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Integrin beta 4 promotes colorectal cancer progression by upregulating Ezrin and activating the Wnt/β‑catenin signaling pathway

  • Authors:
    • Jing Wang
    • Yi Si
    • Mingda Xuan
    • Shuangshuang Han
    • Kunyi Liu
    • Jiao Jiao
    • Xiaoyan Men
    • Hongfei Li
    • Jia Wang
    • Ting Liu
    • Weifang Yu
  • View Affiliations / Copyright

    Affiliations: Department of Endoscopy Center, The First Hospital of Hebei Medical University, Shijiazhuang, Hebei 050031, P.R. China, Department of Infectious Diseases, The First Hospital of Hebei Medical University, Shijiazhuang, Hebei 050031, P.R. China
    Copyright: © Wang et al. This is an open access article distributed under the terms of Creative Commons Attribution License.
  • Article Number: 82
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    Published online on: May 19, 2026
       https://doi.org/10.3892/ijo.2026.5895
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Abstract

Colorectal cancer (CRC) is a major cause of cancer‑related mortality worldwide. Integrin beta 4 (ITGB4) has been previously identified as being overexpressed in CRC; however, its precise oncogenic mechanism remains unclear. The present study aimed to elucidate the functional role of ITGB4 in CRC progression and identify its downstream molecular effectors to provide new insights for targeted therapy. The biological functions of ITGB4 were investigated in CRC cell lines (SW480 and HCT116) using a series of in vitro assays, including Cell Counting Kit‑8, colony formation, Transwell migration and invasion, and flow cytometry for apoptosis following ITGB4 knockdown. An in vivo xenograft mouse model was used to evaluate the effect of ITGB4 on tumor growth. Downstream targets were screened using RNA sequencing (RNA‑seq) and validated by co‑immunoprecipitation and co‑immunofluorescence. The underlying signaling pathway was investigated by western blotting and functional rescue experiments. The results demonstrated that knockdown of ITGB4 significantly suppressed CRC cell proliferation, migration and invasion, while promoting apoptosis in vitro. Similarly, silencing ITGB4 markedly inhibited tumor growth in the in vivo xenograft model. RNA‑seq analysis identified Ezrin (EZR) as a key downstream target of ITGB4, and a direct protein‑protein interaction was confirmed between them. Mechanistically, ITGB4 knockdown decreased the expression of EZR at both the mRNA and protein levels. ITGB4 was demonstrated to exert its pro‑tumorigenic effects through the regulation of EZR, which subsequently activated the Wnt/β‑catenin signaling pathway. Interestingly, EZR overexpression partially restored ITGB4 levels, suggesting a hypothetical positive feedback loop via Wnt/β‑catenin signaling that could amplify this oncogenic axis. Notably, the malignant phenotypes suppressed by ITGB4 silencing were significantly rescued by the overexpression of EZR. In conclusion, the present study identified a novel ITGB4/EZR/Wnt/β‑catenin signaling axis in CRC. ITGB4 promotes CRC progression by modulating EZR expression and subsequently activating the Wnt/β‑catenin pathway. These findings highlight ITGB4 as a potential prognostic biomarker and a promising therapeutic target for CRC.
View Figures

Figure 1

ITGB4 is upregulated in CRC and
associated with poor prognosis. (A-C) Analysis of ITGB4 mRNA
expression in CRC tissues and adjacent normal tissues using The
Cancer Genome Atlas and GEPIA2 databases. (D) Kaplan-Meier analysis
demonstrating the correlation between ITGB4 expression levels and
overall survival in patients with CRC. *P<0.05.
ITGB4, integrin beta 4; CRC, colorectal cancer; COAD, colon
adenocarcinoma; READ, rectum adenocarcinoma; HR, hazard ratio.

Figure 2

Knockdown of ITGB4 inhibits
proliferation, migration and invasion and promotes apoptosis in CRC
cells. (A) RT-qPCR validation of knockdown efficiency for four
different siITGB4 sequences in SW480 cells. (B) Western blot
validation of knockdown efficiency for the four siITGB4 sequences.
(C and D) Verification of siITGB4 knockdown efficiency at the mRNA
level in SW480 and HCT116 cells via RT-qPCR. (E and F) Verification
of siITGB4 knockdown efficiency at the protein level via western
blotting. (G and H) Cell Counting Kit-8 assays showing the effect
of ITGB4 knockdown on the proliferation of SW480 and HCT116 cells.
(I and J) Colony formation assays demonstrating the effect of ITGB4
knockdown on the clonogenic ability of CRC cells. (K and L) Flow
cytometric analysis showing the effect of ITGB4 knockdown on
apoptosis. (M and N) Transwell assays showing the effect of ITGB4
knockdown on the migration and invasion abilities of SW480 and
HCT116 cells (Scale bar, 100 μm). Data are presented as the
mean ± SD. *P<0.05, **P<0.01,
***P<0.001 and ****P<0.0001. ITGB4,
integrin beta 4; CRC, colorectal cancer; RT-qPCR, reverse
transcription-quantitative PCR; si-, small interfering; NC,
negative control.

