Cancer accounts for the highest amounts of disease related premature deaths worldwide with 8.1 million of deaths being associated with malignancy. In the cancer microenvironment, particularly in solid tumours, hypoxia plays a significant role in progression and metastasis by altering signal transduction and gene regulation which leads to aggressive phenotypes, poor prognosis and lower survival rates. Hypoxia-Inducible Factor (HIF) driven gene regulation is well established and believed to promote survival of tumour cells under the hypoxic microenvironment. Accumulating evidence also suggests that Specificity protein (Sp) family transcription factors might also play a role in the hypoxic microenvironment by regulating transcription of key hypoxia responsive genes such as VEGFA in either a HIF dependent or independent manner. In normal cells, Sp transcription factors are ubiquitously expressed and known to regulate numerous genes involved in vital cellular pathways such as cell cycle, apoptosis and angiogenesis. In tumour environments, deregulated Sp protein levels have been demonstrated and shown to correlate with poor prognosis and treatment response. However, the exact role of Sp transcription factors in hypoxic microenvironment is not fully understood. This study aimed to identify the effect of severe (chronic) hypoxia on Sp transcription factors and hypoxia-responsive gene regulation using breast cancer as a cell model. Initial studies measured the expression levels and binding activity of Sp transcription factors. Subsequently, an integrative genomic analysis was performed to identify Sp driven hypoxia-responsive genes in breast cancer cells. The study was further extended to analyse the binding kinetics of Sp protein inhibitors using surface plasmon resonance spectroscopy. Finally, transcriptional changes of hypoxia responsive genes were examined after addition of Sp inhibitors or knockdown of Sp1 level under the hypoxic environment. Expression analysis of Sp family members (Sp1-4) showed that the transcript levels of Sp genes were unaffected due to chronic hypoxic exposure whilst Sp protein levels were induced in all three cell lines. However, expression patterns were dependent on tissue type and severity of hypoxia. Twenty genes were identified as potential Sp driven hypoxia responsive genes which are consist of GC-rich putative Sp binding sites in their promoters. Gene expression analysis validated the hypoxic induction of these genes and their dependency on Sp protein-mediated transcription. Affinity studies of Sp protein inhibitors prove binding of antibiotic derivatives, Mithramycin A and Chromomycin A to GC-rich regions with different binding affinities and kinetics. Different Equilibrium constants (Kd) of Mithramycin A and Chromomycin A were identified which varied according to the promoter sites (10−3 to 10−6 M range). Furthermore, novel data also confirm the Mithramycin-DNA interaction is independent of cation Mg2+ which has been considered obligatory for DNA interaction. Interestingly, nordihydroguaiaretic acid (NDGA) derivative, Terameprocol exhibits no detectable interaction with linear DNA consisting of Sp binding sites. These results emphasise the importance of Sp proteins as regulators of hypoxia-mediated gene transcription. Sp-regulated transcription is vital in altering hypoxia-related cellular pathways and has a potential as biomarkers for solid tumours. Moreover, these results suggest the potential use of Sp antagonists to inhibit expression of key hypoxic genes in the cancer microenvironment. Results also provide solid background knowledge on pharmacokinetics of Sp inhibitors which will be useful in synthesis of new derivatives which can be used in novel therapeutic strategies for cancer and perhaps other diseases. Understanding the molecular mechanisms of Sp mediated hypoxic gene regulation can be further extended to elucidate other cellular stress and cellular adaptive mechanism.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:762091 |
| Date | January 2017 |
| Creators | Wijesinghe, Nishadh P. |
| Contributors | Waby, Jenny ; Greenman, John |
| Publisher | University of Hull |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://hydra.hull.ac.uk/resources/hull:16874 |
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