Nuclear factor of κB (NF-κB) is not a single protein,but a small menagerie of closely related protein dimersthat bind a common sequence motif known as the κBsite1 (BOX 1). The molecular identification of its p50subunit as a member of the reticuloendotheliosis(REL) family provided the first evidence that linkedNF-κB to cancer, as v-REL is an oncoprotein of theREL retrovirus (REV-T)2.

The REL proteins belong to two classes, which aredistinguishable by their mode of synthesis and trans-activation properties (BOX 1). One class consists ofRELA (also known as p65), RELB and c-REL — pro-teins that are synthesized in their mature forms.These proteins contain an amino-terminal RELhomology domain (RHD) that is required for dimer-ization and DNA binding, and transcription-modu-lating domains at their carboxy terminus. The secondclass consists of NF-κB1 (also known as p105) andNF-κB2 (also known as p100), which are synthesizedas large precursors (p105 and p100) with an N-terminal RHD and a C-terminal series of ANKYRIN

REPEATS. Ubiquitin-dependent proteolytic processingremoves this C-terminal domain, resulting in produc-tion of the mature DNA-binding proteins (p50 andp52). The final products contain the RHD, but lack transcription-modulating domains1.

These proteins form various NF-κB homo- and het-erodimers, the activity of which is regulated by twomain pathways (FIG. 1). The first regulatory pathway —the canonical NF-κB activation pathway — applies todimers that are composed of RELA, c-REL and p50,which are held captive in the cytoplasm by specific

inhibitors that are known as the inhibitor of κB (IκB)proteins. IκB proteins consist of an N-terminal regula-tory domain followed by a series of ankyrin repeats,similar to those present within the C-terminal portionsof p100 and p105 (BOX 1). The canonical pathway is nor-mally triggered in response to microbial and viral infec-tions and exposure to proinflammatory cytokines, all ofwhich activate the IκB kinase (IKK) complex (BOX 2).IKK phosphorylates NF-κB-bound IκBs at two con-served serines within the IκB N-terminal regulatorydomain. This targets IκB for ubiquitin-dependentdegradation and allows the liberated NF-κB dimers totranslocate to the nucleus3. IκB phosphorylationdepends mainly on the IKKβ catalytic subunit of theIKK complex4.

The second pathway affects NF-κB2, which prefer-entially dimerizes with RELB5. This processing-depen-dent pathway is triggered by certain members of thetumour-necrosis factor (TNF) cytokine family thatselectively activate the catalytic subunit IKKα, alongwith another protein kinase called NIK. Together,IKKα and NIK induce the phosphorylation-depen-dent proteolytic removal of the IκB-like C-terminaldomain of NF-κB2. This allows RELB–p52 dimers totranslocate to the nucleus6 (FIG. 1).

Once in the nucleus, the transcriptional functionsof NF-κB are further modulated by phosphorylation.Although each NF-κB dimer is likely to have distinctregulatory functions, many of the target genes arecommon to several, if not all, NF-κB proteins. Thesegenes fall into four broad functional categories:immunoregulatory and inflammatory genes;

NF-κB IN CANCER: FROM INNOCENTBYSTANDER TO MAJOR CULPRITMichael Karin, Yixue Cao, Florian R. Greten and Zhi-Wei Li

Nuclear factor of κB (NF-κB) is a sequence-specific transcription factor that is known to beinvolved in the inflammatory and innate immune responses. Although the importance of NF-κB inimmunity is undisputed, recent evidence indicates that NF-κB and the signalling pathways thatare involved in its activation are also important for tumour development. NF-κB should thereforereceive as much attention from cancer researchers as it has already from immunologists.

ANKYRIN REPEAT

A repeating sequence of 30–33amino acids that is found in theankyrin protein. The ankyrinrepeat of IκB proteins isrequired for association with thenuclear localization signal ofNF-κB proteins.

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Laboratory of GeneRegulation and SignalTransduction, Departmentof Pharmacology,School of Medicine,University of CaliforniaSan Diego, 9500 GilmanDrive, La Jolla, California92093, USA.Correspondence to M.K.e-mail:karinoffice@ucsd.edu DOI: 10.1038/nrc780

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Stimulating cell proliferation. NF-κB controls cell prolif-eration by activating target genes such as interleukin(IL)-2, granulocyte–macrophage colony-stimulating fac-tor (GM-CSF) and CD40 ligand (CD40L), which encodegrowth factors that stimulate the proliferation of lym-phoid and myeloid cells. Constitutive production of suchcytokines can chronically stimulate cell proliferation inan autocrine or paracrine fashion. In addition to thisindirect mode of action, NF-κB has also been shown todirectly stimulate the transcription of genes that encodeG1 cyclins. A κB site is present within the cyclin D1 pro-moter10,11, and there is strong evidence that NF-κB-dependent cyclin D1 induction drives the proliferation ofmammary epithelial cells during pregnancy12.

Inhibition of apoptosis. NF-κB is also an inhibitor of pro-grammed cell death13–16. This factor activates the tran-scription of several target genes that are known to blockthe induction of apoptosis by TNF-α and other pro-apoptotic members of this family17. The anti-apoptoticfactors that are induced by NF-κB include cellularinhibitors of apoptosis (cIAPs), caspase-8/FADD (FAS-associated death domain)-like IL-1β-converting enzyme(FLICE) inhibitory protein (c-FLIP) and members of theBCL2 family (such as A1/BFL1 and BCL-X

L)17. NF-κB

can also attenuate the apoptotic response to genotoxicanticancer drugs and ionizing radiation16,18. Tumour cellsin which NF-κB is constitutively active are highly resis-tant to anticancer drugs or ionizing radiation, and inhi-bition of NF-κB activity in these cells greatly increasestheir sensitivity to such treatments18. In addition to con-ferring resistance to cancer therapies, the anti-apoptoticactivity of NF-κB can also have an important role in theemergence of neoplasms, by preventing the death of cellsthat have undergone chromosomal rearrangements orother types of DNA damage. Such cells are normallyeliminated by means of checkpoint controls, such as thep53 pathway19. In fact, there is evidence for TRANSCRIP-

TIONAL ANTAGONISM between NF-κB and p53 (REF. 20).Regardless of mechanism, prevention of apoptosisincreases the pool of genetically altered cells, which willeventually give rise to transformed progeny.

anti-apoptotic genes; genes that positively regulatecell proliferation; and genes that encode negative reg-ulators of NF-κB. Genes of all four categories cancontribute to tumorigenesis.

