The Ras/Raf/ERK signalling pathway drives Schwann cell dedifferentiation

Schwann cells are a regenerative cell type. Following nerve injury, a differentiated myelinating Schwann cell can dedifferentiate and regain the potential to proliferate. These cells then redifferentiate during the repair process. This behaviour is important for successful axonal repair, but the signalling pathways mediating the switch between the two differentiation states remain unclear. Sustained activation of the Ras/Raf/ERK cascade in primary cells results in a cell cycle arrest and has been implicated in the differentiation of certain cell types, in many cases acting to promote differentiation. We therefore investigated its effects on the differentiation state of Schwann cells. Surprisingly, we found that Ras/Raf/ERK signalling drives the dedifferentiation of Schwann cells even in the presence of normal axonal signalling. Furthermore, nerve wounding in vivo results in sustained ERK signalling in associated Schwann cells. Elevated Ras signalling is thought to be important in the development of Schwann cell-derived tumours in neurofibromatosis type 1 patients. Our results suggest that the effects of Ras signalling on the differentiation state of Schwann cells may be important in the pathogenesis of these tumours.     

Single cell Ras-GTP analysis reveals altered Ras activity in a subpopulation of neurofibroma Schwann cells but not fibroblasts

Neurofibromatosis type 1 (NF1) is a common genetic disorder characterized by multiple neurofibromas, peripheral nerve tumors containing mainly Schwann cells and fibroblasts. The NF1 gene encodes neurofibromin, a tumor suppressor postulated to function in part as a Ras GTPase-activating protein. The roles of different cell types and of elevated Ras-GTP in neurofibroma formation are unclear. To determine which neurofibroma cell type has altered Ras-GTP regulation, we developed an immunocytochemical assay for active, GTP-bound Ras. In NIH 3T3 cells, the assay detected overexpressed, constitutively activated K-, N-, and Ha-Ras and insulin-induced endogenous Ras-GTP. In dissociated neurofibroma cells from NF1 patients, Ras-GTP was elevated in Schwann cells but not fibroblasts. Twelve to 62% of tumor Schwann cells showed elevated Ras-GTP, unexpectedly revealing neurofibroma Schwann cell heterogeneity. Increased basal Ras-GTP did not correlate with increased cell proliferation. Normal human Schwann cells, however, did not demonstrate elevated basal Ras activity. Furthermore, compared with cells from wild type littermates, Ras-GTP was elevated in all mouse Nf1(-/-) Schwann cells but never in Nf1(-/-) mouse fibroblasts. Our results indicate that Ras activity is detectably increased in only some neurofibroma Schwann cells and suggest that neurofibromin is not an essential regulator of Ras activity in fibroblasts.     

Poster Sessions BP02: Oligodendrocytes and Schwann Cells

Neurofibromatosis Type 1 tumors are highly vascularized and contain Schwann cells with hyperactivated Ras. In vitro , the NF1-derived neurofibromin deficient Schwann cells have an angiogenic profile, which favors angiogenesis and sustains the growth of the NF1-derived tumors. This study examined the relationship of the activation state of Ras as it related to the expression of angiogenic and antiangiogenic factors in both cultured NF1-derived Schwann cells and normal human Schwann cells. Western blot analysis of normal human Schwann cells revealed low expression of angiogenic vascular endothelial growth factor (VEGF) as well as low expression of the antiangiogenic pigment epithelium derived factor (PEDF). Relative to normal human Schwann cells, NF1-derived Schwann cells have increased RAS activity and a three-fold increase in VEGF expression. Surprisingly, PEDF was also expressed in the NF1-derived Schwann cells at approximately the same level as VEGF expression. Using a retroviral construct, we introduced the GAP-related domain of neurofibromin into the NF1-derived Schwann cells to reduce the level of activated Ras. Relative to the untreated NF1-derived Schwann cells the Schwann cells expressing the GAP-related domain expressed about one-half the VEGF but twice the PEDF. We conclude that decreasing the Ras activity in NF1-drived Schwann cells will not only decrease proliferation, but also slow tumor angiogenesis due to the decreased expression of angiogenic and increased expression of antiangiogenic factors.     

