Journal: International Journal of Molecular Medicine

Article Title: Ebastine targets HER2/HER3 signaling and cancer stem cell traits to overcome trastuzumab resistance in HER2-positive breast cancer

doi: 10.3892/ijmm.2026.5751

Figure Lengend Snippet: EBA impairs cancer stem cell-like properties. (A) BT474 and SKBR3 cells were treated with EBA for 48 h, and ALDH1 activity was assessed by flow cytometry using the Aldefluor assay. DEAB was used to define the baseline of Aldefluor-positive fluorescence. (B) BT474 cells (5x10 4 cells/ml) were plated in ultra-low attachment dishes and cultured in the presence or absence of EBA for 5 days. The number and volume of mammospheres were measured by microscopy. (C) Overall survival of patients with breast cancer stratified by the co-expression of ALDH1A1 and CD44. (D) Spearman correlation analysis of ALDH1A1 and CD44 mRNA levels in patients with HER2-positive breast cancer from The Cancer Genome Atlas cohort (n=76). Kaplan-Meier survival analyses of patients with HER2-overexpressing breast cancer stratified by (E) ALDH1A1 and (F) CD44 expression. Patients were divided into high- and low-expression groups based on the median gene expression. Statistical significance was determined using the log-rank test. (G) JIMT-1 cells were treated with EBA (3 μ M) for 48 h and the CD44 high /CD24 low cell populations were identified by flow cytometry. (H) JIMT-1 cells (1.5x10 4 cells/ml) were cultured under serum-free suspension conditions in the presence of EBA (3 μ M) for 8 days. Mammosphere number and volumes were quantified. ** P<0.01 and **** P<0.0001 vs. vehicle-treated control (0 μ M EBA). EBA, ebastine; ALDH, aldehyde dehydrogenase; DEAB, diethylaminobenzaldehyde; CTL, control; ISO, isotype.

Article Snippet: The human breast cancer cell lines SKBR3, BT474, MDA-MB-453 (American Type Culture Collection) and JIMT-1 (Leibnitz Institute DSMZ-German Collection of Microorganisms and Cell Cultures GmbH) were cultured in DMEM, MEM or RPMI-1640 (all Sigma-Aldrich; Merck KGaA) supplemented with 10% FBS (Gibco; Thermo Fisher Scientific, Inc.) and 100 U/ml penicillin-streptomycin at 37°C in a humidified atmosphere of 5% CO 2 .

Techniques: Activity Assay, Flow Cytometry, Fluorescence, Cell Culture, Microscopy, Expressing, Gene Expression, Suspension, Control

Journal: International Journal of Molecular Medicine

Article Title: Ebastine targets HER2/HER3 signaling and cancer stem cell traits to overcome trastuzumab resistance in HER2-positive breast cancer

doi: 10.3892/ijmm.2026.5751

Figure Lengend Snippet: EBA impairs cancer stem cell-like properties. (A) BT474 and SKBR3 cells were treated with EBA for 48 h, and ALDH1 activity was assessed by flow cytometry using the Aldefluor assay. DEAB was used to define the baseline of Aldefluor-positive fluorescence. (B) BT474 cells (5x10 4 cells/ml) were plated in ultra-low attachment dishes and cultured in the presence or absence of EBA for 5 days. The number and volume of mammospheres were measured by microscopy. (C) Overall survival of patients with breast cancer stratified by the co-expression of ALDH1A1 and CD44. (D) Spearman correlation analysis of ALDH1A1 and CD44 mRNA levels in patients with HER2-positive breast cancer from The Cancer Genome Atlas cohort (n=76). Kaplan-Meier survival analyses of patients with HER2-overexpressing breast cancer stratified by (E) ALDH1A1 and (F) CD44 expression. Patients were divided into high- and low-expression groups based on the median gene expression. Statistical significance was determined using the log-rank test. (G) JIMT-1 cells were treated with EBA (3 μ M) for 48 h and the CD44 high /CD24 low cell populations were identified by flow cytometry. (H) JIMT-1 cells (1.5x10 4 cells/ml) were cultured under serum-free suspension conditions in the presence of EBA (3 μ M) for 8 days. Mammosphere number and volumes were quantified. ** P<0.01 and **** P<0.0001 vs. vehicle-treated control (0 μ M EBA). EBA, ebastine; ALDH, aldehyde dehydrogenase; DEAB, diethylaminobenzaldehyde; CTL, control; ISO, isotype.

Article Snippet: The human breast cancer cell lines SKBR3, BT474, MDA-MB-453 (American Type Culture Collection) and JIMT-1 (Leibnitz Institute DSMZ-German Collection of Microorganisms and Cell Cultures GmbH) were cultured in DMEM, MEM or RPMI-1640 (all Sigma-Aldrich; Merck KGaA) supplemented with 10% FBS (Gibco; Thermo Fisher Scientific, Inc.) and 100 U/ml penicillin-streptomycin at 37°C in a humidified atmosphere of 5% CO 2 .

