Journal: Scientific Reports

Article Title: TRAF6 as a potential target in advanced breast cancer: a systematic review, meta-analysis, and bioinformatics validation

doi: 10.1038/s41598-023-31557-0

Figure Lengend Snippet: Summary of meta-analysis of included studies showing significant association of in vitro breast cancer cell behaviour and modulation of TRAF2/4/6.

Article Snippet: , Proliferation , Pharmacological inhibition , BT549 (4) MCF7 (4) MDA-MB-231 (8) , − 5.78 [− 12.04, 0.47] − 9.50 [− 18.82, − 0.19] − 5.62 [− 10.34, − 0.90] , − 21.46 [− 30.40, − 12.51] , Mean Difference (IV, Random, 95% CI) , Tau 2 = 321.80; Chi 2 = 677.01, df = 15 (P < 0.00001); I 2 = 98% , Z = 4.70 (P < 0.00001).

Techniques: In Vitro, Migration, Inhibition

Journal: Scientific Reports

Article Title: TRAF6 as a potential target in advanced breast cancer: a systematic review, meta-analysis, and bioinformatics validation

doi: 10.1038/s41598-023-31557-0

Figure Lengend Snippet: Summary of meta-analysis of included studies showing significant association of in vivo tumour burden and overt metastasis with modulation of TRAF2/4/6.

Article Snippet: , Proliferation , Pharmacological inhibition , BT549 (4) MCF7 (4) MDA-MB-231 (8) , − 5.78 [− 12.04, 0.47] − 9.50 [− 18.82, − 0.19] − 5.62 [− 10.34, − 0.90] , − 21.46 [− 30.40, − 12.51] , Mean Difference (IV, Random, 95% CI) , Tau 2 = 321.80; Chi 2 = 677.01, df = 15 (P < 0.00001); I 2 = 98% , Z = 4.70 (P < 0.00001).

Techniques: In Vivo, Inhibition

Journal: iScience

Article Title: Conserved role of FOXC1 in TNBC is parallel to FOXA1 in ER+ breast cancer

doi: 10.1016/j.isci.2024.110500

Figure Lengend Snippet: Effect of FOXC1 knockout on phenotype and gene expression (A) Western blot of cell lysate (75 μg) from four parental TNBC cell lines (“WT”) and the corresponding FOXC1 knockout CRISPR clones (“KO”), probed using anti-FOXC1 and anti-β-actin antibodies. (B) Proliferation assays showing growth of FOXC1_KO clones (red) when compared to the parental cell lines (blue). Viable cells were counted every 24 h. Data represent mean and standard deviation (SD) of three biological replicates. Asterisk denotes significant digits in p value derived from unpaired t tests with ∗ p < 0.05, ∗∗ p < 0.005, and ∗∗∗ p < 0.0005. (C) Enrichment using Hallmark gene sets (MSigDB ) showing significantly enriched (padj < 0.01) cancer hallmarks among the DEGs in each of the four TNBC cell lines. (D) Upset plot depicting intersection between up- or downregulated genes among the four cell lines, showing a limited set of DEGs that overlap and are similarly regulated (up- or down-) in all four cell lines. (E) Heatmap depicting log2 (fold change) of the 26 genes significantly (padj < 0.05) regulated upon the loss of FOXC1 in all four cell lines, representing genes repressed by FOXC1 (red) and genes activated by FOXC1 (blue). (F) Log2 (fold change) of selected genes in the RB1/CDK4/6 pathway (FOXC1_KO vs. WT). (G) Colony-forming assays performed in 6-wells in the presence of either DMSO (top row) or 0.5 μM palbociclib (bottom row) for 14 days prior to crystal violet staining. Bar graphs (bottom panel) display relative reduction in the number of colonies compared to the DMSO control in the parental (WT, blue) or the FOXC1_KO (KO, red) groups. Data represents mean ± SD from three biological replicates each. Asterisk denotes significant digits in p value derived from unpaired t tests. ∗∗∗ p < 0.005.

Article Snippet: TNBC cell lines, BT-549 (HTB-122), Hs578t (HTB-126), MDA-MB-231 (HTB-26), and MDA-MB-468 (HTB-132) were purchased from AddexBio (San Diego, CA).

Techniques: Knock-Out, Gene Expression, Western Blot, CRISPR, Clone Assay, Standard Deviation, Derivative Assay, Staining, Control

Journal: iScience

Article Title: Conserved role of FOXC1 in TNBC is parallel to FOXA1 in ER+ breast cancer

