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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.