A role for NF-κB in tumorigenesisAccording to Hanahan and Weinberg, tumorigenesisrequires six essential alterations to normal cell physiology: self-sufficiency in growth signals; insensi-tivity to growth inhibition; evasion of apoptosis;immortalization; sustained angiogenesis; and tissueinvasion and metastasis7. NF-κB is able to induce sev-eral of these cellular alterations (FIG. 2), and has beenshown to be constitutively activated in some types ofcancer cell. There are several mechanisms by whichNF-κB transcription factors are uncoupled from theirnormal modes of regulation, and these have beenassociated with cancer. For example, the avian REV-Toncovirus produces the constitutively active v-RELoncoprotein, which causes rapidly progressing lym-phomas and leukaemias2. The TAX oncoprotein ofhuman T-cell leukaemia virus (HTLV)-1 has beenshown to directly interact with and constitutively acti-vate the IKK complex, which results in the activationof both NF-κB signalling pathways8. Other viral onco-proteins have also been shown to activate NF-κB bymeans of different mechanisms9.

Cancer-associated chromosomal translocations,deletions and mutations might also disrupt genes that encode NF-κB and IκB proteins, uncoupling NF-κB factors from their regulators and causing constitutive NF-κB activation. Finally, AUTOCRINE

and PARACRINE production of proinflammatorycytokines, oncogenic activation of upstream signallingmolecules and chronic infections have been shown to persistently stimulate IKK activity, which leads to constitutive NF-κB activation. Constitutively activatedNF-κB transcription factors have been associated with several aspects of tumorigenesis, including pro-moting cancer-cell proliferation, preventing apopto-sis, and increasing a tumour’s angiogenic and metastatic potential.

AUTOCRINE/PARACRINE

The effect of hormones orgrowth factors that act in thesecretory cell itself is calledautocrine, whereas the effect ofthose that act on adjacent cells iscalled paracrine.

TRANSCRIPTIONAL

ANTAGONISM

The conflicting actions ofmultiple proteins that regulatethe expression of certain genes.

Summary

• Nuclear factor of κB (NF-κB) is a transcriptional regulator that is made up of different protein dimers that bind acommon sequence motif known as the κB site.

• Although NF-κB target genes have been most intensely studied for their involvement in immunity and inflammation,this transcription factor also regulates cell proliferation, apoptosis and cell migration. Therefore, it is not surprisingthat NF-κB has been shown to be constitutively activated in several types of cancer cell.

• NF-κB activity is tightly controlled by several regulatory proteins, and disruption of this process has been associatedwith various haematological malignancies, as well as epithelial tumours such as breast cancer.

• A causal connection between inflammation and cancer has been suspected for many years. Because NF-κB becomesactivated in response to inflammatory stimuli and its constitutive activation has been associated with cancer, NF-κBmight also serve as the missing link between these two processes. Numerous inhibitors of NF-κB are therefore underdevelopment or have been developed.

• Because of the widespread importance of this factor, it has been difficult to develop NF-κB inhibitors that actspecifically in cancer cells. Learning more about the complicated process of NF-κB regulation should lead to bettertherapeutic approaches to target the factor in specific cell types.

has been shown to promote angiogenesis21. In addition,κB sites were identified in the promoters of genes thatencode several matrix metalloproteinases (MMPs) —proteolytic enzymes that promote tumour invasion ofsurrounding tissue. NF-κB activation has been reportedto contribute to extracellular matrix destruction by can-cer cells22–24. Recently, NF-κB activation was found tostimulate angiogenesis, possibly by inducing expressionof IL-8 and vascular endothelial growth factor (VEGF)25.

NF-κB and lymphoid malignanciesLeukaemia and lymphoma — cancers of the bonemarrow and lymph nodes, respectively — are causedby uncontrolled proliferation of blood cells. Given itsimportance in immune-cell function, it is not all thatsurprising that NF-κB is involved in the developmentof such cancers. Initial evidence that linked NF-κB tohaematopoietic malignancies came from the v-Reloncogene, which was shown to cause aggressive lym-phomas and leukaemias in chickens2. The transform-ing activity of v-Rel is much higher than that of itscellular homologue c-Rel, due to accumulation ofmutations that reduce the susceptibility of v-Rel toIκB inhibition, increase v-Rel stability and alter itsDNA-binding properties2.

Genetic alterations that affect the activity and expres-sion of cellular NF-κB/REL proteins have also beenlinked to leukaemia and lymphomas. The human c-RELlocus maps to 2p14–15 — a chromosomal region that isamplified in 23% of extranodal diffuse large B-cell lym-phomas (DBCLs), as well as in other non-Hodgkin’s B-cell lymphomas26–28. This results in a 4–35-foldincrease in c-REL expression. Insertion of a promoterelement into the c-REL locus, which increases its expres-sion, has been detected in a lymphoid tumour cell line29.However, no direct correlation between c-REL expres-sion levels and the progression of lymphoid tumours, ortheir ability to express NF-κB target genes and resistapoptosis, has been made. The only direct evidence thatelevated c-REL expression can result in leukaemogenesisis derived from its ability to transform primary chickenlymphoid cells in culture30. In comparison to c-REL,amplifications or chromosomal rearrangements thataffect the RELA locus, which encodes the p65 subunit ofNF-κB, are rare26,31. No genetic alterations of the RELBlocus have been reported.

