University at Buffalo - The State University of New York
Skip to Content
27132513[PMID] - PMC - NCBI

Display Settings:

Items per page

Search results

Items: 8

1.
Figure 8

Figure 8. Mechanistic model illustrating effects of TNF-α modulation of cREL, ΔNp63α and TAp73 chromatin occupancy and reprogramming of TAp73 from TP53 to AP-1 sites to promote inflammatory and cancer gene programs. From: TNF-α modulates genome-wide redistribution of ΔNp63α/TAp73 and NF-κB c-REL interactive binding on TP53 and AP-1 motifs to promote an oncogenic gene program in squamous cancer.

Most head and neck and solid cancers have high frequencies of TP53 mutation, where TAp73 can serve as a potential tumor suppressor. In this model, without TNF-α, TAp73 predominantly occupies TP53 or p63 binding sites, while NF-κB family member cREL either resides in the cytoplasm or in the nucleus with binding on AP-1 sites, and ΔNp63α is found in the nucleus unbound to DNA (upper left). TNF-α, a major inflammatory cytokine produced in the tumor microenvironment, can promote cREL nuclear translocation, and complexes with ΔNp63α, to occupy TP53/p63 binding sites. TNF-α also induces nuclear displacement or reprogramming of TAp73 to bind neighboring AP-1 sites (upper and bottom right). These dynamic alterations diminish TAp73 tumor suppressor activity, while enhancing cREL/ΔNp63α and TAp73 mediated inflammatory and cancer gene programs (lower left). This model helps explains how TNF-α modulates NF-κB and AP-1 signaling while altering tumor suppressor activity, to promote gene programs implicated in inflammation, survival, and metastasis.

Han Si, et al. Oncogene. ;35(44):5781-5794.
2.
Figure 4

Figure 4. cREL, ΔNp63α, and TAp73 modulate transcriptional regulation and differential gene expression. From: TNF-α modulates genome-wide redistribution of ΔNp63α/TAp73 and NF-κB c-REL interactive binding on TP53 and AP-1 motifs to promote an oncogenic gene program in squamous cancer.

(A) UM-SCC46 cells were transfected with luciferase reporter plasmids containing specific TP53, AP-1, or NF-κB REs (upper panels), or the promoter sequences of CDKN1A (p21) (TP53, TP63 REs), SERPINE1 (AP-1 REs) or IL-6 (AP-1 and NF-κB REs; lower panels). Overexpression of cREL, ΔNp63α, or TAp73α was induced by TNF-α treatment (20 ng/ml) for 48 h. IL-6 binding site-specific point mutant promoter constructs included the deletion mutation of NF-κB or AP-1 binding sites without TNF-α (bottom right panel). The relative reporter activity was normalized to the corresponding β-gal activity and/or compared with the control vectors. Blue, untreated; red, TNF-α treated, except in IL-6 reporter assay (bottom right panel). In IL-6 reporter assay without TNF-α treatment, blue: reporter with full length IL-6 promoter; red: IL-6 promoter with the NF-κB binding motif deleted; green: IL-6 promoter with the AP-1 binding motif deleted. (B) The newly identified target genes were validated by q-RT-PCR 48 h after cREL, ΔNp63α, or TAp73α overexpression under TNF-α treatment (20 ng/ml). Blue, untreated; red, TNF-α treated. The data are presented as the mean ± SD of three replicates from one representative experiment. Statistical significance was calculated using a two-tailed Student's T-Test, p<0.05. * indicates the statistical significance when comparing the conditions with overexpressed plasmids versus control plasmid. # indicates the statistical significance when comparing untreated versus TNF-α treated condition.

Han Si, et al. Oncogene. ;35(44):5781-5794.
3.
Figure 3

Figure 3. Validation of co-localized binding activities of cREL, p63α and TAp73. From: TNF-α modulates genome-wide redistribution of ΔNp63α/TAp73 and NF-κB c-REL interactive binding on TP53 and AP-1 motifs to promote an oncogenic gene program in squamous cancer.

