Glutamine metabolism reprogramming promotes bladder cancer progression via PYCR1: a multi-omics and functional validation study

BACKGROUND: Bladder cancer (BLCA) is a prevalent malignancy worldwide, with advanced stages linked to poor prognosis. Although immune checkpoint inhibitors (ICIs) show clinical promise in treating both operable and advanced BLCA, predicting patient responses remains a major challenge. Glutamine metabolism, a key aspect of metabolic reprogramming, has been implicated in tumor progression and immune modulation. However, the exact role of glutamine metabolism in BLCA remains poorly understood. This study aims to explore its association with clinical outcomes and immunotherapy response while functionally validating key regulatory genes. METHODS: An integrated approach combining targeted metabolomics, single-cell RNA sequencing, and bulk transcriptomic data was used to profile glutamine metabolism in BLCA comprehensively and identify potential metabolic biomarkers. A prognostic model, termed GMscore, based on glutamine metabolism, was constructed using principal component analysis (PCA). Key regulatory genes were identified through random forest analysis. Functional assays, including in vitro proliferation, migration, and metabolic assays, as well as in vivo xenograft models, were employed to validate the findings. RESULTS: Targeted metabolomics revealed increased glutamine metabolism in BLCA cell lines. The GMscore model, developed and validated across multiple cohorts, accurately predicted patient survival. In two immunotherapy cohorts (IMvigor210 and GSE91061), a lower GMscore correlated with improved therapeutic response, suggesting its potential as a predictive biomarker for immunotherapy efficacy. PYCR1 was identified as a key regulatory gene, exhibiting high expression in epithelial cells and cancer-associated fibroblasts (CAFs). Functional assays demonstrated that PYCR1 knockdown inhibited cell proliferation and migration and suppressed tumor growth in vivo. Mechanistically, PYCR1 facilitated proline synthesis through P5CS and activated the PI3K/AKT/mTOR signaling pathway, which modulated glutamine utilization and metabolic reprogramming in BLCA. CONCLUSIONS: This study provides a comprehensive analysis of glutamine metabolism in BLCA and introduces a clinically relevant prognostic model. PYCR1 was identified as a central metabolic regulator, underscoring its critical role in tumor development and progression.