Conversely, chromosomal rearrangements thataffect the NFKB2 locus at chromosomal region 10q24have been associated with a variety of B- and T-celllymphomas, including chronic lymphocytic leukaemia(CLL), multiple myeloma, T-cell lymphoma and cuta-neous B- and T-cell lymphomas32–34. Although they dif-fer molecularly, all of these rearrangements or deletionsresult in removal of the C-terminal IκB-like sequencesof p100 and constitutive production of p52.Interestingly, a targeted 3′ deletion within the mouseNfkb2 locus, which also causes constitutive p52 pro-duction, results in lymphoid hyperplasia, but not can-cer35. So far, none of the lymphoma-derived NFKB2-encoded polypeptides have been shown to haveoncogenic activity in vivo.

Increased metastasis and angiogenesis. Another impor-tant component of tumour growth is angiogenesis — aprocess that requires both migratory and invasive capa-bilities of vascular epithelial cells. Chemokines —chemotactic factors that induce cell migration — aretherefore an important class of NF-κB target-gene prod-ucts. Cells with elevated NF-κB activity deregulate pro-duction of chemokines, which increases migratoryactivity. At least one NF-κB-regulated chemokine, IL-8,

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Box 1 | NF-κB and IκB proteins

There are five mammalian reticuloendotheliosis family (REL)/nuclear factor of κB (NF-κB) proteins that belong to two groups: those that do not do require proteolyticprocessing and those that do require proteolytic processing. The first group consists ofRELA (also known as p65), c-REL and RELB. The second group includes NF-κB1 (alsoknown as p105) and NF-κB2 (also known as p100), which are processed to produce themature p50 and p52 proteins, respectively. These two groups dimerize — the mostcommonly detected NF-κB dimer is p50–RELA. Due to the presence of a strongtranscriptional activation domain, RELA is responsible for most of NF-κBtranscriptional activity. p50–c-REL dimers are less abundant and seem to be activatedwith slower kinetics. Both p50–RELA and p50–c-REL dimers are regulated byinteractions with inhibitor of κB (IκB) proteins, which cause their cytoplasmicretention. RELB, by contrast, mostly associates with p100. The p100–RELB dimers areexclusively cytoplasmic. Proteolytic processing of p100 results in the release ofp52–RELB dimers, which translocate to the nucleus. RELB, unlike RELA and c-REL, canhave both activating and repressing functions.

All NF-κB proteins contain a REL homology domain (RHD) which mediates theirdimerization and binding to DNA. The RHD also contains, at its carboxyl terminus,a nuclear localization signal (NLS) and is recognized by the IκB proteins, thebinding of which to the RHD interferes with the function of the NLS.

The IκB proteins include IκBα , IκBβ and IκBε, which trap NF-κB dimers in thecytoplasm, and BCL3, which acts as a transcriptional co-activator for p50 and p52homodimers. All IκBs contain 6–7 ankyrin repeats, which mediate their binding toRHDs. IκBα, IκBβ and IκBε contain an amino-terminal regulatory domain, withinwhich there are two conserved serines (SS). Phosphorylation at this site targets the IκBsto ubiquitin-dependent degradation. Lysine residues, which are targets forpolyubiquitylation, are also present within the N-terminal regulatory domain. The C-terminal halves of p105 and p100 are similar in sequence, structure and function tothe IκBs. Like the IκBs, the C-terminal portions of p105 and p100 prevent nuclear entry,and are removed by ubiquitin-dependent degradation. Whereas the processing of p105is constitutive, the processing of p100 is regulated. GRR, glycine-rich region; LZ, leucinezipper. The arrows point to the C-terminal residues of p50 and p52 (followingprocessing of p105 and p100, respectively).

IκBα

IκBβ

IκBγ

IκBε

BCL3

IκB

pro

tein

s

446

500

607

361

317

969

898447

433

557

619

551

REL homology region

NF-

κB/R

EL

prot

eins

p100/p52

p105/p50

RELB

c-REL

p65

LZ

GRR

GRR

SS

SS

SS

Ankyrin repeats

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over-expression of BCL3. Unlike other IκB proteins,BCL3 associates with p50 or p52 homodimers in thenucleus to function as a transcriptional co-activator37.Overexpression of Bcl3 in transgenic mice is not sufficientto induce T-cell leukaemogenesis38, but can causesplenomegaly when overexpressed by B cells39. BCL3probably acts in conjunction with other oncogenic mutations or DNA rearrangements to promote cancer.

DBCLs can be further classified into two subtypes— GERMINAL-CENTRE-like and activated-B-cell-like — onthe basis of their gene-expression profiles40.Congruent with their putative origin, DBCLs that dis-play the activated B-cell phenotype have elevatedexpression of NF-κB target genes, which encodecytokines, chemokines and anti-apoptotic proteins (L. M. Staudt, personal communication). Expressionof a non-phosphorylateable IκB mutant — the so-called super-repressor — in these cells inhibits theirproliferation (L. M. Staudt, personal communication).By contrast, DBCLs with a germinal-centre-like gene-expression profile seem to be resistant to the IκBαsuper-repressor. These results indicate that the canon-ical NF-κB signalling pathway, which depends on theIKKβ catalytic subunit and IκB degradation, is consti-tutively active in the activated B-cell-like DBCL cells.As the second NF-κB signalling pathway, whichdepends on IKKα and NF-κB2/p100 processing, isinvolved in germinal-center formation6, it would beinteresting to determine whether it is constitutivelyactivated in germinal-center-like DBCLs.