Poster Sessions BP02: Oligodendrocytes and Schwann Cells. RAS activation regulates angiogenic and anti‐angiogenic factor expression in NF1‐derived Schwann cells

Neurofibromatosis Type 1 tumors are highly vascularized and contain Schwann cells with hyperactivated Ras. In vitro, the NF1‐derived neurofibromin deficient Schwann cells have an angiogenic profile, which favors angiogenesis and sustains the growth of the NF1‐derived tumors. This study examined the relationship of the activation state of Ras as it related to the expression of angiogenic and antiangiogenic factors in both cultured NF1‐derived Schwann cells and normal human Schwann cells. Western blot analysis of normal human Schwann cells revealed low expression of angiogenic vascular endothelial growth factor (VEGF) as well as low expression of the antiangiogenic pigment epithelium derived factor (PEDF). Relative to normal human Schwann cells, NF1‐derived Schwann cells have increased RAS activity and a three‐fold increase in VEGF expression. Surprisingly, PEDF was also expressed in the NF1‐derived Schwann cells at approximately the same level as VEGF expression. Using a retroviral construct, we introduced the GAP‐related domain of neurofibromin into the NF1‐derived Schwann cells to reduce the level of activated Ras. Relative to the untreated NF1‐derived Schwann cells the Schwann cells expressing the GAP‐related domain expressed about one‐half the VEGF but twice the PEDF. We conclude that decreasing the Ras activity in NF1‐drived Schwann cells will not only decrease proliferation, but also slow tumor angiogenesis due to the decreased expression of angiogenic and increased expression of antiangiogenic factors.     

Reconstitution of the NF1 GAP-related domain in NF1-deficient human Schwann cells

Schwann cells derived from peripheral nerve sheath tumors from individuals with Neurofibromatosis Type 1 (NF1) are deficient for the protein neurofibromin, which contains a GAP-related domain (NF1-GRD). Neurofibromin-deficient Schwann cells have increased Ras activation, increased proliferation in response to certain growth stimuli, increased angiogenic potential, and altered cell morphology. This study examined whether expression of functional NF1-GRD can reverse the transformed phenotype of neurofibromin-deficient Schwann cells from both benign and malignant peripheral nerve sheath tumors. We reconstituted the NF1-GRD using retroviral transduction and examined the effects on cell morphology, growth potential, and angiogenic potential. NF1-GRD reconstitution resulted in morphologic changes, a 16-33% reduction in Ras activation, and a 53% decrease in proliferation in neurofibromin-deficient Schwann cells. However, NF1-GRD reconstitution was not sufficient to decrease the in vitro angiogenic potential of the cells. This study demonstrates that reconstitution of the NF1-GRD can at least partially reverse the transformation of human NF1 tumor-derived Schwann cells.     

Brain lipid binding protein in axon-Schwann cell interactions and peripheral nerve tumorigenesis

Loss of axonal contact characterizes Schwann cells in benign and malignant peripheral nerve sheath tumors (MPNST) from neurofibromatosis type 1 (NF1) patients. Tumor Schwann cells demonstrate NF1 mutations, elevated Ras activity, and aberrant epidermal growth factor receptor (EGFR) expression. Using cDNA microarrays, we found that brain lipid binding protein (BLBP) is elevated in an EGFR-positive subpopulation of Nf1 mutant mouse Schwann cells (Nf1(-/-) TXF) that grows away from axons; BLBP expression was not affected by farnesyltransferase inhibitor, an inhibitor of H-Ras. BLBP was also detected in EGFR-positive cell lines derived from Nf1:p53 double mutant mice and human MPNST. BLBP expression was induced in normal Schwann cells following transfection with EGFR but not H-Ras12V. Furthermore, EGFR-mediated BLBP expression was not inhibited by dominant-negative H-Ras, indicating that BLBP expression is downstream of Ras-independent EGFR signaling. BLBP-blocking antibodies enabled process outgrowth from Nf1(-/-) TXF cells and restored interaction with axons, without affecting cell proliferation or migration. Following injury, BLBP expression was induced in normal sciatic nerves when nonmyelinating Schwann cells remodeled their processes. These data suggest that BLBP, stimulated by Ras-independent pathways, regulates Schwann cell-axon interactions in normal peripheral nerve and peripheral nerve tumors.     

