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dc.contributor.advisorBraithwaite, Antony
dc.contributor.advisorKleffmann, Torsten
dc.contributor.advisorWoolley, Adele
dc.contributor.authorAlgie, Michael
dc.date.available2018-04-06T03:44:40Z
dc.date.copyright2018
dc.identifier.citationAlgie, M. (2018). YB-1 oncoprotein in cancer and drug resistance (Thesis, Doctor of Philosophy). University of Otago. Retrieved from http://hdl.handle.net/10523/7996en
dc.identifier.urihttp://hdl.handle.net/10523/7996
dc.description.abstractY-box-binding protein 1 (YB-1) is a biomarker that is predictive of poor prognosis in cancer. Various molecular functions of YB-1 in cancer have been proposed, including the transcriptional regulation of gene expression. YB-1 also binds to RNA transcripts to influence gene expression. In the present study, the status of YB-1 as a biomarker was confirmed by immunohistochemistry using two antibodies against YB-1. However, the prognostic sensitivity of these two antibodies differed. The observed difference in antibody affinity was most likely due to the tertiary structure or protein-protein interactions (PPI) associated with various functions of YB-1 in situ. To gain further insights into the molecular functions and potential mechanisms of YB-1 in cancer biology the state of phosphorylation of YB-1 and the PPI were investigated in the cytoplasm and nucleus of two cancer cell lines. The YB-1 from the cytoplasm and nucleus of the cell lines was extensively phosphorylated. These experiments identified >250 proteins. These binding partners confirmed the multifunctionality of YB-1 as the proteins that co-purify with YB-1 participate in glycolysis, RNA splicing, RNA stabilization, translation, mitochondrial localisation, and chromosomal association. These data suggest that the bulk of YB-1 function may be explained by non-transcriptional mechanisms. Mechanisms of drug resistance were also investigated. Depleting YB-1, using siRNA duplexes, reduced MDA-MB231 cell growth and increased cell death. The loss of YB-1 sensitised MDA-MB231 cells to cisplatin exposure by increasing cell death. Cisplatin exposure altered the distribution of YB-1 protein to perinuclear spots and to foci in the nucleus of many cells. The molecular basis of YB-1 mediated cisplatin resistance was analysed by examining the alterations of YB-1 PPI during cisplatin exposure using co-immunoprecipitation of YB-1 binding partners and mass spectrometry-based protein identification. Quantitative analyses of the co-immunoprecipitated proteins from MDA-MB231 cells indicated that a subset of the proteins, such as TRIM28 and FAM120A, increased markedly after 48 and 96 hours of cisplatin exposure. The chromosomal proteins that interacted with YB-1 were disproportionately affected by cisplatin exposure. The importance of FAM120A, TRIM28, and C1QBP, three YB-1 binding partners identified here, during cisplatin exposure was studied. The subcellular distribution of FAM120A was most similar that of YB-1 in MDA-MB231 cells. Depleting YB-1 or FAM120A, but not TRIM28 or C1QBP, sensitised MDA-MB231 cells to cisplatin exposure. Depleting YB-1 alongside either FAM120A or C1QBP partially restored the growth of MDA-MB231 cells. YB-1 does not appear to participate in the repair of double-strand DNA breaks during cisplatin exposure as depleting YB-1 had no effect on the number of γH2AX foci that formed during cisplatin exposure. This is the first report that integrates findings of protein-binding partners, state of phosphorylation, and subcellular localisation of endogenous YB-1 to understand the complex functions of YB-1. These results confirm the importance of RNA binding to the molecular function of YB-1. The interaction of YB-1 with FAM120A, a novel finding, increases during cisplatin exposure and both proteins together, via an unknown molecular pathway, confer cisplatin resistance to breast cancer cells.
dc.format.mimetypeapplication/pdf
dc.language.isoen
dc.publisherUniversity of Otago
dc.rightsAll items in OUR Archive are provided for private study and research purposes and are protected by copyright with all rights reserved unless otherwise indicated.
dc.subjectcancer
dc.subjectYB-1
dc.subjectprotein interactions
dc.subjectdrug resistance
dc.subjectcisplatin
dc.subjectmass spectrometry
dc.subjectFAM120A
dc.subjectC1QBP1
dc.subjectTRIM28
dc.titleYB-1 oncoprotein in cancer and drug resistance
dc.typeThesis
dc.date.updated2018-04-04T19:59:12Z
dc.language.rfc3066en
thesis.degree.disciplineDepartment of Pathology
thesis.degree.nameDoctor of Philosophy
thesis.degree.grantorUniversity of Otago
thesis.degree.levelDoctoral
otago.openaccessOpen
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