B Members: the Dark Side of NF-Κb

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.

Oncogene (2015) 2279 – 2287 © 2015 Macmillan Publishers Limited Non-conventional functions for NF-κB members L Espinosa et al 2281 derepression by releasing SMRT from the chromatin of specific IKKα ACTIVITY IN CANCER genes (being subsequently degraded by the proteasome), thus Association between NF-κB/IKK and cancer has been extensively allowing binding of transcriptionally active p65–p50 dimer and documented, primarily focused on their role in inflammation.57 gene transcription. Using a comparable mechanism, colorectal However, a more in-depth analysis of the results indicates that cancer (CRC) cells contain aberrantly activated IKKα that localize multiple NF-κB-independent functions that are exerted by nuclear and associated with the promoter of different Notch- elements of the pathways contribute to specific tumor-related dependent genes, including hes1 and herp2. Chromatin-bound capacities (Figure 2). Among others, Michael Karin’s group α IKK constitutively phosphorylates SMRT, leading to its chromatin elegantly demonstrated that elimination of the IKKα kinase release and Notch-dependent gene expression. Conversely, IKK activity (using the IKKαAA mouse model) is sufficient to reduce inhibition, either pharmacologically or by a dominant-negative prostate cancer growth and to prevent metastasis in the TRAMP form of the kinase, restored SMRT chromatin binding, inhibited (transgenic adenocarcinoma of the mouse prostate) mice model Notch-dependent gene expression and prevented tumor growth of prostate cancer.58 Inhibition of metastasis was due to increased 49 α in a xenograft model in nude mice. IKK also phosphorylates expression of the metastasis suppressor Maspin, whose repression NCoR, a nuclear corepressor homologous to SMRT, creating a is mediated by nuclear active IKKα. Accumulation of nuclear active functional 14-3-3-binding domain that imposes its nuclear export α fi 50 IKK in prostate cancer cells signi cantly correlates with meta- in CRC. static progression, reduced Maspin expression and with the presence of RANKL (receptor activator of nuclear factor kappa-B IKKα FUNCTIONS IN CELL CYCLE ligand)-expressing inflammatory cells, in both mouse and human prostate tumors. In this study, it was proposed IKKα might IKKα can also regulate the cell cycle in an NF-κB-independent α β facilitate the recruitment of DNA methyltransferase activity to the manner. For instance, IKK (but not IKK ) is required for estrogen- Maspin promoter, in an NF-κB-independent and cell autonomous induced transcription of E2F1, and the subsequent activation of the manner. A second study from the same group demonstrated that E2F1-responsive genes proliferating cell nuclear antigen, cyclin E androgen ablation (a therapy commonly used for treating prostate and cdc25A in breast cancer cells. Thus, in the absence of IKKα, cells cancer) induced the infiltration of regressing androgen-dependent cannot properly progress through the cell cycle, from the late G1 tumors with B lymphocytes. Then, these cells produce cytokines, into the S phase. Treatment with estrogen increased the association such as lymphotoxin α/β, that activate IKKα and STAT3 in the between IKKα and E2F1 at the gene promoters, and facilitates the prostate cancer cells, thus switching them from hormone acetylation of E2F1 by PCAF (P300/CBP-associated factor).51 IKKα dependent to hormone independent.59 also has a role during the M phase of the cell cycle by 52 In mammary tumorigenesis, IKKα activity is required to support phosphorylating and activating the mitotic kinase Aurora A. the expansion of tumor-initiating cells from premalignant ErbB2- Conversely, IKK activity is regulated during the cell cycle through its α direct association with AKT or Chk1, which in turn contributes to expressing mammary glands. Upon activation, IKK enters the the regulation of cell cycle regulators such as cyclin D1 and c-Myc.53 nucleus and phosphorylates p27/Kip1, inducing its cytoplasmic export.60 In T-cell acute lymphoblastic leukemia, IKKα directly interacts IKK FUNCTIONS IN CELLULAR STRESS with active Notch1 to induce RelB and NFkB2 transcription.61 Most important, IKK is constitutively active and essential for the Autophagy is a cellular process induced by different types of 62 stress, in which IKK activation is also required.54 IKK is important maintenance of Notch-driven leukemia. Nevertheless, the for the transduction of multiple signals that mediate starvation- mechanism supporting this functional requirement, and whether κ induced autophagy, including phosphorylation of AMP-activated it is NF- B and/or Notch dependent is not totally understood. In other cellular models, such as CaSki cervical cancer cells, normal protein kinase and c-Jun-N-terminal kinase 1. The mechanism is 63 64 independent of NF-κB, as inhibition of NF-κB nuclear translocation keratinocytes or MCF7 breast cancer cells, Notch1 modulates α or ablation of p65 failed to suppress IKK-induced autophagy. the activity of IKK both in the cytoplasm and the nucleus. In the α However, cells lacking either IKKα or IKKβ cannot induce latter, Notch1 and IKK accumulate at estrogen-dependent autophagy in response to cellular starvation, a well-known inducer promoters and are required for estrogen-independent transcrip- of this process,55 suggesting that the whole IKK complex is tional activation. In colorectal cancer, the presence of nuclear active IKK required, likely through the regulation of autophagy-related gene 49,65 expression (i.e. IKK is required for basal LC3 expression), significantly correlates with cancer progression. Nevertheless, independent of NF-κB. Nonetheless, starvation also induces the only active form that is found in the nuclear compartment of activation of NF-κB, which in turn upregulates the expression of these tumor cells does not correspond to the canonical 85–87 kDa antiapoptotic genes such as Birc3. Taken together, these results forms of IKKα or IKKβ, but it is a new IKKα isoform with a predicted indicate that IKK has a dual role in response to cellular stress: on molecular weight of 45 kDa (p45-IKKα) that lacks several one hand it controls the expression of autophagy-related genes in regulatory regions at the C-terminal end of the kinase. Active an NF-κB-independent manner; on the other hand, it regulates the p45-IKKα forms a complex with non-phosphorylated full-length NF-κB-dependent transcription of antiapoptotic genes. IKKα and NEMO that phosphorylates SMRT and histone H3. Processing of IKKα into p45-IKKα is required to prevent apoptosis of CRC cells in vitro, and to sustain tumor growth in vivo. Thus, it is IKKα ACTIVITY IN VIRAL INFECTION possible that specific functions previously attributed to nuclear Recently, it has been shown that IKKα is a crucial host factor for IKKα are, in fact, carried out by p45-IKKα, which should be hepatitis C virus (HCV). In brief, interaction of HCV with the DEAD investigated. It is also predictable that there are a variety of box polypeptide 3, X-linked (DDX3X) induces IKKα activation and substoichiometric IKK complexes with different functions, which is its nuclear translocation. Nuclear IKKα activates a CBP/p300- supported by the recent findings that individual elements mediated transcriptional program involving the sterol-regulatory contained in the IKK complex such as NEMO or Rap1 are sufficient element-binding proteins, which activates lipogenic genes, thus to dictate substrate recognition.66,67 It is also unknown whether enhancing core-associated lipid droplet formation to facilitate viral IKKβ can be processed similar to IKKα, giving rise to a functional assembly.56 This specific function is kinase dependent but protein. independent of NF-κB. Chemical inhibitors of the IKKα kinase Taken together, these findings identify nuclear IKKα (either full- activity suppressed HCV infection and IKKα-induced lipogenesis. length or p45-IKK) as a potential therapeutic target for cancer,

