Transforming growth factor-beta (TGF-β) has a dual role in epithelial malignancies, including head and neck squamous cell carcinoma (HNSCC). Attenuation of canonical TGF-β signaling enhances de novo ...tumor development, whereas TGF-β overexpression and signaling paradoxically promotes malignant progression. We recently observed that TGF-β-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-β and NF-κB pathways plays in altered activation of these pathways has not been established. Here, we show TGF-β receptor II and TGF-β-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-β1 treatment induced sequential phosphorylation of TAK1, IKK, IκBα and RELA in human HNSCC lines. TAK1 enhances TGF-β-induced NF-κB activation, as TAK1 siRNA knockdown decreased TGF-β1-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-β1-induced phosphorylation of TAK1 and RELA, and suppressed basal, TGF-β1- 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-β and RELA activation promoted SMAD7 expression. In turn, SMAD7 preferentially suppressed TGF-β-induced SMAD and NF-κB reporters when compared with constitutive or TNF-α-induced NF-κB reporter gene activation. Thus, cross-talk by TGF-β via TAK1 and NF-κB promotes the malignant phenotype of HNSCC. Moreover, NF-κB may contribute to the downstream attenuation of canonical TGF-β signaling through increased SMAD7 expression. Celastrol highlights the therapeutic potential of agents targeting TAK1 as a key node in this pro-oncogenic TGF-β-NF-κB signal pathway.
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.
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 IKKβ or EGFR is observed in head and neck squamous cell carcinomas (HNSCC). Here, we reveal that both IKKα and β 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 IKKβ 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 IKKβ 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/β and EGFR pathways were more active than IKKβ-specific inhibitors in suppressing NF-κB activation and proliferation and inducing cell death. We conclude that IKKα and IKKβ 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.
The molecular mechanisms that contribute to the initiation and progression of head and neck squamous cell carcinoma (HNSCC) have not been completely delineated. Our observations indicate that defects ...in the transforming growth factor-β and PI3K/Akt signaling pathways are common in human HNSCCs. Conditional activation of the PI3K/Akt pathway due to Pten deletion in the mouse head and neck epithelia gives rise to hyperproliferation, but only a few lesions progress to HNSCC. However, Pten-deficient mice developed full-penetrance HNSCC in combination with type I TGF-β receptor (Tgfbr1) deletion. Molecular analysis revealed enhanced cell proliferation, decreased apoptosis, and increased expression of CCND1 in the basal layer of the head and neck epithelia, as well as in the tumors of Tgfbr1/Pten double conditional knockout (2cKO) mice. Furthermore, neoplastic transformation involves senescence evasion, and is associated with an increased number of putative cancer stem cells. In addition, the nuclear factor-κB pathway activation, myeloid-derived suppressor cell infiltration, angiogenesis and immune suppression in the tumor microenvironment, all of which are characteristics of human HNSCCs, contribute significantly to head and neck carcinogenesis in 2cKO mice. These tumors display pathology and multiple molecular alterations resembling human HNSCCs. This suggests that the Tgfbr1/Pten 2cKO mouse model is suitable for preclinical intervention, and that it has significant implications in the development of diagnostic cancer biomarkers and effective strategies for prevention and treatment of HNSCCs.
In order to introduce drought tolerance and improved cell wall digestibility from fescue in fodder ryegrasses, we developed two amphiploid Festulolium synthetics. One is a synthetic composed of three ...selected drought tolerant F1 hybrid genotypes from a cross between tetraploid Lolium multiflorum and hexaploid Festuca arundinacea, further on called LMFA. The other is a synthetic composed of five selected genotypes with soft leaves from a cross between tetraploid Lolium perenne and tetraploid Festuca pratensis, further on called LPFP. We produced seeds in polycrosses of two generations of both amphiploids, i.e., syn1 and syn2, and tested them in plot trials to determine the yield and fodder quality. The syn1 of both Festulolium populations had a higher annual dry matter yield than the reference Lolium cultivars and Festulolium cultivars composed of the same parental species. However, the syn2 of LMFA did not show an improved drought tolerance during a dry growing season compared to other Festulolium cultivars, and the seed yield of LMFA syn1 was low and dropped extremely in syn2. The number of chromosomes of LMFA also decreased gradually from F1 to syn2, and there was a clear shift in chromosome composition towards the Lolium genome. The LPFP synthetic performed better. Although the sugar content was significantly lower than the sugar content of the perennial ryegrass cultivars, organic matter digestibility (OMD) of LPFP was as high as OMD of the tetraploid perennial ryegrass cultivars. The cell wall digestibility (NDFD) of LPFP was significantly higher than the NDFD of both parental species and higher than the NDFD of all tested Festulolium cultivars. The seed yield of LPFP was the same in syn1 and syn2. The chromosome number remained on average the same and no clear shift of the chromosome composition to one of the composing genomes was observed. Overall, chromosome analysis revealed a high number of aneuploidy in syn1 and syn2 generations of both LMFA and LPFP and a lot of variation in number of Lolium, Festuca and recombinant chromosomes, and in the Lolium:Festuca genome ratio was observed among different genotypes of the same population. Therefore, selection for genotypes with a more stable genome composition will be a prerequisite for a sufficient seed yield and a broader exploitation of these new Festulolium synthetics.