Prostaglandin E(2) metabolism is activated in Schwann cell lines derived from human NF1 malignant peripheral nerve sheath tumors

Malignant peripheral nerve sheath tumors (MPNSTs) are characteristic of Neurofibromatosis type 1 (NF1), a human genetic disorder affecting approximately 1 in 3000 individuals. The absence of neurofibromin in Schwann cells results in hyperactivation of Ras, which contributes to Schwann cell hyperplasia. However, additional intracellular abnormalities in Schwann cells might contribute to the malignancy. We now report that cell lines derived from MPNSTs secrete elevated levels of prostaglandin E(2) (PGE(2)), express higher levels of phosphorylated mitogen-activated protein kinase (MAPK), phosphorylated cytosolic phospholipaseA(2) (cPLA(2)) and cyclooxygenase 2 (COX-2) when compared to normal adult human Schwann cells (nhSCs). PCR analysis reveals that NF1 MPNST cell lines express mRNA for both EP2 and EP4 prostaglandin E2 receptors, whereas nhSCs express only the EP4 receptor. COX-2 inhibitors and PGE(2) receptor antagonists decrease the proliferation of MPNST cell lines. These results indicate that prostaglandin metabolism is activated in MPNSTs and might contribute to tumor growth in NF1.     

Angiogenic and invasive properties of neurofibroma Schwann cells

Neurofibromas are benign tumors from patients with von Recklinghausen Neurofibromatosis (NF1) that are comprised primarily of Schwann cells. These Schwann cells are found both in association with axons and in the extracellular matrix that is prevalent in neurofibromas, and in which fibroblasts are also abundant. An unresolved question has been whether cells in neurofibromas are normal cells or are intrinsically abnormal. We have tested the hypothesis that cells in neurofibromas are abnormal and have shown that neurofibroma Schwann cells, unlike normal Schwann cells, promote angiogenesis in the chick chorioallantoic membrane model system, and invade basement membranes in this system. In contrast, neurofibroma fibroblasts neither promote angiogenic reactions nor invade basement membranes. When injected into nude mice, neurofibroma Schwann cells do not form progressive tumors. These results suggest that NF1 Schwann cells differ from normal Schwann cells, that they are preneoplastic, and that genetic and/or epigenetic changes in Schwann cells may be required for development of peripheral nerve tumors in NF1.     

Sensitivity of Malignant Peripheral Nerve Sheath Tumor Cells to TRAIL Is Augmented by Loss of NF1 through Modulation of MYC/MAD and Is Potentiated by Curcumin through Induction of ROS

Malignant peripheral nerve sheath tumor (MPNST) is a rare aggressive form of sarcoma often associated with the tumor syndrome neurofibromatosis type 1 (NF1). We investigated the effects of tumor necrosis factor-related apoptosis inducing ligand (TRAIL) on NF1 associated MPNST and determinants of TRAIL sensitivity. MPNST cell lines with complete neurofibromin deficiency were sensitive to apoptotic cell death induced by TRAIL whereas MPNST cells with retained neurofibromin expression or normal human Schwann cells were resistant. Increased sensitivity to TRAIL was associated with overexpression of death receptors, especially DR5. Re-expression of the GAP related domain of neurofibromin (NF1-GRD) suppressed DR5 expression and decreased sensitivity to TRAIL. We show that death receptor expression and TRAIL sensitivity critically depend on c-MYC and that c-MYC amounts are increased by MEK/ERK and PI3K/AKT signalling pathways which are suppressed by neurofibromin. Furthermore PI3K/AKT signalling strongly suppresses the MYC-antagonist MAD1 which significantly contributes to TRAIL sensitivity. Re-expression of the NF1-GRD decreased c-MYC and increased MAD1 amounts suggesting that neurofibromin influences TRAIL sensitivity at least in part by modulating the MYC/MAX/MAD network. The phytochemical curcumin further increased the sensitivity of neurofibromin deficient MPNST cells to TRAIL. This was presumably mediated by ROS, as it correlated with increased ROS production, was blocked by N-acetylcysteine and mimicked by exogenous ROS.     

