Potential of chopped heath biomass and spent growth media to replace wood chips as bulking agent for composting high N-containing residues.

We investigated the potential of C-rich byproducts to replace wood chips as bulking agent (BA) during composting. The impact of these alternatives on the composting process and on compost stability and characteristics was assessed. Three BA (chopped heath biomass and spent growth media used in strawberry and tomato cultivation) were used for processing leek residues in windrow composting. All BA resulted in stable composts with an organic matter (OM) content suitable for use as soil amendment. Using chopped heath biomass led to high pile temperatures and OM degradation and a nutrient-poor compost with high C/P ratio appropriate for increasing soil organic carbon content in P-rich soils. Spent substrates can replace wood chips, however, due to their dense structure and lower biodegradation potential, adding a more coarse BA is required. Generally, the nutrient content of the composts with growth media was higher than the composts with wood chips and chopped heath biomass.

TNF-α modulates genome-wide redistribution of ΔNp63α/TAp73 and NF-κB cREL interactive binding on TP53 and AP-1 motifs to promote an oncogenic gene program in squamous cancer.

The Cancer Genome Atlas (TCGA) network study of 12 cancer types (PanCancer 12) revealed frequent mutation of TP53, and amplification and expression of related TP63 isoform ΔNp63 in squamous cancers. Further, aberrant expression of inflammatory genes and TP53/p63/p73 targets were detected in the PanCancer 12 project, reminiscent of gene programs comodulated by cREL/ΔNp63/TAp73 transcription factors we uncovered in head and neck squamous cell carcinomas (HNSCCs). However, how inflammatory gene signatures and cREL/p63/p73 targets are comodulated genome wide is unclear. Here, we examined how the inflammatory factor tumor necrosis factor-α (TNF-α) broadly modulates redistribution of cREL with ΔNp63α/TAp73 complexes and signatures genome wide in the HNSCC model UM-SCC46 using chromatin immunoprecipitation sequencing (ChIP-seq). TNF-α enhanced genome-wide co-occupancy of cREL with ΔNp63α on TP53/p63 sites, while unexpectedly promoting redistribution of TAp73 from TP53 to activator protein-1 (AP-1) sites. cREL, ΔNp63α and TAp73 binding and oligomerization on NF-κB-, TP53- or AP-1-specific sequences were independently validated by ChIP-qPCR (quantitative PCR), oligonucleotide-binding assays and analytical ultracentrifugation. Function of the binding activity was confirmed using TP53-, AP-1- and NF-κB-specific REs or p21, SERPINE1 and IL-6 promoter luciferase reporter activities. Concurrently, TNF-α regulated a broad gene network with cobinding activities for cREL, ΔNp63α and TAp73 observed upon array profiling and reverse transcription-PCR. Overlapping target gene signatures were observed in squamous cancer subsets and in inflamed skin of transgenic mice overexpressing ΔNp63α. Furthermore, multiple target genes identified in this study were linked to TP63 and TP73 activity and increased gene expression in large squamous cancer samples from PanCancer 12 TCGA by CircleMap. PARADIGM inferred pathway analysis revealed the network connection of TP63 and NF-κB complexes through an AP-1 hub, further supporting our findings. Thus, inflammatory cytokine TNF-α mediates genome-wide redistribution of the cREL/p63/p73, and AP-1 interactome, to diminish TAp73 tumor suppressor function and reciprocally activate NF-κB and AP-1 gene programs implicated in malignancy.

The PI3K/Akt/mTOR axis in head and neck cancer: functions, aberrations, cross-talk, and therapies.

