Effect of gut microbiota–derived testosterone on distant metastasis in non–small cell lung cancer.

Abstract

8548 Background: Distant metastasis is the major cause of poor prognosis in non–small cell lung cancer (NSCLC). Emerging evidence suggests that gut microbiota and their metabolites influence tumor progression, but their role in NSCLC metastasis remains unclear. This study aimed to characterize gut microbiota and metabolic features associated with NSCLC metastasis and to elucidate the role and mechanism of the key metabolite testosterone. Methods: Fecal samples from 60 NSCLC patients (48 with distant metastasis, 12 without) were analyzed using metagenomic sequencing and untargeted metabolomics. Multi-omics integration with machine learning identified metastasis-associated metabolites. The effects of testosterone on NSCLC cell migration, invasion, and epithelial–mesenchymal transition (EMT) were evaluated in vitro (PC9, A549, H1299) and in vivo using a mouse metastasis model. Molecular mechanisms were investigated by Western blot, transcriptomics, Mendelian randomization analysis, and functional studies of FGF21. Finasteride was used as a pharmacologic antagonist. Results: Patients with metastatic NSCLC showed distinct gut microbiota profiles, with increased alpha diversity and altered community structure compared with non-metastatic patients. Opportunistic pathogens including Oscillospiraceae, Ruminococcus, and Actinobacteria were enriched in metastatic patients, while Fusobacteria was enriched in non-metastatic patients. Metabolomic analysis revealed significant enrichment of steroid-related metabolites in metastatic NSCLC, with markedly elevated testosterone levels. Testosterone demonstrated good predictive value for metastasis (AUC = 0.761). Testosterone significantly enhanced NSCLC cell migration and invasion and induced EMT, characterized by decreased E-cadherin and ZO-1 and increased N-cadherin and Snail expression. In vivo, testosterone promoted liver metastasis and EMT marker expression. Mechanistically, testosterone activated AKT/mTOR signaling, showed a causal association with AKT phosphorylation, and upregulated FGF21. Functional assays confirmed that FGF21 promoted EMT and metastasis via AKT/mTOR activation. Finasteride reversed testosterone-induced EMT, signaling activation, and metastasis by inhibiting the FGF21/AKT/mTOR axis. Conclusions: Metastatic NSCLC is associated with a distinct gut microbiota and metabolic profile. Gut microbiota–derived testosterone is a key metabolite that promotes NSCLC distant metastasis by activating the FGF21/AKT/mTOR pathway and inducing EMT. Finasteride effectively antagonizes this process, suggesting potential therapeutic value. The testosterone/FGF21/AKT/mTOR axis may serve as a biomarker and therapeutic target for metastatic NSCLC.