Neurofibromatosis type 1 (NF1) is an autosomal dominant neurocutaneous disorder affecting 1 in 3000 individuals worldwide 1. The NF1 gene, located on chromosome 17q11.2, was identified by positional cloning, and its protein product, neurofibromin, functions as a tumor suppressor 23. Neurofibromin contains a central domain homologous to the Ras-GTPase-activating protein family (Ras-GAPs), which function as negative regulators of Ras proteins 4.

The main clinical features of NF1 are café au lait macules, skinfold freckling and iris Lisch nodules. Patients are at an increased risk of both benign and malignant tumors, and NF1 is thus classified as a tumor predisposition syndrome. The most common tumors are benign peripheral nerve sheath tumors (neurofibromas), which vary greatly in both number and size, and may be dermal or plexiform 5. In contrast to dermal neurofibromas, which are typically small and grow as discrete lesions in the dermis, plexiform neurofibromas can develop internally along the plexus of major peripheral nerves and become quite large 6. Both dermal and plexiform neurofibromas are heterogeneous tumors mainly composed of Schwann cells (60–80%), together with neurons, fibroblasts, mast cells and other cells.

About 5% of patients with NF1, neurofibromas (mainly plexiform neurofibromas) progress to malignant peripheral nerve sheath tumors (MPNSTs). More than 80% of MPNSTs are high-grade malignant tumors, corresponding to WHO grade III-IV 7. MPNSTs are resistant to conventional therapies, and their deep-seated position and locally invasive growth hinder complete surgical resection. The 5-year survival rate among patients with MPNSTs ranges from 30–50%. Schwann cells are considered to be the progenitors of both neurofibromas and MPNSTs, but recent data suggest that other cell types may contribute to the development of these tumors 8.

The molecular mechanisms responsible for malignant progression of neurofibromas are largely unknown. Only a few relevant genetic alterations have so far been identified 910. In keeping with its role as a classical tumor suppressor gene, NF1 loss of heterozygosity (LOH) has been found in NF1-associated MPNSTs (but also in benign neurofibromas) 11. TP53 mutations have been identified in MPNSTs but not in benign neurofibromas, indicating that the p53-mediated pathway is involved in tumor progression 121314. Consistent with the role of p53 in the progression of MPNSTs, mice that harbor both NF1 and TP53 mutations develop MPNSTs 15. Alterations of other genes (p16/CDKN2A, p14/ARF, p27/KIP1, EGFR) are frequent in MPNSTs but not in neurofibromas 1617181920. Taken together, these studies suggest that loss of NF1 initiates tumor formation, and that malignant progression requires additional genetic lesions.

The recent development of efficient tools for large-scale analysis of gene expression has provided new insights into the involvement of gene networks and regulatory pathways in various tumoral processes 21. These methods include cDNA microarrays, which can be used to analyze the expression of thousands of genes at a time, and real-time RT-PCR assays for more accurate and quantitative expression analysis of smaller numbers of candidate genes 22.

To obtain further insight into the molecular pathogenesis of MPNSTs, we used real-time quantitative RT-PCR to quantify the mRNA expression of a large number of selected genes in pooled MPNST samples, in comparison with pooled plexiform neurofibroma samples. We assessed the expression level of 489 genes involved in various cellular and molecular phenomena associated with tumorigenesis. We focused particularly on the expression of genes related to MPNSTs, and genes expressed during Schwann cell differentiation. Forty genes of interest were further investigated in nine individual MPNSTs (all arising from plexiform neurofibromas), in comparison with 14 plexiform neurofibromas.

We first quantified the mRNA expression level of the 489 genes (see list in annex; Additional file 1) in an MPNST pool, a plexiform neurofibroma pool and a dermal neurofibroma pool. We then selected for further study those genes whose expression in the MPNST pool differed markedly (> 10-fold) from that in both the plexiform neurofibroma pool and the dermal neurofibroma pool. These robust selection criteria (a cutoff of 10-fold expression difference in the MPNST pool) ensure identification of gene with marked interest. The mRNA expression of the genes thus identified was then determined in 9 individual MPNSTs (Table 1) in comparison with 14 plexiform neurofibromas.