Figure 2. IKKα regulates speciﬁc substrates in cancer cells that affect gene transcription.

specifically relevant for patients with refractory CRC or metastatic In breast cancer cells, it was demonstrated that IKKβ prostate cancer. However, a better understanding of the mechan- phosphorylates FOXO3a, thus triggering its cytoplasmic export isms leading to nuclear translocation and/or IKKα processing is and proteasomal degradation, resulting in increased tumor cell required to translate these findings into the clinics. survival and poor patient prognosis. This mechanism was primarily found in tumors where AKT was not activated, as AKT is usually responsible for FOXO3a phosphorylation and degradation.71 ALTERNATIVE FUNCTIONS FOR IKKβ IKKβ is the main responsible kinase for phosphorylating IκBα in response to proinflammatory stimuli, leading to the activation of ALTERNATIVE FUNCTIONS FOR IκBα AND IκBβ canonical NF-κB pathway. However, IKKβ also has specific The primary function of IκB proteins is to sequester NF-κB proteins functions that are independent of its role in canonical NF-κB in the cytoplasm by masking the nuclear localization signal and to (Figure 3). prevent DNA binding. However, a few non-conventional functions Among the non-conventional functions for IKKβ, it was recently have been noticed for these IκB proteins. For example, found that nuclear IKKβ, in association with β-TrCP and the SUMOylated-IκBα has been found accumulated in the nucleus of heterogeneous ribonucleoprotein U, promotes degradation of fibroblast and keratinocytes where it associates with the nuclear IκBα upon ultraviolet (UV) irradiation. Casein kinase 2 and chromatin at specific gene promoters.72,73 This SUMO-modified p38 also bind to IKKβ and participate of the process by inducing IκBα directly binds to histones H2A and H4 and regulates IκBα phosphorylation at C-terminal sites, which are different from Polycomb recruitment both under basal conditions and in the canonical serines 32 and 36 involved in the activation of response to TNFα. Likely related with this function, mutations in canonical NF-κB. Importantly, this mechanism of IκBα degradation the Drosophila IκBα homolog Cactus enhanced the homeotic imposed by nuclear IKKβ result in the suppression of antiapoptotic phenotype of Polycomb mutants, which is not counteracted by genes such as Bcl-XL and x-IAP, thus leading to UV-induced cell mutations of the p65-NF-κB homolog, Dorsal.73 Importantly, death.68 In addition, it was recently demonstrated that DNA accumulation of IκBα in the cytoplasm of keratinocyes highly damage induced by alkylating agents promotes activation and correlated with tumorigenic processes in the skin when patients nuclear translocation of a fraction of IKKβ that directly phosphor- with different degrees of skin lesions were analyzed. ylates ATM.69 This novel mechanism might help to maintain the Not only IκBα but also IκBβ is capable of associating with the antiapoptotic response induced by NF-κB downstream of IKKβ chromatin at specific gene promoters following lipopolysacharide during the process of DNA damage repair mediated by ATM. On stimulation. In this case, it is the newly synthesized non- the other hand, IKKβ participate of the mast cell degranulation, phosphorylated IκBβ that is retained in the nucleus of macro- which is crucial for the proper regulation of immunoglobulin phages to sustain transcription mediated by the p50-p65 NF-κB E-mediated anaphylaxis. This process is achieved in two steps: in heterodimers. Binding of IκBβ to p50-p65 at specific gene the first one, IKKβ phosphorylates SNAP-23, independently of promoters prevents its chromatin release imposed by IκBα leading NF-κB, and is directly involved in mast cell degranulation; and in to a persistent transcription of proinflammatory genes such as the the second, IKKβ regulates the release of proinflammatory TNFα.74 Whether IκBβ associates with the chromatin of other cytokines in an NF-κB-dependent manner, thus having a central NF-κB-dependent or NF-κB-independent genes remains to be role in the allergic response.70 investigated.

