Integrin-linked kinase promotes hepatocellular carcinoma oncogenesis

Abstract

BACKGROUND: Integrin-linked kinase (ILK) was first discovered as an integrin binding protein. It localizes to focal adhesions and facilitates actin polymerization. Accumulating evidences suggest that ILK is a putative oncogene. ILK was over-expressed in various malignancies and its aberrant activation influenced a wide range of cellular functions. In this study, we aimed to elucidate the role of ILK in hepatocarcinogenesis and its clinical significance by assessing ILK expression in human hepatocellular carcinoma (HCC) tissues and functionally characterizing ILK in HCC cell models. MATERIAL AND METHODS: Expression level of ILK in HCC cell lines was examined by Western blotting, while ILK expression in clinical samples was determined by quantitative PCR. ILK knock-down stable clones were established in BEL7402 and HLE using a lentiviral-based short-hairpin knockdown approach. For each cell line, two stable ILK knock-down (shILK) clones and one stable non-target control (shCTL) were established. To functionally characterize ILK in HCC, the knock-down stable clones were subjected to various functional assays including cell proliferation assay, soft agar colony formation assay, cell migration assay, wound-healing assay and cell invasion assay. In vivo tumourigenicity of BEL7402 ILK knock-down stable clones was assessed by subcutaneous injection of the cells into nude mice. RESULTS: Western blotting revealed a higher ILK protein expression in HCC cell lines than in normal liver cell line. In the physiological context, qPCR analysis showed that ILK was over-expressed in 36.9% (21/57) of HCC tissues when compared to the corresponding non-tumourous livers. The overall ILK expression level was significantly higher in tumourous tissues (P = 0.005), with a stepwise increase of expression along tumour stage. Functional characterization of ILK in HCC using the two ILK stable knockdown cell lines showed a reduction in the rate of cell proliferation, migration, invasion and anchorage-independent growth. Knock-down of ILK in BEL7402 also suppressed tumour formation in nude mice, thus decreasing the in vivo tumourigenicity of HCC cells. To probe the underlying mechanism, AKT activity was evaluated in the shILK clones. Western blotting analysis showed a decrease in phospho-AKT(Ser473) level upon ILK silencing. CONCLUSION: Our study suggests that ILK plays a role in the progression of HCC via the activation of the PKB/AKT pathway.