Regulation of EpCAM-positive cancer cells by HIF1α, snail and twist in hypoxic microenvironment of hepatocellular carcinoma

Abstract

The presence of cancer stem cell (CSC) has been reported in hepatocellular carcinoma (HCC), however the regulation mechanism of “stemness” in HCC remains unclear. Under hypoxia, which is common microenvironment of human HCC, the expression of EpCAM (CSC marker), Snail, Twist (EMT related genes) and HIF1α increased in PLC/RPF/5 and SNU423 cell lines. We further studied the effect of Snail, Twist and HIF1α on “stemness” in HCC cell lines and human HCC tissues. EpCAM positive HCC cells showed higher expression of HIF1α, Oct3/4 and Nanog and higher levels of cell proliferation, tumorsphere formation, and invasion compared to EpCAM negative cells under hypoxia. Overexpression of Snail and Twist in HCC cells increased EpCAM positive cells population, in contrast, knockdown of Snail and Twist in HCC cells decreased EpCAM positive cells population. EpCAM-positive HCC cells and promoted in vitro invasion. HIF1α induced PLC/PRF/5 and SNU423 cells exhibited molecular alterations consistent with EMT, characterized by the loss of EMT regulators (Snail and Twist). Co-treatment of HIF1α inhibitor with doxorubicin, cisplatin or sorafenib almost inhibited the chemoresistance effect of EpCAM+ cells. The clinical significance of EpCAM and HIF1α was evaluated using 100 cases of human HCCs. HCCs with high expression of HIF1α showed higher expression of CSC markers (EpCAM, Oct3/4, Nanog) and EMT markers (Snail and Twist). HCCs with HIF1α or EpCAM mRNA high expression showed more frequent microvascular invasion, portal vein invasion, intrahepatic metastasis, and multiple tumor than those with HIF1α or EpCAM mRNA low expression (P<0.001). High expression of HIF1α or EpCAM was significantly correlated with shorter disease free and overall survival of HCC patients (P<0.05). In conclusion, under hypoxia HIF1α high expression HCCs showed significantly increased EpCAM mRNA and

protein expression. EpCAM-positive cells in HCC are induced by HIF1α and contributes to HIF1α-induced invasive potential.