Abstract

Most malignant human tumors display a high degree of intratumoral heterogeneity at the time of diagnosis that contributes to treatment failure. This also applies to malignant peripheral nerve sheath tumors (MPNSTs) and aggressive soft tissue sarcomas that arise sporadically or in the context of neurofibromatosis type 1. On average, MPNSTs measure 10 cm in diameter at diagnosis. To explore molecular changes associated with early malignant progression and that may be present in most, if not all, tumor cells, we generated expression profiles of ethylnitrosourea-induced trigeminal MPNSTs in rats. Because these tumors cause increased intracranial pressure, they become detectable when they are comparatively minuscule. Histologic analyses revealed close resemblance to human MPNSTs. Compared with normal trigeminal nerve tissue, 365 genes were markedly upregulated and 310 genes were consistently downregulated in all MPNST samples. The molecular signature characteristic of early-stage MPNSTs included upregulation of proliferation and tissue remodeling-associated genes, downregulation of genes involved in Schwann cell differentiation, and the absence of transcripts associated with neuronal components. The transforming growth factor-β pathway was consistently upregulated in all tumor samples. These data suggest that the signaling pathways underlying early malignant progression of Schwann cells might be targeted to prevent tumor growth and/or to treat more advanced lesions.

Introduction

At the time of diagnosis, most malignant human tumors display a high degree of intratumoral heterogeneity because of tumor evolution and adaptation that can contribute to treatment failure and drug resistance (1). Large-scale sequencing analyses using multiple samples of a patient primary tumor and metastatic sites have shown that the fraction of mutations found in single biopsies is not present throughout all sampled regions of the same patient's tumor. Furthermore, gene expression profiles indicating a favorable prognosis and molecular signatures pointing to an unfavorable prognosis have been identified in different samples of the same tumor (2). This is particularly discouraging for the search for biomarkers in the context of designing preventive measures, early detection, and/or targeted therapies.

Molecular changes associated with early malignant progression are likely present in every cell or the great majority in small tumors, that is, before diversification has taken place that may mask the early abnormalities. Because they do not manifest clinically before they are large, many human tumors escape early detection. This is often the case for malignant peripheral nerve sheath tumors (MPNSTs), aggressive soft tissue sarcomas that arise either sporadically or in individuals with neurofibromatosis type 1. The prognosis of MPNST is extremely poor. At diagnosis, tumors display an average diameter of 10 cm and exhibit a high degree of intratumoral heterogeneity (3). Therefore, MPNSTs in model organisms that are considerably smaller and more homogeneous can provide useful paradigm for identifying early genetic aberrations that may be targets for prevention and specific therapies.

An animal model of sporadic MPNSTs is provided by ethylnitrosourea (ENU)-induced oncogenesis in the trigeminal nerves of BD rats (4). Because of their anatomic localization, these tumors become evident clinically when they measure less than 0.5 cm in diameter; thus, they fulfill the criteria for early neoplasia. Moreover, the tumor cells have undergone few divisions and, therefore, should be relatively homogeneous. Previously, we were able to establish a large collection of MPNSTs that were induced in different BD strains.

A transversion mutation at nucleotide 2012 in the transmembrane region of the Neu/Erbb2 gene, leading to a constitutional activation of the receptor tyrosine kinase, is a very early event in the development of ENU-induced MPNST in BD rats (5). Dysregulation of neuregulin-1/Erbb signaling has also been observed in human malignant Schwann cell tumors (6, 7). To date, no further genetic alterations in known oncogenes and tumor suppressor genes have been detected in ENU-induced MPNST (8). The Neu/Erbb2 mutation is followed by a loss of heterozygosity on rat chromosome 10, which includes the wild-type allele of the Neu/Erbb2 gene (9). An additional loss of heterozygosity on chromosome 5 could be detected in MPNST of BDIV × BDIX hybrid rats and represents a later step of carcinogenesis (10). In contrast to these structural aberrations, changes in gene expression of trigeminal nerve tissue because of MPNST development have not been investigated.

This study aims at detecting the dysregulation of gene expression in early stages of MPNST as compared with that in normal trigeminal nerve tissue. Because we used tumors that had arisen on different genetic backgrounds, dysregulated expression of distinct genes found in all tumor samples is likely an important, if not necessary, step during the early progression of trigeminal MPNST. Complementary investigation focused on the comprehensive characterization of the morphology of ENU-induced MPNST that should result from the structural and expressional gene alterations observed with regard to the similarity to human tumors.

Materials and Methods

Tissue Samples

Ethylnitrosourea-induced MPNST dissected from BDIV, BDIX, (BDIV × BDIX) F1, BDX.BDIV-Mss4a, and BDX. BDIV-Mss4b rats either embedded in paraffin or frozen at − 80°C were used for this study (11). Trigeminal nerves of three 85-day-old untreated female and 3 male rats, respectively, BDIX, BDIV, and BDX.BDIV-Mss4a rats, were used as control tissue for expression analysis using cDNA Microarrays and real-time polymerase chain reaction (RT-PCR). For this purpose, trigeminal nerves were carefully separated from brain tissue. To avoid contamination with ganglion cells, only 1.5 mm of nerve tissue adjacent to the brain nerve junction, not including the smaller branch of the nerve, were immediately frozen in liquid nitrogen and stored at − 80°C until RNA preparation.

Histopathology

Trigeminal tumors of 27 animals were evaluated by light microscopy of hematoxylin and eosin-stained sections. Tumor classification was carried out according to the World Health Organization Classification of Tumours of the Central Nervous System (12). Grading was performed using the 3-grade system of the Fédération Nationale des Centres de Lutte Contre le Cancer, which is based on mitotic count per 10 high-power fields, the occurrence of tumor necrosis, and differentiation of tumor cells/pleomorphism. Tumors were additionally characterized with regard to cellularity, solid or cystic growth architecture, occurrence of extraneural infiltration, mast cell content, and pigmentation (Table 1).

TABLE 1

Histopathologic Features of Ethylnitrosourea-lnduced Malignant Peripheral Nerve Sheath Tumors

Rat Strain Sex Age, days Necrosis Mitotic Count Grade Type Pleomorphism Cellularity Pigmentation Mast Cells Infiltration Pattern Growth Pattern S100 IRS 
BDIX.BDIV-Mss4b 177 No Conventional Weak Low No No Intraneural increased cellularity Diffuse 
BDIX.BDIV-Mss4a 174 No 12 Conventional Severe High No Yes Extraneural/destructive Solid 
BDIX.BDIV-Mss4b 279 No Conventional Weak Intermediate Yes Yes Extraneural/infiltrative Cystic 
BDIX.BDIV-Mss4a 219 No Conventional Weak Intermediate Yes Yes Extraneural/destructive Cystic 
BDIV 295 No 32 Conventional Moderate High No Yes Extraneural/perineural Solid 
BDIX.BDIV-Mss4a 251 No Conventional Weak Intermediate No No Intraneural increased cellularity Diffuse 
BDIX.BDIV-Mss4b 164 No Conventional Moderate Intermediate Yes Yes No adjacent tissue available Cystic 
BDIX.BDIV-Mss4b 177 No 18 Conventional Moderate Intermediate Yes Yes No adjacent tissue available Cystic 
BDIX.BDIV-Mss4b 189 No 19 Conventional Moderate Intermediate Yes Yes No adjacent tissue available Cystic 
BDIX.BDIV-Mss4b 245 No 57 Conventional Moderate High No No No adjacent tissue available Solid 12 
BDIX.BDIV-Mss4b 221 No 42 Conventional Moderate High No No No adjacent tissue available Cystic 
BDIX.BDIV-Mss4b 184 No 43 Conventional Severe High No No No adjacent tissue available Solid 
BDIX.BDIV-Mss4b 147 No 89 Conventional Severe High No No No adjacent tissue available Cystic 
BDIX.BDIV-Mss4b 189 No Conventional Weak Low Yes No Intraneural increased cellularity Diffuse 
BDIX.BDIV-Mss4b 239 No Conventional Weak Low No No Intraneural increased cellularity Diffuse 
BDIX.BDIV-Mss4b 266 No Conventional Weak Low No No Intraneural increased cellularity Diffuse 
BDIX.BDIV-Mss4b 191 No Conventional Weak Low No No Intraneural increased cellularity Diffuse n.d 
BDIX.BDIV-Mss4b 182 No Conventional Weak Low No No Intraneural increased cellularity Diffuse 
BDIX.BDIV-Mss4b 171 No Conventional Weak Low No No Intraneural increased cellularity Diffuse 12 
BDIX.BDIV-Mss4b 180 No 25 Conventional Weak Low No No Intraneural increased cellularity Diffuse 
BDIX.BDIV-Mss4b 178 No 20 Conventional Moderate High No No Intraneural/infiltrative Diffuse 
BDIX.BDIV-Mss4b 180 No Conventional Moderate Intermediate Yes No Intraneural/infiltrative Cystic 
BDIX.BDIV-Mss4b 221 No 11 Conventional Moderate Low No Yes Intraneural/infiltrative Cystic 
BDIX.BDIV-Mss4b 190 No Conventional Weak Low No No Intraneural/infiltrative Cystic 
BDIX.BDIV-Mss4b 184 No Conventional Weak Low Yes Yes Intraneural/infiltrative Cystic 12 
BDIX.BDIV-Mss4b 185 No 75 Conventional Moderate High No No Intraneural/infiltrative Solid 
BDIX.BDIV-Mss4b 171 No Conventional Weak Intermediate No Yes Intraneural/infiltrative Diffuse 
Rat Strain Sex Age, days Necrosis Mitotic Count Grade Type Pleomorphism Cellularity Pigmentation Mast Cells Infiltration Pattern Growth Pattern S100 IRS 
BDIX.BDIV-Mss4b 177 No Conventional Weak Low No No Intraneural increased cellularity Diffuse 
BDIX.BDIV-Mss4a 174 No 12 Conventional Severe High No Yes Extraneural/destructive Solid 
BDIX.BDIV-Mss4b 279 No Conventional Weak Intermediate Yes Yes Extraneural/infiltrative Cystic 
BDIX.BDIV-Mss4a 219 No Conventional Weak Intermediate Yes Yes Extraneural/destructive Cystic 
BDIV 295 No 32 Conventional Moderate High No Yes Extraneural/perineural Solid 
BDIX.BDIV-Mss4a 251 No Conventional Weak Intermediate No No Intraneural increased cellularity Diffuse 
BDIX.BDIV-Mss4b 164 No Conventional Moderate Intermediate Yes Yes No adjacent tissue available Cystic 
BDIX.BDIV-Mss4b 177 No 18 Conventional Moderate Intermediate Yes Yes No adjacent tissue available Cystic 
BDIX.BDIV-Mss4b 189 No 19 Conventional Moderate Intermediate Yes Yes No adjacent tissue available Cystic 
BDIX.BDIV-Mss4b 245 No 57 Conventional Moderate High No No No adjacent tissue available Solid 12 
BDIX.BDIV-Mss4b 221 No 42 Conventional Moderate High No No No adjacent tissue available Cystic 
BDIX.BDIV-Mss4b 184 No 43 Conventional Severe High No No No adjacent tissue available Solid 
BDIX.BDIV-Mss4b 147 No 89 Conventional Severe High No No No adjacent tissue available Cystic 
BDIX.BDIV-Mss4b 189 No Conventional Weak Low Yes No Intraneural increased cellularity Diffuse 
BDIX.BDIV-Mss4b 239 No Conventional Weak Low No No Intraneural increased cellularity Diffuse 
BDIX.BDIV-Mss4b 266 No Conventional Weak Low No No Intraneural increased cellularity Diffuse 
BDIX.BDIV-Mss4b 191 No Conventional Weak Low No No Intraneural increased cellularity Diffuse n.d 
BDIX.BDIV-Mss4b 182 No Conventional Weak Low No No Intraneural increased cellularity Diffuse 
BDIX.BDIV-Mss4b 171 No Conventional Weak Low No No Intraneural increased cellularity Diffuse 12 
BDIX.BDIV-Mss4b 180 No 25 Conventional Weak Low No No Intraneural increased cellularity Diffuse 
BDIX.BDIV-Mss4b 178 No 20 Conventional Moderate High No No Intraneural/infiltrative Diffuse 
BDIX.BDIV-Mss4b 180 No Conventional Moderate Intermediate Yes No Intraneural/infiltrative Cystic 
BDIX.BDIV-Mss4b 221 No 11 Conventional Moderate Low No Yes Intraneural/infiltrative Cystic 
BDIX.BDIV-Mss4b 190 No Conventional Weak Low No No Intraneural/infiltrative Cystic 
BDIX.BDIV-Mss4b 184 No Conventional Weak Low Yes Yes Intraneural/infiltrative Cystic 12 
BDIX.BDIV-Mss4b 185 No 75 Conventional Moderate High No No Intraneural/infiltrative Solid 
BDIX.BDIV-Mss4b 171 No Conventional Weak Intermediate No Yes Intraneural/infiltrative Diffuse 

