The role of neurofibromin in N-Ras mediated AP-1 regulation in malignant peripheral nerve sheath tumors

Abstract

Plexiform neurofibromas commonly found in patients with Neurofibromatosis type I (NF1) have a 5% risk of being transformed into malignant peripheral nerve sheath tumors (MPNST). Germline mutations in the NF1 gene coding for neurofibromin, which is a Ras GTPase activating protein (RasGAP) and a negative regulator of Ras, result in an upregulation of the Ras pathway. We established a direct connection between neurofibromin deficiency and downstream effectors of Ras in cell lines from MPNST patients by demonstrating that knockdown of NF1 expression using siRNA in a NF1 wild type MPNST cell line, STS-26T, activates the Ras/ERK1,2 pathway and increases AP-1 binding and activity. We believe this is the first time the transactivation of AP-1 has been linked directly to neurofibromin deficiency in a disease relevant MPNST cell line. Previously, we have shown that N-Ras is constitutively activated in cell lines derived from independent MPNSTs from NF1 patients. We therefore sought to analyze the role of the N-Ras pathway in deregulating AP-1 transcriptional activity. We show that STS-26T clones conditionally expressing oncogenic N-Ras show increased phosphorylated ERK1,2 and phosphorylated JNK expression concomitant with increased AP-1 activity. MAP kinase pathways (ERK1,2 and JNK) were further examined in ST88-14, a neurofibromin-deficient MPNST cell line. The basal activity of ERK1,2 but not JNK was found to increase AP-1 activity. These experiments further confirmed the link between the loss of neurofibromin and increased activity of Ras/MAP kinase pathways and the activation of downstream transcriptional mechanisms in MPNSTs from NF1 patients.

RAS/MEK–Independent Gene Expression Reveals BMP2-Related Malignant Phenotypes in the Nf1-Deficient MPNST

“…Phosphorylated ERK1/2 increased by 1.6-fold in siNf1-treated STS26T cells ( Supplementary Fig. S1C), and the transcription factor activity of activator protein 1 (AP1) increased as observed in our previous study (18), indicating that the neurofibromin-NRAS-MEK1/2 axis was functional in STS26T cells.…”

“…S1A). U0126, an inhibitor of MEK1/2 in NF1-associated MPNST cells (1,18), reduced the level of phosphorylated ERK1/2 by approximately 90% after 18 hours treatment at 10 mmol/L (Supplementary Fig. S1B).…”

“…The loss-of-function of neurofibromin and hyperactive NRAS have been characterized in ST88-14 cells (1,18). To identify specific targets of neurofibromin-NRAS axis (DS-5), we defined 2 patterns of gene expression change in the comparisons of ST88-14 versus HSC (DS-4) and siNRas versus scrambled control (DS-2): (i) gene expression upregulated in DS-4 but downregulated in DS-2, or (ii) downregulated in DS-4 but upregulated in DS-2 (Table 1; DS-5 in Supplementary Table S2).…”

“…In brief, genes regulated by NRAS, the predominant RAS protein in MPNST cell lines (1), were identified by siRNA interference, and the mitogen-activated protein/extracellular signal-regulated kinase (MEK)1/2-related genes were characterized by U0126 inhibition in ST88-14 cell line, which is derived from patient with NF1 with mutated neurofibromin (14)(15)(16). Neurofibromin-associated gene changes were first characterized by gene profile comparison between ST88-14 and normal HSCs, and further refined by neurofibromin knockdown in STS26T, a sporadic MPNST cell line with the functional, wild-type neurofibromin (17,18). Using intersection analysis of gene expression profiles under these scenarios, alterations in gene regulation exerted by the neurofibromin-regulated pathways were determined.…”

Expression profiling of 519 kinase genes in matched malignant peripheral nerve sheath tumor/plexiform neurofibroma samples is discriminatory and identifies mitotic regulators BUB1B, PBK and NEK2 as overexpressed with transformation

“…29,33 Neurofibromatosis type 1-associated neurofibromas and malignant peripheral nerve sheath tumors arise in a background of a 'rasopathy' with altered Ras/MAP kinase signaling. 3,14,34,35 We therefore looked for changes occurring with the transformation from plexiform neurofibroma to malignant peripheral nerve sheath tumor in the expression level of kinases in this signaling cascade. Most kinases of this set are again downregulated in malignant peripheral nerve sheath tumors.…”