Abstract
Cutaneous neurofibromas are a hallmark of neurofibromatosis type 1 (NF1), a common genetic disorder that is caused by mutations in the NF1 gene, which functions as a GAP for p21ras. Though the pathogenesis of neurofibroma formation is not completely known, haploinsufficiency of the nonneuronal lineages (fibroblasts, mast cells and endothelial cells) in the tumor microenvironment are required for neurofibroma formation (Zhu, Science 2002). These tumors are characterized by a high concentration of degranulating mast cells (MC) closely associated with fibroblasts, endothelial cells and Schwann cells. We have recently shown that Nf1−/− Schwann cells secrete kit-ligand to recruit Nf1+/− MCs to the tumor microenvironment via hyperactivation of a p21ras-PI3K-Rac dependent pathway (Yang, JCI 2003). Further, Nf1+/− MC also promote Schwann cell invasion and proliferation. Given that collagen is synthesized by fibroblasts and is approximately 80% of the weight of neurofibromas, we tested whether Nf1+/− MC promote the proliferation and collagen synthesis of fibroblasts. Strikingly, the proliferation of Nf1+/− fibroblasts in response to Nf1+/− MC conditioned media (MCCM) was 3 fold higher than any other group tested. In a wound healing assay Nf1+/− MCCM provided potent stimulus for the migration of Nf1+/− but not WT fibroblasts. Similarly, MCCM from primary human NF1+/− MC stimulated the proliferation, migration, and collagen synthesis of human NF1+/− fibroblasts, validating that our observations in Nf1+/− murine cells faithfully phenocopy the biology of human NF1 heterozygous cells. We next established three dimensional collagen lattices containing MC and fibroblasts of the respective genotypes to evaluate extracellular matrix (ECM) reorganization given that remodeling of the ECM by inflammatory cells promotes tumorigenesis. Histological examination revealed that while MC and fibroblasts of both genotypes localized to each other, there was a 2–3 fold quantitative increase in the localization or attachment of Nf1+/− MC to Nf1+/− fibroblasts. Further, Nf1+/− MC preferentially promoted a 2–3 fold increase in the lattice contraction indicative of alteration of the ECM in lattices containing either Nf1+/− or WT fibroblasts. Given that c-kit/kit-ligand interactions between MC and fibroblasts contribute to MC-fibroblast interactions, c-kit blocking antibodies or Gleevec, an antitumor drug that inhibits both BCR/ABL and c-kit tyrosine kinases, were added to MC-fibroblast cultures. Both of these agents blocked the activity of Nf1+/− MC on fibroblast proliferation, collagen remodeling and fibroblast migration. Collectively, these studies demonstrate that murine and human NF1 (Nf1) haploinsufficient MC stimulate the proliferation, migration, collagen synthesis of Nf1+/− fibroblasts as well as remodeling the ECM. This study provides strong evidence that Gleevec may be a candidate therapy for the treatment or prevention of neurofibromas.
Three-Dimensional Characterization of Mechanical Interactions between Endothelial Cells and Extracellular Matrix during Angiogenic Sprouting
