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Article Open Access

Ebastine targets HER2/HER3 signaling and cancer stem cell traits to overcome trastuzumab resistance in HER2‑positive breast cancer

  • Authors:
    • Eunsun Jung
    • Ji Young Kim
    • Dongmi Ko
    • Juyeon Seo
    • Sang Yoon Lee
    • Daeun Lee
    • Subeen Kim
    • Minsu Park
    • Seongjae Kim
    • Soeun Park
    • Kyoungmin Lee
    • Yong Koo Kang
    • Kee Dal Nam
    • Yoon-Jae Kim
    • Jae Hong Seo
  • View Affiliations / Copyright

    Affiliations: Division of Medical Oncology, Department of Internal Medicine, Korea University College of Medicine, Korea University, Seoul 152‑703, Republic of Korea
    Copyright: © Jung et al. This is an open access article distributed under the terms of Creative Commons Attribution License.
  • Article Number: 80
    |
    Published online on: February 2, 2026
       https://doi.org/10.3892/ijmm.2026.5751
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Abstract

Despite advances in HER2‑targeted therapy for HER2‑positive breast cancer, resistance to trastuzumab and tumor recurrence remain major barriers to durable outcomes. The present study evaluated the therapeutic potential of ebastine, a second‑generation H1‑antihistamine, as a repurposing candidate to overcome trastuzumab resistance by targeting HER2 signaling and cancer stem cell (CSC)‑associated phenotypes in HER2‑positive breast cancer cells. Molecular docking studies revealed that ebastine bound to the ATP‑binding site of the HER2 tyrosine kinase domain, thereby suppressing the phosphorylation of HER2, p95HER2 and HER3, as assessed by immunoblotting. Immunoprecipitation assay further demonstrated that this binding disrupted HER2/HER3 and HER2/EGFR heterodimerization, leading to reduced downstream AKT activation. Ebastine significantly decreased aldehyde dehydrogenase (ALDH)1 activity, decreased the CD44high/CD24low CSC‑like population, as assessed by flow cytometry, and inhibited mammosphere formation. In a trastuzumab‑resistant xenograft model, ebastine markedly suppressed tumor growth, decreased the Ki‑67 proliferation index and angiogenesis and induced apoptosis. These effects were accompanied by decreased expression of HER2, HER3, ALDH1, CD44, and vimentin in tumor tissues, as determined by immunohistochemistry. Furthermore, serum biochemical analyses revealed no significant hepatotoxicity or nephrotoxicity, indicating a favorable in vivo safety profile. These findings demonstrated that ebastine effectively disrupts key pathways involved in CSC‑like traits and HER2 activity, even under trastuzumab‑resistant conditions. Its multifaceted inhibitory effects support the repositioning of ebastine as a promising therapeutic strategy for treating refractory HER2‑positive breast cancer.
View Figures

Figure 1

EBA induces apoptosis in
trastuzumab-resistant breast cancer cells. (A) Chemical structure
of EBA. (B) Cell viability and IC50 values were
determined using the MTS assay. JIMT-1 and MDA-MB-453 cells were
treated with EBA (48 h) and analyzed by flow cytometry to assess
(C) sub-G1 phase populations and (D) early and late apoptotic
cells. (E) Morphological changes in JIMT-1 cells following EBA
treatment, observed by phase-contrast microscopy. Magnification,
x200. (F) Effect of EBA on the expression of PARP, cleaved PARP,
caspase-3, -7 and -8 and cleaved caspase-3, -7 and -8 in JIMT-1
cells. GAPDH was used as a loading control. *P<0.05,
**P<0.01, ***P<0.001 and
****P<0.0001 vs. vehicle-treated control (0 μM
EBA). EBA, ebastine.

