Journal: Nature Communications

Article Title: Cooperative interaction between ERα and the EMT-inducer ZEB1 reprograms breast cancer cells for bone metastasis

doi: 10.1038/s41467-022-29723-5

Figure Lengend Snippet: a MCF7-V-ZEB1 cells in the presence of doxycycline (+DOX) express ZEB1. Immunoblots show the levels of EMT markers and ERα after long-term (8–12 weeks) and short-term (1–2 weeks) expression of ZEB1 (results are representative of n = 3 independent experiments). b – i Luciferase reporter assays with transiently transfected cells as indicated, including for ZEB1, which was expressed from plasmid pTRIPz-puro-HA-ZEB1 with DOX treatment. Except for the experiments of b , c , all assays were done with HEK293T cells. The activities of ERα and PR, and the E-cadherin, VEGF, and TGFβ-responsive promoter activities were monitored with the reporter plasmids ERE-Luc, PRE-Luc, and proE-cad670-Luc, VEGFprom-Luc, and SBE4-Luc, respectively. The luciferase activities (RLU) are expressed relative to the activities of the internal transfection standard, Renilla luciferase. Graphs are based on n = 4 for ( b ), n = 6 for ( c ), and n = 3 for ( d – i ) biologically independent experiments. j Expression of ERα target genes in MCF7-V-ZEB1 cells; mRNA levels were analyzed by RT-qPCR following 6 h of treatments as indicated; n = 3 biologically independent experiments, each including n = 2 technical replicates. k Representative images of a three-dimensional (3D) tumor invasion assay with MCF7-V-ZEB1 cells. l Invasion kinetics based on the area of n = 2 independent spheroids examined over three independent experiments as shown in k . veh, E2, FI, and ICI stand for vehicle, 17β-estradiol, forskolin + IBMX, and fulvestrant, respectively. All error bars represent standard errors of the means (mean ± SEM). In b – j , p values are indicated above the bars; statistical significance was determined with one-way ANOVA for f , g , i and a two-way ANOVA for all other panels. Source data are provided as a Source Data file.

Article Snippet: The rabbit polyclonal antiserum against ZEB1 for ChIP experiments and ChIP-seq (10 μg per IP) was from Proteintech (21544-1-AP).

Techniques: Western Blot, Expressing, Luciferase, Transfection, Plasmid Preparation, Quantitative RT-PCR, Invasion Assay

Journal: Nature Communications

Article Title: Cooperative interaction between ERα and the EMT-inducer ZEB1 reprograms breast cancer cells for bone metastasis

doi: 10.1038/s41467-022-29723-5

Figure Lengend Snippet: a ChIP-qPCR of ERα on the GREB1 (+5 kb) and TFF1 (+0.5 kb) binding sites in MCF7-V-ZEB1 cells. ERα ChIP values were normalized to a non-binding region and the input. Recruitment was compared to −DOX with the graph showing the means ± SEM of n = 6 biologically independent experiments. b , c Volcano plots of ERα ChIP-seq with wild-type MCF7-V (data from our previously published data set ) and MCF7-V-ZEB1 cells showing the FDR values as a function of the fold-changes of the normalized ERα values of MCF7-V-ZEB1 cells ( n = 4 biologically independent experiments) compared to the ERα peaks of wild-type MCF7-V cells ( n = 2 biologically independent experiments) treated with E2 ( b ) or FI ( c ). d , e Functional annotations for the biological functions of E2– or FI-only ERα binding sites, respectively, using GREAT. f Table summarizing the number of ERα (ESR1), ZEB1, and ESR1-ZEB1 shared motifs in the ERα ChIP-seq, as found with FIMO (FDR < 0.05; for p values, see Supplementary Data ). g , h ChIP-qPCR of candidate ERα binding sites from top hits of the ChIP-seq data for E2 ( g ) and FI ( h ). g n = 4 biologically independent experiments each including n = 2 technical replicates for TBX2 , and n = 3 biologically independent experiments for ANXA3 . In panel h , n = 3 biologically independent experiments. i ZEB1 ChIP-qPCR with MCF7-V-ZEB1 cells with (+DOX) or without (−DOX) ZEB1 ( n = 3 biologically independent experiments). j Venn diagram shows the intersections between ZEB1-binding sites and E2– or FI-induced ERα binding sites from the ChIP-seq data of MCF7-V-ZEB1 (+DOX) and MCF7-V cells, respectively. k Genome browser views of ZEB1 and ERα binding sites adjacent to the ERα target genes GREB1 , TFF1 , XBP1 , and CDH1 . Highlighted sites were analyzed by re-ChIP-qPCR (see next panel). l Re-ChIP experiment showing that ZEB1 and ERα co-occupy the indicated shared binding sites ( n = 3 biologically independent experiments). The GREB1 (–20 kb) site is a negative control site as highlighted in k . veh, E2, and FI stand for vehicle, 17β-estradiol, and forskolin + IBMX, respectively. Error bars represent the standard errors of the means; p values are indicated above the bars. Statistical significance was determined with a two-way ANOVA. Source data are provided as a Source Data file and Supplementary Data and .

