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Article Open Access

Tumor‑associated macrophages suppress estrogen receptor‑β expression in triple‑negative breast cancer through the PI3K/AKT pathway

  • Authors:
    • Qiulei Zhang
    • Di Guo
    • Gaoran Xu
    • Rong Xie
    • Yuqing Deng
    • Peng Fu
    • Jinnan Wan
  • View Affiliations / Copyright

    Affiliations: Department of Thyroid and Breast Surgery, The Central Hospital of Wuhan, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei 430014, P.R. China, Department of Internal Medicine, Central China Normal University Hospital, Central China Normal University, Wuhan, Hubei 430079, P.R. China
    Copyright: © Zhang et al. This is an open access article distributed under the terms of Creative Commons Attribution License.
  • Article Number: 77
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    Published online on: January 21, 2026
       https://doi.org/10.3892/etm.2026.13072
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Abstract

Triple‑negative breast cancer (TNBC) is a highly aggressive breast cancer subtype with limited therapeutic options. Estrogen receptor (ER)‑β exerts anti‑tumor effects. However, ERβ expression is frequently reduced in TNBC and the mechanisms underlying the downregulation of ERβ in TNBC remain poorly understood. In the present study, findings revealed that tumor‑associated macrophages (TAMs), which are abundant in the tumor microenvironment, suppressed ERβ expression in TNBC cells. The relationship between TAMs and ERβ in TNBC was investigated through bioinformatics analysis, co‑culture and orthotopic mouse models. ERβ expression levels were lower in the TNBC tumors compared with normal tissues. Mechanistically, TAM‑induced activation of the PI3K/AKT pathway suppressed ERβ expression by reducing FOXO3a binding to the ESR2 promoter, whereas inhibition of PI3K/AKT restored ERβ expression. Notably, combined PI3K/AKT pathway inhibition with ERβ activation attenuated TNBC cell metastasis. Overall, findings indicated that TAMs promoted TNBC progression by downregulating ERβ expression through the PI3K/AKT/FOXO3a axis, highlighting the therapeutic potential of combining PI3K/AKT inhibitors with ERβ agonists in TNBC.

View Figures

Figure 1

Clinical value and expression of ERβ
(also known as ESR2) in TNBC. (A) PFS analysis of ESR2 levels in
BRCA. (B) OS analysis of ESR2 levels in BRCA. (C) ESR2 mRNA levels
in different BRCA subtypes. (D) ESR2 mRNA levels in different
breast cancer subtype cell lines. (E) Migratory capacity of 4T1
cells treated with vehicle (ethanol) or ERβ agonist, ERB041 (100
nM), was assessed through Transwell assays. (F) Scatter plot
showing the differentially expressed ESR2 between TNBC tumor and
normal tissues. (G) Immunohistochemical staining from the Human
Protein Atlas database revealed ERβ protein expression in both
normal breast and BRCA tissues. (H) Immunohistochemistry was used
to evaluate the ERβ expression levels in paracancerous and breast
cancer tissues. Scale bar, 100 µm. Magnification, x100.
*P<0.05 and ***P<0.001. Erβ, estrogen
receptor-β; TNBC, triple negative breast cancer; PFS,
progression-free survival; OS, overall survival; BRCA; breast
cancer; HER-2, human epidermal growth factor receptor-2; CON,
control; ns, not significant; TCGA, The Cancer Genome Atlas.

Figure 2

Tumor-associated macrophages enhance
tumor metastasis in TNBC. (A) Boxplots demonstrating the
differential expression of MSR1 between BRCA tumor tissue and
normal tissue. (B) Overall survival analysis of MSR1 levels in
TNBC. (C) Correlation analysis of MSR1 levels with Vim, Mmp-9,
Snai1, Twist1, Fn1 and Mmp2 levels in TNBC. (D) UMAP analysis of
total cells in mouse breast tumors. (E) Gene set enrichment
analysis of the epithelial-mesenchymal transition marker levels in
breast tumor. MSR1, macrophage scavenger receptor ; RSEM,
RNA-sequencing by expectation maximization; BRCA, breast cancer;
TPM, transcripts per million; Mmp-9, matrix metalloproteinase-9;
Vim, vimentin; Fn1, fibronectin 1; MMP2, matrix
metalloproteinase-2; DC, dendritic cells; FDR, false discovery
rate; TNBC, triple negative breast cancer; UMAP, uniform manifold
approximation and projection.