Figure 2

EBA downregulates HER2, p95HER2, HER3
and AKT expression. (A) Immunoblot analysis of HER2, p95HER2 and
p-HER2 (Y1221/1222) in JIMT-1 cells treated with EBA for 48 h. (B)
Immunoblot analysis of HER3, p-HER3 (Y1289), AKT and p-AKT
following treatment with EBA (48 h) in JIMT-1 cells. (C) Immunoblot
analysis of HER2, HER3 and EGFR following IP with anti-HER2
antibody in JIMT-1 cells treated with EBA. In silico
molecular docking of EBA with the crystal structure of HER2-KD. (D)
Surface map of lipophilic and hydrophilic properties at the
ATP-binding site of HER2-KD (red, hydrophobic; blue, hydrophilic).
(E) 2D interaction diagram showing intermolecular interactions
between EBA and HER2-KD. Key amino acid residues within the binding
pocket are shown. (F) Predicted binding pose of EBA (purple stick
model) within the tyrosine kinase domain of HER2 (blue ribbon). (G)
293T cells were treated with DMSO or EBA for 1 h at 37°C, followed
by heating for 3 min. Soluble fractions were collected following
centrifugation and analyzed by immunoblotting using an anti-HER2
antibody. EBA, ebastine; p-, phosphorylated; IP,
immunoprecipitation; IB, immunoblotting; KD, kinase domain PCB,
protein complex binding; TM, transmembrane; a.a., amino acid.

Figure 3

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 (5x104
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 μM)
for 48 h and the CD44high/CD24low cell
populations were identified by flow cytometry. (H) JIMT-1 cells
(1.5x104 cells/ml) were cultured under serum-free
suspension conditions in the presence of EBA (3 μM) for 8
days. Mammosphere number and volumes were quantified.
**P<0.01 and ****P<0.0001 vs.
vehicle-treated control (0 μM EBA). EBA, ebastine; ALDH,
aldehyde dehydrogenase; DEAB, diethylaminobenzaldehyde; CTL,
control; ISO, isotype.

Figure 4

EBA inhibits tumor growth in
trastuzumab-resistant JIMT-1 xenografts. JIMT-1 cells
(3.5x106) were injected into the fourth mammary fat pad
of BALB/c nude mice. Mice were administered EBA intraperitoneally
(20 mg/kg body weight) every other day for 46 days. Tumor (A)
volume and (B) weight were measured. ****P<0.0001 vs.
CTL (vehicle-treated group). (C) Representative xenograft tumors.
(D) No significant alteration in body weight between the
EBA-treated and CTL groups was observed (n=5). (E) Representative
hematoxylin and eosin staining of kidney, liver, lung and tumor
tissue. Red arrows indicate the apoptotic cells with cell shrinkage
and nuclear condensation. (F) Tumor sections were immunostained for
Ki-67 (red) and DAPI (blue). Ki-67-positive cells were quantified.
Apoptosis was assessed by (G) immunostaining for cleaved caspase-3
and (H) TUNEL assay. Arrows indicate TUNEL-positive nuclei) (I)
Tumor angiogenesis was assessed by immunostaining with the
endothelial cell marker CD31 (red) and DAPI (blue). CD31-positive
microvessels in intra- and peritumoral regions were quantified.
CD31-positive staining within the tumor is indicated by white
arrows. The tumor-stroma interface is indicated by a yellow dashed
outline. **P<0.01, ***P<0.001,
****P<0.0001 vs. CTL. EBA, ebastine; CTL, control;
NS, not significant; MVD, microvessel density.

