Evidence that TNF-TNFR1-TRADD-TRAF2-RIP-TAK1-IKK pathway mediates constitutive NF-kappaB activation and proliferation in human head and neck squamous cell carcinoma.
Constitutively activated nuclear factor-kappaB (NF-kappaB) 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-kappaB in a series of HNSCC cell lines. We confirmed that NF-kappaB 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-kappaB-dependent reporter gene expression and its inhibition by dominant-negative (DN)-inhibitory subunit of NF-kappaB (IkappaBalpha), the natural inhibitor of NF-kappaB. Constitutive NF-kappaB activation in HNSCC was found to be due to constitutive activation of IkappaBalpha kinase (IKK); and this correlated with constitutive expression of phosphorylated forms of IkappaBalpha and p65 proteins. All HNSCC showed the expression of p50, p52, p100 and receptor-interacting protein; all linked with NF-kappaB activation. The expression of constitutively active NF-kappaB in HNSCC is mediated through the tumor necrosis factor (TNF) signaling pathway, as NF-kappaB 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-kappaB activation was also associated with the autocrine expression of TNF, TNF receptors and receptor-activator of NF-kappaB and its ligand in HNSCC cells but not interleukin (IL)-1beta. All HNSCC cell lines expressed IL-6, a NF-kappaB-regulated gene product. Furthermore, treatment of HNSCC cells with anti-TNF antibody downregulated constitutively active NF-kappaB, and this was associated with inhibition of IL-6 expression and cell proliferation. Our results clearly demonstrate that constitutive activation of NF-kappaB is mediated through the TRADD-TRAF2-RIP-TAK1-IKK pathway, making TNF a novel target in the treatment of head and neck cancer.
Jackson-Bernitsas
D G
DG
Department of Experimental Therapeutics, The University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA.
Ichikawa
H
H
Takada
Y
Y
Myers
J N
JN
Lin
X L
XL
Darnay
B G
BG
Chaturvedi
M M
MM
Aggarwal
B B
BB
D002294:Carcinoma, Squamous Cell / Q000378:metabolism*; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D006258:Head and Neck Neoplasms / Q000378:metabolism*; D006801:Humans; D051550:I-kappa B Kinase / Q000378:metabolism; D016328:NF-kappa B / Q000096:biosynthesis*; D053422:Receptor-Interacting Protein Serine-Threonine Kinases / Q000378:metabolism; D047888:Receptors, Tumor Necrosis Factor, Type I / Q000378:metabolism; D015398:Signal Transduction; D053199:TNF Receptor-Associated Death Domain Protein / Q000378:metabolism; D047992:TNF Receptor-Associated Factor 2 / Q000378:metabolism; D051996:Transcription Factor RelA; D015291:Transforming Growth Factors / Q000378:metabolism
D016328:NF-kappa B; D047888:Receptors, Tumor Necrosis Factor, Type I; D053199:TNF Receptor-Associated Death Domain Protein; D047992:TNF Receptor-Associated Factor 2; D051996:Transcription Factor RelA; D015291:Transforming Growth Factors; D053422:Receptor-Interacting Protein Serine-Threonine Kinases; D051550:I-kappa B Kinase