Genetic, epigenetic and functional analysis of tumorigenesis in neurofibromatosis type 1 (NF1).

Neurofibromatosis Type 1 (NF1) is an autosomal dominant disorder caused by constitutional inactivation of the NF1 gene. NF1 is associated with extreme phenotypic variability and results in an increased risk of developing benign and malignant peripheral nerve sheath tumours (MPNSTs). The molecular mechanisms that underlie NF1 tumorigenesis are poorly understood although inactivation of additional loci in conjunction with NF1 mutations is postulated to be involved. A combinative approach encompassing genetic, epigenetic and functional analysis was employed to dissect the molecular mechanisms responsible for tumorigenesis and progression to malignancy in NF1. Genetic analysis of benign cutaneous neurofibromas from NF1 patients with a high tumour burden was undertaken involving PCR, sequencing, Loss of Heterozygosity (LOH) and Multiplex Ligation-dependent Probe Amplification (MLPA) at the NF1 locus. In addition, Microsatellite Instability (MSI) analysis and molecular analysis of the TP53, RB1, CDKN2A and MMR genes was performed in a subset of these tumours. It was revealed that a small proportion of tumours from such patients harbour LOH of the TP53 and RB1 genes, a novel finding which indicates that additional modifying loci may underlie the development of multiple cutaneous neurofibromas in patients with a high tumour burden. Furthermore, Schwann cell culture and Laser Capture Microdissection were used to enhance the somatic mutation detection rate in the NF1 gene. Pyrosequencing methylation analysis was employed to determine whether NF1 gene methylation was associated with NF1 tumorigenesis. Promoter methylation of potential modifying genes RASSF1A and MGMT was also assessed. Results of this study demonstrate that methylation of the NF1 and MGMT genes is unlikely to contribute to NF1 tumorigenesis. RASSF1A promoter methylation was significantly higher in tumour samples in comparison to controls. RASSF1A promoter methylation was correlated with a reduction in RASSF1A gene expression, as evidenced by 5aza2dc treatment. Non-synonymous missense mutations account for 15% of lesions in the NF1 germlihe and somatic mutational spectrum although without a suitable functional test, their pathogenicity cannot be defined with certainty. Site directed mutagenesis, a Ras ELISA and bioinformatic analysis were utilised to develop a novel functional assay to assess the potential pathogenicity of NF1 gap related domain (GRD) missense variants. Of the 16 mutations functionally analysed, 11 were found to be pathogenic. A panel of 14 candidate genes were found to be aberrantly expressed in 14 tumours and 5 MPNST tumour-derived cell lines by relative quantification. A number of genes in the Rho-GTPase pathway including RAC1 and ROCK2 are over-expressed in these tumours, suggesting that these genes might be critical for survival of MPNST cells. ShRNA (short hairpin RNA) for RAC1 and ROCK2 were used in adhesion, migration, invasion and wound healing assays on control an d MPNST derived cell lines. RAC1 and ROCK2 knockdown in MPNST cell lines resulted in a significant increase in cell adhesion and a reduction in wound healing, migratory and invasive activity. The cMET gene was also over-expressed in MPNSTs. Treatment of MPNST cell lines with cMET inhibitors also resulted in a reduction of cell growth and migration. This study has identified several novel therapeutic targets for NF1-associated malignancies