Promoter methylation of the secreted frizzled‐related protein 1 gene SFRP1 is frequent in hepatocellular carcinoma

Abstract

BACKGROUND. The secreted frizzled‐related protein 1 gene (SFRP1) encodes a Wnt/β‐catenin signaling antagonist and frequently is inactivated by promoter methylation in many tumors. However, the role of SFRP1 in hepatocellular carcinoma (HCC) is not clear. Therefore, the authors investigated whether methylation of the SFRP1 promoter is common in HCC and whether it may influence SFRP1 expression. METHODS. Four HCC cell lines, 54 HCCs, 42 cirrhotic livers, 21 livers with chronic hepatitis, and 15 normal control tissues were analyzed for 1) SFRP1 promoter methylation by using methylation‐specific polymerase chain reaction analysis and bisulfite sequencing, 2) SFRP1 messenger RNA expression by using quantitative reverse transcriptase‐polymerase chain reaction analysis, and 3) loss of heterozygosity (LOH) by using microsatellite markers flanking the SFRP1 locus. HCC cells were treated with the demethylating agent 5‐aza‐2′‐deoxycytidine to determine whether it could restore SFRP1 expression. RESULTS. SFRP1 promoter methylation was observed in 75%, 48.2%, 21.4%, 14.3% and 0% in HCC cell lines, primary HCCs, cirrhotic livers, livers with chronic hepatitis, and normal control tissues, respectively. Methylation of the SFRP1 promoter region in HCCs increased significantly compared with control tissues. All samples with SFRP1 methylation showed down‐regulation of SFRP1 expression. Demethylation treatment with 5‐aza‐2′‐deoxycytidine in HCC cells restored SFRP1 expression. Moreover, LOH of markers D8S505 and D8S1722 was found in 25% and 27.6% of the informative samples, respectively. CONCLUSIONS. The current results suggested that promoter hypermethylation of SFRP1 is a common event in HCC and plays an important role in the regulation of SFRP1 expression. In addition to methylation‐mediated down‐regulation of SFRP1, LOH also may play a role. Cancer 2006. © 2006 American Cancer Society.

Involvement of genetic instability in the downregulation of sFRP1 in Chinese patients with hepatocellular carcinoma

“…The finding is consistent with the previous studies (Shih et al, 2006;Huang et al, 2007) where downregulation of the sFRP1 has been detected at both the mRNA and protein levels in HCC samples. The percentage of sFRP1 positive tumor tissues appears to be correlated with the degree of tumor differentiation, as a higher positive rate of sFRP1 expression was in the highly differentiated HCC cases than in the moderately and poorly differentiated cases.…”

“…It has been reported that reconstitution of sFRP1 inhibits HCC growth in vitro and in vivo, and the sFRP1 restoration offers a potential therapeutic approach for treatment of HCC (Hu et al, 2009;Jiang et al, 2010). Promoter hypermethylation has been documented to be at least partially accounting for the reduction of sFRP1 levels in cancers (Shih et al, 2006;Huang et al, 2007;Shih et al, 2007). Given that the sFRP1 gene is located in a frequent LOH region (8p12-p11.1), in this study, we focused on the influence of LOH and MSI events on the expression of sFRP1.…”

“…The PCR conditions for both loci D8S532 and D8S1722 were similar except the annealing temperature: that is, preincubation at 95 C for 5 min, 35 cycles of denaturation at 94 C for 1 min, annealing for 1 min at 59 C (D8S532) or 55 C (D8S1722), extension at 72 C for 1 min, and a final extension at 72 C for 4 min. As shown in previous reports (Shih et al, 2006;Huang et al, 2007), the primers for amplification of both loci were as follows: 5 0 -GCTCAAAGCCTCCAATGAC-3 0 and 5 0 -GA CTTCGTGATCCACCTGC-3 0 for the D8S532 locus, and 5 0 -CCTTGCTGGGAATTGTG-3 0 and 5 0 -AGCTGCCTGGCT AAGAG-3 0 for the D8S1722 locus. The primers were synthesized by Shanghai Bioengineering, China.…”

“…The Wnt signaling pathway has recently been documented to be associated with cancer development (Moon et al, 2002;Shih et al, 2006Shih et al, , 2007. Aberrant activation of this pathway prevents the phosphorylation and degradation of b-catenin, which translocates into the nucleus and forms a complex with members of the T-cell factor/ lymphocyte enhanced factor family, inducing numerous target genes associated with cell proliferation and survival.…”

“…Decrease in the sFRP1 expression due to promoter hypermethylation may result in the activation of the Wnt pathway and consequent tumor development. Interestingly, reduced expression of sFRP1 has been found in many malignancies, including colorectal (Caldwell et al, 2004;Suzuki et al, 2004), lung (Fukui et al, 2005), ovarian (Takada et al, 2004), breast (Veeck et al, 2006), esophageal (Zou et al, 2005;Clement et al, 2006), and liver (Shih et al, 2006) cancers.…”