Opinion

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Vol.14 No.2 February 2004

NF-kB: tumor promoter or suppressor? Neil D. Perkins Division of Gene Regulation and Expression, School of Life Sciences, MSI/WTB Complex, Dow Street, University of Dundee, Dundee, UK DD1 5EH

A role for the NF-kB family of transcription factors as tumor promoters is firmly established. However, other data suggest that NF-kB can also inhibit tumor growth. Moreover, NF-kB activity is modulated by tumor suppressors, such as p53 and ARF, whereby NF-kB subunits repress, rather than activate, the expression of tumorpromoting genes. This suggests a dual function of NF-kB during tumor progression – in the early stages, NF-kB inhibits tumor growth but, as further mutations lead to a loss of tumor suppressor expression, the oncogenic functions of NF-kB become unleashed, allowing it to actively contribute to tumorigenesis. Here, I discuss this hypothesis, its implications for NF-kB function, and how this might influence the use of NF-kB-based anticancer therapies. A reader of the scientific literature might easily conclude that inhibitors of the nuclear factor kB (NF-kB) family of transcription factors could be the secret to the treatment of many chronic human diseases. Indeed, with any disease that includes an inflammatory component, there are excellent reasons for believing that downregulating aberrantly active NF-kB would have a beneficial effect [1]. NF-kB induces the expression of inflammatory cytokines and chemokines and, in turn, is induced by them [1,2]. This establishes the basis for a positive feedback mechanism, which although usually kept in check, has the potential, when NF-kB becomes aberrantly active, to produce the chronic or excessive inflammation associated with diseases such as rheumatoid arthritis, asthma and inflammatory bowel disease [1]. Furthermore, conditions such as atherosclerosis also include an inflammatory NF-kBregulated step [3]. In addition, it is well established that NF-kB plays an important role in some virally induced diseases [4]; in particular, it regulates the replication of HIV-1, the causative agent of AIDS [4]. NF-kB has also been implicated in pathological conditions such as Alzheimer’s disease [5]. For all these reasons, there has been much interest, especially in the pharmaceutical and biotechnology sectors, in developing inhibitors of NF-kB [6,7], with particular emphasis on inhibitors of IkB kinase b (IKK) activity (Box 1); it is possible that these IKK inhibitors could soon enter clinical trials. Although such inhibitors have great potential, given that the normal function of NF-kB is regulation of cellular processes, such as the immune response, cell adhesion, apoptosis and proliferation, it is hoped that they will function without significant side effects, especially over a long-term course

of treatment (Box 2) [2,8– 11]. Perhaps the area that has generated the greatest interest over the past few years is the role of NF-kB in cancer [12 – 14]. In this article, I will discuss NF-kB function in tumorigenesis and propose that NF-kB does not always promote tumor growth. In fact, there is good evidence that, under some circumstances, NF-kB contributes to pathways that inhibit cancer development. NF-kB is a tumor promoter The suggestion that the members of NF-kB family might behave as tumorigenic transcription factors was first put forward upon the cloning of the p50– p105 (NF-kB1) subunit [15,16]. Analysis of the NF-kB1 sequence immediately revealed homology to v-rel, a potent transforming oncogene of the avian reticuloendotheliosis virus [17], and its cellular counterpart the protooncogene c-rel. Further support for a role of NF-kB subunits in cancer came with the discovery of the gene encoding p52 – p100 (NF-kB2) – in some B- and T-cell lymphomas the gene encoding p100 is