Techniques: Activity Assay, Flow Cytometry, Fluorescence, Cell Culture, Microscopy, Expressing, Gene Expression, Suspension, Control

Journal: BMC Cancer

Article Title: GCNT3 and ST3GAL1 expression correlates with HER2 status and MUC1/β-catenin/Cyclin D1 axis in breast cancer

doi: 10.1186/s12885-026-15821-w

Figure Lengend Snippet: In silico analysis showing differential expression of glycogenes in the progression of BC: A Gene Set Enrichment Analysis (GSEA) showing the heatmap for different glycogenes in the HER2 high vs low phenotype. B Pie chart made using PANTHER software representing: cellular pathways of genes overexpressed in glycosylation geneset, and biological pathways of genes overexpressed in glycosylation geneset, C Cellular pathways of genes overexpressed in KRAS geneset and biological pathways of genes overexpressed in KRAS geneset. Major pathways highlighted in these pie charts have been indicated

Article Snippet: HER2 positive cell line SKBR3 (ATCC Catalog HTB-30) was cultured in McCoy’s 5 A medium.

Techniques: In Silico, Quantitative Proteomics, Software, Glycoproteomics

Journal: BMC Cancer

Article Title: GCNT3 and ST3GAL1 expression correlates with HER2 status and MUC1/β-catenin/Cyclin D1 axis in breast cancer

doi: 10.1186/s12885-026-15821-w

Figure Lengend Snippet: In vitro analysis of ST3GAL1’s association with HER2: A Bar plot showing qRT-PCR analysis of ST3GAL1 in HER2 -ve and HER2 +ve BC cells. T-test based p-value obtained were as follows: SKBR3 vs MCF7 (p=0.0045, n=3), SKBR3 vs MDAMB231 (p=0.0034, n=3) and SKBR3 vs MDAMB435 (p=0.05, n=3). B Representative images of immunohistochemical analysis for ST3GAL1 in Grade 2 and 3 of Stage I, II and III BC tissues ( C ) ST3GAL1 expression in HER2 positive BC tissue. D Mean intensity value calculated as average pixel intensity using ImageJ, for ST3GAL1. Two-way ANOVA p-value exhibited p<0.05 (n=3) for Grade 2, Stage I vs Stage II vs Stage III. Similarly, p <0.05 (n=3) was obtained for Grade 3, Stage I vs Stage II vs Stage III. Moreover, t-test based p<0.05 (n=3) was obtained in the HER2 positive and HER2 negative BC tissues

Article Snippet: HER2 positive cell line SKBR3 (ATCC Catalog HTB-30) was cultured in McCoy’s 5 A medium.

Techniques: In Vitro, Quantitative RT-PCR, Immunohistochemical staining, Expressing

Journal: BMC Cancer

Article Title: GCNT3 and ST3GAL1 expression correlates with HER2 status and MUC1/β-catenin/Cyclin D1 axis in breast cancer

doi: 10.1186/s12885-026-15821-w

Figure Lengend Snippet: In silico and in vitro analyses showing inverse association of GCNT3 with HER2: A Survival plot representing the gene expression of GCNT3 in BC cohorts. B Differential expression analysis of GCNT3 using the RNAseq data for the tumor vs control BC. C Differential expression analysis for the control vs tumor samples from the TCGA BC dataset for GCNT3 using GEPIA. D Bar plot showing qRT-PCR analysis for GCNT3 using the HER2 -ve and HER2 +ve BC cells with T-test based p-value obtained as follows: SKBR3 vs MCF7 (p=0.045, n=3), SKBR3 vs MDAMB231 (p=0.012, n=3) and SKBR3 vs MDAMB435 (p=0.00069, n=3). E Representative images of immunohistochemical analysis for GCNT3 in Grade 2 and 3 of Stage I, II and III BC tissues. F , G Mean intensity values calculated as average pixel intensity using ImageJ, for GCNT3. Two-way ANOVA p-value exhibited p<0.05 for Grade 2, Stage I vs Stage II vs Stage III. Similarly, p<0.05 (n=3) was obtained for Grade 3, Stage I vs Stage II vs Stage III. Additionally, t-test based p<0.05 (n=3) was obtained in the HER2 negative vs positive BC tissues

Article Snippet: HER2 positive cell line SKBR3 (ATCC Catalog HTB-30) was cultured in McCoy’s 5 A medium.

