Journal: Cell death & disease

Article Title: ETV7 reduces inflammatory responses in breast cancer cells by repressing the TNFR1/NF-κB axis.

doi: 10.1038/s41419-023-05718-y

Figure Lengend Snippet: Fig. 6 An inverse correlation between ETV7 and TNFR1 in BC patients. Three samples from BC patients with invasive ductal carcinoma were analyzed by IHC. Tissues were stained with antibodies against ETV7 and TNFR1 proteins and studied at ×10 magnification. A ×20 magnification insert is shown for each image. Top panels: tissues from a HER2-positive BC; middle panel: tissues from a triple-negative BC; bottom panels: tissues from a luminal A BC.

Article Snippet: Then, the samples were diluted and incubated with 2 μg the appropriate antibody targeting ETV7 (Santa Cruz, TEL2, E-1), pSTAT3 (Cell Signaling Technologies, 124H6), H3K9me3 (Cell Signaling Technologies, 13969P), H3K4me3 (Abcam, ab8580), H3ac (Abcam, ab47919) or IgG (Santa Cruz Biotechnologies, mouse or rabbit according to the antibody used) and Dynabeads with protein G or A (Life Technologies) overnight at 4 °C in a rotator.

Techniques: Staining

Journal: Cell death & disease

Article Title: ETV7 reduces inflammatory responses in breast cancer cells by repressing the TNFR1/NF-κB axis.

doi: 10.1038/s41419-023-05718-y

Figure Lengend Snippet: Fig. 7 ETV7 can compete with STAT3 in the regulation of the TNFRSF1A gene influencing the NF-κB regulatory pathway. A The canonical STAT3/TNF-α/NF-κB regulatory pathway. STAT3 binds to its regulatory element in the first intron of the TNFRSF1A gene and induces its expression by recruiting chromatin remodelers that result in an “active” state. Consequently, the TNF-α receptor 1 is produced. TNF-α molecules bind the TNFR1 receptor and activate the NF-κB signaling pathway. B In the context where ETV7 expression is increased, ETV7 can displace STAT3 from its binding sites on the Intron 1 of TNFRSF1A and directly represses its expression by altering the deposition of histone marks. This ETV7-mediated repression leads to the reduced activation of NF-κB signaling and, hence, reduces the expression of pro- inflammatory genes.

Article Snippet: Then, the samples were diluted and incubated with 2 μg the appropriate antibody targeting ETV7 (Santa Cruz, TEL2, E-1), pSTAT3 (Cell Signaling Technologies, 124H6), H3K9me3 (Cell Signaling Technologies, 13969P), H3K4me3 (Abcam, ab8580), H3ac (Abcam, ab47919) or IgG (Santa Cruz Biotechnologies, mouse or rabbit according to the antibody used) and Dynabeads with protein G or A (Life Technologies) overnight at 4 °C in a rotator.

Techniques: Expressing, Produced, Binding Assay, Activation Assay

Journal: Cell Death & Disease

Article Title: ETV7 reduces inflammatory responses in breast cancer cells by repressing the TNFR1/NF-κB axis

doi: 10.1038/s41419-023-05718-y

Figure Lengend Snippet: A Gene Set Enrichment Analysis of MCF7 cells over-expressing ETV7 or its Empty counterpart. Enrichment plot for the inflammatory response in MCF7 cells (on the left) and TNFA signaling via NF-κB in MCF7 cells (on the right) gene sets of the Hallmark Collection. The Normalized Enrichment Score (NES) shows the degree of the enrichment of the gene set; the negative sign indicates that the gene set is down-regulated in cells over-expressing ETV7. FDR = False Discovery Rate. B RT-qPCR analysis for the validation of genes involved in inflammation and immune response in MCF7 (on the left) and T47D (on the right) cells over-expressing ETV7 or Empty vector. Bars represent the averages and standard deviations of at least three biological replicates. C The expression of TNFRSF1A at the protein level in MCF7 and T47D cells over-expressing ETV7 or its empty counterpart. On the right of each blot is indicated the approximate observed molecular weight. HSP70 was used as a loading control. D RT-qPCR analysis of the normalized expression of the TNFRSF1A gene in MDA-MB-231 and SK-BR-3 cells transiently over-expressing ETV7 or harboring its Empty counterpart. Bars demonstrate the averages and standard deviations of at least three biological replicates. E RT-qPCR analysis of the normalized expression of the TNFRSF1A gene in MCF7, T47D, MDA-MB-231, and ZR-75-1 cells transfected with ETV7 targeting siRNA #1 and siRNA #2 or the scrambled control. F On the left, a dot plot demonstrating the differential expression analysis for the TNFRSF1A gene in a BRCA-matched patients’ dataset from TCGA (The Cancer Genome Atlas) database. Tumor (light red), normal (light blue). On the right, a dot plot of TNFRSF1A expression in ER-positive and triple-negative breast cancer patient cohort when comparing tumor tissue (light red) with matched adjacent tissue (light blue). ER+—Estrogen Receptor-positive; TNBC—triple-negative breast cancer; Adj—adjacent tissue. Indicated are the means and the SEMs. G TNFRSF1A expression in TCGA BRCA samples classified by PAM50 molecular subtypes. H Correlation analysis between ETV7 and TNFRSF1A mRNA level in TNBC patients from University Medical Center Hamburg-Eppendorf. Significance and numerosity are indicated. I Kaplan–Meier curves for RFS from a breast cancer cohort according to the relative expression of TNFRSF1A obtained with the KM plotter tool. The number of patients is shown below the graph. HR (Hazardous Ratio) and the statistical analyses are reported in the right corner of the graph. Whole panel: * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; n.s. not significant.