Head and neck squamous cell carcinoma (HNSCC) is one of the most morbid, mortal, and genetically diverse malignancies. Although HNSCC is heterogeneous in nature, alterations in major components of the PI3K/Akt/mTOR pathway are consistently observed throughout the majority of HNSCC cases. These alterations include genetic aberrations, such as mutations or DNA copy number variations, and dysregulation of mRNA or protein expression. In normal physiology, the PI3K/Akt/mTOR axis regulates cell survival, growth, and metabolism. However, alterations in this pathway lead to the malignant phenotype which characterizes HNSCC, among many other cancers. For this reason, both pharmaceutical companies and academic institutions are actively developing and investigating inhibitors of PI3K, Akt, and mTOR in preclinical and clinical studies of HNSCC. Many of these inhibitors have shown promise, while the effects of others are tempered by the mechanisms through which HNSCC can evade therapy. As such, current research aimed at elucidating the interactions between PI3K/Akt/mTOR and other important signaling pathways which may drive resistance in HNSCC, such as p53, NF-κB, and MAPK, has become a prominent focus toward better understanding how to most effectively treat HNSCC.

Aberrant IKKα and IKKß cooperatively activate NF-κB and induce EGFR/AP1 signaling to promote survival and migration of head and neck cancer.

The inhibitor-κB kinase-nuclear factor-κB (IKK-NF-κB) and epidermal growth factor receptor-activator protein-1 (EGFR-AP1) pathways are often co-activated and promote malignant behavior, but the underlying basis for this relationship is unclear. Resistance to inhibitors of IKKß or EGFR is observed in head and neck squamous cell carcinomas (HNSCC). Here, we reveal that both IKKα and ß contribute to nuclear activation of canonical and alternate NF-κB/REL family transcription factors, and overexpression of signal components that enhance co-activation of the EGFR-AP1 pathway. We observed that IKKα and IKKß exhibit increased protein expression, nuclear localization, and phosphorylation in HNSCC tissues and cell lines. Individually, IKK activity varied among different cell lines, but overexpression of both IKKs induced the strongest NF-κB activation. Conversely, siRNA knock down of both IKKs significantly decreased nuclear localization and phosphorylation of canonical RELA and IκBα and alternative p52 and RELB subunits. Knock down of both IKKs more effectively inhibited NF-κB activation, broadly modulated gene expression and suppressed cell proliferation and migration. Global expression profiling revealed that NF-κB, cytokine, inflammatory response and growth factor signaling are among the top pathways and networks regulated by IKKs. Importantly, IKKα and IKKß together promoted the expression and activity of transforming growth factor α, EGFR and AP1 transcription factors cJun, JunB and Fra1. Knock down of AP1 subunits individually decreased 8/15 (53%) of IKK-targeted genes sampled and similarly inhibited cell proliferation and migration. Mutations of NF-κB and AP1-binding sites abolished or decreased IKK-induced interleukin-8 (IL-8) promoter activity. Compounds such as wedelactone with dual IKK inhibitory activity and geldanomycins that block IKKα/ß and EGFR pathways were more active than IKKß-specific inhibitors in suppressing NF-κB activation and proliferation and inducing cell death. We conclude that IKKα and IKKß cooperatively activate NF-κB and EGFR/AP1 networks of signaling pathways and contribute to the malignant phenotype and the intrinsic or acquired therapeutic resistance of HNSCC.

TGF-ß and NF-κB signal pathway cross-talk is mediated through TAK1 and SMAD7 in a subset of head and neck cancers.