Based on overlapped binding peaks in the regulatory regions, we predicted core binding sequences containing potential p63/TP53 or NF-κB/cREL binding motifs using a bioinformatics approach. PCR primer sequences were designed to flank the regions containing the binding motifs (). In the genes of interest, the overlapping binding peaks were located in promoter/enhancer regions (SERPINE1, BCL3, CEBPA, HBEGF) or in first (CDKN1A, FOSL1, TNFSF10) or other (GADD45A) introns. Quantitative PCR was performed in independent ChIP experiments, and isotype antibody served as the negative control. (A-C) ChIP binding activity of cREL (A), p63α (B), and TAp73 (C). Blue, untreated; red, TNF-α treated. (D) The DNA sequences were extracted from ChIP-seq peaks and the core p53, p63, AP-1, NF-κB, cREL consensus motifs were predicted and depicted. Nuclear extracts were isolated from UM-SCC46 cells and the binding assays were performed using a 96-well colorimetric binding assay with 50-70mer oligos containing the 10-20bp motifs synthesized and labeled with biotin (). Open bars, anti-p63α antibody; dashed bars, anti-TAp73 antibody. Neg: negative control using p21 control oligo without lysate. p21-P: positive control using a p21 oligo containing the known TP53/TP63 site. The data are presented as the mean ± SD calculated from three replicates from one representative experiment.

Han Si, et al. Oncogene. ;35(44):5781-5794.
4.
Figure 5

Figure 5. TNF-α modulates expression of a global gene repertoire bound by cREL, p63α, and TAp73. From: TNF-α modulates genome-wide redistribution of ΔNp63α/TAp73 and NF-κB c-REL interactive binding on TP53 and AP-1 motifs to promote an oncogenic gene program in squamous cancer.

RNA was isolated from cells treated with TNF-α for 1, 3, 6, 12, and 24 h and the differential gene expression was examined by an Illumina bead-based array. (A) Differential expression of up- and down-regulated genes in UM-SCC46 cells under basal level conditions when compared with normal human oral keratinocyte cells, and (B) altered gene expression of UM-SCC46 cells upon TNF-α treatment. The colored sections represent the percentages of differentially expressed genes with binding activities for cREL, p63, or TAp73. (C) Gene numbers with individual and intersecting binding activities among the three TFs at the basal level, or (D) after TNF-α treatment. Top Venn diagrams represent up-regulated genes, and bottom Venn diagrams represent down-regulated genes. (E) Heat maps of hierarchical cluster analysis of 46 up-regulated (left) and 27 down-regulated (right) genes with overlapped TF binding activities induced by TNF-α. Red, increased expression; blue, decreased expression (compared with untreated controls). Color key, Z-score, reflects the relationship of the value of gene expression in a specific sample to the mean of the expression values of the same gene in all the samples. Euclidean distance with complete linkage was used to constitute the gene cluster.

Han Si, et al. Oncogene. ;35(44):5781-5794.
5.
Figure 1

Figure 1. TNF-α promotes genome-wide cREL, p63α and TAp73 binding activities in the regulatory regions. From: TNF-α modulates genome-wide redistribution of ΔNp63α/TAp73 and NF-κB c-REL interactive binding on TP53 and AP-1 motifs to promote an oncogenic gene program in squamous cancer.

(A) UM-SCC46 cells were treated with TNF-α (20 ng/ml) for 1 h to induce altered cREL, p63, and p73 protein expression in the nucleus. Oct-1 was used as the loading control. (B) ChIP-seq was performed using antibodies against cREL, p63α, and TAp73 for the UM-SCC46 cells treated (TNF) and untreated (NT) with TNF-α. The pulled-down DNAs were sequenced by the high-throughput sequencer GAIIx from Illumina. Total peak numbers (left) and peak-bound genes (right) are presented in the bar graph. (C) Characterization of the modulation of TF binding in the regulatory regions. The distribution of binding peaks in different regions of the genome is presented in the pie charts, and peak numbers are labeled at the top. The upper panel shows the percentages of the peaks distributed among regulatory (promoter, TTS, intragenic) and intergenic regions. The lower panel shows the percentages of the peaks distributed only among the different intragenic regions. (D) The binding peak numbers of each TF were plotted within 20 kb upstream (−) and downstream of the TSSs across the whole genome. The blue line is the basal transcription factor binding without TNF-α treatment; while the green line is after TNF-α treatment. (E) The transcription factor binding sites within 1 kb distance were identified, and the number of overlapping sites are presented in the Venn diagrams. (F) Distance relationships between two peak sets under different conditions in the ChIP-seq experiments were analyzed. Intersected peaks were defined to be within 1kb distance on the same chromosome. Y axis shows the quantity of intersected peaks at different intersection distance in bp (x axis). Blue, untreated; green, TNF-α treated.

