Entwistle, Susannah J.
(2024)
Investigating the role of Rab GTPases in medulloblastoma pathogenesis.
PhD thesis, University of Nottingham.
Abstract
Introduction: Medulloblastoma is the most common malignant paediatric brain cancer, accounting for 20% of paediatric brain tumours and 10% of all cancer deaths. It is located within the cerebellum and is defined by four molecular subgroups: Wingless (WNT), Sonic Hedgehog (SHH), group 3 and group 4. Whilst WNT and SHH are named after the pathways which drive these tumours, the mechanisms underlying groups 3 and 4 are less well understood. Group 3 tumours are the group most likely to be metastatic at diagnosis and are therefore associated with the poorest prognosis. There is therefore a particular need to investigate the mechanisms that drive these tumours, to enhance diagnostic methods and treatment strategies and eventually improve patient outcomes. Exosomes are small membrane-bound extracellular vesicles (EV) of endosomal origin. They contain a variety of cargo, including RNA and proteins. Increased exosome release has been associated with disease progression and metastasis in multiple cancers. Rab GTPases (Rabs) are a family of small GTPases (66 in humans) which regulate vesicle trafficking. Several Rabs have been shown to regulate exosome biogenesis and secretion and may consequently contribute to cancer progression. Rabs also have non-exosomal associations with tumourigenesis through roles in intracellular vesicular trafficking. The role of Rabs in medulloblastoma pathogenesis has not yet been investigated. This study aims to therefore explore the pathways in which Rabs may contribute to medulloblastoma pathogenesis.
Methods and results: Analysis of medulloblastoma patient datasets such as the Cavalli dataset on the R2 genomics analysis and visualisation platform, showed an association between high expression of specific Rabs and poor prognosis. This included Rab11A and Rab40B in group 3 medulloblastoma (p values ranging from 0.00075 to 0.03). RT-qPCR analysis showed no subgroup-specific enrichment of Rab11A expression, but Rab40B had significantly higher expression in the group 3 cell line HD-MB03 (p<0.01). CRISPR-Cas9 technology was then used disrupt the Rab11A and Rab40B genes to generate stable protein knockout in the group 3 HD-MB03 cell line. Nine Rab11A knockout lines were fully validated, validation of Rab40B knockout lines was however inconclusive. From then on exploring the function of Rab11A in group 3 medulloblastoma became the focus of the study.
Analysis of EV secretion showed no statistical difference in median diameter and concentration of EVs secreted between Rab11A knockout and non-targeting CRISPR control cell lines. The effect of Rab11A knockout on the endocytic pathways through analysis of transferrin uptake was inconclusive. Proteomic analysis using SWATH-MS was then conducted on Rab11A knockout cell lysates and EVs. Differential protein expression analysis of cell lysates identified statistically significant downregulation of the mitochondrial metabolism pathways, oxidative phosphorylation and the TCA cycle, which occur in the inner mitochondrial membrane and mitochondrial matrix respectively. This finding is particularly interesting since recent studies showed that the HD-MB03 cell line has high TCA cycle and oxidative phosphorylation activity, a finding that was also present in other group 3 cell lines with a MYC amplification. Thereby suggesting that Rab11A is a regulator of mitochondrial metabolism in MYC amplified group 3 medulloblastoma. In EVs from Rab11A knockout versus control cells, proteomic analysis identified protein sidekick 2 (SDK2) as being significantly downregulated. SDK2 is associated with several functional pathways, including maintenance of neural circuits in the retina, that have recently been associated with genetic signatures of the cell of origin for group 3 medulloblastoma, thus linking Rab11A-regulated EV cargo and fundamental mechanisms driving the formation of the disease.
Conclusions: This study has identified, for the first time, a prognostic association between Rab GTPases and medulloblastoma. It has also identified subgroup-specific Rab targets for further exploration. Including a potential novel role for one Rab, Rab11A, in regulation of mitochondrial metabolism pathways. In addition to this, an association has been made between Rab11A-regulated EV cargo and mechanisms that are thought to drive group 3 medulloblastoma. Further exploration of the role of Rab11A may therefore give valuable insight into mechanisms driving pathogenesis and tumour initiation in group 3 medulloblastoma.
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