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Article Open Access

Induction of CXCL8 by endoplasmic reticulum stress promotes migration and invasion of esophageal squamous cell carcinoma through activation of SMAD2/3

  • Authors:
    • Junhong Wu
    • Fangyu Su
    • Juntao Lu
    • Huanchen Xu
    • Xia Yang
    • Fei Li
    • Lei Liu
    • Wei Guo
  • View Affiliations / Copyright

    Affiliations: Laboratory of Pathology, Hebei Cancer Institute, The Fourth Hospital of Hebei Medical University, Shijiazhuang, Hebei 050011, P.R. China, Department of Thoracic Surgery, The Fourth Hospital of Hebei Medical University, Shijiazhuang, Hebei 050011, P.R. China
    Copyright: © Wu et al. This is an open access article distributed under the terms of Creative Commons Attribution License.
  • Article Number: 311
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    Published online on: September 5, 2025
       https://doi.org/10.3892/mmr.2025.13676
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Abstract

Endoplasmic reticulum stress (ERS) is a protective stress response aimed at mitigating its own abnormal proteins, which is closely associated with tumors. However, the molecular mechanism of ERS in the pathogenesis of esophageal squamous cell carcinoma (ESCC) remains unclear. In the present study, RNA sequencing was carried out in the ESCC ERS cell model in vitro, and differentially expressed genes were screened, among which CXCL8 with exhibited differential expression which was studied. CXCL8 was significantly upregulated after thapsigargin (TG; an ERS inducer) treatment in ESCC cells. A marked elevated expression of CXCL8 and its receptors were observed in ESCC cells. CXCL8 was induced by the IRE1α and PERK pathways of ERS, transcription of which was activated by the downstream transcription factors XBP1 and ATF4. TG and rh‑CXCL8 facilitated migration and invasion of ESCC cells, and the migration and invasion effect of TG on ESCC cells could be partially prevented by knockdown of CXCR1. Furthermore, CXCL8‑CXCR1 could activate SMAD2/3 and the activation of SMAD2/3 directly or indirectly regulated the transcription of SNAI2 and ZEB1 to promote the progression of epithelial‑mesenchymal transition (EMT) in ESCC. Both in vivo experiments and immunohistochemical analyses further demonstrated the oncogenic effects of CXCL8. In conclusion, the data obtained in the present study indicated that CXCL8 may be induced via the IRE1α/XBP1 and PERK/ATF4 pathways, and that the CXCL8‑CXCR1/2‑SMAD2/3‑SNAI2/ZEB1 axis is involved in the EMT process of ER‑stressed ESCC cells. Thus, blocking the CXCL8‑CXCR1/2 axis may disrupt ERS‑induced migration and invasion of ESCC cells, thereby improving the prognosis of patients with ESCC.
View Figures

Figure 1

Endoplasmic reticulum stress induces
CXCL8 expression in ESCC cells. KYSE-150 and TE-1 cells were
treated with 100 nM TG for 0, 3, 6, 12 and 24 h, and expression
levels of effectors in the UPR were examined by (A) RT-qPCR and (B)
western blot analysis. (C) The volcano plot shows differentially
expressed genes in TE-1 cells treated with 100 nM TG for 12 h. The
x-axis represents the log2-transformed of FC ratios and the y-axis
is the log10-transformed adjusted P-value. Red/yellow dots
represent significantly up/downregulated genes (|log2FC|≥1,
adj.P<0.05). The blue arrow indicates the possible location of
CXCL8. The (D) mRNA and (E) protein expression levels of CXCL8 in
ESCC cells treated with 100 nM TG for 24 h. (F) The CXCL8
expression profile across all tumor samples and corresponding
normal tissues was obtained from the GEPIA. (G) The mRNA expression
levels of CXCL8 in ESCC tissues (n=182) and normal tissues (n=286)
were obtained from the GEPIA database. (H) Expression levels of
CXCL8, the receptors CXCR1 and CXCR2 in the normal esophageal
epithelial cell line and ESCC cell lines were determined by RT-qPCR
method. All data are expressed as mean ± SD, n=3/group. *P<0.05
vs. TG-treated 0 h group. CXCL8, C-X-C motif chemokine ligand;
ESCC, esophageal squamous cell carcinoma; TG, thapsigargin; FC,
fold change; GEPIA, Gene Expression Profiling Interactive
£Analysis, RT-qPCR, reverse transcription-quantitative PCR; HEEC,
human normal esophageal epithelial cell; XBP1, X-box binding
protein 1; ATF, activating transcription factor; TCGA-GTEx; The
Cancer Genome Atlas-Genotype-Tissue Expression; sign, significance;
up, upregulated; no, no significance; down, downregulated; TPM,
transcript per million.