Article Snippet: The rabbit polyclonal antiserum against ZEB1 for ChIP experiments and ChIP-seq (10 μg per IP) was from Proteintech (21544-1-AP).

Techniques: ChIP-qPCR, Binding Assay, ChIP-sequencing, Functional Assay, Negative Control

Journal: Nature Communications

Article Title: Cooperative interaction between ERα and the EMT-inducer ZEB1 reprograms breast cancer cells for bone metastasis

doi: 10.1038/s41467-022-29723-5

Figure Lengend Snippet: a Immunoblots of an ERα and ZEB1 co-immunoprecipitation experiment. IPs with extracts from MCF7-V-ZEB1 without or with DOX treatment (for 1 week) were performed with antibodies specific to the exogenously expressed ZEB1 (left) or to the endogenous ERα (right). A control immunoprecipitation was performed in parallel with IgG, blotted, and exposed simultaneously (results are representative of n = 3 independent experiments). b , c Venn diagrams showing overlap of AP2γ ( b ) or FOXA1( c ) binding sites with ZEB1 and ERBSs from the ChIP-seq data. d Aggregation plot of the binding sites of ZEB1, ERα, AP2γ, FOXA1, GATA3, and P300, and the open chromatin histone marks H3K27ac, H3K4me1, and H3K9me3. Except for ERα, ChIP-seq data were from published data sets: GSE21234 (TFAP2C), GSE25315 (FOXA1), GSE60270 (GATA3, P300, H3K27ac, H3K4me1, and H3K9me3). e Genome browser snapshots of ZEB1, ERα, AP2γ, and FOXA1 enhancers of indicated genes. Highlights show shared binding sites for indicated factors. f Luciferase reporter assays with ERE-Luc in HEK293T cells infected with lentiviral constructs for shRNAs targeting FOXA1 , TFAP2C , or both mRNAs; scrambled shRNA (shScr) was used as negative control (mean ± SEM, n = 3 biologically independent experiments). g Co-IPs with HEK293T cells co-transfected with ZEB1 and ERα expression vectors. A control IP was performed with an IgG antibody (results are representative of n = 3 independent experiments). h Cells infected with viruses for expression of shScr, shTFAP2C, or shFOXA1. ERα ChIP-qPCR values are represented as the fold of the shScr in −DOX (mean ± SEM, n = 4 biologically independent experiments). i – j ERα ChIP-qPCR of binding sites associated with the genes LGALS1 and RAP1GAP2 ( i ), and SIRT5 and CD276 ( j ) in MCF7-V-ZEB1 cells infected with shScr or shTFAP2C. k ChIP-qPCR of ERBS at the TFAP2C 5′-UTR (means ± SEM, n = 4 and n = 3 biologically independent experiments in i and k , and j , respectively). l Immunoblots of extracts from MCF7-V-ZEB1 cells. veh, E2, and FI stand for vehicle, 17β-estradiol, and forskolin + IBMX, respectively. For bar graphs, p values are indicated above the bars; statistical significance was determined with a two-way ANOVA. Source data are provided as a Source Data file.

Article Snippet: The rabbit polyclonal antiserum against ZEB1 for ChIP experiments and ChIP-seq (10 μg per IP) was from Proteintech (21544-1-AP).

Techniques: Western Blot, Immunoprecipitation, Control, Binding Assay, ChIP-sequencing, Luciferase, Infection, Construct, shRNA, Negative Control, Transfection, Expressing, ChIP-qPCR

Journal: Nature Communications

Article Title: Cooperative interaction between ERα and the EMT-inducer ZEB1 reprograms breast cancer cells for bone metastasis

doi: 10.1038/s41467-022-29723-5

Figure Lengend Snippet: a Venn diagram showing the overlap of the differentially expressed genes for MCF7-V-ZEB1 cells with (+DOX) or without (−DOX) ZEB1 expression in presence of indicated treatments. Fold changes were calculated relative to values from −DOX (veh) ( n = 2 biologically independent experiments). b Venn diagram of the veh, E2- or FI-induced transcriptome changes dependent on ZEB1 expression. c – f Bar plots of a GSEA showing the –log10 ( p value) of the top 20 unique GO terms associated with significantly altered mRNA expression levels upon E2– or FI-induced ERα activation in cells with (+DOX) or without (−DOX) ZEB1. E2 and FI stand for 17β-estradiol and forskolin + IBMX, respectively. In all panels only genes up/downregulated by at least 1.3-fold were considered for the analysis. Only the genes that had an FDR < 0.05 were included in the analyses. Source data are provided in Supplementary Data – .