Transforming growth factor-beta (TGF-ß) has a dual role in epithelial malignancies, including head and neck squamous cell carcinoma (HNSCC). Attenuation of canonical TGF-ß signaling enhances de novo tumor development, whereas TGF-ß overexpression and signaling paradoxically promotes malignant progression. We recently observed that TGF-ß-induced growth arrest response is attenuated, in association with aberrant activation of nuclear factor-κB (NF-κB), a transcription factor, which promotes malignant progression in HNSCC. However, what role cross-talk between components of the TGF-ß and NF-κB pathways plays in altered activation of these pathways has not been established. Here, we show TGF-ß receptor II and TGF-ß-activated kinase 1 (TAK1) are predominantly expressed in a subset of HNSCC tumors with nuclear activation of NF-κB family member RELA (p65). Further, TGF-ß1 treatment induced sequential phosphorylation of TAK1, IKK, IκBα and RELA in human HNSCC lines. TAK1 enhances TGF-ß-induced NF-κB activation, as TAK1 siRNA knockdown decreased TGF-ß1-induced phosphorylation of IKK, IκB and RELA, degradation of IκBα, RELA nuclear translocation and DNA binding, and NF-κB-induced reporter and target gene transcription. Functionally, TAK1 siRNA inhibited cell proliferation, migration and invasion. Celastrol, a TAK1 inhibitor and anti-inflammatory compound used in traditional Chinese medicine, also decreased TGF-ß1-induced phosphorylation of TAK1 and RELA, and suppressed basal, TGF-ß1- and tumor necrosis factor-alpha (TNF-α)-induced NF-κB reporter gene activity. Celastrol also inhibited cell proliferation, while increasing sub-G0 DNA fragmentation and Annexin V markers of apoptosis. Furthermore, TGF-ß and RELA activation promoted SMAD7 expression. In turn, SMAD7 preferentially suppressed TGF-ß-induced SMAD and NF-κB reporters when compared with constitutive or TNF-α-induced NF-κB reporter gene activation. Thus, cross-talk by TGF-ß via TAK1 and NF-κB promotes the malignant phenotype of HNSCC. Moreover, NF-κB may contribute to the downstream attenuation of canonical TGF-ß signaling through increased SMAD7 expression. Celastrol highlights the therapeutic potential of agents targeting TAK1 as a key node in this pro-oncogenic TGF-ß-NF-κB signal pathway.

YAP dysregulation by phosphorylation or ΔNp63-mediated gene repression promotes proliferation, survival and migration in head and neck cancer subsets.

Overexpression of the Yes-associated protein (YAP), and TP53 family members ΔNp63 and p73, have been independently detected in subsets of head and neck squamous cell carcinomas (HNSCCs). YAP may serve as a nuclear cofactor with ΔNp63 and p73, but the functional role of YAP and their potential relationship in HNSCCs are unknown. In this study, we show that in a subset of HNSCC lines and tumors, YAP expression is increased but localized in the cytoplasm in association with increased AKT and YAP phosphorylation, and with decreased expression of ΔNp63 and p73. In another subset, YAP expression is decreased but detectable in the nucleus in association with lower AKT and YAP phosphorylation, and with increased ΔNp63 and p73 expression. Inhibiting AKT decreased serine-127 phosphorylation and enhanced nuclear translocation of YAP. ΔNp63 bound to the YAP promoter and suppressed its expression. Transfection of a YAP-serine-127-alanine phosphoacceptor-site mutant or ΔNp63 knockdown significantly increased nuclear YAP and cell death. Conversely, YAP knockdown enhanced cell proliferation, survival, migration and cisplatin chemoresistance. Thus, YAP function as a tumor suppressor may alternatively be dysregulated by AKT phosphorylation at serine-127 and cytoplasmic sequestration, or by transcriptional repression by ΔNp63, in different subsets of HNSCC. AKT and/or ΔNp63 are potential targets for enhancing YAP-mediated apoptosis and chemosensitivity in HNSCCs.

Influence of damaging and wilting red clover on lipid metabolism during ensiling and in vitro rumen incubation.

