Decreased ASPP2 Expression Promotes Tumor Growth And Metastasis In HCC

The Ankyrin-Repeat-Containing, SH3-Domain-Containing, and Proline-Rich-Region-Containing Protein (ASPP) family of proteins regulates apoptosis through interaction with p53 and its family members. We have previously reported that ASPP2 was frequently down-regulated by DNA methylation in HCC cell lines. This study analysis the molecular mechanism of ASPP2's epigenetic regulation in HBV-positive hepatocellular carcinoma (HCC) and explores the effects of down-regulation of ASPP1 and ASPP2 on the development of HCC. Also we investigate the role of ASPP2 in epithelial-mesenchymal transition (EMT) and metastasis of HCC.The expression of ASPP1 and ASPP2 was analysed in 51 HCC patients and compared with the clinicopathological features of the patients. ASPP1 and ASPP2 expression levels were examined by real-time RT-PCR and Westernblotting analyses. The methylation status of ASPP1 and ASPP2 was examined by methyl-specific polymerase chain reaction and bisulfite sequencing. ChIP analysis was performed to analyze the binding of transcriptional regulators on ASPP1 and ASPP2 promoters. We applied RNA interference mediated gene silence to inhibit the expression of ASPP1 and ASPP2 in HCC cells. Tumorigenicity potentials of HCC cells were studied in vitro and in nude mice. Apoptosis were examined by TUNEL assay and Annexin V-FITC kit. Wound-healing and Matrigel invasion assays were used to determine migration and invasion potentials of HCC cells. RT-PCR,Westernblotting and Immunofluorscence were used to analysis EMT. Co-IP were used to verify the binding of ASPP2 and Csk.The expression of ASPP1 and ASPP2 was analysed in 51 HCC patients and compared with the clinicopathological features of the patients. The expression of ASPP2 was correlated with metastasis(P=0.010) and AJCC stages (P=0.036) in HCC patients..ASPP2 became methylated upon HBV X protein (HBx) expression. The expression of HBx enhanced the recruitment of DNMT1 and DNMT3A on ASPP2 promoter to initiate DNA methylation, and subsequent increased the binding of methyl-CpG binding proteins on its promoter to suppress ASPP2 expression. Down-regulation of ASPP1 and ASPP2 promoted the growth of HCC cells in soft agar and in nude mice, and decreased the sensitivity of HCC cells to apoptotic stimuli. Down-regulation of ASPP2 by short hairpin RNA made cells undergoing morphological changes from a classic epithelial morphology to a spindle-like shape. Down-regulation of ASPP2 also enhanced cell migration and invasion through Matrigel in vitro, promoted lung metastasis in HCC xenografts in nude mice. Co-IP showed ASPP2 binding with Csk Further studies revealed that down-regulation of ASPP2 activated the non-receptor tyrosine kinase c-Src and then promoted P-catenin translocating from cytomembrane to cytoplasm and nucleus.Our results characterized the epigenetic regulation of ASPPl and ASPP2 genes by HBx, as well as down-regulation of ASPP2 promotes EMT and metastatic progression of HCC. These findings provide new insight into the molecular mechanisms leading to matastasis of HCC and may have potential therapeutic applications.