Dysregulation of aquaporin-1 in human CCA cells triggers epithelial-mesenchymal transition

GASPARI, CÉSAR I.; ARBELAIZ ANDER; LAURA FOUASSIER; VAQUERO JAVIER; MARINELLI RA; RICHARD SETH; MARRONE, JULIETA; SERGIO GRADILONE1

Praga

Congreso; Liver Cancer Summit; 2020

European Association for the Study of Liver

Background and aims: Cholangiocarcinoma (CCA) is a tumor arising from biliary epithelial cells. Theoverall prognosis is very poor due to the ability of the tumor to develop chemoresistance and metastasis.Epithelial-mesenchymal transition (EMT) is a multistep process during which epithelial cells graduallyadopt structural and functional characteristics of mesenchymal cells. EMT is required for tumor cellmigration, invasion and has been considered an early event of metastasis. EMT occurs in CCA whereis associated with progression and chemoresistance. The function of aquaporin (AQP) water channelsin tumors has been the focus of substantial research efforts. However, the role of AQPs in liver cancersand specifically in CCA remains unknown. Our aim was to study the role of aquaporin-1 (AQP1) in theregulation of EMT in CCA.Method: Two established human CCA cell lines, HuCCT1 and KMCH, were used as experimentalmodels. We generated KMCH AQP1 shRNA knock-down and HuCCT1 AQP1 KO by CRISPR/Cas9system. Cell proliferation and migration were assessed by the Incucyte live-cell imaging system. Weperformed an unbiased discovering experiment using RNAseq comparing wild-type and AQP1 KO CCAcells. EMT and stemness markers expression were evaluated by RT-qPCR, western-blot and confocalmicroscopyResults: Both CCA cell lines express AQP1 largely located in intracellular compartments. Dysregulationof AQP1 in tumor cells significantly induces proliferation and migration, suggesting a tumor suppressorrole of AQP1 in CCA. RNAseq of AQP1 KO cells showed a total of 2702 differentially expressed genes.The Ingenuity Pathway Analysis showed Cancer (p-value range 3.36E-04 - 9.16E-34), Cellularmovement (p-value range 2.54E-04 - 1.71E-20), and Cellular Growth and Proliferation (p-value range2.23E-04 - 3.34E-11) as the top affected Diseases and Biological functions. In addition, the epithelialmarkers genes were significantly downregulated upon AQP1 silencing (E-cadherin gene CDH1 by -133fold change; p < 0.001, cytokeratin19 gene KRT19 by -8 fold change; p < 0.001), while mesenchymalmarkers genes were significantly upregulated (vimentin gene VIM by 4 fold change; p < 0.001,fibronectin gene FN1 by 3.51 fold change; p < 0.001), suggesting that the loss of AQP1 induced EMT.Furthermore, these changes were corroborated at mRNA and protein levels, with a major E-cadherindownregulation along with an upregulation of mesenchymal markers vimentin, fibronectin and EMTinducingtranscription factors ZEB1 and ZEB2.Conclusion: Our data suggest that AQP1 acts as a tumour suppressor in CCA cells by acting both onproliferation and migration through an EMT process. Therefore, AQP1 might have an important role by restraining CCA progression.