2. Academic Validation
  3. Afatinib inhibits esophageal squamous cell carcinoma by regulating ferroptosis and NRF2 protein homeostasis

Afatinib inhibits esophageal squamous cell carcinoma by regulating ferroptosis and NRF2 protein homeostasis

  • Int Immunopharmacol. 2026 Mar 1:172:116131. doi: 10.1016/j.intimp.2025.116131.
Yingying Liao 1,  Wei Chen 2,  Zequn Sun 3,  Qun Zhao 3,  Changlong Ruan 3,  Shuixiang He 4
Affiliations

Affiliations

  • 1 Department of Gastroenterology, First Affiliated Hospital of Xi'an Jiaotong University, Xi'an, Shaanxi, China; Department of Gastroenterology, Renmin Hospital, Hubei University of Medicine, Shiyan, Hubei, China.
  • 2 Department of Gastroenterology, Taihe Hospital, Hubei University of Medicine, Shiyan, Hubei, China.
  • 3 Department of Gastroenterology, Renmin Hospital, Hubei University of Medicine, Shiyan, Hubei, China.
  • 4 Department of Gastroenterology, First Affiliated Hospital of Xi'an Jiaotong University, Xi'an, Shaanxi, China. Electronic address: [email protected].
PMID: 41520560 DOI: 10.1016/j.intimp.2025.116131
Abstract

Aim: To investigate the potential mechanism of afatinib in inhibiting esophageal squamous cell carcinoma (ESCC).

Methods: Two cell lines, KYSE-150 and Eca-109, were used to verify the inhibitory effect of afatinib on the malignant growth of ESCC, and the effects of afatinib on Ferroptosis indicators such as Fe iron, GSH, MDA, lipid ROS, GPX4 and xCT levels were detected. Treatment of the cells with a Ferroptosis inhibitor reversed the effect of afatinib, which was further validated in xenograft tumors. Ubiquitination assay and Proteasome Inhibitor MG132 treatment confirmed the effect of afatinib on Nrf2 in ESCC cells. For further mechanism study, Nrf2 overexpression plasmid was used to transfect ESCC cells, and CCK-8, colony formation, flow cytometry, Transwell, qRT-PCR and Western Blots were used to confirm the reversal effect of Nrf2 overexpression on afatinib-induced Ferroptosis inhibiting ESCC cell function.

Results: Afatinib treatment could effectively reduce ESCC cell viability and induce Apoptosis, inhibit cell migration, invasion and epithelial-mesenchymal transition (EMT), and induce Ferroptosis. Ferroptosis inhibitor can reverse the inhibition of afatinib on the progression of ESCC in vitro and in vivo. Further mechanistic studies showed that afatinib interacted with Nrf2 and promoted its degradation, and regulated xCT/GPX4 to induce Ferroptosis, which ultimately inhibited the malignant progression of ESCC.

Conclusion: This study is the first to demonstrate that afatinib inhibits ESCC cell growth in vivo and in vitro by inducing Ferroptosis, and that the regulatory axis Nrf2/xCT/GPX4 is involved in this process. The results of this study provide a novel mechanism for afatinib mediated anti-ESCC activity.

Keywords

Afatinib; Esophageal squamous cell carcinoma (ESCC); Ferroptosis; NRF2; xCT/GPX4.

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