Figure 3

TAMs suppress ERβ expression in TNBC.
(A) Bubble plot demonstrating the correlation between ESR2 mRNA
levels and 24 immune cell type infiltrates in BRCA. Bubble size
correlates with FDR. Black outline border indicates FDR ≤0.05. (B)
Correlation between macrophages infiltration and ESR2 expression.
(C) Relative mRNA levels of ESR2 in different groups. (D)
Representative flow cytometric analysis of ERβ expression levels in
4T1 cells co-cultured with different proportion of TAMs (low,
median and high) for 24 h. Mean fluorescence intensity of ERβ in
the 4T1 cells of each group shown on the right (n=3). (E)
Representative cellular immunofluorescence images of ERβ (red) and
nuclei stained with DAPI (blue). (F) Immunofluorescence staining
for MSR1 (green) and ERβ (red) in the tumor tissues of the control
(left) and clodronate liposome-treated (right) groups. Original
magnification, x200. Scale bars, 50 µm. *P<0.05,
**P<0.01 and ***P<0.001. Comp-BV421-A
represents F4/80 staining, and Comp-PE-A represents ERβ staining.
ns, no significant; ERβ, estrogen receptor-β; TNBC, triple-negative
breast cancer; BRCA, breast cancer; FDR, false discovery rate; TAM,
tumor-associated macrophage; Cor., correlation; CON, control; CM,
conditioned medium; SSC-A, side scatter-area; FSC-A, forward
scatter-area; MFI, mean fluorescence intensity.

Figure 4

Tumor-associated macrophages suppress
ERβ expression through the PI3K/AKT pathway. (A) Top 10 Kyoto
Encyclopedia of Genes and Genomes enrichment pathways. (B) PI3K/AKT
pathway activity in high and low breast cancer MSR1 expression
groups. (C) Immunohistochemistry for MSR1 and p-AKT levels in the
control and clodronate-treated groups. Original magnification,
x200, scale bars, 100 µm. (D) 4T1 cells cultured in CM then treated
DMSO or 100 nM, 1 µM or 10 µM of LY294002 for 24 h. Relative mRNA
levels of ESR2 in different groups (n=4). (E) Co-localization of
p-AKT and ER β in 4T1 cells. Representative cellular
immunofluorescence images of 4T1 cells treated with CM or CM +
LY294002 (1 µM), ERβ (green), p-AKT (red) and DAPI (blue). Original
magnification, x400, scale bar, 20 µm. (F) Binding motif of FOXO3a
(from JASPAR). (G) ChIP-PCR shows the binding of FOXO3a on the
promoter of ESR2. Agarose gel electrophoresis of ChIP-PCR products.
(H) ChIP analysis for binding of FOXO3a to the ESR2 promoter in 4T1
cells upon CM, CON or CM + LY294002 treatment for 24 h. Data are
expressed as enrichment relative to the input.
**P<0.01 and ***P<0.001; ns, no
significant; CM, conditioned medium; ChiP; chromatin
immunoprecipitation; Erβ, estrogen receptor-β; CON, control; p-AKT;
phosphorylated AKT; MSR1, macrophage scavenger receptor 1; Expr.,
expression; FDR, false discovery rate.