Oncogene (2015) 2279 – 2287 © 2015 Macmillan Publishers Limited Non-conventional functions for NF-κB members L Espinosa et al 2283 In neurons, phosphorylation of IκBβ at Tyr-161 increases its SPECIFIC FUNCTIONS OF IKK IN THE SKIN binding to p65 producing an increase in NF-κB DNA-binding The first indication that IKKs can exert additional NF-κB- and activity. This specific modification is reduced during low kinase-independent functions came from the skin-specific pheno- potassium-induced neuronal apoptosis, which is counteracted by type observed in the IKKα mutant mice.38,76–78 Subsequently, overexpression of wild-type IκBβ.75 a complex functional network regulating skin homeostasis

Figure 3. IKKβ exert non-conventional functions that are both dependent and independent of the NF-κB pathway.

Figure 4. Summary of non-conventional IKKα and IκBα functions in normal and transformed keratinocytes.

© 2015 Macmillan Publishers Limited Oncogene (2015) 2279 – 2287 Non-conventional functions for NF-κB members L Espinosa et al 2284 has been demonstrated, with individual NF-kB elements having different Myc antagonists, such as Mad1, Mad2 and Ovol1, speciﬁc roles.79–81 through the interaction with the SMAD2/3 transcription factors, Among others (Figure 4), epidermal IKKα was found to interact and nuclear translocation of IKKα and its association with with SMAD2/3 after TGF-β stimulation to facilitate transcriptional SMAD2/3 is required for epidermal differentiation. Epidermal IKKα activation of cell cycle regulators promoting cell growth arrest and also regulates 14-3-3σ expression through direct binding to its keratinocyte differentiation.82 IKKα also controls the expression of promoter, thereby preventing recruitment of Suv39h1, an enzyme

Table 1. Table summarizing published data on non-conventional functions for NF-κB pathway elements

Protein Effect/substrate Function Cell type References

IKKα Phosphorylation of S10 histone H3 Activation of NF-κB-dependent MEFs Anest et al.,41 transcription Yamamoto et al.42 Activation of NF-κB-dependent Macrophages Park et al.43 transcription Activation of c-Fos Fibroblasts Anest et al.45 Transcription of hormone-responsive Breast cancer Park et al.46 genes Transcriptional activation of IL17a T cells Li et al.47 Phosphorylation of SMRT and NCoR Activation of NF-κB-dependent genes Prostate cancer Hoberg et al.48 Regulation of Notch-dependent gene CRC Fernández-Majada transcription et al.,49 Fernández- Majada et al.50 E2F1 transcription Activation of the E2F1-responsive Breast cancer Tu et al.51 genes Phosphorylation of Aurora A Cell cycle progression HeLa Prajapati et al.52 Regulation of cyclin D1 and c-Myc Cell cycle progression U2OS Barré and Perkins53 Phosphorylation of AMPK and JNK1 Starvation-induced autophagy Fibroblast Criollo et al.54 Regulation of autophagy-related gene Starvation-induced autophagy MEFs Comb et al.55 expression Activation of lipogenic genes in Facilitation of viral assembly Huh7.5.1 Li et al.56 hepatitis C infection Maspin gene repression Metastasis induction Prostate cancer Luo et al.58 Lymphotoxin α/β-mediated signaling Stimulation of metastasis Prostate cancer Ammirante et al.59 Phosphorylation of p27 Expansion of tumor-initiating cells Breast cancer Zhang et al.60 Interaction with Notch1 Induction of RelB and NF-κB2 T-ALL Vilimas et al.61 Induction of NF-κB Maintenance of Notch-driven T-ALL Espinosa et al.62 leukemia Accumulation with Notch1 at ER- Estrogen-independent transcriptional Breast cancer Hao et al.64 dependent promoters activation Interaction with the TGF-β transducers Facilitation of transcriptional Skin Descargues et al.82 SMAD2/3 activation of cell cycle regulators Regulation of 14-3-3σ expression Inhibition of proliferation Skin Zhu et al.78

p45-IKKα Phosphorylation of SMRT Apoptosis inhibition CRC Margalef et al.65 and histone H3

IKKβ Degradation of nuclear IκBα Suppression of antiapoptotic genes Fibroblasts Tsuchiya et al.68 upon UV irradiation Phosphorylation of ATM Maintenance of the antiapoptotic AGS and HeLa Sakamoto et al.69 response Phosphorylation of SNAP-23 Mast cell degranulation Mast cells Suzuki and Verma70 Phosphorylation of FOXO3a Increased cell survival Breast cancer Hu et al.71

IκBα PRC2 recruitment Regulation of Hox and Irx Fibroblasts and Aguilera et al.,72 Mulero transcriptional program skin et al.73

IκBβ Prevents IκBα binding and Persistent transcription of Macrophages Rao et al.74 dissociation of p65–p50 dimers from proinﬂammatory genes the chromatin Increase in NF-κB DNA-binding Apoptosis prevention Neurons Liu and D’Mello75 activity TAK1 Suppression of speciﬁc NEMO Suppression of the pro-oncogenic/ Hepatocytes Bettermann et al.93 function pronecrotic pathway

NEMO Binding to RIP1 Blocking of necrotic death pathway T cells O’Donnell et al.94 Binding to p45-IKKα Inhibition of apoptosis CRC Margalef et al.65 Abbreviations: AMPK, AMP-activated protein kinase; ATM, ataxia telangiectasia mutated; CRC, colorectal cancer; FOXO3, forkhead box O3; IKK, IκB kinase; IL, interleukin; JNK, c-Jun-N-terminal kinase; MEF, mouse embryo ﬁbroblast; NCoR, nuclear corepressor; NEMO, NF-κB essential modulator; NF-κB, nuclear facor-κB; PRC2, polycomb repressive complex 2; SMRT, silencing mediator for retinoic acid and thyroid hormone receptor; SNAP, synaptosomal-associated protein; T-ALL, T-cell acute lymphoblastic leukemia; TAK1, transforming growth factor-β activated kinase 1; UV, ultraviolet.

