AQP3 has been established as a critical determinant of tumor growth and spread of human GC in previous studies. It has been speculated to promote GC cell migration and metastasis by inducing EMT. We found that AQP3 was up-regulated, and E-cadherin was repressed in cancer tissues. Vimentin immunoactivity was observed in 14 carcinoma tissues where AQP3 was overexpressed and E-cadherin was lacking. Over-expression of AQP3 correlated with repression of E-cadherin, and expression of vimentin. Loss of E-cadherin is regarded as a key step of EMT, while vimentin is a marker of mesenchymal differentiation. EMT is thought to be transient and occurs during progression towards metastases in several types of solid tumors. Our findings suggest that AQP3 is associated with EMT induction in human GC cases.
With respect to the clinical significance of AQP3 over-expression, E-cadherin repression, and vimentin expression, we showed that they were all associated with lymphovascular invasion. In particular, AQP3 and E-cadherin were associated with lymph node metastasis, while AQP3 and vimentin were associated with Lauren classification, and E-cadherin was associated with depth of tumor invasion. Patients with AQP3 over-expression exhibited worse OS compared with those lacking AQP3 expression. Repression of E-cadherin, and vimentin expression predicted poor prognosis for GC. These results are consistent with those reported by Zhou and Corso. However, our findings demonstrate for the first time the role of AQP3 in the prognosis of patients with GC.
Our previous results have shown that AQP3 promotes GC cell proliferation and migration. Because EMT of tumor cells is accepted to be closely associated with cancer invasion and metastasis[10, 11], we investigated the effects of AQP3 on GC cell proliferation, migration, and invasion using EdU incorporation assays and transwell assays. AQP3 over-expression enhanced cell proliferation, migration and invasion, implying that AQP3 has a role in facilitating GC progression.
To determine whether AQP3 promotes GC progression through the induction of EMT, we investigated the effects of AQP3 on the expression of E-cadherin, vimentin, and fibronectin (all EMT-related proteins) in human GC cells. Expression of E-cadherin was down-regulated upon AQP3 over-expression, and up-regulated upon AQP3 silencing. Additionally, expression levels of mesenchymal markers (vimentin and fibronectin) correlated with AQP3 expression, suggesting that AQP3 is capable of inducing EMT in human GC. We postulated that the effects of AQP3 could be attributed to its induction of EMT in cases of human GC.
PI3K signaling plays a key role in inducing and maintaining EMT. Cells expressing a constitutively active form of PKB/AKT, the most important downstream effector of PI3K signaling, induces the expression of Snail-1, which in turn represses E-cadherin gene transcription and induces EMT. In the present study, we showed that AQP3 over-expression enhanced the phosphorylation of AKT in cells, whereas AQP3 down-regulation had the opposite effect. Consistently, the expression of Snail correlated with AQP3 expression levels. A specific PI3K/AKT inhibitor attenuated AQP3-induced phosphorylation of AKT and Snail expression. These preliminary results reveal that the PI3K/AKT/Snail signaling pathway is likely involved in AQP3-mediated EMT of human GC cells.