NF-κB signalling pathways also seem to be involvedin RNA and DNA virus-induced leukaemias and lym-phomas. TAX, the transforming protein of HTLV-1,directly interacts with and activates IKK8. In addition,TAX can divert IKKα into new complexes that stimu-late the phosphorylation-dependent processing ofNF-κB2/p100 (REF. 8). So, TAX is capable of activatingboth the canonical and the processing-based NF-κBactivation pathways. NF-κB activation has been shownto be essential for TAX-mediated transformation41.Epstein–Barr virus (EBV) — a DNA tumour virus thathas been implicated in Burkitt’s and Hodgkin’s lym-phomas, as well as in B-cell lymphomas in immuno-compromised hosts42 — is also capable of inducingpersistent NF-κB activation9. At least two EBV-encodedproteins — EBV nuclear antigen (EBNA)-2 and latentmembrane protein-1 (LMP-1) — increase both NF-κBDNA binding and transcriptional activities byunknown mechanisms.

BCR–ABL is another oncoprotein — albeit of cellu-lar origin — that is capable of activating NF-κB43.Although the tumorigenic mechanisms of BCR–ABL,which is associated with myelogenous leukaemia, arenot well understood, its ability to activate NF-κB hasbeen proposed to be an essential component of itsoncogenic activity43. B-cell maturation antigen(BCMA) — a member of the TNF receptor (TNFR)family — was first identified as the product of t(4;16) inT-cell lymphoma cells44. BCMA has been proposed toactivate IKK, leading to constitutive NF-κB activation.Similarly, chronic activation of another TNFR family

No rearrangements, amplifications or deletions of thehuman NFKB1 locus, located at 4q24, have been associ-ated with leukaemia or lymphoma. However, BCL3,a gene that is located at chromosomal region 19q13.1 and that encodes an IκB family member, was originallycloned as a rearranged locus from a B-cell lymphocyticleukaemia (B-CLL)36. This translocation causes

GERMINAL CENTRE

A site in secondary lymphoidtissue where B cells are exposedto antigen, and are induced toeither proliferate, mature orundergo cell death.

NF-κB

Negative feedback Immunity Anti-apoptosis

Chemokines, cytokines,antimicrobial peptides,adhesion molecules,iNOS, COX2

cIAPs, A1/BFL1,BCL-XL, c-FLIP

IκBα, IκBβ, A20

Proliferation

Cyclin D1,c-MYC

Figure 2 | NF-κB contributes to the induction of four classes of genes. The genes that areinduced in the response to nuclear factor of κB (NF-κB) activation can be divided into fourfunctional classes: genes that have products that are involved in negative-feedback control of NF-κB activity; genes that have products that serve various immunoregulatory functions; genesthat have products that inhibit caspase activation and apoptosis; and genes that promote cellproliferation. COX2, cyclooxygenase-2; FLIP, FLICE (FAS-associated death domain (FADD)-like IL-1β-converting enzyme) inhibitory protein; cIAPs, cellular inhibitors of apoptosis; iκBα, inhibitor of κBα; IκBβ, inhibitor of κBβ; iNOS, inducible nitric oxide synthase.

IKKγ

IKKβ IKKα

TNF-α, CD40LIL-1, LPS

LTβ, BAFF

RELA

RELA

p50IKβ

NF-κB226S

RELB

p52

PP

PP

NIK

p50

RELBp52

RELB

Figure 1 | The IKK complex controls two distinct NF-κBactivation pathways. The inhibitor of κB (IκB) kinase (IKK)complex is composed of two catalytic subunits, IKKα andIKKβ, and one regulatory subunit, IKKγ. In response to stimulisuch as tumour-necrosis factor-α (TNF-α), CD40 ligand(CD40L), interleukin-1 (IL-1) or lipopolysaccharide (LPS), theIKKβ subunit is activated, and phosphorylates the IκB proteins(bound to the NF-κB heterodimers) at two conserved serines.This phosphorylation event triggers the ubiquitin-dependentdegradation of IκB by the 26S proteasome, resulting in thenuclear translocation of RELA–p50 (or c-REL–p50)heterodimers and transcriptional activation of target genes. Inresponse to other stimuli, such as the TNF family memberslymphotoxin B (LTβ) and BAFF, IKKα is activated to induce thephosphorylation of p100 (bound to RELB) at two serineresidues at its carboxyl terminus. This phosphorylation eventtriggers the ubiquitin-dependent degradation of the carboxy-terminal half of p100, releasing its amino-terminal half, the p52polypeptide, which together with its heterodimer partner,RELB, translocates to the nucleus to activate transcription.

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viruses constitutively activate this transcription factor toprevent host-cell apoptosis, leading to oncogenesis.Different mechanisms by which NF-κB can contributeto leukaemia and lymphogenesis are illustrated in FIG. 3.

NF-κB and breast cancerNF-κB has also been shown to be involved in thedevelopment of carcinomas — cancers of epithelial

member, CD40, by autocrine production of its ligand,CD40L, has been linked to the genesis of CLL45.

A particularly interesting example of how continu-ous IKK activation can promote lymphoma develop-ment is the case of mucosa-associated lymphoid tissue(MALT) lymphomas. These lymphomas, which areassociated with t(1;14) and t(11;18), are the most com-mon subset of extranodal non-Hodgkin’s lymphoma46.The t(1;14) moves the IgG promoter upstream of thegene that encodes BCL10, which results in overexpres-sion of a truncated BCL10 protein47. Somehow, overex-pression of truncated BCL10 leads to constitutive NF-κB activation and, eventually, lymphoma. Loss ofBCL10, by contrast, as shown in knockout mice, pre-vents IKK and NF-κB activation in lymphocytes inresponse to antigen-receptor stimulation48. The t(11;18)results in generation of a different fusion protein —API2–MALT1 (REF. 49) — which also activates NF-κB. Infact, expression of MALT1 alone can enhance the activa-tion of NF-κB by BCL10 (REF. 50). So, MALT1 andBCL10 seem to be components of an IKK-dependentNF-κB activation pathway that is initiated by antigen-receptor activation. The chromosomal translocationsthat are associated with MALT lymphoma create fusionproteins that induce constitutitve activation of thispathway, which results in lymphoma.

