Neurofibromatosis type 1 (NF1) is a common autosomal dominant disorder affecting 1 in 3,500 individuals worldwide. Typical clinical features of NF1 include multiple café-au-lait spots, axillary freckling, iris Lisch nodules, and neurofibromas. Neurofibromas are benign peripheral nerve sheath tumors designated as either dermal neurofibromas (DNFs) or plexiform neurofibromas (PNFs). PNFs are regarded as congenital benign tumors that often develop in association with major nerve tracts. About 30 to 50% of NF1 patients develop clinically visible PNFs 1 . Although PNFs are considered as benign tumors, they can be life threatening when they develop deeply and compress internal organs. PNFs are complex tumors, heterogeneous at the cellular level, mainly composed of Schwann cells, which are the likely pathogenic cell type in neurofibromas, together with fibroblasts, mast cells, neurons, vascular elements, and perineurial cells. In contrast to DNFs, PNFs can transform in malignant peripheral nerve sheath tumors (MPNST) in about 10% of the cases. MPNSTs are highly invasive sarcomas that metastasize widely and display a poor prognosis.

NF1 is caused by germline heterozygous loss-of-function mutations in the tumor suppressor gene NF1 located at 17q11.2. NF1 encodes neurofibromin, a RAS-GTPase-activating protein (RAS-GAP). Neurofibromin negatively regulates the RAS signalling pathways (i.e. MAPK and PI3K/AKT/mTOR) involved in proliferation, survival, and differentiation. According to the Knudson’s two-hit model, NF1 tumorigenesis results from a somatic mutation disrupting the second functional copy of the NF1 gene. This complete inactivation of NF1 induces RAS signaling pathway activation and seems required but not sufficient for tumorigenesis promotion. Rare additional molecular alterations have been described in PNFs, including CDKN2A/B locus deletions 2 . In contrast, MPNSTs are characterized by complexe genomic changes including inactivation of TP53 and RB1 and amplification of EGFR, HGF, and MET 3 . Expression studies have reported differential expression profile in MPNSTs of genes involved in cell proliferation, apoptosis, invasion, extracellular remodeling and Schwann cell differentiation such as TP53, RB1, CDKN2A, TWIST1, BIRC5, TOP2A, and SOX9 4 5 6 . Finally, transgenic mouse models have provided evidences for TP53 and PTEN implication in PNFs formation and Schwann cells malignant transformation 7 8 .

Since their initial discovery in Caenorhabditis elegans in 1993 9 , the highly conserved small non-coding RNAs called microRNAs (miRNAs) have been extensively implicated in human physiology and pathology. In the last few years, miRNAs have revealed major roles in regulating critical biological processes such as development, proliferation, differentiation, and apoptosis. MiRNAs aberrant expression has also been characterized in many human cancer types. However, the involvement of miRNAs deregulation in the formation of benign neurofibromas and malignant progression from PNFs to MPNSTs remains largely unknown. Here, we used real-time quantitative reverse transcription-PCR (RT-PCR) assays to quantify the expression of a panel of 377 well-validated miRNAs in a large series of NF1-associated tumors (including nine DNFs, 41 PNFs, and 15 MPNSTs), two normal peripheral nerve samples, and two MPNST cell lines.

We quantified the expression of 377 miRNAs in nine DNFs, 41 PNFs, and 15 MPNSTs. We also analyzed miRNAs expression in two adult peripheral nerves as a non-tumorigenic control tissue and in two MPNST cell lines (ST88.14 and 90–8) as malignant controls. A significant number (122/377; 32.4%) of miRNAs were below the detection level of the assay (median Ct ≥ 40) in MPNSTs, PNFs, and DNFs and were consequently regarded as “not expressed”. Eighty-four (84/377; 22.3%) miRNAs were considered as detectable but not reliably quantifiable (32 < median Ct < 40) in the three groups of tumors. Thereby, more than half of miRNAs (206/377; 54.6%) were not further analyzed in our study. In each sample, the negative control assay unrelated to mammalian species, ath-miR159a, was not expressed (Ct ≥ 40).

Recent evidences indicate that miRNA network play a critical role in the regulation of gene expression involved in tumor development and progression. In this study, we applied a RT-PCR analysis to comprehensively characterize the expression pattern of 377 miRNAs in a large series of benign and malignant NF1-related nerve sheath tumors.

