Taylor, Louisa
(2022)
Investigating the role of Y-box binding protein 1 in medulloblastoma tumour progression and drug resistance.
PhD thesis, University of Nottingham.
Abstract
Introduction: Medulloblastoma relapse represents the most significant unmet clinical challenge in childhood cancer. In spite of this, our understanding of the molecular biology of relapsed medulloblastoma is limited. Evidence is emerging, however, that the biology of medulloblastoma at relapse differs significantly from the disease at diagnosis. Such genetic divergence may arise through the emergence and expansion of a therapy-resistant sub-population of cells. Thus, the identification of targets that either mark therapy resistant cell populations, or drive the proliferation of resistant medulloblastoma cells, may improve treatment strategies for relapsed disease. YB-1, a transcription factor encoded by the YBX1 gene, has been extensively researched in non-CNS tumours where elevated expression is associated with tumour progression and drug resistance. However, little is known regarding the significance of YB-1 in paediatric brain tumours. This study, therefore, set out to examine the functional role of YB-1 in medulloblastoma tumourigenesis. Particular attention was paid to the multi-drug transporter ABCB1, a putative YB-1 target, which our studies have shown to be associated with cancer cell therapy resistance and high-risk medulloblastoma.
Methods and Results: Genomic analysis of medulloblastoma patient datasets demonstrated elevated YBX1 expression across all four core molecular medulloblastoma subgroups, which was associated with poor overall survival and metastasis. In support of this, genetic knockdown of YBX1 diminished the invasive capability of medulloblastoma cells in 3D in vitro invasion assays. To build a global picture of YB-1 transcriptional control in medulloblastoma, whole transcriptome sequencing of YBX1 knockdown cell lines was employed. Ingenuity Pathway Analysis indicated YB-1 involvement in key cellular processes including lipid metabolism, the Sirtuin signalling pathway and the activation of MYC and mTOR pathway components; revealing novel, pro-tumourigenic functions for YB-1 in medulloblastoma cells.
To assess the association between YB-1 and ABCB1 transcriptional regulation, chromatin immunoprecipitation (ChIP) assays were undertaken, confirming strong YB-1 interaction within the distal ABCB1 promoter region. Accordingly, YB-1 depletion was associated with decreased ABCB1 expression and concomitant increased sensitivity to vincristine – an ABCB1 substrate. Further investigation of the association between YB-1 and drug response, this time with cisplatin, a non-ABCB1 substrate, revealed YB-1 nuclear expression was elevated in medulloblastoma cells following short-term cisplatin treatment, indicative of YB-1 involvement in the acute cisplatin stress response. ChIP sequencing of cisplatin-tolerant cell lines was used to identify YB-1 target genes associated with a chronic cisplatin-tolerant phenotype. This highlighted an additional function for YB-1 in the acquisition of cisplatin resistance in medulloblastoma cells.
In order to better understand general mechanisms surrounding drug resistance in medulloblastoma, 3’mRNA sequencing of stable cisplatin- and vincristine tolerant cell lines was also undertaken. Notably, differential gene expression analysis identified the existence of a drug-tolerant gene expression signature – a gene set common to all sequenced drug-tolerant lines, irrespective of subgroup or treatment type. These genes likely represent novel mediators of the medulloblastoma drug-tolerant state and thus may present new opportunities for therapeutic targeting.
Conclusion: This study has characterised the role of YB-1 in medulloblastoma and has demonstrated clear associations between YB-1 expression and various aspects of medulloblastoma tumourigenesis, including invasion, MYC activity and lipid metabolism. Importantly, we also provide evidence to support a function for YB-1 in both acute drug response and acquired drug resistance and identify YB-1 as a transcriptional regulator of drug resistance-related gene ABCB1. Finally, we identify therapeutically targetable hits implicated in the acquisition of drug-tolerance, both directly regulated by and independent from YB-1, which may inform future pre-clinical investigations to establish more effective therapeutic options for the treatment of high-risk medulloblastoma.
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