Figure 3

ITGB4 silencing suppresses colorectal
cancer growth in vivo. (A and B) Reverse
transcription-quantitative PCR and western blot analysis verifying
the stable knockdown efficiency of shITGB4 in HCT116 cells. (C)
Representative images of tumor-bearing nude mice from the shNC and
shITGB4 groups. (D) Tumor growth curves for both groups over 21
days. (E and F) Images and weights of the excised xenograft tumors.
(G and H) Verification of ITGB4 knockdown in xenograft tumor
tissues. (I) Representative H&E staining of tumor sections
(scale bars, 50 μm). (J and K) Representative
immunohistochemical staining and quantification of Ki-67 in tumor
sections (scale bars, 50 μm). Data are presented as the mean
± SD. *P<0.05, **P<0.01,
***P<0.001 and ****P<0.0001. ITGB4,
integrin beta 4; sh-, short hairpin; NC, negative control; AOD,
average optical density.

Figure 4

Systematic screening identifies
candidate downstream targets of ITGB4. (A) Heatmap of DEGs
identified by RNA-seq analysis of SW480 cells after ITGB4
knockdown. (B) Volcano plot illustrating the distribution of DEGs.
(C) Schematic flowchart illustrating the multi-step filtering
strategy. A Venn diagram was used to intersect ITGB4-downregulated
genes (from RNA-seq) with genes co-expressed with ITGB4 and
upregulated in colorectal cancer (from public databases),
identifying 35 candidate genes. These candidates were further
filtered by subcellular localization to identify the final five
targets. (D and E) Reverse transcription-quantitative PCR
validation of the mRNA levels of the five candidate target genes
following ITGB4 knockdown in SW480 and HCT116 cells.
**P<0.01, ***P<0.001 and
****P<0.0001. ITGB4, integrin beta 4; DEGs,
differentially expressed genes; RNA-seq, RNA sequencing; si-, small
interfering; NC, negative control; EZR, Ezrin.

Figure 5

Bioinformatic analysis highlights EZR
as a prime candidate downstream of ITGB4. (A) Subcellular
localization of ITGB4 and the five candidate genes as annotated in
the GeneCards database. (B) Expression analysis of the five
candidate genes in CRC vs. adjacent normal tissues using the GEPIA2
database. (C) Correlation analysis of ITGB4 expression with each of
the five candidate genes in The Cancer Genome Atlas-Colon
Adenocarcinoma cohort. (D) Kaplan-Meier survival analysis showing
the prognostic significance of the five candidate genes in patients
with CRC. *P<0.05. EZR, Ezrin; ITGB4, integrin beta
4; CRC, colorectal cancer; HR, hazard ratio.

Figure 6

ITGB4 interacts with EZR and
regulates its expression. (A) Co-immunoprecipitation assay in SW480
cells demonstrating the interaction between endogenous ITGB4 and
EZR proteins. (B) Co-immunofluorescence staining in SW480 cells
showing the co-localization of ITGB4 (green) and EZR (red) at the
cell membrane. Nuclei were stained with DAPI (blue). (C)
Protein-protein interaction network from the STRING database
predicting an association between ITGB4 and EZR. (D and E)
Verification of EZR overexpression efficiency in SW480 and HCT116
cells by RT-qPCR and western blot after transfection with
pcDNA3.1-EZR. (F and G) RT-qPCR and western blot analysis showing
decreased EZR mRNA and protein levels in SW480 cells after ITGB4
knockdown. (H and I) Western blot and RT-qPCR analysis confirming
that EZR overexpression does not rescue the siRNA-mediated
knockdown of ITGB4, indicating EZR acts downstream of ITGB4. Data
are presented as the mean ± SD. *P<0.05,
**P<0.01, ***P<0.001 and
****P<0.0001. ITGB4, integrin beta 4; EZR, Ezrin;
RT-qPCR, reverse transcription-quantitative PCR; si-, small
interfering; NC, negative control.