F, female; IRS, Remmele immunoreactive score (13); M, male; n.d., not done.

Immunohistochemistry

For immunohistochemistry, 4-µm-thick sections were cut from paraffin-embedded tissue blocks. We used a rabbit polyclonal antibody against S100 (dilution, 1:400; IgG; Dako Cytomation, Glostrup, Denmark) and a mouse monoclonal antibody against cyclin D1 (dilution, 1:2000; IgG1, clone K-2; Zytomed Systems, Berlin, Germany) together with a highly sensitive and specific polymer detection system using horseradish peroxidase (ZytoChem-Plus HRP Polymer-Kit, Zytomed Systems). Development was carried out with a permanent brown chromogen substrate system (Permanent AEC Kit, Zytomed Systems). Finally, nuclei were counterstained with hematoxylin for 5 minutes.

Staining intensity was assessed with the Remmele immunoreactive score by multiplying the level of staining intensity (0–3 points) with the percentage of positive tumor cells (0–4 points) (13). Intensity of staining was judged according to the following scale: negative (0 points); weak (1–3 points); moderate (4–8 points); and strong (9–12 points).

RNA Extraction

For preparation of total RNA, frozen tissue was homogenized in TRIzol Reagent (Invitrogen, Karlsruhe, Germany). RNA was purified according to the manufacturer's instructions and quantified using a Nanodrop spectrophotometer (Thermo Fisher Scientific, Waltham, MA).

RT-PCR

Complementary DNA synthesis was performed with the SuperScript III Platinum Two-Step qPCR Kit with SYBR Green (Invitrogen) using 0.5 µg RNA per reaction. Kit-included random hexamer and oligo(dT)20 primers were applied for first strand cDNA synthesis according to the following protocol: 10 minutes at 25°C for primer annealing; 50 minutes at 42°C for the reverse transcription step; 5 minutes at 85°C for inactivation of SuperScript III RT; then cooling on ice. Tubes were stored at −20°C until used for subsequent RT-PCR, which was performed with the 7500-Fast-Real-Time-System Instrument in standard mode (Applied Biosystems, Darmstadt, Germany). Hprt was used as a reference gene for relative mRNA quantification of target genes. Two microliters of cDNA samples served as template in a 20-µL RT-PCR reaction. The PCR conditions for mRNA quantification were as follows: 50°C for 2 minutes, 95°C for 2 minutes followed by 40 cycles of 95°C for 15 seconds, and 60°C for 30 seconds. Primer sequences used for mRNA quantification are listed in Table, Supplemental Digital Content 1 ().

Microarray Hybridization, Scanning, and Data Analysis

RNA samples were processed for hybridization to microarrays on an Illumina Bead Station in the Max-Planck Institute for Molecular Genetics, Berlin, Germany, according to the manufacturer's instructions.

Qualitative integrity tests were carried out on a Bioanalyzer 2100 System (Agilent Technologies, Palo Alto, CA). One hundred nanograms of biotin-labeled cRNA, which was produced using a linear amplification kit (Ambion, Austin, TX), was hybridized to Sentrix Rat-Ref-12-v1 Expression Bead Chips containing gene-specific oligonucleotides (~22.000 genes; Illumina, Inc., San Diego, CA). Hybridization was detected with 1 µg/mL of Cy3-Strepavidin (Amersham Bioscience, Piscataway, NJ). The chips were scanned using an Illumina Bead Reader. All basic expression data analysis was carried out using the BeadStudio software 3.0 (Macrogen, Seoul, Korea). To define genes consistently upregulated or downregulated between tumor and normal nerve tissue, data from each group were combined respectively, and the means of expression values were calculated and compared. Genes with at least 2-fold differential expression between tumor and regular nerve tissue were identified. The resulting gene list was additionally selected for candidates that did not show overlapping expression values between tumor and normal nerve tissue in any of the samples used. Microarray data were deposited at the NCBI Geo repository in accordance with the MIAME standards and can be accessed via http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?token=vbcldkmaacmkovu&acc=GSE25929.

Results

Histologic Characterization of ENU-Induced Trigeminal MPNST

Median age of the 27 animals carrying trigeminal MPNSTs induced by ENU on postnatal day 1 was 185 days (mean, 202 ± 37.0 [SD] days; range, 147–295 days). The tumor tissue examined originated from MPNSTs induced in BDIX.BDIV-Mss4b (n = 23), BDIX.BDIV-Mss4a (n = 3), and BDIV (n = 1) rats. Regardless of the genetic background, we exclusively found the conventional spindle cell-type histopathologic pattern of MPNSTs in different stages of development (Fig. 1A). Extraneural spread of MPNSTs was mostly observed in older animals (n = 4). In those cases, there was either a predominantly perineural or infiltrative/ destructive phenotype of tumor spread; no adjacent tissue had been dissected in 7 cases. Tumors were exclusively of low- or intermediate-grade malignancy. Necroses were not detected and a severe nuclear pleomorphism was focally seen in only 3 tumors, whereas 10 MPNSTs displayed moderate and 15 displayed weak pleomorphism. Median mitotic count per 10 high-power field was 7 (mean, 18 ± 24 [SD]; range, 0–89). The occurrence of intratumoral mast cells was recognized in all 4 MPNSTs with extraneural spread, in 3 tumors with missing adjacent tissue, and in 3 (21%) of 14 intraneural tumors. Nineteen (70%) tumors expressed the S100 protein, as detected by immunohistochemical investigation (n = 1, weak; n = 14, intermediate; n = 4, strong; Fig. 1B). Results of the morphologic analyses of all tumors are summarized in Table 1.

FIGURE 1

Ethylnitrosourea-induced malignant peripheral nerve sheath tumor (MPNST) with sweeping fascicles formed by tumor cells that have enlarged and hyperchromatic nuclei. The tumor displays destruction and invasion of adjacent bone (left). (B) Same MPNST with a typical patchy expression of the S-100 protein. Original magnification for both: 400×.

FIGURE 1

Ethylnitrosourea-induced malignant peripheral nerve sheath tumor (MPNST) with sweeping fascicles formed by tumor cells that have enlarged and hyperchromatic nuclei. The tumor displays destruction and invasion of adjacent bone (left). (B) Same MPNST with a typical patchy expression of the S-100 protein. Original magnification for both: 400×.