Box 1. The NF-kB family and its activation NF-kB is a collective name for the complexes formed by the multigene NF-kB-Rel family. In mammalian cells, there are five NF-kB subunits RelA(p65), RelB, c-Rel, p105 –p50 (NF-kB1) and p100 –p52 (NF-kB2 or lyt-10). All contain an , 300-amino-acid region of extensive homology in their N-termini, termed the Rel homology domain (RHD), which mediates their DNA-binding and dimerization [8,11]. Most combinations of homo- and heterodimers are possible [36]. RelA(p65), RelB and c-Rel contain nonhomologous transactivation domains in their C-termini. p105 and p100 require proteolytic processing to generate their active nuclear-DNA-binding forms – p50 and p52. Typically, in unstimulated normal cells, NF-kB subunits are held in an inactive cytoplasmic form bound to a member of IkB protein family – IkB a, b or 1 [8,11]. The unprocessed forms of p100 and p105, which contain ankyrin repeats in their C-termini, similar to those seen in the IkBs, can also function as IkB-like proteins, retaining their dimeric NF-kB partners in the cytoplasm of the cell [8,11]. The Bcl-3 protein has the appearance of an IkB but is actually a transcriptional coactivator for p52 and p50 [8,11]. In response to stimulation, typical examples of which are exposure to the inflammatory cytokines tumor necrosis factor a (TNFa) and interleukin 1 (IL-1), signaling pathways are initiated that result in the activation of the IkB kinase (IKK) complex [37]. IKK, which consists of two catalytic subunits – IKKa and IKKb (or IKK1 and IKK2) – in addition to a regulatory subunit IKK g (or NEMO), phosphorylates members of the IkB family (a, b or 1), promoting their ubiquitination and degradation by the proteasome [37]. The degradation process releases NF-kB, allowing it to translocate to the nucleus where it can regulate gene expression. IKK b appears to be the primary IkB kinase, whereas IKK a has other roles, including the phosphorylation of the p100 subunit of NF-kB and a function as a histone kinase [38].

Corresponding author: Neil D. Perkins ([email protected]). www.sciencedirect.com 0962-8924/$ - see front matter q 2003 Elsevier Ltd. All rights reserved. doi:10.1016/j.tcb.2003.12.004

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Box 2. The function and complexity of the NF-kB response The central role played by NF-kB as a regulator of the immune response is illustrated by its conservation throughout evolution [8]. Drosophila melanogaster, for example, contains NF-kB-related proteins that regulate its immune system [39]. Gene knockouts in mice have confirmed the importance of NF-kB as a regulator of immunecell function [9]. These experiments also revealed that RelA(p65) subunit of NF-kB is a crucial regulator of apoptosis – as a result of TNFa-induced apoptosis of liver cells, rela 2/2 mice die in utero [9]. NF-kB also plays a central role in the inflammatory response, because it is induced by inflammatory molecules and also induces the expression of many cytokines and chemokines [2]. The regulation and function of NF-kB is highly complex and it is also an important regulator of many aspects of the cellular response to stimulation such as the stress response, cell adhesion and proliferation. However, hundreds of inducers of NF-kB DNA-binding have been described [2]. Furthermore, given the almost ubiquitous presence of NF-kB in most cell types and the possibility that there will be hundreds of NF-kB target genes to regulate [2], the mechanisms controlling the NF-kB response are by necessity diverse and achieving the required specificity and selectivity with which it acts is complex. Therefore, the nature of the NF-kB response will differ, depending on the context in which it is found. This specificity is achieved through a combination of mechanisms [36]. Differential activation of NF-kB subunits can result in the regulation of different target genes through differences in DNA-binding affinity. Moreover, interactions with heterologous DNA-binding proteins, often resulting in cooperative DNA-binding, can determine which genes become activated by NF-kB. Finally, coactivator and co-repressor complexes are required for NF-kB function and their regulation will have effects on NF-kB itself. Therefore, NF-kB does not work alone; it functions as part of a network of coordinately regulated DNA-binding proteins and transcription factors that, together, determine the pattern of gene expression required for the response to a particular cellular stimulus. This complexity has led to many apparent contradictions in the scientific literature. There are many reports of both proapoptotic and antiapoptotic activity of NF-kB [10]. Under some circumstances, NF-kB can also induce proapoptotic genes such as Fas and Fas ligand [2,10]. The ability to perform these apparently opposing functions is an example of how context, and possibly posttranslational modifications, can profoundly affect NF-kB function and the specificity of target genes. Interestingly, the b-catenin proto-oncogene represses NF-kB-mediated activation of Fas expression [40].