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.
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.
Agricultural soils in northwest Europe as well as in parts of the USA have excessive P levels. To meet European environmental standards on nutrient leaching in soils with a high P load, the total P ...content of manures and composts limits their application rate. The aim of the present study was to determine the effect of feedstock composition on C/P ratio and P availability in mature compost. A functional compost with a high C/P ratio and a high organic matter content is a valuable tool for maintaining soil fertility. We produced composts with C/P ratios of 60–180 and high organic matter contents, i.e., 43–72% on dry matter basis. P concentration in the compost increased and C/P ratio decreased due to mass reduction during the composting process, which was determined by the biochemical composition, i.e., the biodegradation potential of the feedstock mixture. The feedstock selection also had indirect effects on P availability due to its effect on compost characteristics such as pH, which strongly reduced the readily available P concentrations at
pH
H
2
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> 8.5. Compost pH was found to be an important indicator for readily available P concentrations in the studied composts with
pH
H
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between 7.1 and 9.4, and was positively related to total Ca content in the compost, indicating an interaction between Ca and P. In contrast, P availability was not related to the organic matter content of the composts. Mixing biochar (pyrolyzed organic matter) into the compost further reduced the readily available P in two types of compost by 10–64%, even after addition of a small amount of additional mineral P. This was observed for two types of biochar with different chemical composition. The reduction was higher for the biochar with the highest Ca, Fe and Al concentrations, indicating the role of these elements for P binding in the biochar-blended compost.
Graphical Abstract
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.
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•Chopped heath and spent growth media can be used as bulking agents.•Chopped heath compost has a high OM content and C/P ratio.•Clear differences in N immobilization between bulking agents were observed.•NIRS can monitor quickly the chemical properties and stability during composting.
Agroforestry systems (AFS) are considered to be a sustainable agricultural practice, as they deliver a wide range of ecosystem services (ES) while maintaining (agricultural) primary production. To ...optimize the productivity of AFS, the recommendation is to use well adapted tree-crop combinations, thereby limiting competition for resources and maximizing synergies. However, yield and quality data on arable crops in temperate AFS are scarce, in particular for AFS with a mature tree component. Here we assessed the influence of tree rows of contrasting age on yield and quality of key western European arable crops. We focused on (forage) maize, potato, winter wheat and winter barley during three consecutive years (2015–2017) on a set of 16 arable agroforestry fields in Belgium comprising 6 young (2–7 year old) alley cropping fields and 10 fields bordered by a row of deciduous trees of moderate to older age (15–48 years old). Both tree age and crop type were key determinants of yield and quality of the investigated arable crops. While effects on crop yield were limited for all crops near young tree rows, substantial yield reductions were observed near mature trees, in particular for maize and potato (both summer crops). Effects on crop quality were limited for all crops under study, with substantial effects only arising near the oldest tree rows. To optimize the provisioning service of AFS, the cultivation of winter cereals may be advisable over maize and potato towards the end of the rotation of an AFS. In addition, poplar trees should be harvested when they reach their target diameter for industrial processing. If tree rows are preserved for the delivery of other ES, however, substantial impacts on crop yield and quality should be taken into account.
•Effects of trees on crop yield were limited on young alley cropping fields.•Near to mature trees, yield and quality effects strongly varied depending on the crop.•Effects of trees on crop quality were smaller than on crop yield.•Near to mature trees, cultivation of winter cereals is preferable to maize and potato.
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