Esophageal cancer is one of the most common malignant tumors and the seventh leading cause of cancer-related deaths worldwide (Bray et al., 2024). Over half of the global esophageal cancer cases occur in China, where esophageal squamous cell carcinoma (ESCC) accounts for more than 90 % of the cases (Codipilly and Wang, 2022). Some studies have reported that neoadjuvant chemoradiotherapy in combination with surgical resection can significantly prolong the long-term survival of patients with locally advanced esophageal cancer. However, the 5-year overall survival (OS) rate of patients with esophageal cancer remains at only 25 %–47 % (Shah et al., 2020, Sjoquist et al., 2011, van Hagen et al., 2012). For patients with advanced ESCC, treatment options become significantly limited following the failure of first-line therapy, leading to a poorer prognosis (Zhang et al., 2024b). After neoadjuvant chemoradiotherapy, only 20 %–48 % of patients with ESCC achieve a pathologic complete response (pCR) (Mariette et al., 2014, Scheer et al., 2011, Yang et al., 2018), highlighting the substantial individual variations in sensitivity to chemoradiotherapy. Therefore, there is an urgent need for new strategies to overcome therapeutic resistance in patients with ESCC.

Upon exposure to ionizing radiation (IR) and chemotherapy, cells undergo various cell death mechanisms, including apoptosis, necrosis, and ferroptosis (Lei et al., 2020, Tang et al., 2019). Ferroptosis, a form of cell death driven by iron-dependent lipid peroxidation, is a crucial mechanism by which radiotherapy kills tumor cells. Additionally, ferroptosis plays a significant role in chemoradiotherapy resistance (Lei et al., 2020, Stockwell et al., 2017, Zhou et al., 2024). In radiation-insensitive renal cancers, ferroptosis does not occur following radiotherapy (Lei et al., 2020), indicating that tumor cells may resist radiotherapy-induced damage by inhibiting ferroptosis. In addition, there is increasing evidence showing that chemotherapy drugs such as cisplatin (DDP), which is one of the most commonly used chemotherapy drugs for ESCC, kill tumor cells by inducing ferroptosis (Liang et al., 2024, Roh et al., 2017, Zhang et al., 2023). Therefore, targeting ferroptosis could improve the effectiveness of chemoradiotherapy for the treatment of tumors. Nuclear factor erythroid 2-related factor 2 (NRF2) functions as a key regulator of ferroptosis inhibition, whereas kelch like ECH associated protein 1 (KEAP1) acts as a negative regulator of NRF2, targeting it for degradation (Adinolfi et al., 2023, Dodson et al., 2019). However, the mechanism underlying the activation of the KEAP1-NRF2 pathway under radiation stress in ESCC remains unclear.

E3 ubiquitin ligases influence tumorigenesis by adding ubiquitin chains to various substrate proteins. Currently, around 700 human E3 ubiquitin ligases have been identified, some of which are promising therapeutic targets (Cruz Walma et al., 2022, Zhang et al., 2024a, Zheng and Shabek, 2017). In this study, we established two radioresistant ESCC cell lines and observed a decrease in ferroptosis levels in these resistant cells under radiotherapy stress. This was accompanied by a significant upregulation of NRF2 protein levels and substantial degradation of KEAP1. Using mass spectrometry and RNA-seq, we identified that the ring finger protein 217 (RNF217), an E3 ubiquitin ligase, interacts with KEAP1 and is specifically upregulated in Kyse30R cells. RNF217 mediates protein degradation through ubiquitination (Jiang et al., 2021), and its dysregulation is associated with the occurrence and progression of human diseases (Fontanari Krause et al., 2014, Jiang et al., 2021). However, the relationship between RNF217 and radioresistance in ESCC or other tumors has not been previously explored.

In this study, we showed that RNF217 upregulation correlated with chemoradiotherapy resistance in ESCC by inhibiting ferroptosis. The underlying molecular mechanism involves RNF217 promoting degradation of KEAP1 through K48- and K63-linked ubiquitination modifications, thereby enhancing the protein stability of NRF2, an important molecule for inhibiting ferroptosis. Furthermore, we found that NRF2 promoted the expression of RNF217 through transcriptional activation during the long-term radiation treatment of cancer cells. Our study revealed a potential mechanism of abnormal NRF2 activation during chemoradiotherapy in ESCC and suggested that the RNF217-KEAP1-NRF2 axis could induce therapeutic resistance by negatively regulating ferroptosis. The results of this study will provide potential strategies for overcoming chemoradiotherapy resistance in ESCC.