Han Si, et al. Oncogene. ;35(44):5781-5794.
6.
Figure 2

Figure 2. De novo motif search identified TP53 and AP-1 consensus sequences. From: TNF-α modulates genome-wide redistribution of ΔNp63α/TAp73 and NF-κB c-REL interactive binding on TP53 and AP-1 motifs to promote an oncogenic gene program in squamous cancer.

The motifs most frequently bound by p63, TAp73 and c-REL transcription factors were identified by MEME-ChIP () or Gibbs Motif Sampler (). They are shown as the sequence logos with known consensus (A-D left). (A, left) The predominant TP53/p63 motifs consistent with the TP53.02 consensus sequences were identified in basal binding of TAp73. (B, left) The predominant motif of TP63 was identified by TP63α binding activities after TNF-α treatment. (C, left) AP-1 motif was identified in basal cREL binding. (D, left) The AP-1 motif was observed in TNF-α induced TAp73 binding. (A-D, right) Distance relationships between different motifs detected by ChIP-Seq were examined. Motifs are considered to be intersecting if they are located within 1 kb distance. The corresponding motifs were mapped back to peak sequences. Motif density (y axis) was plotted against the distance from the center of the binding peak (x axis), showing the distribution pattern of specific motifs in peaks (scales are labeled as ×102). (E) The relative interaction on the TP53 motif (y axis) was plotted against the distance of basal TAp73 binding vs. TP63α after TNF-α treatment. Under TNF treated condition, the relative interaction (x axis) of TP63 binding on TP53 motifs and TAp73 binding on AP-1 motif was plotted against the distance between the two binding motifs (x axis, right). (F) Sedimentation coefficient distribution of purified cRel Rel homology domain (RHD, left) and p73 DNA binding domain (DBD, right) binding to fluorescein-labeled AP-1 (black), cREL (red) and TP53 (blue) response elements (REs). Fluorescein absorbance was detected at 488 nm. The peaks corresponding to the protein dimers and tetramers are indicated. No tetrameric binding of p73 DBD was observed to the AP-1 and cREL REs. For clarity the peak at 2 S for the unbound DNA species is not displayed.

Han Si, et al. Oncogene. ;35(44):5781-5794.
7.
Figure 7

Figure 7. Squamous cancer signatures by CircleMaps and PARADIGM SuperPathway analyses. From: TNF-α modulates genome-wide redistribution of ΔNp63α/TAp73 and NF-κB c-REL interactive binding on TP53 and AP-1 motifs to promote an oncogenic gene program in squamous cancer.