doi: 10.1016/j.isci.2024.110500

Figure Lengend Snippet: Genome-wide binding site analysis of FOXC1 in TNBC (A) Log2 (fold change) of normalized mRNA expression of 38 core, conserved gene targets that associate with a FOXC1 peak and are differentially regulated upon FOXC1 KO in all four TNBC cell lines. Dots represent literature-based evidence of the gene’s function, either as an oncogene (blue dots) or a tumor suppressor (red dots) in breast cancer. Nine genes ( THRB , CREBRF , SLC35B2 , CAV2 , C11orf24 , WWC3 , SLC7A5 , CENPV , and LONRF1 ) overlap with those in Figure 1 E. (B) Schema illustrating the tumor suppressors and oncogenes directly regulated by FOXC1 and their respective role in tumorigenesis in TNBC. Created with BioRender. (C) ChIP-seq traces showing FOXC1 peaks near oncogene CRABP2 and tumor-suppressor CREBRF . Bar plots depict normalized gene expression value in parental (WT, yellow) or FOXC1_KO (KO, blue) cell lines. Statistical significance determined using DESeq2 (∗ p adj < 0.05, ∗∗ p adj < 0.005, ∗∗∗ p adj ≤ 0.0005). (D) Kaplan-Meier survival analysis of a panel of 17 core upregulated targets of FOXC1 of ER-(IHC), PR-(IHC) and HER2 negative (array) in 220 breast cancer patient samples from kmplot.com . (E) Heatmap of FOXC1 peaks associated with a H3K27ac signal in each of the four TNBC cell lines. (F) Averaged quantification of FOXC1 and H3K27ac signal intensity in heatmap in (D). (G) FOXC1 ChIP-seq peak traces near the super-enhancer 128 kb upstream of FOXC1 gene (orange box), and FOXC1 peaks unique to MDA-MB-468 (purple boxes). (H) Bar plots showing the normalized expression of FOXC1 mRNA in parental (blue, WT) and FOXC1_KO (red, KO) in TNBC cell lines. Statistical significance determined using DESeq2 (ns = not significant, ∗∗∗ p adj < 0.0005).

Article Snippet: TNBC cell lines, BT-549 (HTB-122), Hs578t (HTB-126), MDA-MB-231 (HTB-26), and MDA-MB-468 (HTB-132) were purchased from AddexBio (San Diego, CA).

Techniques: Genome Wide, Binding Assay, Expressing, ChIP-sequencing, Gene Expression

Journal: iScience

Article Title: Conserved role of FOXC1 in TNBC is parallel to FOXA1 in ER+ breast cancer

doi: 10.1016/j.isci.2024.110500

Figure Lengend Snippet: Core, conserved FOXC1 gene targets in TNBC (A) Unsupervised k-means clustering of peaks among the four TNBC cell lines into 5 clusters: cluster 1, peaks in all cell lines; clusters 2–5, peaks dominant in a particular cell line. (B) Number of peaks in each cluster identified in (A). (C) Quantification of the FOXC1 signal in each of the five clusters. One-way ANOVA followed by Tukey’s post-hoc test, comparing cell lines within each peak cluster. (D) Location of FOXC1 peaks near genome features in each of the five clusters. (E) The average intensity of FOXC1 peaks in Fujioka (AML) cell lines overlaid onto the five clusters of FOXC1 binding sites in TNBC from (A). (F) Top enriched transcription factors that have similar gene targets as core targets of FOXC1, using the TF target (ENCODE and ChEA consensus from ChIP-X experiments), generated using ShinyGO.

Article Snippet: TNBC cell lines, BT-549 (HTB-122), Hs578t (HTB-126), MDA-MB-231 (HTB-26), and MDA-MB-468 (HTB-132) were purchased from AddexBio (San Diego, CA).

Techniques: Binding Assay, Generated

Journal: iScience

Article Title: Conserved role of FOXC1 in TNBC is parallel to FOXA1 in ER+ breast cancer

doi: 10.1016/j.isci.2024.110500

Figure Lengend Snippet: Core FOXC1 binding sites in TNBC are bound by FOXA1/NR2F2 in luminal breast cancer (A) Heatmaps depicting overlap of ChIP-seq data of transcription factors (GATA3, ESR1, NR2F2, and FOXA1) from MCF-7 luminal breast cancer cell lines with the five TNBC clusters of FOXC1 peaks identified in Figure 3 A. (B) Average ChIP-seq tag intensity of transcription factors (GATA3, ESR1, NR2F2, and FOXA1) in MCF-7 over TNBC peak clusters from previous panel. (C) Average intensity of FOXC1 peaks in MDA-MB-231 at sites that are bound by FOXA1+NR2F2 versus GATA3 (cognate sites) in MCF-7. Adjusted p value calculated using a Tukey post-hoc test. (D) Proximity of FOXC1 to GATA3 binding sites in MDA-MB-231 and the number of peaks overlapping between FOXC1 and over-expressed GATA3 in MDA-MB-231. (E) Average intensity of GATA3 over FOXC1 peaks in each of the five clusters in Figure 3 A. (F) ChIP-seq traces (above) showing FOXC1 peaks in TNBC and AML cell lines and FOXA1 peaks in MCF-7 near MCM7 and RXRA genes, two critical genes in breast cancer and barplots (below) show changes in normalized gene expression from RNA-seq of the genes in WT (blue) or FOXC1_KO (red). Statistical significance determined using DESeq2 (ns = not significant, ∗ p adj < 0.05, ∗∗∗∗ p adj < 0.00005). (G) Change in mean expression of up- or downregulated core genes in all breast invasive carcinomas based on RNA-seq data from the GEO database in tumor, adjacent normal or metastatic samples ( TNMplot.com ). p value shown calculated using Kruskal-Wallis tests. (H) Kaplan-Meier survival analysis of upregulated (left) and downregulated (right) core gene targets of FOXC1 on 2032 breast cancer patient samples from kmplot.com .

Article Snippet: TNBC cell lines, BT-549 (HTB-122), Hs578t (HTB-126), MDA-MB-231 (HTB-26), and MDA-MB-468 (HTB-132) were purchased from AddexBio (San Diego, CA).

Techniques: Binding Assay, ChIP-sequencing, Gene Expression, RNA Sequencing, Expressing