Wade, Philippa
(2024)
Exploring the role of multidrug exporters and extracellular vesicles in medulloblastoma chemoresistance.
PhD thesis, University of Nottingham.
Abstract
Introduction: Medulloblastoma is the most common malignant paediatric brain tumour; patients harbouring relapsed and metastatic tumours exhibit a dismal 5-year survival prognosis, where less than 10% of patients respond to current treatments. Whilst there have been considerable steps forward in our understanding of the molecular biology underpinning medulloblastoma, our understanding of relapsed medulloblastoma remains incredibly limited. The high frequency of recurrence observed in patients not only highlights an urgent and unmet need to identify new therapeutic targets, but it also indicates the presence of intrinsic drug resistance mechanisms. Multidrug exporters and extracellular vesicles have been implicated in chemoresistance in several cancers, the former exerting effects through drug efflux and the latter through the transfer of biologically active cargo. Whilst multidrug exporter ABCB1 has previously been associated with high-risk medulloblastoma and therapy resistance, the roles of additional multidrug exporters and extracellular vesicles in medulloblastoma chemoresistance are yet to be explored. This study, therefore, set out to examine both the role of extracellular vesicles and the contribution of multidrug transporters ABCB1, ABCC1 and ABCG2 to medulloblastoma chemoresistance.
Methods and results: Gene and protein expression analysis of medulloblastoma cell lines revealed sub-group specific differential expression patterns. ABCB1 was highly expressed in the SHH subgroup, whilst ABCC1 expression was highest in the Group 3 and Group 4 subgroups. Subsequent analysis of drug-tolerant cell lines (cisplatin and vincristine) revealed that, of the three multidrug exporters, only ABCB1 exhibited elevated expression in a SHH cell line that was resistant to vincristine. In support of this, complete re-sensitisation of this cell line to vincristine was achieved upon small molecule inhibition of ABCB1 function. Moreover, flow cytometry of extracellular vesicles isolated from the resistant cell line exhibited greater ABCB1 expression in comparison to control extracellular vesicles. Additionally, these extracellular vesicles demonstrated the capacity to transfer resistance to sensitive parental cells, an effect that could be eliminated by ABCB1 inhibition.
Whilst we established a role for ABCB1 and extracellular vesicles in vincristine resistance within the SHH subgroup, this was not generalisable, therefore we sought to explore alternative mechanisms to cisplatin resistance in Group 3 medulloblastoma. This was achieved using LC-MS/MS proteomic analysis to identify differentially expressed proteins between cisplatin-resistant and control cells. KEGG pathway analysis of cisplatin resistant cell lines highlighted an enrichment of proteins associated with metabolic pathways, including nucleotide, amino acid and cholesterol biosynthesis pathways.
Conclusion: This study establishes a role for ABCB1 in vincristine resistance in the SHH subgroup, re-highlighting its potential as a therapeutic target for medulloblastoma treatment. Additionally, we provide evidence that ABCB1 can be transferred on extracellular vesicles, enabling the spread of drug resistance within tumours. Finally, we identify alterations in metabolic pathways that are associated with cisplatin resistance in Group 3 medulloblastoma. This work makes a sizable contribution to the identification and development of therapeutic targets in high-risk medulloblastoma.
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