Ras signals principally via Erk in G1 but cooperates with PI3K/Akt for Cyclin D induction and S-phase entry

Numerous studies exploring oncogenic Ras or manipulating physiological Ras signalling have established an irrefutable role for Ras as driver of cell cycle progression. Despite this wealth of information the precise signalling timeline and effectors engaged by Ras, particularly during G1, remain obscure as approaches for Ras inhibition are slow-acting and ill-suited for charting discrete Ras signalling episodes along the cell cycle. We have developed an approach based on the inducible recruitment of a Ras-GAP that enforces endogenous Ras inhibition within minutes. Applying this strategy to inhibit Ras stepwise in synchronous cell populations revealed that Ras signaling was required well into G1 for Cyclin D induction, pocket protein phosphorylation and S-phase entry, irrespective of whether cells emerged from quiescence or G2/M. Unexpectedly, Erk, and not PI3K/Akt or Ral was activated by Ras at mid-G1, albeit PI3K/Akt signalling was a necessary companion of Ras/Erk for sustaining cyclin-D levels and G1/S transition. Our findings chart mitogenic signaling by endogenous Ras during G1 and identify limited effector engagement restricted to Raf/MEK/Erk as a cogent distinction from oncogenic Ras signalling.     

Aberrant cAMP Metabolism in NF1 Malignant Peripheral Nerve Sheath Tumor Cells

Malignant peripheral nerve sheath (MPNST) cell lines derived from patients with neurofibromatosis type 1 (NF!) were found to have basal cAMP levels which are two-fold higher than cAMP levels in normal human adult Schwann cells (nHSC). PCR analysis also revealed that normal adult human Schwann cells express mRNA for types Ill, IV, and IX adenylyl cyclase (AC) while NF1 MPNST cells express AC mRNA of types II, V, and VIII in addition to expressing all the isoforms of normal adult human Schwann cells. Further PCR analysis revealed that NF1 MPNST lines express mRNA for EP2 and EP4 prostaglandin receptors whereas nHSC only express mRNA for the EP2 receptor. Exogenous prostaglandins alone or in combination with PDGF BB induced greater increases in cAMP levels and proliferation of NF1 MPNST cells compared to nHSC. We conclude that aberrant cAMP signaling in NF1 MPNST cells contributes to tumor formation in NF1 patients.     

Neurofibromatosis type I tumor suppressor neurofibromin regulates neuronal differentiation via its GTPase-activating protein function toward Ras

Neurofibromin, the neurofibromatosis type 1 (NF1) gene product, contains a central domain homologous to a family of proteins known as Ras-GTPase-activating proteins (Ras-GAPs), which function as negative regulators of Ras. The loss of neurofibromin function has been thought to be implicated in the abnormal regulation of Ras in NF1-related pathogenesis. In this study, we found a novel role of neurofibromin in neuronal differentiation in conjunction with the regulation of Ras activity via its GAP-related domain (GRD) in neuronal cells. In PC12 cells, time-dependent increases in the GAP activity of cellular neurofibromin (NF1-GAP) were detected after NGF stimulation, which were correlated with the down-regulation of Ras activity during neurite elongation. Interestingly, the NF1-GAP increase was due to the induction of alternative splicing of NF1-GRD type I triggered by the NGF-induced Ras activation. Dominant-negative (DN) forms of NF1-GRD type I significantly inhibited the neurite extension of PC12 cells via regulation of the Ras state. NF1-GRD-DN also reduced axonal and dendritic branching/extension of rat embryonic hippocampal neurons. These results demonstrate that the mutual regulation of Ras and NF1-GAP is essential for normal neuronal differentiation and that abnormal regulation in neuronal cells may be implicated in NF1-related learning and memory disturbance.     

