Abstract 1505: E-cigarette aerosols increase NRF2 expression and enhance cancer stemness in human oral epithelial cells

Abstract Background: The popularity of e-cigarettes has surged among cigarette and non-cigarette users, in part due to their promotion as a safer choice compared to traditional smoking. Yet, the true health implications of vaping are still uncertain and need to be explored. E-cigarette aerosols contain harmful substances that can cause oxidative stress. We and others have reported that exposure to e-cigarette aerosols increases reactive oxygen species (ROS) and reduces total antioxidant capacity, leading to a significant increase in DNA damage in oral epithelial cells. This raises important questions about the long-term health effects. Nuclear factor erythroid 2-related factor 2 (NRF2), the master regulator of antioxidant signaling, is crucial for managing oxidative stress and regulating cell stemness. In this study, we examined how exposure of oral epithelial cells to e-cigarette aerosols affect the expression of NRF2 and its downstream targets, which play a critical role in stem cell regulation. Methods: Human oral epithelial cancer (UM-SCC-1) cells were treated with aerosol extracts of e-cigarettes (tobacco flavor) from two brands, every other day for two weeks at 30 ng/ml nicotine. Standard combustible tobacco extracts were used as a positive control. Changes in gene and protein expression were assessed by qRT-PCR and western blotting analysis, respectively. The stemness of cancer cells was evaluated by spheroid assay, and the ALDH+ve cancer stem cell population was measured by the ALDEFLUOR assay. Data analysis was performed using Student's t-test. Results: Exposure to e-cigarette aerosol extracts significantly increased the mRNA and proteins levels of NRF2, SOD2, CAT, and HMOX1, along with a decrease in KEAP1 protein. We observed enhanced spheroid formation in oral epithelial cells exposed to e-cigarette extracts. Notably, a significant rise in the cancer stem cell population was observed in oral cancer cells exposed to e-cigarette aerosols. Furthermore, we observed a marked increase in the expression levels of specific mRNAs related to stemness factors (e.g., BMI1, OCT4), which are crucial for maintaining stem cells. Conclusion: Our findings indicate that exposure to e-cigarette aerosols significantly activates the NRF2 signaling pathway and promotes stemness of oral epithelial cells. The increased expression of NRF2 and its downstream targets suggests an enhanced response to oxidative stress. Additionally, the upregulation of stemness markers highlights the potential of e-cigarette aerosols to enhance cancer stem cell characteristics and tumor progression. These results raise concerns about the role of e-cigarette aerosols in oral carcinogenesis and imply that the perceived safety of e-cigarettes may be misleading, thus warranting further investigation into their long-term effects on human health. Funding: TSET HPRC (Ganapathy), NIH/NCI (R01CA242168, Queimado). Citation Format: Vengatesh Ganapathy, Jimmy Manyanga, Gautham Chengizkhan, Mayilvanan Chinnaiyan, Balaji Sadhasivam, Ilangovan Ramachandran, David A. Rubenstein, Lurdes Queimado. E-cigarette aerosols increase NRF2 expression and enhance cancer stemness in human oral epithelial cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 1505.