Inhibition of transcription factor nuclear factor-kappa B by a mutant inhibitor-kappa B alpha attenuates resistance of human head and neck squamous cell carcinoma to TNF-alpha caspase-mediated cell death

Tumour necrosis factor-alpha (TNF-(alpha) is a cytokine that can induce cel l death of different cancers via a cellular cascade of proteases, the caspa ses. However, TNF-alpha has been detected in tumour and serum of patients w ith head and neck squamous cell carcinoma (HNSCC), and tumour cell lines de rived from this environment often exhibit resistance to TNF-alpha -induced cell death. Cell death mediated by TNF-alpha and caspases may be inhibited by cytoprotective genes regulated by transcription factor nuclear factor-ka ppaB (NF-kappaB). We recently showed that NF kappaB is constitutively activ ated in HNSCC, and that inhibition of NF-kappaB by expression of a nondegra dable mutant inhibitor of NF-kappaB, I kappa -B alphaM, markedly decreased survival and growth of HNSCC cells in vivo. In the present study, we examin ed the TNF-alpha sensitivity and response of HNSCC with constitutively acti ve NF-kappaB, and of HNSCC cells in which NF-kappaB is inhibited by stable expression of a dominant negative mutant inhibitor, I kappaB alphaM. Human lines UM-SCC-9, 11B and 38, previously shown to exhibit constitutive activa tion of NF-kappaB, were found to be highly resistant to growth inhibition b y up to 10(4) U/ml of TNF-alpha in 5 day MTT assay. These TNF-a resistant H NSCC lines expressed TNF receptor I, and exhibited constitutive and TNF-a-i nducible activation of NF-kappaB as demonstrated by nuclear localization of NF-kappaB p65 by immunohistochemistry. UM-SCC-9 I11 cells which stably exp ressed an inhibitor of NF-kappaB, I kappaB alpham, were susceptible to TNF- alpha -induced growth inhibition. DNA cell cycle analysis revealed that TNF -alpha induced growth inhibition was associated with accumulation of cells with sub-G0/G1 DNA content. Cell death was demonstrated by trypan blue stai ning, and was blocked by caspase Inhibitor. We conclude that HNSCC that exh ibit constitutive and TNF-alpha -inducible activation of transcription fact or NF-kappaB are resistant to TNF-alpha, and that inhibition of NF-kappaB s ensitizes HNSCC to TNF-alpha caspase-mediated cytotoxicity. The demonstrati on of the role of activation of NF-kappaB in resistance of HNSCC to TNF-alp ha may be helpful in the identification of potential targets for pharmacolo gical, molecular and immune therapy of HNSCC. (C) 2000 Cancer Research Camp aign.