Nuclear factor-KB (NF-KB) is constitutively activated in diverse human malignancies. The mucin 1 (MUC1) oncoprotein is overexpressed in human carcinomas and, like NF-KB, blocks cell death and induces transformation. The present studies show that MUC1 constitutively associates with NF-KB p65 in carcinoma cells. The MUC1 COOH-terminal subunit (MUC1-C) cytoplasmic domain binds directly to NF-KB p65 and, importantly, blocks the interaction between NF-KB p65 and its inhibitor IKBA. We show that NF-KB p65 and MUC1-C constitutively occupy the promoter of the Bcl-xL gene in carcinoma cells and that MUC1-C contributes to NF-KBmediated transcriptional activation. Studies in nonmalignant epithelial cells show that MUC1-C interacts with NF-KB in the response to tumor necrosis factor-A stimulation. Moreover, tumor necrosis factor-A induces the recruitment of NF-KB p65-MUC1-C complexes to NF-KB target genes, including the promoter of the MUC1 gene itself. We also show that an inhibitor of MUC1-C oligomerization blocks the interaction with NF-KB p65 in vitro and in cells. The MUC1-C inhibitor decreases MUC1-C and NF-KB p65 promoter occupancy and expression of NF-KB target genes. These findings indicate that MUC1-C is a direct activator of NF-KB p65 and that an inhibitor of MUC1 function is effective in blocking activation of the NF-KB pathway. [Cancer Res 2009;69(17):7013-21]

The mucin 1 (MUC1) oncoprotein is aberrantly overexpressed by ∼90% of human breast cancers. However, there are no effective agents that directly inhibit MUC1 and induce death of breast cancer cells. We have synthesized a MUC1 inhibitor (called ) that binds to the MUC1 cytoplasmic domain and blocks the formation of MUC1 oligomers in cells. GO-201, and not an altered version, attenuates targeting of MUC1 to the nucleus of human breast cancer cells, disrupts redox balance, and activates the DNA damage response. GO-201 also arrests growth and induces necrotic death. By contrast, the MUC1 inhibitor has no effect on cells null for MUC1 expression or nonmalignant mammary epithelial cells. Administration of GO-201 to nude mice bearing human breast tumor xenografts was associated with loss of tumorigenicity and extensive necrosis, which results in prolonged regression of tumor growth. These findings show that targeting the MUC1 oncoprotein is effective in inducing death of human breast cancer cells in vitro and in tumor models. [Cancer Res 2009;69(12):5133-41]

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...