Oncogene (2015) 2279 – 2287 © 2015 Macmillan Publishers Limited Non-conventional functions for NF-κB members L Espinosa et al 2285 that is responsible for histone H3 lysine 9 trimethylation, leading plethora of studies, the interplay of NF-κB with other pathways, its to transcriptional activation. Because 14-3-3σ negatively regulates connection with cancer independently of inflammation and the activity of the cell cycle phosphatase CDC25, in the absence of NF-κB-independent functions for specific elements are still poorly functional IKKα, cells aberrantly proliferate, which results in the understood. Our interpretation is that non-conventional functions loss of skin homeostasis and increased cell transformation.78 for NF-κB signaling components are used for tissue homeostasis to Moreover, IKKα protects the 14-3-3σ locus from hypermethylation, accommodate changes imposed by fluctuations in the cellular which serves as a mechanism for maintaining genomic stability in environment, DNA damage or the signals related to inflammation keratinocytes. These two mechanisms that contribute to preserve or pathogen aggressions. the accurate balance between proliferation and differentiation We here have revised some of the recently identified non- in the skin38,77,78 are NF-κB and kinase independent.83 conventional functions for specific NF-κB family members (see However, mutations that impair chromatin binding or impose Table 1) with the aim of broadening the scope of future IKKα cytoplasmic localization promote epidermal squamous cell investigations in the field. This would permit having a wider carcinomas and skin papillomas.84,85 image of the downstream effects of NF-κB activation in particular Recently, Mulero et al.73 demonstrated that chromatin-bound systems and under specific circumstances such as embryonic IκBα regulate a whole transcriptional program in the skin involving development, tissue/cell differentiation or aging. These considera- several developmental and differentiation-related genes, such tions get special importance when studying the IKK kinase those belonging to the Hox and Irx families. Most importantly, complex, which exert both cytoplasmic and nuclear functions – both IκBα deletion86 88 and persistent localization of IκBα in the that are either kinase dependent or independent. A better nucleus of the keratinocytes89 result in altered proliferation and understanding of the particularities of NF-κB/IKK members will differentiation in the mouse skin. Oncogenic transformation of be crucial not only from an academic point of view but, most primary keratinocytes by H-Ras and ΔNp63 leads to the loss of importantly, for future clinical applications. nuclear IκBα, which accumulates in the cytoplasm leading to a massive activation of Hox and Irx genes, comparable to that observed in human epidermal squamous cell carcinoma CONFLICT OF INTEREST 73 samples. In the same direction, non-degradable IκBα super The authors declare no conflict of interest. repressor, which preferentially localizes in the cytoplasm of the cells, cooperates with Ras in keratinocyte transformation,90 whereas mice expressing this IκBα mutant in the skin develop ACKNOWLEDGEMENTS squamous cell carcinoma.91 Interestingly, most of the skin We thank the members of our laboratory for helpful discussions. This work has been phenotypes associated with alterations on IκBα function are supported by AGAUR (SGR23), ISCIII (PI13/00448) and RTICC (RD12/0036/0054). rescued after removal of TNFα signaling,92 indicating that epidermal IκBα acts as a sensor that integrates inflammatory REFERENCES signals with skin homeostasis. 1 Sen R, Baltimore D. Multiple nuclear factors interact with the immunoglobulin enhancer sequences. Cell 1986; 46:705–716. ALTERNATIVE FUNCTIONS FOR OTHER MEMBERS 2 Pahl HL. Activators and target genes of Rel/NF-kappaB transcription factors. 