Investigating the role of TWIST1 in medulloblastoma metastasis

Introduction Medulloblastoma is the most common malignant paediatric brain tumour. Many patients present with metastatic disease and sadly this remains a powerful predictor for poor outcome. Metastasis is a complex multi-step process and the underlying molecular mechanisms behind medulloblastoma metastasis are poorly understood. One of the initial stages of this process involves the invasion of tumour cells through the basement membrane and into the leptomeningeal space. The transcription factor TWIST1 has been extensively studied in epithelial tumours and is considered as one of the master regulators initiating the first stage of this process. An in-depth analysis of the functional role of TWIST1 in initiating medulloblastoma metastasis remains to be explored. This project therefore aimed to assess the functional role of TWIST1 and whether it could play an essential part in driving medulloblastoma metastasis. Methods TWIST1 gene and protein expression was analysed in medulloblastoma patient samples to investigate if expression correlated with metastatic disease and patient outcome. Metastatic cell lines representing two out of the four medulloblastoma molecular subgroups (SHH and Group 3) were employed for in vitro analysis of TWIST1 function. A 3D transwell assay was used to quantify cellular migration and invasion through a simple basement membrane. Inhibition of TWIST1 using genetic (shRNA) and chemical (harmine, a beta-carboline alkaloid) methods was investigated. We then explored the presence of a TWIST1-ABCB1 axis, using chromatin immunoprecipitation (ChIP) and the specific ABCB1 inhibitor vardenafil. Finally, ChIP-Sequencing was carried out to define additional members of TWIST1’s transcriptional regulatory network. Results TWIST1 was highly expressed across the four medulloblastoma molecular subgroups compared to the normal cerebellum. Analysis of patient tissue microarrays revealed that high expression was a strong indicator for worse event-free survival and for driving metastatic disease in medulloblastoma patients. Reduced TWIST1 expression significantly decreased medulloblastoma cell migration in the 3D transwell migration and invasion assay. ChIP analysis confirmed TWIST1 bound to the ABCB1 promoter and inhibition of ABCB1 with vardenafil reduced cell migration. ChIP-Sequencing identified many TWIST1 downstream target genes and pathways that are important in initiating the metastatic process. This included pathways and genes that are involved in interacting with cell adhesion molecules, focal adhesions and the extracellular matrix. Conclusions This thesis has shown that TWIST1 expression is crucial for initiating medulloblastoma metastasis through its ability to target pathways and genes that are essential in initiating the metastatic process. Therapeutically targeting TWIST1 in the primary tumour offers a novel approach to prevent primary tumour cells from invading the extracellular matrix and ultimately halt metastatic dissemination and, thereby greatly improving patient outcome