Differential bortezomib sensitivity in head and neck cancer lines corresponds to proteasome, nuclear factor-kappaB and activator protein-1 related mechanisms.

Head and neck squamous cell carcinomas (HNSCC) exhibit constitutive activation of transcription factors nuclear factor-kappaB (NF-kappaB) and activator protein-1 (AP-1), which are modulated by the proteasome and promote resistance to cell death. HNSCC show variable sensitivity to the proteasome inhibitor bortezomib in vitro as well as in murine xenografts and patient tumors in vivo, and the mechanisms are not well understood. To address this question, the sensitivities of nine HNSCC cell lines to bortezomib were determined using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays, and the potential relationship between the sensitivity and bortezomib effects on biological processes was examined in HNSCC lines of differential bortezomib sensitivity. The most sensitive cell line (UM-SCC-11B) underwent cell death at 10(-9) mol/L in vitro and tumor regression at a maximally tolerated dose of bortezomib in a murine xenograft model. The differential sensitivity between UM-SCC-11A and UM-SCC-11B cells corresponded to differences in the extent of suppression of proteasome activity, ubiquitinated protein degradation, and NF-kappaB and AP-1 activation. Lower concentrations of bortezomib transiently increased NF-kappaB and sustained AP-1 activation in UM-SCC-11A cells. AP-1 reporter activity and cell density of UM-SCC-11A were suppressed when bortezomib was combined with c-Jun NH(2)-terminal kinase and p38 kinase pathways inhibitors. Thus, the differential sensitivities to bortezomib corresponded to dissimilar effects on the proteasome, NF-kappaB and AP-1 activities. Inhibition of c-Jun NH(2)-terminal kinase and p38 pathways blocked AP-1 activity and enhanced the antitumor effects. These findings revealed molecular mechanisms of bortezomib sensitivity and resistance, which are under development as biomarkers for clinical trials in patients with HNSCC.

A signal network involving coactivated NF-kappaB and STAT3 and altered p53 modulates BAX/BCL-XL expression and promotes cell survival of head and neck squamous cell carcinomas.

Abrogation of apoptosis to sustain cell survival is an essential step in development of cancer. Aberrant activation of signal transcription factors NF-kappaB 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-kappaB, STAT3 and p53 modulates expression of proapoptotic BAX and antiapoptotic BCL-XL proteins, and promotes cell survival of HNSCC. We found that NF-kappaB 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 over-expressing mt p53 protein suggested that decreased p53 expression might enhance activation of NF-kappaB, STAT3 and BCL-XL. Reexpression of wt p53 suppressed NF-kappaB and STAT3 nuclear binding activity, and BCL-XL expression, while inducing p21 and BAX. Over-expression of p53 together with inhibition of NF-kappaB or STAT3 induced greater increase in the BAX/BCL-XL ratio and apoptosis than modulation of these transcription factors individually. Conversely, NF-kappaB or STAT3 inducing cytokines decreased the BAX/BCL-XL ratio. Thus, a network involving signal coactivation of NF-kappaB 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-kappaB 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.

A novel nuclear factor-kappaB gene signature is differentially expressed in head and neck squamous cell carcinomas in association with TP53 status.

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-kappaB (NF-kappaB) and TP53 previously associated with decreased cell death, response to therapy, and worse prognosis. 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-kappaB signature, NF-kappaB p65, and TP53 were examined in HNSCC tissue specimens by immunostaining. Predicted promoter binding and modulation of expression of candidate NF-kappaB 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-kappaB regulatory motifs. Coexpression of cluster B proteins was observed with strong NF-kappaB phospho-p65 and weak TP53 staining, and NF-kappaB phospho-p65 was inversely associated with TP53 (P = 0.02). Promoter binding of the NF-kappaB signature genes was confirmed by p65 ChIP, and down-modulation of their expression and cell death were induced by p65 siRNA. CONCLUSION: NF-kappaB promotes expression of a novel NF-kappaB-related gene signature and cell survival in HNSCC that weakly express TP53, a subset previously associated with inactivated wild-type TP53, greater resistance to chemoradiotherapy, and worse prognosis.