Differential Gene Expression Profiles of MPNSTs and Trigeminal Nerve Tissue

In this study, oligonucleotide microarrays containing probe sets for 22,000 rat genes were used to obtain gene expression profiles of 12 full-blown ENU-induced MPNSTs and 1 microtumor. Most tumors had developed in F1 hybrids of BDIV and BDIX rats (T1, T2, T5, T7, T8, T9, T10); the remaining cases were from BDIV (T11, T12), BDIX (T3, T4), and congenic BDIX.BDIV-Mss4a (T6, MT) rats (11). Trigeminal nerves of 85-day-old BDIV, BDIX, and BDIX.BDIV-Mss4a rats of both sexes were used as reference tissue.

Genes that were at least 2-fold upregulated or down-regulated in tumors compared with normal nerve tissue and showing no overlapping expression values in tumor or normal tissue in any of the samples used were identified. Using these criteria, the expression of 365 genes was significantly upregulated, whereas 310 genes exhibited significantly decreased expression in tumor tissue (Table, Supplemental Digital Content 2, and Table, Supplemental Digital Content 3, ). The most pronounced gene expression differences between normal trigeminal nerve tissue and MPNSTs were visualized by performing a hierarchal cluster analysis (Fig. 2). To gain insight into the biologic mechanisms underlying malignant transformation in the peripheral nervous system, these genes were classified regarding their function by using the Gene Ontology database and literature searches (Tables 2,3).

TABLE 2

Genes Significantly Upregulated In Ethylnitorosourea-Induced Malignant Peripheral Nerve Sheath Tumors Compared With Normal Trigeminal Nerve Tissue