This paper describes the relationship between protein-bound phenols in red clover, induced by different degrees of damaging before wilting and varying wilting duration, and in silo lipid metabolism. The ultimate effect of these changes on rumen biohydrogenation is the second focus of this paper. For this experiment, red clover, damaged to different degrees (not damaged (ND), crushing or frozen/thawing (FT)) before wilting (4 or 24 h) was ensiled. Different degrees of damaging and wilting duration lead to differences in polyphenol oxidase (PPO) activity, measured as increase in protein-bound phenols. Treatment effects on fatty acid (FA) content and composition, lipid fractions (free FAs, membrane lipids (ML) and neutral fraction) and lipolysis were further studied in the silage. In FT, red clover lipolysis was markedly lower in the first days after ensiling, but this largely disappeared after 60 days of ensiling, regardless of wilting duration. This suggests an inhibition of plant lipases in FT silages. After 60 days of ensiling no differences in lipid fractions could be found between any of the treatments and differences in lipolysis were caused by reduced FA proportions in ML of wilted FT red clover. Fresh, wilted (24 h) after damaging (ND or FT) and ensiled (4 or 60 days; wilted 24 h; ND or FT) red clover were also incubated in rumen fluid to study the biohydrogenation of C18:3n-3 and C18:2n-6 in vitro. Silages (both 60 days and to a lower degree 4 days) showed a lower biohydrogenation compared with fresh and wilted forages, regardless of damaging. This suggests that lipids in ensiled red clover were more protected, but this protection was not enhanced by a higher amount of protein-bound phenols in wilted FT compared with ND red clover. The reduction of rumen microbial biohydrogenation with duration of red clover ensiling seems in contrast to what is expected, namely a higher biohydrogenation when a higher amount of FFA is present. This merits further investigation in relation to strategies to activate PPO toward the embedding of lipids in phenol-protein complexes.

Lipoblastoma of the back; a case of a 7-month-old boy.

Lipoblastomatous tumours are rare, benign tumours of embryonic adipose tissue which primarily occur in children younger than 3 years. Most common, these tumours can be found in the extremities, yet cases involving other locations, such as the trunk, face, neck and mediastinum, have been described and they have an excellent prognosis despite its potential to local invasion and rapid growth. In medical literature two types are known, the focal well circumscribed lipoblastoma, and the more diffusely infiltrating lipoblastomatosis. The authors report a case of a 7-months old Ethiopian boy with a congenital lipoblastoma on the back. Despite surgical intervention, we were confronted with recurrences. In conclusion, we stress the morbidity of possible surgical management and the difficulty in decision-making. Therefore, awareness in young children with a fast growing lipomatous mass for this clinical and histopathological entity is crucial.

Expression of proangiogenic chemokine Gro 1 in low and high metastatic variants of Pam murine squamous cell carcinoma is differentially regulated by IL-1alpha, EGF and TGF-beta1 through NF-kappaB dependent and independent mechanisms.

We previously reported that chemokine Growth Regulated Oncogene 1 (Gro 1) is over-expressed in murine squamous cell carcinoma (SCC) with metastatic tumor progression. The enhanced expression of Gro-1 gene by SCC is regulated by activation of nuclear factor-kappaB (NF-kappaB), leading to accelerated tumor growth, angiogenesis and metastasis in vivo. In our study, we investigated the effect of the regulatory cytokines, IL-1alpha, EGF and TGF-beta1 on activation of NF-kappaB and Gro1 in primary and metastatic sublines of the murine SCC Pam 212. We found that Gro 1 expression could be induced by IL-1alpha or EGF in the low cytokine producing Pam 212 cells, but no significant induction was observed in high cytokine producing and metastatic LY-2 cells. Conditioned medium from LY-2 containing functional IL-1alpha induced Gro 1 expression in Pam 212 cells, which can be blocked by IL-1 receptor antagonist (IL-1RA). IL-1RA, however, had a minimal effect on constitutive Gro 1 production by LY-2 cells. TGF-beta1 suppressed constitutive as well as IL-1alpha and EGF-inducible Gro 1 production in both Pam 212 and LY-2 cells. IL-1alpha and EGF, but not TGF-beta1, were found to activate NF-kappaB in Pam 212, whereas none of the stimulants showed a significant effect on constitutive activation of NF-kappaB in LY-2 cells. Overexpression of a super repressor IkappaBalphaM in Pam 212 inhibited NF-kappaB binding activity, which led to impaired Gro 1 induction by IL-1alpha and EGF. These results demonstrate that IL-1alpha, EGF, and TGF-beta1 are important modulators of Gro 1 expression in SCC. Different responses to these modulators observed along with SCC metastatic progression may suggest a transition mechanism(s) for Gro 1 expression from host factor dependent to an independent stage involving NF-kappaB activation. Published 2001 Wiley-Liss, Inc.