In addition to EBV, other factors are thought to beinvolved in the persistent activation of NF-κB and IKKin the Reed–Sternberg (HRS) cells of Hodgkin’s lym-phomas51. Although much remains to be learned aboutthe exact mechanisms of IKK and NF-κB activation inHRS cells, inhibition of NF-κB has been shown toinduce apoptosis in these cells52. Similarly, inhibition ofNF-κB causes the spontaneous apoptosis of EBV-trans-formed lymphoblastoid cells53, Kaposi’s-sarcoma-asso-ciated herpesvirus-associated lymphomas54 and B-CLL45. These are further indications that pathogenic

NF-κB

ApoptosisG1 S

Viral oncoproteins

Gene rearrangements

CD40L

CD40

Cyclin D1, D2

Anti-apoptoticgenes

Figure 3 | Different mechanisms by which NF-κBactivation can contribute to leukaemia andlymphogenesis. Nuclear factor of κB (NF-κB) can beconstitutively activated in myeloid and lymphoid cells inresponse to growth factors and cytokines or the expression ofcertain viral oncoproteins. Persistent NF-κB activation can alsobe brought about by chromosomal rearrangements that affectgenes that encode NF-κB or inhibitor of κB (IκB) proteins.Once NF-κB is activated, it can lead to the production ofcytokines and growth factors, such as CD40 ligand (CD40L),that further propagates its activation. It also activates thetranscription of cell-cycle regulators, such as cyclins D1 andD2, which promote G1- to S-phase transition, or inhibitors ofapoptosis, such as BCL-XL, cIAPs and A1/BFL1.

Box 2 | NF-κB regulators

Inhibitor of κB (IκB) kinase (IKK) is a protein complex that is composed of three subunits: IKKα and IKKβ are catalytic(protein kinases) and IKKγ(NEMO) is regulatory. IKKα and IKKβ have protein kinase activity towards their substratesin vitro, but in intact cells, their activity and/or activation depend on the IKKγsubunit. IKKγis a bifunctional proteinthat is required for the assembly of a high-order IKK complex, in which two IKKα and IKKβ homo- or heterodimers areheld together by two or four IKKγsubunits. In addition, IKKγis required for the activation of IKKα and IKKβ, which ismediated through their phosphorylation at two conserved serine residues that are located within their activation loops.Whereas IKKβ is required for activation of the canonical nuclear factor of κB (NF-κB) pathway that is based on thephosphorylation-induced degradation of IκBs, IKKα is required for activation of a second NF-κB pathway that is basedon the phosphorylation-induced processing of p100. The exact mechanisms by which IKKα and IKKβ are activated inresponse to extracellular stimuli are not clear, but it seems that IKKα and IKKβ are differentially regulated. The IKKα-mediated p100 processing pathway is activated by two specific members of the TNF family: lymphotoxin B (LTβ) andBAFF (BLYS). The IKKβ-dependent, canonical NF-κB pathway is activated by many other stimuli, including TNF-α andmost members of its family, IL-1, and various innate immune stimuli, such as lipopolysaccharide (LPS) and double-stranded RNA, which activate Toll-like receptors (TLRs).

Once the IκBs are phosphorylated at their N-terminal regulatory serines by the IKK complex, they are recognized by asecond protein complex, E3IκBα. This complex consists of at least four subunits: CUL1, SKP1, ROD and the F-box proteinβ-TrCP. The latter is responsible for the recognition of the dually phosphorylated form of IκBs. The binding of the E3IκBα

complex to the phosphorylated IκBs results in the recruitment of a specific E2-ubiquitin-conjugating enzyme, UBC5,which catalyses the transfer of ubiquitin chains from E1 ubiquitin conjugates by an enzyme — thioester intermediate —to the IκBs. It is not known whether the ubiquitylation of p100 and p105, which is required for their processing, isdependent on the E3IκBα complex or a different ubiquitin ligase.

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NF-κB1/p50–RELA(p65) heterodimers12, and mam-mary carcinomas. High levels of NF-κB2 expression(both p100 and p52) were observed in mammary carcinoma cell lines and primary tumours55,59. Althoughoverexpresion of NF-κB2/p100 inhibits NF-κB DNA-binding activity59, the presence of elevated NF-κB activ-ity in most breast cancers55–57 indicates that either theexpression levels of NF-κB2/p100 are not very high,or that most of this protein is processed to p52.NF-κB2/p100 interacts with RELB5, and elevated expres-sion of RELB has also been reported in primary breastcarcinomas55. However, the same study also reportedincreased expression of c-REL and NF-κB1/p50.

Apart from increased expression of NF-κB compo-nents, the exact cause of elevated NF-κB activity inbreast cancer is not known. The mechanisms thatunderlie the elevated expression of NF-κB familymembers in some cancer cells are also not clear. It ispossible that oncoproteins that are known to be acti-vated in breast cancer cells, such as ERBB2(HER2/neu) or HRAS, trigger signalling cascades thatlead to NF-κB activation60–62.

Another possible mechanism for NF-κB upregula-tion in breast cancer cells has been revealed by a recentreport of the importance of IKKα and NF-κB in mam-mary-gland development12. NF-κB (primarilyp50–RELA heterodimers) is activated during twophases of mouse mammary-gland development —pregnancy (peaking around days 15–16 post-coitus)and involution. Involution is the stage when most ofthe epithelial network regresses in size by means ofapoptosis, and tissue is remodelled to that resembling aNULLIPAROUS female63,64.