18 – κ fi Oncogene 1999; : 6853 6866. Finally, few other NF- B elements also display speci c non- 3 Hayden MS, Ghosh S. Signaling to NF-kappaB. Genes Dev 2004; 18: 2195–2224. conventional functions: TAK1, the kinase responsible for the 4 Kucharczak J, Simmons MJ, Fan Y, Gélinas C. To be, or not to be: NF-kappaB is the activation of the IKK complex and a critical element in the answer--role of Rel/NF-kappaB in the regulation of apoptosis. Oncogene 2003; 22: regulation of innate and adaptive immune responses, has an 8961–8982. important role in the regulation of liver homeostasis. Conditional 5 Gerondakis S, Grossmann M, Nakamura Y, Pohl T, Grumont R. Genetic approaches ablation of TAK1 in liver parenchymal cells induces hepatocyte in mice to understand Rel/NF-kappaB and IkappaB function: transgenics and dysplasia and early onset of hepatocarcinogenesis, concomitant knockouts. Oncogene 1999; 18: 6888–6895. with biliary ductopenia and cholestasis. Mechanistically, TAK1 6 Staudt LM, Singh H, Sen R, Wirth T, Sharp PA, Baltimore D. A lymphoid-specific mediates activation of NF-κB in response to TNF, thus precluding protein binding to the octamer motif of immunoglobulin genes. Nature 1986; 323:640–643. caspase-3-dependent hepatocyte apoptosis and liver cancer. On 7 Beg AA, Sha WC, Bronson RT, Ghosh S, Baltimore D. Embryonic lethality and liver the other hand, TAK1 suppresses a procarcinogenic/pronecrotic degeneration in mice lacking the RelA component of NF-kappa B. Nature 1995; pathway that is NEMO dependent but NF-κB independent, and 376:167–170. 93 likely mediated through c-Jun-N-terminal kinase and FOXO3a. 8 Brown K, Park S, Kanno T, Franzoso G, Siebenlist U. Mutual regulation of the NEMO-deficient T cells are hypersensitive to programmed transcriptional activator NF-kappa B and its inhibitor, I kappa B-alpha. Proc Natl necrosis when stimulated with TNF in the presence of caspase Acad Sci USA 1993; 90: 2532–2536. inhibitors.94 It was previously shown that some cell types can 9 Sun SC, Ganchi PA, Ballard DW, Greene WC. NF-kappa B controls expression of switch from apoptosis to necrosis when treated with these inhibitor I kappa B alpha: evidence for an inducible autoregulatory pathway. 259 – inhibitors.95,96 Interestingly, prosurvival activity of NEMO in T cells Science 1993; : 1912 1915. κ 10 Arenzana-Seisdedos F, Turpin P, Rodriguez M, Thomas D, Hay RTVirelizier JL et al. is not dependent on NF- B-mediated gene transcription, but Nuclear localization of I kappa B alpha promotes active transport of NF-kappa B depend on the ability of NEMO to bind ubiquitinated RIP1, which from the nucleus to the cytoplasm. J Cell Sci 1997; 110(Part 3): 369–378. restrains its ability to engage the necrotic death pathway. In the 11 DiDonato JA, Hayakawa M, Rothwarf DM, Zandi E, Karin M. A cytokine-responsive absence of NEMO, or in conditions where RIP1 ubiquitination is IkappaB kinase that activates the transcription factor NF-kappaB. Nature 1997; blocked, caspase inhibition induces the necrosis pathway, which is 388:548–554. dependent on the deubiquitinase CYLD. 12 Mercurio F, Zhu H, Murray BW, Shevchenko A, Bennett BL, Li J et al. IKK-1 and IKK-2: cytokine-activated IkappaB kinases essential for NF-kappaB activation. Science 1997; 278:860–866. CONCLUSIONS AND PERSPECTIVES 13 Zandi E, Rothwarf DM, Delhase M, Hayakawa M, Karin M. The IkappaB kinase κ complex (IKK) contains two kinase subunits, IKKalpha and IKKbeta, necessary for NF- B is a complex pathway that has evolved linked to the 91 – κ IkappaB phosphorylation and NF-kappaB activation. Cell 1997; :243 252. development of the immune system. Thus, NF- B has become 14 Rothwarf DM, Zandi E, Natoli G, Karin M. IKK-gamma is an essential regulatory essential for multicellular organisms to defend from external subunit of the IkappaB kinase complex. Nature 1998; 395:297–300. insults and survive in a variable environment. While immunity- 15 Chen ZJ, Parent L, Maniatis T. Site-specific phosphorylation of IkappaBalpha by a related NF-κB functions (conventional) have been the focus of a novel ubiquitination-dependent protein kinase activity. Cell 1996; 84:853–862.