Burkitt's/Burkitt's-like lymphoma presenting as bacterial sinusitis in two HIV-infected children.

Two children (ages 12 and 13 years) with transfusion-acquired human immunodeficiency virus (HIV) infection presented with facial pain and rhinorrhea. Radiographic imaging showed extensive paranasal sinus disease, presumed to be bacterial sinusitis, and the patients were treated with broad-spectrum oral antibiotics. Both patients were unresponsive to oral agents and were switched to intravenous antibiotics. Despite aggressive antimicrobial therapy, one patient (case 1) developed increased periorbital swelling and proptosis, and the other patient (case 2) developed symptoms of nasopharyngeal obstruction. Repeat imaging showed progression of the infiltrative process extending from the paranasal sinuses into the orbit (case 1), and nasopharynx (case 2). Surgical exploration and tissue biopsies were performed on both patients and the histopathology was consistent with Burkitt's/Burkitt's-like lymphoma. Combination systemic and intrathecal chemotherapy resulted in a complete remission in both patients. These reports illustrate the fact that Burkitt's/Burkitt's-like lymphoma in the paranasal sinuses may initially masquerade as an acute bacterial sinusitis. The ability of the tumor to extend rapidly from the sinuses into the orbit and nasopharynx reinforces the importance of early diagnosis and treatment. Burkitt's/Burkitt's-like lymphoma in the paranasal sinuses has not been previously described in HIV-infected children.

Hepatocyte growth factor/scatter factor-induced activation of MEK and PI3K signal pathways contributes to expression of proangiogenic cytokines interleukin-8 and vascular endothelial growth factor in head and neck squamous cell carcinoma.

The proangiogenic activity of hepatocyte growth factor (HGF)/scatter factor has been closely associated with its ability to stimulate endothelial cell chemotaxis, migration, proliferation, and capillary formation. However, the potential of HGF as a paracrine factor in regulating the expression of angiogenesis factors by tumor cells is not widely appreciated. We observed that increased HGF was correlated with higher levels of angiogenesis factors interleukin (IL)-8 and vascular endothelial growth factor (VEGF) in serum of patients with head and neck squamous cell carcinoma (HNSCC) as compared with that in normal volunteers and hypothesized that HGF may regulate angiogenesis factor production by tumor cells through the activation of its receptor c-Met, which is expressed by HNSCC cells. To test this hypothesis, we examined the effect of HGF treatment on IL-8 and VEGF expression by a panel of primary keratinocytes and HNSCC lines. HGF induced a significant dose-dependent increase in IL-8 and/or VEGF cytokine production in eight HNSCC lines tested, which is not observed in normal keratinocytes. In addition, HGF increased mRNA expression of IL-8 in 3 of 6 and VEGF in 5 of 6 HNSCC lines. The increase in induction of these factors by HGF corresponded to an increase in phosphorylation of c-Met in HNSCC. HGF-induced phosphorylation of mitogen-activated protein/extracellular signal-regulated kinase kinase (MEK) pathway substrate p42/p44(erk) and phosphatidylinositol 3'-kinase (PI3K) pathway substrate Akt provided evidence for downstream activation of MEK and PI3K pathways in HNSCC. Inhibitors of MEK (U0126) and PI3K (LY294002) blocked p42/p44(erk) and Akt, respectively, and partially blocked HGF-induced production of IL-8 and VEGF, whereas the combination of U0126 and LY294002 completely inhibited expression of IL-8 and VEGF by UMSCC-11A. Our results demonstrate that HGF can promote expression of angiogenesis factors in tumor cells through both MEK- and PI3K-dependent pathways. Understanding HGF/Met paracrine regulatory mechanisms between tumor and host cells may provide critical information for targeting of therapies against angiogenesis.

