Key words: EGFR; NF-B; AP-1; IL-8; VEGF; PI3K; MEK; HNSCCIncreased angiogenesis is critical to tumor progression and metastasis, and we and others have shown that expression of members of the C-X-C cytokine and vascular endothelial growth factor families such as IL-8, growth regulated oncogene-1 (Gro 1) and VEGF can promote angiogenesis, tumorigenesis and metastatic tumor progression. 1-3 The expression of multiple factors with proangiogenic activity by cancer cells poses a significant obstacle to effective therapy with agents targeted toward individual factors and receptors. Identification of common mechanism(s) underlying expression of such a diversity of factors could guide the development of therapy using fewer selective agents. We previously observed that IL-8, VEGF and other cytokines are coexpressed and often vary concurrently in serum and supernatants from cell lines from different patients with head and neck squamous cell carcinoma (HNSCC), 4,5 suggesting that these factors could be regulated by common signal pathways or transcription factors.Examination of the regulatory region of many proinflammatory cytokines and proangiogenic factors reveals that they share common promoter sites for transcription factors such as nuclear factor-B (NF-B) and/or activator protein-1 (AP-1), which can be activated by injury, cytokines and growth factors. 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, 7-10 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. 10 These results provided evidence that at least 2 signal pathways upstream of NF-B and AP-1 make important contributions to expression of these angiogenesis factors.We have recently examined the contribution of several factors that may contribute to upstream signal activation of NF-B and AP-1 and expression of IL-8 and VEGF. We found that IL-1␣ expressed by HNSCC promotes autocrine activation of NF-B and AP-1, expression of IL-8 and cell survival. 11 Expression of IL-1 receptor antagonist inhibited NF-B reporter activity and IL-8 expression by 60 -80%, indicating that IL-1␣ is a major contributor to constitutive activation of NF-B and IL-8. HNSCC were also found to express c-MET, a receptor tyrosine kinase for hepatocyte growth factor/scatter factor (HGF/SF). 12 Increased expression of HGF/SF was detected together with IL-8 and VEGF in serum of patients with HNSCC, and recombinant HGF and HGF from stromal fibroblasts was found to further induce IL-8 and VEGF expression by human HNSCC lines. Inhibitors of MEK and Abbreviations: AP-1, activator...

Purpose: To determine if gene signatures differentially expressed in head and neck squamous cell carcinomas (HNSCC) are related to alterations in transcription factors nuclear factor-nB (NF-nB) andTP53previouslyassociatedwithdecreasedcelldeath,responsetotherapy,andworseprognosis. Experimental Design: Unique gene signatures expressed by HNSCC lines were identified by cDNA microarray, principal components, and cluster analyses and validated by quantitative reverse transcription-PCR (RT-PCR) and in situ hybridization. Bioinformatic analysis of the promoters and ontogeny of these clustered genes was done. Expression of proteins encoded by genes of a putative NF-nB signature, NF-nB p65, and TP53 were examined in HNSCC tissue specimens by immunostaining. Predicted promoter binding and modulation of expression of candidate NF-nB genes and cell survival were evaluated by p65 chromatin immunoprecipitation (ChIP) and small interfering RNA (siRNA) knockdown. Results: Two groups of HNSCC exhibiting distinct gene signatures were identified: cluster A enriched for histone genes, with a higher prevalence of TP53 promoter binding motifs; and cluster B enriched for injury response genes with NF-nB regulatory motifs. Coexpression of cluster B proteins was observed with strong NF-nB phospho-p65 and weak TP53 staining, and NF-nB phospho-p65 was inversely associated with TP53 (P = 0.02). Promoter binding of the NF-nB signature genes was confirmed by p65 ChIP, and down-modulation of their expression and cell death were induced by p65 siRNA. Conclusion: NF-nB promotes expression of a novel NF-nB^related gene signature and cell survival in HNSCC that weakly express TP53, a subset previously associated with inactivated wild-typeTP53, greater resistance to chemoradiotherapy, and worse prognosis.

Abrogation of apoptosis to sustain cell survival is an essential step in development of cancer. Aberrant activation of signal transcription factors NF-jB or STAT3, alterations in p53 status, or BCL/ BAX family expression have each been reported to affect cell survival in cancer, including head and neck squamous cell carcinomas (HNSCC). However, molecular targeting of these alterations individually has yielded disappointing results. In our study, we examined the hypothesis that alterations in a signal network involving NF-jB, STAT3 and p53 modulates expression of proapoptotic BAX and antiapoptotic BCL-XL proteins, and promotes cell survival of HNSCC. We found that NF-jB and STAT3 are coactivated together, and with cytokine stimulation or siRNA knock-down, both modulate BAX/BCL-XL. Greater modulation among HNSCC lines expressing low wt p53 than those overexpressing mt p53 protein suggested that decreased p53 expression might enhance activation of NF-jB, STAT3 and BCL-XL. Reexpression of wt p53 suppressed NF-jB and STAT3 nuclear binding activity, and BCL-XL expression, while inducing p21 and BAX. Over-expression of p53 together with inhibition of NF-jB or STAT3 induced greater increase in the BAX/BCL-XL ratio and apoptosis than modulation of these transcription factors individually. Conversely, NF-jB or STAT3 inducing cytokines decreased the BAX/BCL-XL ratio. Thus, a network involving signal coactivation of NF-jB and STAT3, differentially modified by p53 inactivation or mutation, promotes altered BAX/BCL-XL expression and cell survival in HNSCC. Inhibition of signal activation of both NF-jB and STAT3 together with reexpression of p53 could be the most effective strategy to restore BAX/BCL-XL regulation and for cytotoxic therapy of HNSCC.