Article Snippet: The rabbit polyclonal antiserum against ZEB1 for ChIP experiments and ChIP-seq (10 μg per IP) was from Proteintech (21544-1-AP).

Techniques: Expressing, Activation Assay

Journal: Nature Communications

Article Title: Cooperative interaction between ERα and the EMT-inducer ZEB1 reprograms breast cancer cells for bone metastasis

doi: 10.1038/s41467-022-29723-5

Figure Lengend Snippet: a Heatmap showing the gene expression levels of the top 20 genes from the RNA-seq data analysis, grouped to highlight treatment-specific signatures (as indicated on the side). Red rectangles around gene names indicate the candidate genes selected for validation and functional assays. b , c mRNA levels of candidate genes determined by RT-qPCR (mean ± SEM, n = 3 biologically independent experiments). d Bar graphs representing the changes in the 3D invasion ability of spheroids ( n = 3 biologically independent samples, each including n = 2 replicate wells) formed from cells with (+DOX) or without (−DOX) ZEB1 expression upon knocking down the expression of MUC16 , ESR2 , DSCAM , and P2RX7 with the respective shRNAs. Note that the values for shScr in −DOX/+DOX spheroids (gray and orange, respectively) are from the same data points in all graphs. veh, E2, FI, and ICI stand for vehicle, 17β-estradiol, forskolin + IBMX, and fulvestrant, respectively. Error bars represent the standard errors of the means. All time points are shown in Supplementary Fig. . Source data are provided as a Source Data file.

Article Snippet: The rabbit polyclonal antiserum against ZEB1 for ChIP experiments and ChIP-seq (10 μg per IP) was from Proteintech (21544-1-AP).

Techniques: Gene Expression, RNA Sequencing, Biomarker Discovery, Functional Assay, Quantitative RT-PCR, Expressing

Journal: Nature Communications

Article Title: Cooperative interaction between ERα and the EMT-inducer ZEB1 reprograms breast cancer cells for bone metastasis

doi: 10.1038/s41467-022-29723-5

Figure Lengend Snippet: a – h show different aspects of scRNA-seq analysis of MCF7-V cells expressing ZEB1 after induction with DOX for 5 weeks to obtain EpCAM high and EpCAM low cell populations. a Visualization of the distribution of the merged data sets of both EpCAM high (red rectangle) and EpCAM low (blue rectangle) cell populations, based on the comparison of the transcriptomes of individual cells; the graph was generated by a Uniform Manifold Approximation and Projection (UMAP); the clusters are color-coded and numbered, and each dot represents a single cell. b Single cells expressing EPCAM in different clusters. c Violin plot of the EPCAM transcript levels across different clusters; note that the steps at low levels of expression are due to rounding off after normalization. d Single cells expressing ESR1 in different clusters. e Violin plot for ESR1 transcripts with the same color code and numbering as in a . f – h Expression of top markers of epithelial ( f ), hybrid EMT ( g ), and mesenchymal-like ( h ) states in breast cancer cells upon induction of an EMT by ZEB1 expression. The legend shows a color gradient of normalized expression. The accompanying scheme of the EMT on the right was created with BioRender.com. i Expression of the mRNA for the CD151 cell surface marker across various subpopulations. j Relative proliferation of −DOX and +DOX cells with or without CD151 knockdown exposed to veh or E2 for 72 h (means ± SEM, n = 4 biologically independent experiments averaged from a total of n = 16 technical replicates). Statistical significance was determined with two-way ANOVA. p values are indicated above the bars. k Quantification of the effect of CD151 knockdown on the migration of MCF7-V-ZEB1 cells treated with veh or E2 in a wound-healing assay; the Y axis shows the remaining wound area into which cells have not yet migrated (means ± SEM and n = 3 biological replicates each including n = 2 technical replicates). Statistical significance was determined with a two-way ANOVA. p values are indicated above the bars. Source data are provided as a Source Data file.

Article Snippet: The rabbit polyclonal antiserum against ZEB1 for ChIP experiments and ChIP-seq (10 μg per IP) was from Proteintech (21544-1-AP).

