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Mechanistic role of the ILK‑S100A4 axis in modulating invasion and EMT in salivary adenoid cystic carcinoma

  • Authors:
    • Yang Yang
    • Jia-Xin Luo
    • Yuan-Yang Li
    • Ke-Hong Guo
    • Lin Ye
    • Dan Zhao
  • View Affiliations / Copyright

    Affiliations: Department of Oral and Maxillofacial Surgery, The Affiliated Stomatological Hospital, Southwest Medical University, Luzhou, Sichuan 646000, P.R. China
    Copyright: © Yang et al. This is an open access article distributed under the terms of Creative Commons Attribution License.
  • Article Number: 597
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    Published online on: October 16, 2025
       https://doi.org/10.3892/ol.2025.15343
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Abstract

Salivary adenoid cystic carcinoma (SACC) is a prevalent malignant tumor of the salivary glands, characterized by invasive growth and perineural invasion, resulting in high rates of local recurrence, distant metastasis and poor long‑term survival. Thus, elucidating the molecular mechanisms underlying SACC invasion and identifying effective therapeutic targets are of clinical importance. Functional cellular assays including proliferation, migration, and flow cytometry, and molecular experiments, such as western blot, were conducted in vitro to explore the effects of integrin‑linked kinase (ILK) knockdown on the biological behavior of SACC cells and the expression of epithelial‑mesenchymal transition (EMT) markers. Transcriptome sequencing identified S100 calcium‑binding protein A4 (S100A4) as a key downstream effector regulated by ILK. Additionally, 52 clinical SACC specimens were analyzed to evaluate the correlation between S100A4 expression and clinicopathological features, as well as ILK expression. Rescue experiments validated the role of S100A4 in mediating the effects of ILK. ILK knockdown significantly suppressed the malignant behavior of SACC cells, including migration and invasion, and reversed EMT phenotypes. S100A4 expression was significantly associated with clinical features such as clinical stage and perineural invasion, along with ILK expression. Overexpression of S100A4 restored Snail expression, promoted the EMT process and rescued the impaired migration and invasion capabilities of SACC cells caused by ILK knockdown. ILK may facilitate EMT‑mediated invasion in SACC cells via the Glycogen synthase kinase‑3 β) signaling pathway by modulating the transcription factor Snail. S100A4 may contribute to this mechanism by modulating Snail protein. These findings imply that simultaneously targeting both ILK and S100A4 represents a novel and promising therapeutic strategy to suppress SACC progression.
View Figures

Figure 1

ILK contributes to the malignant
biological behavior of SACC. (A) Western blot analysis confirmed
effective ILK knockdown using shRNA. (B) Cell Counting Kit-8 assay
to determine the inhibitory effect of ILK knockdown on SACC cell
proliferation. (C) Colony formation assay demonstrated decreased
colony-forming ability upon ILK knockdown. (D) Flow cytometry
analysis showed ILK knockdown-induced cell cycle arrest in SACC
cells. (E) Wound healing assay demonstrated reduced migratory
capacity after ILK knockdown. (F) Transwell invasion assay revealed
decreased invasive potential following ILK knockdown. Results are
shown as the mean ± SD of three independent assays. **P<0.01,
***P<0.001, ****P<0.0001 vs. control. ns, not significant.
ILK, integrin-linked kinase; sh, short hairpin; SACC, salivary
adenoid cystic carcinoma; CON, control; NC, negative control.

Figure 2

Western blot analysis of the effects
of ILK knockdown on EMT markers and signaling. Results are shown as
the mean ± SD of three independent assays. *P<0.05, **P<0.01,
***P<0.001, ****P<0.0001 vs. control group. ns, not
significant.

Figure 3

Bioinformatics profiling and western
blotting identified S100A4 as a downstream effector of ILK. (A)
GSEA (P<0.05, false discovery rate <0.25) revealed that ILK
knockdown downregulated cell cycle pathways while upregulating
epithelial phenotype-associated pathways. (B) Venn diagram analysis
identified eight overlapping genes between DEGs and 1,153 EMT-core
genes from the EMTome database: CFH, CPA4, FST, ANGPTL2, LUM,
ADGRF1, ILK and S100A4. (C) Protein-protein interaction network
constructed via STRING database highlighted ILK as the central hub
node. (D) Western blot analysis validated that ILK knockdown
downregulated the protein expression of S100A4 in both cell lines.
***P<0.001, ****P<0.0001. ns not significant; ILK,
integrin-linked kinase; GSEA, Gene Set Enrichment Analysis; DEG,
differentially expressed gene; CFH, Complement Factor H; CPA,
Carboxypeptidase A; FST, Follistatin; ANGPTL, Angiopoietin-like
Protein; LUM, Lumican; ADGRF, Adhesion G protein-coupled receptor
F; NC, negative control; CON, blank control; sh, short hairpin;
EMT, epithelial-mesenchymal transition; GO, Gene Ontology; KEGG,
Kyoto Encyclopedia of Genes and Genomes; SACC, salivary adenoid
cystic carcinoma.

