Journal: Frontiers in Oncology

Article Title: FOXA2-Interacting FOXP2 Prevents Epithelial-Mesenchymal Transition of Breast Cancer Cells by Stimulating E-Cadherin and PHF2 Transcription

doi: 10.3389/fonc.2021.605025

Figure Lengend Snippet: Identification of FOXA2-interacting transcription factor FOXP2 in breast cancer cells. (A) The lysates of MCF-7 cells transfected with pAvi-FOXA2, pEF1-BirA, or both, were incubated with streptavidin resin. The pull-down proteins were separated by PAGE and visualized with coomassie brilliant blue staining. The position of FOXP2 was pointed out in the lane of the FOXA2+BirA sample on the gel. (B, C) The interaction between FOXA2 and FOXP2 in cells. The lysates of cells transfected with pAvi-FOXA2 and pHis-FOXP2 or pEF1-BirA, were pulled down with streptavidin resin (B) or Ni beads (C) . Western blotting was performed with anti-FOXA2 antibody, anti-FOXP2 antibody, or streptavidin-conjugated HRP (SA-HRP) respectively. (D, E) Mapping the regions mediating FOXA2-FOXP2 interaction in the proteins. The lysates of cells transfected with pHis-FOXP2 plus one of expressing plasmid vectors containing Flag-tagged FOXA2 1-165aa, 166-324aa, and 409-633aa (D) , or the lysates of cells transfected with pCMV-FOXA2 plus one of expressing plasmid vectors containing Flag-tagged FOXP2 1-196aa, 197-408aa, 325-463aa (E) , were immunoprecipitated with anti-Flag antibody. Western blotting was performed with anti-FOXP2 antibody, anti-FOXA2 antibody, or anti-Flag antibody respectively. The top panel showed a schematic representation of the regions in FOXA2 protein (D) and FOXP2 protein (E) . (F) The interaction of endogenous FOXA2 and FOXP2 was detected by the immunofluorescent microscopy in MCF-7 cells. The staining for FOXA2 (red), for FOXP2 (green), for DAPI (blue), and the merge of the FOXA2 and FOXP2 staining were shown. The cells were pictured by TE2000S (Nikon, 200×). (G) The levels of FOXP2 in the four subgroups (Basal, Her2, LumA, and LumB) of breast cancer and normal breast tissue in the clinical breast cancer samples (the BRCA data set, n=394). (H, I) The gene expression correlation analysis of FOXA2 and FOXP2 in the BRCA data set (n=394) (H) and in the Basal subgroup (n=98) (I) were executed by using ggstatsplot package through R project. The pink and green histograms represented the distribution of BRCA samples based on the levels of FOXA2 and FOXP2 expression respectively.

Article Snippet: Human FOXA2 siRNA (Santa cruz, sc-35569) and control siRNA (Santa cruz, sc-37007) were purchased from Santa Cruz, USA.

Techniques: Transfection, Incubation, Staining, Western Blot, Expressing, Plasmid Preparation, Immunoprecipitation, Microscopy

Journal: Frontiers in Oncology

Article Title: FOXA2-Interacting FOXP2 Prevents Epithelial-Mesenchymal Transition of Breast Cancer Cells by Stimulating E-Cadherin and PHF2 Transcription

doi: 10.3389/fonc.2021.605025

Figure Lengend Snippet: List of candidate FOXA2-interacting proteins from the mass spectrometry screens.

Article Snippet: Human FOXA2 siRNA (Santa cruz, sc-35569) and control siRNA (Santa cruz, sc-37007) were purchased from Santa Cruz, USA.

Techniques: Mass Spectrometry, Histone Deacetylase Assay

Journal: Frontiers in Oncology

Article Title: FOXA2-Interacting FOXP2 Prevents Epithelial-Mesenchymal Transition of Breast Cancer Cells by Stimulating E-Cadherin and PHF2 Transcription