Figure 5

PI3K/AKT inhibition and ERβ
activation inhibit tumor metastasis. (A) Representative images of
Transwell migration assays after treatment with LY294002 (1 µM) or
a combination of LY294002 and ERB041 (10-500 nM). Graph of
Transwell assays (n=3). (B) Representative images of wound healing
assay at 0 and 24 h after treatment with CM, LY294002 (1 µM) or a
combination of LY294002 and ERB041 (100 nM). (C) Graph of wound
healing assay (n=3). (D) 4T1 cells (1x106) were injected
into the fat pads of BALB/c mice, then treated with DMSO, LY294002
(50 mg/kg intraperitoneally twice a week for five weeks) or
LY294002 combined with ERB041 (5 mg/kg subcutaneously daily for
five weeks). Representative images showing the H&E staining of
lung metastatic nodules in 4T1 mice model. Scale bar, 5 mm (E) Lung
metastatic nodule counts in different groups (n=4). (F) The volume
of the 4T1 tumor across all groups (n=4). (G) Representative
immunohistochemical staining images of p-AKT and ERβ in the breast
tumors of different treatment groups. Scale bar, 50 µm.
Magnification, x200. *P<0.05, **P<0.01
and ***P<0.001; ns, not significant. Erβ, estrogen
receptor-β; CM, conditioned medium; p-AKT, phosphorylated AKT; Con,
control.
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Copy and paste a formatted citation
Spandidos Publications style
Zhang Q, Guo D, Xu G, Xie R, Deng Y, Fu P and Wan J: <p>Tumor‑associated macrophages suppress estrogen receptor‑&beta; expression in triple‑negative breast cancer through the PI3K/AKT pathway</p>. Exp Ther Med 31: 77, 2026.
APA
Zhang, Q., Guo, D., Xu, G., Xie, R., Deng, Y., Fu, P., & Wan, J. (2026). <p>Tumor‑associated macrophages suppress estrogen receptor‑&beta; expression in triple‑negative breast cancer through the PI3K/AKT pathway</p>. Experimental and Therapeutic Medicine, 31, 77. https://doi.org/10.3892/etm.2026.13072
MLA
Zhang, Q., Guo, D., Xu, G., Xie, R., Deng, Y., Fu, P., Wan, J."<p>Tumor‑associated macrophages suppress estrogen receptor‑&beta; expression in triple‑negative breast cancer through the PI3K/AKT pathway</p>". Experimental and Therapeutic Medicine 31.3 (2026): 77.
Chicago
Zhang, Q., Guo, D., Xu, G., Xie, R., Deng, Y., Fu, P., Wan, J."<p>Tumor‑associated macrophages suppress estrogen receptor‑&beta; expression in triple‑negative breast cancer through the PI3K/AKT pathway</p>". Experimental and Therapeutic Medicine 31, no. 3 (2026): 77. https://doi.org/10.3892/etm.2026.13072
Copy and paste a formatted citation
x
Spandidos Publications style
Zhang Q, Guo D, Xu G, Xie R, Deng Y, Fu P and Wan J: <p>Tumor‑associated macrophages suppress estrogen receptor‑&beta; expression in triple‑negative breast cancer through the PI3K/AKT pathway</p>. Exp Ther Med 31: 77, 2026.
APA
Zhang, Q., Guo, D., Xu, G., Xie, R., Deng, Y., Fu, P., & Wan, J. (2026). <p>Tumor‑associated macrophages suppress estrogen receptor‑&beta; expression in triple‑negative breast cancer through the PI3K/AKT pathway</p>. Experimental and Therapeutic Medicine, 31, 77. https://doi.org/10.3892/etm.2026.13072
MLA
Zhang, Q., Guo, D., Xu, G., Xie, R., Deng, Y., Fu, P., Wan, J."<p>Tumor‑associated macrophages suppress estrogen receptor‑&beta; expression in triple‑negative breast cancer through the PI3K/AKT pathway</p>". Experimental and Therapeutic Medicine 31.3 (2026): 77.
Chicago
Zhang, Q., Guo, D., Xu, G., Xie, R., Deng, Y., Fu, P., Wan, J."<p>Tumor‑associated macrophages suppress estrogen receptor‑&beta; expression in triple‑negative breast cancer through the PI3K/AKT pathway</p>". Experimental and Therapeutic Medicine 31, no. 3 (2026): 77. https://doi.org/10.3892/etm.2026.13072
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