Patients with neurofibromatosis type 1 (NF1) develop peripheral nerve sheath tumors, neurofibromas that consist mainly of Schwann cells and fibroblasts. NF1 patients have mutations in the NF1 gene, which encodes a protein called neurofibromin. Neurofibromin is a GTPase-activating protein (GAP) for Ras proteins. In the last granting period we identified numerous effects of NF1 loss on Schwann cells and fibroblasts, including changes in proliferation, cell invasion, and matrix deposition. Surprisingly, while some of these changes can be attributed to altered Ras-GAP activity within cells, others appear to be H-Ras independent. To explain these findings we hypothesize that neurofibromin is a key Ras-GAP in peripheral nerve cells for less studied Ras family members, including R-Ras. Preliminary data support a role for R-Ras in Nf1-deficient Schwann cells. To test this hypothesis we plan to use cells from Nf1 mutant mice and from human neurofibromas in in vitro assays we developed. We will express activated R-Ras or H-Ras and test if loss of neurofibromin function is mimicked. We will express the NF1 GAP-related domain (GRD) to test if this domain reverses defects of NF1 mutant cells. We will also create a transgenic mouse model for neurofibroma formation based on this hypothesis, by expressing either H-Ras-GTP or R-Ras-GTP, or both, in Schwann cells. In addition, we have defined differences in expression of proteins (EGF receptor) and transcripts (by cDNA microarray analysis) in Nf1 mutant Schwann cells. We plan to test if these specific changes are sufficient to initiate tumorigenesis. We will also test if H-Ras and/or R-Ras can regulate expression of individual cDNAs. Together, these studies are anticipated to identify novel intervention pathways to treat human NF1 disease.
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