MUC1 oncoprotein activates the IκB kinase β complex and constitutive NF-κB signalling

Abstract

Nuclear factor-κB (NF-κB) is constitutively activated in diverse human malignancies by mechanisms that are not understood 1,2 . The MUC1 oncoprotein is aberrantly overexpressed by most human carcinomas and, similarly to NF-κB, blocks apoptosis and induces transformation [3][4][5][6] . This study demonstrates that overexpression of MUC1 in human carcinoma cells is associated with constitutive activation of NF-κB p65. We show that MUC1 interacts with the highmolecular-weight IκB kinase (IKK) complex in vivo and that the MUC1 cytoplasmic domain binds directly to IKKβ and IKKγ. Interaction of MUC1 with both IKKβ and IKKγ is necessary for IKKβ activation, resulting in phosphorylation and degradation of IκBα. Studies in non-malignant epithelial cells show that MUC1 is recruited to the TNF-R1 complex and interacts with IKKβ-IKKγ in response to TNFα stimulation. TNFα-induced recruitment of MUC1 is dependent on TRADD and TRAF2, but not the death-domain kinase RIP1. In addition, MUC1-mediated activation of IKKβ is dependent on TAK1 and TAB2. These findings indicate that MUC1 is important for physiological activation of IKKβ and that overexpression of MUC1, as found in human cancers, confers sustained induction of the IKKβ-NF-κB p65 pathway.Nuclear localization of NF-κB p65 was studied in HCT116 colon cancer and HeLa cervical cancer cells that stably express either an empty vector or MUC1 (ref. 4, also see Supplementary Information, Fig. S1a). Levels of nuclear NF-κB p65 were lower in vector cells than in cells expressing MUC1 (Fig. 1a). Human ZR-75-1 and MCF-7 breast cancer cells that express endogenous MUC1 were stably transfected to express either an empty vector or a MUC1 siRNA 4 ( Supplementary Information, Fig. S1a). Silencing of MUC1 in ZR-75-1 (ref. 4) and MCF-7 cells 7 decreased nuclear NF-κB p65 (Fig. 1b). MUC1 expression was also associated with a decrease in cytosolic NF-κB p65 levels in HeLa and ZR-75-1 cells ( Supplementary Information, Fig. S1b). To determine whether MUC1 is associated with activation of the NF-κB p65 transcription function, HeLa and ZR-75-1 cells were transfected with a construct containing a NF-κB-binding site upstream of the luciferase reporter (pNF-κB-Luc). MUC1 expression was associated with activation of pNF-κB-Luc (Fig. 1c). In contrast, MUC1 had no effect on activation of a pNF-κB-Luc construct that was mutated at the NF-κB p65 binding site (Fig. 1c). In addition, expression of Bclx L , a gene activated by NF-κB, was higher in cells expressing MUC1 (Fig. 1d). To determine whether MUC1 affects IκBα phosphorylation (as phosphorylated IκBα is targeted for ubiquitination and proteosomal degradation) cytosolic lysates were immunoblotted with an anti-phospho-IκBα antibody. Indeed, phospho-IκBα levels were significantly higher in cells expressing MUC1 (Fig. 1e). Assessment of IκBα stability indicated that MUC1 expression increases degradation of IκBα (Fig. 1f). The half-lives of IκBα in the absence and presence of MUC1, were 6.7 ± 0.5 h and 3. Fig. 2a). In vitro studies with puri...

Muc1 Cell Surface Mucin Attenuates Epithelial Inflammation in Response to a Common Mucosal Pathogen

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