Cellular and molecular events in the progression of medulloblastoma

Childhood brain tumours are the leading cause of death in paediatric oncology in Australia. Medulloblastoma (MB) is an embryonal malignant brain tumour that predominantly affects the cerebellum of children. MB is classified into four molecular subgroups (WNT, SHH, Groups 3 and 4) based on MB’s transcriptional profiles. Compared to WNT/SHH subgroups, Groups 3 and 4 are the most aggressive subgroups of the disease, with an increased risk of metastasis and shorter survival for patients. In addition, MB’s current therapy approaches are not entirely successful and are accompanied by severe cognitive and neurological adverse effects. Thus, there is an urge to develop better treatments for MB patients by understanding the biology of MB to identify novel pharmacological therapeutic targets for controlling high?risk MBs. Calcium signalling and glycolytic metabolic pathways are crucial to various cancer?related processes. Dysregulations in calcium signalling and glycolysis pathways have been investigated extensively in many malignancies; nevertheless, MB remains under-explored primarily in this area.
In this PhD study, first, I investigated the landscape of calcium signalling and glycolysis regulators to identify potential important candidates in MB tumours. Through publicly available datasets, I identified the expression of regulator genes involved in calcium signalling and glycolysis in MB patients. Consequently, the expression levels of these genes were stratified within the molecular subgroups of MB, metastasis and overall patient survival to identify associations between gene expression and clinical characteristics. Second, I assessed the expression levels of the identified genes of interest in human MB tissues and cell lines and determined the impact of their pharmacological inhibition and siRNA-mediated silencing on MB cell proliferation, metabolism, and stemness in vitro. Third, I utilised proteomics analysis, compelled with cell-based assays, to further understand the mechanism of action of my identified pathways in MB.
For the in silico analysis, 169 regulating genes involved in calcium signalling (92 genes) and glycolysis (77 genes) pathways were collected for the initial assessment. Across the ten datasets used in this thesis, CACNA1H (from calcium regulator genes) and LIN28B (from glycolysis regulator genes) were of particular interest, in which both genes showed consistent significant upregulation in MB patients, especially in the harsh Groups 3 and 4 MB subgroups, compared to healthy brain tissues. This upregulation was also significantly correlated with the incidence of metastasis and the overall survival of MB patients. For CACNA1H, another PhD candidate in our team carried out further in vitro pharmacological studies to understand the role and importance of this pathway in MB cells. I conducted in vitro studies on LIN28B to better comprehend its function and mechanism of action in MB.
My studies showed that inhibition of LIN28 activity using its pharmacological antagonist, Lin28 1632, reduced MB cell growth and sphere formation, and the expression of LIN28B and stemness marker CD133 proteins in a concentration-dependent manner. Likewise, selective knockdown of LIN28B using siRNA, significantly suppressed MB cell viability and metabolism. In addition, data from proteomics analysis demonstrated that silencing LIN28B regulated proteins involved in ribosomal biogenesis and lipid metabolic processes. These findings were confirmed by further cell-based assays, where LIN28B silencing reduced nucleolar size and phosphoprotein B23 protein expression and promoted lipid accumulation in MB cells.
In conclusion, this project led to the identification of two important proteins in MB, CACNA1H and LIN28B. This study showed the importance of these proteins as potential prognostic and diagnostic markers, as well as promising targets for the control of MB. Further studies are warranted to confirm and explore the roles of LIN28B and CACNA1H in other MB preclinical models to assess their targeting as potential novel treatment strategies against MB.