1. ¹Department of Experimental Therapeutics, The University of Texas MD Anderson Cancer Center, Houston, TX, USA
2. ²Department of Head and Neck Surgery, The University of Texas MD Anderson Cancer Center, Houston, TX, USA
3. ³Department of Molecular and Cellular Oncology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA

Correspondence: Professor BB Aggarwal, Cytokine Research Laboratory, Department of Experimental Therapeutics, Unit 143, The University of Texas MD Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, TX 77030, USA. E-mail: aggarwal@mdanderson.org

⁴On leave from the Department of Zoology, University of Delhi, Delhi 110007, India

Received 2 May 2006; Revised 18 July 2006; Accepted 21 July 2006; Published online 4 September 2006.

Abstract

Constitutively activated nuclear factor-B (NF-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-B in a series of HNSCC cell lines. We confirmed that NF-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-B-dependent reporter gene expression and its inhibition by dominant-negative (DN)-inhibitory subunit of NF-B (IB), the natural inhibitor of NF-B. Constitutive NF-B activation in HNSCC was found to be due to constitutive activation of IB kinase (IKK); and this correlated with constitutive expression of phosphorylated forms of IB and p65 proteins. All HNSCC showed the expression of p50, p52, p100 and receptor-interacting protein; all linked with NF-B activation. The expression of constitutively active NF-B in HNSCC is mediated through the tumor necrosis factor (TNF) signaling pathway, as NF-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--activated kinase 1 (TAK1), DN--Ras, DN-AKT and DN-IKK but not by DN-TRAF5 or DN-TRAF6. Constitutive NF-B activation was also associated with the autocrine expression of TNF, TNF receptors and receptor-activator of NF-B and its ligand in HNSCC cells but not interleukin (IL)-1. All HNSCC cell lines expressed IL-6, a NF-B-regulated gene product. Furthermore, treatment of HNSCC cells with anti-TNF antibody downregulated constitutively active NF-B, and this was associated with inhibition of IL-6 expression and cell proliferation. Our results clearly demonstrate that constitutive activation of NF-B is mediated through the TRADD-TRAF2-RIP-TAK1-IKK pathway, making TNF a novel target in the treatment of head and neck cancer.