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Harnessing TP73‑targeted nintedanib: A novel strategy to halt triple‑negative breast cancer via p53‑PPARα/PI3K‑Akt pathway suppression

  • Authors:
    • Xiaomeng Zou
    • Shiyu Li
    • Sisi Huang
    • Ruilan Niu
    • Gang Liu
    • Zhili Wang
  • View Affiliations / Copyright

    Affiliations: Department of Ultrasound, The First Medical Center, Chinese People's Liberation Army General Hospital, Beijing 100853, P.R. China, Department of Radiology, The First Medical Center, Chinese People's Liberation Army General Hospital, Beijing 100853, P.R. China
    Copyright: © Zou et al. This is an open access article distributed under the terms of Creative Commons Attribution License.
  • Article Number: 88
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    Published online on: August 22, 2025
       https://doi.org/10.3892/ijo.2025.5794
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Abstract

Triple‑negative breast cancer (TNBC) is an aggressive malignancy with limited treatment options, leading to poor clinical outcomes and the need for novel therapeutic approaches. Nintedanib, a United States Food and Drug Administration‑approved multi‑kinase inhibitor with anti‑fibrotic and anti‑angiogenic properties, has shown promise in cancer treatment. However, its precise molecular effects on TNBC have not yet been fully elucidated. Therefore, the present study aimed to investigate the therapeutic potential of nintedanib in TNBC using in vitro and in vivo models, specifically focusing on its regulatory effects on key oncogenic pathways. The present study utilized TNBC cell lines (MDA‑MB‑231 and 4T1) and BALB/c mice to evaluate the antitumor efficacy of nintedanib. Cell viability and clonogenic capacity were assessed using Cell Counting Kit‑8 and colony formation assays. Subsequently, apoptosis induction and cell cycle progression were determined by flow cytometry, and cell migration and invasion were analyzed through scratch and Transwell assays. To identify underlying mechanisms, potential molecular targets were identified via bioinformatics and network pharmacology, and were validated through western blotting, immunofluorescence and immunohistochemistry. Finally, an orthotopic TNBC mouse model was established and monitored in real time by multimodal ultrasound imaging. The results revealed that nintedanib significantly inhibited TNBC cell proliferation and suppressed stem cell‑like properties. Furthermore, it induced cell cycle arrest at the G2/M phase and promoted apoptosis. Mechanistic analysis revealed that nintedanib activated tumor protein p73 (TP73), leading to the disruption of the p53‑peroxisome proliferator‑activated receptor α (PPARα)/PI3K‑Akt signaling axis. Additionally, it downregulated epithelial‑mesenchymal transition (EMT) markers, including Snail and zinc finger E‑box‑binding homeobox protein 1, thereby mitigating tumor invasiveness. In vivo, nintedanib treatment effectively reduced tumor growth, angiogenesis and stiffness, indicating its potential as a viable therapeutic agent for TNBC. In conclusion, nintedanib exerts potent anti‑TNBC effects by modulating TP73, disrupting oncogenic signaling via the p53‑PPARα/PI3K‑Akt axis, and attenuating EMT‑associated transcription factors. These findings highlight its potential as a promising targeted therapy for TNBC, warranting further clinical exploration.
View Figures

Figure 1

Nintedanib suppresses the viability
of triple-negative breast cancer cells. Viability of (A) MDA-MB-231
and (B) 4T1 cells was assessed using the Cell Counting Kit-8 assay
following treatment with nintedanib for 24, 48 and 72 h. (C) Colony
formation assays were performed to further confirm the inhibitory
effects of nintedanib on cell proliferation. (D) Quantification of
colony formation rates based on statistical analysis. (E) Western
blot analysis of protein expression levels in TNBC cells after
nintedanib treatment. (F) Statistical analysis of relative protein
expression levels. (G) Cell cycle distribution of MDA-MB-231 and
4T1 cells analyzed after nintedanib treatment by flow cytometry.
(H) Quantitative analysis of cell cycle progression. All data are
presented as the mean ± SD from three independent experiments.
**P<0.01 and ****P<0.0001 vs. control.
CDC2, cell division control protein 2; PCNA, proliferating cell
nuclear antigen.