   Chromosomal Location   
Symbol Gene Name GenBank ID RNO Mb Fold Change 
Cell adhesion       
CDH17 Cadherin 17 NM_053977.1 26.1 2.8 0.000696 
COL16A1 Procollagen, type XVI, alpha 1 XM_345584.3 149.1 3.8 0.000001 
ITGA1 Integrin alpha 1 NM_030994.1 Unknown 19.8 0.000001 
ITGA4 Integrin alpha 4 XM_230033.4 62.1 14.3 0.000003 
NINJ1 Ninjurin 1 NM_012867.1 17 21.4 3.1 0.000001 
PCDHB5 Protocadherin beta 5 XM_001055177.1 18 30.1 15.1 0.000020 
PCDHGC3 Protocadherin gamma subfamily C, 3 NM_053943.1 18 30.7 8.1 0.000007 
Cell cycle       
BUB1 Budding uninhibited by benzimidazoles 1 homolog XM_215849.4 115.3 23.5 0.000001 
BUB1B Budding uninhibited by benzimidazoles 1 homolog, beta XM_342494.3 105.1 15.3 0.000001 
CCNA2 Cyclin A2 NM_053702.1 123.1 17.5 0.000002 
CCNB2 Cyclin B2 NM_001009470.1 74.9 23.4 0.000001 
CCND1 Cyclin D1 NM_171992.2 205.4 10.3 0.000001 
CDC2A Cell division cycle 2 homolog A NM_019296.1 20 20.0 36.9 0.000001 
CDK4 Cyclin-dependent kinase 4 NM_053593.2 Unknown 2.9 0.000001 
CDKN3 Cyclin-dependent kinase inhibitor 3 XM_214152.4 15 22.6 13.5 0.000007 
MCM3 Minichromosome maintenance deficient 3 XM_236988.4 19.5 18.5 0.000001 
MCM5 Minichromosome maintenance deficient 5 XM_001064207.1 19 13.4 4.4 0.000001 
MCM6 Minichromosome maintenance deficient 6 XM_001055953.1 13 40.2 13.0 0.000001 
RGD1562047 Cyclin-dependent kinases regulatory subunit 2, similar XM_001054024.1 17 13.3 23.4 0.000001 
TGFB1 Transforming growth factor, beta 1 NM_021578.1 80.9 4.8 0.000001 
TGFB2 Transforming growth factor, beta 2 NM_031131.1 13 102.7 6.6 0.000025 
TTK Ttk protein kinase XM_001062174.1 84.4 34.9 0.000006 
Chromosomal organization       
ASF1B ASF1 anti-silencing function 1 homolog B XM_001072446.1 19 23.7 11.7 0.000001 
Cytoskeleton       
ACTG2 Actin, gamma 2 NM_012893.1 117.7 33.1 0.003450 
ADAM17 A disintegrin and metalloproteinase domain 17 NM_020306.1 41.9 4.1 0.000001 
ASPM Asp-like, microcephaly associated XM_213891.4 13 52.9 27.2 0.000001 
AURKB Aurora kinase B NM_053749.1 10 55.8 8.9 0.000003 
COTL1 Coactosin-like 1 XM_341700.3 19 50.1 7.2 0.000001 
DLG7 Discs, large homolog 7 XM_223937.3 15 23.3 9.7 0.000001 
KIF11 Kinesin family member 11 XM_001060913.1 241.7 19.9 0.000003 
KIF15 Kinesin family member 15 NM_181635.2 127.8 8.3 0.000005 
KIF20A Kinesin family member 20A XM_341592.3 18 27.1 19.9 0.000001 
KIF22 Kinesin family member 22 NM_001009645.1 186.2 10.9 0.000001 
MLPH Melanophilin NM_001012135.1 90.1 85.9 0.000200 
MYL9 Myosin, light polypeptide 9, regulatory XM_001067182.1 144.0 10.6 0.000004 
NDE1 Nuclear distribution gene E homolog 1 NM_053347.1 10 0.8 3.8 0.000001 
PDLIM7 PDZ and LIM domain 7 NM_173125.1 17 15.2 5.9 0.000002 
RGD1566336 Similar to RIKEN cDNA 4933440J22 XM_217737.4 30.1 9.8 0.000001 
RHOC ras homolog gene family, member C XM_215659.4 200.1 4.2 0.000001 
TPX2 Microtubule-associated protein homolog 2 XM_001060351.1 140.1 20.2 0.000002 
Extracellular matrix       
ADAMTS1 Adam metallopeptidse with thrombospondin type 1 motif 1 NM_024400.1 11 25.4 14.9 0.000001 
AGRN Agrin NM_175754.1 173.0 8.1 0.000005 
COL18A1 Procollagen, type XVIII, alpha 1 XM_241632.4 20 12.0 8.2 0.000001 
CSPG2 Versican/chondroitin sulfate proteoglycan 2 XM_215451.4 19.7 31.7 0.000001 
(Continued on next page) 
CTGF Connective tissue growth factor NM_022266.2 21.3 7.1 0.000022 
ECM1 Extracellular matrix protein 1 NM_053882.1 190.5 7.4 0.000116 
EMILIN1 Elastin microfibril interfacer 1 XM_001064749.1 24.9 13.4 0.000092 
FBN2 Fibrillin 2 NM_031826.1 18 Unknown 22.8 0.000003 
LGALS3BP Lectin, galactoside-binding, soluble, 3 binding protein NM_139096.1 10 108.4 3.3 0.000003 
LOX Lysyl oxidase NM_017061.1 18 47.9 7.7 0.000761 
MMP11 Matrix metallopeptidase 11 NM_012980.1 20 13.1 29.7 0.011806 
MMP16 Matrix metalloproteinase 16 NM_080776.1 32.6 11.1 0.000099 
MMP17 Matrix metallopeptidase 17 XM_001072688.1 12 28.8 13.9 0.000002 
POSTN Periostin, osteoblast specific factor XM_342245.3 143.6 25.8 0.000195 
RGD1560062 Similar to Laminin alpha-4 chain precursor XM_001061207.1 20 43.2 7.9 0.000001 
SPON1 Spondin 1 XM_579693.1 172.0 14.0 0.000001 
ACPL2 Acid phosphatase-like 2 NM_001007710.1 101.9 4.8 0.000001 
ANGPT2 Angiopoietin 2 XM_001065522.1 16 69.0 19.7 0.000055 
C1QB Complement component 1, q subcomponent, beta NM_019262.1 155.6 4.4 0.000007 
C1QG Complement component 1, q subcomponent, gamma NM_001008524.1 155.7 4.7 0.000021 
GDF15 Growth differentiation factor 15 NM_019216.1 16 19.3 11.4 0.000299 
PDGFA Platelet-derived growth factor, alpha NM_012801.1 12 16.2 3.5 0.000001 
PNLIP Pancreatic lipase NM_013161.1 265.1 17.2 0.021419 
PTHLH Parathyroid hormone-like peptide NM_012636.1 Unknown 15.2 0.000003 
SERPINE1 Serine peptidase inhibitor, clade E, member 1 NM_012620.1 12 20.9 11.2 0.000131 
Golgi apparatus       
B4GALT6 UDP-Gal:betaGlcNAc beta 1,4-galactosyltransferase 6 NM_031740.1 18 12.4 4.5 0.000001 
GNAS GNAS complex locus XM_575296.2 165.2 7.7 0.003738 
HIP1 Huntingtin interacting protein 1, transcript variant 2 XM_001071106.1 12 23.0 6.4 0.000001 
LOC311716 Similar to Protein KIAA1510 precursor XM_230973.3 170.1 33.2 0.000007 
PTGFRN Prostaglandin F2 receptor negative regulator NM_019243.1 196.1 6.0 0.000012 
ST6GALNAC2 Sialyltransferase 7B NM_001031652.1 10 106.8 23.8 0.000038 
SULF2 Sulfatase 2 XM_001072989.1 153.4 9.2 0.000001 
Ion binding       
MOXD1 Monooxygenase, DBH-like 1 XM_220095.4 21.6 161.8 0.000001 
Lysosome       
CD68 CD68 antigen XM_001079491.1 10 53.5 5.1 0.000118 
Melanin metabolic process      
SILV Silver homolog XM_343146.3 2.0 37.2 0.000234 
Phosphorylation       
PBK PDZ binding kinase XM_224300.4 15 45.2 34.9 0.000001 
Regulation of apoptosis      
ALDH1A3 Aldehyde dehydrogenase family 1, subfamily A3 NM_153300.1 120.8 46.3 0.000040 
CASP7 Caspase 7 NM_022260.2 262.7 5.6 0.000001 
IGFBP3 Insulin-like growth factor binding protein 3 NM_012588.1 14 88.0 11.3 0.000016 
Regulation of T-cell activation      
BLM Bloom syndrome homolog XM_218837.4 136.3 6.5 0.000063 
CASP3 Caspase 3, apoptosis related cysteine protease NM_012922.2 16 48.9 3.3 0.000001 
RARA Retinoic acid receptor, alpha NM_031528.1 10 87.8 4.5 0.000001 
Regulation of transcription      
CASK Calcium/calmodulin-dependent serine protein kinase NM_022184.1 Unknown 3.0 0.000034 
FOXM1 Forkhead box M1 NM_031633.1 165.4 7.7 0.000007 
FZD1 Frizzled homolog 1 NM_021266.2 26.0 7.0 0.000001 
MXD3 Max dimerization protein 3 NM_145773.1 17 15.3 29.7 0.000001 
MYCN v-myc myelocytomatosis viral related oncogene XM_234025.3 36.5 8.8 0.000193 
NKX2-2 NK2 transcription factor related, locus 2 XM_001056116.1 133.5 15.5 0.000027 
OLIG1 Oligodendrocyte transcription factor 1 NM_021770.2 11 31.2 24.8 0.045772 
TCFAP2B Transcription factor AP-2 beta XM_217356.4 18.1 17.6 0.010974 
Tube development       
AARD Alanine and arginine rich domain containing protein NM_145093.1 88.4 39.3 0.000001 
Miscellaneous       
CDCA2 Cell division cycle associated 2, transcript variant 1 XM_001068286.1 15 41.6 14.0 0.000066 
DHRS7C Dehydrogenase/reductase member 7C XM_001078936.1 10 51.7 27.9 0.028976 
ECT2 Ect2 oncogene XM_342220.3 113.0 14.4 0.000001 
FBLIM1 Filamin binding LIM protein 1 NM_001007554.1 160.5 21.7 0.000001 
KCNN4 Potassium conductance calcium-activated channel, N 4 NM_023021.1 79.6 11.8 0.000001 
KIF23 Kinesin family member 23 XM_001073723.1 61.8 13.2 0.000032 
KIF4 Kinesin family member 4 XM_343797.3 88.7 24.8 0.000001 
PCDH20 Protocadherin 20 XM_001074783.1 15 63.9 43.3 0.000814 
PCDHB15 Protocadherin beta 15 XM_001055818.1 18 30.3 12.4 0.000002 
PRC1 Protein regulator of cytokinesis 1 XM_001061201.1 126.3 26.2 0.000001 
TACSTD1 Tumor-associated calcium signal transducer 1 NM_138541.1 11.2 15.9 0.000018 
TRAF4AF1 TRAF4 associated factor 1 NM_001004264.1 105.3 29.1 0.000001 
CDCA1 Cell division cycle associated 1 NM_001012028.1 13 85.3 12.0 0.000001 
CMTM3 CKLF-like MARVEL transmembrane domain containing 3 XM_226200.3 19 0.6 7.7 0.000001 
DUSP6 Dual-specificity phosphatase 6 NM_053883.2 36.9 5.1 0.000001 
EDG5/S1PR2 Sphingosine-1-phosphate receptor 2 NM_017192.1 20.0 8.7 0.000001 
EDNRB Endothelin receptor type B NM_017333.1 15 Unknown 8.6 0.000001 
EIF4E2 Eukaryotic translation initiation factor 4E member 2 XM_343616.2 86.0 3.0 0.000001 
EMP1 Epithelial membrane protein 1 NM_012843.2 172.3 6.3 0.000064 
GPR37 G protein-coupled receptor 37 NM_057201.1 52.2 5.8 0.000030 
IER5L Immediate early response 5-like XM_001079588.1 8.5 3.6 0.000001 
MAN2B1 Mannosidase 2, alpha B1 NM_199404.1 19 24.7 4.0 0.000001 
MARCH3 Membrane-associated ring finger 3 NM_001007759.1 18 52.5 4.3 0.000001 
MGMT O-6-methylguanine-DNA methyltransferase NM_012861.1 196.9 3.2 0.008704 
MKLN1 Muskelin 1 NM_031359.1 58.6 3.7 0.000010 
MPZL1 Myelin protein zero-like 1 NM_001007728.1 13 81.3 3.3 0.000001 
NNT Nicotinamide nucleotide transhydrogenase NM_001013157.1 51.5 3.2 0.000001 
PLOD2 Procollagen lysine, 2-oxoglutarate 5-dioxygenase 2 NM_175869.2 97.6 3.1 0.000039 
UBE2T Ubiquitin-conjugating enzyme E2T XM_001062580.1 13 46.7 30.2 0.000005 
   Chromosomal Location   
Symbol Gene Name GenBank ID RNO Mb Fold Change 
Cell adhesion       
CDH17 Cadherin 17 NM_053977.1 26.1 2.8 0.000696 
COL16A1 Procollagen, type XVI, alpha 1 XM_345584.3 149.1 3.8 0.000001 
ITGA1 Integrin alpha 1 NM_030994.1 Unknown 19.8 0.000001 
ITGA4 Integrin alpha 4 XM_230033.4 62.1 14.3 0.000003 
NINJ1 Ninjurin 1 NM_012867.1 17 21.4 3.1 0.000001 
PCDHB5 Protocadherin beta 5 XM_001055177.1 18 30.1 15.1 0.000020 
PCDHGC3 Protocadherin gamma subfamily C, 3 NM_053943.1 18 30.7 8.1 0.000007 
Cell cycle       
BUB1 Budding uninhibited by benzimidazoles 1 homolog XM_215849.4 115.3 23.5 0.000001 
BUB1B Budding uninhibited by benzimidazoles 1 homolog, beta XM_342494.3 105.1 15.3 0.000001 
CCNA2 Cyclin A2 NM_053702.1 123.1 17.5 0.000002 
CCNB2 Cyclin B2 NM_001009470.1 74.9 23.4 0.000001 
CCND1 Cyclin D1 NM_171992.2 205.4 10.3 0.000001 
CDC2A Cell division cycle 2 homolog A NM_019296.1 20 20.0 36.9 0.000001 
CDK4 Cyclin-dependent kinase 4 NM_053593.2 Unknown 2.9 0.000001 
CDKN3 Cyclin-dependent kinase inhibitor 3 XM_214152.4 15 22.6 13.5 0.000007 
MCM3 Minichromosome maintenance deficient 3 XM_236988.4 19.5 18.5 0.000001 
MCM5 Minichromosome maintenance deficient 5 XM_001064207.1 19 13.4 4.4 0.000001 
MCM6 Minichromosome maintenance deficient 6 XM_001055953.1 13 40.2 13.0 0.000001 
RGD1562047 Cyclin-dependent kinases regulatory subunit 2, similar XM_001054024.1 17 13.3 23.4 0.000001 
TGFB1 Transforming growth factor, beta 1 NM_021578.1 80.9 4.8 0.000001 
TGFB2 Transforming growth factor, beta 2 NM_031131.1 13 102.7 6.6 0.000025 
TTK Ttk protein kinase XM_001062174.1 84.4 34.9 0.000006 
Chromosomal organization       
ASF1B ASF1 anti-silencing function 1 homolog B XM_001072446.1 19 23.7 11.7 0.000001 
Cytoskeleton       
ACTG2 Actin, gamma 2 NM_012893.1 117.7 33.1 0.003450 
ADAM17 A disintegrin and metalloproteinase domain 17 NM_020306.1 41.9 4.1 0.000001 
ASPM Asp-like, microcephaly associated XM_213891.4 13 52.9 27.2 0.000001 
AURKB Aurora kinase B NM_053749.1 10 55.8 8.9 0.000003 
COTL1 Coactosin-like 1 XM_341700.3 19 50.1 7.2 0.000001 
DLG7 Discs, large homolog 7 XM_223937.3 15 23.3 9.7 0.000001 
KIF11 Kinesin family member 11 XM_001060913.1 241.7 19.9 0.000003 
KIF15 Kinesin family member 15 NM_181635.2 127.8 8.3 0.000005 
KIF20A Kinesin family member 20A XM_341592.3 18 27.1 19.9 0.000001 
KIF22 Kinesin family member 22 NM_001009645.1 186.2 10.9 0.000001 
MLPH Melanophilin NM_001012135.1 90.1 85.9 0.000200 
MYL9 Myosin, light polypeptide 9, regulatory XM_001067182.1 144.0 10.6 0.000004 
NDE1 Nuclear distribution gene E homolog 1 NM_053347.1 10 0.8 3.8 0.000001 
PDLIM7 PDZ and LIM domain 7 NM_173125.1 17 15.2 5.9 0.000002 
RGD1566336 Similar to RIKEN cDNA 4933440J22 XM_217737.4 30.1 9.8 0.000001 
RHOC ras homolog gene family, member C XM_215659.4 200.1 4.2 0.000001 
TPX2 Microtubule-associated protein homolog 2 XM_001060351.1 140.1 20.2 0.000002 
Extracellular matrix       
ADAMTS1 Adam metallopeptidse with thrombospondin type 1 motif 1 NM_024400.1 11 25.4 14.9 0.000001 
AGRN Agrin NM_175754.1 173.0 8.1 0.000005 
COL18A1 Procollagen, type XVIII, alpha 1 XM_241632.4 20 12.0 8.2 0.000001 
CSPG2 Versican/chondroitin sulfate proteoglycan 2 XM_215451.4 19.7 31.7 0.000001 
(Continued on next page) 
CTGF Connective tissue growth factor NM_022266.2 21.3 7.1 0.000022 
ECM1 Extracellular matrix protein 1 NM_053882.1 190.5 7.4 0.000116 
EMILIN1 Elastin microfibril interfacer 1 XM_001064749.1 24.9 13.4 0.000092 
FBN2 Fibrillin 2 NM_031826.1 18 Unknown 22.8 0.000003 
LGALS3BP Lectin, galactoside-binding, soluble, 3 binding protein NM_139096.1 10 108.4 3.3 0.000003 
LOX Lysyl oxidase NM_017061.1 18 47.9 7.7 0.000761 
MMP11 Matrix metallopeptidase 11 NM_012980.1 20 13.1 29.7 0.011806 
MMP16 Matrix metalloproteinase 16 NM_080776.1 32.6 11.1 0.000099 
MMP17 Matrix metallopeptidase 17 XM_001072688.1 12 28.8 13.9 0.000002 
POSTN Periostin, osteoblast specific factor XM_342245.3 143.6 25.8 0.000195 
RGD1560062 Similar to Laminin alpha-4 chain precursor XM_001061207.1 20 43.2 7.9 0.000001 
SPON1 Spondin 1 XM_579693.1 172.0 14.0 0.000001 
ACPL2 Acid phosphatase-like 2 NM_001007710.1 101.9 4.8 0.000001 
ANGPT2 Angiopoietin 2 XM_001065522.1 16 69.0 19.7 0.000055 
C1QB Complement component 1, q subcomponent, beta NM_019262.1 155.6 4.4 0.000007 
C1QG Complement component 1, q subcomponent, gamma NM_001008524.1 155.7 4.7 0.000021 
GDF15 Growth differentiation factor 15 NM_019216.1 16 19.3 11.4 0.000299 
PDGFA Platelet-derived growth factor, alpha NM_012801.1 12 16.2 3.5 0.000001 
PNLIP Pancreatic lipase NM_013161.1 265.1 17.2 0.021419 
PTHLH Parathyroid hormone-like peptide NM_012636.1 Unknown 15.2 0.000003 
SERPINE1 Serine peptidase inhibitor, clade E, member 1 NM_012620.1 12 20.9 11.2 0.000131 
Golgi apparatus       
B4GALT6 UDP-Gal:betaGlcNAc beta 1,4-galactosyltransferase 6 NM_031740.1 18 12.4 4.5 0.000001 
GNAS GNAS complex locus XM_575296.2 165.2 7.7 0.003738 
HIP1 Huntingtin interacting protein 1, transcript variant 2 XM_001071106.1 12 23.0 6.4 0.000001 
LOC311716 Similar to Protein KIAA1510 precursor XM_230973.3 170.1 33.2 0.000007 
PTGFRN Prostaglandin F2 receptor negative regulator NM_019243.1 196.1 6.0 0.000012 
ST6GALNAC2 Sialyltransferase 7B NM_001031652.1 10 106.8 23.8 0.000038 
SULF2 Sulfatase 2 XM_001072989.1 153.4 9.2 0.000001 
Ion binding       
MOXD1 Monooxygenase, DBH-like 1 XM_220095.4 21.6 161.8 0.000001 
Lysosome       
CD68 CD68 antigen XM_001079491.1 10 53.5 5.1 0.000118 
Melanin metabolic process      
SILV Silver homolog XM_343146.3 2.0 37.2 0.000234 
Phosphorylation       
PBK PDZ binding kinase XM_224300.4 15 45.2 34.9 0.000001 
Regulation of apoptosis      
ALDH1A3 Aldehyde dehydrogenase family 1, subfamily A3 NM_153300.1 120.8 46.3 0.000040 
CASP7 Caspase 7 NM_022260.2 262.7 5.6 0.000001 
IGFBP3 Insulin-like growth factor binding protein 3 NM_012588.1 14 88.0 11.3 0.000016 
Regulation of T-cell activation      
BLM Bloom syndrome homolog XM_218837.4 136.3 6.5 0.000063 
CASP3 Caspase 3, apoptosis related cysteine protease NM_012922.2 16 48.9 3.3 0.000001 
RARA Retinoic acid receptor, alpha NM_031528.1 10 87.8 4.5 0.000001 
Regulation of transcription      
CASK Calcium/calmodulin-dependent serine protein kinase NM_022184.1 Unknown 3.0 0.000034 
FOXM1 Forkhead box M1 NM_031633.1 165.4 7.7 0.000007 
FZD1 Frizzled homolog 1 NM_021266.2 26.0 7.0 0.000001 
MXD3 Max dimerization protein 3 NM_145773.1 17 15.3 29.7 0.000001 
MYCN v-myc myelocytomatosis viral related oncogene XM_234025.3 36.5 8.8 0.000193 
NKX2-2 NK2 transcription factor related, locus 2 XM_001056116.1 133.5 15.5 0.000027 
OLIG1 Oligodendrocyte transcription factor 1 NM_021770.2 11 31.2 24.8 0.045772 
TCFAP2B Transcription factor AP-2 beta XM_217356.4 18.1 17.6 0.010974 
Tube development       
AARD Alanine and arginine rich domain containing protein NM_145093.1 88.4 39.3 0.000001 
Miscellaneous       
CDCA2 Cell division cycle associated 2, transcript variant 1 XM_001068286.1 15 41.6 14.0 0.000066 
DHRS7C Dehydrogenase/reductase member 7C XM_001078936.1 10 51.7 27.9 0.028976 
ECT2 Ect2 oncogene XM_342220.3 113.0 14.4 0.000001 
FBLIM1 Filamin binding LIM protein 1 NM_001007554.1 160.5 21.7 0.000001 
KCNN4 Potassium conductance calcium-activated channel, N 4 NM_023021.1 79.6 11.8 0.000001 
KIF23 Kinesin family member 23 XM_001073723.1 61.8 13.2 0.000032 
KIF4 Kinesin family member 4 XM_343797.3 88.7 24.8 0.000001 
PCDH20 Protocadherin 20 XM_001074783.1 15 63.9 43.3 0.000814 
PCDHB15 Protocadherin beta 15 XM_001055818.1 18 30.3 12.4 0.000002 
PRC1 Protein regulator of cytokinesis 1 XM_001061201.1 126.3 26.2 0.000001 
TACSTD1 Tumor-associated calcium signal transducer 1 NM_138541.1 11.2 15.9 0.000018 
TRAF4AF1 TRAF4 associated factor 1 NM_001004264.1 105.3 29.1 0.000001 
CDCA1 Cell division cycle associated 1 NM_001012028.1 13 85.3 12.0 0.000001 
CMTM3 CKLF-like MARVEL transmembrane domain containing 3 XM_226200.3 19 0.6 7.7 0.000001 
DUSP6 Dual-specificity phosphatase 6 NM_053883.2 36.9 5.1 0.000001 
EDG5/S1PR2 Sphingosine-1-phosphate receptor 2 NM_017192.1 20.0 8.7 0.000001 
EDNRB Endothelin receptor type B NM_017333.1 15 Unknown 8.6 0.000001 
EIF4E2 Eukaryotic translation initiation factor 4E member 2 XM_343616.2 86.0 3.0 0.000001 
EMP1 Epithelial membrane protein 1 NM_012843.2 172.3 6.3 0.000064 
GPR37 G protein-coupled receptor 37 NM_057201.1 52.2 5.8 0.000030 
IER5L Immediate early response 5-like XM_001079588.1 8.5 3.6 0.000001 
MAN2B1 Mannosidase 2, alpha B1 NM_199404.1 19 24.7 4.0 0.000001 
MARCH3 Membrane-associated ring finger 3 NM_001007759.1 18 52.5 4.3 0.000001 
MGMT O-6-methylguanine-DNA methyltransferase NM_012861.1 196.9 3.2 0.008704 
MKLN1 Muskelin 1 NM_031359.1 58.6 3.7 0.000010 
MPZL1 Myelin protein zero-like 1 NM_001007728.1 13 81.3 3.3 0.000001 
NNT Nicotinamide nucleotide transhydrogenase NM_001013157.1 51.5 3.2 0.000001 
PLOD2 Procollagen lysine, 2-oxoglutarate 5-dioxygenase 2 NM_175869.2 97.6 3.1 0.000039 
UBE2T Ubiquitin-conjugating enzyme E2T XM_001062580.1 13 46.7 30.2 0.000005 

