Investigating novel transcriptional regulators of AURKB and their implications in cancer

Abstract

The Aurora B kinase regulates key processes and checkpoints during mitosis. Abnormalities of Aurora B result in aneuploidy and chromosomal instability. It is overexpressed in many cancers and associated with earlier metastasis and poorer prognosis. The work in this thesis sought to investigate the promoter and 5' region of AURKB, between it and neighbouring gene CTC1, to test the hypothesis that it contains sequences that can highlight novel regulators of the gene. In silico analysis of the region followed by experimental validation of candidate factors was carried out. The results propose lincRNA, Linc00324 as a novel regulator of AURKB, and able to reduce promoter activity. Analysis of datasets from the Cancer Genome Atlas revealed a highly statistically significant link between Linc00324 and AURKB mRNA expression in breast invasive carcinoma, with a tendency towards under-expression of this lncRNA in samples where AURKB was overexpressed. Transcription factors (TFs), AHR, BACH1, EGR1, and HIF1- were identified to have predicted transcription factor binding sites (TFBS) in both the AURKB promoter and Linc00324. The results of EMSA experiments, supported the binding of EGR1 and BACH1 to predicted TFBSs within the AURKB promoter. Analysis of ENCODE ChIP-seq data confirmed enrichment of EGR1 and BACH1 at the AURKB promoter region and at Linc00324. These transcription factors also caused a dose-dependent increase of AURKB promoter activity, in gene reporter assays. The candidate TFs are all, either hypoxia-responsive or involved in orchestrating the adaptive response to hypoxia through transcriptional regulation of target genes. Cobalt chloride was used to mimic hypoxia in experiments and results showed reduced activity of the AURKB promoter. Hypoxia is therefore proposed as a condition that can influence AURKB promoter activity and provides a mechanistic link to the identified TFs and potentially Linc00324. This has implications for the potential dysregulation of AURKB in cancer, within hypoxic intra-tumoral environments.