doi: 10.3389/fonc.2021.605025

Figure Lengend Snippet: The activation of the E-cadherin promoter by FOXP2 relied on the participation of FOXA2. (A) The E-cadherin promoter was activated by FOXP2 in breast cancer cells. A luciferase reporter plasmid (1.5 μg) containing the fragment of -733bp to +60 bp of E-cadherin promoter or a control luciferase reporter plasmid (1.5 μg) containing the fragment of -233 bp to +52 bp of E-cadherin promoter and loading control pRL-CMV luciferase reporter plasmid (20 ng) were transfected into MCF-7 cells with pHis-FOXP2 or pCMV-EGFP (1 μg). (B) FOXP2 bound to the endogenous E-cadherin promoter. Gene sequence analysis was performed to predict positions of putative FOXP2 binding sites in -1 kb human E-cadherin promoter and the primers for ChIP assays were designed. The chromatin of MCF-7 cells was cross-linked, sonicated, and immunoprecipitated (IP) with either FOXP2 antibody or rabbit IgG. The amount of promoter DNA associated with the IP chromatin was measured by qPCR with primers specific to E-cadherin promoter regions -760 bp to -610 bp and -414 bp to -280 bp. (C) FOXP2 bound to E-cadherin promoter region -701 bp to -691 bp. Nuclear extracts were prepared from pHis-FOXP2-transfected cells and used for EMSAs with a FAM-labeled DNA probe synthesized from E-cadherin promoter sequence -706 bp to −686 bp (E-cad pro). The unlabeled probe (50×) or 1 μg of FOXP2 antibody (α-FOXP2) was added to the reaction to show the specificity of FOXP2/DNA complex formation. EMSAs with a FAM-labeled mutated probe were also performed (Mut pro). (D) FOXP2 mediated the binding of FOXA2 to the E-cadherin promoter. Nuclear extracts were prepared from pHis-FOXP2-transfected, pCMV-FOXA2-transfected, or both-transfected cells (48 h) and used for EMSAs with a FAM-labeled DNA probe synthesized from E-cadherin promoter sequence -706 bp to -686 (E-cad probe). (E) Knockdown of FOXA2 abolished the activation of the E-cadherin promoter by FOXP2. A luciferase reporter plasmid (1.5 μg) containing the fragment of -733 bp to +60 bp of E-cadherin promoter and loading control pRL-CMV luciferase reporter plasmid (20 ng) were transfected into MCF-7 cells with the pHis-FOXP2 or pCMV-EGFP (1 μg) plus FOXA2 siRNA or Control siRNA (200 nM). Protein lysates were prepared at 36 h following transfection and used to measure dual luciferase enzyme activities. (F, G) The knockdown of FOXP2 and FOXA2 together showed a synergistic repression of the E-cadherin expression in breast cancer cells. The shCon MCF-7 cells and shFOXP2 MCF-7 cells were transfected with Control siRNA or FOXA2 siRNA (200 nM) and examined by qPCR for mRNA levels (F) and by Western blotting for protein levels (G) . (H) Overexpression of FOXA2 enhanced the activation of the E-cadherin promoter by FOXP2 in MDA-MB-231 cells. A luciferase reporter plasmid (1.5 μg) containing the fragment of -733 bp to +60 bp of E-cadherin promoter and loading control pRL-CMV luciferase reporter plasmid (20 ng) were transfected into MDA-MB-231 cells with the pCMV-FOXA2 (1 μg), pHis-FOXP2 (1 μg), or both (balanced with the pCMV-EGFP vector). Protein lysates were prepared at 36 h following transfection and used to measure dual luciferase enzyme activities. (I, J) The overexpression of FOXP2 and FOXA2 together showed a synergistic activation of the E-cadherin expression in MDA-MB-231 cells. The MDA-MB-231 cells were transfected with FOXA2 expression vector (pCMV-FOXA2), FOXP2 expression vector (pHis-FOXP2), or both (balanced with the pCMV-EGFP vector) and examined by Western blotting for protein levels (I) and by qPCR for mRNA levels (J) . The asterisks indicate statistically significant changes: **P ≤ 0.01, ***P ≤0.001.

Article Snippet: Human FOXA2 siRNA (Santa cruz, sc-35569) and control siRNA (Santa cruz, sc-37007) were purchased from Santa Cruz, USA.

Techniques: Activation Assay, Luciferase, Plasmid Preparation, Transfection, Sequencing, Binding Assay, Sonication, Immunoprecipitation, Labeling, Synthesized, Expressing, Western Blot, Over Expression

Journal: Frontiers in Oncology

Article Title: FOXA2-Interacting FOXP2 Prevents Epithelial-Mesenchymal Transition of Breast Cancer Cells by Stimulating E-Cadherin and PHF2 Transcription

doi: 10.3389/fonc.2021.605025

Figure Lengend Snippet: The roles of FOXP2 in EMT of breast cancer cells. (A) The Oncomine boxplots of FOXP2 levels were analyzed between invasive breast tissues and normal breast tissues from the oncomine.org website. The p-value of the TCGA RNA-seq data of normal breast samples (n=61) versus invasive breast carcinoma samples (n=76) was 7.76E -22 . (B) The FOXP2-improved survival in breast cancer patients relied on the participation of FOXA2. The FOXA2 High subgroup (n=131) or the FOXA2 Low subgroup (n=131) contained either the top 1/3 or the bottom 1/3 of the collected BRCA data set (n=394), respectively. FOXP2-related survival of patients in the FOXA2 High and FOXA2 Low subgroups was fitted by the “survfit” function, and Kaplan–Meier curves were drawn by the “ggsurv” function in the R package “survival”. (C) The roles of FOXP2 in epithelial-mesenchymal transition (EMT) of breast cancer cells. FOXP2 prevented EMT of breast cancer cells by regulating the transcription of multiple EMT-related genes: FOXP2 could bind to certain promoters and stimulate the transcription of genes such as PHF2 and E-cadherin. The transcriptional activation by FOXP2 could be mediated by FOXA2. FOXP2 could also repress the transcription of certain genes such as SRPX2 and uPAR, through recruiting co-repressors such as CtBP-1.