RNO, rat chromosome.

Genes stimulating proliferation through their role in cell cycle regulation would be expected to be upregulated also in early-stage tumor tissue. Indeed, 15 genes that have various roles during cell proliferation displayed highly elevated expression in ENU-induced rat MPNSTs (Table 2). Among them, cyclin D1 (Ccnd1), which mediates the proliferative response to cAMP in Schwann cells, showed a 200-fold up-regulation compared with trigeminal nerve tissue and was strongly expressed in all ENU-induced MPNST on the protein level (Fig. 4) (16). Interestingly, a marked upregulation of the Tgfb1 gene was also detected. This was accompanied by elevated expression of a group of genes, namely Cspg2/Vcan (17), Postn/Osf2 (18), or Tgfbi, a paralog of Postn (19), all of which have been described as induced by transforming growth factor-β1 (Tgfb1), indicating an activation of the Tgfb pathway. Apart from its role in cell proliferation, the TGFs (Tgfb1, Tgfb2, and Tgfb3) have also been described to be present in the cyst fluid of transplanted ENU-induced rat MPNST and may play an immunosuppressive role by inhibiting lymphocyte proliferation so that tumor cells escape immuno-surveillance (20, 21). Because we previously showed that T lymphocytes invade trigeminal nerves as a consequence of tumor initiation and seemed to be involved in the resistance mechanism against ENU-induced MPNST development in BDIV rats, upregulation of Tgfb1 in MPNST seems plausible (22, 23).