IL (interleukin)-1alpha promotes nuclear factor-kappaB and AP-1-induced IL-8 expression, cell survival, and proliferation in head and neck squamous cell carcinomas.

Interleukin 1alpha (IL-1alpha) is an important regulatory cytokine, the release of which after an injury can induce activation of transcription factors nuclear factor (NF)kappaB and activator protein (AP-1), which promote expression of genes involved in cell survival, proliferation, and angiogenesis. IL-1alpha is expressed autonomously by head and neck squamous cell carcinomas (HNSCCs) and a variety of other cancers, raising the possibility that IL-1alpha may serve as an autocrine factor that stimulates the activation of prosurvival transcription factors and target genes in cancer. In this study, we examined the role of IL-1alpha in the activation of NFkappaB and AP-1, the expression of proangiogenic cytokine IL-8, and in the survival and proliferation of HNSCC cell lines. HNSCCs were found to secrete and respond to functional IL-1alpha, in that culture supernatant from a high IL-1alpha-secreting line, UM-SCC-11B, could induce secretion of cytokine IL-8 by a low IL-1alpha-secreting line, UM-SCC-9; and the induction of IL-8 secretion could be blocked by the anti-IL-1alpha-neutralizing antibody or the IL-1 receptor antagonist (IL-1RA). Furthermore, IL-1alpha could induce the expression of IL-8 through an autocrine mechanism, in that transfection of UM-SCC-9 cells with a plasmid encoding IL-1alpha resulted in the increased coexpression of IL-1alpha and IL-8; whereas transfection with a plasmid encoding IL-1RA lacking the secretory leader sequence led to the decreased coexpression of IL-1alpha and IL-8. IL-1alpha was found to induce coexpression of IL-8 through the activation of NFkappaB and AP-1, in that mutation of the NFkappaB site within the IL-8 promoter abolished autocrine- and recombinant IL-1alpha-induced IL-8 reporter gene activity, whereas mutation in AP-1 partially decreased IL-8 reporter gene activity in UM-SCC-9 cells. Intracellular expression of IL-1RA decreased NFkappaB reporter gene activity, indicating that endogenously expressed IL-1alpha contributes to constitutive NFkappaB activation in this HNSCC line. Expression of IL-1alpha affected survival of UM-SCC-9, inasmuch as transfection of cells with plasmid encoding IL-1alpha or IL-1RA led to the increased or decreased survival of cells cotransfected with a beta-galactosidase reporter gene, respectively. IL-1alpha was also found to promote the increased growth of UM-SCC-9 cells in vitro. We demonstrate that exogenous and endogenous IL-1alpha contributes to the transcriptional activation of NFkappaB and AP-1, to the expression of IL-8, and to cell survival and the growth of HNSCC in vitro.

Molecular profiling of transformed and metastatic murine squamous carcinoma cells by differential display and cDNA microarray reveals altered expression of multiple genes related to growth, apoptosis, angiogenesis, and the NF-kappaB signal pathway.

