Function of the NF1 Tumor Suppressor Gene in Angiogenesis.
Authors: Munchhof Amy M. , Li Fang , Mead Laura , White Hilary , Fenoglio Amy , Yang Feng-Chun , Ingram David A. 
Published: April 2020

Abstract
Mutations in the NF1 tumor suppressor gene cause neurofibromatosis type I. Neurofibromin, the protein encoded by NF1, is a negative regulator of Ras activity. 95% of NF1 patients develop neurofibromas. Haploinsufficiency of Nf1 in the non-neuronal cell lineages of the neurofibroma microenvironment is essential for tumor progression in genetically engineered mice (Science 2002). Thus, understanding the effect of haploinsufficiency of Nf1 in cell lineages of the tumor microenvironment is critical for understanding disease pathogenesis. Neurofibromas are highly vascular and express mRNA for vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF), which are growth factors linked to induction of the “angiogenic” switch required for tumor development. Both local proliferation and migration of endothelial cells (ECs) and recruitment of circulating endothelial progenitor cells (EPCs) from peripheral blood contribute to tumor angiogenesis. To determine the role of neurofibromin in regulating EC function, we isolated murine ECs from Nf1 +/− and wildtype (WT) mice by FACS for cells expressing the EC antigen CD31 but not the hematopoietic specific cell antigen, CD45. We also isolated EPCs from the peripheral blood of NF1 patients and aged matched controls utilizing recently established techniques by our laboratory (BLOOD, 2004). Murine Nf1+/− ECs demonstrated a 2-fold increase in migration in response to both bFGF and VEGF in Boyden chamber assays compared to WT controls (n=5, p&lt;.01). Further, Nf1 +/− mice demonstrated a 2-fold increase in angiogenesis in vivo compared to WT controls after matrigel plugs containing either VEGF or bFGF were inserted into the ventral groins of both murine genotypes (n=5, p&lt;.01). Isolation of sufficient numbers of primary murine ECs for biochemical studies is problematic. Therefore, we used siRNAs to genetically silence neurofibromin in primary human ECs to determine the biochemical mechanisms for the Nf1 +/− EC phenotype. Human ECs transfected with a NF1 siRNA demonstrated a 2-fold increase in both migration and proliferation compared to ECs transfected with a control siRNA in reponse to both VEGF and bFGF (n=3 p&lt;.01). Human ECs transfected with a NF1 siRNA had a 2–3-fold increase in Ras and ERK activity compared to controls (n=3 p&lt;.01). Pharmacological blockade of the Ras-ERK signaling axis with a MEK inhibitor inhibited the migration and proliferation of human ECs transfected with either the NF1 siRNA, demonstrating that hyperactivation of this pathway is directly linked to the Nf1 +/− cellular phenotype (n=3 p&lt;.01). Based on this result, we next tested whether local administration of a MEK inhibitor in a matrigel plug would inhibit the angiogenic response of Nf1 +/− mice to either VEGF or bFGF in vivo (n=3 p&lt;.01). Strikingly, local administration of the MEK inhibitor completely inhibited the angiogenic response of Nf1 +/− mice to VEGF and bFGF confirming that the biochemical mechanism identified in vitro for the neurofibromin deficient EC phenotype was operative in vivo. Finally, EPCs isolated from three different NF1 patients demonstrated an increase in both basal and growth factor induced proliferation and migration compared to EPCs isolated from healthy adults. Thus, these studies demonstrate that loss of neurofibromin alters EC functions via hyperactivation of distinct Ras effector pathway identifying a potential therapeutic target in the neurofibroma microenvironment.