Clinical Cancer Research 13, 4956-4959, September 1, 2007. doi: 10.1158/1078-0432.CCR-07-1287
© 2007 American Association for Cancer Research
Inhibitor-
B Kinase in Tumor Promotion and Suppression During Progression of Squamous Cell Carcinoma
Carter Van Waes1,
Ming Yu1,
Liesl Nottingham1 and
Michael Karin2
Authors' Affiliations: 1 Head and Neck Surgery Branch, National Institute on Deafness and Other Communication Disorders, NIH, Bethesda, Maryland and 2 Laboratory of Gene Regulation and Signal Transduction, Department of Pharmacology and Cancer Center, School of Medicine, University of California, San Diego, La Jolla, California
Requests for reprints: Carter Van Waes, Head and Neck Surgery Branch, National Institute on Deafness and Other Communication Disorders, NIH, Room 4-2732, CRC Building, 10 Center Drive, Bethesda, MD 20892. Phone: 301-402-4216; Fax: 301-402-1140; E-mail: vanwaesc{at}nidcd.nih.gov.
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Novel Role of Inhibitor- B Kinase- as a Tumor Suppressor Gene in Squamous Cell Carcinoma
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Previous studies have provided evidence that activation of inhibitor-B kinases (IKK) leads to nuclear translocation and activation of nuclear factor-B (NF-B), a transcription factor that can promote cell survival and malignant phenotypic changes important in development and progression of squamous cell carcinomas (SCC) and other cancers (Fig. 1
; refs. 1, 2). Recent findings in this issue of Clinical Cancer Research (3) and elsewhere (4) provide evidence for an important new role for one of the IKK subunits, IKK, as a tumor suppressor in SCC. In this issue, Maeda et al. (3) report that loss or decreased nuclear expression of IKK occurs in 20 of 64 (
33%) of oral SCCs and is significantly associated with decreased histologic differentiation and prognosis. Using HaCaT immortalized keratinocytes, they show that decreased expression of IKK, involucrin, and keratins are functionally linked during dedifferentiation, which occurs with calcium deprivation. Based on the observation that the gene encoding IKK is located near 10q24, a locus altered in a subset of SCC, they sought evidence for genetic or epigenetic alterations of the IKK gene. Although in oral SCC allelic/biallelic loss of the gene was found in only a few cases, they showed that microsatellite instability and epigenetic inactivation by promoter hypermethylation occurs with high frequency in those SCCs that exhibit loss of nuclear IKK.
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Fig. 1. IKK in cancer pathogenesis. A, IKK and NF-B activation in cancer development and progression has been linked to activation of cytokine and growth factor receptors and exposure to bacteria, viral products, chemical promoters, carcinogens, and reactive oxygen species (ROS), which cause repeated DNA damage. NF-B activation of cytokine and growth factor receptors and also occurs in response to activation of tumor-associated macrophages induced by necrotic cell death of cancer cells, cytotoxic chemotherapy, and ionizing radiation. B, canonical NF-B pathway activation occurs in response to these stimuli through aberrant cytokine production, and integrin, growth factor, and cytokine receptor activation [e.g., tumor necrosis factor receptor (TNFR), interleukin-1R (IL-1R), integrin 6ß4, and epidermal growth factor receptor (EGFR)]; expression of viral proteins (e.g., EBV LMP 1); or aberrant activation by intermediate kinases (e.g., phosphatidylinositol 3-kinase, casein kinase 2, and AKT). These signals converge on the tripartite IKK, IKKß, and IKK complex, whose IKKß subunit is important for phosphorylation of IBs, marking them for ubiquitylation by the E3 ligase ßTrCP (SCFIB), resulting in proteasomal degradation. This is followed by nuclear entry of NF-B1/RELA (p50/p65) or p50/cREL dimers, which bind promoters of genes regulating proliferation, apoptosis, migration, inflammation, angiogenesis, and innate immunity. C, alternative pathway. The alternative pathway may be activated by several tumor necrosis factor and tumor necrosis factor receptor family members via NF-B–inducing kinase and IKK/IKK homodimers, which mark NF-B2/p100/RELB for processing into p52/RELB heterodimers. The p52/RELB heterodimer then translocates into the nucleus to bind promoters of genes that are important for the malignant phenotype in some cancers, B-cell development, and secondary lymphoid organ morphogenesis. D, IKK as a nuclear cofactor. IKK may also translocate to the nucleus, associate with chromatin, and promote coactivation of NF-B and retinoid receptor target genes while inhibiting transcription of other genes. E, IKK in keratinocyte differentiation and tumor suppression. IKK may induce keratinocyte differentiation in response to yet unidentified signals and thereby act as a tumor suppressor in SCC. In this case, IKK is proposed to act in the nucleus with other, yet to be identified, transcription factors. Loss by epigenetic inactivation or allelic loss is associated with loss of differentiation, accelerated proliferation, and decreased prognosis.