Article Snippet: After 24 h, cells were treated with 20 ng/ml IL-6 and 4 h post-treatment lysed using CHAPS buffer and incubated overnight with 2 μg of an anti-ETV7 antibody (TEL2, Santa Cruz Biotechnologies) or normal mouse IgG (Santa Cruz Biotechnologies) previously bound with Dynabeads protein G magnetic beads (Life Technologies).

Techniques: Expressing, Quantitative RT-PCR, Biomarker Discovery, Plasmid Preparation, Molecular Weight, Control, Transfection, Quantitative Proteomics

Journal: Cell Death & Disease

Article Title: ETV7 reduces inflammatory responses in breast cancer cells by repressing the TNFR1/NF-κB axis

doi: 10.1038/s41419-023-05718-y

Figure Lengend Snippet: A A schematic view of the TNFRSF1A Intron 1 and the studied ETV7 binding sites. TNFRSF1A BS#1 is located +5483 bp from the Transcription Start Site (TSS); BS#2 + 5627 bp from TSS; BS#3 + 6069 bp from TSS. B , C ChIP-qPCR of TNSFRSF1A Intron 1 in MCF7 ( B ) or T47D ( C ) cells over-expressing ETV7. The percentage of the enrichment of ETV7 or control (normal mouse IgG) bound to TNFRSF1A Intron 1 with respect to input DNA is shown. NSB—non-specific binding, the ACTB promoter. D , E Chip-qPCR assessing H3K9me3, H3K4me3, and H3ac (pan-acetyl) deposition at the TNFRSF1A Intron 1 binding site #1 ( D , F ) and binding site #2 ( E , G ) in MCF7 and T47D over-expressing ETV7 or its empty counterpart. The percentage of the enrichment of ETV7 or control (normal rabbit IgG) bound to TNFRSF1A Intron 1 with respect to input DNA is shown. Whole panel: Bars represent the averages and standard deviations of at least three biological replicates. * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001, n.s. not significant.

Article Snippet: After 24 h, cells were treated with 20 ng/ml IL-6 and 4 h post-treatment lysed using CHAPS buffer and incubated overnight with 2 μg of an anti-ETV7 antibody (TEL2, Santa Cruz Biotechnologies) or normal mouse IgG (Santa Cruz Biotechnologies) previously bound with Dynabeads protein G magnetic beads (Life Technologies).

Techniques: Binding Assay, ChIP-qPCR, Expressing, Control

Journal: Cell Death & Disease

Article Title: ETV7 reduces inflammatory responses in breast cancer cells by repressing the TNFR1/NF-κB axis

doi: 10.1038/s41419-023-05718-y

Figure Lengend Snippet: A Gene reporter assays in MCF7 and T47D cells over-expressing ETV7 or its empty counterpart transiently transfected with the pGL3-NF-κB reporter plasmid. Data is normalized using the Renilla reniformis luciferase reporter vector pRL-SV40 and shown as fold of induction relative to the Empty control. B Gene reporter assays in MCF7 (on the left) and T47D (on the right) cells over-expressing ETV7 or its empty counterpart transfected with the pGL3-NF-κB reporter plasmid and stimulated with TNF-α (10 ng/ml for MCF7 and 15 ng/ml for T47D), IL-6 (20 ng/ml) or combination of both for 4 h. Data are normalized using the Renilla reniformis luciferase reporter vector pRL-SV40 and shown as fold of induction relative to the Empty control. C – F RT-qPCR analysis of known NF-κB target genes: A20 ( C ), IL-8 ( D ), IL-6 ( E ), and TNF-α ( F ) in MCF7 ETV7 or Empty cells treated with TNF-α (10 ng/ml) for 4 h. Bars represent the averages and standard deviations of at least three biological replicates. G RT-qPCR analysis of the normalized expression of NF-κB target genes in MCF7 parental cells transfected with ETV7 targeting siRNA #1 and siRNA #2 or the scrambled control. Whole panel: Bars represent the averages and standard deviations of at least three biological replicates. * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001, n.s. not significant.