Neurofibromin GTPase-activating protein-related domains restore normal growth in Nf1-/- cells

Members of the Ras superfamily of signaling proteins modulate fundamental cellular processes by cycling between an active GTP-bound conformation and an inactive GDP-bound form. Neurofibromin, the protein product of the NF1 tumor suppressor gene, and p120GAP are GTPase-activating proteins (GAPs) for p21(Ras) (Ras) and negatively regulate output by accelerating GTP hydrolysis on Ras. Neurofibromin and p120GAP differ markedly outside of their conserved GAP-related domains (GRDs), and it is therefore unknown if the respective GRDs contribute functional specificity. To address this question, we expressed the GRDs of neurofibromin and p120GAP in primary cells from Nf1 mutant mice in vitro and in vivo. Here we show that expression of neurofibromin GRD, but not the p120GAP GRD, restores normal growth and cytokine signaling in three lineages of primary Nf1-deficient cells that have been implicated in the pathogenesis of neurofibromatosis type 1 (NF1). Furthermore, utilizing a GAP-inactive mutant NF1 GRD identified in a family with NF1, we demonstrate that growth restoration is a function of NF1 GRD GAP activity on p21(Ras). Thus, the GRDs of neurofibromin and p120GAP specify nonoverlapping functions in multiple primary cell types.     

Klinik und Genetik der Neurofibromatose Typ 1

Neurofibromatosis type 1 (NF1) is an autosomal dominant disorder caused by mutations in the NF1 gene located on chromosome 17q11.2. NF1 is fully penetrant, meaning that every individual carrying the mutation exhibits symptoms of the disease, although with some considerably variable expressivity. NF1 is characterised by the eponymous neurofibromas, which are benign Schwann cell tumours. Among the other main characteristic features of NF1 are pigmentary anomalies such as café-au-lait spots, axillary or inguinal freckling, and Lisch nodules. NF1 is a member of the class of hereditary cancer syndromes, and patients with NF1 are at increased risk of developing specific NF1-associated tumours. These tumours are caused by the biallelic inactivation of the NF1 tumour suppressor gene, resulting in aberrant Ras regulation. Over the last few years, significant progress has been made in identifying and managing the clinical symptoms of NF1 as well as in developing novel therapeutic approaches.     

A Genetic Screen for Anchorage-Independent Proliferation in Mammalian Cells Identifies a Membrane-Bound Neuregulin

Anchorage-independent proliferation is a hallmark of oncogenic transformation and is thought to be conducive to proliferation of cancer cells away from their site of origin. We have previously reported that primary Schwann cells expressing the SV40 Large T antigen (LT) are not fully transformed in that they maintain a strict requirement for attachment, requiring a further genetic change, such as oncogenic Ras, to gain anchorage-independence. Using the LT-expressing cells, we performed a genetic screen for anchorage-independent proliferation and identified Sensory and Motor Neuron Derived Factor (SMDF), a transmembrane class III isoform of Neuregulin 1. In contrast to oncogenic Ras, SMDF induced enhanced proliferation in normal primary Schwann cells but did not trigger cellular senescence. In cooperation with LT, SMDF drove anchorage-independent proliferation, loss of contact inhibition and tumourigenicity. This transforming ability was shared with membrane-bound class III but not secreted class I isoforms of Neuregulin, indicating a distinct mechanism of action. Importantly, we show that despite being membrane-bound signalling molecules, class III neuregulins transform via a cell intrinsic mechanism, as a result of constitutive, elevated levels of ErbB signalling at high cell density and in anchorage-free conditions. This novel transforming mechanism may provide new targets for cancer therapy.     

