Mechanism Of Anoikis Resistance Induced By AEG-1in Hepatocellular Carcinoma Cells And Effects Of Autophagy In This Process

Aims: Liver cancer is the most5common cancer in china, and the metastasis and rapid growth of hepatocellular carcinoma are the main cause of recurrence and poor prognosis. The invasion and metastasis of tumor cells is a complex and continuous process including multiple steps. After invading into the vessel lomen, most cells occur anoikis due to losing of connection between cells and extracellular matrix. Only a few cells which own the ability of anoikis resistance survived and finally formed distant metastasis. Thus, anoikis resistance has been considered as a prerequisite of tumor metastasis. This project is to investigate that AEG-1could promote anoikis resistance by activating PI3K/Akt signaling in hepatocellular carcinoma cells and effects of autophagy on anoikis resistance enhanced by AEG-1.Methods:Thirty-six pairs of liver cancer and their adjacent normal liver tissues were obtained from the patients with histologically confirmed HCC after informed consent was given. The mRNA and protein levels of AEG-1expression in liver cancer and their adjacent normal liver tissues and four liver cancer cell lines (including SMMC-7721, HepG2, MHCC-97H, and HCC-LM3) were examined by Real-time PCR and Western blot, and the relationship between AEG-1expression and metastasis of liver cancer was assayed. The pcDNA3.1-AEG-1plasmid which contains the AEG-1CDS sequence was transfected into SMMC-7721cells to obtain stable cell line with AEG-1over-expression; and psilencer2.0-AEG-1-shRNAs against AEG-1was transfected into HCC-LM3cells to obtain stable cell line with AEG-1knockdown. Cell cycle, proliferation, colony formation and apoptosis were detected by flow cytometry assay, MTT assay, soft agar colony formation assay. The anoikis ratio of AEG-1-overexpressing and-knockdown cells was examied by flow cytometry. Activated caspase-3was detected by Western blot to confirm the occurrence of anoikis. The activation of PI3K/Akt and ERK1/2pathways was confirmed by protein detection of key molecule by Western blot. LY294002was used to inhibit the activation of PI3K/Akt signaling and ERK1/2signaling was block by U0126. Bcl-2and Bad, downstream of PI3K/Akt, was examined by Western blot. Autophagy level of anoikis-resistant HCC cells was confirmed by detection of LC3I/II transform by Western blot. Atg5siRNA was transfected into cells to down-regulate the autophagy level in AEG-1-overexpressing cells.Results:We detected the expression of AEG-1in thirty-six pairs of human hepatocellular carcinoma and their corresponding para-carcinoma tissues. In twenty-three pairs of samples, the mRNA and protein levels of AEG-1expression were significantly higher (≥2fold) than that in adjacent para-carcinoma tissues. And the expression of AEG-1was closely related to tumor size (p<0.05), differentiation degree (p<0.05), portal invasion (p<0.05) and lymph node metastases (p<0.05). The expression levels of AEG-1mRNA and protein in HCC cells with high metastasis potential (MHCC-97H, HCC-LM3) were higher than that in HCC cells with low and moderate metastasis potential (HepG2, SMMC-7721). Compared with SMMC-7721cells with pcDNA3.1empty vector plasmid transfection, the mRNA and protein levels of AEG-1expression were significantly enhanced in pcDNA3.1-AEG-1plasmid transfected SMMC-7721cells. Compared with HCC-LM3cells with psilencer2.0empty vector plasmid transfection, the mRNA and protein levels of AEG-1expression were significantly reduced in psilencer2.0-AEG-1-shRNAs plasmid transfected HCC-LM3cells. There were no significant differences in cell cycle, cell proliferation (p>0.05), colony formation (p>0.05) and apoptosis (p>0.05) between SMMC-7721-AEG-1cells and SMMC-7721-Vec cells. While, the soft agar colony formation of SMMC-7721-AEG-1cells was enhanced compared with SMMC-7721-vec cells (p<0.05). The cell cycle(p<0.05), proliferation(p<0.05), colony formation(p<0.05), and soft agar colony formation(p<0.05) were significantly inhibited in HCC-LM3-shAEG-1cells compared with HCC-LM3-Vec cells. The apoptosis ratio of AEG-1-knockdown HCC-LM3cells was increased compared to empty plasmid transfected cells (p<0.05). After cells were suspension cultured for24h, the anoikis ratio of SMMC-7721-AEG-1cells was significantly reduced compared with SMMC-7721-Vec cells, and the anoikis ratio of HCC-LM3-shAEG-1cells was remarkably increased compared with HCC-LM3-Vec cells. Also, cleaved-caspase-3expression was down-regulated in SMMC-7721-AEG-1cells and up-regulated in HCC-LM3-shAEG-1cells compared with their corresponding control group. p-ERKl/2, p-p85, p-Akt, Bcl-2, and p-Bad protein expressions were increased in suspension cultured SMMC-7721-AEG-1cells compared with vector control group; and these proteins were reduced in suspension cultured HCC-LM3-shAEG-1cells compared to vector control group. PI3K/Akt inhibitor LY294002could promote the anoikis ratio of SMMC-7721-AEG-1cells and down-regulate the expression of Bc1-2and p-Bad. But ERK1/2inhibitor U0126can not change the anoikis ratio of SMMC-7721-AEG1cells. The autophagy levels of anokis-resistant cells were confirmed by detection of autophagy related LC3Ⅰ/Ⅱ transform. LC3Ⅱ/LC3I was increased in suspension cultured SMMC-7721cells compared with normal cultured cells. After suspension cultured for24h, LC3II/LC3I further increased in SMMC-7721-AEG-1cells compared with SMMC-7721-Vec cells. After SMMC-7721-AEG-1cells were transfected with Atg5siRNA, and suspension cultured for24h, the anoikis ratio was increased compared with non-transfected SMMC-7721-AEG-1cells (p<0.05).Conclusion:AEG-1enhances the ability of anoikis resistance in hepatocellular carcinoma cells via activating PI3K/Akt signaling and up-regulating Bcl-2and phosphorylated Bad, which involved in the process of liver cancer metastasis; up-regulated autophagy level may also mediated anoikis resistance induced by AEG-1of HCC cells.