AEG1 Promoting Autophagy Inhibitis Anoikis In Hepatocellular Carcinoma

OBJECTIVE: Hepatocellular carcinoma(HCC) is the fifth commonest malignancy worldwide without effective treatments. Its early vascular invasion results poor prognosis.Tumor cells dissociated from the organ of origin, which infiltrate in blood vessels, are known as circulation tumor cells(CTC). Because of CTC lossing contact with other cells or matrix, it would develop a special kind of apoptosis, which is named as anoikis. Different kinds of tumor cells have different ability to resistant anoikis, and the stronger the resistance,the stronger ability of its metastasis. In recent years, AEG1 has been proved to have an important role in invasion and metastasis of HCC as an remarkable oncogene.Simultaneously, autophagy is also found to play a crucial protecting role in cells survival especially while cells suffering from adverse conditions such as anoikis. So it is supposed that AEG1 and autophagy may paly a role in HCC CTC. In this study, we aimed to explore the function of AEG1 and autophagy in HCC anoikis resistance. Furthermore, we studied the relationship between AEG1 and autophagy, and the related signaling pathways.METHODS: Build the HCC anoikis models in vitro by HCC-LM3 with relatively high expression of AEG1 and SMMC-7721 with low expression of AEG1. Over-expression AEG1 in SMMC-7721 and knockdown AEG1 in HCC-LM3 were constructed by using plasmid and small interference RNA. The changes in protein expression, including cleaved-caspase-3, LC3 I/II transform, p-ULK1, ULK1 were detected by western blot.Apoptosis of cell lines with different AEG1 expression was analyzed by flow cytometry.Autophagy was evaluated by electron microscope and GFP-RFP double fluorescence system in above cell lines. Knockdown autophagy genes in SMMC-7721 with over-expression AEG1, cells apoptosis was then analyzed by western blot and flow cytometry. Finally, theactivation of ER stress pathways was detected by western blot in cell lines with different AEG1 expression.RESULTS: SMMC-7721 cells with AEG1 overexpression underwent less cell death compared to the empty vector control cells after suspension culture for 24 h. HCC-LM3 cells with AEG-1 knockdown underwent more apoptosis compared to negative control. The LC3I/LC3 II transform and autophagy flux are increased in SMMC-7721 cells with AEG1 overexpression, which are decreased in HCC-LM3 cells with AEG-1 knockdown. The apoptosis of SMMC-7721 and HCC-LM3 cells in suspension culture for 24 h was significantly higher while ATG knockdown compared with their controls. Finally,endoplasmic reticulum stress response pathway is activated in HCC cells cultured in suspension with AEG-1 overexpression or AEG-1 knockdown.CONCLUSIONS: AEG1 could enhance anoikis resistance to promote metastasis by up-regulating autophagy level in HCC. ER stress response pathway would be involved in this biological process probably.