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These results are consistent with the recent demonstration by Liu et al. (4) that marked reduction of expression and mutation of the IKK locus is associated with poorly differentiated human SCC of the skin and that transgenic mice overexpressing normal IKK in the epidermis develop fewer SCCs and metastases when subjected to a standard two-stage 7,12-dimethylbenz(a)anthraacene/12-O-tetradecanoylphorbol-13-acetate tumor induction protocol. In their murine model, expression of IKK promoted terminal differentiation and reduced proliferation, angiogenesis, and metastasis. Furthermore, SCC that appeared in IKK+/– mice showed loss of heterozygosity, a clear indication of a tumor suppressor function.3 Down-regulation of nuclear IKK has also been observed in association with decreased differentiation and invasive and metastatic progression of SCC of the skin, lung, and head and neck.4 These data emerging from independent laboratories indicate that nuclear IKK is a major SCC tumor suppressor in mice and men. Such loss of nuclear IKK is associated with decreased differentiation and epithelial to mesenchymal transition, a phenotype linked with SCC progression, invasion, and worse prognosis.
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Cytoplasmic Functions of IKK in Canonical and Alternative NF-B Pathway Activation
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Early studies revealed that IKK together with IKKß and IKK forms a cytoplasmic complex that integrates numerous signaling pathways important in activation of NF-B transcription factors (Fig. 1A and B), which together have been implicated in protective responses to injury, promotion of inflammation, and oncogenesis (1, 2). This IKK-NF-B pathway has become known as the canonical pathway (Fig. 1B). This tripartite IKK complex has been shown to phosphorylate via IKKß Ser32 and Ser36 of inhibitor of B (IB), leading to its polyubiquitylation and proteasomal degradation, releasing NF-B for nuclear translocation and binding to target gene promoters. This IKK complex was also shown to phosphorylate Ser536 of the RELA subunit of the NF-B1/RELA (p50/p65) heterodimer (5), important for transactivation of genes by bound NF-B, as well as for termination of the induction response (6). Genes controlled by NF-B execute a diverse program of cellular functions, including survival, proliferation, inflammation, angiogenesis, and innate and adaptive immunity, many of the processes altered during development of cancer (7).