subject to rearrangement, resulting in truncated and constitutively nuclear forms of the protein [18,19]. Furthermore, Bcl-3, a coactivator of the p50 and p52 subunits of NF-kB, is also a protooncogene and was originally identified as part of a translocation event in chronic lymphocytic leukemia [18,19]. Loss-of-function mutations in IkB proteins, which result in constitutively active NF-kB, have also been found in cases of Hodgkin’s lymphoma [18]. Furthermore, rearrangements, amplifications and missense mutations have been reported for NF-kB subunits in both solid and hematopoietic tumors [18]. Despite these findings, genetic alterations in NF-kB and IkB subunits are relatively rare phenomena, especially when compared with those in well-known oncogenes and tumor suppressors such as c-Myc, Ras and p53. What has emerged is that aberrant activation and nuclear localization of NF-kB in cancer is actually quite frequent but most often results from defects in the pathways regulating NF-kB [12– 14]. Constitutive signaling of the upstream signaling pathways can cause high levels of IKK activity, resulting in NF-kB activation [12– 14]. Alternatively, the loss of negative www.sciencedirect.com

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feedback mechanisms, which usually serve to limit the NF-kB response, could result in its aberrant activity. An example of this is the CYLD tumor suppressor gene and deubiquitinating enzyme. Loss of CYLD, which is associated with a predisposition to familial cylindromatosis (tumors of skin appendages), leads to aberrant NF-kB activation [20]. In addition, the microenvironment of a solid tumor frequently contains high levels of inflammatory cytokines and/or hypoxic conditions, which both stimulate nuclear translocation of NF-kB [12 – 14]. Aberrant activation of NF-kB does not always involve increased nuclear localization, however. Activated cellular oncoproteins, such as Bcr– Abl and Ras, stimulate RelA(p65) transactivation, independent of effects on NF-kB DNAbinding and IkB degradation [13]. Importantly, NF-kB activity is required for cellular transformation induced by such oncogenes [13]. Finally, many viral oncoproteins, such as Tax, which is encoded by the human T-cell lymphotrophic virus I, and latent membrane protein 1 (LMP1), encoded by the Epstein – Barr Virus, also induce NF-kB activity [4]. Such viruses either can be carcinogenic themselves or are associated with tumor development. So why would aberrant activation of NF-kB have such a profound effect on tumor growth and development, especially when it is a transcription factor previously associated with the immune response and inflammation? The answer to this lies in the multitude of cellular functions regulated by NF-kB subunits, which directly affect the growth and survival of a malignant tumor. Resistance to apoptosis is one of the principal characteristics acquired by tumor cells [21], and a plethora of studies have now demonstrated that, in many types of tumor, NF-kB activity can confer resistance to cell death [10]. In particular, NF-kB can inhibit apoptosis induced by many common chemotherapeutic drugs and ionizing radiation [10,13]. Therefore, because these treatments also stimulate NF-kB activity, even in those tumors that do not display intrinsically aberrant levels of NF-kB activity, its inhibition could provide a method of enhancing the effectiveness of cancer therapy. Furthermore, NF-kB can stimulate angiogenesis, metastasis, cellular proliferation and the expression of many genes associated with tumor growth and survival (Box 3) [12 – 14]. Inhibiting NF-kB function would, therefore, target many characteristics of tumor cells, and animal models have supported the possible beneficial effects of such treatments [12 – 14]. NF-kB inhibitors could be used in isolation but probably will prove most effective in conjunction with other new and specific treatments or as a method of increasing the efficacy of existing treatment regimes. Taken together, the argument that NF-kB is a potent and important tumor promoter appears conclusive, as does the notion that inhibiting its activity in many clinical settings will be beneficial. Indeed, this is almost certainly the case, but not necessarily all the time! Is NF-kB a tumor suppressor? DNA damage, oncogene activation and cellular stress are well-established activators of the p53 tumor suppressor; however, they also induce DNA-binding and transcriptional activity of NF-kB [2]. Activation of p53 can result in