Article Snippet: Human FOXA2 siRNA (Santa cruz, sc-35569) and control siRNA (Santa cruz, sc-37007) were purchased from Santa Cruz, USA.

Techniques: RNA Sequencing Assay, Activation Assay

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: The FOXA1 transcriptional network coordinates key functions of primary human airway epithelial cells

doi: 10.1152/ajplung.00023.2020

Figure Lengend Snippet: Chromatin immunoprecipitation-sequencing (ChIP-seq) of Forkhead box A1 and A2 (FOXA1 and FOXA2) in primary human bronchial epithelial (HBE) cells. A: genomic region and gene association for FOXA1 and FOXA2 peaks ± 500 kb from transcriptional start sites. Data from two biological replicates subjected to irreproducible discovery rate (IDR) analysis are shown for each factor. B: genomic feature annotation for all peaks passing IDR false discovery rate (FDR) threshold of 0.01. C: heat map of ChIP-seq signal intensity for FOXA1 or FOXA2 around peaks of the other, or H3K27ac, ±750 bp of binding peak center. D: transcription factor DNA binding motifs ranked by −log10(P value) under FOXA1 or FOXA2 peaks. E: de novo motif analysis sequence logos and percent enrichment.

Article Snippet: HBE cells were treated with either negative control A siRNA (sc-37007; Santa Cruz Biotechnology), FOXA1 (HNF3α) siRNA (sc-37930; Santa Cruz Biotechnology), FOXA2 (HNF3β) siRNA (sc-35569), or a combination of the latter, each at 30 nM using RNAiMax transfection reagent (Life Technologies).

Techniques: ChIP-sequencing, Binding Assay, Sequencing

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: The FOXA1 transcriptional network coordinates key functions of primary human airway epithelial cells

doi: 10.1152/ajplung.00023.2020

Figure Lengend Snippet: RNA-seq of primary human bronchial epithelial (HBE) cells following depletion of Forkhead box A1 (FOXA1) and/or FOXA2. A: RNA-seq read counts of FOXA1 or FOXA2 normalized for sequencing depth in the FOXA1 depletion, FOXA2 depletion, or combined depletion. ***P < 0.0001; **P < 0.001; ns, not significant. B: principal component analysis for each triplicate treatment. C: Venn diagram comparing the number of differentially expressed genes (DEGs) with fold change ≥2 shared between the three treatment groups. D: heat map and dendrogram of the relative expression profiles of all DEGs identified in the complete transcriptome analysis. KD, knockdown.

Article Snippet: HBE cells were treated with either negative control A siRNA (sc-37007; Santa Cruz Biotechnology), FOXA1 (HNF3α) siRNA (sc-37930; Santa Cruz Biotechnology), FOXA2 (HNF3β) siRNA (sc-35569), or a combination of the latter, each at 30 nM using RNAiMax transfection reagent (Life Technologies).

Techniques: RNA Sequencing Assay, Sequencing, Expressing

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: The FOXA1 transcriptional network coordinates key functions of primary human airway epithelial cells

doi: 10.1152/ajplung.00023.2020

Figure Lengend Snippet: Specific differential gene expression following Forkhead box A1 (FOXA1) depletion. A: volcano plot of RNA-seq expression analysis of FOXA1-depleted human bronchial epithelial (HBE) versus negative control (NC)-treated cells. Plots show −log10 adjusted P value versus log2 fold change. Genes with ≥2-fold absolute value change passing a 0.001 adjusted P value threshold are noted in red. B: RT-quantitative PCR measurements for transcript abundance normalized to β-2-microglobulin (n = 6). ***P < 0.0001; **P < 0.001; *P < 0.01. C: heat map displaying relative expression of all statistically significant upregulated or downregulated transcription factors. D: integrated genomics viewer genome browser image of FOXA1 peaks identified by irreproducible discovery rate (IDR) in HBE cells (blue, scaled by signal intensity), and the FOXA1 Chromatin immunoprecipitation-sequencing (ChIP-seq) tag counts (maroon) for the HOPX (i), SPDEF (ii), and FOXA2 (iii) genes.

Article Snippet: HBE cells were treated with either negative control A siRNA (sc-37007; Santa Cruz Biotechnology), FOXA1 (HNF3α) siRNA (sc-37930; Santa Cruz Biotechnology), FOXA2 (HNF3β) siRNA (sc-35569), or a combination of the latter, each at 30 nM using RNAiMax transfection reagent (Life Technologies).

Techniques: Expressing, RNA Sequencing Assay, Negative Control, Real-time Polymerase Chain Reaction, ChIP-sequencing