Several studies have provided evidence that the canonical IKK and NF-B pathway plays an important role in the pathogenesis of SCC. One of our laboratories discovered that NF-B1/RELA (p50/p65), the NF-B heterodimer activated by the canonical pathway, is aberrantly activated in murine and human SCC lines derived from advanced tumors (8, 9). Zhang et al. (10) confirmed that increased nuclear activation of RELA/p65 frequently occurs in development of squamous dysplasias and carcinomas and is associated with malignant progression and decreased prognosis. Increased expression of NF-B–related gene signatures is observed in SCC lines and tumor specimens in association with tumor progression, metastasis, epithelial-mesenchymal transition, and decreased prognosis (11–13). Consistent with a functional role for the IKK and NF-B canonical pathway in aberrant gene expression and the malignant phenotype, we showed that forced expression of IB mutated at the IKK phospho-acceptor sites inhibits altered gene expression together with cell survival, proliferation, angiogenesis, tumorigenesis, and therapeutic resistance by SCC (12, 14–16). Tamatani et al. (17) provided further evidence that IKK complex immunoprecipitated by anti-IKK antibody contributes to phosphorylation of IB activation in head and neck SCC. Consistent with a role of IKK in NF-B activation, we found that expression of either IKK- or IKKß K44A–kinase deficient mutants can attenuate NF-B reporter gene activation in SCC (18).
IKK has been shown to have critical function distinct from its role as part of the canonical IKK pathway. Besides the trimeric IKK complex involved in the canonical pathway, a cytosolic homodimeric IKK complex acts as the lynchpin in an alternative pathway that activates NF-B2/RELB dimers important in development of B lymphocytes and secondary lymphoid organs (Fig. 1C; refs. 1, 2, 19). This pathway has been shown to mediate processing of NF-B2 p100 to p52, which has also been shown to be deregulated and activate NF-B target genes in some cancers (19). Although we have detected nuclear localization of NF-B2 in head and neck SCC lines (8),5 its functional role has not yet been defined, and these lines may represent the subset of well-differentiated SCC that have not lost nuclear IKK (3, 4).
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IKK as a Nuclear Factor
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IKK has been shown recently to function in the nucleus both as transcriptional regulator and protein kinase (Fig. 1D). Epidermal growth factor has been shown to induce the recruitment of IKK to the c-fos promoter to regulate promoter-specific histone H3 Ser(10) phosphorylation. This occurs in a manner that is independent of p65/RELA, but together with p65/RELA, IKK up-regulates c-fos expression (20). Park et al. (21) detected similar bacterial lipopolysaccharide-induced IKK binding and chromatin modification in association with RELA in promoting expression of a variety of NF-B modulated genes in macrophage lines. Although this mechanism has not been shown in keratinocytes or SCC, epidermal growth factor ligand and receptor overexpression and lipopolysaccharide exposure are common in head and neck SCC. It should be noted that the study by Maeda et al. in this issue did not examine whether the loss of nuclear IKK affects nuclear localization of NF-B or transactivation of its target genes, information that would be helpful in determining if the alterations associated with IKK loss may affect and be mediated via NF-B–dependent or NF-B–independent mechanisms.
Now, nuclear IKK has also been shown to function as a tumor suppressor gene (Fig. 1E; refs. 3, 4). Perhaps most critical and relevant to its role as a tumor suppressor in SCC is the observation that IKK controls the proliferation and differentiation of epidermal keratinocytes (22, 23). IKK-deficient mice show dysregulated hyperproliferation and defective differentiation of the epidermis, as well as increased NF-B activity due to loss of an inhibitor function (23). This augmentation of NF-B activation was shown to occur in keratinocytes stimulated with TNF, a cytokine found previously to play an important role in promoting SCC formation in mice (24). The increased activation of NF-B is most likely due to formation of IKK complexes containing only IKKß as the kinase subunit, with IKKß being more active than IKK (23). The control of keratinocyte proliferation and differentiation, however, is not dependent on IKK protein kinase activity but requires its nuclear translocation (25). Thus, nuclear IKK is critical for keratinocyte differentiation, and its loss promotes proliferation while preventing the differentiation of the very cell type that causes SCC.
