Transcriptional silencing of the Secreted Frizzled-Related Protein gene family via promoter hypermethylation in human hepatocellular carcinoma

• Main Authors: Shih Yu-Lueng, 施宇隆
• Other Authors: Lin Ya-Wen
• Format: Others
• Language: zh-TW
• Published: 2007
• Online Access: http://ndltd.ncl.edu.tw/handle/59576306742524399923

博士 === 國防醫學院 === 醫學科學研究所 === 95 === Hepatocellular carcinoma (HCC) is the most frequent primary malignancy of the liver and accounts for as many as 1 million deaths annually worldwide. The Wnt/β-catenin signaling pathway, initially discovered by genetic analysis in the wing development of Drosophila, has been implicated in colon carcinogenesis. Recently, the focus was placed on the link between aberrant Wnt pathway activation and human cancer, including hepatocellular carcinoma (HCC). However, this signaling pathway can be modulated by a group of soluble proteins, the secreted frizzled-related proteins (SFRPs). The SFRPs encode Wnt/β-catenin signaling antagonists and are frequently inactivated by promoter methylation in many tumors. However, the role of SFRPs in hepatocellular carcinoma (HCC) is not clear. Therefore, we investigated whether methylation of the SFRP1 promoter is common in HCC and whether it may influence SFRP1 expression. Epigenetic inhibition of SFRP1 transcription was investigated, and increased methylation of the promoter region was demonstrated in eleven HCC cell lines, 54 HCCs, 42 cirrhosis livers, 21 chronic hepatitis livers and 15 normal controls. SFRP1 promoter methylation was observed in 81.2%, 48.2%, 21.4%, 14.3% and 0% in HCC cell lines, primary HCCs, cirrhosis livers, chronic hepatitis livers and normal controls, respectively. Methylation of SFRP1 promoter region in HCCs increased significantly compared with control tissues. All samples with SFRP1 methylation showed downregulation of SFRP1 expression. Demethylation treatment with 5-aza-2´-deoxycytidine in HCC cells restored the SFRP1 expression. Moreover, LOH of markers D8S505 and D8S1722 was found to be 25% and 27.6% of the informative cases, respectively. Our data suggest that promoter hypermethylation of SFRP1 is a common event in HCCs, and play an important role in regulation of SFRP1 expression. In addition to methylation-mediated downregulation of SFRP1, LOH may also play a role. To confirm and extend these findings, the methylation status of the other SFRP members, including SFRP2, SFRP4 and SFRP5, was examined by methylation-specific polymerase chain reaction (MS-PCR). Hypermethylation of SFRP genes, except for SFRP4, is frequent in HCCs and the levels found here were significantly higher than those seen in cirrhotic livers, chronic hepatitis livers and normal controls (P < 0.0001 for SFRP1 and SFRP2, P < 0.05 for SFRP5). To elucidate the function of SFRPs in hepatoma cells, we used reexpression of SFRP1 in β-catenin-dependent HCC cell lines: Huh6 and HepG2. Restoration of SFRP1 attenuated Wnt signaling in those Huh6 hepatoma cells with a β-catenin gene point mutation, decreased abnormal accumulation of β-catenin in the nucleus and suppressed cell growth. Conversely, restoration of SFRP1 in HepG2 hepatoma cells with truncated β-catenin could not block the Wnt signaling pathway. Furthermore, knocking down SFRP1 by RNA interference in β-catenin-deficient cell lines (SK-Hep1) stimulated Wnt signaling and promoted cell growth. Our data suggested that SFRP1 suppressed liver cancer cells growth through Wnt canonical signaling. Moreover, β-catenin-independent noncanonical pathway might be involved in Wnt signaling activation through unknown molecules in HCC.