Article Snippet: After 24 h, cells were treated with 20 ng/ml IL-6 and 4 h post-treatment lysed using CHAPS buffer and incubated overnight with 2 μg of an anti-ETV7 antibody (TEL2, Santa Cruz Biotechnologies) or normal mouse IgG (Santa Cruz Biotechnologies) previously bound with Dynabeads protein G magnetic beads (Life Technologies).

Techniques: Expressing, Transfection, Plasmid Preparation, Luciferase, Control, Quantitative RT-PCR

Journal: Cell Death & Disease

Article Title: ETV7 reduces inflammatory responses in breast cancer cells by repressing the TNFR1/NF-κB axis

doi: 10.1038/s41419-023-05718-y

Figure Lengend Snippet: A Immunofluorescence analysis for the p65 (green signal) nuclear translocation in MCF7 Empty and MCF7 ETV7 cells. Nuclei are stained in blue. MCF7 cells were left untreated or stimulated with 10 ng/ml TNF-α for 60 min. Shown are representative data for at least three biological replicates. Images were acquired with a ×20 magnification. The arrows indicate the nuclear localization of p65. B Quantification of nuclear:cytoplasmic ratios of p65 fluorescence intensity in MCF7 Empty and MCF7 ETV7 cells. Bars represent mean ± standard deviation from analysis of 10 (per each biological replicate; n = 3) separated field images. C Western blot analysis of phosphorylated IκBα in MCF7 Empty and ETV7 cells in response to 10 ng/ml TNF-α treatment for 1 h. On the right of each blot is indicated the approximate observed molecular weight. GAPDH was used as a loading control. D The secretion of IL-8 and IL-6 in the supernatant of MCF-7 Empty and MCF7 ETV7 cells without or with stimulation with TNF-α (10 ng/ml), measured by ELISA. Bars represent the mean and the standard deviation of three biological replicates. E Gene reporter assays in MCF7 Empty or MCF7 ETV7 cells transfected with the pGL3-NF-κB reporter vector and pcDNA3.1-TNFR1 or pcDNA3.1-Empty plasmids and untreated or treated with 10 ng/ml TNF-α for 4 h. Data is normalized using the Renilla reniformis luciferase reporter vector pRL-SV40 and shown as fold of induction relative to the Empty untreated control. F A dot plot demonstrating the differential expression analysis for the TNF_SIGNALING_VIA_NFkB gene set in a BRCA-matched patients’ dataset from TCGA (The Cancer Genome Atlas) database. Tumor (light red), Normal (light blue). G Kaplan–Meier curves for TCGA breast cancer patients stratified according to the average expression of TNFA_SIGNALING_VIA_NFkB gene signature. Curves represent the probability of disease-specific survival (DSS). p -values are calculated with the log-rank test. H TNFA_SIGNALING_VIA_NFkB gene set expression in TCGA BRCA samples classified by PAM50 molecular subtypes. p -values are shown when significant. Whole panel: * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001.

Article Snippet: After 24 h, cells were treated with 20 ng/ml IL-6 and 4 h post-treatment lysed using CHAPS buffer and incubated overnight with 2 μg of an anti-ETV7 antibody (TEL2, Santa Cruz Biotechnologies) or normal mouse IgG (Santa Cruz Biotechnologies) previously bound with Dynabeads protein G magnetic beads (Life Technologies).

Techniques: Immunofluorescence, Translocation Assay, Staining, Fluorescence, Standard Deviation, Western Blot, Molecular Weight, Control, Enzyme-linked Immunosorbent Assay, Transfection, Plasmid Preparation, Luciferase, Quantitative Proteomics, Expressing

