Investigating IKKα-dependent NF-kB signalling and gene regulation in a human bone cancer cell line

IKKα, a serine threonine kinase, has been consistently implicated in a range of inflammation-driven diseases and cancers. In the non-canonical NF-κβ signalling pathway, IKKα homodimers function in a NIK-dependent manner, activated by specific members of the TNF superfamily, including lymphotoxin and LIGHT ligands. This activation led to phosphorylation of p100 and the subsequent nuclear translocation of the NF-κB heterodimer RelB:p52. In contrast, the canonical NF-κB pathway was activated by various pro-inflammatory stimuli, including IL-1β and TNFα, and relies on TAK1 and the IKK complex, which is composed of IKKβ, IKKα, and IKKγ. Within this complex, IKKβ plays the dominant role in initiating downstream signalling, resulting in phosphorylation and degradation of IκBα and the nuclear translocation of NF-κB dimers, most commonly p50:p65. Previously, a novel signalling mechanism was described in which IL-1β induced early p100 phosphorylation in an IKKα-dependent manner in human endothelial cells and in the human osteosarcoma cell line U2OS. In this study, potential downstream functions of p100 phosphorylation in this context were investigated, and IL-1β-mediated IKKα-dependent genes were explored. This study validated that IL-1β-induced p100 phosphorylation is IKKα-dependent in U2OS cells and demonstrated the selectivity of two novel first-in-class IKKα inhibitors, SU1261 and SU1349, and their ability to inhibit this signalling mechanism. Furthermore, the TAK1 inhibitor 5Z-7-oxozeaenol was used to show that both p100 and IKKα phosphorylation was inhibited by TAK1 inhibition, and that this occurred at lower inhibitor concentrations than those required to suppress IKKB-dependent responses following IL-1β stimulation. Additionally, downstream p52 and p65 nuclear translocation were inhibited at concentrations similar to those affecting IKKβ- dependent responses rather than IKKα-dependent responses, indicating that p52 was not downstream of IKKα-mediated p100 phosphorylation in this model. Therefore, Illumina RNA sequencing was subsequently employed to identify potential IKKα- dependent genes using IKKα CRISPR-Cas9 knockdown in this signalling system. These included the gene encoding the osteoclast-associated receptor, OSCAR, which was upregulated in the transcriptomic analysis of IKKα CRISPR-Cas9 knockdown cells but downregulated at the mRNA level following IKKα siRNA knockdown independently of IL-1B stimulation. The gene encoding DHCR7, a key enzyme in the final stages of cholesterol biosynthesis, was found to be downregulated in IKKα CRISPR-Cas9 knockdown U2OS cells in the transcriptomic data, which was also IL1B-independent. This was subsequently validated by RT-qPCR, where similar findings were observed in both CRISPR-Cas9 and siRNA knockdown models. Red Amplex cholesterol assays also demonstrated significantly lower cholesterol production in IKKα CRISPR-Cas9 knockdown U2OS cells compared with wild-type cells. A smaller, yet significant, reduction in cholesterol production was also observed in IKKα siRNA-transfected cells compared with non-targeting siRNA controls. Finally, this study examined IL-1B-mediated expression of CXCL5, a gene associated with osteosarcoma progression and osteogenesis that contributed to tumour development. CXCL5 mRNA expression was shown to be IKKα-dependent using both IKKα CRISPR-Cas9 and siRNA knockdown methods, as well as through treatment with the novel IKKα inhibitors (SU1261 and SU1349). IL-1B-mediated CXCL8 expression was also investigated as a comparator, as it is a well-established as an IKKB-dependent gene. Like CXCL5, CXCL8 binds to CXCR2 to promote osteogenesis and osteosarcoma progression. This study demonstrated that IKKα inhibition increased IL-1B-induced CXCL8 mRNA expression, whereas treatment with a dual IKKα/IKKβ inhibitor, SU1266, decreased both CXCL5 and CXCL8 expression. Collectively, this study provided the first evidence that CXCL5 is a bona fide downstream IKKα-dependent target and provides an insight into several other functional roles which IKKα may play in osteosarcoma cells.

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