Hepatocyte growth factor/scatter factor differentially regulates expression of proangiogenic factors through Egr-1 in head and neck squamous cell carcinoma.

Hepatocyte growth factor/scatter factor (HGF) and the angiogenesis factors platelet-derived growth factors (PDGF), vascular endothelial growth factor (VEGF), and interleukin-8 (IL-8) are found in elevated concentrations in serum or tumor tissue of patients with head and neck squamous cell carcinomas (HNSCC), suggesting these factors may be coregulated. A cDNA microarray analysis for HGF-inducible genes revealed that HGF also modulates PDGFA expression, a gene recently shown to be inducible by the transcription factor, early growth response-1 (Egr-1). In the present study, we investigated the potential role of HGF-induced Egr-1 in expression of PDGF, VEGF, and IL-8. HGF induced expression of all three factors and Egr-1 expression and DNA-binding activity. The analysis of promoter sequences showed putative Egr-1 binding sites in the PDGFA or VEGF but not in the IL-8 promoter, and HGF-induced Egr-1-binding activity was confirmed by chromatin immunoprecipitation (ChIP) assay. The maximal basal and HGF-induced promoter activity for the PDGFA gene existed within -630 bp of the promoter region, and overexpression of Egr-1 significantly increased such activity. Consistent with this, expression of PDGFA and VEGF but not IL-8 showed corresponding differences with Egr-1 expression in HNSCC tumor specimens and were strongly suppressed by transfection of Egr-1-antisense or small interference RNA (siRNA) oligonucleotides. HGF-induced expression of Egr-1, PDGFA, and VEGF was suppressed by pharmacologic and siRNA inhibitors of mitogen-activated protein kinase kinase 1/2 (MEK1/2) and protein kinase C (PKC) pathways. We conclude that the HGF-induced activation of transcription factor Egr-1 by MEK1/2- and PKC-dependent mechanisms differentially contributes to expression of PDGF and VEGF, which are important angiogenesis factors and targets for HNSCC therapy.

Metastatic squamous cell carcinoma cells that overexpress c-Met exhibit enhanced angiogenesis factor expression, scattering and metastasis in response to hepatocyte growth factor.

We previously performed gene expression profiling in a multistep squamous cell carcinoma (SCC) progression model, and identified growth-regulated oncogene-1 (Gro-1/KC) as a factor that contributes to enhanced angiogenesis, tumorigenesis and metastasis. In the present study, we explored molecular pathways coactivated with Gro-1/KC, and identified a transcript that encodes c-Met, the receptor for hepatocyte growth factor/scatter factor (HGF). Northern, Western blot, and immunohistochemical analyses confirm that expression of c-Met mRNA and protein is increased with SCC progression. In vitro, HGF preferentially promoted scattering in the metastatic LY-1 and LY-2 lines, and enhanced angiogenesis factors Gro-1/KC and vascular endothelial growth factor (VEGF) production by all tumor cell lines. In vivo, tumor growth and lung metastasis were promoted by transfection and overexpression of HGF cDNA in metastatic LY-1 cells. Our data indicate that metastatic SCC cells that overexpress c-Met exhibit angiogenesis factor expression and enhanced scattering in response to HGF in vitro, and tumorigenesis and metastasis in response to HGF in the tumor microenvironment in vivo.

A pilot study of longitudinal serum cytokine and angiogenesis factor levels as markers of therapeutic response and survival in patients with head and neck squamous cell carcinoma.