One of the major questions that pertain to the function of nuclear IKK in keratinocytes and its loss in SCC is how it controls cell differentiation and proliferation. It was shown that reintroduction of IKK into IKK-deficient keratinocytes induces cell cycle arrest (23), but the mechanism by which IKK controls cell cycle progression is still being unraveled. IKK seems to promote expression of a keratinocyte differentiation-inducing factor, independent of its role in activation of NF-B (23, 26). Keratinocytes with targeted deletion of IKK were also reported to be defective in calcium-induced differentiation and barrier function, and this was shown to be mediated via effects on retinoic acid receptor gene expression (26). These functions of IKK in normal squamous epithelia seem most consistent with the associated loss of IKK expression and differentiation observed in the subset of cutaneous and head and neck SCC described by Maeda et al. (3) and Liu et al. (4). These results suggest that in addition to its cytoplasmic role in modulation of NF-B activation, nuclear IKK may be a key mediator of another mechanism or pathway that represses more pathogenic phenotypic changes along the spectrum of SCC.
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Is IKK Part of a Pathway Suppressing Malignant Progression?
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At this writing, this mechanism is unknown, but based on previous reports, there are several intriguing candidates. One of our laboratories reported previously that stepwise progression in a murine SCC model is associated with increased activation of NF-B and target genes (9, 11), including cytokines, such as Gro-1, which promote inflammation, angiogenesis, and increased tumorigenesis (12, 27). In a subsequent study, we found that NF-B activation and Gro-1 expression could be repressed by transforming growth factor ß1 (TGFß1) via NF-B–dependent and NF-B–independent mechanisms in early-stage SCC, but not in the more advanced metastatic SCC (28). This occurred without apparent loss of TGFß receptor, suggesting that dysregulation of other intermediates downstream of the receptor could be involved in disruption of TGFß signaling and malignant progression in SCC. Li et al. (29) report that dysregulation of the TGFß signaling pathway at the level of TGFß receptor 2 and SMADs is common in SCC and, as with loss of IKK, is associated with increased inflammation, angiogenesis, proliferation, and increased skin-derived SCC formation. Additionally, Kobielak et al. (30) have reported that targeted knockout of -catenin, another common alteration in SCC, results in increased NF-B activation and increased SCC carcinogenesis of the skin. Interestingly, dysregulation of TGFß signaling has been linked to cadherin/catenin expression in squamous epithelia. It will be interesting if malignant progression is linked to NF-B–dependent and NF-B–independent pathways altered by dysregulation of IKK or other components of the TGFß pathway. Thus, loss of IKK can result in defective differentiation and aberrant proliferation as well as up-regulation of NF-B signaling due to loss of several inhibitory loops. Together, IKK loss could result in increased cell survival and angiogenesis, loss of differentiation, epithelial to mesenchymal transition, and malignant progression. Further studies of the mechanisms regulating IKK nuclear localization, loss, and function will be important to understanding the potential for therapeutic targeting of these mechanisms.
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Therapeutic Implications
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There are potential therapeutic implications suggested by the studies of Maeda et al. The current results indicating hypermethylation may be a common mechanism for inactivation of IKK in head and neck SCC raise the potential that IKK expression and at least part of its tumor suppressor function could be restored by treatment with demethylating agents, which have been shown to reactivate other hypermethylated tumor suppressor genes. Such an approach could be tested alone, and together with TGFß, in the subset of SCC found to be deficient for IKK expression.
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Footnotes
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Grant support: National Institute on Deafness and Other Communication Disorders intramural research project Z01-DC-00016.
Commentary on Maeda et al., p. 5041
3 Y. Hu, personal communication. 
4 A. Costanzo and M. Karin, unpublished data.
5 L. Nottingham, unpublished results.
Received 5/24/07;
revised 5/31/07;
accepted 6/ 1/07.