To identify changes in gene expression with transformation and metastasis, we investigated differential gene expression in a squamous carcinoma model established in syngeneic mice. We used mRNA differential display (DD) to detect global differences and cDNA arrays enriched for cancer-associated genes using mRNA from primary keratinocytes, transformed Pam 212 squamous carcinoma cells, and metastases of Pam 212. After DD, 72 candidate cDNAs expressed primarily in transformed and metastatic cells were selected and cloned. Fifty-seven were detected, and 32 were confirmed to be differentially expressed by Northern blot analysis. mRNA expression profiles were also generated using a mouse cDNA array composed of 4000 elements representing known genes and expressed sequence tags plus the 57 DD candidate cDNAs detected by Northern analysis to facilitate data validation. cDNA array detected 76.9% of the differentially expressed mRNAs selected from DD and confirmed by Northern blot, whereas low-abundance mRNAs did not reach the threshold for detection by the lower-sensitivity array method. Clustering analysis of DD and array results from transformed and metastatic cells identified genes that exhibited decreased or increased expression with transformation and metastasis. Alterations in the expression of several genes detected during tumor progression were consistent with their functional activities involving growth (p21, p27, and cyclin D1), resistance and apoptosis (glutathione-S-transferase, cIAP-1, PEA-15, and Fas ligand), inflammation and angiogenesis [chemokine growth-regulated oncogene 1 (also called KC)], and signal transduction (c-Met, yes-associated protein, and syk). Strikingly, 10 of 22 genes in the cluster expressed in metastases have been associated with activation of the nuclear factor (NF)-kappaB signal pathway. The NF-kappaB-inducible cytokine Gro-1 was recently shown to promote tumor growth, metastasis, and angiogenesis of squamous cell carcinomas in vivo (Loukinova et al., Oncogene, 19: 3477-3486, 2000). The results demonstrate that early response genes related to NF-kappaB contribute to metastatic tumor progression. Comparison of cell lines and tumor tissue revealed a concordance of approximately 50% by array, and 70% for Northern-confirmed, metastasis-related genes. Functional genomic approaches comparing expression among cell lines and tumor tissue may promote a better understanding of the genes expressed by malignant and host cells during tumor progression and metastasis.

Novel proteasome inhibitor PS-341 inhibits activation of nuclear factor-kappa B, cell survival, tumor growth, and angiogenesis in squamous cell carcinoma.

We have shown that activation of nuclear factor-kappa B (NF-kappa B) promotes cell survival and expression of cytokines such as growth-regulated oncogene-alpha, which can modulate angiogenesis, growth, and metastasis of squamous cell carcinoma (SCC). Activation of NF-kappa B and cytoprotective genes in cancer may result from signal-induced phosphorylation and proteasome-dependent degradation of inhibitor-kappa B. In this study, we examined the effects of the novel proteasome inhibitor PS-341 on activation of NF-kappa B and cell survival, growth, and angiogenesis in murine and human SCC cell lines. PS-341 inhibited activation of NF-kappa B DNA binding and functional reporter activity at concentrations between 10(-8) and 10(-7) M. Cytotoxicity was observed at 10(-7) M in four murine and two human SCC lines, and followed early cleavage of poly(ADP-ribose) polymerase, a marker of caspase-mediated apoptosis. In vivo, PS-341 inhibited growth of murine and human SCC in mice at doses of 1--2 mg/kg given three times weekly, and dose-limiting toxicity was encountered at 2 mg/kg. Tumor growth inhibition was associated with a marked decrease in vessel density. PS-341 inhibited expression of the proangiogenic cytokines growth-regulated oncogene-alpha and vascular endothelial growth factor by SCC in the range at which PS-341 inhibits NF-kappa B. We conclude that PS-341 inhibits activation of NF-kappa B pathway components related to cell survival, tumor growth, and angiogenesis in SCC.

NIH symposium summary: organ preservation therapies for squamous cancers of the head and neck.

This symposium, sponsored by the NIH Office of Rare Diseases, the National Cancer Institute, the National Institute of Deafness and Other Communication Disorders, and the National Institute of Dental and Craniofacial Diseases, reviewed the current status of organ preservation therapies for head and neck cancers, as well as promising newer approaches for therapy and for toxicity amelioration.

Coexpression of proangiogenic factors IL-8 and VEGF by human head and neck squamous cell carcinoma involves coactivation by MEK-MAPK and IKK-NF-kappaB signal pathways.