Eleven miRNAs were found to be differentially expressed between DNFs and PNFs (Table 1). Interestingly, the most significantly upregulated miRNA in PNFs, miR-486-3p, targets the major tumor suppressor gene, PTEN 10 . PTEN expression is frequently decreased in a wide spectrum of human cancers with several miRNAs being validated as PTEN regulators 37 . Using murine conditional deletion of Pten and activation of Kras, Gregorian at al. suggested that PTEN dosage is critical for formation of PNFs 8 . MiRNAs regulation typically allows such subtle modulations in gene expression dosage. It is important to note that the two tested peripheral nerve samples (non-tumorigenic samples) showed similar level expression of miR-486-3p than DNF samples (Table 1). Overexpression of miR-486-3p in PNFs may therefore specifically reflect an abnormal upregulation in these tumors. We further hypothesised that miR-486-3p may be a major onco-miR by downregulating PTEN in PNFs development.

Our results also reveal overexpression of miR181a in PNFs. MiR181a targets ATM 14 , a tumor suppressor that regulates the p53 pathway 38 that has been shown to be essential for PNFs formation in transgenic mouse models 7 . Similar level expression of miR181a was found in both peripheral nerve samples and in DNFs samples (Table 1).

Finally, three other markedly overexpressed miRNAs in PNFs compared to DNFs have previously been demonstrated to negatively regulate the RAS signaling pathway: miR-370 targets MAP3K8 11 , miR-143 targets KRAS 12 13 , and miR-145 targets RREB1 15 . Biallelic loss of function of the NF1 gene in neurofibromas promotes activation of the RAS signaling pathway. We assume that overexpression of these four miRNAs may reveal a feedback loop that attempts to correct hyperactivation of RAS signaling. Interestingly, miR-143 and miR-145 are both located at 5q32 and have positive correlated expressions, suggesting a common transcription unit, allowing a concerted action on RAS pathway.

We also investigated the 113 differentially expressed miRNAs between PNFs and MPNSTs. Among the 103 upregulated and the ten downregulated miRNAs in MPNSTs, we further analyzed the most markedly differentially expressed miRNAs (Table 2). A subset of miRNAs (miR-301a 26 , miR19a 29 , and miR-106b 31 32 ) previously demonstrated to directly target the suppressor PTEN was found to be overexpressed in MPNSTs. Recent transgenic mice model with Pten and Nf1 conditional knock out in Schwann cells, implicated the synergistic role of Pten and Nf1 inactivation in MPNSTs development 23 . We confirmed that PTEN expression was inversely correlated with miR-301a, miR-19a, and miR-106b. Interestingly, we showed that paralogs miR-301a and miR-301b have significantly positive correlated expression suggesting that these two miRNAs may synergistically target PTEN.

We also identified a panel of five deregulated miRNAs (miR-135a, miR-135b, miR-9, miR-200c, and let-7b) that are major regulators of epithelial-mesenchymal transition (EMT). MiR-135b and miR-135a represent the most overexpressed miRNAs in MPNSTs compared to PNFs (Table 2). Members of the miR-135 family target the tumor suppressor APC, a key negative regulator of the WNT canonical pathway involved in tumorigenesis 39 . E-cadherin 27 is a major epithelial adherens junction protein which plays a critical role in EMT. MiR-9 is also overexpressed in MPNSTs and it has been previously demonstrated that miR-9 is upregulated in breast cancer cells and targets the E-cadherin gene (also named CDH1) 40 .

MiR-200c and let-7b are both downregulated in MPNSTs compared to PNFs. Members of miR-200 family act as critical regulators of EMT and contribute to the acquisition of invasive behavior in many types of cancer 22 . They negatively regulate expression of ZEB1 19 and ZEB2, two E-box binding transcription factors that are powerful regulators of E-cadherin expression and several other effectors involved in epithelial polarity. Let-7 family miRNAs regulate cell proliferation and differentiation and are frequently downregulated in many human cancers 21 . They are considered as tumor suppressor miRNAs, notably through targeting genes with oncogenic activity such as RAS 21 and HMGA2 41 . We confirmed that HMGA2 expression is inversely correlated with let-7b. HMGA2 enhances TWIST (whom expression is regulated by WNT signaling) and inhibits CDKN2A/CDKN2B gene expression, corresponding with aggressive behavior 42 43 44 . Hyperactivation of RAS signaling pathway, overexpression of TWIST1 6 and CDKN2A inactivation 3 have frequently been reported in MPNSTs. Together, upregulation of miR-135a, miR-135b, and miR-9 and downregulation of miR-200c and let-7b in MPNSTs may thus contribute to mesenchymal transition with acquisition of an invasive behavior in these malignant aggressive tumors.