TABLE 3

Genes Significantly Downregulated in Ethylnitrosourea-Induced Malignant Peripheral Nerve Sheath Tumors Compared With Normal Trigeminal Nerve Tissue

   Chromosomal Location   
Symbol Gene Name GenBank ID RNO Mb Fold Change p-Value 
Axon       
CNTF Ciliary neurotrophic factor NM_013166.1 215.8 20.8 0.000136 
MBP Myelin basic protein NM_001025293.1 18 79.0 35.9 0.014727 
MAP1B Microtubule-associated protein 1b XM_215469.4 30.4 10.7 0.000195 
SNCA Synuclein, alpha NM_019169.2 89.6 32.8 0.000493 
TAC1 Tachykinin 1 NM_012666.1 32.7 36.8 0.001130 
Cytosol       
AOX1 Aldehyde oxidase 1 NM_019363.2 56.8 17.6 0.002535 
Extracellular region       
TGFA Transforming growth factor alpha NM_012671.1 120.4 6.3 0.000420 
CHGB Chromogranin B NM_012526.1 120.6 5.9 0.008129 
TUBA4 Tubulin, alpha 4 NM_001007004.1 74.5 25.9 0.013571 
LECT1 Leukocyte cell-derived chemotaxin 1 NM_030854.1 15 60.9 35.6 0.011099 
Glutathione metabolism      
GSTM3 Glutathione S-transferase, mu type 3 NM_031154.1 203.5 16.8 0.000387 
Golgi membrane       
NTRK1 Neurotrophic tyrosine kinase, receptor, type 1 NM_021589.1 179.8 41.6 0.000691 
FUT8 Fucosyltransferase 8 fucosyltransferase) NM_001002289.1 100.0 7.1 0.006179 
Heterotrimeric G-protein complex      
RGS7 Regulator of G-protein signaling 7 NM_019343.1 13 90.7 15.0 0.006259 
Ion channel complex      
KCNS3 Potassium voltage-gated channel S member 3 NM_031778.2 34.5 21.8 0.007466 
SCN10A Sodium channel, voltage-gated, type 10, alpha NM_017247.1 124.6 21.2 0.000576 
SCN11A Sodium channel, voltage-gated, type XI, alpha NM_019265.2 124.7 21.1 0.002923 
SCN4B Sodium channel, voltage-gated, type IV, beta NM_001008880.1 48.1 36.8 0.005867 
Membrane fraction       
SLC17A6 Solute carrier family 17, member 6 NM_053427.1 101.5 21.3 0.000322 
FDFT1 Farnesyl diphosphate farnesyl transferase 1 NM_019238.2 15 42.4 5.0 0.000759 
CD24 CD24 antigen NM_012752.2 20 47.5 24.2 0.005478 
MAL mal, T-cell differentiation protein NM_012798.1 115.2 43.9 0.000865 
GNAO Guanine nucleotide binding protein, alpha o NM_017327.1 19 11.5 15.8 0.006533 
NBL1 Neuroblastoma, suppression of tumorigenicity 1 NM_031609.1 Unknown 12.0 0.000001 
Neuron projection       
FBXO2 F-box only protein 2 NM_053511.1 165.2 22.3 0.006879 
SNAP25 Synaptosomal-associated protein 25 NM_030991.1 124.9 39.3 0.000268 
ANK1 Ankyrin 1, erythroid XM_240464.3 16 73.3 25.5 0.000317 
PIB5PA Phosphatidylinositol bisphosphate 5-phosphatase, A NM_133562.1 14 84.1 20.9 0.008625 
SNCG Synuclein, gamma NM_031688.1 16 10.0 20.6 0.000395 
CABP1 Calcium-binding protein 1, transcript variant 3 NM_001033676.1 12 42.7 22.2 0.002197 
STMN2 Stathmin-like 2 NM_053440.2 95.3 27.6 0.003760 
GABBR2 Gamma-aminobutyric acid B receptor 2 NM_031802.1 63.2 36.3 0.001984 
P2RXL1 Purinergic receptor P2X-like 1, orphan receptor NM_012721.1 11 85.4 20.1 0.009438 
NEF3 Neurofilament 3, medium NM_017029.1 15 47.7 20.0 0.001899 
CHRNA3 Cholinergic receptor, nicotinic, alpha polypeptide 3 XM_001072823.1 54.9 40.2 0.003179 
DDN Dendrin NM_030993.1 137.6 20.9 0.005056 
Synapse       
MGLL Monoglyceride lipase NM_138502.2 122.9 24.6 0.000276 
SYNPR Synaptoporin NM_023974.1 15 13.1 20.4 0.007072 
HTR3A 5-Hydroxytryptamine receptor 3a NM_024394.1 Unknown 38.5 0.001119 
RGD1559440 Ca2+-dependent activator for secretion protein 2 XM_231528.4 49.7 23.1 0.000367 
Tight junction       
CLDN19 Claudin 19 NM_001008514.1 139.8 40.8 0.001479 
Transcription       
EEF1A2 Eukaryotic translation elongation factor 1 alpha 2 NM_012660.2 170.3 16.0 0.001032 
   Chromosomal Location   
Symbol Gene Name GenBank ID RNO Mb Fold Change p-Value 
Axon       
CNTF Ciliary neurotrophic factor NM_013166.1 215.8 20.8 0.000136 
MBP Myelin basic protein NM_001025293.1 18 79.0 35.9 0.014727 
MAP1B Microtubule-associated protein 1b XM_215469.4 30.4 10.7 0.000195 
SNCA Synuclein, alpha NM_019169.2 89.6 32.8 0.000493 
TAC1 Tachykinin 1 NM_012666.1 32.7 36.8 0.001130 
Cytosol       
AOX1 Aldehyde oxidase 1 NM_019363.2 56.8 17.6 0.002535 
Extracellular region       
TGFA Transforming growth factor alpha NM_012671.1 120.4 6.3 0.000420 
CHGB Chromogranin B NM_012526.1 120.6 5.9 0.008129 
TUBA4 Tubulin, alpha 4 NM_001007004.1 74.5 25.9 0.013571 
LECT1 Leukocyte cell-derived chemotaxin 1 NM_030854.1 15 60.9 35.6 0.011099 
Glutathione metabolism      
GSTM3 Glutathione S-transferase, mu type 3 NM_031154.1 203.5 16.8 0.000387 
Golgi membrane       
NTRK1 Neurotrophic tyrosine kinase, receptor, type 1 NM_021589.1 179.8 41.6 0.000691 
FUT8 Fucosyltransferase 8 fucosyltransferase) NM_001002289.1 100.0 7.1 0.006179 
Heterotrimeric G-protein complex      
RGS7 Regulator of G-protein signaling 7 NM_019343.1 13 90.7 15.0 0.006259 
Ion channel complex      
KCNS3 Potassium voltage-gated channel S member 3 NM_031778.2 34.5 21.8 0.007466 
SCN10A Sodium channel, voltage-gated, type 10, alpha NM_017247.1 124.6 21.2 0.000576 
SCN11A Sodium channel, voltage-gated, type XI, alpha NM_019265.2 124.7 21.1 0.002923 
SCN4B Sodium channel, voltage-gated, type IV, beta NM_001008880.1 48.1 36.8 0.005867 
Membrane fraction       
SLC17A6 Solute carrier family 17, member 6 NM_053427.1 101.5 21.3 0.000322 
FDFT1 Farnesyl diphosphate farnesyl transferase 1 NM_019238.2 15 42.4 5.0 0.000759 
CD24 CD24 antigen NM_012752.2 20 47.5 24.2 0.005478 
MAL mal, T-cell differentiation protein NM_012798.1 115.2 43.9 0.000865 
GNAO Guanine nucleotide binding protein, alpha o NM_017327.1 19 11.5 15.8 0.006533 
NBL1 Neuroblastoma, suppression of tumorigenicity 1 NM_031609.1 Unknown 12.0 0.000001 
Neuron projection       
FBXO2 F-box only protein 2 NM_053511.1 165.2 22.3 0.006879 
SNAP25 Synaptosomal-associated protein 25 NM_030991.1 124.9 39.3 0.000268 
ANK1 Ankyrin 1, erythroid XM_240464.3 16 73.3 25.5 0.000317 
PIB5PA Phosphatidylinositol bisphosphate 5-phosphatase, A NM_133562.1 14 84.1 20.9 0.008625 
SNCG Synuclein, gamma NM_031688.1 16 10.0 20.6 0.000395 
CABP1 Calcium-binding protein 1, transcript variant 3 NM_001033676.1 12 42.7 22.2 0.002197 
STMN2 Stathmin-like 2 NM_053440.2 95.3 27.6 0.003760 
GABBR2 Gamma-aminobutyric acid B receptor 2 NM_031802.1 63.2 36.3 0.001984 
P2RXL1 Purinergic receptor P2X-like 1, orphan receptor NM_012721.1 11 85.4 20.1 0.009438 
NEF3 Neurofilament 3, medium NM_017029.1 15 47.7 20.0 0.001899 
CHRNA3 Cholinergic receptor, nicotinic, alpha polypeptide 3 XM_001072823.1 54.9 40.2 0.003179 
DDN Dendrin NM_030993.1 137.6 20.9 0.005056 
Synapse       
MGLL Monoglyceride lipase NM_138502.2 122.9 24.6 0.000276 
SYNPR Synaptoporin NM_023974.1 15 13.1 20.4 0.007072 
HTR3A 5-Hydroxytryptamine receptor 3a NM_024394.1 Unknown 38.5 0.001119 
RGD1559440 Ca2+-dependent activator for secretion protein 2 XM_231528.4 49.7 23.1 0.000367 
Tight junction       
CLDN19 Claudin 19 NM_001008514.1 139.8 40.8 0.001479 
Transcription       
EEF1A2 Eukaryotic translation elongation factor 1 alpha 2 NM_012660.2 170.3 16.0 0.001032 

RNO, rat chromosome.