We used real-time quantitative RT-PCR to quantify the mRNA expression of 489 selected genes in pooled MPNST samples, in comparison with pooled plexiform neurofibroma and pooled dermal neurofibroma samples. Forty genes of interest were then investigated in 9 individual MPNSTs and 14 plexiform neurofibromas. Comparison of pool values with the mean of the corresponding individual values showed that RNA pooling was an appropriate initial screening approach, significantly limiting the required number of PCR experiments. Using the same approach, we have also shown the involvement of several molecular pathways in the genesis of plexiform neurofibroma 23, and other human diseases 24.

Real-time quantitative RT-PCR is a promising alternative to cDNA microarrays for molecular tumor profiling, being far more precise, reproducible and quantitative. Real-time RT-PCR is more useful for analyzing weakly expressed genes (such as TERT, CXCL5, FOXM1 and FOXA2/HNF3B in the present study). Finally, real-time RT-PCR requires smaller amounts of total RNA (about 2 ng per target gene), and is therefore suitable for analyzing small tumors (as in the present study) or microdissected samples.

We included a number of genes known to be involved in various cellular and molecular mechanisms associated with tumorigenesis, and known to be altered (mainly at the transcriptional level) in various cancers. These genes encode proteins involved in cell cycle control, cell-cell interactions, signal transduction pathways, apoptosis, angiogenesis, etc. (about 10–20 genes were selected per pathway). After scrutinizing the literature, we also included most genes reported to be involved in neurofibromas and MPNSTs, and also genes expressed during Schwann cell differentiation.

Among the 489 genes analyzed, 28 (5.7%) showed a significantly different level of expression in MPNSTs relative to plexiform neurofibromas, suggesting that several signaling pathways are specifically involved in MPNST (Tables 2 and 3).

Some of our results support those reported in the literature on NF1-associated MPNSTs. First, several genes associated with cell cycle control (MKI67, TOP2A, CCNE2) were over-expressed in MPNSTs, suggesting higher cell proliferation rates than in plexiform neurofibromas, as previously reported by Kindblom et al. 25. MKI67 encodes Ki-67, a large protein of unknown function used as a classical histopathological marker of cell proliferation. Other proliferation-associated genes (CCND1, CCNE1, etc) were also upregulated in MPNSTs, but less markedly than MKI67, TOP2A and CCNE2. Second, most of mast cell-specific genes (CMA1/Chymase 1 and TPSB/Tryptase beta) and Schwann cell-specific genes (L1CAM, MPZ, S100B, SOX10) tested here were markedly under-expressed in MPNSTs, probably owing either to a lower abundance of a particular cell type (likely mast cells) in MPNSTs relative to plexiform neurofibromas, or to dedifferentiation of a particular cell type (likely Schwann cells). Schwann cell dedifferentiation could also explain the observed under-expression of ITGB4 and ERBB3, key genes in the Schwann cell lineage 2627. DeClue et al. 19 suggested that, following acquisition of ERBB1/EGFR overexpression, Schwann cells may begin to dedifferentiate and lose markers such as ERBB3. In agreement with these data, we observed slight over-expression of ERBB1/EGFR in MPNSTs, whereas the two other ERBB family members (ERBB2 and ERBB4) were normally expressed (Figure 3A). It is noteworthy that, in Schwann cells, ERBB3 could be regulated by SOX10 (found here to be under-expressed in MPNSTs), as in peripheral glial cells 28.

Some of our results for MPNSTs are new, but are in keeping with general concepts of tumorigenesis, in which altered genes are involved in senescence, apoptosis and extracellular matrix remodeling. First, TERT and TERC/hTR, the two main components of human telomerase, were upregulated in MPNSTs. The expression patterns of TERT and TERC/hTR were very similar (r = +0.793, P = 0.00001; Spearman rank correlation test). TERT (human telomerase revere transcriptase) is the rate-limiting factor for telomerase activity, whereas TERC/hTR (human telomerase RNA) also seems to correlate to a certain extent with telomerase reactivation 24. TERT and TERC/hTR are upregulated in almost all malignancies but not in benign tumors 29.