Techniques: Expressing, Comparison, Generated, Marker, Knockdown, Migration, Wound Healing Assay

Journal: Nature Communications

Article Title: Cooperative interaction between ERα and the EMT-inducer ZEB1 reprograms breast cancer cells for bone metastasis

doi: 10.1038/s41467-022-29723-5

Figure Lengend Snippet: a Left panel shows the Bioluminescent images (BLI) of primary tumors of mice injected with one million ZsGreen- or ZEB1-expressing wild-type MCF7 cells into the mammary fat pad. The panel on the right is the corresponding quantitative analysis of the average radiance of multiple primary tumors. An unpaired and two-tailed Student’s t test was applied to determine whether tumor sizes are different ( n = 8 independent animals for ZsGreen cells and n = 4 for ZEB1 cells; means ± SEM). b Left panel is the BLI images showing metastatic lesions in hindlimb bones of ZsGreen- or ZEB1-expressing wild-type MCF7 cells, respectively. The corresponding panel on the right is a bar graph of the metastatic indices (the ratio of the average radiance of metastases over that of the corresponding primary tumor; see Methods for details) for bones ( n = 8 independent animals for ZsGreen cells and n = 4 for ZEB1 cells; means ± SEM); note that the data are shown after log10 transformation; an unpaired and two-tailed Student’s t test was used for the statistical analysis. c – f IF images of EpCAM, vimentin, ERα, and ZEB1, and DAPI staining of resected primary tumors formed by MCF7-ZEB1 cells (+DOX) following their orthotopic injection into mouse mammary fat pads (Scale bar = 10 μm). g , h IF images of EpCAM and ZEB1, and DAPI staining of metastatic lesions in femurs of mice xenografted with ZEB1-expressing ( g ) and ZsGreen-expressing ( h ) cells (scale bar = 10 μm). i Multiphoton confocal microscopy images of an ex vivo bone invasion assay showing the bone explant stained in red with alizarin red and the invading ZsGreen-labeled (green) control (Ctl) or ZEB1-expressing MCF7-V cells. Without ZEB1, the cells remain on top of the bone tissue, whereas ZEB1-expressing cells invade it. The upper left of each panel shows a projection of a Z-stack with 20 images, and in the upper right of each panel a slice of the area delimited by a yellow rectangle. Images were processed and generated with the software Imaris 9.7 to illustrate representative slides and 3D views of bone tissue. Scale bar = 50 μm. j Transwell assay to evaluate the migration of ZsGreen-labeled control (Ctl) or ZEB1-expressing MCF7-V cells stimulated by bone and muscle tissues. Crystal violet dye was used to stain the migrated cells. Decellularized bone served as a negative control. Images are representative of three independent experiments. Source data are provided as a Source Data file. Scale bar = 50 μm.

Article Snippet: The rabbit polyclonal antiserum against ZEB1 for ChIP experiments and ChIP-seq (10 μg per IP) was from Proteintech (21544-1-AP).

Techniques: Injection, Expressing, Two Tailed Test, Transformation Assay, Staining, Confocal Microscopy, Ex Vivo, Invasion Assay, Labeling, Control, Generated, Software, Transwell Assay, Migration, Negative Control

Journal: Nature Communications

Article Title: Cooperative interaction between ERα and the EMT-inducer ZEB1 reprograms breast cancer cells for bone metastasis

doi: 10.1038/s41467-022-29723-5

Figure Lengend Snippet: Non-invasive primary epithelial breast tumors (EpCAM high ) that express high levels of ERα and are negative for ZEB1 expression are formed by the abnormal proliferation of luminal mammary epithelial cells. FOXA1 acts as the main pioneer factor for the recruitment of ERα for transcription of genes involved in cell proliferation. In early/hybrid states of EMT, AP2γ becomes a determining pioneer factor promoting the formation of a ZEB1-ERα complex at ERα binding sites, which enhances ERα target gene expression. Without AP2γ recruitment, FOXA1 and/or other factors may partially sustain ERα recruitment to ERBSs and ERα-stimulated transcription. This complex reprograms the ERα cistrome and transcriptome towards the activation of genes involved in partial EMT and metastatic dissemination. Expression of specific factors such as CD151 marks the partial EMT state. CD151 could potentially be targeted to prevent cancer cell proliferation, migration, and invasion. The illustration was created with BioRender.com.

Article Snippet: The rabbit polyclonal antiserum against ZEB1 for ChIP experiments and ChIP-seq (10 μg per IP) was from Proteintech (21544-1-AP).

Techniques: Expressing, Binding Assay, Targeted Gene Expression, Activation Assay, Migration