FIGURE 2

Hierarchical cluster analysis of gene expression comparing ethylnitrosourea-induced malignant peripheral nerve sheath tumors (T1–T12), microtumor (MT), and normal trigem-inal nerve (WT) tissue. See dendrogram above the heat map. RNAs used for hybridizations are arranged in columns; individual genes (91 unique probe sets) are in rows. Expression signal strength is indicated by color (red, high expression; green, low expression). Color scale of arbitrary signal strength expression is shown on the right. Red asterisks indicate gene expression data that were confirmed by real-time polymerase chain reaction; green asterisks indicate additional immunohistochemistry.

FIGURE 2

Hierarchical cluster analysis of gene expression comparing ethylnitrosourea-induced malignant peripheral nerve sheath tumors (T1–T12), microtumor (MT), and normal trigem-inal nerve (WT) tissue. See dendrogram above the heat map. RNAs used for hybridizations are arranged in columns; individual genes (91 unique probe sets) are in rows. Expression signal strength is indicated by color (red, high expression; green, low expression). Color scale of arbitrary signal strength expression is shown on the right. Red asterisks indicate gene expression data that were confirmed by real-time polymerase chain reaction; green asterisks indicate additional immunohistochemistry.

Validation of Microarray Data by RT-PCR

To confirm the gene expression data obtained by microarray analysis, we conducted quantitative RT-PCR assays for 20 genes displaying the strongest expression differences between MPNST and normal trigeminal nerve tissue (Fig. 2). According to the expression array data, Aard, Cspg2, Ednrb, Itga1, Mlph, Moxd1, Postn, Prc1, Silv, Spon1 were significantly upregulated in MPNST compared with trigeminal nerve tissue, whereas Aox1, CD24, Cldn19, Cntf, Fxyd2, Gstm3, Lect1, Mal, Mbp, Nbl1 exhibited higher expression in control nerve tissue than in the tumors. These data could be reproduced by RT-PCR (Fig. 3).

FIGURE 3

Validation of microarray data by real-time polymerase chain reaction. The mRNA-expression of 10 genes was examined in ethylnitrosourea-induced malignant peripheral nerve sheath tumors (MPNSTs) (T1–T9) versus untreated trigeminal nerve tissue (WT). Bar plots show expression in each MPNST and normal trigeminal nerve, box plots show median values (horizontal bars in red boxes: MPNST; black boxes represent normal nerve tissue). (A) Genes upregulated in MPNST. (B) Genes downregulated in MPNSTs. According to the Wilcoxon signed-rank test, the expression of these genes was significantly different between MPNST and WT, p < 0.01.

FIGURE 3

Validation of microarray data by real-time polymerase chain reaction. The mRNA-expression of 10 genes was examined in ethylnitrosourea-induced malignant peripheral nerve sheath tumors (MPNSTs) (T1–T9) versus untreated trigeminal nerve tissue (WT). Bar plots show expression in each MPNST and normal trigeminal nerve, box plots show median values (horizontal bars in red boxes: MPNST; black boxes represent normal nerve tissue). (A) Genes upregulated in MPNST. (B) Genes downregulated in MPNSTs. According to the Wilcoxon signed-rank test, the expression of these genes was significantly different between MPNST and WT, p < 0.01.

Immunolocalization of the Cyclin D1 Protein in MPNST Versus Trigeminal Nerve Tissue

Abundant amounts of Ccnd1 mRNA in all MPNSTs investigated in contrast to low levels in trigeminal nerve tissue point to an important role this gene may play in PNS onco-genesis. Therefore, we investigated whether this difference is reflected by a corresponding protein expression pattern. Immunohistochemical investigations on MPNST induced in BDIX.BDIV-Mss4b using an anti-cyclin D antibody showed strong, mainly nuclear staining in every tumor (n = 8); this staining was almost totally absent in 85-day-old trigeminal nerve tissue (n = 7) (Fig. 4 A, B).

FIGURE 4

(A) Ethylnitrosourea-induced malignant peripheral nerve sheath tumors strongly express cyclin D1, as predicted by elevated levels of mRNA detected by expression profiling and real-time polymerase chain reaction. (B) A trigeminal nerve from an 85-day-old rat lacks cyclin D1 expression. Original magnification for both: 400×.

FIGURE 4

(A) Ethylnitrosourea-induced malignant peripheral nerve sheath tumors strongly express cyclin D1, as predicted by elevated levels of mRNA detected by expression profiling and real-time polymerase chain reaction. (B) A trigeminal nerve from an 85-day-old rat lacks cyclin D1 expression. Original magnification for both: 400×.

Discussion

Preventive measures and curative cancer therapies counteracting the abnormal biology of cancer cells would ideally require target signal transduction pathways aberrant in every preneoplastic and/or full-blown tumor cell. However, it is evident that most somatic genetic mutations are not present ubiquitously within a tumor, and molecular signatures that are meant to predict a patient's outcome vary within the same tumor (2).

With this study, we aimed at detecting molecular alterations at the level of gene transcription tightly associated with early stages of tumor progression in MPNST. Ethylnitrosourea-induced MPNSTs in rats of the BD strains predominantly arise in the trigeminal nerves close to the brain-nerve junction. They invite analysis of early genetic and epigenetic alterations propelling tumor progression. Because of their anatomic localization in the skull, they become clinically evident early when they measure about a few millimeters in diameter and when the tumor cells still are comparatively homogeneous. This surpasses the possibilities offered by human MPNSTs, which usually display a several thousandfold larger volume at detection and have a high degree of intratumoral heterogeneity as a result of secondary cellular diversification caused by tumor evolution and adaption (3).

Before global gene expression profiling, we performed a detailed histologic analysis of ENU-induced MPNST to investigate the morphologic correlates of dysregulated gene expression and the similarity to that in human MPNST. The MPNSTs investigated histologically had arisen in different BDIX-derived rat strains and in a BDIV rat (11). The light microscopic findings differed regarding tumor stage but did not vary depending on the genetic background. In the great majority of tumors, there was a conventional spindle cell phenotype with variable degrees of cellularity, pleomorphism, and mitotic activity. This is in accordance with findings of other authors (5, 14). Most tumors expressed the S100 protein focally, as is seen in human MPNST (15). As expected, only the histology of the few most advanced ENU-induced rat MPNSTs resembled the average human tumor; all other ENU-induced tumors of the trigeminal nerves represented early tumor stages.

Studies of global gene expression in early-stage MPNSTs that arose on various genetic backgrounds compared with genetically matched normal trigeminal nerve tissue of adult rats should allow identification of genes that are crucial for early malignant progression of Schwann cells. These genes should be expressed to a similar extent in all MPNSTs independent of the genetic background, and the strength of expression should differ significantly from that of wild-type nerve tissue. Accordingly, we identified 2 sets of genes that were either markedly upregulated or downregulated in all MPNST specimens. These differentially expressed genes were functionally classified into different biologic categories. Several of them are involved in signaling pathways playing important roles in malignant transformation.

Tissue remodeling in ENU-induced MPNST first indicated by a marked softening of tumor tissue compared with normal nerve and later by complete destruction of normal nerve structure was reflected by increased expression of genes belonging to the functional classes of extracellular matrix molecules, cell adhesion proteins, and proteases in all ENU-induced MPNSTs investigated. Many of these are known to play an important role in human cancer, including Col16a1, Col18a1, Itga1, Itga4, Ecm1 fibrillin/Fbn2, Spon1, Mmp11, Mmp16, and Mmp17 (Table 2).

In addition, there were genes with unexplained functions in carcinogenesis that were upregulated in ENU-induced MPNSTs, such as monooxygenase1 (Moxd1), which was more than 160-fold overexpressed, and Aard, a gene of unknown function that is highly expressed in mouse testis that has never previously been associated with the malignant phenotype (24).

Most of the genes that were downregulated in ENU-induced MPNSTs are associated with the differentiated state of normal nerve tissue, that is, myelinating and nonmyelinating Schwann cells, the precursor cells which are thought to represent the cells of origin for MPNSTs (25). Another group of genes that were downregulated are normally expressed in nerve tissue components but not represented in the tumors, for example, ganglion cells with their axons and synapses (Table 3). Transcripts of genes such as Mbp, which encodes a structural myelin protein, and Scn4b, which is responsible for action potential initiation and propagation in excitable cells, were essentially not present in MPNST tissue; those involved in syn-aptic processes, such as transcripts of Synpr, Snap25, and Htr3a, were also not present.

In addition, the telomeric region on chromosome 10 (107–110, 7 Mb), which is frequently deleted in ENU-induced rat MPNSTs (10), harbors 2 genes that are significantly down-regulated: neuronal pentraxin 1 (LOC497675/NPTX1) and Fructosamine 3 kinase (Fnsk/Fn3k).

In summary, the molecular signature characteristic of very early stage MPNST includes upregulation of proliferation-associated genes, genes involved in tissue remodeling and of different partially unknown function as well as the down-regulation of genes maintaining the differentiated Schwann cell phenotype and the absence of transcripts being associated with nerve tissue components not represented in MPNST-like ganglion cells, axons, and synapses. In comparison with the gene expression profiles of human MPNSTs, it is not surprising that the expression of only a few genes in the rat MPNSTs were dysregulated in a similar way.

