Activation of the cGAS–STING pathway by clofoctol through mitochondrial damage in triple-negative breast cancer

Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer that lacks effective targeted therapies. Drug repurposing offers a promising avenue for accelerating the discovery of novel treatments. Clofoctol, a synthetic antibacterial agent, has recently emerged as a potential anticancer candidate; however, its mechanisms of action in TNBC remain largely unclear. Potential targets of clofoctol in breast cancer were initially identified through network pharmacology and enrichment analysis. The predicted pathways were validated using a series of in vitro assays, including MTT and colony formation assays for cell proliferation, and flow cytometry for cell cycle distribution, apoptosis, mitochondrial membrane potential (MMP), and reactive oxygen species (ROS) levels. Western blotting was used to assess key regulatory proteins, while mitochondrial ultrastructure was examined via transmission electron microscopy. In vivo antitumor efficacy and systemic safety were evaluated using a 4T1 xenograft mouse model by monitoring tumor growth, body weight, and histopathological changes. Clofoctol significantly inhibited TNBC cell proliferation, colony formation, and metastatic behaviors. Mechanistically, clofoctol induced G0/G1 phase arrest by downregulating CDK4 and Cyclin D1 and promoted apoptosis via mitochondrial dysfunction, characterized by MMP loss, elevated ROS generation, and cytochrome c release. Furthermore, clofoctol triggered mitochondrial DNA (mtDNA) leakage, which activated the cGAS–STING signaling pathway and led to phosphorylation of TBK1 and IRF3. In vivo, clofoctol markedly suppressed tumor growth in TNBC xenograft models without inducing notable systemic toxicity, while further validating that clofoctol activates antitumor immune responses. Clofoctol exerts potent anti-TNBC activity by integrating DNA damage response activation, mitochondrial apoptosis, and cGAS–STING pathway stimulation. Its dual cytotoxic and immunomodulatory effects, together with a favorable safety profile, underscore clofoctol’s promise as a repurposed therapeutic candidate for TNBC and warrant further evaluation in patient-derived and immunocompetent models.