RBX1+ CAFs Drives Pancreatic Ductal Adenocarcinoma Progression Through Tenascin C Overexpression

<b>Background:</b> Pancreatic ductal adenocarcinoma (PDAC) is characterized by a dense desmoplastic stroma that actively drives malignant progression. However, the specific contributions of E3 ubiquitin ligases within the cancer-associated fibroblast (CAFs) compartment to the PDAC landscape remain largely elusive. <b>Methods:</b> Pancreatic tissue samples were collected from the First Affiliated Hospital of Harbin Medical University. Gene expression was analyzed by RT-PCR, and single-cell RNA sequencing (scRNA-seq) data were integrated for cell subtype identification. Kaplan-Meier survival analysis assessed gene expression and survival. Pseudotime analysis and CellChat evaluated fibroblast transitions and intercellular communication. Cell lines were transfected with RBX1 siRNAs, and protein levels were measured by Western blotting. Proliferation was assessed using colony formation and EdU staining. Statistical analyses were performed using R (v4.4) and GraphPad Prism 8.0. <b>Results:</b> Thirteen E3 ubiquitin ligases were significantly upregulated in PDAC and correlated with unfavorable clinical outcomes. Among these, RBX1 was identified as a candidate preferentially expressed in CAF populations and strongly associated with poor prognosis. Single-cell transcriptomic profiling and pseudotime analysis further revealed that RBX1-positive CAFs were predominantly involved in extracellular matrix remodeling and pro-tumorigenic pathways. Functional assays demonstrated that silencing RBX1 markedly inhibited PAAD cell proliferation and tumor growth both in vitro and in xenograft models. Mechanistically, RBX1 was found to upregulate Tenascin C (TNC) expression, while ectopic overexpression of TNC partially rescued the growth suppression induced by RBX1 knockdown. <b>Conclusions:</b> Our findings suggest that RBX1 facilitates PDAC progression through a CAF-related mechanism and TNC regulation, positioning RBX1 as a potential therapeutic target for PDAC intervention.