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Notch3 mediated TGF‑β1 activation enhances epithelial‑mesenchymal transition and cancer stemness in non‑small lung cancer

  • Authors:
    • Fang Wang
    • Siqi Hu
    • Jiangrong Bian
    • Qing Gao
    • Liuzhao Cao
    • Linli Sang
    • Junjun Yang
    • Xingxiang Xu
  • View Affiliations / Copyright

    Affiliations: Department of Respiration and Critical Care Medicine, Northern Jiangsu People's Hospital Affiliated to Yangzhou University, Yangzhou, Jiangsu 225001, P.R. China
    Copyright: © Wang et al. This is an open access article distributed under the terms of Creative Commons Attribution License.
  • Article Number: 85
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    Published online on: August 20, 2025
       https://doi.org/10.3892/ijo.2025.5791
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Abstract

Notch3 is a key regulator in various cancers, playing a crucial role in maintaining stemness and promoting epithelial‑mesenchymal transition (EMT). However, its differential expression and regulatory mechanisms in non‑small cell lung cancer (NSCLC) and cancer stem cells remain poorly understood. To investigate this, the present study examined Notch3 expression in NSCLC through Oncomine, The Cancer Genome Atlas and Gene Expression Omnibus databases and validated the results with immunohistochemistry, reverse transcription‑quantitative PCR and western blotting. EMT was induced by TGF‑β1 in NSCLC cells and functional assays (Transwell, wound healing and sphere formation) were performed to assess cellular changes. In vivo experiments using a xenograft mouse model were conducted to evaluate tumor growth and metastasis. The results showed that high Notch3 expression was associated with poor prognosis in NSCLC patients. Downregulation of Notch3 inhibited TGF‑β1‑induced EMT and CSC characteristics, resulting in reduced tumorigenic potential, whereas overexpression of the Notch3 intracellular domain enhanced these effects. Silencing Notch3 suppressed EMT and markedly inhibited tumor growth and metastasis in vivo. These findings demonstrated that Notch3 regulated EMT and CSC properties in NSCLC, promoting tumor recurrence and metastasis. Notch3 thus represents a promising therapeutic target and prognostic marker for NSCLC.
View Figures

Figure 1

Notch3 expression in the lung cancer
and related to unfavorable prognosis in NSCLC patients. (A)
Expression of Notch3 protein in the indicated cell lines was
determined by western blotting. (B) Relative Notch3 gene expression
was examined in the indicated cell lines. (C) Differential
expression of Notch3 between tumor (n=979) and adjacent normal
(n=685) tissues in NSCLC. (D) Notch3 protein expression in normal
lung tissue and NSCLC specimens. Images were obtained from the
Human Protein Atlas online database. (E) Correlation between Notch3
and prognosis of lung cancer in the Kaplan-Meier plotter database.
NSCLC, non-small cell lung cancer; HR, hazard ratio.

Figure 2

A549 and H1975 cells were transfected
with Notch3-expressing or empty vector and A549 cells were
transfected with Notch3 knock-down lentivirus. (A) A549 and H1975
cells were transfected with Notch3 expressing or empty vector, at
48 h after transfection, protein expression of Notch3 were analyzed
by WB. (B) Cell viability was measured after CCK-8 staining. (C) WB
and (D) RT-qPCR validate the efficiency of constructed Notch3
knockdown A549 cells. (E) Cell viability was measured after CCK-8
staining. Data are presented as mean ± SD. *P<0.05
and ***P<0.001. WB, western blotting; CCK-8, Cell
Counting Kit-8; RT-qPCR, reverse transcription quantitative
polymerase chain reaction; sh, short hairpin; NC, negative
control.

Figure 3

Notch3 drives the EMT through TGF-β1
signaling. (A) Gene Ontology analysis of differentially expressed
genes after Notch3 knockdown. (B) Volcano plots of differentially
expressed genes. Red dots and blue dots represent up- and
downregulated genes, respectively. (C) Heat map of the indicated
target genes in Notch3-silenced and control cells. The target genes
of TGF-β1 signaling were decreased in Notch3-silenced cells. (D)
Using the STRING program to analyses the Notch3, TGF-β1, Vimentin
and E-Cadherin (CDH1). (E) Bioinformatic analysis revealed a
correlation between Notch3 and TGF-β1 and EMT-related genes
(E-Cadherin and Vimentin) expression. (F) Transfection of H1975 and
A549 cells with Notch3 vector or Notch3 shRNA. After transfection,
TGF-β1 production in the culture supernatant was measured using an
ELISA. (G) Notch3 was either overexpressed or silenced in H1975 and
A549 cell lines, followed by treatment with or without 10 ng/ml
TGF-β1 48 h. EMT markers E-Cadherin and Vimentin were analyzed by
WB. Data are presented as mean ± SD. **P<0.01 and
***P<0. 001. EMT, epithelial-mesenchymal transition;
TGF-β1, transforming growth factor-β; sh, short hairpin; NC,
negative control; ELISA, enzyme-linked immunosorbent assay; WB,
western blotting.

Figure 4

Notch3 mediates TGF-β1 induced
proliferation, migration and invasion of H1975 and A549 cell lines.
Notch3 was either overexpressed or silenced in H1975 and A549 cell
lines, followed by treatment with or without 10 ng/ml TGF-β1 48 h.
(A) Cell proliferation was detected by the CCK-8 detection kit. (B)
Cell migration invasion assay using Transwell. Scale bar, 200
μm. (C) A wound healing assay. Scale bar, 500 μm.
Data are presented as mean ± SD. *P<0.05,
**P<0.01 and ***P<0.001. TGF-β1,
transforming growth factor-β; CCK-8, Cell Counting Kit-8.

