Aberrant activation of nuclear factor-κB (NF-κB) transcription factors has been implicated in the pathogenesis of breast cancer. We previously showed elevated activity of IκB kinase α (IKKα), IKKβ, and protein kinase CK2 in primary human breast cancer specimens and cultured cells. A novel inducible IKK protein termed IKK-i/IKKε has been characterized as a potential NF-κB activator. Here, we provide evidence that implicates IKK-i/IKKε in the pathogenesis of breast cancer. We show IKK-i/IKKε expression in primary human breast cancer specimens and carcinogen-induced mouse mammary tumors. Multiple breast cancer cell lines showed higher levels of IKK-i/IKKε and kinase activity compared with untransformed MCF-10F breast epithelial cells. Interestingly, IKK-i/IKKε expression correlated with CK2α expression in mammary glands and breast tumors derived from MMTV-CK2α transgenic mice. Ectopic CK2 expression in untransformed cells led to increased IKK-i/IKKε mRNA and protein levels. Inhibition of CK2α via the pharmacologic inhibitor apigenin or upon transfection of a CK2 kinase-inactive subunit reduced IKK-i/IKKε levels. Expression of a kinase-inactive IKK-i/IKKε mutant in breast cancer cells reduced NF-κB activity as judged by transfection assays of reporters driven either by NF-κB elements or the promoters of two NF-κB target genes, cyclin D1 and relB. Importantly, the kinase-inactive IKK-i/IKKε mutant reduced the endogenous levels of these genes as well as the ability of breast cancer cells to grow in soft agar or form invasive colonies in Matrigel. Thus, CK2 induces functional IKK-i/IKKε, which is an important mediator of the activation of NF-κB that plays a critical role in the pathogenesis of breast cancer. (Cancer Res 2005; 65(24): 11375-83)