Figure 2

CXCL8 is induced by the PERK and
IRE1α pathways of the unfolded protein response in esophageal
squamous cell carcinoma cells. Evaluation of the indicated (A) mRNA
and (B) protein expression levels in siRNA-transfected KYSE-150 and
TE-1 cells, which were treated with 100 nM TG or DMSO for 24 h.
Evaluation of (C) mRNA and (D) protein expression levels of CXCL8
in transfected KYSE-150 and TE-1 cells by reverse
transcription-quantitative PCR and western blot analysis.
Dual-luciferase reporter assays were conducted in KYSE-150 cells to
verify the direct binding effects of (E) XBP1 and (F) ATF4 on the
CXCL8 promoter. All data are expressed as mean ± SD, n=3/group.
*P<0.05, vs. siNS + DMSO group, siNS + TG group or pcDNA3.1
group; #P<0.05 vs. siNS + TG group. n.s., not
significant; IRE1α, inositol-requiring enzyme 1α; PERK, protein
kinase R (PKR)-like endoplasmic reticulum kinase; si, small
interfering; ERN, endoplasmic reticulum to nucleus signaling;
EIF2AK3, ATF, eukaryotic translation initiation factor 2 alpha
kinase 3; TG, thapsigargin; NS, non-silencing; CXCL, C-X-C motif
chemokine ligand.

Figure 3

CXCL8 facilitates migration and
invasion of ESCC cells. Evaluation of (A) mRNA and (B) protein
expression levels of CXCR1 and CXCR2 in KYSE-150 and TE-1 cells
treated with 0, 10, 20, 49, 80 and 160 mg/ml rh-CXCL8 for 24 h.
Evaluation of (C) mRNA and (D) protein expression levels of CXCR1
and CXCR2 in KYSE-150 and TE-1 cells treated with 10 ng/ml rh-CXCL8
for 0, 12, 24 and 48 h. (E) Cell proliferation was analyzed with an
MTS assay in KYSE-150 and TE-1 cells treated with 10 ng/ml rh-CXCL8
for 24 h. (F) Wound healing and (G) Transwell assays were conducted
to assess the migration and invasion abilities in KYSE-150 and TE-1
cells treated with 10 ng/ml rh-CXCL8 for 24 h. All data are
expressed as mean ± SD, n=3/group. *P<0.05 vs. 0 ng/ml
rh-CXCL8-treated group, rh-CXCL8-treated 0 h group, NC-treated 0 h
group or NC group. n.s., not significant; rh-CXCL, recombinant
human-C-X-C motif chemokine ligand; CXCR, chemokine receptor; NC,
negative control.

Figure 4

Effect of CXCR1 knockdown on
esophageal squamous cell carcinoma cell migration and invasion
under an endoplasmic reticulum stress state. (A) Wound healing and
(B) Transwell assays were conducted to explore the migration and
invasion ability KYSE-150 and TE-1 cells that underwent various
treatments (magnification, ×100). All data are expressed as mean ±
SD, n=3/group. *P<0.05 vs. NC-treated 24 h group, TG-treated 24
h group, NC-treated group or TG-treated group. NC, negative
control; TG, thapsigargin; si, small interfering; CXCR, chemokine
receptor.

Figure 5

CXCL8 participates in the EMT process
by regulating the expression of SNAI2 and ZEB1. KYSE-150 cells were
treated with specified concentrations of rh-CXCL8 for 24 h, and
then (A) mRNA expression of SNAI2, ZEB1, TWIST1, SNAIL, FN1,
TWIST2, CDH2, VIMENTIN and ZEB2 was detected by RT-qPCR, and (B)
protein expression of SNAI2 and ZEB1 was detected by western
blotting. (C) KYSE-150 and TE-1 cells were treated with specified
concentrations of rh-CXCL8 for 24 h and the protein expression
levels of EMT-related pathway genes were examined by Western blot
assay. (D) KYSE-150 and TE-1 cells were treated with 10 ng/ml
rh-CXCL8 for indicated times, and the protein expression of SMAD2/3
and p-SMAD2/3 was detected by Western blot assay. All data are
expressed as mean ± SD, n=3/group. *P<0.05 vs. 0 ng/ml
rh-CXCL8-treated group or rh-CXCL8-treated 0 h group. n.s., not
significant; rh-CXCL, recombinant human C-X-C motif chemokine
ligand; EMT, epithelial-mesenchymal transition.