Figure 5

Spherical cells derived from lung
cancer cells exhibit cancer stem cell characteristics. (A) The
morphologies of adherent A549 cells and A549 cell spheres. Scale
bar, 100 μm (left) and 50 μm (right). (B)
Immunofluorescence of the stemness markers in A549 spheres. The
nuclei were stained with DAPI. Scale bar, 50 μm. (C) The
mRNA expression levels of stem cell markers in A549 and A549
spheres. (D) Western blotting of stemness markers (OCT4, CD44,
CD133 and ALDHA1) in A549 and A549 spheres. (E) Transwell migration
invasion assay of parental cells and sphere cells. Scale bar, 200
μm. Data are presented as mean ± SD. *P<0.05
and ***P<0.001.

Figure 6

Molecular properties of A549 CSCs.
(A) Immunofluorescence staining for E-Cadherin and Vimentin
expression in A549 cells and A549-spheres. Scale bar, 100
μm. (B) WB and (C) RT-qPCR show E-Cadherin and Vimentin
expressions in A549 cells and A549 CSCs. (D) TGF-β1 production in
the culture supernatant was measured using an ELISA. (E) Western
blotting shows Notch3 expression in A549 cells and A549 CSCs. Data
are presented as mean ± SD. ***P<0.001. CSCs, cancer
stem cells; RT-qPCR, reverse transcription quantitative polymerase
chain reaction; ELISA, enzyme-linked immunosorbent assay.

Figure 7

Notch3 promotes the stemness of A549
CSCs via TGF-β1. (A) Sphere-formation ability was detected in A549
CSCs with Notch3 overexpression or knockdown after 10 ng/ml TGF-β1
stimulation. Scale bar, 500 μm (B) The expression of
stemness markers (OCT4, CD44, CD133 and ALDHA1) was examined in
A549 CSCs with Notch3 overexpression or knockdown after TGF-β1
stimulation. Data are presented as mean ± SD.
**P<0.01. CSCs, cancer stem cell stemness; TGF-β1,
transforming growth factor-β; sh, short hairpin; NC, negative
control.

Figure 8

Notch3 promotes TGF-β1-driven
tumorigenicity and the metastasis of lung cancer in nude mice. (A)
Xenograft tumor volume and weight of the xenograft tumors were
measured after mice were sacrificed, Notch3 knockdown suppressed
tumor growth (n=6 per group). (B) Immunohistochemistry analysis of
E-Cadherin, Vimentin, CD44, CD133 and Ki67 expression in tumor
tissues. Scale bar, 20 μm. (C) Notch3 knockdown reduced the
numbers of metastatic nodules stained with hematoxylin-eosin. Scale
bar, 500 μm. Data are presented as mean ± SD.
***P<0.001. TGF-β1, transforming growth factor-β; sh,
short hairpin; NC, negative control.
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Copy and paste a formatted citation
Spandidos Publications style
Wang F, Hu S, Bian J, Gao Q, Cao L, Sang L, Yang J and Xu X: Notch3 mediated TGF‑&beta;1 activation enhances epithelial‑mesenchymal transition and cancer stemness in non‑small lung cancer. Int J Oncol 67: 85, 2025.
APA
Wang, F., Hu, S., Bian, J., Gao, Q., Cao, L., Sang, L. ... Xu, X. (2025). Notch3 mediated TGF‑&beta;1 activation enhances epithelial‑mesenchymal transition and cancer stemness in non‑small lung cancer. International Journal of Oncology, 67, 85. https://doi.org/10.3892/ijo.2025.5791
MLA
Wang, F., Hu, S., Bian, J., Gao, Q., Cao, L., Sang, L., Yang, J., Xu, X."Notch3 mediated TGF‑&beta;1 activation enhances epithelial‑mesenchymal transition and cancer stemness in non‑small lung cancer". International Journal of Oncology 67.4 (2025): 85.
Chicago
Wang, F., Hu, S., Bian, J., Gao, Q., Cao, L., Sang, L., Yang, J., Xu, X."Notch3 mediated TGF‑&beta;1 activation enhances epithelial‑mesenchymal transition and cancer stemness in non‑small lung cancer". International Journal of Oncology 67, no. 4 (2025): 85. https://doi.org/10.3892/ijo.2025.5791
Copy and paste a formatted citation
x
Spandidos Publications style
Wang F, Hu S, Bian J, Gao Q, Cao L, Sang L, Yang J and Xu X: Notch3 mediated TGF‑&beta;1 activation enhances epithelial‑mesenchymal transition and cancer stemness in non‑small lung cancer. Int J Oncol 67: 85, 2025.
APA
Wang, F., Hu, S., Bian, J., Gao, Q., Cao, L., Sang, L. ... Xu, X. (2025). Notch3 mediated TGF‑&beta;1 activation enhances epithelial‑mesenchymal transition and cancer stemness in non‑small lung cancer. International Journal of Oncology, 67, 85. https://doi.org/10.3892/ijo.2025.5791
MLA
Wang, F., Hu, S., Bian, J., Gao, Q., Cao, L., Sang, L., Yang, J., Xu, X."Notch3 mediated TGF‑&beta;1 activation enhances epithelial‑mesenchymal transition and cancer stemness in non‑small lung cancer". International Journal of Oncology 67.4 (2025): 85.
Chicago
Wang, F., Hu, S., Bian, J., Gao, Q., Cao, L., Sang, L., Yang, J., Xu, X."Notch3 mediated TGF‑&beta;1 activation enhances epithelial‑mesenchymal transition and cancer stemness in non‑small lung cancer". International Journal of Oncology 67, no. 4 (2025): 85. https://doi.org/10.3892/ijo.2025.5791
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