Oncogene (2015) 34, 2279–2287 © 2015 Macmillan Publishers Limited All rights reserved 0950-9232/15 www.nature.com/onc REVIEW Non-conventional functions for NF-κB members: the dark side of NF-κB L Espinosa, P Margalef and A Bigas NF-κB pathway exerts an essential function in the regulation of the immune response, which has been the nucleus of numerous studies for the past 25 years. Both activation of the pathway and termination of the NF-κB response are tightly regulated events, which is essential to prevent exacerbated inflammatory responses. Thus, alterations in NF-κB regulatory elements might result in tissue damage and cancer in different systems. In addition, several of the proteins involved in NF-κB regulation display additional, and much less studied, functions that connect with specific NF-κB-unrelated pathways. Many of these pathways are in turn regulators of particular physiologic and/or pathologic responses. Which are the principal non-conventional functions that have been identified for specific NF-κB elements, how they connect with other signaling pathways and what is their potential impact on cancer is the focus of this review. Oncogene (2015) 34, 2279–2287; doi:10.1038/onc.2014.188; published online 30 June 2014 INTRODUCTION multiple stimuli such as TNF-α, interleukin-1β (IL1β), pathogen- More than 25 years ago, NF-κB was discovered in the laboratory of associated molecular patterns and others. Association of TNFα Sen and Baltimore.1 After that, five members of the transcription with its receptor (TNFR1) localized at the cell surface leads to the factor NF-κB have been identified in mammals: RelA (p65), RelB recruitment of the adaptor proteins TRADD, TRAF2, cIAP1, cIAP2 and c-Rel, the p50 precursor NF-κB1 (p105) and NF-κB2 (p100) that and RIP1. Assembly of this complex to the TNFR1 promotes RIP1 can be processed to p52. These family of proteins are polyubiquitination by non-degradative K63-linked polyubiquitin characterized by a highly conserved Rel homology domain, which chains, which then serve as docking sites for TAB2 and TAB3, mediates dimerization, DNA binding and their interaction with which are essential components of the TAK1 complex. The IKK inhibitor of κB(IκB) proteins.2,3 Targeted disruption of different complex is also recruited to the TNFR1 signalosome through NF-κB family genes in mice has revealed a certain degree of NEMO, which recognizes the ubiquitinated RIP1, thus bringing redundancy,4,5 likely due to their ability to form homo- and TAK1 and IKK into close proximity, and also facilitating IKK heterodimers that recognize a common DNA sequence motif, the phosphorylation by TAK1. Recently, a novel ubiquitin E3 ligase κB consensus.6 One of the more striking phenotypes is that complex, LUBAC, was found to be recruited to TNFR1 in response obtained following p65 deletion, which leads to embryonic death to stimulation and required for NF-κB activation.16 LUBAC by massive tumor necrosis factor-α (TNFα)-induced apoptosis in (composed of HOIP, HOIL and Sharpin) and Ubc5 catalyze linear the liver.7 polyubiquitination of NEMO in vitro,17 and depletion of a single – Activation of different NF-κB dimers (except for p52:RelB) is LUBAC element impairs NF-κB activation.18 20 In general, different controlled by IκB proteins, which prevent nuclear entry and DNA E3 ubiquitin ligases participate of the TNFR1 signaling by binding through masking the nuclear localization signal of Rel promoting non-degradative ubiquitination of specific NF-κB proteins. A critical regulatory event in NF-κB signaling is the site- elements that is counteracted by the activity of the CYLD- and – specific phosphorylation of IκB(α, β and ε) by the IκB kinase (IKK) A20-deubiquitinating enzymes.21 26 complex, which results in their subsequent polyubiquitination and Other stimuli such as BAFF or the CD40 ligand are involved in – proteasomal degradation. Then, IκBs are rapidly resynthesized in the activation of the alternative NF-κB pathway.27 31 TRAF2, TRAF3 response to NF-κB activation, leading to a robust negative and cIAP1/2 inhibit alternative NF-κB in resting cells by targeting regulation of the pathway that is crucial for the proper termination the NIK kinase for ubiquitin-dependent degradation.32 Upon CD40 of the NF-κB response.8–10 or BAFF-R activation, TRAF2 ubiquitinates and activates cIAP1– The IKK complex consists of the IKKα and IKKβ kinases, and the cIAP2, which in turn induce a degradative K48-linked polyubiqui- regulatory subunit IKKγ, also called NEMO (for NF-κB essential tination of TRAF3. In the absence of TRAF3, newly synthesized NIK modulator).11–15 Activation of IKKα and IKKβ is triggered by fails to associate with the TRAF2–cIAP1–cIAP2 complex. This allows phosphorylation at specific residues (serines 176–180 for IKKα and NIK accumulation and its activation by autophosphorylation. Then, 177–181 for IKKβ)11 that is catalyzed by NF-κB-inducing kinase activated NIK phosphorylates IKKα,33,34 thus promoting p100 (NIK) and transforming growth factor-β