NRF2-WNT5A promotes stem cell persistence and radioresistance in esophageal squamous cancer

Abstract

The transcription factor nuclear factor erythroid 2-related factor 2 (NRF2) drives radioresistance in esophageal squamous cell carcinoma (ESCC), thereby compromising therapeutic efficacy; however, the underlying mechanisms remain elusive. Cancer stem cells (CSCs) are recognized to harbor intrinsic radioresistance regulated by complex oncogenic signaling networks. Here, integrating 3D cancer spheroid models with RNA-seq analysis, we investigated NRF2 hyperactivation in CSCs and its role as a core mediator of CSC-associated radioresistance and stemness via the canonical WNT/β-catenin pathway. Subsequent ChIP-seq and ATAC-seq analyses identified the noncanonical WNT ligand WNT5A as a direct target of NRF2, with its activity closely linked to chromatin accessibility. Quantitative genome-wide analysis showed that radiation treatment significantly augmented NRF2 binding peaks, whereas NRF2 knockdown markedly attenuated these peaks. Clinical analysis further uncovered a strong positive correlation between NRF2 and WNT5A expression levels in ESCC tissues. Comprehensive in vitro and in vivo validations systematically confirmed that WNT5A depletion impaired NRF2 overexpression-induced WNT/β-catenin activation, cancer stemness and radioresistance, while WNT5A overexpression rescued these effects. Combined with clinical data, these findings reinforce the critical role of WNT5A in maintaining the stemness phenotypes of esophageal epithelia and establishing it as an oncogenic driver associated with poor prognosis. Mechanistically, under NRF2 hyperactivation, NRF2-regulated WNT5A interacts with glycogen synthase kinase-3β (GSK-3β) to increase its phosphorylation at Ser9; this inactivates GSK-3β, relieving its inhibitory effect on β-catenin nuclear accumulation and thus activating the canonical WNT pathway. Collectively, we identify the NRF2/WNT5A/β-catenin axis as a critical regulator of ESCC stemness and radioresistance, validated across cellular assays, xenograft models, and clinical cohorts. These findings highlight WNT5A as a promising biomarker and therapeutic target for improving clinical outcomes in ESCC patients.