© 2015 Macmillan Publishers Limited Oncogene (2015) 2279 – 2287 Non-conventional functions for NF-κB members L Espinosa et al 2286 16 Haas TL, Emmerich CH, Gerlach B, Schmukle AC, Cordier SM, Rieser E et al. 42 Yamamoto Y, Verma UN, Prajapati S, Kwak Y-T, Gaynor RB. Histone H3 phos- Recruitment of the linear ubiquitin chain assembly complex stabilizes the TNF-R1 phorylation by IKK-alpha is critical for cytokine-induced gene expression. Nature signaling complex and is required for TNF-mediated gene induction. Mol Cell 2003; 423:655–659. 2009; 36: 831–844. 43 Park GY, Wang X, Hu N, Pedchenko T V, Blackwell TS, Christman JW. NIK is 17 Tokunaga F, Sakata S, Saeki Y, Satomi Y, Kirisako T, Kamei K et al. Involvement of involved in nucleosomal regulation by enhancing histone H3 phosphorylation by linear polyubiquitylation of NEMO in NF-kappaB activation. Nat Cell Biol 2009; 11: IKKalpha. J Biol Chem 2006; 281: 18684–18690. 123–132. 44 Temmerman ST, Ma CA, Zhao Y, Keenan J, Aksentijevich I, Fessler M et al. 18 Gerlach B, Cordier SM, Schmukle AC, Emmerich CH, Rieser E, Haas TL et al. Linear Defective nuclear IKKα function in patients with ectodermal dysplasia with ubiquitination prevents inflammation and regulates immune signalling. Nature immune deficiency. J Clin Invest 2012; 122: 315–326. 2011; 471: 591–596. 45 Anest V, Cogswell PC, Baldwin AS. IkappaB kinase alpha and p65/RelA contribute 19 Ikeda F, Deribe YL, Skånland SS, Stieglitz B, Grabbe C, Franz-Wachtel M et al. to optimal epidermal growth factor-induced c-fos gene expression independent SHARPIN forms a linear ubiquitin ligase complex regulating NF-κB activity and of IkappaBalpha degradation. J Biol Chem 2004; 279: 31183–31189. apoptosis. Nature 2011; 471:637–641. 46 Park K-J, Krishnan V, O’Malley BW, Yamamoto Y, Gaynor RB. Formation of an 20 Tokunaga F, Nakagawa T, Nakahara M, Saeki Y, Taniguchi M, Sakata S et al. IKKalpha-dependent transcription complex is required for estrogen receptor- SHARPIN is a component of the NF-κB-activating linear ubiquitin chain assembly mediated gene activation. Mol Cell 2005; 18:71–82. complex. Nature 2011; 471:633–636. 47 Li L, Ruan Q, Hilliard B, Devirgiliis J, Karin M, Chen YH. Transcriptional regulation of 21 Kovalenko A, Chable-Bessia C, Cantarella G, Israël A, Wallach D, Courtois G. The the Th17 immune response by IKK(alpha). J Exp Med 2011; 208:787–796. tumour suppressor CYLD negatively regulates NF-kappaB signalling by deubi- 48 Hoberg JE, Yeung F, Mayo MW. SMRT derepression by the IkappaB kinase alpha: a quitination. Nature 2003; 424: 801–805. prerequisite to NF-kappaB transcription and survival. Mol Cell 2004; 16: 245–255. 22 Trompouki E, Hatzivassiliou E, Tsichritzis T, Farmer H, Ashworth A, Mosialos G. 49 Fernández-Majada V, Aguilera C, Villanueva A, Vilardell F, Robert-Moreno A, CYLD is a deubiquitinating enzyme that negatively regulates NF-kappaB Aytés A et al. Nuclear IKK activity leads to dysregulated notch-dependent gene activation by TNFR family members. Nature 2003; 424:793–796. expression in colorectal cancer. Proc Natl Acad Sci USA 2007; 104:276–281. 23 Brummelkamp TR, Nijman SMB, Dirac AMG, Bernards R. Loss of the cylin- 50 Fernández-Majada V, Pujadas J, Vilardell F, Capella G, Mayo MW, Bigas A et al. dromatosis tumour suppressor inhibits apoptosis by activating NF-kappaB. Nature Aberrant cytoplasmic localization of N-CoR in colorectal tumors. Cell Cycle 2007; 6: 2003; 424: 797–801. 1748–1752. 24 Wertz IE, O’Rourke KM, Zhou H, Eby M, Aravind L, Seshagiri S et al. 51 Tu Z, Prajapati S, Park K-J, Kelly NJ, Yamamoto Y, Gaynor RB. IKK alpha regulates De-ubiquitination and ubiquitin ligase domains of A20 downregulate NF-kappaB estrogen-induced cell cycle progression by modulating E2F1 expression. J Biol signalling. Nature 2004; 430: 694–699. Chem 2006; 281: 6699–6706. 25 Boone DL, Turer EE, Lee EG, Ahmad R-C, Wheeler MT, Tsui C et al. The ubiquitin- 52 Prajapati S, Tu Z, Yamamoto Y, Gaynor RB. IKKalpha regulates the mitotic phase of modifying enzyme A20 is required for termination of Toll-like receptor responses. the cell cycle by modulating Aurora A phosphorylation. Cell Cycle 2006; 5: Nat Immunol 2004; 5: 1052–1060. 2371–2380. 26 Wang L, Du F, Wang X. TNF-alpha induces two distinct caspase-8 activation 53 Barré B, Perkins ND. A cell cycle regulatory network controlling NF-kappaB sub- pathways. Cell 2008; 133:693–703. unit activity and function. EMBO J 2007; 26: 4841–4855. 27 Dejardin E, Droin NM, Delhase M, Haas E, Cao Y, Makris C et al. The lymphotoxin- 54 Criollo A, Senovilla L, Authier H, Maiuri MC, Morselli E, Vitale I et al. The IKK beta receptor induces different patterns of gene expression via two NF-kappaB complex contributes to the induction of autophagy. EMBO J 2010; 29: 619–631. pathways. Immunity 2002; 17: 525–535. 