The activation of NF-κB during pregnancy is dri-ven by a member of the TNF cytokine family known asreceptor activator of NF-κB (RANK) ligand(RANKL)12. RANKL is produced by mammary epithe-lial cells during pregnancy in response to hormonalstimuli and acts in an autocrine manner through itsreceptor RANK, which is also expressed in the mam-mary epithelium65. A deficiency in either RANKL orRANK arrests the mammary gland at the nulliparousstage, preventing the extensive lobuloalveolar prolifera-tion that occurs during pregnancy65. Unlike TNF-αreceptor 1 (TNFR1), which signals to NF-κB by IKKβ4,RANKL signals to NF-κB by IKKα12. Interference withIKKα kinase activity, or a specific inhibition of NF-κBwithin the mammary epithelium, results in the samelobuloalveolar proliferative defect that is caused byRANKL or RANK deficiency12.

Apparently, the only function of NF-κB in mam-mary epithelial cells during pregnancy is to stimulatecell proliferation by increasing transcription of thecyclin D1 gene12. Maintenance of normal cyclin D1levels by expression of a mammary-specific cyclinD1 transgene obliterates the requirement for IKKαor NF-κB. These studies have established the role ofthe IKKα-dependent RANK–NF-κB pathway in con-trolling the proliferation of the mammary epithe-lium during pregnancy (FIG. 4). These findings alsoraise the possibility that deregulated production of

origin, such as breast cancer. Numerous studies have documented elevated or constitutive NF-κB DNA-binding activity both in mammary carcinomacell lines and primary breast cancer cells of humanand rodent origin55–57. Almost all chemically inducedrat mammary carcinomas have high levels of NF-κBactivity56, and in human breast cancer cells, NF-κBactivity was reported to correlate with oestrogen inde-pendence57. Subsequent studies have shown that NF-κB is activated in most human breast cancer cells,regardless of hormone-dependency status55. ElevatedNF-κB DNA-binding activity was also detected inmammary glands of carcinogen-treated rats withinthree weeks of exposure — well before the appearanceof detectable tumours58. This indicates that constitu-tive NF-κB activation might be one of the early eventsin breast cancer pathogenesis.

It is not clear, however, whether there is a differ-ence in the composition of NF-κB between normalmammary epithelial cells, which primarily use

NULLIPAROUS

A female that has never borneoffspring.

GF

RTK

RAS

MAPK

AP1

RANKL

RANK

IKKα

IκBα

NF-κB (p50–p65)

?

AP1 κB

Cyclin D1

G1 S

?

Figure 4 | Signalling pathways that stimulate theproliferation of mammary epithelial cells by induction ofcyclin D1 gene transcription. At least two signallingpathways can contribute to the induction of cyclin D1transcription in mammary epithelial cells. One pathway, whichleads to activation of transcription factor AP1, is activated bygrowth factors (GF), which bind to receptor tyrosine kinases(RTK). This pathway relies on activation of RAS and mitogen-activated protein kinase (MAPK) cascades. The secondpathway is activated by the tumour-necrosis factor familymember receptor activator of NF-κB ligand (RANKL), whichbinds to the receptor activator of NF-κB (RANK). Thispathway, which leads to activation of NF-κB, depends on theIKKα subunit of the inhibitor of κB (IκB) kinase (IKK) complex.IKKα activation is required for the induction of IκBdegradation, freeing NF-κB to translocate to the nucleus.There, it activates cyclin D1 expression, leading to cell-cycleprogression. The expression of GFs and RANKL is regulatedby various hormonal stimuli during mammary-glanddevelopment. Aberrant and persistent activation of eitherpathway can lead to deregulated proliferation of mammaryepithelial cells.

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with cancer, NF-κB might be the missing linkbetween these two processes. By virtue of its anti-apoptotic activity, the persistent activation of NF-κBthat occurs during chronic inflammation or infectionmight prevent the elimination of genetically altered,precancerous cells. In addition, by stimulating thetranscription of cyclin D1 and other G1 cyclins, con-stitutively active NF-κB might cause enhanced cellproliferation. In inflammatory cells, continuous NF-κB activity could promote the production of reactiveoxygen species, thereby damaging DNA of surround-ing epithelial cells. Some of the best circumstantialevidence that supports such a role for NF-κB comesfrom various gastrointestinal cancers (FIG. 5).

One of the main risk factors that is linked to gas-tric cancer — the second most common malignancyworldwide — is Helicobacter pylori infection72. Single-nucleotide polymorphisms in the IL1 gene, which arebelieved to increase its expression levels, are anotherrisk factor for gastric cancer73. Both IL-1 and H. pyloriare potent NF-κB activators. Not only can H. pylori activate NF-κB in gastric epithelial cells, butactivated NF-κB was also found in cells of gastricbiopsy specimens that were infected with H. pylori 74.The activation of NF-κB by H. pylori requires genes inthe CAG PATHOGENICITY ISLAND72. Once NF-κB is activatedin gastric epithelial cells, it activates the transcriptionof IL-1, IL-6, IL-8, TNF-α, cyclooxygenase-2 (COX2)and, probably, other mediators of inflammation74,75,some of which can further propagate the NF-κB activation response76.

Knockout mice that lack the inhibitory C-terminaldomain of NF-κB1/p100, and therefore constitutivelyexpress NF-κB2/p52, have dramatic hyperplasia of thegastric epithelium35. So, in addition to promotinginflammation, NF-κB (probably p52–RELB het-erodimers) can stimulate the proliferation of the gastricepithelium, and thereby increase susceptibility to car-cinogenesis by ingested environmental and naturallyoccurring mutagens.

RANKL, or constitutive RANK or IKKα activation,might underlie the elevation in NF-κB activity inbreast cancer.