BACKGROUND: Head and neck squamous cell carcinomas (HNSCCs) were previously shown to express a repertoire of cytokines and angiogenesis factors that contribute to malignant pathogenesis and are detectable in serum. Pretreatment and posttreatment serum levels of cytokines and angiogenesis factors were evaluated as markers for outcome in patients with HNSCC. METHODS: Baseline cytokine and factor levels of 29 patients with HNSCC were compared with those of 15 age-matched and sex-matched controls, and pretreatment and posttreatment levels of 22 of the patients eligible for treatment and followed for a median of 37 months were compared. RESULTS: Mean serum concentrations of interleukin (IL)-6, IL-8, hepatocyte growth factor (HGF), vascular endothelial growth factor (VEGF), and growth regulated oncogene 1 (GRO-1) were increased in patients with HNSCC, but elevation of these factors was not associated with clinical outcome. However, changes in first posttreatment serum cytokine levels were observed for many of the patients consistent with response, progression, and survival. Later increases in IL-6 or HGF were observed in patients who had a relapse and inflammatory or infectious complications. A relationship between the change in the pretreatment and first posttreatment cytokine measurement with survival was detected for HGF, IL-8, IL-6, and VEGF using a Cox-proportional hazards model (p = .004, p = .06, p = .10, and p = .11). The association between longitudinal decreases in IL-6, IL-8, VEGF, and HGF throughout the follow-up with survival was detected with a time-dependent Cox model (p = .01, .07, .08, and .05, respectively). CONCLUSIONS: Longitudinal changes in serum HGF, IL-6, IL-8, and VEGF were detected with treatment response, relapse, or complications in individual patients and were associated with survival, with HGF showing the strongest relationship with survival. HGF, IL-6, IL-8, and VEGF merit investigation as markers of response, survival, and recurrence in larger prospective studies.

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

We previously reported that expression of angiogenesis factors interleukin-8 (IL-8) and vascular endothelial growth factor (VEGF) is promoted by coactivation of transcription factors nuclear factor-kappaB (NF-kappaB) and activator protein-1 (AP-1) by interleukin-1alpha in human head and neck squamous cell carcinomas (HNSCC). However, expression of IL-1 receptor antagonist incompletely blocked reporter gene activity and cytokine expression, suggesting that other upstream signals may contribute to activation. Overexpression and autocrine activation of epidermal growth factor receptor (EGFR) is detected in 90% of HNSCC, and EGFR inhibitors have been reported to inhibit IL-8 and VEGF expression, but the intermediary signal pathways and transcription factors by which EGFR modulates proangiogenic factors is unknown. EGFR can activate the phosphotidylinositol-3 kinase (PI3K) and mitogen-activated/extracellular signal-regulated kinase (MEK) pathways, which can potentially modulate activation of NF-kappaB and AP-1, respectively. In our study, we examined the effect of EGF and antagonists of EGFR, PI3K and MEK on NF-kappaB and AP-1 activation and IL-8 and VEGF expression in HNSCC cell lines UM-SCC-9 and 11B in which EGFR is overexpressed and activated. Recombinant EGF induced EGFR phosphorylation, activation of NF-kappaB and AP-1 reporter genes and IL-8 and VEGF expression, indicating that EGFR can mediate coactivation of both transcription factors and cytokine genes in HNSCC. EGFR antagonist PD153035 and anti-EGFR antibody C225 completely inhibited EGF-induced reporter activity and cytokine expression, but only partially inhibited constitutive activity. MEK inhibitor U0126 preferentially blocked AP-1 activity and expression of both IL-8 and VEGF, while PI3K inhibitor LY-294002 or a dominant negative inhibitor-kappaB preferentially blocked NF-kappaB activation and expression of IL-8 but not VEGF. EGFR, PI3K and MEK antagonists inhibited growth of HNSCC. We conclude that antagonists of EGFR, PI3K and MEK signal pathways have inhibitory activity against EGFR-induced NF-kappaB and AP-1 activation, IL-8 and VEGF expression and growth by HNSCC. Published 2002 Wiley-Liss, Inc.