The lethality of esophageal squamous cell carcinoma (ESCC), and other epithelial cancers, is primarily due to its aggressive nature and frequent lymphatic metastasis, both of which impact prognosis. In this review, we explore the underlying molecular mechanisms of ESCC lymphatic metastasis, specifically, the functional role of NRF2 and therapeutic strategies. Current data suggest that NRF2 hyperactivation (NRF2^high) may promote lymphatic metastasis of ESCC by affecting the extracellular matrix (ECM), epithelial–mesenchymal transition (EMT), lymphangiogenesis, immune evasion, metabolic programming, and Hippo signaling. We also update the latest developments in NRF2 inhibitors, their mechanisms of action, screening strategies, and approaches for evaluating compound efficacy. Finally, we highlight the utility of animal models for mechanistic studies and therapeutic development. We believe elucidation of the functional role of NRF2 in ESCC lymphatic metastasis and developing proper NRF2 inhibitors will greatly improve the clinical prognosis of ESCC in human patients.

Background: Esophageal squamous cell carcinoma (ESCC) remains a highly lethal malignancy with limited therapeutic options, in part due to frequent activation of nuclear factor erythroid 2-related factor 2 (NFE2L2 or NRF2). Gain-of-function mutations in NRF2 disrupt its negative regulation by Kelch-like ECH-associated protein 1 (KEAP1), resulting in sustained NRF2 signaling that promotes tumor growth and resistance to chemotherapy and radiation. We previously identified the FDA-approved drug pyrimethamine (PYR) as an NRF2 inhibitor and demonstrated that inhibition of dihydrofolate reductase (DHFR) represents the primary mechanism underlying its NRF2-suppressive activity, supporting its advancement into a Phase I window-of-opportunity clinical trial (NCT 05678348). Meanwhile, in NRF2^W24C-KYSE70 and NRF2^D77V-KYSE180 cells, PYR promoted NRF2^Mut ubiquitination and proteasomal degradation and shortened its half-life. This study aims to explore additional modes of action by which PYR inhibits NRF2. Methods: Cell cycle analysis was performed by flow cytometry. Cell proliferation, apoptosis and chemosensitivity were assessed by Live-Cell Analysis System, while radiosensitivity was evaluated using X-ray irradiation and the CellTiter-Glo assay. Molecular interactions between NRF2 and KEAP1 were examined through Co-IP and PLA, and the direct binding of PYR to KEAP1 was quantified using ITC and SPR. Molecular docking and dynamic simulations were employed to predict potential PYR-binding pockets within the Kelch domain. Results: Using genetically defined isogenic ESCC cell models, we show that activation of mutant NRF2 (NRF2^Mut) or wild-type NRF2 (NRF2^WT) produces distinct, context-dependent effects on squamous differentiation, proliferation, and therapeutic response. We further demonstrate that PYR restores sensitivity to chemotherapy and ionizing radiation in NRF2^Mut ESCC cells. Mechanistically, short-term PYR treatment promotes KEAP1-dependent proteasome-mediated degradation of NRF2^W24C. Biochemical and biophysical assays indicate that PYR enhances the interaction between KEAP1 and NRF2^W24C in a manner associated with KEAP1-dependent proteasomal degradation. Computational modeling further suggests that PYR may engage a pocket within the Kelch domain to facilitate the NRF2^W24C-KEAP1 interaction. Conclusions: These findings show that PYR functionally restores KEAP1-mediated NRF2 degradation of select NRF2^Mut through a glue-like effect and overcomes therapy resistance in ESCC. Although the proposed glue-like mechanism remains hypothetical, this work supports further investigation into the NRF2–KEAP1 interaction and may inform the development of KEAP1-targeted strategies for NRF2^Mut cancers, including ESCC.