Lung cancer is the leading cause of cancer-related mortality worldwide, with cigarette smoking identified as the primary risk factor. Beyond promoting tumorigenesis, chronic smoking exposure has been associated with reduced chemotherapy efficacy, but the molecular mechanisms linking smoking to chemoresistance are incompletely understood. The authors sought to model long-term smoking exposure in lung cancer cells and to identify epigenetic or signaling mediators that connect chronic smoke exposure to chemotherapy resistance.
To mimic chronic smoking, the investigators repeatedly treated Lewis lung carcinoma (LLC) cells with cigarette smoke extract (CSE) over a prolonged period. After four months of repeated CSE exposure, treated LLC cells displayed several altered phenotypes compared with control cells: enhanced proliferation, increased migration, and resistance to chemotherapy-induced cell death. These phenotypic changes established a preclinical model in which to probe mechanisms underlying smoking-associated chemoresistance.
Mechanistic studies in the CSE-exposed cells identified upregulation of the epigenetic regulator EZH2 as a central change induced by chronic smoke exposure. Increased EZH2 activity was associated with repressive histone methylation at the promoter region of RIPK3, leading to transcriptional suppression of RIPK3 expression. Thus, chronic CSE exposure reduced cellular RIPK3 levels via an EZH2-dependent epigenetic mechanism.
The authors investigated how reduced RIPK3 expression affected response to chemotherapy. Loss of RIPK3 impaired chemotherapy-induced cell death in this model. Importantly, the impairment was attributed primarily to inhibition of ferroptosis rather than necroptosis, indicating that RIPK3 contributes to the execution or facilitation of ferroptotic cell death in these lung cancer cells exposed to chemotherapy. This finding highlights a previously underappreciated role for RIPK3 in regulating ferroptosis within the context of smoking-associated chemoresistance.
Targeting the upstream epigenetic driver reversed the suppression of RIPK3 and restored chemosensitivity. Both genetic depletion of EZH2 and pharmacological inhibition of EZH2 activity re-established RIPK3 expression in the CSE-treated lung cancer cells. Restoration of RIPK3 sensitized the cells to treatment with gemcitabine in vitro, and similar sensitization was observed in in vivo models. These experiments connect EZH2 inhibition, RIPK3 re-expression, and enhanced chemotherapy response in the smoking-exposed preclinical system.
Analysis of human lung cancer datasets reported by the authors demonstrated an inverse correlation between EZH2 and RIPK3 expression across samples. Additionally, RIPK3 expression was reported to decrease progressively with increasing smoking history. These human data are presented as consistent with the preclinical findings that chronic cigarette exposure elevates EZH2 and suppresses RIPK3, and that this axis associates with features of chemoresistance.
Collectively, the results identify an EZH2/RIPK3 axis as a mediator of cigarette smoking–associated chemoresistance in lung cancer. The data suggest that pharmacologic or genetic inhibition of EZH2 may restore RIPK3 expression and resensitize tumors to chemotherapeutic agents such as gemcitabine, particularly in patients with a history of smoking. The study further reveals a role for RIPK3 in promoting chemotherapy-induced ferroptosis, expanding potential mechanisms to exploit therapeutically.
The article is presented as a preprint and has not undergone peer review. The authors declared no competing interests. Reported funders include the National Heart, Lung, and Blood Institute (R56HL162749), the Tobacco-Related Disease Research Program (T35KT9504), and the American Cancer Society (IRG-22-144-60).
This work is a preprint posted to bioRxiv and has not been certified by peer review. The authors reported no competing interests. Funding sources listed in the report included NHLBI, the Tobacco-Related Disease Research Program, and the American Cancer Society.
Note: Specific experimental details, quantitative results, statistical metrics, dosing regimens, in vivo model descriptions, and supplementary data were not reproduced here because the summary is limited to the findings and statements provided in the source preprint abstract and article metadata.