Withaferin A disrupts ubiquitin-based NEMO reorganization induced by canonical NF-κB signaling

The NF-kappa B family of transcription factors regulates numerous cellular processes, including cell proliferation and survival responses. The constitutive activation of NF-kappa B has also emerged as an important oncogenic driver in many malignancies, such as activated B-cell like diffuse large B cell lymphoma, among others. In this study, we investigated the impact and mechanisms of action of Withaferin A, a naturally produced steroidal lactone, against both signal-inducible as well as constitutive NF-kappa B activities. We found that Withaferin A is a robust inhibitor of canonical and constitutive NF-kappa B activities, leading to apoptosis of certain lymphoma lines. In the canonical pathway induced by TNF, Withaferin A did not disrupt RIP1 polyubiquitination or NEMO-IKK beta interaction and was a poor direct IKK beta inhibitor, but prevented the formation of TNF-induced NEMO foci which colocalized with TNF ligand. While GFP-NEMO efficiently formed TNF-induced foci, a GFP-NEMOY3085 mutant that is defective in binding to polyubiquitin chains did not form foci. Our study reveals that Withaferin A is a novel type of IKK inhibitor which acts by disrupting NEMO reorganization into ubiquitin-based signaling structures in vivo. (C) 2014 Elsevier Inc. All rights reserved.