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Title: Disrupting the inhibitory Kappa B kinase (IKK)-Aurora A axis in cancer
Author: Wilson, Alastair
Awarding Body: University of Strathclyde
Current Institution: University of Strathclyde
Date of Award: 2013
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The IKK complex is an essential regulator of the NF-κB- signalling pathway. It is comprised of three sub-units; catalytic subunits IKKα and IKKβ and the catalytically inactive scaffolding protein IKKγ/NF-κB Essential Modulator (NEMO). The complex can be assembled in multiple conformations as either hetero- or homo-dimers of IKKα/β with or without the scaffolding protein IKKγ. This regulates NF-κB signalling and related cellular inflammatory responses that are often constitutively active in many cancer cells. Beyond regulation of NF-κB signalling a number of other potential IKK substrates have been proposed including the mitotic kinase Aurora A, one of three serine/threonine kinases of the Aurora kinase family that mediate mitotic progression in cells. Aurora A kinase specifically has several roles in the cell cycle, regulating spindle formation/attachment, centrosome maturation and cytokinesis, and has been observed to be overexpressed in several cancers. Several studies have linked IKK complex to Aurora A signalling, as either a substrate of IKKα (Prajapati et al, 2006) or as a target for IKKβ-mediated βTRCP-regulated degradation (Irelan et al, 2007) both linked to cell cycle progression independent of NF-κB signalling. Aurora A kinase has however also been linked to NF-κB signalling through the phosphorylation and degradation of IκBα in primary breast tumours in a subgroup of patients exhibiting Aurora A gene amplification (Briassouli et al, 2007). Prostate cancer is a significant worldwide health issue and is the 2nd most prevalent cancer in males and 5th most prevalent cancer overall with over 900,000 new cases diagnosed in 2008 (Ferlay et al, 2010). The current therapies treating prostate cancer are far from ideal and have significant side effects. Both Aurora A and NF-κB-related kinases have been shown to be over-expressed or constitutively active in prostate cancer and therefore the suggested novel interaction may serve as a potential mechanism for intervention in cancer. This study aimed to validate IKK-Aurora A interactions and potentially identify novel targets for the development of therapeutics in prostate cancer. Using recombinant protein methodologies the direct interaction of IKKα and IKKβ with Aurora A were confirmed. Utilising peptide array technology, it has been possible to further elucidate the nature of the interaction between Aurora A and IKKα/β. The interaction of Aurora A and IKKα/β was mapped to two key regions of the IKKs, the kinase domain and the NEMO binding domain. The NEMO-Binding Domain is a conserved hexapeptide sequence, L-D-W-S-W-L, across both IKKα and IKKβ mediating the interaction with the scaffolding protein IKKγ/NEMO. This highlighted a novel role of the NBD as a multi-protein docking site. Binding of these proteins was further confirmed endogenously in a cellular setting, by means of co-immunoprecipitation and also the kinetic profiles of these interactions were characterised using recombinant proteins and Surface Plasmon Resonance. Thereafter Cell Permeable Peptides (CPPs) based on the IKK NBD were utilised as pharmacological tools, to examine potential competitive perturbation of Aurora A-IKK interactions mediated by the NBD in prostate cancer cells. This identified in a cellular setting treatment with NBD CPPs resulted in the inhibition of Aurora A phosphorylation and induced Aurora A degradation, which correlated with cell cycle arrest. It was also suggested through molecular modeling that the IKK NBD peptides could bind to the Aurora A protein, potentially at sites engaged by TPX2, an activation accessory protein for Aurora A. This purported mechanism may account for the observed IKK NBD peptide-mediated de-phosphorylation and subsequent degradation of Aurora A. Therefore, this study has identified the NEMO binding d omain as a potential multi-protein binding site and has identified novel functionality of the IKK NBD cell permeable peptides. This may represent a novel target for the intervention of IKK-mediated regulation of the cell cycle and serve as the basis for the development of novel peptidomimetics and related small molecules for the treatment of Prostate cancer.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available