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Ephrin‑B2 promotes gastric cancer growth by inhibiting apoptosis and regulating the cell cycle via the Wnt/β‑catenin signaling pathway

  • Authors:
    • Ding Ding
    • Xiaoshan Wang
    • Ran Xuan
    • Rui Li
    • Yalu Zhang
    • Zhengguang Wang
  • View Affiliations / Copyright

    Affiliations: Department of General Surgery, The First Affiliated Hospital of Anhui Medical University, Hefei, Anhui 230022, P.R. China, Department of General Surgery, The Third Affiliated Hospital of Anhui Medical University (The First People's Hospital of Hefei), Hefei, Anhui 230061, P.R. China, Department of General Surgery, The First Affiliated Hospital of University of Science and Technology of China, Division of Life Science and Medicine, University of Science and Technology of China, Hefei, Anhui 230001, P.R. China
    Copyright: © Ding et al. This is an open access article distributed under the terms of Creative Commons Attribution License.
  • Article Number: 8
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    Published online on: November 18, 2025
       https://doi.org/10.3892/ijo.2025.5821
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Abstract

Gastric cancer (GC) ranks among the most prevalent malignancies worldwide and is associated with high mortality rates. Ephrin‑B2 (EFNB2), a membrane‑bound ligand that interacts with Eph receptor tyrosine kinases, has been implicated in various cancer‑related biological processes; however, its precise role in GC remains poorly understood. By integrating data from multiple public databases with immunohistochemical analyses of tissue microarrays, significant upregulation of EFNB2 expression in GC specimens compared with paired adjacent normal tissue was demonstrated. Elevated EFNB2 levels were associated with the poor overall survival and disease‑free survival in patients with GC. EFNB2 knockdown inhibited cellular proliferation and viability, increased apoptosis, and induced cell cycle arrest at the G0/G1 phase in GC cells. By contrast, EFNB2 overexpression resulted in the opposite oncogenic effects. Mechanistically, rescue experiments identified the Wnt/β‑catenin signaling cascade as the primary molecular pathway mediating EFNB2‑driven tumorigenic effects. These results were further validated in vivo using cell‑derived xenograft models, which confirmed the key role of Wnt/β‑catenin pathway activation in EFNB2‑induced tumor progression. Collectively, these results suggested that EFNB2 represents a promising molecular target for therapeutic intervention in GC.
View Figures

Figure 1

EFNB2 is highly expressed in GC
tissues and is associated with tumor markers. (A) Expression levels
of EFNB2 in GC tissues (n=408) and normal gastric mucosa tissues
(n=211) in the GEPIA database (unpaired Student's t-test). (B)
Analysis of data from the GEPIA database indicated the levels of
EFNB2 in different pathological stages of GC (one-way ANOVA). The
expression levels of EFNB2 in different (C) pathological stages and
(D) T stages of GC analyzed using the Linkedomics database (one-way
ANOVA). (E) Representative IHC staining images for EFNB2 in ANT and
GC tissues. The magnification is indicated in the figure (×100 or
×200). (F) IHC scores of EFNB2 were evaluated in 96 GC tissues and
84 ANTs (Mann-Whitney U test). The levels of different tumor
markers were compared between the EFNB2-high expression group and
the EFNB2-low expression group, including (G) CA199, (H) CEA and
(I) CA50 (Mann-Whitney U test). *P<0.05,
***P<0.001. GC, gastric cancer; GEPIA, Gene
Expression Profiling Interactive Analysis; IHC,
immunohistochemical; ANT, adjacent normal tissue; EFNB2,
ephrin-B2.

Figure 2

Upregulated EFNB2 expression is
associated with poor prognosis of patients with GC. The
Kaplan-Meier plotter demonstrated that EFNB2 was not only
associated with (A) OS (P=0.028), but also served as a negative
factor for (B) DFS in GC (P=0.014), when excluding the dataset
GSE62254. In the GEPIA database, the EFNB2-high expression group
exhibited shorter (C) OS and (D) DFS compared with the EFNB2-low
expression group, with the conditions set as 'Cutoff-High vs.
Cutoff-Low: 30 vs. 30' (P=0.0078 and 0.008, respectively). (E)
Analysis of the cohort of patients whose samples were included in
the tissue microarray (n=96) demonstrated that patients with GC
with high EFNB2 expression had a worse clinical outcome compared
with those with low EFNB2 expression (P=0.0007). OS, overall
survival; DFS, disease-free survival; HR, hazard ratio; EFNB2,
ephrin-B2; GC, gastric cancer; GEPIA, Gene Expression Profiling
Interactive Analysis; TPM, transcript per million.

