IQGAP3 as a gatekeeper of epithelial integrity and prognostic biomarker in extrahepatic cholangiocarcinoma

Background: Biliary tract cancer (BTC) is an aggressive malignancy for which reliable prognostic biomarkers remain scarce. Although recent efforts have advanced the molecular characterization of intrahepatic cholangiocarcinoma (iCCA), the biology of extrahepatic CCA (eCCA) is still insufficiently understood. IQ motif-containing GTPase-activating protein 3 (IQGAP3) is a scaffold protein involved in cytoskeletal regulation, cell-cycle control, and epithelial junctional stability. Because epithelial-mesenchymal transition (EMT) drives invasion and metastasis in BTC, we hypothesized that IQGAP3 may influence epithelial identity and clinical behavior in eCCA. This study sought to clarify the clinicopathological significance of IQGAP3 and examine its biological role using integrated pathological and functional analyses. Methods: We retrospectively analyzed 100 patients who underwent curative resection for perihilar or distal CCA between 2016 and 2020. IQGAP3 expression was assessed by immunohistochemistry and quantified using QuPath-based H-scores; tumor regions were manually annotated in QuPath to ensure that H-scores reflected staining intensity exclusively in cancer cells. Associations with clinicopathological factors and overall survival (OS) were evaluated using standard statistical approaches. Functional studies were conducted in HuCCT1 and TFK-1 cells with shRNA-mediated IQGAP3 knockdown. RNA sequencing and gene set enrichment analysis (GSEA) were performed to identify transcriptional changes associated with IQGAP3 loss. Expression of epithelial markers, including E-cadherin (CDH1), was examined in cell models and resected tissues. Results: High IQGAP3 expression was associated with significantly longer OS than low expression (median 102.7 vs 40.2 months; hazard ratio 0.51; 95% CI 0.30-0.88; P = 0.017). Low IQGAP3 levels correlated with lymph-node metastasis, advanced stage, and positive resection margins. Functional assays showed that IQGAP3 knockdown activated EMT-related transcriptional programs in both cell lines (normalized enrichment score 1.43 and 1.44; P < 0.01; false discovery rate q < 0.25) and reduced CDH1 expression, suggesting impaired epithelial cohesion. In clinical samples, IQGAP3 and CDH1 mRNA levels demonstrated a strong positive correlation (r = 0.74), supporting a biological link between IQGAP3 loss and disruption of epithelial integrity. Conclusions: IQGAP3 plays a key role in maintaining epithelial identity in CCA. Its loss promotes EMT activation and is associated with more aggressive disease features, whereas high expression confers favorable prognosis in eCCA. IQGAP3 may serve as a practical biomarker for risk stratification and represents a potential target for strategies aimed at limiting EMT-driven tumor progression.