activated kinase 1 ( TAK1), phosphorylation, association with SCFβTrCP and its subsequent respectively, which determine the signaling through alternative or proteasome-dependent processing into p52.35 Generation of canonical NF-κB pathway. Canonical NF-κB can be induced by mature p52 by IKKα is IKKβ and NEMO independent,27 and is Stem Cells and Cancer Research Laboratory, Institut Hospital del Mar d'Investigacions Mèdiques (IMIM), Barcelona, Spain. Correspondence: Dr L Espinosa, Stem Cells and Cancer Research Laboratory, Institut Hospital del Mar d'Investigacions Mèdiques (IMIM), Dr Aiguader 88, Barcelona 08003, Spain. E-mail: [email protected] Received 16 April 2014; revised 19 May 2014; accepted 23 May 2014; published online 30 June 2014 Non-conventional functions for NF-κB members L Espinosa et al 2280 sufficient to promote nuclear translocation of the p52/RelB dimer impaired IL-12 activation, suggesting that this nuclear IKKα leading to alternative NF-κB signaling.36,37 function is also dependent on NEMO.44 On the other hand, phosphorylation of histone H3 by IKKα is not restricted to NF-κB- regulated gene promoters. For example, IKKα mediates the NON-CONVENTIONAL FUNCTIONS FOR IKKα activation of c-fos, a mitogenic transducer, in response to As mentioned, although most IKKα is located in the cytoplasm as epidermal growth factor, and in the absence of NF-κB part of the IKK complex, its kinase activity is not essential for the activation.45 IKKα also promotes transcription of hormone- activation of canonical NF-κB by most proinflammatory responsive genes in breast cancer cells through its direct stimuli.38,39 However, its activity is primarily required for the interaction with the nuclear hormone coactivator SRC-3.46 activation of the alternative pathway, which participates on Specifically, IKKα facilitates the recruitment of ERα and SRC-3 to adaptative immunity and lymphoid organogenesis.40 On the the cyclin D1 and c-myc gene promoters, leading to gene other hand, several stimuli promote IKKα to accumulate in the transcription, whereas reduction of IKKβ, p65, c-Rel, or p100 levels nuclear compartment of the cells, where it phosphorylates specific do not have any effect. IKKα-dependent activation of these genes substrates resulting in particular outcomes (Figure 1). results in increased proliferation of breast cancer cells. In T cells, IKKα regulates IL-2, interferon-γ and IL-17 transcription in the T helper type 17 lineage, a subset of T cells with a prominent role PHOSPHORYLATION OF HISTONE H3 BY IKKα in many inflammatory diseases. A kinase dead form of IKKα failed The first indication that IKKα exerted additional nuclear functions to promote inflammatory-related transcription and blocked the was the demonstration that IKKα accumulates in the nucleus and development of autoimmune encephalomyelitis.47 Physiologically, phosphorylates serine 10 of histone H3 at NF-κB-dependent IKKα is present in the nucleus of CD4+ T cells and recruited to the promoters following TNFα stimulation (for a review, see Il17a promoter upon T helper type 17 differentiation, thus Anest et al.41 and Yamamoto et al.42). Thus, IKKα-deficient cells promoting histone H3 phosphorylation and a transcriptional do not show any defect on IκBα phosphorylation or degradation, activation of the locus, in an NF-κB-independent manner. but they display a reduced capacity to activate NF-κB-dependent transcription (i.e. IκBα or IL-8 genes). Mechanistically, IKKα not only phosphorylates histone H3 but it also interacts with PHOSPHORYLATION OF NUCLEAR COREPRESSORS the coactivator CBP facilitating histone acetylation and Other substrates for IKKα kinase are the silencing mediator for chromatin relaxation. A similar function is exerted by IKKα in retinoic acid and thyroid hormone receptor (SMRT) and the lipopolysaccharide-treated macrophages. However, lipopolysac- nuclear corepressor (NCoR). Phosphorylation of SMRT by IKKα at charide not only affect IKKα localization but also that NIK shows serine 2410 induces its dissociation from the chromatin, and is a nuclear distribution in endotoxin-treated macrophages, associated prerequisite for the activation of NF-κB-dependent genes, such as with an increase in the levels of IKKα and histone H3 ciap-2 and IL-8, following laminin attachment in androgen- phosphorylation. Reduction of NIK levels by small interfering insensitive DU145 prostate cancer cells.48 In basal conditions, RNA reduced IKKα activity and histone H3 phosphorylation in SMRT and HDAC3 are bound to the chromatin together with p50 these cells.43 Patients with mutations in NEMO show reduced homodimers leading to basal gene repression. Upon stimulation, levels of phosphorylated (S10) histone H3 associated with IKKα-mediated phosphorylation of SMRT initiates transcriptional Figure 1. IKKα kinase activity is exerted on both cytoplasmic and nuclear substrates. The schematic diagram represents a summary of known non-NF-κB substrates that are phosphorylated by IKKα under physiologic conditions.