1,4-Dioxane Induces Epithelial-Mesenchymal Transition and Carcinogenesis in an Nrf2-Dependent Manner

The carcinogenic potential of the environmental pollutant 1,4-dioxane (1,4-D) in humans is not yet fully understood or recognised. In this study, we provide evidence that 1,4-D acts as a carcinogen in human epithelial cells. Using the human bronchial epithelial cell line BEAS-2B, with or without CRISPR-Cas9-mediated Nrf2 knockout, we demonstrate that continuous exposure to environmentally relevant concentrations of 1.25-20 ppm 1,4-D over 2 months induces malignant transformation in an Nrf2-dependent manner. Transformed cells exhibit enhanced anchorage-independent growth in soft agar, increased migration and invasion, and tumorigenic potential in a xenograft mouse model. Integrated RNA sequencing and proteomics analyses reveal that 1,4-D robustly activates Nrf2 signalling, driving extracellular vesicle (EV) biogenesis and cargo loading with syndecan 4 (SDC4) and other proteins, including COL12A1, CAPG and NNMT, which are associated with epithelial-mesenchymal transition (EMT) and cancer metastasis. Nrf2 knockout reduces SDC4 expression and its incorporation into EVs, leading to decreased EV uptake by recipient cells. Unlike EVs from 1,4-D-transformed WT cells, which enhance the proliferation, migration and invasion of recipient cells, EVs from 1,4-D-transformed Nrf2 KO cells exhibit a diminished capacity to promote these EMT properties. Furthermore, we demonstrate that the Nrf2 target gene SDC4, induced by 1,4-D and enriched in EVs, plays a critical role in EV uptake by recipient cells, thereby facilitating EMT propagation. Collectively, our findings suggest that 1,4-D is a human carcinogen, with its carcinogenicity largely dependent on Nrf2 activation, which orchestrates the biogenesis of EVs with EMT-promoting functions.