Constitutive activation of transcription factors NF-?B, AP-1, and NF-IL6 in human head and neck squamous cell carcinoma cell lines that express pro-inflammatory and pro-angiogenic cytokines

Abstract

We previously reported that human head and neck squamous cell carcinomas (HNSCCs) express the pro-inflammatory and pro-angiogenic cytokines interleukin (IL)-1alpha, IL-6, IL-8, and granulocyte-macrophage colony-stimulating factor in vitro and in vivo. The promoter region of the genes encoding these cytokines include binding sites for the transcription factors nuclear factor (NF) kappaB/Rel A, activator protein-1 (AP-1), and CCAAT enhancer-binding protein beta (C/EBPbeta, or NF-IL6), which have been reported to contribute to activation of these cytokine genes. In the study presented here, we examined the activation, composition, and function of these transcription factors in HNSCC cell lines that express pro-inflammatory cytokines, by using electrophoretic mobility shift and reporter-gene assays. Constitutive activation of NF-kappaB, AP-1, and NF-IL6 DNA-binding proteins was detected. Supershift analysis with antibodies specific for NF-kappaB, AP-1, and NF-IL6 binding proteins showed that the NF-kappaB-binding protein included p65/Rel A and p50; AP-1 activity included c-jun, junB, junD, and Fra-1; and NF-IL6 included C/EBPbeta. Mutational analysis of the NF-kappaB, AP-1, and NF-IL6 sites in the IL-8 promoter region showed that NF-kappaB and AP-1 sites contributed to constitutive IL-8 reporter activity in HNSCC. HNSCC lines that exhibited increased IL-8 secretion relative to simian virus 40-immortalized and primary keratinocyte cell lines also demonstrated a concordant increase in NF-kappaB reporter activity relative to nonmalignant keratinocytes. We concluded that the early transcription factors NF-kappaB, AP-1, and NF-IL6 are constitutively activated in human HNSCC cell lines and that NF-kappaB and AP-1 promote expression of the pro-inflammatory and pro-angiogenic cytokine IL-8 in HNSCC. The demonstration of the activation of these transcription factors will be helpful in defining the identity and role of these and other early gene products that contribute to pathogenesis of the malignant phenotype in HNSCC and in defining potential targets for pharmacologic and molecular therapy of HNSCC. Mol. Carcinog. 26:119-129, 1999. Published 1999 Wiley-Liss, Inc.

Effects of pharmacologic antagonists of epidermal growth factor receptor, PI3K and MEK signal kinases on NF‐κB and AP‐1 activation and IL‐8 and VEGF expression in human head and neck squamous cell carcinoma lines

“…13 These results indicated that cytokine IL-1␣ and EGF promote expression of Gro 1 through activation of NF-B in murine SCC. In human HNSCC, differences in expression of IL-1␣, a potential inducer of NF-B and AP-1, were noted to correlate with differences in constitutive activation of NF-B and AP-1 and expression of IL-8 in the low and high cytokine producers UM-SCC-9 and 11B, 6 consistent with the hypothesis that IL-1␣ could regulate constitutive activation of these transcription and angiogenesis factors. IL-1␣ was shown to induce NF-B and AP-1 activation, while mutations of the NF-B, and to a lesser extent, the AP-1 promoter sites, attenuated IL-1␣-mediated IL-8 reporter gene activity.…”

“…These data provide evidence for a link between EGFR activation, 18 coactivation of NF-B and AP-1 6 and coexpression of IL-8 and VEGF 5,10 in HNSCC. Our data indicate that inhibition of NF-B and AP-1 activation and proangiogenic factor expression may be an important mechanism underlying the activity of anti-EGFR antibodies and tyrosine kinase inhibitors in HNSCC and other cancers that overexpress EGFR.…”

“…Basal and EGF-inducible IL-8 production was partially inhibited by both LY-294002 and U0126, consistent with dependence of IL-8 expression on both NF-B and AP-1 activation, which we demonstrated previously by mutational analysis of the IL-8 promoter. 6,11 However, U0126 had a greater inhibitory effect than LY-294002 on constitutive and EGF-inducible VEGF production. Figure 5c also shows that combining LY-294002 and U0126 had a greater inhibitory effect on cytokine production than either FIGURE 4 -Effect of EGF and PD153035 on EGFR, Akt and ERK1/2 phosphorylation.…”

“…6 We found that differences in expression of these cytokines in different cancers was often related to differences in constitutive activation of both NF-B and AP-1. 6 Inhibition of NF-B activation by expression of a dominant negative inhibitor-B or pharmacologic agents was found to inhibit expression of IL-8 and other proinflammatory and proangiogenic cytokines, [7][8][9][10] as well as tumorigenesis and angiogenesis in vivo. 7,9 Inhibition of activation of extracellular signalregulated kinase (ERK) and AP-1 with antagonists of mitogenactivated/extracellular signal-regulated kinase (MEK) partially inhibited expression of both IL-8 and VEGF.…”

15-PGJ2, but not thiazolidinediones, inhibits cell growth, induces apoptosis, and causes downregulation of Stat3 in human oral SCCa cells

“…Inhibition of cytokine expression by 15-PGJ 2 would conceivably result in decreased activation of STAT molecules that mediate the transduction of the cytokine signal from the cell surface to the nucleus. Head and neck cancer cells have been shown to express a variety of pro-inflammatory and pro-angiogenic cytokines Ondrey et al, 1999) and to respond to IL-6 stimulation with upregulation of Stat3 phosphorylation and promotion of cell growth; reversal of this process could participate in the observed effects of 15-PGJ 2 on oral SCCa cells. In that aberrant tumour growth factor-a/ epidermal growth factor receptor signalling has been demonstrated to play a major role in Stat3 constitutive activation of head and neck SCCa cells, possible interference of 15-PGJ 2 with this pathway, at the level of the ligand or the receptor, constitutes another distinct possibility.…”