Figure 2

Nintedanib induces apoptosis in TNBC
cells. (A) Flow cytometric analysis of apoptosis in TNBC cells
following nintedanib treatment. (B) Quantification of apoptosis
rates based on flow cytometry data. (C) Western blot analysis of
Bcl-2 and Bax protein expression in TNBC cells after nintedanib
treatment. (D) Statistical analysis of relative protein expression
levels. All data are presented as the mean ± SD from three
independent experiments. **P<0.01,
***P<0.001 and ****P<0.0001 vs.
control. PI, propidium iodide; TNBC, triple-negative breast
cancer.

Figure 3

Nintedanib inhibits the migration and
invasion of TNBC cells. (A) Wound-healing assays were performed on
MDA-MB-231 and 4T1 cells following nintedanib treatment (scale bar,
200 µm). (B) Quantification of relative wound closure rates.
(C) Transwell migration and invasion assays were conducted to
evaluate the effects of nintedanib on TNBC cell motility (scale
bar, 200 µm). (D) Statistical analysis of the number of TNBC
cells that migrated or invaded through the membrane. All data are
presented as the mean ± SD from three independent experiments.
***P<0.001 and ****P<0.0001 vs.
control. TNBC, triple-negative breast cancer.

Figure 4

Nintedanib inhibits
epithelial-mesenchymal transition in MDA-MB-231 and 4T1 cells. (A)
Western blot analysis of N-cadherin, E-cadherin and vimentin
expression in TNBC cells following nintedanib treatment. (B)
Semi-quantification of relative protein expression levels.
Immunofluorescence analysis of E-cadherin and vimentin expression
in (C) MDA-MB-231 and (D) 4T1 TNBC cells after nintedanib treatment
(scale bar: 50 µm). Data are presented as the mean ± SD from
three independent experiments. *P<0.05,
**P<0.01 and ****P<0.0001 vs. control.
TNBC, triple-negative breast cancer.

Figure 5

Bioinformatics and network
pharmacology analysis of the effects of nintedanib on TNBC. (A)
Principal component analysis scatter plot comparing control and
nintedanib-treated groups. (B) Volcano plot displaying DEGs
identified from RNA-seq data. (C) Heatmap of DEGs across three
nintedanib-treated and three control samples, with high expression
levels shown in red and low levels in blue. (D) Gene Ontology
enrichment analysis of DEGs related to TNBC. (E) Kyoto Encyclopedia
of Genes and Genomes pathway analysis of DEGs associated with TNBC.
(F) Venn diagram highlighting overlapping genes between DEGs from
RNA-seq and nintedanib targets identified via the PharmMapper
database. (G) Chemical structure of nintedanib. (H) Molecular
docking visualization of the interaction between nintedanib and
tumor protein p73. BP, biological process; CC, cellular component;
DEGs, differentially expressed genes; MF, molecular function;
RNA-seq, RNA sequencing; TNBC, triple-negative breast cancer.

Figure 6

Expression of signaling pathway
proteins in TNBC cells following nintedanib treatment. (A) Western
blot analysis of key signaling pathway proteins after nintedanib
treatment. (B) Semi-quantification of relative protein expression
levels. (C) Western blot analysis of EMT regulatory protein
expression in TNBC cells after nintedanib treatment. (D)
Statistical analysis of relative EMT protein expression levels. All
data are presented as the mean ± SD from three independent
experiments. *P<0.05, **P<0.01,
***P<0.001 and ****P<0.0001 vs.
control. EMT, epithelial-mesenchymal transition; mTOR, mammalian
target of rapamycin; p-, phosphorylated; PPARα, peroxisome
proliferator-activated receptor α; TNBC, triple-negative breast
cancer; TP73, tumor protein p73; ZEB, zinc finger E-box-binding
homeobox protein.