Figure 7

ITGB4 exerts its oncogenic function
through the EZR-mediated activation of the Wnt/β-catenin signaling
pathway. (A) Kyoto Encyclopedia of Genes and Genomes pathway
enrichment analysis of differentially expressed genes following
ITGB4 knockdown, highlighting the Wnt signaling pathway. (B)
Western blot analysis and corresponding quantification showing that
ITGB4 knockdown suppresses the expression of β-catenin, c-Myc and
Cyclin D1, and this effect is rescued by EZR overexpression in
HCT116 cells. (C) Cell Counting Kit-8 assays demonstrating that EZR
overexpression rescues the proliferation defect caused by ITGB4
knockdown in SW480 and HCT116 cells. (D-G) Wound healing assays
showing that EZR overexpression restores the migratory capacity
inhibited by ITGB4 knockdown (magnification, ×200). (H and I)
Transwell migration assays confirming the rescue of cell migration
by EZR overexpression (crystal violet staining; scale bar, 100
μm). (J and K) Transwell invasion assays showing that EZR
overexpression rescues the invasive potential suppressed by ITGB4
knockdown (crystal violet staining; scale bar, 100 μm). Data
are presented as the mean ± SD. *P<0.05,
**P<0.01, ***P<0.001 and
****P<0.0001. ITGB4, integrin beta 4; EZR, Ezrin;
si-, small interfering; NC, negative control.
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Copy and paste a formatted citation
Spandidos Publications style
Wang J, Si Y, Xuan M, Han S, Liu K, Jiao J, Men X, Li H, Wang J, Liu T, Liu T, et al: Integrin beta 4 promotes colorectal cancer progression by upregulating Ezrin and activating the Wnt/&beta;‑catenin signaling pathway. Int J Oncol 69: 82, 2026.
APA
Wang, J., Si, Y., Xuan, M., Han, S., Liu, K., Jiao, J. ... Yu, W. (2026). Integrin beta 4 promotes colorectal cancer progression by upregulating Ezrin and activating the Wnt/&beta;‑catenin signaling pathway. International Journal of Oncology, 69, 82. https://doi.org/10.3892/ijo.2026.5895
MLA
Wang, J., Si, Y., Xuan, M., Han, S., Liu, K., Jiao, J., Men, X., Li, H., Wang, J., Liu, T., Yu, W."Integrin beta 4 promotes colorectal cancer progression by upregulating Ezrin and activating the Wnt/&beta;‑catenin signaling pathway". International Journal of Oncology 69.1 (2026): 82.
Chicago
Wang, J., Si, Y., Xuan, M., Han, S., Liu, K., Jiao, J., Men, X., Li, H., Wang, J., Liu, T., Yu, W."Integrin beta 4 promotes colorectal cancer progression by upregulating Ezrin and activating the Wnt/&beta;‑catenin signaling pathway". International Journal of Oncology 69, no. 1 (2026): 82. https://doi.org/10.3892/ijo.2026.5895
Copy and paste a formatted citation
x
Spandidos Publications style
Wang J, Si Y, Xuan M, Han S, Liu K, Jiao J, Men X, Li H, Wang J, Liu T, Liu T, et al: Integrin beta 4 promotes colorectal cancer progression by upregulating Ezrin and activating the Wnt/&beta;‑catenin signaling pathway. Int J Oncol 69: 82, 2026.
APA
Wang, J., Si, Y., Xuan, M., Han, S., Liu, K., Jiao, J. ... Yu, W. (2026). Integrin beta 4 promotes colorectal cancer progression by upregulating Ezrin and activating the Wnt/&beta;‑catenin signaling pathway. International Journal of Oncology, 69, 82. https://doi.org/10.3892/ijo.2026.5895
MLA
Wang, J., Si, Y., Xuan, M., Han, S., Liu, K., Jiao, J., Men, X., Li, H., Wang, J., Liu, T., Yu, W."Integrin beta 4 promotes colorectal cancer progression by upregulating Ezrin and activating the Wnt/&beta;‑catenin signaling pathway". International Journal of Oncology 69.1 (2026): 82.
Chicago
Wang, J., Si, Y., Xuan, M., Han, S., Liu, K., Jiao, J., Men, X., Li, H., Wang, J., Liu, T., Yu, W."Integrin beta 4 promotes colorectal cancer progression by upregulating Ezrin and activating the Wnt/&beta;‑catenin signaling pathway". International Journal of Oncology 69, no. 1 (2026): 82. https://doi.org/10.3892/ijo.2026.5895
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