Figure 5

EBA downregulates HER2, ICD-HER2,
HER3, ALDH1A1, CD44 and vimentin in JIMT-1 xenograft tumors.
Immunofluorescence staining of JIMT-1 xenograft tumor tissue for
(A) full-length HER2 (green), (B) ICD-HER2 (green) and (C) HER3.
Immunohistochemical analysis of (D) ALDH1A1 (green) and (E) CD44
(red) in tumor tissue. (F) Tumor sections were immunostained for
vimentin (red). Magnification, x500. Fluorescence intensities were
quantified. Serum biochemical analysis for (G) liver and (H) kidney
function in EBA-treated or CTL mice (n=5). Serum levels of ALT,
AST, TBL, BUN and creatinine were assessed.
***P<0.001, ****P<0.0001. ALT, alanine
aminotransferase; AST, aspartate aminotransferase; BUN, blood urea
nitrogen; EBA, ebastine; ICD, intracellular domain; ALDH, aldehyde
dehydrogenase; CTL, control; NS, not significant.
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Copy and paste a formatted citation
Spandidos Publications style
Jung E, Kim JY, Ko D, Seo J, Lee SY, Lee D, Kim S, Park M, Kim S, Park S, Park S, et al: Ebastine targets HER2/HER3 signaling and cancer stem cell traits to overcome trastuzumab resistance in HER2‑positive breast cancer. Int J Mol Med 57: 80, 2026.
APA
Jung, E., Kim, J.Y., Ko, D., Seo, J., Lee, S.Y., Lee, D. ... Seo, J.H. (2026). Ebastine targets HER2/HER3 signaling and cancer stem cell traits to overcome trastuzumab resistance in HER2‑positive breast cancer. International Journal of Molecular Medicine, 57, 80. https://doi.org/10.3892/ijmm.2026.5751
MLA
Jung, E., Kim, J. Y., Ko, D., Seo, J., Lee, S. Y., Lee, D., Kim, S., Park, M., Kim, S., Park, S., Lee, K., Kang, Y. K., Nam, K. D., Kim, Y., Seo, J. H."Ebastine targets HER2/HER3 signaling and cancer stem cell traits to overcome trastuzumab resistance in HER2‑positive breast cancer". International Journal of Molecular Medicine 57.4 (2026): 80.
Chicago
Jung, E., Kim, J. Y., Ko, D., Seo, J., Lee, S. Y., Lee, D., Kim, S., Park, M., Kim, S., Park, S., Lee, K., Kang, Y. K., Nam, K. D., Kim, Y., Seo, J. H."Ebastine targets HER2/HER3 signaling and cancer stem cell traits to overcome trastuzumab resistance in HER2‑positive breast cancer". International Journal of Molecular Medicine 57, no. 4 (2026): 80. https://doi.org/10.3892/ijmm.2026.5751
Copy and paste a formatted citation
x
Spandidos Publications style
Jung E, Kim JY, Ko D, Seo J, Lee SY, Lee D, Kim S, Park M, Kim S, Park S, Park S, et al: Ebastine targets HER2/HER3 signaling and cancer stem cell traits to overcome trastuzumab resistance in HER2‑positive breast cancer. Int J Mol Med 57: 80, 2026.
APA
Jung, E., Kim, J.Y., Ko, D., Seo, J., Lee, S.Y., Lee, D. ... Seo, J.H. (2026). Ebastine targets HER2/HER3 signaling and cancer stem cell traits to overcome trastuzumab resistance in HER2‑positive breast cancer. International Journal of Molecular Medicine, 57, 80. https://doi.org/10.3892/ijmm.2026.5751
MLA
Jung, E., Kim, J. Y., Ko, D., Seo, J., Lee, S. Y., Lee, D., Kim, S., Park, M., Kim, S., Park, S., Lee, K., Kang, Y. K., Nam, K. D., Kim, Y., Seo, J. H."Ebastine targets HER2/HER3 signaling and cancer stem cell traits to overcome trastuzumab resistance in HER2‑positive breast cancer". International Journal of Molecular Medicine 57.4 (2026): 80.
Chicago
Jung, E., Kim, J. Y., Ko, D., Seo, J., Lee, S. Y., Lee, D., Kim, S., Park, M., Kim, S., Park, S., Lee, K., Kang, Y. K., Nam, K. D., Kim, Y., Seo, J. H."Ebastine targets HER2/HER3 signaling and cancer stem cell traits to overcome trastuzumab resistance in HER2‑positive breast cancer". International Journal of Molecular Medicine 57, no. 4 (2026): 80. https://doi.org/10.3892/ijmm.2026.5751
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