Figure 7

Nintedanib inhibits the growth of 4T1
orthotopic tumors in BALB/c mice. (A) Ultrasound-guided injection
of TNBC cell suspension into the MFP: (a) Localization of the mouse
MFP before implantation (arrow); (b) needle insertion into the MFP
(arrow); (c) injection of the cell suspension into the MFP; (d)
needle removal. (B) Multi-modal ultrasound imaging of tumors. (C)
Histological analysis of 4T1 orthotopic tumors treated with or
without nintedanib, including H&E staining,
immunohistochemistry and TUNEL staining (scale bar, 20 µm).
(D) Tumor volume measurements following nintedanib treatment. (E)
Body weight changes in mice. (F) H&E staining of major organs
from TNBC-bearing mice treated with nintedanib (scale bar, 50
µm). All data are presented as the mean ± SD from three
independent experiments. ****P<0.0001 vs. control.
H&E, hematoxylin and eosin; MFP, mammary fat pad; PCNA,
proliferating cell nuclear antigen; TNBC, triple-negative breast
cancer; CDFI, color Doppler flow imaging; CPA, color power
angiography; USE, ultrasound strain elastography.
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Copy and paste a formatted citation
Spandidos Publications style
Zou X, Li S, Huang S, Niu R, Liu G and Wang Z: Harnessing TP73‑targeted nintedanib: A novel strategy to halt triple‑negative breast cancer via p53‑PPAR&alpha;/PI3K‑Akt pathway suppression. Int J Oncol 67: 88, 2025.
APA
Zou, X., Li, S., Huang, S., Niu, R., Liu, G., & Wang, Z. (2025). Harnessing TP73‑targeted nintedanib: A novel strategy to halt triple‑negative breast cancer via p53‑PPAR&alpha;/PI3K‑Akt pathway suppression. International Journal of Oncology, 67, 88. https://doi.org/10.3892/ijo.2025.5794
MLA
Zou, X., Li, S., Huang, S., Niu, R., Liu, G., Wang, Z."Harnessing TP73‑targeted nintedanib: A novel strategy to halt triple‑negative breast cancer via p53‑PPAR&alpha;/PI3K‑Akt pathway suppression". International Journal of Oncology 67.5 (2025): 88.
Chicago
Zou, X., Li, S., Huang, S., Niu, R., Liu, G., Wang, Z."Harnessing TP73‑targeted nintedanib: A novel strategy to halt triple‑negative breast cancer via p53‑PPAR&alpha;/PI3K‑Akt pathway suppression". International Journal of Oncology 67, no. 5 (2025): 88. https://doi.org/10.3892/ijo.2025.5794
Copy and paste a formatted citation
x
Spandidos Publications style
Zou X, Li S, Huang S, Niu R, Liu G and Wang Z: Harnessing TP73‑targeted nintedanib: A novel strategy to halt triple‑negative breast cancer via p53‑PPAR&alpha;/PI3K‑Akt pathway suppression. Int J Oncol 67: 88, 2025.
APA
Zou, X., Li, S., Huang, S., Niu, R., Liu, G., & Wang, Z. (2025). Harnessing TP73‑targeted nintedanib: A novel strategy to halt triple‑negative breast cancer via p53‑PPAR&alpha;/PI3K‑Akt pathway suppression. International Journal of Oncology, 67, 88. https://doi.org/10.3892/ijo.2025.5794
MLA
Zou, X., Li, S., Huang, S., Niu, R., Liu, G., Wang, Z."Harnessing TP73‑targeted nintedanib: A novel strategy to halt triple‑negative breast cancer via p53‑PPAR&alpha;/PI3K‑Akt pathway suppression". International Journal of Oncology 67.5 (2025): 88.
Chicago
Zou, X., Li, S., Huang, S., Niu, R., Liu, G., Wang, Z."Harnessing TP73‑targeted nintedanib: A novel strategy to halt triple‑negative breast cancer via p53‑PPAR&alpha;/PI3K‑Akt pathway suppression". International Journal of Oncology 67, no. 5 (2025): 88. https://doi.org/10.3892/ijo.2025.5794
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