Loss of Aquaporin-1 in Tumor Cells Fosters Intrahepatic Cholangiocarcinoma Progression

Aquaporin-1 (AQP1) is a water channel expressed by cholangiocytes described to modulate cell proliferation and invasion in several cancers. But the role of AQP1 in cholangiocarcinoma (CCA) remains unknown. The aim was to study the function of AQP1 in CCA. AQP1 expression was evaluated in 39 intrahepatic CCA (iCCA) specimens from transcriptomic data. AQP1-knockout in HuCCT1 iCCA cells was achieved by clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9). Next-generation sequencing (RNA sequencing) was performed to study the consequences of AQP1 inhibition on cell phenotype. Epithelial-mesenchymal transition (EMT), cell migration and proliferation, and signaling pathways were evaluated by live-cell imaging system, quantitative RT-PCR, Western blot, and immunostaining. In vivo experiments were performed using a xenograft CCA model. In human iCCA, low AQP1 expression correlated with reduced overall survival. In vivo, CCA cells depleted for AQP1 displayed a higher tumorigenic potential than control cells. In vitro, RNA-sequencing analysis of AQP1-depleted CCA cells showed signatures of tumor progression, including EMT. Indeed, AQP1-depleted cells showed a cell dispersion phenotype, loss of the junction protein E-cadherin, and higher expression of vimentin and zinc finger E-box binding homeobox 1 (ZEB1), along with stemness traits. Functionally, loss of AQP1 is associated with increased cell migration and proliferation. Moreover, an activation of the insulin-like growth factor 2/insulin-like growth factor 1 receptor/insulin receptor pathway was found in AQP1-depleted CCA cells. The data suggest that AQP1 acts as a tumor suppressor in iCCA.