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References
|
|---|
- Luo JL, Kamata H, Karin M. IKK/NF-B signaling: balancing life and death—a new approach to cancer therapy. J Clin Invest 2005;115:2625–32.[CrossRef][Medline]
- Van Waes C. Nuclear factor-B in development, prevention, and therapy of cancer. Clin Cancer Res 2007;13:1076–82.[Abstract/Free Full Text]
- Maeda G, Chiba T, Kawashiri S, Satoh T, Imai K. Epigenetic inactivation of IB kinase- in oral carcinomas and tumor progression. Clin Cancer Res 2007;13:5041–7.[Abstract/Free Full Text]
- Liu B, Park E, Zhu F, et al. A critical role for IB kinase in the development of human and mouse squamous cell carcinomas. Proc Natl Acad Sci U S A 2006;103:17202–7.[Abstract/Free Full Text]
- Sakurai H, Suzuki S, Kawasaki N, et al. Tumor necrosis factor--induced IKK phosphorylation of NF-B p65 on serine 536 is mediated through the TRAF2, TRAF5, and TAK1 signaling pathway. J Biol Chem 2003;278:36916–23.[Abstract/Free Full Text]
- Lawrence T, Bebien M, Liu GY, Nizet V, Karin M. IKK limits macrophage NF-B activation and contributes to the resolution of inflammation. Nature 2005;434:1138–43.[CrossRef][Medline]
- Karin M. Nuclear factor-B in cancer development and progression. Nature 2006;441:431–6.[CrossRef][Medline]
- Ondrey FG, Dong G, Sunwoo J, et al. Constitutive activation of transcription factors NF-()B, AP-1, and NF-IL6 in human head and neck squamous cell carcinoma cell lines that express pro-inflammatory and pro-angiogenic cytokines. Mol Carcinog 1999;26:119–29.[CrossRef][Medline]
- Dong G, Chen Z, Kato T, Van Waes C. The host environment promotes the constitutive activation of nuclear factor-B and proinflammatory cytokine expression during metastatic tumor progression of murine squamous cell carcinoma. Cancer Res 1999;59:3495–504.[Abstract/Free Full Text]
- Zhang PL, Pellitteri PK, Law A, et al. Overexpression of phosphorylated nuclear factor-B in tonsillar squamous cell carcinoma and high-grade dysplasia is associated with poor prognosis. Mod Pathol 2005;18:924–32.[CrossRef][Medline]
- Dong G, Loukinova E, Chen Z, et al. Molecular profiling of transformed and metastatic murine squamous carcinoma cells by differential display and cDNA microarray reveals altered expression of multiple genes related to growth, apoptosis, angiogenesis, and the NF-B signal pathway. Cancer Res 2001;61:4797–808.[Abstract/Free Full Text]
- Loercher A, Lee TL, Ricker JL, et al. Nuclear factor-B is an important modulator of the altered gene expression profile and malignant phenotype in squamous cell carcinoma. Cancer Res 2004;64:6511–23. Erratum in: Cancer Res 2004;64:8130–2.[Abstract/Free Full Text]
- Chung CH, Parker JS, Ely K, et al. Gene Expression profiles identify epithelial-to-mesenchymal transition and activation of nuclear factor-B signaling as characteristics of a high-risk head and neck squamous cell carcinoma. Cancer Res 2006;66:8210–8.[Abstract/Free Full Text]
- Duffey DC, Chen Z, Dong G, et al. Expression of a dominant-negative mutant inhibitor-B of nuclear factor-B in human head and neck squamous cell carcinoma inhibits survival, proinflammatory cytokine expression, and tumor growth in vivo. Cancer Res 1999;59:3468–74.[Abstract/Free Full Text]
- Duffey DC, Crowl-Bancroft CV, Chen Z, et al. Inhibition of transcription factor nuclear factor-B by a mutant inhibitor-B attenuates resistance of human head and neck squamous cell carcinoma to TNF- caspase-mediated cell death. Br J Cancer 2000;83:1367–74.[CrossRef][Medline]
