The NF-kB signalling pathway is a critical mediator of the cellular responses to inflammatory cytokines. The IκB kinase (IKK) complex, which is composed of two catalytic subunits (IKKα and IKKβ) and one regulatory subunit (IKKγ/NEMO) acts as the master regulator of NF-κB transcription factor activity. Seminal genetic studies in knockout (KO) mouse embryonic fibroblasts (MEFs) have defined two pathways of NF-κB activation; a canonical pathway, activated in response to cytokines such as TNFα/IL-1β, that requires NEMO and predominantly IKKβ catalytic activity; and a non-canonical pathway, activated in response to a subset of TNF-family cytokines, which requires IKKα and NIK kinase. We have generated and validated CRISPR-Cas9 IKKα, IKKβ and IKKα/β DKO HCT116 colorectal cancer cell lines to interrogate novel functions of the I kappa B kinases in colorectal cancer, including the relative contributions of these kinases to the activation of NF-κB signalling pathways downstream of TNFα induction. Contrary to the seminal studies in KO MEFs, IKKα appeared to make a more significant contribution to canonical NF-κB induction in these cells than IKKβ. Western blot studies demonstrated that both IKKs contributed to the phosphorylation and degradation of IκB and the phosphorylation of the NF-κB subunit, p65 at Serine 536. However, high-content immunofluorescence studies demonstrated that IKKα KO cells were defective in TNFα-induced nuclear translocation of p65 compared to WT and IKKβ KO cells. Additionally, NF-κB-driven luciferase reporter assays showed that IKKα, but not IKKβ, KO cells exhibited significantly reduced NF-κB-dependent gene expression following TNFα stimulation. We also have evidence to suggest that the phosphorylation site at Serine 468 on p65, previously defined as an IKKβ-dependent site, is in-fact an IKKα-dependent site in these cells. Furthermore, IKKα knockout revealed a potentially important role for IKKα activity in preventing the stabilisation of NIK protein following prolonged TNFα stimulation. RNA sequencing analysis of wild-type, IKKα KO, IKKβ KO and IKKα/β DKO cells stimulated with TNFα was performed to identify genes whose expression were differentially deregulated by IKK KO. These analyses confirmed the importance of IKKα for canonical NF-κB gene expression. Furthermore, IKKβ knockout had unexpected effects on the expression of a broad range of genes involved in chromatin organisation, cytoskeletal organisation, mitotic cell cycle control and the DNA damage response. During the characterisation of IKK KO cells it was discovered that the expression of NEMO was downregulated at the protein, but not mRNA level by approximately 50% in IKKα KO cells and 90% in IKKα/β DKO cells. IKKβ KO cells, meanwhile, exhibited wild-type NEMO expression. Emetine-chase and radioactive pulse chase labelling experiments demonstrated that the half-life of NEMO in IKKα and IKKα/β DKO cells was significantly shortened due to enhanced proteasomal turnover. Bioinformatics analyses predicted significant regions of intrinsic structural disorder within NEMO, particularly at the N- and C-termini, the former of which overlapped with the IKK binding domain. On this basis, the susceptibility of NEMO to in vitro degradation by the 20S proteasome was examined, with NEMO proving be a highly effective substrate of the 20S proteasome. Importantly, IKKα and IKKβ were both shown to protect NEMO from proteasomal degradation, leading us to propose a model whereby interaction with IKK kinase subunits sequesters/masks intrinsically disordered regions in NEMO that would otherwise make NEMO a highly effective substrate for ubiquitin-dependent and/or ubiquitin-independent proteasomal degradation. BMS-345541 is a commercially available allosteric inhibitor of IKKβ that has been used extensively in numerous studies, including a report that proposed novel functions for IKKβ in mitotic cell cycle progression (Blazkova et al., 2007). Similar antiproliferative effects to those reported by Blazkova et al., were observed during the characterisation of a novel ATP-competitive inhibitor of IKKβ, AZD2230. In depth characterisation of the selectivity of AZD2230 and BMS-345541, however, revealed that the antiproliferative effects of AZD2230 and BMS-345541 are, in fact, due to off-target inhibition, potentially at the level of RNA Polymerase II C-terminal domain phosphorylation, and hence general transcription. Collectively, these studies reveal novel functions of the IKK kinases in NF-κB signalling and inform therapeutic strategies for targeting chronic canonical NF-κB activation in colorectal cancer.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:744930 |
| Date | January 2018 |
| Creators | Prescott, Jack |
| Contributors | Cook, Simon ; Smith, Paul |
| Publisher | University of Cambridge |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | https://www.repository.cam.ac.uk/handle/1810/277025 |
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