(A) CircleMap of PARADIGM-Shift differences associated with SCC tissue origins and TP53 mutation status were created for TP53, TP63, TP73 and target genes identified in this study. Samples were ordered first by tissue origin of SCC (innermost ring), then by TP53 mutation status (second ring), GISTIC score (indicating CNV), mRNA expression level, and finally by PARADIGM activities (outer ring). The Red-blue color intensity reflects magnitude of CNV, expression and PARADIGM activities (red: high, blue: low). TP53 mutation is highlighted (black: truncation, gray: missense). Samples were restricted to the C2-Squamous-like cluster-of-cluster-assignments (COCA), including 156 lung SSC, 293 HNSCC, and 24 bladder SCC samples. Each plot illustrates multiple data types across many samples for a given gene. (B) The PARADIGM activity of ΔNp63 or TP73, and expression of eight target genes are higher across SCC tumors compared with other cancer types as shown in (A). Pearson correlation coefficients between ΔNp63 or TP73 PARADIGM activity and eight target genes presented in (A) were calculated, and the significance of p value was presented in y axis. The PARADIGM activity of ΔNp63 exhibited significant positive correlations with expression of five target genes bound and inducible by TNF-α, and a negative correlation with CEBPA expression. The PARADIGM activity of TP73 exhibited negative associations with with IL-6 and SERPINE1, and a positive association with CDKN1A (p21). (C) PARADIGM SuperPathway subnetwork defining C2-Squamous-like Pan-Cancer 12 integrative subtype. Zoom-in view of network neighborhood surrounding the ΔNp63α tetramer, TAp63γ tetramer, RelA/p50 and Jun/Fos complexes. Color of the nodes reflects activation (red) or repression (blue) within the squamous subtype when compared with the mean of other tumor types. Edge color denotes interaction type: inhibitory (green) and activating (yellow). Node shape reflects feature type: protein (circle), complex (diamond), family or miRNA or RNA (square), abstract concepts (arrowhead). The target genes with cRel, p63 and TAp73 binding identified in this study are highlighted with different colored outer rings as showing in .

Han Si, et al. Oncogene. ;35(44):5781-5794.
8.
Figure 6

Figure 6. cREL, ΔNp63, and TAp73 nuclear and cytoplasmic localization and target gene expression in human HNSCC tissues and skin of ΔNp63α transgenic mice. From: TNF-α modulates genome-wide redistribution of ΔNp63α/TAp73 and NF-κB c-REL interactive binding on TP53 and AP-1 motifs to promote an oncogenic gene program in squamous cancer.

(A) 46 up-regulated and 27 down-regulated genes from were queried using the mRNA expression dataset from TCGA HNSCC project, which includes 279 tumor and 16 mucosa specimens. Significant up-regulation of 13/46 activated genes (red), and down-regulation of 10/27 repressed genes (blue) are detected in TCGA tumors compared to normal samples (fold change ≥1.5, student t-Test with FDR < 1% multivariate comparison correction cut off). (B) Three publicly available datasets of gene profiling microarrays were investigated, which included a total of 125 (67 metastatic and 58 non-metastatic) HNSCC tissues. Pearson correlation coefficients of gene expression between p63 and potential target genes were calculated, and genes exhibiting statistically significant correlation are presented. The line indicates p<0.05. (C) RNA was isolated from the skins of ΔNp63α transgenic mice (red). qRT-PCR was performed to quantify gene expression levels with a statistically significant increase compared with their age-matched non-transgenic littermates (blue, p<0.05). The data were calculated from triplicates of one representative experiment and presented as the mean ± SD. (D) Human HNSCC and normal mucosa frozen sections were stained for cREL, ΔNp63, and TAp73, and intensities within nuclei or cytoplasm in three 200X fields per slide were acquired and quantified using an Aperio Scanscope and Cell Quantification Software (Vista, CA, USA), and presented as mean histoscores ± SD for 8-13 samples. * p<0.05, HNSCC vs normal mucosa by t-tests. (E) Immunohistochemistry comparing transcription factors JUNB and FOSL1 nuclear staining in human HNSCC tissue array. Images were acquired using an Aperio Scanscope at 200X magnification, and staining intensity was quantified using Aperio Cell Quantification Software. Tumor protein expression of evaluable specimens for JUNB and FOSL1 (n=66) and mucosa (n=11) samples. Student t-test, p<0.05 (F) Associations in expression levels between the transcription factors nuclear cREL with targets nuclear JUNB and IKKε (stage III tumors), nuclear cREL with nuclear FOSL1 (in all tumor stages), and nuclear TP63 with nuclear SERPINE1/PAI1 (metastatic tumors). A non-directional test for the significance of the Pearson Product-Moment Correlation Coefficient with the computed histoscores for each protein was used.

Han Si, et al. Oncogene. ;35(44):5781-5794.

Display Settings:

Items per page

Supplemental Content

Recent activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...
Support Center