Abstract 4977: Identification of transcriptional mechanisms specific to of NF1 gene deficiency

Abstract

Abstract Neurofibomatosis type 1 (NF1) is an autosomal dominant disease (1:3000) resulting from the loss-of-function mutation of the NF1 gene that encodes neurofibromin. Although deregulation of Ras and mTOR has been reported, the role of neurofibromin in NF1 disease pathogenesis is not fully understood. In this study, we developed a systems approach to identify aberrant gene regulation and transcription factor (TF) binding activities associated with loss-of-function of neurofibromin. Two malignant peripheral nerve sheath tumor (MPNST) cell lines were used. STS26T (NF1+/+) has the wild type of NF1 gene and ST88-14(NF1−/−) that has a mutant NF1 gene. Specifically, TF binding activities and gene expression changes related to N-Ras, active ERK and neurofibromin deficiency were determined by using Protein/DNA arrays and gene expression arrays, respectively. Three comparison systems have been employed in STS26T (NF1+/+): 1) inducible oncogenic N-Ras expression, 2) MAPK pathway inhibition by U0126, 3) NF1 gene siRNA knockdown. Similarly, we applied three systems to ST88-14(NF1−/−): 1) N-Ras siRNA knockdown, 2) MAPK pathway inhibition by U0126, 3) normal human Schwann cells vs. ST88-14(NF1−/−). Based on these comparisons, the significant TF binding activity changes due to activation of N-Ras, MAPK and neurofibromin deficiency were for the first time revealed as critical effector pathways in NF1 disease. We identified many TF binding activity changes and their possible relationships to N-Ras, MAPK pathway and neurofibromin deficiency. For example, the activity changes for MEF3 and GATA1/2 only occurred when NF1 gene is siRNA knockdown in STS26T (NF1+/+). Thus, neurofibromin may regulate the pathway independent of MAPK and N-Ras. On the other hand, the binding activity of Pax3 increased only in U0126 treated ST88-14(NF1−/−) indicating that the MAPK pathway regulates its function directly. All these changes were verified by electrophoretic mobility shift assays. Gene expression arrays are being used to confirm target gene expression due to TF banding activity changes and comparative genome hybridization will used to exclude gene expression changes resulting from the gene amplification. A method to study transcription regulatory pathways based on the relationship between transcription factor binding profiles and gene expression profiles will be established and used to identify aberrant gene regulation uniquely related to N-Ras, MAPK and neurofibromin, respectively. This systematic analysis of NF1 deficiency will provide an understanding of the gene dysregulation in this disease, which can facilitate novel drug target discovery. At same time, new functions of neurofibromin other than its activity as a Ras-GAP are being revealed through these analyses. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4977.