We also found that two genes involved in apoptosis – BIRC5/Survivin and TP73 – were upregulated in MPNSTs. BIRC5/Survivin encodes an antiapoptotic protein overexpressed in common human cancers 30. It is noteworthy that coexpression of BIRC5/Survivin and TERT transcripts is associated with a high risk of tumor-related death among patients with soft-tissue sarcomas 31. TP73, the second apoptosis-related candidate gene identified here, encodes two different proteins that are expressed under the control of two independent promoters and have opposite activities, namely the transcriptionally active full-length protein TAp73, and the amino-terminally truncated dominant-negative protein Δ Np73 32. Unlike TP53, TP73 is mainly regulated at the transcriptional level. TAp73 induces cell-cycle arrest and apoptosis, whereas ΔNp73 inhibits both TAp73-induced and p53-induced apoptosis. Furthermore, ΔNp73 is induced by TAp73 and p53, in a dominant-negative feedback loop that regulates p53 and p73 function 32. Interesting, we found that TAp73 transcription was significantly upregulated in MPNSTs (P < 0.01; AUC-ROC, 0.952), while ΔNp73 transcription was significantly down-regulated (P < 0.01; AUC-ROC, 0.917) (data not shown).

MPNSTs showed upregulation of two matrix metalloproteinase genes (MMP9 and MMP13) and down-regulation of a tissue inhibitor of metalloproteinases (TIMP4). MMPs are associated with extracellular matrix turnover, a process that plays a very active role in tumor invasion and metastasis 33. For their part, TIMPs play a key role in extracellular matrix homeostasis by regulating MMP activity 34. Many reports suggest that an MMP-TIMP imbalance may contribute to the malignant phenotype 3334. The other extracellular matrix-related genes tested in the present studies showed no statistically differentially expression levels between MPNSTs and plexiform neurofibromas (Figures 3B and 3C). We have previously identified MMP9 as a major upregulated gene in plexiform neurofibromas compared to dermal neurofibromas 23 (Figure 3B), suggesting that MMP9 upregulation may be an early event in MPNST tumorigenesis.

Finally, our results suggest that inappropriate activation of molecular signaling pathways occurs specifically in NF1-associated MPNST or in limited human tumors, particularly brain and skin tumors. MPNSTs showed upregulation of CXCL5, the gene encoding the chemokine ENA-78 (epithelial cell-derived neutrophil-activating peptide) 35. Chemokines play fundamental roles in the development, homeostasis and functioning of the immune system, but also have effects on endothelial cells and cells of the nervous system 36. CXCL5 is physically linked, in chromosomal region 4q12-q13, to other chemokine genes with markedly similar nucleotide sequences, including CXCL6, IL8 and GRO1/CXCL1 37. We found that CXCL6 was slightly upregulated (trend toward statistical significance) in MPNSTs. We have previously detected IL8 and GRO1/CXCL1 upregulation in the early stages of plexiform neurofibroma tumorigenesis 23 (Figure 3D). Our results thus point to a role of paracrine and autocrine signaling defects involving these chemokines, located in 4q12-q13, in the tumorigenesis of plexiform neurofibromas and/or MPNSTs. Comparative genomic hybridization analysis has shown gains of chromosome arm 4q12-ter in both sporadic and NF1-associated MPNSTs 38, suggesting co-overexpression of these chemokines by a DNA amplification mechanism.