Techniques: In Silico, In Vitro, Gene Expression, Quantitative Proteomics, RNA sequencing, Control, Quantitative RT-PCR, Immunohistochemical staining

Journal: BMC Cancer

Article Title: GCNT3 and ST3GAL1 expression correlates with HER2 status and MUC1/β-catenin/Cyclin D1 axis in breast cancer

doi: 10.1186/s12885-026-15821-w

Figure Lengend Snippet: In silico analysis of ST3GAL1’s association with HER2: A Survival plots representing gene expression of HER2 and ST3GAL1 for these genes in BC cohorts. B Oncomine extracted data represented by box plots on ST3GAL1 with increasing IHC score ( C ) Violin plots representing differential expression analysis of HER2 and ST3GAL for the tumor vs control using the RNAseq data for the BC samples. D Correlation plot between ST3GAL1 and HER2 on R2 dataset. E Differential expression analysis for the control vs tumor samples from the TCGA BC dataset for HER2 and ST3GAL1 using GEPIA. F Correlation plot between HER2 and ST3GAL1 on the cbioportal database

Article Snippet: HER2 positive cell line SKBR3 (ATCC Catalog HTB-30) was cultured in McCoy’s 5 A medium.

Techniques: In Silico, Gene Expression, Quantitative Proteomics, Control, RNA sequencing

Journal: BMC Cancer

Article Title: GCNT3 and ST3GAL1 expression correlates with HER2 status and MUC1/β-catenin/Cyclin D1 axis in breast cancer

doi: 10.1186/s12885-026-15821-w

Figure Lengend Snippet: Functional analysis using GCNT3 inhibitor (talniflumate) in HER2 -ve and HER2 +ve BC cells: Representative images and quantification using talniflumate for ( A ) wound healing on MCF7 cells, t-test based p-value for control vs treatment at 24h and 48h were p=0.38 (n=3) and p=0.17 respectively, B colony formation on MCF7 cells, C wound healing on MDAMB231 cells, t-test based p-value for control vs treatment at 24h and 48h were p=0.023 (n=3) and p=0.084 (n=3) respectively, D colony formation on MDAMB231 cells, E wound healing on MDAMB435 cells, t-test based p-value for control vs treatment at 24h and 48h were p=0.09 (n=3) and p=0.40 (n=3) respectively, F colony formation on MDAMB435 cells, G . wound healing for SKBR3 cells, t-test based p-value for control vs treatment at 12h and 24h were p=0.49 (n=3) and p=0.32 (n=3) respectively and H colony formation for SKBR3 cells. NOTE: T-test based p-value calculation for colony formation assay were found to be p<0.05 (n=3) for all the cell lines

Article Snippet: HER2 positive cell line SKBR3 (ATCC Catalog HTB-30) was cultured in McCoy’s 5 A medium.

Techniques: Functional Assay, Control, Colony Assay

Journal: BMC Cancer

Article Title: GCNT3 and ST3GAL1 expression correlates with HER2 status and MUC1/β-catenin/Cyclin D1 axis in breast cancer

doi: 10.1186/s12885-026-15821-w

Figure Lengend Snippet: Clinical significance of MUC1, β-catenin, Cyclin D1 in BC specimens: Representative images of immunohistochemical analysis in different stages and grades of BC for ( A ) β-catenin ( B ) MUC1 and C Cyclin D1. Mean intensity calculated of ( D ) β-catenin analyzed using two-way ANOVA p-value exhibited p<0.05 (n=3) for Grade 2, Stage I vs Stage II vs Stage III and Grade 3, Stage I vs Stage II vs Stage III, E MUC1 analyzed usingtwo-way ANOVA p-value exhibited p<0.05 (n=3) for Grade 2, Stage I vs Stage II, III and p>0.05 (n=3) for Grade 3, Stage I vs Stage II vs Stage III and F Cyclin D1 analyzed using two-way ANOVA p-value exhibited p<0.05 (n=3) for Grade 2, Stage I vs Stage II vs Stage III; however p>0.05 (n=3) was found for Grade 3 tissues. All the BC tissues herein were HER2 -ve

Article Snippet: HER2 positive cell line SKBR3 (ATCC Catalog HTB-30) was cultured in McCoy’s 5 A medium.

Techniques: Immunohistochemical staining

Journal: BMC Cancer

Article Title: GCNT3 and ST3GAL1 expression correlates with HER2 status and MUC1/β-catenin/Cyclin D1 axis in breast cancer

doi: 10.1186/s12885-026-15821-w

Figure Lengend Snippet: Multiple analyses of GCNT3 in driving the MUC1/β-catenin/Cyclin D1 axis in BC: Representative immunohistochemistry images and mean intensity plots of HER2 +ve and HER2 -ve BC samples for (A ) β-catenin ( B ) MUC1 and C Cyclin D1. D Correlation plots of MUC1 with GCNT3, β-catenin, Cyclin D1 and ST3GAL1. E Gene interacting plot for GCNT3 with top 20 interacting nodes using STRING. F Scheme of the mechanistic link between GCNT3 and the β-catenin/Cyclin D1 axis in breast cancer. NOTE: T-test based p-value was found to be significant only for the Cyclin D1 stained HER2 positive vs negative BC tissues (p<0.05, n=3) whereas they were insignificant for the β-catenin and MUC1

Article Snippet: HER2 positive cell line SKBR3 (ATCC Catalog HTB-30) was cultured in McCoy’s 5 A medium.

Techniques: Immunohistochemistry, Staining