Figure 4

Immunohistochemical staining of
S100A4 and HE staining in SACC subtypes and normal salivary gland
tissue. (A) S100A4 expression in normal salivary gland tissue. (B)
SACC tissue with negative S100A4 expression. (C) HE staining of
cribriform adenoid cystic carcinoma. (D) Weak S100A4 expression in
cribriform SACC subtype. (E) HE staining of tubular adenoid cystic
carcinoma. (F) Moderate S100A4 expression in tubular SACC subtype.
(G) HE staining of solid adenoid cystic carcinoma. (H) Strong
S100A4 expression in solid SACC subtype. Magnification, ×20. HE,
hematoxylin-eosin; SACC, salivary adenoid cystic carcinoma.

Figure 5

Interaction analysis of ILK and
S100A4 in perineural invasion of SACC. HE staining demonstrated
perineural invasion in SACC at (A) low (4X) and (B) high (40X)
magnification. (C) Dense S100A4 expression is observed in the
perineural invasion focus. (D) Strong ILK expression is detected at
the perineural invasion site. (E) Loss of membranous E-cadherin in
tumor cells in the region of neural invasion. (F) Positive
N-cadherin expression in tumor cells in the perineural invasion
region. (G) Intense Snail expression in tumor cells at perineural
invasion site. Magnification, ×40. (H) Significant positive
correlation between ILK and S100A4 protein expression levels
(quantified by H-score) in SACC specimens (Spearman's ρ=0.563,
P<0.001). ILK, integrin-linked kinase; SACC, salivary adenoid
cystic carcinoma.

Figure 6

S100A4 OE rescues the suppression of
epithelial-mesenchymal transition caused by ILK knockdown.
*P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. ns not
significant; ILK, integrin-linked kinase; OE, overexpression; sh,
short hairpin; NC, negative control; CON, blank control.

Figure 7

S100A4 OE rescues the inhibitory
effects of ILK knockdown on the migration and invasion of adenoid
cystic carcinoma. (A) Wound healing assays (10X magnification)
demonstrate that S100A4 OE restores migratory ability of SACC
cells, with sh + OE cells showing closure rates comparable with NC.
(B) Transwell assays revealed restoration of invasive capacity in
sh + OE cells suggests a reversal of ILK knockdown-induced
inhibition. ***P<0.001, ****P<0.0001. ns, not significant;
OE, overexpression; ILK, integrin-linked kinase; SACC, salivary
adenoid cystic carcinoma; sh, short hairpin; NC, negative control;
CON, blank control.
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Copy and paste a formatted citation
Spandidos Publications style
Yang Y, Luo J, Li Y, Guo K, Ye L and Zhao D: Mechanistic role of the ILK‑S100A4 axis in modulating invasion and EMT in salivary adenoid cystic carcinoma. Oncol Lett 30: 597, 2025.
APA
Yang, Y., Luo, J., Li, Y., Guo, K., Ye, L., & Zhao, D. (2025). Mechanistic role of the ILK‑S100A4 axis in modulating invasion and EMT in salivary adenoid cystic carcinoma. Oncology Letters, 30, 597. https://doi.org/10.3892/ol.2025.15343
MLA
Yang, Y., Luo, J., Li, Y., Guo, K., Ye, L., Zhao, D."Mechanistic role of the ILK‑S100A4 axis in modulating invasion and EMT in salivary adenoid cystic carcinoma". Oncology Letters 30.6 (2025): 597.
Chicago
Yang, Y., Luo, J., Li, Y., Guo, K., Ye, L., Zhao, D."Mechanistic role of the ILK‑S100A4 axis in modulating invasion and EMT in salivary adenoid cystic carcinoma". Oncology Letters 30, no. 6 (2025): 597. https://doi.org/10.3892/ol.2025.15343
Copy and paste a formatted citation
x
Spandidos Publications style
Yang Y, Luo J, Li Y, Guo K, Ye L and Zhao D: Mechanistic role of the ILK‑S100A4 axis in modulating invasion and EMT in salivary adenoid cystic carcinoma. Oncol Lett 30: 597, 2025.
APA
Yang, Y., Luo, J., Li, Y., Guo, K., Ye, L., & Zhao, D. (2025). Mechanistic role of the ILK‑S100A4 axis in modulating invasion and EMT in salivary adenoid cystic carcinoma. Oncology Letters, 30, 597. https://doi.org/10.3892/ol.2025.15343
MLA
Yang, Y., Luo, J., Li, Y., Guo, K., Ye, L., Zhao, D."Mechanistic role of the ILK‑S100A4 axis in modulating invasion and EMT in salivary adenoid cystic carcinoma". Oncology Letters 30.6 (2025): 597.
Chicago
Yang, Y., Luo, J., Li, Y., Guo, K., Ye, L., Zhao, D."Mechanistic role of the ILK‑S100A4 axis in modulating invasion and EMT in salivary adenoid cystic carcinoma". Oncology Letters 30, no. 6 (2025): 597. https://doi.org/10.3892/ol.2025.15343
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