We identified two genes (RASSF2, HMMR/RHAMM) putatively involved in the Ras signaling pathway; RASSF2 was down-regulated, whereas HMMR/RHAMM was upregulated. RASSF2 is a member of the RASSF family genes that encode for proteins which bind directly to Ras protein in a GTP-dependent manner via a Ras effector domain. This family currently has six members, RASSF1, RASSF2, RASSF3, RIN2, AD037 and NORE1. RASSF proteins promote both cell cycle arrest and apoptosis; they behave as tumor suppressor genes, and their down-regulation might play a key role in tumorigenesis. RASSF genes are a new class of tumor suppressor genes for which epigenetic silencing is an overwhelming mechanism of inactivation, and somatic mutations are rare 3940. Vos et al. 41recently obtained evidence that RASSF2 is specific to K-Ras, as only a weak interaction with H-ras was detected. The other five RASSF genes were also tested here, and showed no difference in their expression levels between MPNSTs and plexiform neurofibromas (Figure 3E). The second Ras signaling-associated gene identified here, HMMR/RHAMM, is an oncogene that regulates signaling through Ras and controls mitogen-activated protein kinase (ERK protein) expression 42. However, HMMR/RHAMM has also recently been characterized as a centrosomal protein that interacts with dynein and maintains spindle pole stability 43. Finally, HMMR/RHAMM has been identified as a major cell-cycle-regulated gene by cDNA microarray analysis 44.

Interesting, seven of the 28 genes showing significant differential expression between MPNSTs and plexiform neurofibromas are involved in the Hedgehog-Gli signaling pathway, namely DHH, PATCH2 and five downstream target genes of Gli transcription factors (SPP1/Osteopontin, FOXA2/HNF3B, FOXM1, OSF-2/Periostin and EPHA7) 45464748. The Hedgehog-Gli signaling pathway is important in regulating patterning, proliferation, survival and growth in both embryos and adults 49. Inappropriate activation of the Hedgehog-Gli signaling pathway occurs in several tumor types, including brain and skin tumors 50. PTCH2 functions as a tumor suppressor gene, as its product normally inhibits SMOH (which functions as an oncogene), resulting in Gli inhibition. Mutations of human PTCH1 and PTCH2 have been detected in basal cell carcinomas and medulloblastomas, and result in Gli signaling activation 50. Other genes associated with the Hedgehog-Gli signaling pathway were slightly (but not significantly) upregulated in MPNSTs, including SMOH, HHIP (a Hedgehog-interacting protein), three human GLI genes (GLI2, GLI3 and GLI4; Figures 3F) and the major Gli-regulated genes IGF2, PTHR1 and H19 (Figures 3G) 474851. The eight Gli-regulated genes that we found altered in MPNSTs encode growth factors (IGF2, SPP1/Osteopontin, OSF-2/Periostin), growth factor receptors (PTHR1, EPHA7) and transcription factors (FOXA2/HNF3B, FOXM1), whereas H19 yields an untranslated mRNA. Several of these genes are altered in various tumors 45525354. The positive correlation between H19 and IGF2 expression in our tumor series argues against abnormal IGF2 imprinting as a source of IGF2 over-expression in MPNSTs. Indeed, a reduction or loss of H19 expression would be expected to result in increased IGF2 transcription, as expression of these two genes is functionally linked by their competition for a common enhancer 55.

We have previously found that SHH, GLI1, IGF2 and H19 are up-regulated in plexiform neurofibromas 23 (Figures 3F and 3G). Taken together, our results suggest that activation of the Hedgehog-Gli signaling pathway is not only an early event in the genesis of benign plexiform neurofibromas, but that it is also required for malignant transformation.

In conclusion, this study points to the involvement of several altered molecular pathways, and especially the Hedgehog-Gli signaling pathway, in the tumorigenesis of NF1-associated MPNST. Further studies are necessary to elucidate the genetic (or epigenetic) mechanisms responsible for the altered gene expression. It will be of interest to study the genes identified here in sporadic MPNSTs and in other neurological tumors (medulloblastomas, oligodendrogliomas, astrocytomas, neuroblastomas and schwannomas).

PW was responsible for patients screening. KL and JW determined the histological diagnosis. PL, IL, BP and GB designed the 489 primers. Real-time RT-PCR have been carried out by IL. IB, IL and PL interpreted the result, performed bioinformatics and statistical analyses. IB managed the limiting factors. This study has been supervised by DV, MV and IB.