title: Withaferin A disrupts ubiquitin-based NEMO reorganization induced by canonical NF-κB signaling.
abstract: The NF-κB family of transcription factors regulates numerous cellular processes, including cell proliferation and survival responses. The constitutive activation of NF-κB 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-κB activities. We found that Withaferin A is a robust inhibitor of canonical and constitutive NF-κB activities, leading to apoptosis of certain lymphoma lines. In the canonical pathway induced by TNF, Withaferin A did not disrupt RIP1 polyubiquitination or NEMO-IKKβ interaction and was a poor direct IKKβ 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-NEMO(Y308S) 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.
authors.affiliation: McArdle Laboratory for Cancer Research, Department of Oncology, University of Wisconsin-Madison, 6159 Wisconsin Institute for Medical Research, 1111 Highland Avenue, Madison, WI 53705, USA; Medical Scientist Training Program, University of Wisconsin-Madison, 1111 Highland Avenue, Madison, WI 53705, USA; Cellular and Molecular Biology Program, University of Wisconsin-Madison, 1111 Highland Avenue, Madison, WI 53705, USA.
authors.forename: Shawn S
authors.initials: SS
authors.lastname: Jackson
authors.affiliation: McArdle Laboratory for Cancer Research, Department of Oncology, University of Wisconsin-Madison, 6159 Wisconsin Institute for Medical Research, 1111 Highland Avenue, Madison, WI 53705, USA.
authors.forename: Christopher
authors.initials: C
authors.lastname: Oberley
authors.affiliation: McArdle Laboratory for Cancer Research, Department of Oncology, University of Wisconsin-Madison, 6159 Wisconsin Institute for Medical Research, 1111 Highland Avenue, Madison, WI 53705, USA; Cellular and Molecular Biology Program, University of Wisconsin-Madison, 1111 Highland Avenue, Madison, WI 53705, USA.
authors.forename: Christopher P
authors.initials: CP
authors.lastname: Hooper
authors.affiliation: Department of Medicine, Division of Hematology and Oncology, University of Wisconsin-Madison, 1111 Highland Avenue, Madison, WI 53705, USA.
authors.forename: Kreg
authors.initials: K
authors.lastname: Grindle
authors.affiliation: McArdle Laboratory for Cancer Research, Department of Oncology, University of Wisconsin-Madison, 6159 Wisconsin Institute for Medical Research, 1111 Highland Avenue, Madison, WI 53705, USA.
authors.forename: Shelly
authors.initials: S
authors.lastname: Wuerzberger-Davis
authors.affiliation: Department of Medicine, Division of Hematology and Oncology, University of Wisconsin-Madison, 1111 Highland Avenue, Madison, WI 53705, USA.
authors.forename: Jared
authors.initials: J
authors.lastname: Wolff
authors.affiliation: McArdle Laboratory for Cancer Research, Department of Oncology, University of Wisconsin-Madison, 6159 Wisconsin Institute for Medical Research, 1111 Highland Avenue, Madison, WI 53705, USA; Medical Scientist Training Program, University of Wisconsin-Madison, 1111 Highland Avenue, Madison, WI 53705, USA; Molecular and Cellular Pharmacology Program, University of Wisconsin-Madison, 1111 Highland Avenue, Madison, WI 53705, USA.
authors.forename: Kevin
authors.initials: K
authors.lastname: McCool
authors.affiliation: Department of Medicine, Division of Hematology and Oncology, University of Wisconsin-Madison, 1111 Highland Avenue, Madison, WI 53705, USA.
authors.forename: Lixin
authors.initials: L
authors.lastname: Rui
authors.affiliation: McArdle Laboratory for Cancer Research, Department of Oncology, University of Wisconsin-Madison, 6159 Wisconsin Institute for Medical Research, 1111 Highland Avenue, Madison, WI 53705, USA; Medical Scientist Training Program, University of Wisconsin-Madison, 1111 Highland Avenue, Madison, WI 53705, USA; Cellular and Molecular Biology Program, University of Wisconsin-Madison, 1111 Highland Avenue, Madison, WI 53705, USA. Electronic address: smiyamot@wisc.edu.
authors.forename: Shigeki
authors.initials: S
authors.lastname: Miyamoto
mesh_terms: D000818:Animals; D015153:Blotting, Western; D049109:Cell Proliferation / Q000187:drug effects; D002478:Cells, Cultured; D024202:Electrophoretic Mobility Shift Assay; D053595:Embryonic Stem Cells / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D005347:Fibroblasts / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D005434:Flow Cytometry; D005455:Fluorescent Antibody Technique; D006801:Humans; D051550:I-kappa B Kinase / Q000378:metabolism; D007124:Immunoenzyme Techniques; D047468:Immunoprecipitation; D047908:Intracellular Signaling Peptides and Proteins / Q000737:chemistry / Q000502:physiology*; D051379:Mice; D018345:Mice, Knockout; D016328:NF-kappa B / Q000378:metabolism*; D054448:Precursor Cells, B-Lymphoid / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D015398:Signal Transduction; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology; D025801:Ubiquitin / Q000378:metabolism*; D054875:Ubiquitination; D054358:Withanolides / Q000494:pharmacology*
chemical_list: D047908:Intracellular Signaling Peptides and Proteins; C514981:NEMO protein, mouse; D016328:NF-kappa B; D014409:Tumor Necrosis Factor-alpha; D025801:Ubiquitin; D054358:Withanolides; D051550:I-kappa B Kinase; C009684:withaferin A
keywords: ABC type diffuse large B-cell lymphoma; Apoptosis; IKK; NEMO; NEMO foci; NF-κB; Ubiquitin; Withaferin A