Fig. 8

Tetraspanin 1 (TSPAN1) enhances phosphoinositide-3-kinase (PI3K)/AKT/glycogen synthase kinase (GSK)-3β/snail family transcriptional repressor (Snail)/phosphatase and tensin homolog feedback loop. (a) FAK, Src, extracellular signal-regulated kinase 1 and 2 (ERK1/2), PI3K, AKT and p38 and their phosphorylation were assayed in modified CCLP1 and HCCC9810 and their control cells. (b) CCLP1-Con and CCLP1-TSPAN1 cells were treated with laminin 5 (5 μg/ml) for 0, 15, 30, 60 min, and time-response experiment showed significant difference in p-AKT. (c) Phosphorylation of GSK-3β and Snail were examined in indicated cells. (d) Western blot analysis demonstrated that the PI3K inhibitor LY294002 effectively decreased expression levels of p-AKT, p-GSK-3β, and Snail induced by TSPAN1. (e) Stable CCLP1-TSPAN1 cells were treated with LY294002 and subjected to migration and invasion assays (100× magnification). (f) In vivo lung metastatic assay. Nude mice were injected with eight luciferase-expressing cell lines (CCLP1-TSPAN1 and CCLP1-TSPAN1 plus LV-shSnail, HCCC9810-shTSPAN1 and HCCC9810-shTSPAN1 plus LV-Snail, and their respective control cells) via the tail vein. Tumors were monitored using bioluminescence imaging. (g) Histological analyses of lung metastatic tumors by hematoxylin and eosin (H&E) staining. Images showing representative H&E staining of lung tissue samples from the different experimental groups. (h) Western blot analyses showed PTEN expression altered inversely with Snail change in CCLP1 and HCCC9810 cells. Knockdown of Snail expression using LV-shSnail in CCLP1-TSPAN1 cells significantly enhanced PTEN expression and attenuated the AKT/GSK-3β pathway induced by TSPAN1. In contrast, upregulation of Snail using LV-Snail markedly decreased PTEN and enhanced the pathway in HCCC9810-shTSPAN1 cells. (i) Schematic presentation of mechanism underlying TSPAN1-mediated CCA metastasis. Experiments were performed three times and data are means ± SD