Integrated Genomic and Single-Cell Analysis Identifies Notch1 as a Metastasis Suppressor in Nasopharyngeal Carcinoma Targeted by the AKT/Smad3 Axis.

backgroundNotch1 exhibits a high mutation rate in head and neck squamous cell carcinoma (HNSCC), but its functional role in nasopharyngeal carcinoma (NPC) invasion and metastasis remains incompletely understood. This study systematically investigates the function and molecular mechanisms of Notch1 as a metastatic suppressor in NPC.

methodsNotch1 expression levels were assessed in NPC tissue microarrays using immunohistochemistry, and correlations with tumor differentiation and metastasis were analyzed. TCGA databases were mined to explore mutation rates and sample clustering based on EMT-related gene signatures. Integrated analysis of TCGA and GEO datasets identified Notch1-associated genes, enabling molecular classification of patients. Additionally, scRNA-seq of NPC clinical samples was performed to validate the association between Notch1, stemness, and EMT features in the tumor microenvironment. The biological functions of Notch1 were further examined in vitro using proliferation (CCK-8, colony formation), migration and invasion (Transwell, wound healing) assays, as well as in vivo xenograft and metastatic models. Molecular mechanisms were probed by examining EMT markers, Smad3 phosphorylation and nuclear translocation, and protein-protein interactions via co-immunoprecipitation.

resultsImmunohistochemistry revealed significantly reduced Notch1 expression in NPC tissues, closely associated with poor differentiation and lymph node metastasis. TCGA analysis showed a 16.8% Notch1 mutation rate in HNSCC. Consensus clustering based on EMT-related genes identified subgroups with significant differences in Notch1 mutation status. Using 181 Notch1-related genes from integrated datasets, patients were further classified into three molecular subtypes (C1, C2, C3) with distinct prognosis, Notch1 mutation rates, EMT status, immune landscapes, and ferroptosis-related gene expression. Notably, the C2 subgroup exhibited the highest Notch1 mutation rate, enhanced EMT, poorest prognosis, and immune suppression.Consistent with these multidimensional datasets, scRNA‑seq analysis of primary NPC samples showed that Notch1‑high tumor cells preferentially retained a differentiated epithelial, low‑EMT/low‑stemness phenotype, further supporting an EMT‑ and metastasis‑suppressive role of Notch1 in clinical tumors. Functionally, Notch1 was downregulated across multiple NPC cell lines; knockout of Notch1 significantly promoted NPC cell proliferation, migration, invasion, and EMT both in vitro and in nude mouse models. Mechanistically, Notch1 loss increased EMT marker expression, augmented Smad3 phosphorylation and nuclear translocation, all without altering total Smad3 levels. Notch1 intracellular domain was found to interact directly with Smad3, while AKT and Smad3 interaction decreased upon Notch1 depletion, resulting in elevated Smad3 phosphorylation and EMT gene activation.

conclusionsNotch1 suppresses EMT and metastasis in NPC through regulation of the AKT/Smad3 pathway. These findings provide novel insights for molecular classification and targeted treatment strategies in NPC.