Expression of a dominant-negative mutant inhibitor-kappaBalpha of nuclear factor-kappaB in human head and neck squamous cell carcinoma inhibits survival, proinflammatory cytokine expression, and tumor growth in vivo.
We demonstrated recently that constitutive expression of proinflammatory cytokines interleukin (IL)-1alpha, IL-6, IL-8, and granulocyte-macrophage colony-stimulating factor in head and neck squamous cell carcinoma is correlated with activation of transcription factor nuclear factor (NF)-kappaB/Rel A (p50/p65), which binds the promoter region within each of the genes encoding this repertoire of cytokines. NF-kappaB can be activated after signal-dependent phosphorylation and degradation of inhibitor-kappaBalpha and has been reported to promote cell survival and growth. In the present study, we expressed a phosphorylation site mutant of inhibitor-kappaBalpha (IkappaBalphaM) in head and neck squamous cell carcinoma lines UM-SCC-9, -11B, and -38 to determine the effect of inhibition of NF-kappaB on cytokine expression, cell survival in vitro, and growth in vivo. After transfection with IKBalphaM, only a few UM-SCC-9 clones were obtained that stably expressed the mutant IkappaB, suggesting that expression of a mutant IkappaBalpha may affect survival of the transfected UM-SCC cell lines. After cotransfection of IkappaBalphaM with a Lac-Z reporter, we found that the number of surviving beta-galactosidase-positive cells in the three cell lines was reduced by 70-90% when compared with controls transfected with vector lacking the insert. In UM-SCC-9 cells that stably expressed IkappaBalphaM, inhibition of constitutive and tumor necrosis factor-a induced NF-kappaB activation, and production of all four cytokines was observed. Although UM-SCC-9 IkappaBalphaM-transfected cells proliferated at the same rate as vector-transfected cells in vitro, a significant reduction in growth of tumor xenografts was observed in SCID mice in vivo. The decreased growth of UM-SCC-9 IkappaBalphaM-transfected tumor cells accompanied decreased immunohistochemical detection of the activated form of NF-kappaB in situ. These results provide evidence that NF-KB and IkappaBalpha play an important role in survival, constitutive and inducible expression of proinflammatory cytokines, and growth of squamous cell carcinoma. NF-kappaB could serve as a potential target for therapeutic intervention against cytokine and other immediate-early gene responses that contribute to the survival, growth, and pathogenesis of these cancers.
Tumor Biology Section, Head and Neck Surgery Branch, National Institute on Deafness and Other Communication Disorders, NIH, Bethesda, Maryland 20892, USA.
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Duffey
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Chen
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Dong
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Ondrey
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Wolf
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Brown
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Siebenlist
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Van Waes
D000818:Animals; D002294:Carcinoma, Squamous Cell / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D016207:Cytokines / Q000096:biosynthesis* / Q000235:genetics; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000502:physiology; D015972:Gene Expression Regulation, Neoplastic; D005799:Genes, Dominant; D017930:Genes, Reporter; D006258:Head and Neck Neoplasms / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D006801:Humans; D020672:I-kappa B Proteins; D007763:Lac Operon; D051379:Mice; D016513:Mice, SCID; D000072000:NF-KappaB Inhibitor alpha; D016328:NF-kappa B / Q000502:physiology; D009363:Neoplasm Proteins / Q000235:genetics* / Q000502:physiology; D009368:Neoplasm Transplantation; D010766:Phosphorylation; D017354:Point Mutation; D011401:Promoter Regions, Genetic; D011499:Protein Processing, Post-Translational; D011993:Recombinant Fusion Proteins / Q000502:physiology; D014158:Transcription, Genetic; D014162:Transfection; D014407:Tumor Cells, Cultured / Q000378:metabolism; D014409:Tumor Necrosis Factor-alpha / Q000502:physiology
D016207:Cytokines; D004268:DNA-Binding Proteins; D020672:I-kappa B Proteins; D016328:NF-kappa B; C000606204:NFKBIA protein, human; D009363:Neoplasm Proteins; C000606205:Nfkbia protein, mouse; D011993:Recombinant Fusion Proteins; D014409:Tumor Necrosis Factor-alpha; D000072000:NF-KappaB Inhibitor alpha