55 Comb WC, Cogswell P, Sitcheran R, Baldwin AS. IKK-dependent, NF-κB-indepen- 28 Kayagaki N, Yan M, Seshasayee D, Wang H, Lee W, French DM et al. BAFF/BLyS dent control of autophagic gene expression. Oncogene 2011; 30: 1727–1732. receptor 3 binds the B cell survival factor BAFF ligand through a discrete surface 56 Li Q, Pène V, Krishnamurthy S, Cha H, Liang TJ. Hepatitis C virus infection activates loop and promotes processing of NF-kappaB2. Immunity 2002; 17:515–524. an innate pathway involving IKK-α in lipogenesis and viral assembly. Nat Med 29 Claudio E, Brown K, Park S, Wang H, Siebenlist U. BAFF-induced NEMO-inde- 2013; 19: 722–729. pendent processing of NF-kappa B2 in maturing B cells. Nat Immunol 2002; 3: 57 Grivennikov SI, Greten FR, Karin M. Immunity, inflammation, and cancer. Cell 2010; 958–965. 140:883–899. 30 Coope HJ, Atkinson PGP, Huhse B, Belich M, Janzen J, Holman MJ et al. CD40 58 Luo J-L, Tan W, Ricono JM, Korchynskyi O, Zhang M, Gonias SL et al. Nuclear regulates the processing of NF-kappaB2 p100 to p52. EMBO J 2002; 21: cytokine-activated IKKalpha controls prostate cancer metastasis by 5375–5385. repressing Maspin. Nature 2007; 446: 690–694. 31 Novack DV, Yin L, Hagen-Stapleton A, Schreiber RD, Goeddel D V, Ross FP et al. 59 Ammirante M, Luo J-L, Grivennikov S, Nedospasov S, Karin M. B-cell-derived The IkappaB function of NF-kappaB2 p100 controls stimulated osteoclastogenesis. lymphotoxin promotes castration-resistant prostate cancer. Nature 2010; 464: J Exp Med 2003; 198: 771–781. 302–305. 32 Vallabhapurapu S, Matsuzawa A, Zhang W, Tseng P-H, Keats JJ, Wang H et al. 60 Zhang W, Tan W, Wu X, Poustovoitov M, Strasner A, Li W et al. A NIK-IKKα module Nonredundant and complementary functions of TRAF2 and TRAF3 in a ubiquiti- expands ErbB2-induced tumor-initiating cells by stimulating nuclear export of nation cascade that activates NIK-dependent alternative NF-kappaB signaling. Nat p27/Kip1. Cancer Cell 2013; 23:647–659. Immunol 2008; 9: 1364–1370. 61 Vilimas T, Mascarenhas J, Palomero T, Mandal M, Buonamici S, Meng F et al. 33 Senftleben U, Cao Y, Xiao G, Greten FR, Krähn G, Bonizzi G et al. Activation by Targeting the NF-kappaB signaling pathway in Notch1-induced T-cell leukemia. IKKalpha of a second, evolutionary conserved, NF-kappa B signaling pathway. Nat Med 2007; 13:70–77. Science 2001; 293: 1495–1499. 62 Espinosa L, Cathelin S, D’Altri T, Trimarchi T, Statnikov A, Guiu J et al. The Notch/ 34 Xiao G, Harhaj EW, Sun SC. NF-kappaB-inducing kinase regulates the processing of Hes1 pathway sustains NF-κB activation through CYLD repression in T cell leu- NF-kappaB2 p100. Mol Cell 2001; 7:401–409. kemia. Cancer Cell 2010; 18:268–281. 35 Amir RE, Haecker H, Karin M, Ciechanover A. Mechanism of processing of the 63 Song LL, Peng Y, Yun J, Rizzo P, Chaturvedi V, Weijzen S et al. Notch-1 associates NF-kappa B2 p100 precursor: identification of the specific polyubiquitin chain- with IKKalpha and regulates IKK activity in cervical cancer cells. Oncogene 2008; anchoring lysine residue and analysis of the role of NEDD8-modification on the 27: 5833–5844. SCF(beta-TrCP) ubiquitin ligase. Oncogene 2004; 23: 2540–2547. 64 Hao L, Rizzo P, Osipo C, Pannuti A, Wyatt D, Cheung LW-K et al. Notch-1 activates 36 Betts JC, Nabel GJ. Differential regulation of NF-kappaB2(p100) processing and estrogen receptor-alpha-dependent transcription via IKKalpha in breast control by amino-terminal sequences. Mol Cell Biol 1996; 16: 6363–6371. cancer cells. Oncogene 2010; 29:201–213. 37 Solan NJ, Miyoshi H, Carmona EM, Bren GD, Paya CV. RelB cellular regulation and 65 Margalef P, Fernández-Majada V, Villanueva A, Garcia-Carbonell R, Iglesias M, transcriptional activity are regulated by p100. J Biol Chem 2002; 277: 1405–1418. López L et al. A truncated form of IKKα is responsible for specific nuclear IKK 38 Hu Y, Baud V, Delhase M, Zhang P, Deerinck T, Ellisman M et al. Abnormal mor- activity in colorectal cancer. Cell Rep 2012; 2:840–854. phogenesis but intact IKK activation in mice lacking the IKKalpha subunit of 66 Schröfelbauer B, Polley S, Behar M, Ghosh G, Hoffmann A. NEMO ensures sig- IkappaB kinase. Science 1999; 284:316–320. naling specificity of the pleiotropic IKKβ by directing its kinase activity toward 39 Takeda K, Takeuchi O, Tsujimura T, Itami S, Adachi O, Kawai T et al. Limb and skin IκBα. Mol Cell 2012; 47: 111–121. abnormalities in mice lacking IKKalpha. Science 1999; 284: 313–316. 67 Teo H, Ghosh S, Luesch H, Ghosh A, Wong ET, Malik N et al. Telomere- 40 Bonizzi G, Karin M. The two NF-kappaB activation pathways and their role in independent Rap1 is an IKK adaptor and regulates NF-kappaB-dependent gene innate and adaptive immunity. Trends Immunol 2004; 25:280–288. expression. Nat Cell Biol 2010; 12: 758–767. 41 Anest V, Hanson JL, Cogswell PC, Steinbrecher KA, Strahl BD, Baldwin AS. 68 Tsuchiya Y, Asano T, Nakayama K, Kato T, Karin M, Kamata H. Nuclear IKKbeta is an A nucleosomal function for IkappaB kinase-alpha in NF-kappaB-dependent gene adaptor protein for IkappaBalpha ubiquitination and degradation in UV-induced expression. Nature 2003; 423:659–663. NF-kappaB activation. Mol Cell 2010; 39:570–582.

