Journal: Oncology Research

Article Title: FOXA2 as a SETD1A-Regulated Driver of Tamoxifen Resistance in Breast Cancer

doi: 10.32604/or.2025.072592

Figure Lengend Snippet: SET Domain Containing 1A (SETD1A) regulates the expression of the FOXA2 gene. ( A ) MCF-7 cells were treated with different concentrations of tamoxifen for 24 h, and FOXA2 levels were assessed by western blotting analysis. β-Actin was used as the loading control. The FOXA2 levels in MDA-MB-231 and TamR cells were used as a positive control. ( B ) ERα or FOXA1 was knocked down in MCF-7 cells. ERα and FOXA2 levels were assessed by western blotting analysis. β-Actin was used as the loading control. ( C ) Analysis of mRNA and nascent mRNA levels of FOXA2 in SETD1A-depleted TamR cells. ( D ) Effects of SETD1A on FOXA2 protein expression. Protein levels of SETD1A and FOXA2 in TamR cells depleted of SETD1A using two independent shRNAs were evaluated using western blotting, with β-actin serving as the loading control. ( E ) Overexpression of SETD1A (ovSETD1A) or SOX2 (ovSOX2) in MCF-7 cells and FOXA2 levels were assessed by western blotting analysis. β-Actin was used as the loading control. ( F ) SETD1A (red) and FOXA2 (green) immunofluorescence in TamR cells transfected with shSETD1A (n = 3). Fluorescence intensities are plotted in the right panel, and the correlation between SETD1A and FOXA2 expression was assessed via Pearson analysis using GraphPad Prism v.8.0.2 (n = 23). ( G ) ChIP assays were performed in TamR cells to examine SETD1A recruitment at the FOXA2 locus. Quantification of the precipitated FOXA2 region by anti-SETD1A antibody was conducted via quantitative PCR. ( H ) Investigation of SETD1A’s role in H3K4me3 methylation at the FOXA2 locus. SETD1A expression was knocked down in TamR cells using shRNA. ( I ) Investigation of SETD1A-mediated chromatin accessibility at the FOXA2 gene via formaldehyde-assisted isolation of regulatory elements (FAIRE)-qPCR. ( J ) Effects of SETD1A depletion on Pol II binding to the FOXA2 promoter (n = 3). * p < 0.05; ** p < 0.01; *** p < 0.001

Article Snippet: SETD1A , 1:1000 , Bethyl Laboratories (Montgomery, AL, USA) , A300-289A.

Techniques: Expressing, Western Blot, Control, Positive Control, Over Expression, Immunofluorescence, Transfection, Fluorescence, Real-time Polymerase Chain Reaction, Methylation, shRNA, Isolation, Binding Assay

Journal: Oncology Research

Article Title: FOXA2 as a SETD1A-Regulated Driver of Tamoxifen Resistance in Breast Cancer

doi: 10.32604/or.2025.072592

Figure Lengend Snippet: Clinical significance of FOXA2 in patients with tamoxifen-resistant breast cancer. ( A ) mRNA expression of SETD1A and FOXA2 in ERα-positive breast cancer patients treated with tamoxifen ( GSE9893 ). The closed box indicates values between the 25th and 75th percentiles. ( B ) FOXA2 expression analysis predicts poor outcomes in patients with breast cancer treated with chemotherapy. The area under the curve (AUC) and corresponding p -value were determined for the FOXA2 gene signature. ( C ) Kaplan–Meier overall survival curves illustrate the prognostic significance of FOXA2 signature. Patients with FOXA2 expression above the cohort median were classified as the high-expression group, while the rest were designated as the low-expression group. ( D ) Representative images depict SETD1A and FOXA2 expressions in breast cancer tissue samples. Immunofluorescence staining was performed on tissue microarrays of malignant human breast cancer samples (breast, n = 36) and matched metastatic lymph node tissues (lymph node, n = 36). Protein expression levels for each sample were quantified as integrated optical density (IOD). ( E ) Correlation between SETD1A and FOXA2 expression in breast cancer tissues was evaluated using scatter plots of quantified protein levels in malignant breast and lymph node tissues. Pearson’s correlation coefficient was calculated using GraphPad Prism v8.0.2. ( F) Proposed mechanism by which FOXA2 acts as a SETD1A-regulated driver of tamoxifen resistance in breast cancer

Article Snippet: SETD1A , 1:1000 , Bethyl Laboratories (Montgomery, AL, USA) , A300-289A.

Techniques: Expressing, Immunofluorescence, Staining