Evidence that TNF-TNFR1-TRADD-TRAF2-RIP-TAK1-IKK pathway mediates constitutive NF-κB activation and proliferation in human head and neck squamous cell carcinoma

Constitutively activated nuclear factor-kappa B (NF-kappa B) has been associated with a variety of aggressive tumor types, including head and neck squamous cell carcinoma (HNSCC); however, the mechanism of its activation is not fully understood. Therefore, we investigated the molecular pathway that mediates constitutive activation of NF-kappa B in a series of HNSCC cell lines. We confirmed that NF-kappa B was constitutively active in all HNSCC cell lines (FaDu, LICR-LON-HN5 and SCC4) examined as indicated by DNA binding, immunocytochemical localization of p65, by NF-kappa B-dependent reporter gene expression and its inhibition by dominant-negative (DN)-inhibitory subunit of NF-kappa B (I kappa B alpha), the natural inhibitor of NF-kappa B. Constitutive NF-kappa B activation in HNSCC was found to be due to constitutive activation of IjBa kinase (IKK); and this correlated with constitutive expression of phosphorylated forms of I kappa B alpha and p65 proteins. All HNSCC showed the expression of p50, p52, p100 and receptor-interacting protein; all linked with NF-kappa B activation. The expression of constitutively active NF-kappa B in HNSCC is mediated through the tumor necrosis factor (TNF) signaling pathway, as NF-kappa B reporter activity was inhibited by DN-TNF receptor-associated death domain ( TRADD), DN-TNF receptor-associated factor ( TRAF) 2, DN-receptor-interacting protein ( RIP), DN-transforming growth factor-beta-activated kinase 1 (TAK1), DN-kappa-Ras, DN-AKT and DN-IKK but not by DN-TRAF5 or DN-TRAF6. Constitutive NF-kappa B activation was also associated with the autocrine expression of TNF, TNF receptors and receptor-activator of NF-kappa B and its ligand in HNSCC cells but not interleukin (IL)-1 beta. All HNSCC cell lines expressed IL-6, a NF-kappa B-regulated gene product. Furthermore, treatment of HNSCC cells with anti-TNF antibody downregulated constitutively active NF-kappa B, and this was associated with inhibition of IL-6 expression and cell proliferation. Our results clearly demonstrate that constitutive activation of NF-kappa B is mediated through the TRADD-TRAF2-RIP-TAK1-IKK pathway, making TNF a novel target in the treatment of head and neck cancer.