Oncogene (2015) 2279 – 2287 © 2015 Macmillan Publishers Limited Non-conventional functions for NF-κB members L Espinosa et al 2287 69 Sakamoto K, Hikiba Y, Nakagawa H, Hirata Y, Hayakawa Y, Kinoshita H et al. 84 Liu B, Park E, Zhu F, Bustos T, Liu J, Shen J et al. A critical role for I kappaB kinase Promotion of DNA repair by nuclear IKKβ phosphorylation of ATM in response to alpha in the development of human and mouse squamous cell carcinomas. Proc genotoxic stimuli. Oncogene 2013; 32: 1854–1862. Natl Acad Sci USA 2006; 103: 17202–17207. 70 Suzuki K, Verma IM. Phosphorylation of SNAP-23 by IkappaB kinase 2 regulates 85 Park E, Zhu F, Liu B, Xia X, Shen J, Bustos T et al. Reduction in IkappaB kinase alpha mast cell degranulation. Cell 2008; 134:485–495. expression promotes the development of skin papillomas and carcinomas. Cancer 71 Hu MC-T, Lee D-F, Xia W, Golfman LS, Ou-Yang F, Yang J-Y et al. IkappaB kinase Res 2007; 67: 9158–9168. promotes tumorigenesis through inhibition of forkhead FOXO3a. Cell 2004; 117: 86 Beg AA, Sha WC, Bronson RT, Baltimore D. Constitutive NF-kappa B activation, 225–237. enhanced granulopoiesis, and neonatal lethality in I kappa B alpha-deficient mice. 72 Aguilera C, Hoya-Arias R, Haegeman G, Espinosa L, Bigas A. Recruitment of Genes Dev 1995; 9: 2736–2746. IkappaBalpha to the hes1 promoter is associated with transcriptional repression. 87 Klement JF, Rice NR, Car BD, Abbondanzo SJ, Powers GD, Bhatt PH et al. Ikap- Proc Natl Acad Sci USA 2004; 101: 16537–16542. paBalpha deficiency results in a sustained NF-kappaB response and severe 73 Mulero MC, Ferres-Marco D, Islam A, Margalef P, Pecoraro M, Toll A et al. widespread dermatitis in mice. Mol Cell Biol 1996; 16: 2341–2349. Chromatin-bound IκBα regulates a subset of polycomb target genes in differ- 88 Rebholz B, Haase I, Eckelt B, Paxian S, Flaig MJ, Ghoreschi K et al. Crosstalk entiation and cancer. Cancer Cell 2013; 24:1–16. between keratinocytes and adaptive immune cells in an IkappaBalpha protein- 74 Rao P, Hayden MS, Long M, Scott ML, West AP, Zhang D et al. IkappaBbeta acts to mediated inflammatory disease of the skin. Immunity 2007; 27: 296–307. inhibit and activate gene expression during the inflammatory response. Nature 89 Wuerzberger-Davis SM, Chen Y, Yang DT, Kearns JD, Bates PW, Lynch C et al. 2010; 466: 1115–1119. Nuclear export of the NF-κB inhibitor IκBα is required for proper B cell and 75 Liu L, D’Mello SR. Phosphorylation of IkappaB-beta is necessary for neuronal secondary lymphoid tissue formation. Immunity 2011; 34: 188–200. survival. J Biol Chem 2006; 281: 1506–1515. 90 Dajee M, Lazarov M, Zhang JY, Cai T, Green CL, Russell AJ et al. NF-kappaB 76 Hu Y, Baud V, Oga T, Kim KI, Yoshida K, Karin M. IKKalpha controls formation of the blockade and oncogenic Ras trigger invasive human epidermal neoplasia. Nature epidermis independently of NF-kappaB. Nature 2001; 410:710–714. 2003; 421: 639–643. 77 Marinari B, Moretti F, Botti E, Giustizieri ML, Descargues P, Giunta A et al. The tumor 91 Van Hogerlinden M, Rozell BL, Ahrlund-Richter L, Toftgård R. Squamous cell car- suppressor activity of IKKalpha in stratified epithelia is exerted in part via the cinomas and increased apoptosis in skin with inhibited Rel/nuclear factor-kappaB TGF-beta antiproliferative pathway. Proc Natl Acad Sci USA 2008; 105: 17091–17096. signaling. Cancer Res 1999; 59: 3299–3303. 78 Zhu F, Xia X, Liu B, Shen J, Hu Y, Person M et al. IKKalpha shields 14-3-3sigma, a G 92 Shih VF-S, Kearns JD, Basak S, Savinova O V, Ghosh G, Hoffmann A. Kinetic control (2)/M cell cycle checkpoint gene, from hypermethylation, preventing its silencing. of negative feedback regulators of NF-kappaB/RelA determines their pathogen- Mol Cell 2007; 27: 214–227. and cytokine-receptor signaling specificity. Proc Natl Acad Sci USA 2009; 106: 79 Seitz CS, Freiberg RA, Hinata K, Khavari PA. NF-kappaB determines localization 9619–9624. and features of cell death in epidermis. J Clin Invest 2000; 105:253–260. 93 Bettermann K, Vucur M, Haybaeck J, Koppe C, Janssen J, Heymann F et al. TAK1 80 Seitz CS, Lin Q, Deng H, Khavari PA. Alterations in NF-kappaB function in trans- suppresses a NEMO-dependent but NF-kappaB-independent pathway to genic epithelial tissue demonstrate a growth inhibitory role for NF-kappaB. Proc liver cancer. Cancer Cell 2010; 17: 481–496. Natl Acad Sci USA 1998; 95: 2307–2312. 94 O’Donnell MA, Hase H, Legarda D, Ting AT. NEMO inhibits programmed 81 Seitz CS, Deng H, Hinata K, Lin Q, Khavari PA. Nuclear factor kappaB subunits necrosis in an NFκB-independent manner by restraining RIP1. PLoS ONE 2012; 7: induce epithelial cell growth arrest. Cancer Res 2000; 60: 4085–4092. e41238. 82 Descargues P, Sil AK, Sano Y, Korchynskyi O, Han G, Owens P et al. IKKalpha is a critical 95 Li M, Beg AA. Induction of necrotic-like cell death by tumor necrosis factor alpha coregulator of a Smad4-independent TGFbeta-Smad2/3 signaling pathway that and caspase inhibitors: novel mechanism for killing virus-infected cells. J Virol controls keratinocyte differentiation. Proc Natl Acad Sci USA 2008; 105: 2487–2492. 2000; 74:7470–7477. 83 Sil AK, Maeda S, Sano Y, Roop DR, Karin M. IkappaB kinase-alpha acts in the 96 Vercammen D, Beyaert R, Denecker G, Goossens V, Van Loo G, Declercq W et al. epidermis to control skeletal and craniofacial morphogenesis. Nature 2004; 428: Inhibition of caspases increases the sensitivity of L929 cells to necrosis mediated 660–664. by tumor necrosis factor. J Exp Med 1998; 187: 1477–1485.