normal donors. Further study is needed to assess the impact of NB cancer cells on the immune response of MSCs.

In summary, we present initial data of a genome-wide analysis of MSCs from NB patients. Our data suggest that the microarray approach is a useful tool to identify deregulated genes in cultured MSCs isolated from NB patients. We provide preliminary genetic evidence of the interaction between MSCs and NB cancer cells in BM. Furthermore, we identifed relevant biological processes potentially altered in MSCs in response to NB. Future studies are necessary to connect these and other differentially expressed genes into their biological roles.

Acknowledgements

This study was supported by grants from the Fondo de Investigaciones Sanitarias (FIS; PI05/2217 and PI08/0029 to J.G.C.), MICINN (PLE2009-0115) and the Madrid Regional Government (S-BIO-0204-2006 and P2010/BMD-2420) in Spain. The experiments were approved by the appropriate committees.

References

1. 

A EhningerA TrumppThe bone marrow stem cell niche grows up: mesenchymal stem cells and macrophages move inJ Exp Med208421428201110.1084/jem.2011013221402747

2. 

PJ MishraJW GlodD BanerjeeMesenchymal stem cells: flip side of the coinCancer Res6912551258200910.1158/0008-5472.CAN-08-356219208837

3. 

E HiyamaK HiyamaH YamaokaT SuedaCP ReynoldsT YokoyamaExpression profiling of favorable and unfavorable neuroblastomasPediatr Surg Int203338200410.1007/s00383-003-1077-314691637

4. 

N KameiK HiyamaH YamaokaEvaluation of genes identified by microarray analysis in favorable neuroblastomaPediatr Surg Int25931937200910.1007/s00383-009-2448-119701758

5. 

QR ChenYK SongLR YuGlobal genomic and proteomic analysis identifies biological pathways related to high-risk neuroblastomaJ Proteome Res9373382201010.1021/pr900701v19921788

6. 

F AbelD DaleviM NethanderA 6-gene signature identifies four molecular subgroups of neuroblastomaCancer Cell Int119201110.1186/1475-2867-11-921492432

7. 

A AbarrategiL Marinas-PardoI MironesE RinconJ Garcia-CastroMesenchymal niches of bone marrow in cancerClin Transl Oncol13611616201110.1007/s12094-011-0706-x21865132

8. 

G GrisendiR BussolariE VeronesiUnderstanding tumor-stroma interplays for targeted therapies by armed mesenchymal stromal progenitors: the MesenkillersAm J Cancer Res1787805201122016827

9. 

M MaJY YeR DengCM DeeGC ChanMesenchymal stromal cells may enhance metastasis of neuroblastoma via SDF-1/CXCR4 and SDF-1/CXCR7 signalingCancer Lett312110201110.1016/j.canlet.2011.06.02821906874

10. 

I AiroldiC CoccoF MorandiI PrigioneV PistoiaCXCR5 may be involved in the attraction of human metastatic neuroblastoma cells to the bone marrowCancer Immunol Immunother57541548200810.1007/s00262-007-0392-217786442

11. 

M DominiciK Le BlancI MuellerMinimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statementCytotherapy8315317200610.1080/14653240600855905

12. 

VG TusherR TibshiraniG ChuSignificance analysis of microarrays applied to the ionizing radiation responseProc Natl Acad Sci USA9851165121200110.1073/pnas.09106249811309499

13. 

AI SaeedV SharovJ WhiteTM4: a free, open-source system for microarray data management and analysisBiotechniques34374378200312613259

14. 

dW HuangBT ShermanQ TanDAVID Bioinformatics Resources: expanded annotation database and novel algorithms to better extract biology from large gene listsNucleic Acids Res35W169W1752007

15. 

T HahnR OrG BrucheltControl of neuroblastoma cell proliferation and differentiation by human bone marrowCancer7726142621199610.1002/(SICI)1097-0142(19960615)77:12%3C2614::AID-CNCR27%3E3.0.CO;2-U8640713

16. 

JD WaltonBA SpenglerJL BiedlerWL GeraldNKV CheungRA RossMicroarray analyses of human neuroblastoma stem cells identifies genes associated with malignancyProc Amer Assoc Cancer Res20056882005

17. 

C RozzoV ChiesaM PonzoniIntegrin upregulation as marker of neuroblastoma cell differentiation: correlation with neurite extensionCell Death Differ4713724199710.1038/sj.cdd.440030416465284

18. 

I MiyakeM OhiraA NakagawaraR SakaiDistinct role of ShcC docking protein in the differentiation of neuroblastomaOncogene28662673200910.1038/onc.2008.41318997821

19. 

S GeryCL SawyersDB AgusJW SaidHP KoefflerTMEFF2 is an androgen-regulated gene exhibiting antiproliferative effects in prostate cancer cellsOncogene2147394746200210.1038/sj.onc.120514212101412

20. 

K LinJR TaylorTD WuTMEFF2 is a PDGF-AA binding protein with methylation-associated gene silencing in multiple cancer types including gliomaPLoS One6e18608201110.1371/journal.pone.001860821559523

21. 