Unlike other cell types that express similar levels of allthree G1 cyclins, mammary epithelial cells express mostlycyclin D1, and their proliferation is therefore highlydependent on this particular protein66,67. The cyclin D1promoter contains binding sites for several transcriptionfactors, the activity of which is induced in response toextracellular stimuli, including AP1 and NF-κB (FIG. 4).Whereas the NF-κB site10,11 is likely to be responsive to theRANK-generated signal12, the AP1 site seems to beresponsive to signals that are generated by receptor tyro-sine kinases, such as ERBB2 (REF. 68). Importantly, cyclinD1 expression is also required for breast carcinogenesisfollowing mammary-specific expression of the ERBB2 orHRAS oncogenes69. Future studies should examinewhether transmisison of these oncogenic signals to thecyclin D1 promoter depends on IKKα and NF-κB, orwhether it relies solely on activation of AP1.

Support for a possible involvement of IKK in breastcarcinogenesis is provided by a study that showed ele-vated IKK activity in both breast carcinoma cell linesand primary tumours70. Overexpression of catalyticallyinactive IKKα or IKKβ in such cell lines resulted in inhi-bition of NF-κB activity70, loss of tumorigenic potentialand increased sensitivity to apoptosis-inducing anti-cancer drugs71. It is not clear whether the inhibitoryeffect of the catalytically inactive IKKβ reflects its directinvolvement in the tumorigenic process, or whether theoverexpressed IKKβ mutant somehow interferes withIKKα activity.

Inflammation and cancerA causal connection between inflammation and can-cer has been suspected for many years. However, themechanistic link between inflammation and tumori-genesis is not well understood. Because NF-κBbecomes activated in response to inflammatory stim-uli and its constitutive activation has been associated

CAG PATHOGENICITY ISLAND

An Helicobacter pylori locus ofapproximately 40 kb thatcontains 31 genes. Several cagisland genes have homology togenes that encode type IVsecretion system proteins, whichexport proteins from bacterialcells. The terminal gene in theisland, cagA, is commonly used asa marker for the entire cag locus.

NF-κB

Normalgastric/intestinalepithelial cell

Gastric/intestinalepithelial cellwith DNA damage

Environmentalcarcinogen

H. pylori,inflammation NF-κBNF-κB

NF-κB

NF-κB

p53-mediated apoptosis

ROS

Figure 5 | Role of NF-κB in gastric and colorectal cancers. Exposure to environmental carcinogens in the diet can cause DNAdamage to gastric or intestinal epithelial cells. Such cells are normally eliminated by p53-mediated apoptosis, but oncogene activationor loss of tumour-suppressor activity, coupled with Helicobacter pylori infection or inflammation, can lead to constitutive nuclear factorof κB (NF-κB) activation. NF-κB activation leads to production of enzymes such as inducible nitric oxide synthase (iNOS) andcyclooxygenase-2, which enhance the production of reactive oxygen species (ROS), leading to additional DNA damage. Finally,through enhanced production of growth factors and cytokines, NF-κB can lead to further proliferation of transformed cells.

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large body of tantalizing circumstantial evidence, somedirect (preferably genetic) evidence that NF-κB andIKK are involved in colorectal and gastric cancerpathogenesis is required.

Implications and future directionsAn impressive body of evidence implicates NF-κB acti-vation in the development of lymphoid-, myeloid- andepithelial-derived malignancies. However, certainaspects of NF-κB biology indicate that it can also havea tumour-suppressor function. Given the ability ofNF-κB to activate innate and adaptive immuneresponses, its persistent activation in tumours seemsparadoxical — its ability to stimulate the production ofchemokines and cytokines would lead to recruitmentof immune cells to the tumour and contribute to itsrejection. If NF-κB does promote tumour develop-ment, it is likely that some of its immune and inflam-mation-related target genes might not always be acti-vated. If a cancer cell is able to alter its profile of NF-κB target genes, this might allow a cancer cell to suppress apoptosis without expressing thechemokines and cytokines that broadcast its presenceto the immune system.

Numerous inhibitors of NF-κB are under develop-ment or have been developed. Small molecules and viralvectors that inhibit IKK, or other aspects of the NF-κBactivation pathway, have been shown to induce apopto-sis and inhibit the proliferation of tumours or tumour-derived cell lines. Unfortunately, none of the small mol-ecules have proven to be completely specific for IKK orNF-κB, and viral vectors are not yet practical for clinicalapplications. Clearly, more specific and potent inhibitorsare needed.

Given the two distinct modes of NF-κB activationby the two catalytic subunits of the IKK complex,IKKα and IKKβ, selective inhibitors that target onesubunit and not the other would be of great thera-peutic and basic research value. Reagents such asthese would help differentiate the exact biologicalroles of each pathway. Based on our current knowl-edge, we can speculate that IKKα-specific inhibitorsmight offer a selective therapy for germinal-center-derived DBCL and, possibly, breast cancer, whereasIKKβ-specific inhibitors might be useful for inducingapoptosis in all types of tumours that have constitu-tively active NF-κB. An IKKβ inhibitor, however,would also inhibit innate and adaptive immunity, andit would increase the sensitivity of many normal cellsto TNF-α-induced apoptosis.

To investigate the role of NF-κB proteins in cancerdevelopment, we also need to further analyse the largecollection of genetically altered mouse strains that carrydeletions or other genetic alterations in genes thatencode NF-κB, IκB and IKK components. The suscepti-bility of such mouse strains to a variety of cancers andcancer treatments needs to be examined. The identifica-tion of NF-κB target genes in different types of normalcell and their transformed derivatives is another impor-tant area for future research. As we begin to understandwhich genes are activated by NF-κB under different

NF-κB activation is also associated with colorectalcancer. Colon cancer cell lines and human tumoursamples, as well as nuclei of stromal macrophages insporadic adenomatous polyps, were found to haveincreased NF-κB activity77,78. Most cases of colorectalcancer are sporadic, but 10–15% of cases are caused byhereditary syndromes, such as familial adenomatouspolyposis (FAP) or hereditary non-polyposis cancer(HNPCC) and colitis-associated cancer79,80. Ulcerativecolitis is a chronic inflammatory bowel disease (IBD),which, together with Crohn’s disease (another type ofIBD), is associated with persistent NF-κB activation intissue macrophages and epithelial cells of the colonicmucosa81,82. Crohn’s disease also increases the risk ofcolorectal cancer, but not as much as ulcerative colitis83.

