835 Background: Bladder cancer (BLCA) is one of the most common malignancies of the urinary system, with muscle-invasive bladder cancer (MIBC) characterized by high aggressiveness and a strong tendency for lymph node metastasis. However, the molecular mechanisms underlying lymph node metastasis remain largely unclear, and there is currently a lack of safe and effective therapeutic strategies targeting this process. Methods: We established a murine popliteal lymph node metastasis model using MB49 bladder cancer cells. Through iterative in vivo selection over five consecutive rounds, we successfully generated a highly metastatic subline, MB49-LN5. Subsequently, we performed integrative analyses combining full-length transcriptomic sequencing, proteomic profiling, clinical BLCA tissue samples, and patient prognostic data, and identified ACSL5 as a potential key gene involved in lymph node metastasis. Further mechanistic studies were conducted using MB49 cells stably overexpressing wild-type or enzymatically inactive ACSL5. These cells were subjected to the popliteal lymph node metastasis model, along with in vivo tumor assays and flow cytometry analysis, to systematically assess the function and molecular mechanism of ACSL5 in BLCA metastasis. Results: Downregulation of ACSL5 markedly promoted lymph node metastasis of BLCA and was accompanied by a significant increase in tumor-infiltrating B cells within both primary tumors and metastatic lymph nodes. Functional assays demonstrated that the inhibitory effect of ACSL5 on lymph node metastasis was dependent on its enzymatic activity. Mechanistically, loss or inactivation of ACSL5 led to the accumulation of palmitic acid (PA) in the tumor microenvironment, which activated B cells and enhanced VEGF-C secretion, thereby promoting lymphangiogenesis and accelerating metastatic dissemination. Further molecular analyses revealed that PA induced palmitoylation of FOXO1, altering its subcellular localization and enhancing its cytoplasmic colocalization with AKT, leading to increased FOXO1 phosphorylation and suppression of its transcriptional activity. Reduced FOXO1 activity resulted in the downregulation of pro-apoptotic and cell cycle arrest–related genes, thereby facilitating B-cell proliferation and activation and accelerating lymph node metastasis. Conversely, mutation of the Cys111 site on FOXO1 reduced its palmitoylation, restored transcriptional activity, suppressed B-cell activation and lymphangiogenesis, and consequently diminished metastatic potential. Conclusions: Collectively, We revealed that ACSL5 modulates palmitic acid metabolism through its enzymatic activity, thereby shaping the tumor microenvironment by promoting B-cell proliferation and activation to facilitate lymphangiogenesis and metastasis.
Sun et al. (Sun,) studied this question.