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Box 3. How NF-kB can stimulate tumor growth and survival The most obvious mechanism through which NF-kB, and its RelA(p65) subunit in particular, can promote tumorigenesis is through its ability to induce the expression of antiapoptotic genes such as Bcl-xL, XIAP and IEX-1L [10,41]. But there are many other aspects of NF-kB function that can promote tumor growth and survival. For example, the cyclin D1 promoter is a target of NF-kB [42,43], an observation of potential significance to the role of NF-kB in breast cancer, in which cyclin D1 is frequently overexpressed [19]. Furthermore, NF-kB stimulates the activity of the c-Myc promoter [2]; through activating such genes, NF-kB can stimulate cellular proliferation. However, the ability of tumors to invade surrounding tissues, enter the bloodstream and metastasize to distant sites is ultimately one of the most significant factors contributing to the mortality of a cancer patient [21]. Here, NF-kB also appears to play a role through its ability to regulate the expression of cellular adhesion molecules, such as ICAM-1 and VCAM-1, matrix metalloproteinases, such as MMP-9, chemokine receptors, such as CXCR4, and urokinase-type plasminogen activator (uPA) [2,12,13]. Solid tumors can also induce the growth of blood vessels and therefore overcome the effects of hypoxia and nutrient starvation induced by rapid increases in proliferation [21]. Again, NF-kB can contribute to this process through inducing vascular endothelial growth factor (VEGF) [2,12,13]. Finally, NF-kB can also promote tumor growth by inducing the expression of genes such as cyclooxygenase 2 (COX2) [2,12,13]. In addition to those discussed in the main text, there are other reports linking NF-kB with p53 and other tumor suppressors. For example, NF-kB has been reported to induce the p53 inhibitor, Mdm2, in a p53-dependent manner, thus inhibiting the p53 response [44]. This provides another mechanism through which NF-kB can promote resistance to cancer therapy. By contrast, NF-kB has also been described as an inducer of the p53 promoter [45], whereas Mdm2 has been described as an inducer of the RelA(p65) promoter [46]. In addition, an NF-kB inhibitory protein, HSCO, which promotes translocation of RelA from the nucleus to the cytoplasm, was also found to inhibit p53-induced apoptosis [47]. Finally, p53 directly interacts with IkBa [48,49]. If these observations emerge as a common mechanism, results relying on the inhibition of NF-kB with superrepressor versions of IkBa should possibly be viewed with caution because IkBa might have NF-kB-independent effects on tumor growth. Indeed, IkBa can also inhibit the function of HIV-1 Rev, a protein involved in the nuclear export of HIV-1 RNA [50]. It is possible that IkBa might also affect other proteins regulating nuclear export. Thus, IkBa is perhaps not the NF-kB-specific inhibitor it is usually assumed to be.

either cell-cycle arrest or induction of apoptosis, whereas as discussed above, the induction of NF-kB is generally associated with resistance to apoptosis and proliferation. This suggested that mechanisms must exist within the cell to integrate the activities of these two divergent pathways. Consistent with this hypothesis, my laboratory reported that p53 and the RelA(p65) subunit of NF-kB could mutually inhibit the transcriptional activity of each other [22]; similar results were reported by the laboratories of Bedi and Collins [23,24]. In addition, a more recent study from the Culmsee laboratory [25] indicates that, in neurons, reciprocal inhibition of transcriptional activity by p53 and NF-kB determines cell fate. These observations provided a somewhat satisfactory explanation for the parallel induction of p53 and NF-kB, which was also consistent with the tumor suppressor status of the former and the role of the latter as a tumor promoter; each could fulfill its ascribed functions by inhibiting the activity of the other. www.sciencedirect.com