- Kato T, Duffey DC, Ondrey FG, et al. Cisplatin and radiation sensitivity in human head and neck squamous carcinomas are independently modulated by glutathione and transcription factor NF-B. Head Neck 2000;22:748–59.[CrossRef][Medline]
- Tamatani T, Azuma M, Aota K, Yamashita T, Bando T, Sato M. Enhanced IB kinase activity is responsible for the augmented activity of NF-B in human head and neck carcinoma cells. Cancer Lett 2001;171:165–72.[CrossRef][Medline]
- Yu M, Yeh J, Van Waes C. Protein kinase casein kinase 2 mediates inhibitor-B kinase and aberrant nuclear factor-B activation by serum factor(s) in head and neck squamous carcinoma cells. Cancer Res 2006;66:6722–31.[Abstract/Free Full Text]
- Xiao G, Rabson AB, Young W, Qing G, Qu Z. Alternative pathways of NF-B activation: a double-edged sword in health and disease. Cytokine Growth Factor Rev 2006;17:281–93.[CrossRef][Medline]
- Anest V, Cogswell PC, Baldwin AS, Jr. IB kinase and p65/RelA contribute to optimal epidermal growth factor-induced c-fos gene expression independent of IB degradation. J Biol Chem 2004;279:31183–9.[Abstract/Free Full Text]
- Park GY, Wang X, Hu N, Pedchenko TV, Blackwell TS, Christman JW. NIK is involved in nucleosomal regulation by enhancing histone H3 phosphorylation by IKK. J Biol Chem 2006;281:18684–90.[Abstract/Free Full Text]
- Hu Y, Baud V, Delhase M, et al. Abnormal morphogenesis but intact IKK activation in mice lacking the IKK subunit of IB kinase. Science 1999;284:316–20.[Abstract/Free Full Text]
- Hu Y, Baud V, Oga T, Kim KI, Yoshida K, Karin M. IKK controls formation of the epidermis independently of NF-B. Nature 2001;410:710–4.[CrossRef][Medline]
- Moore RJ, Owens DM, Stamp G, et al. Mice deficient in tumor necrosis factor- are resistant to skin carcinogenesis. Nat Med 1999;5:828–31.[CrossRef][Medline]
- Sil AK, Maeda S, Sano Y, Roop DR, Karin M. IB kinase- acts in the epidermis to control skeletal and craniofacial morphogenesis. Nature 2004;428:660–4.[CrossRef][Medline]
- Gareus R, Huth M, Breiden B, et al. Normal epidermal differentiation but impaired skin-barrier formation upon keratinocyte-restricted IKK1 ablation. Nat Cell Biol 2007;9:461–9.[CrossRef][Medline]
- Loukinova E, Dong G, Enamorado-Ayalya I, et al. Growth regulated oncogene- expression by murine squamous cell carcinoma promotes tumor growth, metastasis, leukocyte infiltration, and angiogenesis by a host CXC receptor-2 dependent mechanism. Oncogene 2000;19:3477–86.[CrossRef][Medline]
- Loukinova E, Chen Z, Van Waes C, Dong G. Expression of proangiogenic chemokine Gro 1 in low and high metastatic variants of Pam murine squamous cell carcinoma is differentially regulated by IL-1, EGF, and TGF-ß1 through NF-B dependent and independent mechanisms. Int J Cancer 2001;94:637–44.[CrossRef][Medline]
- Li AG, Lu SL, Han G, Hoot KE, Wang XJ. Role of TGFß in skin inflammation and carcinogenesis. Mol Carcinog 2006;45:389–96.[CrossRef][Medline]
- Kobielak A, Fuchs E. Links between -catenin, NF-B, and squamous cell carcinoma in skin. Proc Natl Acad Sci U S A 2006;103:2322–7.[Abstract/Free Full Text]
Key Article
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Epigenetic Inactivation of IB Kinase- in Oral Carcinomas and Tumor Progression
- Genta Maeda, Tadashige Chiba, Shuichi Kawashiri, Tazuko Satoh, and Kazushi Imai
Clin. Cancer Res. 2007 13: 5041-5047.
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Novel Role of Inhibitor-{kappa}B...
Cytoplasmic Functions of IKK...