Figure 3

EFNB2 promotes the proliferation and
viability of GC cells in vitro. (A) Representative western
blot bands. (B) Expression levels of EFNB2 were detected in
multiple GC cell lines and human GES-1 normal gastric mucosal
epithelial cells using western blotting (one-way ANOVA). (C)
Representative western blot bands. (D) The effects of EFNB2
knockdown and overexpression were verified by western blotting
(one-way ANOVA for AGS cells; unpaired Student's test for HGC-27
cells). (E) Cell Counting Kit-8 assays revealed the OD values at
450 nm after 0, 24, 48 and 72 h (one-way ANOVA). (F) Representative
colony formation assay images. (G) Colony formation was assessed by
counting the number of colonies in various groups (one-way ANOVA
for AGS cells; unpaired Student's test for HGC-27 cells). Data are
presented as the mean ± SD. *P<0.05,
**P<0.01, ***P<0.001. GC, gastric
cancer; EFNB2, ephrin-B2; OD, optical density; OE, overexpression
vector; sh, short hairpin RNA; NC, negative control.

Figure 4

EFNB2 inhibits apoptosis and alters
the cell cycle distribution in gastric cancer cells in
vitro. (A) Representative flow cytometry plots and (B)
quantification of apoptosis in AGS and HGC-27 cells (one-way ANOVA
for AGS cells; unpaired Student's test for HGC-27 cells). (C)
Representative cell cycle distribution plots and (D) quantification
for the EFNB2 knockdown and overexpression groups as assessed by
flow cytometry (one-way ANOVA). (E) Representative western blot
bands and semi-quantification of protein expression levels of
apoptosis-related proteins (Bcl-2 and Bax) and cell cycle-related
proteins (CyclinD1 and CDK4) detected in the context of EFNB2 (F)
knockdown or (G) overexpression (one-way ANOVA for AGS cells;
unpaired Student's test for HGC-27 cells). Data are presented as
the mean ± SD. *P<0.05, **P<0.01,
***P<0.001 vs. sh-NC or vector group. EFNB2,
ephrin-B2; AAD, amino-actinomycin D; APC, allophycocyanin; OE,
overexpression vector; sh, short hairpin RNA; NC, negative
control.

Figure 5

Wnt/β-catenin signaling pathway is
responsible for EFNB2-mediated biological effects in gastric cancer
cells. (A) Gene Expression Profiling Interactive Analysis and (B)
Tumor Immune Estimation Resource databases were employed to explore
the transcriptional correlation between EFNB2 and CTNNB1, GSK3β and
downstream MYC (Spearman). Representative western blot bands and
semi-quantification of protein expression levels of p-GSK3β, GSK3β,
β-catenin and c-myc detected under the condition of EFNB2 (C)
knockdown and (D) overexpression (one-way ANOVA for AGS cells;
unpaired Student's test for HGC-27 cells). Representative western
blot bands and semi-quantification of protein expression levels in
the presence of (E) an agonist (CHIR99021) and (F) an inhibitor
(DIF-3) of the Wnt/β-catenin signaling pathway in the EFNB2
knockdown or overexpression groups, respectively. Protein levels of
p-GSK3β, GSK3β, β-catenin and c-myc were detected by western
blotting (one-way ANOVA). Data are presented as the mean ± SD.
*P<0.05, **P<0.01,
***P<0.001 vs. sh-NC or vector group. ns, not
significant; NC, negative control; DIF-3, differentiation-inducing
factor-3; p-, phosphorylated; OE, overexpression vector; sh, short
hairpin RNA; CTNNB1, catenin β1; TPM, transcript per million;
EFNB2, ephrin-B2.

Figure 6

DIF-3 abrogates EFNB2-mediated
biological effects in gastric cancer cells. (A) A Cell Counting
Kit-8 was used to assess the impact of DIF-3 on HGC-27 cells
transfected with EFNB2 overexpression vector in terms of
proliferation (one-way ANOVA). (B) Representative images of colony
formation assays. (C) Quantitative analysis of colony formation was
performed to evaluate the effect of DIF-3 on EFNB2-overexpressing
cells. (D and F) Representative flow cytometry plots and
quantification of (E) apoptosis and (G) cell cycle distribution for
the evaluation of the effect of DIF-3 on EFNB2-overexpressing cells
(one-way ANOVA). Data are presented as the mean ± SD.
*P<0.05, **P<0.01 vs. vector; ns, DIF-3
vs. OE-EFNB2 + DIF-3. ns, not significant; EFNB2, ephrin-B2; AAD,
amino-actinomycin D; APC, allophycocyanin; DIF-3,
differentiation-inducing factor-3; OD, optical density; OE,
overexpression vector.