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This simple model did not last long. In the year 2000, Ryan and colleagues [26] demonstrated that NF-kB, and its RelA(p65) subunit in particular, was actually required for p53-dependent apoptosis. This surprising and controversial result implied a completely different way of thinking about NF-kB – that is, in addition to functioning as a tumor promoter, NF-kB could potentially contribute to tumor suppression. This report prompted a re-examination of the relationship between NF-kB and tumor suppressors, which resulted in the discovery that p53 could also regulate the activity of the p52 subunit of NF-kB [27]. However, rather than just inhibiting its activity, we found that p53 induced a switch from p52 – Bcl-3 activator complexes to p52 – HDAC (histone deacetylase) repressor complexes, resulting in the inhibition of the cyclin D1 promoter [27]. In a second study, the role of ARF tumor suppressor was investigated. In response to oncogene expression, ARF mediates p53 activation by binding to and sequestering the p53 inhibitor Mdm2. Here, we reported that ARF could also induce the association of RelA(p65) with HDAC1, thereby turning it into a repressor of gene expression [28]. These effects were independent of p53 and Mdm2. Furthermore, the ability of NF-kB to activate transcription, in response to the Bcr– Abl oncogene, was dependent upon ARF expression. Importantly, NF-kB was not proapoptotic under these circumstances; rather, it acted as a facilitator of apoptosis by repressing antiapoptotic gene expression. The apoptotic stimulus itself needed to come from an outside source such as TNFa [28] or etoposide treatment (S. Rocha, and N. Perkins, unpublished). It remains to be seen how widespread these effects are and how many NF-kB-regulated genes will be affected by this mechanism. It is possible that such a mechanism of NF-kB regulation might also be used in other situations, in which the NF-kB response needs to be curtailed, for example during the inhibition of an inflammatory response. Taken together, I propose that tumor suppressors can recruit NF-kB subunits to pathways that inhibit cancer development by converting them from activators to repressors of genes that promote tumor growth (Figure 1). In other words, NF-kB can actively contribute to tumor

Tumor promoter NF-κB

Bcl-3 p52 p52

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HDAC1 p52 p52

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TRENDS in Cell Biology

Figure 1. Regulation of NF-kB activity by tumor suppressors. NF-kB subunits are inherently oncogenic and tumor promoting because of their ability to induce the expression of genes that promote resistance to apoptosis (e.g. Bcl-xL) and induce cellular proliferation (e.g. cyclin D1). However, tumor suppressor proteins, such as p53 and ARF, can induce the association of NF-kB subunits with histone deacetylase 1 (HDAC1) co-repressor complexes. This results in NF-kB-dependent repression of target gene expression, providing a model through which NF-kB subunits can facilitate apoptosis and cell-cycle arrest, and thus functioning as tumor suppressors themselves.

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Loss of tumor suppressor genes Malignant cancer cells

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Figure 2. Two roles for NF-kB in tumorigenesis. In this model, the broken arrows represent the progression of a normal cell into a transformed and malignant cancer cell. In normal cells, when an oncogenic signal, such as DNA damage, oncogene activation or infection by oncogenic viruses, is received, tumor suppressors, such as p53 and ARF, are activated. These tumor suppressors can modulate the activity of NF-kB complexes, which are either preexisting within the cell or are activated by the oncogenic stimuli. Consequently, the tumor-promoting characteristics of NF-kB are neutralized, ‘recruiting’ it to the tumor suppressor program (Figure 1). However, as the potential tumor cell accumulates additional mutations, tumor suppressor activity of NF-kB is lost, its oncogenic functions are now unleashed, and it can actively contribute to the growth and development of the cancer cell.

suppression rather than just being passively inhibited, as was suggested by the previous model. It is possible that such a scenario was also occurring, at least in part, in the study by Ryan et al. [26], with p53 converting NF-kB to a repressor of antiapoptotic gene expression, thus facilitating p53-induced apoptosis through established mechanisms. Indeed, in these experiments, p53 did not induce the transcriptional activity of NF-kB, as measured by standard reporter plasmid assays (K. Ryan and K. Vousden, pers. commun.). This is consistent with the earlier reports of both p53-induced inhibition of RelA(p65) transactivation and NF-kB-mediated repression of transcription. It is also noteworthy that other tumor suppressors, such as p16INK4a and PTEN, inhibit the transcriptional activity of NF-kB [29,30]. Whether these proteins can also induce association of NF-kB subunits with co-repressor complexes is not known; but, it is possible that this represents a common mechanism through which many tumor suppressor proteins function. These observations suggest that drugs capable of re-establishing the tumor suppressor form of NF-kB might have much greater potential for the treatment of cancer than compounds that merely inhibit its nuclear localization. Apart from studies in cell lines, is there any evidence for NF-kB actually functioning as a physiological suppressor of tumors? Several reports suggest that there is. Gapuzan et al. [31], for example, demonstrated that immortalized rela 2/2 mouse fibroblasts have many characteristics of transformed cells, including a spindled morphology, reduced adherence and an ability to grow to a higher cell density and form colonies in soft agar. Significantly, rela 2/2 cells formed tumors in immunodeficient mice, although these regressed, indicating that other mutations would be required for a more malignant phenotype [31]. A tumor suppressor function for NF-kB in skin cells has also been proposed. In these studies, the inhibition of NF-kB was found to induce epithelial cell proliferation [32] and stimulate the development of squamous cell carcinomas [33,34]. In addition, the overexpression of RelA(p65) was found to reduce the tumorigenicity of MCF7 cells in www.sciencedirect.com