Figure 6

CXCL8 regulates the expression of
SNAI2 and ZEB1 through phosphorylated activation of SMAD2/3. (A)
Influence of knocking down CXCR1 on the protein expression of
SMAD2/3 and p-SMAD2/3 in KYSE-150 and TE-1 cells, treated with 10
ng/ml rh-CXCL8 for 24 h. (B) Influence of knocking down CXCR1 on
protein expression of SMAD2/3 and p-SMAD2/3 in TG (100 nM; 24 h)
treated KYSE-150 and TE-1 cells. (C) mRNA and (D) protein
expression levels of ZEB1 and SNAI2 were examined in KYSE-150 and
TE-1 cells treated with TGF-β1 (10 ng/ml; 48 h) using reverse
transcription-quantitative PCR and western blot analysis.
Dual-luciferase reporter assays were conducted in KYSE-150 cells to
detect the transcriptional regulatory effect of SMAD3 on (E) SNAI2
and (F) ZEB1. (G) Venn diagram showing the bioinformatics
identification of SMAD3-regulated miRNAs that target ZEB1. All data
are expressed as mean ± SD, n=3/group. *P<0.05 vs. rh-CXCL8
group, DMSO + siNS group, TG + siNS group or DMSO group. n.s., not
significant; rh-CXCL, recombinant human-C-X-C motif chemokine
ligand; CXCR, chemokine receptor; p-, phosphorylated; TG,
thapsigargin; si, small interfering; NS, non-silencing; Luc,
luciferase; miRNA, microRNA; UTR, untranslated region.

Figure 7

Animal experiments validate the role
of the CXCL8-CXCR1/2-SMAD2/3-SNAI2/ZEB1 axis in tumor growth and
metastasis. (A) Image of subcutaneous xenograft tumors following
injection of CXCL8-overexpressing KYSE-150 cells (n=5) or control
cells (n=5) in BALB/c-nude mice. The volume (B) and weight (C) of
harvested xenograft tumors were measured (n=5). (D) Representative
immunohistochemical images of CXCR1/2, p-SMAD2/3, SNAI2 and ZEB1
expression in CXCL8-overexpressing ESCC tissues and corresponding
control tissues (n=5). Scale bar, 50 µm. (E) Mechanistic diagram of
CXCL8 in ESCC cells under an ER stress state. All data are
expressed as mean ± SD. *P < 0.05. vs. PCDH-Vector group. CXCL,
C-X-C motif chemokine ligand; IRE1α, inositol-requiring enzyme 1α;
PERK, protein kinase R (PKR)-like endoplasmic reticulum kinase;
ATF, activating transcription factor; XBP, X-box binding protein;
ER, endoplasmic reticulum; p-, phosphorylated; ESCC, esophageal
squamous cell carcinoma.
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Copy and paste a formatted citation
Spandidos Publications style
Wu J, Su F, Lu J, Xu H, Yang X, Li F, Liu L and Guo W: Induction of CXCL8 by endoplasmic reticulum stress promotes migration and invasion of esophageal squamous cell carcinoma through activation of SMAD2/3. Mol Med Rep 32: 311, 2025.
APA
Wu, J., Su, F., Lu, J., Xu, H., Yang, X., Li, F. ... Guo, W. (2025). Induction of CXCL8 by endoplasmic reticulum stress promotes migration and invasion of esophageal squamous cell carcinoma through activation of SMAD2/3. Molecular Medicine Reports, 32, 311. https://doi.org/10.3892/mmr.2025.13676
MLA
Wu, J., Su, F., Lu, J., Xu, H., Yang, X., Li, F., Liu, L., Guo, W."Induction of CXCL8 by endoplasmic reticulum stress promotes migration and invasion of esophageal squamous cell carcinoma through activation of SMAD2/3". Molecular Medicine Reports 32.6 (2025): 311.
Chicago
Wu, J., Su, F., Lu, J., Xu, H., Yang, X., Li, F., Liu, L., Guo, W."Induction of CXCL8 by endoplasmic reticulum stress promotes migration and invasion of esophageal squamous cell carcinoma through activation of SMAD2/3". Molecular Medicine Reports 32, no. 6 (2025): 311. https://doi.org/10.3892/mmr.2025.13676
Copy and paste a formatted citation
x
Spandidos Publications style
Wu J, Su F, Lu J, Xu H, Yang X, Li F, Liu L and Guo W: Induction of CXCL8 by endoplasmic reticulum stress promotes migration and invasion of esophageal squamous cell carcinoma through activation of SMAD2/3. Mol Med Rep 32: 311, 2025.
APA
Wu, J., Su, F., Lu, J., Xu, H., Yang, X., Li, F. ... Guo, W. (2025). Induction of CXCL8 by endoplasmic reticulum stress promotes migration and invasion of esophageal squamous cell carcinoma through activation of SMAD2/3. Molecular Medicine Reports, 32, 311. https://doi.org/10.3892/mmr.2025.13676
MLA
Wu, J., Su, F., Lu, J., Xu, H., Yang, X., Li, F., Liu, L., Guo, W."Induction of CXCL8 by endoplasmic reticulum stress promotes migration and invasion of esophageal squamous cell carcinoma through activation of SMAD2/3". Molecular Medicine Reports 32.6 (2025): 311.
Chicago
Wu, J., Su, F., Lu, J., Xu, H., Yang, X., Li, F., Liu, L., Guo, W."Induction of CXCL8 by endoplasmic reticulum stress promotes migration and invasion of esophageal squamous cell carcinoma through activation of SMAD2/3". Molecular Medicine Reports 32, no. 6 (2025): 311. https://doi.org/10.3892/mmr.2025.13676
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