YB-1 oncoprotein in cancer and drug resistance
Y-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.
Advisor: Braithwaite, Antony; Kleffmann, Torsten; Woolley, Adele
Degree Name: Doctor of Philosophy
Degree Discipline: Department of Pathology
Publisher: University of Otago
Keywords: cancer; YB-1; protein interactions; drug resistance; cisplatin; mass spectrometry; FAM120A; C1QBP1; TRIM28
Research Type: Thesis