nude mice [35]. However, from knockout mouse studies or human tumors, no clear evidence for active NF-kBdependent tumor suppression currently exists. The complexity and functional redundancy of the NF-kB response might preclude a clear cancer phenotype emerging. It is more probable, however, that the appropriate experiments have not been performed and the relevant transgenic mice containing the additional loss- or gain-of-function mutations that would allow the emergence of a NF-kB tumor suppressor phenotype have not been developed. A revised model for the role of NF-kB in cancer An important implication of the hypothesis that NF-kB can function as a tumor suppressor is that its behavior in normal untransformed cells might be quite different from that in transformed and malignant tumor cells. Oncogenic stimulation of untransformed cells will not only activate DNA-binding and transcriptional activity of NF-kB but will also activate the tumor suppressor programs of the cell (Figure 2) [12,13,21]. These tumor suppressors, in particular p53 and ARF, can then act to inhibit the tumorigenic functions of NF-kB. In fact, the data suggest that they will actively utilize NF-kB subunits to repress the potentially tumorigenic genes normally induced by NF-kB activity [27,28]. Thus, in the early stages of cancer, NF-kB might be tumor-suppressing rather than tumorpromoting. However, as the potential cancer cells accumulate more mutations, there will be selective pressure on them to lose the expression of tumor-suppressor genes. The effect on NF-kB will be a reversal of its role. The mechanisms for keeping the tumorigenic functions of NF-kB in check will no longer be in place. Rather its tumorpromoting activity will be unleashed, with NF-kB subunits becoming free to induce the expression of a wide range of genes that can promote the development of malignant and metastatic tumors. It should be acknowledged that, similar to the requirements for different tumor suppressors and oncogenes, this two-step mechanism for NF-kB function in cancer development is probably both tumorand cell-type specific.

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This hypothesis has implications for future NF-kBbased therapy. It could be important to make sure that the right form of NF-kB is present in the tumor for the treatment to be effective. Indeed, if reagents can be developed, such as phosphospecific antibodies that can distinguish between the different forms of RelA, it will be important to investigate whether there is any correlation between the functional status of NF-kB and which tumors do or do not respond to both conventional and new forms of cancer therapy. Will drugs that inhibit NF-kB actually cause cancer? At this time, it seems improbable that NF-kB is a sufficiently potent tumor suppressor, whereby its inhibition would result in the formation of cancer cells in humans. It is possible, however, that such drugs might stimulate the growth of tumors still undetected but present at early stages of development. Therefore, although inhibiting the activity of NF-kB represents a potentially exciting new therapy, it should be remembered that NF-kB performs functions that we might not want to inhibit and thus appropriate caution should be taken. Acknowledgements N.D.P. is the recipient of a Royal Society University Research Fellowship. Many thanks to Sonia Rocha and Kirsteen Campbell for their critical reading of this manuscript and helpful suggestions.

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NF-kB inhibitor (IkBm) represses p53-dependent apoptosis in acute lymphoblastic leukemia cells: interaction of IkBm and p53. Oncogene 22, 8137 – 8144 50 Wu, B.Y. et al. (1997) Distinct domains of IkB-a inhibit human immunodeficiency virus type 1 replication through NF-kB and Rev. J. Virol. 71, 3161– 3167

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