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(2022)ThesisNeuroblastoma (NB) is a solid tumor of the developing sympathetic nervous system that occurs during childhood and has a less than 50% survival rate for high-risk patients. Amplification of the MYCN oncogene remains the single most important genetic predictor of poor prognosis. Many of the protumorigenic functions of the oncogene MYCN are attributed to its regulation of global gene expression programs. Alternative splicing is another important regulator of gene expression and has been implicated in NB development, however, the molecular mechanisms behind this remain unknown. In this thesis, the core spliceosomal protein, SNRPD3, was found to be significantly up-regulated in cell and animal models of NB initiation and progression. Moreover, high SNRPD3 expression correlated with poor patient prognosis and is an independent prognostic factor. Further investigation revealed that SNRPD3 was not only a MYCN transcriptional target but together with MYCN formed a protein complex with PRMT5, the protein responsible for methylating SNRPD3. RNA-sequencing revealed an overall increase in the number of genes being differentially spliced when MYCN was overexpressed, while depletion of SNRPD3 in the presence of overexpressed MYCN was sufficient to induce additional increases in differential splicing, particularly genes involved in the cell cycle. SNRPD3 knockdown in the presence of MYCN was accompanied with marked loss of cell viability, suggesting that SNRPD3 maintains a splicing balance for MYCN, and prevents detrimental differential splicing of cell cycle genes in NB. Further analysis revealed BIRC5 and CDK10 to be among the topmost differentially spliced cell cycle genes, both of which play a role in the G2/M phase of the cell cycle. Flow cytometry analysis revealed that depletion of SNRPD3 resulted in G2/M cell cycle arrest. Furthermore, SNRPD3 was an oncogenic co-factor for MYCN in vitro and in vivo. As such, SNRPD3 represents a therapeutic vulnerability for NB cells in the presence of high MYCN expression. Indeed, the PRMT5 inhibitor, JNJ64619178 and GSK3326595, that prevents SNRPD3 methylation was cytotoxic in NB cell lines with high SNRPD3 and MYCN expression. Overall, this thesis provides mechanistic insight into the function of SNRPD3 in MYCN-driven NB and highlights the importance of SNRPD3-dependent balanced splicing. Furthermore, this thesis presents preliminary data for a novel therapeutic approach for neuroblastoma patients using PRMT5 inhibitors, currently in clinical trials.
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(2024)ThesisHigh-grade gliomas (HGG) are highly aggressive brain tumours with dismal prognosis for adult and paediatric patients. Current therapeutic options (surgical resection, radiotherapy and chemotherapy with temozolomide) show limited efficacy. Therefore, elucidating new therapeutic targets, selective to cancer cells, is highly critical for this incurable disease. Characterisation of cell surface proteins is an attractive approach, as they are readily accessible targets for therapeutics. Recently, attention has been drawn to endoplasmic reticulum (ER) chaperones as surface antigens in cancers, including HGG. The overexpression of ER chaperones upon stress activation has been associated with cell surface translocation, exhibiting pro-cancer functions. Hence, this project aimed to unravel the tumour cell surface proteome (surfaceome) of adult and paediatric HGG and examine the expression and translocation of various ER chaperones in adult and paediatric HGG models compared to non-neoplastic brain cells. Initially, a bioinformatic analysis of the mRNA and protein expression of 7 ER chaperones (CALR, CANX, HSP47, GRP78, GRP94, GRP170 and PDI) showed an upregulation (p<0.05) in adult HGG patient samples, compared to normal tissues (Chapter 3). Previous evidence of the translocation of these chaperones from the ER to the cell surface was demonstrated in a small cohort of adult HGG cell line models. Therefore, to expand our understanding of the surfaceome, surface proteins of adult and paediatric HGG cells were labelled with biotin, isolated and analysed using label-free quantitative shotgun proteomics. The surfaceome of 3 cell lines (Chapter 4) and 16 patient-derived cell (PDC) models (Chapter 5), including adult and paediatric samples, was obtained using this proteomic methodology. Moreover, the effects of the ER stressor thapsigargin or irradiation treatment on the surfaceome composition of HGG were examined for the first time. The expression of the ER chaperones was confirmed in adult and paediatric HGG cell lines and PDCs. Specifically, GRP94, CALR and CANX were upregulated in PDCs compared to non-neoplastic brain cells. Additionally, this approach identified typical and non-typical HGG-specific surface targets. Finally, the role of the ER chaperone GRP170 (Chapter 6) was studied in adult HGG cells for the first time, finding a significant (p<0.05) change in HGG proliferation, invasion and clonogenicity with differential effects for the different mutants evaluated. Collectively, this work broadened the understanding of ER chaperones as potential targets in adult and paediatric HGG and described the role of GRP170 in this cancer type. Moreover, typical and non-typical membrane proteins were exclusively identified in HGG samples, setting the path for further validation of novel therapeutic targets to treat this deadly cancer.