Journal: Cell Death & Disease

Article Title: ETV7 reduces inflammatory responses in breast cancer cells by repressing the TNFR1/NF-κB axis

doi: 10.1038/s41419-023-05718-y

Figure Lengend Snippet: A Canonical ETV7 and STAT3 binding sites known from the literature. B Gene Set Enrichment Analysis of MCF7 cells over-expressing ETV7 or its Empty counterpart. Enrichment plot for IL6_JAK_STAT3 signaling in MCF7 cells gene sets of the Hallmark Collection. The Normalized Enrichment Score (NES) shows the degree of the enrichment of the gene set; the negative sign indicates that the gene set is down-regulated in cells over-expressing ETV7. FDR = False Discovery Rate. C Western blot analysis of subcellular fractionation from MCF7 Empty and MCF7 ETV7 cells untreated or treated with IL-6 (20 ng/ml) for 4 h. On the right of each blot is indicated the approximate observed molecular weight. Cyt—cytoplasmic protein fraction, Chr—chromatin-enriched protein fraction. GAPDH was used as a loading control for the cytoplasmic fraction. Histone H3 was used as a loading control for chromatin-enriched protein fraction. D , E ChIP-qPCR of TNSFRSF1A Intron 1 Binding site #1 ( D ) and Binding site #2 ( E ) in MCF7 cells over-expressing ETV7 untreated or treated with IL-6 (20 ng/ml) for 4 h. The percentage of the enrichment of pSTAT3 or control (normal rabbit IgG) bound to TNFRSF1A Intron 1 with respect to Input DNA is shown. NSB—non-specific binding, a region within the ACTB promoter. BS —binding site. Bars represent the averages and standard deviations of at least three biological replicates. F) Gene reporter assays in MCF7 Empty/ETV7 (on the left) and T47D Empty/ETV7 (on the right) cells transfected with M67-STAT3 reporter untreated or treated with IL-6 (20 ng/ml) for 4 h. Data are normalized using the Renilla reniformis luciferase reporter vector pRL-SV40 and shown as fold of induction relative to the Empty untreated control. Whole panel: * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001.

Article Snippet: After 24 h, cells were treated with 20 ng/ml IL-6 and 4 h post-treatment lysed using CHAPS buffer and incubated overnight with 2 μg of an anti-ETV7 antibody (TEL2, Santa Cruz Biotechnologies) or normal mouse IgG (Santa Cruz Biotechnologies) previously bound with Dynabeads protein G magnetic beads (Life Technologies).

Techniques: Binding Assay, Expressing, Western Blot, Fractionation, Molecular Weight, Control, ChIP-qPCR, Transfection, Luciferase, Plasmid Preparation

Journal: Cell Death & Disease

Article Title: ETV7 reduces inflammatory responses in breast cancer cells by repressing the TNFR1/NF-κB axis

doi: 10.1038/s41419-023-05718-y

Figure Lengend Snippet: Three samples from BC patients with invasive ductal carcinoma were analyzed by IHC. Tissues were stained with antibodies against ETV7 and TNFR1 proteins and studied at ×10 magnification. A ×20 magnification insert is shown for each image. Top panels: tissues from a HER2-positive BC; middle panel: tissues from a triple-negative BC; bottom panels: tissues from a luminal A BC.

Article Snippet: After 24 h, cells were treated with 20 ng/ml IL-6 and 4 h post-treatment lysed using CHAPS buffer and incubated overnight with 2 μg of an anti-ETV7 antibody (TEL2, Santa Cruz Biotechnologies) or normal mouse IgG (Santa Cruz Biotechnologies) previously bound with Dynabeads protein G magnetic beads (Life Technologies).

Techniques: Staining

Journal: Cell Death & Disease

Article Title: ETV7 reduces inflammatory responses in breast cancer cells by repressing the TNFR1/NF-κB axis

doi: 10.1038/s41419-023-05718-y

Figure Lengend Snippet: A The canonical STAT3/TNF-α/NF-κB regulatory pathway. STAT3 binds to its regulatory element in the first intron of the TNFRSF1A gene and induces its expression by recruiting chromatin remodelers that result in an “active” state. Consequently, the TNF-α receptor 1 is produced. TNF-α molecules bind the TNFR1 receptor and activate the NF-κB signaling pathway. B In the context where ETV7 expression is increased, ETV7 can displace STAT3 from its binding sites on the Intron 1 of TNFRSF1A and directly represses its expression by altering the deposition of histone marks. This ETV7-mediated repression leads to the reduced activation of NF-κB signaling and, hence, reduces the expression of pro-inflammatory genes.

Article Snippet: After 24 h, cells were treated with 20 ng/ml IL-6 and 4 h post-treatment lysed using CHAPS buffer and incubated overnight with 2 μg of an anti-ETV7 antibody (TEL2, Santa Cruz Biotechnologies) or normal mouse IgG (Santa Cruz Biotechnologies) previously bound with Dynabeads protein G magnetic beads (Life Technologies).

Techniques: Expressing, Produced, Binding Assay, Activation Assay