Loss of aquaporin-1 triggers epithelial-mesenchymal transition in human cholangiocarcinoma
Background and Aims: Epithelial-mesenchymal transition (EMT) is involved in metastatic dissemination and associated with the progression of cholangiocarcinoma (CCA). Aquaporin-1 (AQP1) is a water channel expressed by cholangiocytes described to modulate cell proliferation and invasion in several cancers. However, the role of AQP1 in CCA remains unknown. Our aim was to study the function of AQP1 in CCA. Material and methods: AQP1 expression was evaluated in 39 human intrahepatic CCA (iCCA) by transcriptomic analysis. AQP1-KO in CCA cells, HucCCT1 was achieved by CRISPR/Cas9. Incucyte live-cell imaging system was used for functional studies. Next-generation sequencing (RNAseq) was used to further study the consequences of AQP1 KO. EMT and signaling pathways were evaluated by RT-qPCR, western blot, and immunostaining. In vivo experiments were performed using a xenograft CCA model.  Results: AQP1 expression correlated positively with overall survival in patients with iCCA and negatively with EMT, stemness, and proliferative signatures. In vitro, RNA-seq analysis of CCA cells depleted for AQP1 showed a downregulation of epithelial markers (CDH1 and KRT19; p< 0.001) and an upregulation of mesenchymal markers (VIM and FN1; p < 0.001). In these cells, we observed a phenotypic change with induction of cell scattering, a loss of cell-cell junction protein E-cadherin, and a higher expression of vimentin and ZEB1, an EMT-inducing transcription factor. Functionally, loss of AQP1 is associated with increased cell migration and proliferation. Moreover, we found an activation of the IGF2/IGF1R/IR pathway in AQP1-depleted CCA cells. In vivo, AQP1-depleted CCA cells displayed a higher tumorigenic potential and tumor burden than control cells.  Conclusion: Our data suggest that AQP1 acts as a tumor suppressor in CCA by acting both on cell proliferation and migration through an EMT process. Therefore, AQP1 might have an important role in the regulation of CCA progression. Overall design: AQP1-KO in CCA cells, HucCCT1 was achieved by CRISPR/Cas9. Next-generation sequencing (RNAseq) was used to further study the consequences of AQP1 KO.