CPNE8 Suppresses Hepatocellular Carcinoma Progression Through Downregulation of ITGB1/CDC42/PAK1 Signaling and an Altered Immune-Marker Profile

Background: Hepatocellular carcinoma (HCC) is among the most lethal malignancies, and its immunosuppressive microenvironment limits the efficacy of current therapies. The integrin β1 (ITGB1)-CDC42-p21-activated kinase 1 (PAK1) axis is an established driver of HCC progression; however, the role of copine 8 (CPNE8), a calcium-dependent phospholipid-binding protein previously reported to have oncogenic effects in gastric cancer, has not been defined in HCC. The aim of this study was to investigate the expression, function, and mechanistic relevance of CPNE8 in HCC, as well as its association with the tumor immune microenvironment.Methods: CPNE8 expression was assessed in the TCGA-LIHC cohort and HCC cell lines. Stable CPNE8-overexpression (Huh7) and -knockdown (Hep3B) cell lines were generated and evaluated using cell counting kit-8 (CCK-8) and Transwell migration assays. Colony-formation and Transwell invasion assays were additionally performed in the overexpression cell lines. ITGB1 and CDC42 protein levels and PAK1 phosphorylation (p-PAK1/total PAK1 ratio) were examined by Western blotting, ITGB1 mRNA expression was measured by quantitative real-time PCR (qRT-PCR), and an ITGB1 rescue experiment was performed. Cpne8-overexpressing Hepa1-6 cells were implanted subcutaneously into immunocompetent C57BL/6J mice, and immune markers were evaluated by immunohistochemistry.Results: CPNE8 was consistently downregulated in HCC (TCGA-LIHC, 371 tumors vs. 50 adjacent normal tissues; Wilcoxon rank-sum test, p < 0.0001) and showed a weak inverse correlation with estimated regulatory T-cell infiltration (Spearman r = –0.20, p = 1.03 ×10-4). CPNE8 overexpression suppressed cell viability, colony formation, migration, and invasion (all p < 0.001), whereas knockdown increased cell viability and migration (both p < 0.001). CPNE8 overexpression reduced ITGB1 and CDC42 protein levels and the phospho-PAK1/total PAK1 ratio (all p < 0.001), while ITGB1 re-expression reversed these effects (all p < 0.001). In vivo, Cpne8 overexpression suppressed tumor growth, increased the CPNE8-positive area, and reduced the ITGB1-positive area and Ki67+ nuclear density (all p < 0.001). These changes were accompanied by higher densities of CD8+ and Granzyme B+ cells, lower FOXP3+ cell density, and a reduced PD-L1-positive area (all p < 0.001).Conclusion: CPNE8 is downregulated in HCC and suppresses malignant phenotypes through reduced ITGB1 and CDC42 protein levels and decreased PAK1 phosphorylation. Its restoration is also associated with an altered immune-marker profile in a syngeneic model, although the present study design cannot distinguish a direct effect of CPNE8 on the tumor microenvironment from secondary changes resulting from reduced tumor burden. These findings identify CPNE8 as a candidate tumor suppressor in HCC and warrant further investigation.