Interleukin 8 (IL-8) and vascular endothelial growth factor (VEGF) promote tumor angiogenesis, growth, and metastasis and are coexpressed by human head and neck squamous cell carcinomas (HNSCCs) and a variety of other cancers. The promoters of the IL-8 and VEGF genes contain different recognition sites for transcription factors nuclear factor (NF)-kappaB and activator protein-1 (AP-1), which we showed previously are coactivated in HNSCCs. NF-kappaB and AP-1 may be modulated by the inhibitor kappaB kinase (IKK) and mitogen-activated protein kinase (MAPK) signal pathways, but the contribution of these pathways to expression of IL-8 and VEGF and as potential targets for antiangiogenesis therapy in HNSCC is not known. In this study, we examined the effects of modulation of the MAPK and IKK pathways on expression of IL-8 and VEGF by UM-SCC-9 and UM-SCC-11B cell lines. Interruption of IKK-mediated activation of NF-kappaB by expression of an inhibitor kappaB alpha mutant (IkappaB alphaM) in UM-SCC-9 cells resulted in partial inhibition of expression of IL-8 but not VEGF. Analysis of possible alternative pathways for induction of these genes revealed activation of the MAPK extracellular signal-regulated kinase (ERK1/2) in cell lines UM-SCC-9 and UM-SCC-11B. Basal and tumor necrosis factor-alpha-inducible phosphorylation of ERK1/2 and secretion of IL-8 and VEGF could be specifically inhibited by a MEK inhibitor, U0126. Expression of IL-8 and VEGF in the cell lines was associated with coactivation of both NF-kappaB and AP-1, and U0126 inhibited both NF-kappaB and AP-1 reporter activity in UM-SCC-9 and UM-SCC-11B cells. The ERK pathway appears to contribute to expression of IL-8 and VEGF and transactivation of NF-kappaB as well as AP-1 in HNSCC. Combined inhibition of both MAPK and IKK pathways may be needed for suppression of the signal transduction mechanism(s) regulating VEGF and IL-8 secretion and angiogenesis by human HNSCC.

Cisplatin and radiation sensitivity in human head and neck squamous carcinomas are independently modulated by glutathione and transcription factor NF-kappaB.

BACKGROUND: Response to neoadjuvant cisplatin-based chemotherapy has been used to predict overall response to chemoradiation therapy and to select patients with head and neck squamous cell carcinoma (HNSCC) for organ preservation therapy in NCI and VA cooperative group trials. However, different molecular determinants have been reported to contribute to sensitivity of cells to cisplatin and radiation, including glutathione (GSH), and activation of nuclear factor-kappaB (NF-kappaB), a transcription factor that regulates cytoprotective genes. We have reported that NF-kappaB is constitutively activated in HNSCC, but the relationship of NF-kappaB to GSH and to cisplatin and radiation sensitivity in HNSCC is unknown. METHODS: We examined human HNSCC lines to define the relationship of cisplatin and radiation sensitivity to intracellular GSH and NF-kappaB and determined whether HNSCC could be sensitized to these modalities by lowering the concentration of glutathione with L-buthionine sulfoximine or inhibiting activation of NF-kappaB by expression of a degradation-resistant mutant inhibitor-kappaBalpha. RESULTS: Cisplatin resistance did not predict radiation resistance in three HNSCC cell lines, UM-SCC-9, 11B, and, 38. Resistance to cisplatin correlated with intracellular GSH, and depletion of GSH by treatment with L-BSO sensitized UM-SCC-9 cells to cisplatin but not radiation. Conversely, radiation resistance was correlated with activation of NF-kappaB. Expression of a mutant Inhibitor-kappaB after gene transfer inhibited NF-kappaB and sensitized UM-SCC-9 cells to radiation but not cisplatin. CONCLUSIONS: GSH and transcription factor NF-alphaB can contribute independently to cisplatin and radiation sensitivity of human HNSCC. These results highlight the need to define molecular determinants of chemotherapy and radiation sensitivity for use in the selection of patients and as novel targets for therapy in future chemoradiation therapy trials for organ preservation in patients with HNSCC.