TCGA-based identification and functional validation of PBK in intrahepatic cholangiocarcinoma progression with involvement of AKT signaling

Abstract

Intrahepatic cholangiocarcinoma (ICC) is an aggressive malignancy with limited molecular targets. This study aimed to identify candidate genes involved in ICC progression and to examine the functional role of PBK. Integrated bioinformatics analysis was performed on TCGA-CHOL data. PBK expression was assessed in ICC clinical samples and cell lines. Gain- and loss-of-function experiments were performed in HUCC-T1 and HCCC-9810 cells. AKT-IN-1 was used to evaluate the involvement of AKT signaling, and preliminary assays were performed to support the use of 1 μM AKT-IN-1 in rescue experiments. A subcutaneous xenograft model was used for in vivo validation. DEG analysis revealed distinct expression profiles between CHOL and non-tumor tissues, and WGCNA identified three tumor-associated modules: METurquoise, MEblue, and MEyellow. PBK was selected for further validation based on its upregulation, survival association, kinase-related background, and limited characterization in ICC. PBK expression was increased in ICC tissues and cell lines and was associated with TNM stage and shorter OS and DFS in the clinical cohort. Internal ROC analysis provided only an exploratory estimate of discriminative ability in the TCGA-CHOL cohort, and this finding requires validation in independent cohorts. PBK knockdown reduced ICC cell proliferation, migration, and invasion, whereas PBK overexpression enhanced these phenotypes. PBK overexpression increased p-AKT (Ser473), and AKT-IN-1 attenuated PBK-induced colony formation, EdU incorporation, migration, and invasion. In vivo, PBK knockdown reduced xenograft growth and was accompanied by decreased p-AKT, KI67, and PCNA expression. These findings suggest that PBK contributes to ICC malignant phenotypes, with AKT signaling involved in this process.