Shemais, TAH; (2015) The epigenetic differences between neurofibromas and malignant peripheral nerve sheath tumours. Doctoral thesis , UCL (University College London.

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Abstract

Malignant peripheral nerve sheath tumours (MPNSTs) are rare soft tissue sarcomas, which can arise de novo in a peripheral nerve or from a neurofibroma. The molecular mechanisms leading to progression of neurofibroma to MPNST remain largely unknown. In this thesis, I examined the role played by microRNAs (miRNAs) in the pathogenesis of MPNSTs from neurofibromas. I analysed miRNA microarray expression profiles from 10 MPNSTs and 10 neurofibromas and identified sixteen differentially expressed miRNAs; fourteen were downregulated and two upregulated in MPNSTs. One of these, miR-29c, was confirmed as downregulated in MPNSTs by qRT-PCR. Its expression was also shown to be relatively low in other sarcomas and low-grade tumours. In vitro functional studies revealed miR-29c inhibits cell migration following its ectopic expression in MPSNT-derived sNF96.2 cells. Using in silico prediction analyses, I identified extracellular matrix (ECM) genes COL1A1, COL1A2, COL4A1, COL4A2, COL5A2, COL21A1 and MMP2 as well as TGFB3 and DNA methyltransferases DNMT3A and DNMT3B as potential targets of miR-29c. I demonstrated direct inhibition of these genes and partial inhibition of MMP9 via an indirect mechanism in miR-29c-transfected sNF96.2 cells. DNMT3B mRNA expression was higher in MPNSTs vs neurofibromas and inversely correlated with miR-29c expression. Using previous methylome and microarray data, I identified CDKN2A, WT1, and S100B as possible targets for epigenetic regulation in MPNSTs. I demonstrated hypermethylation of these genes’ regulatory regions in MPNSTs by pyrosequencing and, in a larger cohort of 80 MPNSTs and 80 neurofibromas archival samples, I revealed increased levels of WT1 and S100B gene methylation in high-grade MPNSTs compared to low-grade MPNSTs and neurofibromas. S100B protein expression by immunohistochemistry was also shown to be downregulated in MPNSTs compared to neurofibromas and its expression inversely correlated with S100B gene methylation. Furthermore, introduction of S100B cDNA using an adenoviral expression vector resulted in reduced proliferation of sNF96.2 cells. My results suggest miR-29c may function as a tumour suppressor in MPNSTs by targeting ECM genes as well as DNA methyltransferases and TGFB3. S100B was also identified as a potential tumour suppressor whose expression is epigenetically regulated in MPNSTs. These findings lead to a better understanding of MPNST pathogenesis and identify S100B and miR-29c as potential targets for future intervention in MPNST patients.

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