Abstract
Constitutively activated nuclear factor-κB (NF-κB) 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-κB in a series of HNSCC cell lines. We confirmed that NF-κB 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-κB-dependent reporter gene expression and its inhibition by dominant-negative (DN)-inhibitory subunit of NF-κB (IκBα), the natural inhibitor of NF-κB. Constitutive NF-κB activation in HNSCC was found to be due to constitutive activation of IκBα kinase (IKK); and this correlated with constitutive expression of phosphorylated forms of IκBα and p65 proteins. All HNSCC showed the expression of p50, p52, p100 and receptor-interacting protein; all linked with NF-κB activation. The expression of constitutively active NF-κB in HNSCC is mediated through the tumor necrosis factor (TNF) signaling pathway, as NF-κB 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-β-activated kinase 1 (TAK1), DN-κ-Ras, DN-AKT and DN-IKK but not by DN-TRAF5 or DN-TRAF6. Constitutive NF-κB activation was also associated with the autocrine expression of TNF, TNF receptors and receptor-activator of NF-κB and its ligand in HNSCC cells but not interleukin (IL)-1β. All HNSCC cell lines expressed IL-6, a NF-κB-regulated gene product. Furthermore, treatment of HNSCC cells with anti-TNF antibody downregulated constitutively active NF-κB, and this was associated with inhibition of IL-6 expression and cell proliferation. Our results clearly demonstrate that constitutive activation of NF-κB is mediated through the TRADD-TRAF2-RIP-TAK1-IKK pathway, making TNF a novel target in the treatment of head and neck cancer.
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Abbreviations
- ALLN:
-
N-acetyl-leu-leu-norleucinal
- IκB:
-
inhibitory subunit of NF-κB
- IKK:
-
IκBα kinase
- JNK:
-
c-Jun N terminal kinase
- MAPK:
-
mitogen-activated protein kinase
- MEKK:
-
mitogen-activated protein kinase/extracellular signal-regulated kinase kinase kinase
- NF-κB:
-
nuclear factor-κB
- PBS:
-
phosphate-buffered saline
- TRAF:
-
TNF receptor-associated factor
- RANK:
-
receptor activator of NF-κB
- RANKL:
-
receptor activator of NF-κB ligand
- TNF:
-
tumor necrosis factor
- TRADD:
-
TNF receptor-associated death domain
- RIP:
-
receptor-interacting protein
- TAK1:
-
transforming growth factor-β-activated kinase 1
- TAB1:
-
TAK1-binding protein
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Acknowledgements
We thank Ann Sutton for her critical review of this manuscript. Supported in part by the Clayton Foundation for Research (BBA), Department of Defense US Army Breast Cancer Research Program grant BC010610 (BBA), grant P01 CA91844 from the National Institutes of Health (NIH) on lung chemoprevention (BBA) and a P50 Head and Neck Specialized Programs of Research Excellence grant from the NIH (BBA).
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Jackson-Bernitsas, D., Ichikawa, H., Takada, Y. et al. 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. Oncogene 26, 1385–1397 (2007). https://doi.org/10.1038/sj.onc.1209945
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DOI: https://doi.org/10.1038/sj.onc.1209945
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