Constitutive NF-κB activation has also beenobserved in Il-10-deficient mice, which provide anexcellent animal model for IBD81. Downregulation ofRelA expression in such mice, by use of antisenseoligonucleotides, resulted in a significant reduction ofIBD symptoms81,84. NF-κB inhibitors might, therefore,be useful in treating human IBD. Most of the drugs thatare commonly used for treating acute IBD and for con-trolling remission, including sulphasalazine,mesalamine, glucocorticoids and methotrexate, wereshown to inhibit NF-κB or IKK76,85–88. It is notable thatmost of these drugs act through different mechanismsand have different molecular targets, so their only com-mon denominator is an ability to inhibit NF-κB.

NF-κB inhibitors might also prevent progressionto colorectal cancer by preventing expression of COX2— another NF-κB target gene. COX2 is responsiblefor inducible prostaglandin synthesis during inflam-mation. The link between COX2 and colorectal canceris supported strongly by epidemiological and experi-mental evidence. COX2 is overexpressed in colon ade-nomas and carcinomas of human and mouse ori-gin89,90, and Cox2-null mice are resistant to colorectalcancer91,92. Long-term consumption of aspirin orother COX inhibitors over a period of 10–15 years hasbeen reported to reduce the relative risk of colorectalcancer by 40–50% (REFS 93,94). Aspirin and other non-steroidal anti-inflammatory drugs (NSAIDs) alsoreduce the incidence of colorectal cancer in animalmodels95,96 and the risk of gastric cancer inhumans97,98. Although such drugs inhibit both COX1and COX2, the latter is the more relevant target forsuppression of colorectal cancer91.

Given the ability of both aspirin and sulindac toinhibit IKK99,100, it is possible that some of theirchemopreventive activity is derived from their abilityto prevent NF-κB activation. Inhibition of IKK activityusing sulindac sulphide was shown to induce theapoptosis of a colorectal cancer cell line100. Curcumin— another less potent and even less specific inhibitorof IKK — is another anti-inflammatory compound101.Curcumin has been shown to reduce colon carcino-genesis in several animal models95,102 and to inhibit theproliferation of colon cancer cells103. Its extensive con-sumption in the Indian subcontinent has been linkedto low incidence of colorectal cancer104. Despite the

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conditions, and which transcription factors and sig-nalling pathways are involved in NF-κB activation, weshould be able to design new therapeutic strategies thatwill allow the blocking of certain NF-κB target genesand not others. Furthermore, drugs that specifically dis-rupt post-translational modification of NF-κB subunits,and therefore inhibit only subsets of subunit-specifictarget genes, seem to be another attractive therapeuticoption. However, little is known so far about the path-ways that are responsible for these modifications, andsome of them might have other substrates.

In the future, drugs might be designed to inhibitNF-κB activation of anti-apoptotic or pro-prolifera-tion target genes without affecting the induction ofgenes that are required for immunity, or for protect-ing normal cells from killing by members of theTNF-α family. Given the rapid progress in under-standing the mechanisms that are involved in activa-tion of NF-κB and its function, what might seem to be a pipe dream today will become a reality in the next decade — in the form of a widely used anticancer drug.

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AcknowledgementsM. K. is the Frank and Else Schilling American Cancer SocietyResearch Professor. Research in his laboratory is supported by theNational Institutes of Health and the State of California CancerResearch Program, and the Breast Cancer Basic ResearchProgram. Y. C., F. R. G. and Z.-W. L. are supported by postdoctoralfellowships from the California Breast Cancer Research Program,the Deutsche Forschungsgemeinschaft and the Cancer ResearchInstitute, respectively.

Online links

DATABASESThe following terms in this article are linked online to:Cancer.gov: http://www.cancer.gov/cancer_information/breast cancer | Hodgkin’s lymphomas | chronic lymphocyticleukaemia | colorectal cancer | gastric cancer | gastrointestinalcancer | Kaposi’s sarcoma | multiple myeloma | non-Hodgkin’slymphomasGenBank: http://www.ncbi.nih.gov/Genbank/EBNA2 | EBV | LMP1LocusLink: http://www.ncbi.nlm.nih.gov/LocusLink/ABL | API2 | BCL2 | BCL3 | Bcl3 | BCL10 | BCL-XL | BCMA | BCR |BFL1 | BLYS | β-TrCP | caspase-8 | CD40 | CD40L | COX2 | Cox2 |CUL1 | cyclin D1 | cyclin D2 | cyclin G1 | ERBB2 | FLIP | GM-CSF |HRAS | cIAPs | IgG | IκBα | IκBβ | IκBε | IL1 | IL-2 | IL-6 | IL-8 |iNOS | LTβ | MALT1 | MAPK | MMPs | NEMO | NF-κB1 | Nfkb2 |NF-κB2 | NIK | p53 | RANK | RANKL | Rel | REL | RelA | RELA |RELB | SKP1 | TAX | TLRs | TNF-α | TNFR | TNFR1 | VEGFMedscape DrugInfo:http://promini.medscape.com/drugdb/search.aspaspirin | mesalamine | methotrexate | sulindac | sulphasalazineOMIM: http://www.ncbi.nlm.nih.gov/Omim/familial adenomatous polyposis | hereditary non-polyposis cancer |ulcerative colitis

FURTHER INFORMATIONBiology of the Mammary Gland: http://mammary.nih.govThe Mouse Models of Human Cancers Consortium:http://emice.nci.nih.gov Access to this interactive links box is free online.