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.
Duffey
D C
DC
Tumor Biology Section, Head and Neck Surgery Branch, National Institute on Deafness and Other Communication Disorders, NIH, Bethesda, Maryland 20892, USA.
Chen
Z
Z
Dong
G
G
Ondrey
F G
FG
Wolf
J S
JS
Brown
K
K
Siebenlist
U
U
Van Waes
C
C
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