Figure 7

EFNB2 promotes the tumor growth of
gastric cancer via the Wnt/β-catenin signaling pathway in
vivo. (A) Tumors from the immunodeficient nude mice in the four
experimental groups were excised and imaged. (B) Subcutaneous tumor
growth (mm3) was measured every 3-4 days following
various treatments (one-way ANOVA). (C) Excised tumor masses (g)
were weighed and compared across groups (one-way ANOVA). (D)
Histopathological evaluation of xenograft tumors included H&E
staining to examine tissue morphology, IHC analysis of EFNB2,
β-catenin and Ki67 expression, and TUNEL staining for apoptotic
cell evaluation. All images were captured at a magnification of
×200. (E) Quantification of IHC scores for EFNB2 and β-catenin, and
the percentage of positive cells for Ki67 and TUNEL staining
(Kruskal-Wallis H test for EFNB2 and β-catenin; one-way ANOVA for
Ki67 and TUNEL staining). Data are presented as the mean ± SD.
*P<0.05, **P<0.01,
***P<0.001. ns, not significant; EFNB2, ephrin-B2;
DIF-3, differentiation-inducing factor-3; OE, overexpression
vector; IHC, immunohistochemical.
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Copy and paste a formatted citation
Spandidos Publications style
Ding D, Wang X, Xuan R, Li R, Zhang Y and Wang Z: Ephrin‑B2 promotes gastric cancer growth by inhibiting apoptosis and regulating the cell cycle via the Wnt/&beta;‑catenin signaling pathway. Int J Oncol 68: 8, 2026.
APA
Ding, D., Wang, X., Xuan, R., Li, R., Zhang, Y., & Wang, Z. (2026). Ephrin‑B2 promotes gastric cancer growth by inhibiting apoptosis and regulating the cell cycle via the Wnt/&beta;‑catenin signaling pathway. International Journal of Oncology, 68, 8. https://doi.org/10.3892/ijo.2025.5821
MLA
Ding, D., Wang, X., Xuan, R., Li, R., Zhang, Y., Wang, Z."Ephrin‑B2 promotes gastric cancer growth by inhibiting apoptosis and regulating the cell cycle via the Wnt/&beta;‑catenin signaling pathway". International Journal of Oncology 68.1 (2026): 8.
Chicago
Ding, D., Wang, X., Xuan, R., Li, R., Zhang, Y., Wang, Z."Ephrin‑B2 promotes gastric cancer growth by inhibiting apoptosis and regulating the cell cycle via the Wnt/&beta;‑catenin signaling pathway". International Journal of Oncology 68, no. 1 (2026): 8. https://doi.org/10.3892/ijo.2025.5821
Copy and paste a formatted citation
x
Spandidos Publications style
Ding D, Wang X, Xuan R, Li R, Zhang Y and Wang Z: Ephrin‑B2 promotes gastric cancer growth by inhibiting apoptosis and regulating the cell cycle via the Wnt/&beta;‑catenin signaling pathway. Int J Oncol 68: 8, 2026.
APA
Ding, D., Wang, X., Xuan, R., Li, R., Zhang, Y., & Wang, Z. (2026). Ephrin‑B2 promotes gastric cancer growth by inhibiting apoptosis and regulating the cell cycle via the Wnt/&beta;‑catenin signaling pathway. International Journal of Oncology, 68, 8. https://doi.org/10.3892/ijo.2025.5821
MLA
Ding, D., Wang, X., Xuan, R., Li, R., Zhang, Y., Wang, Z."Ephrin‑B2 promotes gastric cancer growth by inhibiting apoptosis and regulating the cell cycle via the Wnt/&beta;‑catenin signaling pathway". International Journal of Oncology 68.1 (2026): 8.
Chicago
Ding, D., Wang, X., Xuan, R., Li, R., Zhang, Y., Wang, Z."Ephrin‑B2 promotes gastric cancer growth by inhibiting apoptosis and regulating the cell cycle via the Wnt/&beta;‑catenin signaling pathway". International Journal of Oncology 68, no. 1 (2026): 8. https://doi.org/10.3892/ijo.2025.5821
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