Nf1-Deficient Mouse Schwann Cells Are Angiogenic and Invasive and Can Be Induced To Hyperproliferate: Reversion of Some Phenotypes by an Inhibitor of Farnesyl Protein Transferase

Abstract

We have developed a potential model of Schwann cell tumor formation in neurofibromatosis type 1 (NF1). We show that mouse Schwann cells heterozygous or null at Nf1 display angiogenic and invasive properties, mimicking the behavior of Schwann cells from human neurofibromas. Mutations at Nf1 are insufficient to promote Schwann cell hyperplasia. Here we show that Schwann cell hyperplasia can be induced by protein kinase A activation in mutant cells. Removal of serum from the culture medium also stimulates hyperplasia, but only in some mutant cells. After serum removal, clones of hyperproliferating Schwann cells lose contact with axons in vitro, develop growth factor-independent proliferation, and exhibit decreased expression of the cell differentiation marker P0 protein; hyperproliferating cells develop after a 1-week lag in Schwann cells heterozygous at Nf1. The experiments suggest that events subsequent to Nf1 mutations are required for development of Schwann cell hyperplasia. Finally, an anti-Ras farnesyl protein transferase inhibitor greatly diminished both clone formation and hyperproliferation of null mutant cells, but not invasion; farnesyl transferase inhibitors could be useful in treating benign manifestations of NF1.Neurofibromatosis type 1 (NF1) is one of the most common inherited human autosomal dominant diseases, with a worldwide incidence of 1 in 3,500 individuals (56). Benign manifestations frequently associated with NF1 include pigmented lesions of the skin (café au lait spots), hamartomas of the iris (Lisch nodules), learning disabilities, formation of optic pathway gliomas, and neurofibromas (22). Neurofibromas are one of the major defining features of NF1. Cutaneous neurofibromas (benign peripheral nerve sheath tumors) are associated with small nerve branches, plexiform neurofibromas develop along major peripheral nerves, and malignant peripheral nerve sheath tumors (MPNST) develop in about 4% of NF1 patients (56). Genetic studies show that malignant tumors in NF1, including MPNSTs, contain cells with mutations in both the constitutionally inactivated and previously normal somatic allele, consistent with NF1 acting as a tumor suppressor (19,36,64). The mechanism of neurofibroma formation is less clear. While recent studies demonstrated loss of heterozygosity (LOH) at the NF1 locus in some neurofibromas (12, 60), LOH could be associated with either of the major cell types in the benign tumors, fibroblasts or Schwann cells.Schwann cells may be the primary pathogenic cells in neurofibromas. The majority (40 to 85%) of cells in neurofibromas are Schwann cells (18,49), and in contrast to Schwann cells in normal nerve, neurofibroma Schwann cells are found without association with axons (28, 50). Furthermore, in vitro, Schwann cells, but not fibroblasts, from neurofibromas show angiogenic and invasive properties (63). Because the genetic status of neurofibroma Schwann cells is unknown, it remains unclear if a single NF1 mutation is sufficient for manifestation of some neurofibroma Schwann cell phenotype...

Contribution of Src and Ras pathways in FGF-2 induced endothelial cell differentiation

“…In accordance with several studies in other cell systems (Haklai et al, 1998;Kim et al, 1997;Nagase et al, 1996), treatment of the IBEC with Rasinhibitors inhibited FGF-2 induced cell proliferation. Our data suggest an obligatory signaling cascade to IBEC proliferation that starts with FGFR-1 activation, phosphorylation of the adaptor proteins Shc and FRS2 and subsequent activation of Ras followed by the linear Raf1/MEK/MAPK pathway.…”

Overexpression of activated neu/erbB2 initiates immortalization and malignant transformation of immature Schwann cells in vitro

“…Cells were grown in the presence and absence of either drug for 48 h then assayed for cell proliferation and invasion as described above. We found that both drugs signi®cantly inhibited NID cell [ 3 H]thymidine incorporation ( Figure 6a) and cell invasion ( Figure 6b) at concentrations previously shown to block Ras processing (Kim et al, 1997b) and Src kinase activity (Sobko et al, 1998) in Schwann cells. Both drugs inhibited cell invasion to a similar degree, while herbimycin A also inhibited cell motility as indicated by the small number of cells that reached the bottom of the ®lter (Figure 6b).…”

“…While herbimycin A and L-739,749 may a ect additional signaling molecules, they were used at concentrations previously shown to block Src kinase activity and Ras farnesylation (which is required for Ras to become active), respectively, in Schwann cells (Sobko et al, 1998;Kim et al, 1997b). Both drugs were e ective in reversing the ability of NID cells to invade matrix and to proliferate in the absence of added growth factors.…”

“…It is therefore likely that signaling cascades involving Ras and Src are involved in maintaining the phenotype of neu-infected Schwann cells. We tested this notion using either an inhibitor of farnesyl protein transferase (L-739,749) to block cellular Ras activity (Kim et al, 1997b;Prendergast et al, 1994;James et al, 1993), or herbimycin A to block src activity (Uehara et al, 1989;Fukazawa et al, 1991). Cells were grown in the presence and absence of either drug for 48 h then assayed for cell proliferation and invasion as described above.…”

“…Cells were treated with 5 mM herbimycin A (Sigma; Sobko et al, 1998) for 16 ± 24 h or with 10 mM L739,749 (Merck; Kim et al, 1997b) for 48 h prior to proliferation and invasion assays.…”