S GesierichC ParetD HildebrandColocalization of the tetraspanins, CO-029 and CD151, with integrins in human pancreatic adenocarcinoma: impact on cell motilityClin Cancer Res1128402852200510.1158/1078-0432.CCR-04-193515837731

22. 

M ZöllerTetraspanins: push and pull in suppressing and promoting metastasisNat Rev Cancer94055200919078974

23. 

K KoshikawaH OsadaK KozakiSignificant upregulation of a novel gene, CLCP1, in a highly metastatic lung cancer subline as well as in lung cancers in vivoOncogene2128222828200210.1038/sj.onc.120540511973641

24. 

M KimKT LeeHR JangEpigenetic downregulation and suppressive role of DCBLD2 in gastric cancer cell proliferation and invasionMol Cancer Res6222230200810.1158/1541-7786.MCR-07-014218314483

25. 

AA RizvanovME YalvaçAK ShafigullinaInteraction and self-organization of human mesenchymal stem cells and neuro-blastoma SH-SY5Y cells under co-culture conditions: A novel system for modeling cancer cell micro-environmentEur J Pharm Biopharm76253259201010.1016/j.ejpb.2010.05.012

26. 

DJ MaltmanSA HardySA PrzyborskiRole of mesenchymal stem cells in neurogenesis and nervous system repairNeurochem Int59347356201121718735

27. 

W DuN HozumiM SakamotoJ HataT YamadaReconstitution of Schwannian stroma in neuroblastomas using human bone marrow stromal cellsAm J Pathol17311531164200810.2353/ajpath.2008.07030918772334

28. 

Y TakashimaT EraK NakaoNeuroepithelial cells supply an initial transient wave of MSC differentiationCell12913771388200710.1016/j.cell.2007.04.02817604725

29. 

S YonaHH LinWO SiuS GordonM StaceyAdhesion-GPCRs: emerging roles for novel receptorsTrends Biochem Sci33491500200810.1016/j.tibs.2008.07.00518789697

30. 

T IguchiK SakataK YoshizakiK TagoN MizunoH ItohOrphan G protein-coupled receptor GPR56 regulates neural progenitor cell migration via a G alpha 12/13 and Rho pathwayJ Biol Chem2831446914478200810.1074/jbc.M70891920018378689

31. 

L XuRO HynesGPR56 and TG2: possible roles in suppression of tumor growth by the microenvironmentCell Cycle6160165200710.4161/cc.6.2.376017314516

32. 

S ShashidharG LorenteU NagavarapuGPR56 is a GPCR that is overexpressed in gliomas and functions in tumor cell adhesionOncogene2416731682200510.1038/sj.onc.120839515674329

33. 

WA LamL CaoV UmeshAJ KeungS SenS KumarExtracellular matrix rigidity modulates neuroblastoma cell differentiation and N-myc expressionMol Cancer935201010.1186/1476-4598-9-3520144241

34. 

S MussunoorGI MurrayThe role of annexins in tumor development and progressionJ Pathol216131140200810.1002/path.240018698663

35. 

Y SoharaH ShimadaC MinkinA Erdreich-EpsteinJA NoltaYA DeClerckBone marrow mesenchymal stem cells provide an alternate pathway of osteoclast activation and bone destruction by cancer cellsCancer Res6511291135200510.1158/0008-5472.CAN-04-285315734993

36. 

PD JohannM VaeglerF GiesekeTumor stromal cells derived from paediatric malignancies display MSC-like properties and impair NK cell cytotoxicityBMC Cancer10501201010.1186/1471-2407-10-50120858262

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August 2012
Volume 30 Issue 2

Print ISSN: 1107-3756
Online ISSN:1791-244X

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Spandidos Publications style
Rodriguez-Milla MÁ, Mirones I, Mariñas-Pardo L, Melen GJ, Cubillo I, Ramírez M and García-Castro J: Enrichment of neural-related genes in human mesenchymal stem cells from neuroblastoma patients. Int J Mol Med 30: 365-373, 2012
APA
Rodriguez-Milla, M.Á., Mirones, I., Mariñas-Pardo, L., Melen, G.J., Cubillo, I., Ramírez, M., & García-Castro, J. (2012). Enrichment of neural-related genes in human mesenchymal stem cells from neuroblastoma patients. International Journal of Molecular Medicine, 30, 365-373. https://doi.org/10.3892/ijmm.2012.1008
MLA
Rodriguez-Milla, M. Á., Mirones, I., Mariñas-Pardo, L., Melen, G. J., Cubillo, I., Ramírez, M., García-Castro, J."Enrichment of neural-related genes in human mesenchymal stem cells from neuroblastoma patients". International Journal of Molecular Medicine 30.2 (2012): 365-373.
Chicago
Rodriguez-Milla, M. Á., Mirones, I., Mariñas-Pardo, L., Melen, G. J., Cubillo, I., Ramírez, M., García-Castro, J."Enrichment of neural-related genes in human mesenchymal stem cells from neuroblastoma patients". International Journal of Molecular Medicine 30, no. 2 (2012): 365-373. https://doi.org/10.3892/ijmm.2012.1008