Our results also independently confirm that miR-10b is significantly upregulated in MPNSTs compared to PNFs. Expression of miR-10b is induced by the metastasis-promoting transcription factor TWIST 44 . Chai et al. previously showed overexpression of miR-10b in MPNSTs and demonstrated that miR-10b targets NF1 mRNA 44 . MiR-10b controls expression of the pro-metastatic RhoC via directly targeting HOXD10 mRNA 33 . HOXD10 encodes a homeobox (HOX) protein that act as a transcriptional repressor of RHOC. Aberrant expression of RHO GTPases or RHO effectors has been described in various cancer types, suggesting the implication of RHO signaling pathway in NF1 tumorigenesis 34 . Interestingly, our analysis of miRNAs expression in MPNSTs revealed a significant upregulation of others miRNAs that target HOX genes (Table 2): miR-196b targets HOXB8 35 , miR-210 targets HOXA1, HOXA9, and HOXA3 25 , miR-10a and miR-10b target HOXA3 and HOXD10 28 , and miR-9 target CDX2 45 . HOX genes represent a large group of related genes encoding transcription factors with crucial roles in development, apoptosis, differentiation, and cell motility 46 .

Among the most markedly differentially expressed miRNAs in MPNSTs (Table 2), we also identified a panel of significantly deregulated miRNAs (including miR-195, let-7b, miR-210, miR-20a, miR-19a, miR-449a, miR-129-3p, and miR-106b) that target major regulators of cell cycle. MiR-195 is downregulated in MPNSTs and has recently been described as a tumor suppressor in various types of cancers 16 . MiR-195 targets several G1/S transition-related genes such as cyclin D1 (CCND1), CDK6, E2F3 17 , and CCNE1 20 . Let-7b (downregulated in MPNSTs, Table 2) has also been shown to repress CCND1 expression 30 . In addition miR-210 targets E2F3 25 , miR-20a targets CCND1 and E2F1 31 , miR-19a and miR-449a target CCND1 24 , miR-129-3p targets CDK6 47 , and miR-106b targets CCND1 and E2F1 31 . Interestingly, NF1 inactivation has previously been shown to enhance CCND1 expression in Schwann cells 18 . Overexpression of CCND1 could thus account for a major feature of the Schwann cells proliferation in MPNSTs formation. Moreover, miR-210 has been described as a key regulator of the hypoxia response in tumor onset identified as a major HIF1A-induced miRNA, and makes link between hypoxia and the regulation of cell cycle 25 . We confirmed that miR-210 has a significant positive correlated expression with HIF1A (r = +0.57; P = 5.8.10^-6).

A subset of significantly deregulated miRNAs in MPNSTs (let-7b, miR-195, and miR-10b) has previously been described to target members of the RAS-MAPK pathway. Let-7b is a member of the let-7 miRNAs family which target RAS 21 . Recent data report that miR-195 targets RAF1 48 , a direct downstream effector of RAS. Interestingly, let-7b and miR-195 are both downregulated in MPNSTs compared to PNFs and could subsequently act as indirect activator of the RAS-MAPK pathway. Additionally, miR-10b is upregulated in MPNSTs and has been previously demonstrated to target NF1 44 .

In conclusion, miRNAome profiling of NF1-associated peripheral nerve sheath tumors suggests intricately deregulated associated molecular networks. We identified several subsets of markedly deregulated miRNAs in PNFs, in particular miR-486-3p that targets PTEN, a major component of the RAS-PI3K-AKT signaling pathway. Our work highlights the key role of miRNAs in NF1-associated tumorigenesis, leading to subtle reductions in PTEN levels. Our result also confirmed the major role of PTEN in MPNSTs formation. Our study identifies miRNAs clearly deregulated in MPNSTs and involved in oncogenic events including loss of cell adhesion and metastatic behavior mediated by EMT, deregulation of HOX genes expression, increased cell cycle progression, and RAS-MAPK aberrant signaling. All of these miRNAs should be further investigated using in vitro or in vivo experiments to fully understand their role in the formation of neurofibromas and MPNSTs and to identify mechanisms involved in their deregulation. Finally, our results may serve for the basis of potential diagnostic and predictive biomarkers and represent novel strategies for effective pharmacological therapies of NF1 tumors.

The authors declare that they have no competing interests.

MV, DV, IB, and PW participated in the design of the study, and interpretation of data. KL participated in tumor samples collection. NO performed tumors anatomopathologic analysis. LVA, and PW provided clinical follow-up information of the patient cohort. MH, VD, and LL performed tumor surgical excision. JV, IL, and AL performed the experiments and JMP and IB were responsible for data analysis. IB, JMP, and EP have been involved in writing the manuscript with assistance from BP, and MH. All authors read and approved the final manuscript.