Sporadic and Nf1-associated human MPNSTs could not be distinguished by gene expression profiling (26). Thus, the different etiology of ENU-induced rat MPNSTs versus human tumors might be more indicative of the different tumor stages. We performed the present study because we intended to detect pathways that are deregulated in early malignant progression and as a consequence would be detectable in most if not all tumor cells. Therefore, the molecular signature of ENU-induced rat MPNSTs at the mRNA level might correspond to the expression profile of a small subset of human MPNSTs that might possibly be detected in the early stages only by accident.

Moreover, in most studies, gene expression of MPNSTs or MPNST cell lines is compared with profiles of Schwann cell lines or primary cultured Schwann cells (25, 27, 28). In contrast, gene expression profiles obtained for primary ENU-induced trigeminal MPNST tissue in this study were compared with those of regular trigeminal nerve tissue. Because the 1.5 mm of nerve tissue adjacent to the brain-nerve junction that was obtained was devoid of ganglion cells and blood vessels, these pieces of tissue seemed to constitute an appropriate control.

With the strategy used in this study, we meant to rule out expression artifacts caused by culture conditions. It is known that cells that are part of a 3-dimensional tissue network show different expression patterns than they do in monolayer tissue culture. Although it was previously shown that the expression of many genes in MPNSTs and MPNST cell lines corresponded to each other, the expression of stem cell and mature cell markers varied considerably in the MPNST cell line S462, depending on whether it was growing adherently or in spheres (27, 29). Whereas spheres seemed to be enriched in stemlike cells and exhibited a neural crest signature, adherent cells seemed to be more differentiated. Nevertheless, some genes found upregulated in a fraction of human MPNSTs, such as BUB 1, CASP3, CCNB2, CCND1, CDC6, CDC20, CTGF, FOXM1, KIF4, MCM6, PBK, POLE, POSTN, or TTK, were also identified in the present study.

Having gained insight into critical pathways underlying early malignant progression of rat Schwann cells will enable us to target, for example, Tgfb signaling in the same rat model to prevent ENU-induced MPNST growth and/or to treat more advanced lesions. These experiments will determine whether targets defined in early lesions represent effective therapies.

Acknowledgement

The authors thank Aydah Sabah at the Max Planck Institute of Molecular Genetics, Berlin, Germany, for hybridization of expression arrays.

References

1.
Mullighan
CG
Phillips
LA
Su
X
et al
.
Genomic analysis of the clonal origins of relapsed acute lymphoblastic leukemia
.
Science
 
2008
;
322
:
1377
80
2.
Gerlinger
M
Rowan
AJ
Horswell
S
et al
.
Intratumor heterogeneity and branched evolution revealed by multiregion sequencing
.
N Engl J Med
 
2012
;
366
:
883
92
3.
LaFemina
J
Qin
LX
Moraco
NH
et al
.
Oncologic outcomes of sporadic, neurofibromatosis-associated, and radiation-induced malignant peripheral nerve sheath tumors
.
Ann Surg Oncol
 
2013
;
20
:
66
72
4.
Druckrey
H
Landschutz
C
Ivankovic
S
.
[Transplacental induction of malignant tumours of the nervous system. II. Ethyl-nitrosurea in 10 genetically defined strains of rats]
.
Z Krebsforsch
 
1970
;
73
:
371
86
5.
Nikitin
A
Ballering
LA
Lyons
J
et al
.
Early mutation of the neu (erbB-2) gene during ethylnitrosourea-induced oncogenesis in the rat Schwann cell lineage
.
Proc Nat Acad Sci USA
 
1991
;
88
:
9939
43
6.
Holtkamp
N
Malzer
E
Zietsch
J
et al
.
EGFR and erbB2 in malignant peripheral nerve sheath tumors and implications for targeted therapy
.
Neuro Oncol
 
2008
;
10
:
946
57
7.
Stonecypher
MS
Byer
SJ
Grizzle
WE
et al
.
Activation of the neuregulin-1/ErbB signaling pathway promotes the proliferation of neoplastic Schwann cells in human malignant peripheral nerve sheath tumors
.
Oncogene
 
2005
;
24
:
5589
605
8.
Sukumar
S
Barbacid
M
.
Specific patterns of oncogene activation in transplacentally induced tumors
.
Proc Nat Acad Sci USA
 
1990
;
87
:
718
22
9.
Kindler-Rohrborn
A
Kolsch
BU
Fischer
C
et al
.
Ethylnitrosourea-induced development of malignant schwannomas in the rat: Two distinct loci on chromosome of 10 involved in tumor susceptibility and oncogenesis
.
Cancer Res
 
1999
;
59
:
1109
14
10.
Koelsch
BU
Kindler-Rohrborn
A
Held
S
et al
.
Loss of heterozygosity in malignant rat schwannomas chemically induced in hybrids of inbred rat strains with differential tumor susceptibility
.
Carcinogenesis
 
2002
;
23
:
1033
37
11.
Koelsch
B
Winzen-Reichert
B
Fischer
C
et al
.
Sex-biased suppression of chemically induced neural carcinogenesis in congenic BDIX.BDIV-Mss4a rats
.
Physiol Genomics
 
2011
;
43
:
631
39
12.
Louis
DN
Ohgaki
H
Wiestler
OD
, eds.
WHO Classification of Tumours of the Central Nervous System
 .
Lyon, France
:
IARC Press
,
2007
:
160
62
13.
Remmele
W
Stegner
HE
.
[Recommendation for uniform definition of an immunoreactive score (IRS) for immunohistochemical estrogen receptor detection (ER-ICA) in breast cancer tissue]
.
Der Pathologe
 
1987
;
8
:
138
40
14.
Swenberg
JA
Clendenon
N
Denlinger
R
et al
.
Sequential development of ethylnitrosourea-induced neurinomas: Morphology, biochemistry, and transplantability
.
J Natl Cancer Inst
 
1975
;
55
:
147
52
15.
Woodruff
JM
Kourea
HP
Louis
DN
et al
.
Schwannoma
. In:
Kleihues
P
Cavenee
WK
, eds.
Pathology and Genetics of Tumours of the Nervous System
 .
Lyon, France
:
IARC Press
,
2000
:
164
66
16.
Kim
HA
Ratner
N
Roberts
TM
et al
.
Schwann cell proliferative responses to cAMP and Nf1 are mediated by cyclin D1
.
J Neurosci
 
2001e
;
21
:
1110
16
17.
Soikkeli
J
Podlasz
P
Yin
M
et al
.
Metastatic outgrowth encompasses COL-I, FN1, and POSTN up-regulation and assembly to fibrillar networks regulating cell adhesion, migration, and growth
.
Am J Pathol
 
2010
;
177
:
387
403
18.
Wen
W
Chau
E
Jackson-Boeters
L
et al
.
TGF-ss1 and FAK regulate periostin expression in PDL fibroblasts
.
J Dent Res
 
2010
;
89
:
1439
43
19.
Hoersch
S
Andrade-Navarro
MA
.
Periostin shows increased evolutionary plasticity in its alternatively spliced region
.
BMC Evol Biol
 
2010
;
10
:
30
20.
Altenschmidt
U
Kahl
R
Klundt
E
et al
.
Schwannoma cells induce a tumor cell-specific cytotoxic T-cell response upon transplantation into syngeneic rats but escape elimination through the secretion of immuno-suppressive factors
.
Int J Cancer
 
1997
;
70
:
542
50
21.
Donkor
MK
Sarkar
A
Savage
PA
et al
.
T cell surveillance of oncogene-induced prostate cancer is impeded by T cell-derived TGF-beta1 cytokine
.
Immunity
 
2011
;
35
:
123
34
22.
Gering
KM
Marx
JA
Lennartz
K
et al
.
The interaction mode of premalignant Schwann and immune effector cells during chemically induced carcinogenesis in the rat peripheral nervous system is strongly influenced by genetic background
.
Cancer Res
 
2006
;
66
:
4708
14
23.
Marx
JA
Rohrborn
AJ
Koelsch
BU
et al
.
Ablation of T-cell immunity differentially influences tumor risk in inbred BD rat strains
.
Cancer Immunol Immunother
 
2009
;
58
:
1287
95
24.
Svingen
T
Beverdam
A
Verma
P
et al
.
Aard is specifically up-regulated in Sertoli cells during mouse testis differentiation
.
Int J Dev Biol
 
2007
;
51
:
255
58
25.
Miller
SJ
Jessen
WJ
Mehta
T
et al
.
Integrative genomic analyses of neurofibromatosis tumours identify SOX9 as a biomarker and survival gene
.
EMBO Mol Med
 
2009
;
1
:
236
48
26.
Holtkamp
N
Mautner
VF
Friedrich
RE
et al
.
Differentially expressed genes in neurofibromatosis 1-associated neurofibromas and malignant peripheral nerve sheath tumors
.
Acta Neuropathol
 
2004
;
107
:
159
68
27.
Miller
SJ
Rangwala
F
Williams
J
et al
.
Large-scale molecular comparison of human Schwann cells to malignant peripheral nerve sheath tumor cell lines and tissues
.
Cancer Res
 
2006
;
66
:
2584
91
28.
Lee
PR
Cohen
JE
Tendi
EA
et al
.
Transcriptional profiling in an MPNST-derived cell line and normal human Schwann cells
.
Neuron Glia Biol
 
2004
;
1
:
135
47
29.
Spyra
M
Kluwe
L
Hagel
C
et al
.
Cancer stem cell-like cells derived from malignant peripheral nerve sheath tumors
.
PLoS One
 
2011
;
6
:
e21099

Supporting Information

Author notes

This work was supported by the Wilhelm Sander Stiftung, Munich, Germany (Grant No. 2005.093.1 to Andrea Kindler-Röhrborn).
Supplemental digital content is available for this article. Direct URL citations appear in the printed text and are provided in the HTML and PDF versions of this article on the journal's Web site (http://jnen.oxfordjournals.org/).