Voltage-Gated ion currents of schwann cells in cell culture models of human neurofibromatosis

K(+) (K) channels play a role in the proliferation of many cell types in normal cells and certain disease states. Several laboratories have studied K currents in cultured Schwann cells from models of the human diseases, neurofibromatosis type 1 (NF1) and neurofibromatosis type 2 (NF2). These diseases are characterized by the growth of Schwann cell tumors. In all cell culture NF models the K current properties differ in tumor-derived and normal Schwann cells. Depending on the model however, the type of K channel abnormality differs. K channels appear to play a role in the proliferation of Schwann cell cultures of these disease models, because a link has been established between K current blockade and the inhibition of Schwann cell proliferation in NF1 and NF2. Differences in the proliferation response of normal Schwann cells to K channel blockers suggest that in vitro regulation of proliferation in neoplastic and normal Schwann cells is complex.     

Coupling of ras p21 signalling and GTP hydrolysis by GTPase activating proteins.

Ras p21 proteins cycle between inactive, GDP-bound forms and active GTP-bound forms. Hydrolysis of bound GTP to GDP is mediated by proteins referred to as GAPs, two forms of which have been described. The first, p120-GAP, contains regions of homologies with tyrosine kinase oncogenes, and interacts with tyrosine phosphoproteins as well as with ras proteins; p120-GAP may therefore connect signalling pathways that involve tyrosine kinase and ras p21 proteins. The second type of GAP is the product of the neurofibromatosis type 1 gene (NF1-GAP). This is a protein of 325,000 Da that is defective in patients with NF1; NF1-GAP is regulated by signalling lipids, and may serve to connect ras p21 with phospholipid second messenger systems. The significance of ras p21 interaction with distinct GAPs is discussed.     

Astrocyte-specific neurofibromatosis 1 gene deletion is insufficient for astrocytoma formation in mice

To gain insight into the role of the NF1 (Neurofibromatosis type 1) gene during neural development and in tumorigenesis, we have utilized the bacteriophage P1, Cre/loxP system to generate a conditional allele at the NF1 locus (NF1 flox) that permits temporal and spatial ablation of function through Cre-mediated recombination. We have been using these mice to assess the scope of NF1 requirement in distinct cell types. At the center of this approach is to identify the cells that give origin to the tumors most frequently found in NF1 patients: neurofibromas, neurofibrosarcomas, and astrocytomas. We have hypothesized that specific stem cells must lose NF1 by LOH to begin this process. I will discuss the consequences of NF1 loss in neurons, Schwann cells, and neural precursors. Distinct tumor phenotypes appear in each case. In malignant tumors, our mouse models indicate that the p53 pathway must also become mutated to cooperate with loss of NF1. Additionally, we have genetic evidence that the haploin-sufficient state is essential for tumor appearance. These data suggest that profilactic therapies preceding tumor appearance should be considered for NF1. Acknowledgements:  Funded by NINDS, NNFF, and DOD.     

Symposium 2: The Genetic,Molecular and Cellular Bases of Neurofibromatosis Type 1. A role of NF1 as a tumor suppressor

To gain insight into the role of the NF1 (Neurofibromatosis type 1) gene during neural development and in tumorigenesis, we have utilized the bacteriophage P1, Cre/loxP system to generate a conditional allele at the NF1 locus (NF1 flox) that permits temporal and spatial ablation of function through Cre‐mediated recombination. We have been using these mice to assess the scope of NF1 requirement in distinct cell types. At the center of this approach is to identify the cells that give origin to the tumors most frequently found in NF1 patients: neurofibromas, neurofibrosarcomas, and astrocytomas. We have hypothesized that specific stem cells must lose NF1 by LOH to begin this process. I will discuss the consequences of NF1 loss in neurons, Schwann cells, and neural precursors. Distinct tumor phenotypes appear in each case. In malignant tumors, our mouse models indicate that the p53 pathway must also become mutated to cooperate with loss of NF1. Additionally, we have genetic evidence that the haploin‐sufficient state is essential for tumor appearance. These data suggest that profilactic therapies preceding tumor appearance should be considered for NF1. Acknowledgements: Funded by NINDS, NNFF, and DOD.