Nordihydroguaiaretic acid inhibits bladder cancer metastasis through suppression of α1,3-mannosyltransferase expression and LRFN4 N-glycosylation.

Abstract

<h4>Background</h4>Metastasis remains a predominant contributor to cancer-related mortality worldwide. Elucidating the molecular mechanisms underlying cancer metastasis is crucial for developing strategies to inhibit tumor progression and improve clinical outcomes. Protein glycosylation, a hallmark of cancer pathogenesis mediated by specific glycosyltransferases, has emerged as a critical regulatory mechanism. This study investigates the functional role of glycosylation in cancer progression and explores its therapeutic potential.<h4>Methods</h4>We employed an integrated approach combining bioinformatics analysis of datasets, in vitro biological assays, transcriptional profiling, immunoprecipitation, and lectin pull-down assays to characterize α1,3-mannosyltransferase (ALG3)-dependent glycosylation mechanism in bladder cancer metastasis. Therapeutic targeting was investigated through virtual screening, molecular docking, molecular dynamics simulation, cellular thermal shift assay (CETSA), and validated in nude mice model using the Tranditional Chinese Medicine (TCM) monomer (NDGA).<h4>Results</h4>Our analysis revealed significantly elevated ALG3 expression in bladder cancer patients. Combined measurement of ALG3 in urine and serum samples demonstrated strong diagnostic potential, with higher AUC, sensitivity and specificity. Mechanistically, ALG3 promoted oncogenic cell behaviors through Ras signaling pathway activation. Immunoprecipitation and lectin pull-down assays identified LRFN4 as a novel ALG3 target, with ALG3 mediated N-glycosylation of LRFN4 being essential for its oncogenic function. Virtual screening identified nordihydroguaiaretic acid (NDGA) as a potent ALG3 inhibitor, which was validated through molecular docking, molecular dynamics simulation and CETSA. NDGA exhibited significant anti-tumor effects in both in vitro and in vivo models.<h4>Conclusions</h4>Our findings established ALG3 as a promising detection glycobiomarker for bladder cancer, and a key regulator for metastasis through LRFN4 N-glycosylation and Ras signaling pathway activation. The identified ALG3 inhibitor-NDGA demonstrated significant therapeutic potential, offering a foundation for developing personalized treatment strategies against bladder cancer metastasis.