Activation of NF-κB following detachment delays apoptosis in intestinal epithelial cells

We reported earlier that IL-1β, an NF-κB-regulated cytokine, was made by intestinal epithelial cells during detachment-induced apoptosis (anoikis) and that IL-1 was antiapoptotic for detached cells. Since surviving anoikis is a prerequisite for cancer progression and metastases, we are further exploring the link between anoikis and cytokines. Here we determined that multiple genes are expressed following detachment including a number of NF-κB-regulated products and therefore aimed to determine whether NF-κB signalling plays any role in regulating apoptosis. Using Western blotting, we detected that IκBα becomes phosphorylated immediately following detachment and that levels of phospho-IκBα peaked within 20 min. Phosphorylation of IκBα was followed by Rel A (p65) nuclear translocation. Increased NF-κB activity following detachment was confirmed using the detection of NF-κB-promoted luciferase gene expression delivered by adenovirus infection. Infection of cells with adenovirus expressing a super-repressor IκBα protein and pharmacological inhibitors of NF-κB resulted in the failure to phosphorylate IκBα, a more rapid activation of caspases and earlier apoptosis. We also detected that IκB kinase α (IKKα) and not IKKβ became phosphorylated following detachment. Since IKKα is activated by NF-κB-inducing kinase (NIK), we overexpressed native NIK using an adenovirus vector that resulted in enhanced phospho-IκBα and nuclear p65 in detached cells compared to control detached cells but did not result in a significantly greater number of cells surviving to 24h. We conclude that detachment directly activates NF-κB, which, in addition to launching an inflammatory cytokine wave, contributes to a delay in apoptosis in intestinal epithelial cells.