Expression of a Dominant-Negative Mutant Inhibitor-kBa of Nuclear Factor-kB 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)-1a, IL-6, IL-8, and granulocytemacrophage colony-stimulating factor in head and neck squamous cell carcinoma is correlated with activation of transcription factor nuclear factor (NF)-kB/Rel A (p50/p65), which binds the promoter region within each of the genes encoding this repertoire of cytokines. NF-kB can be activated after signal-dependent phosphorylation and degradation of inhibitor-kBa and has been reported to promote cell survival and growth. In the present study, we expressed a phosphorylation site mutant of inhibitor-kBa (IkBaM) in head and neck squamous cell carcinoma lines UM-SCC-9, -11B, and -38 to determine the effect of inhibition of NF-kB on cytokine expression, cell survival in vitro, and growth in vivo. After transfection with IkBaM, only a few UM-SCC-9 clones were obtained that stably expressed the mutant IkB, suggesting that expression of a mutant IkBa may affect survival of the transfected UM-SCC cell lines. After cotransfection of IkBaM with a Lac-Z reporter, we found that the number of surviving b-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 IkBaM, inhibition of constitutive and tumor necrosis factor-a induced NF-kB activation, and production of all four cytokines was observed. Although UM-SCC-9 IkBaM-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 IkBaM-transfected tumor cells accompanied decreased immunohistochemical detection of the activated form of NF-kB in situ. These results provide evidence that NF-kB and IkBa play an important role in survival, constitutive and inducible expression of proinflammatory cytokines, and growth of squamous cell carcinoma. NF-kB 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.