Abstract
Global DNA hypomethylation is a characteristic feature of cancer cells that closely associates with chromosomal instability (CIN). However, the association between these characteristics during hepatocarcinogenesis remains unclear. Herein, we determined the relationship between hypomethylation and CIN in human hepatocellular carcinoma (HCC) by analyzing 179 HCCs, 178 matched non-tumor livers and 23 normal liver tissues. Hypomethylation at three different repetitive DNA (rDNA) sequences and hypermethylation of 12 CpG loci, including 11 tumor suppressor gene (TSG) promoters, were quantified using MethyLight or combined bisulfite restriction analysis. Fractional allelic loss (FAL) was used as a marker for CIN, calculated by analyzing 400 microsatellite markers. Gains and losses at each chromosome were also determined using semi-quantitative microsatellite analysis. The associations between rDNA hypomethylation and FAL, as well as between TSG hypermethylation and FAL were investigated. Significantly more hypomethylation was observed in HCC tissues than in normal liver samples. Progression of hypomethylation during carcinogenesis was more prominent in hepatitis C virus (HCV)-negative cases, which was in contrast to our previous reports of significantly increased TSG methylation levels in HCV-positive tumors. Absence of liver cirrhosis and higher FAL scores were identified as independent contributors to significant hypomethylation of rDNA in HCC. Among the chromosomal alterations frequently observed in HCC, loss of 8p, which was unique in the earliest stages of hepatocarcinogenesis, was significantly associated with hypomethylation of rDNA by multivariable analysis (p = 0.0153). rDNA hypomethylation was also associated with a high FAL score regardless of tumor differentiation (p = 0.0011, well-differentiated; p = 0.0089, moderately/poorly-differentiated HCCs). We conclude that DNA hypomethylation is an important cause of CIN in the earliest step of HCC, especially in a background of non-cirrhotic liver.
Highlights
Several reports suggest that promoter hypermethylation accounts for inactivation of the corresponding tumor suppressor genes (TSGs) [1]
hepatocellular carcinoma (HCC) tumors at all stages of development had less repetitive DNA (rDNA) methylation compared to normal liver and non-cancerous liver tissue at all three sequences, suggesting that hypomethylation is specific to carcinogenesis (Table 2 and Fig. S1)
Less methylation was observed in non-cancerous liver tissues compared to normal liver tissues (p = 0.0076, post-hoc Turkey-Kramer honestly significant difference (HSD) multiple comparison), and methylation levels in HCC tissues were significantly decreased compared to normal liver samples even at the earliest stages of tumor development (p,0.0001: Fig. 1A)
Summary
Several reports suggest that promoter hypermethylation accounts for inactivation of the corresponding tumor suppressor genes (TSGs) [1]. Global DNA hypomethylation commonly found in cancer is thought to induce activation of potential oncogenes as well as chromosomal alterations, thereby contributing to carcinogenesis [2,3]. A significant link between global DNA hypomethylation and chromosomal aberrations has been reported in several cancers, implying that global hypomethylation may play an important role in inducing chromosomal instability (CIN) [4,5,6]. We reported that inactivation of TSGs by regional hypermethylation in their promoters is a major mechanism driving human hepatocarcinogenesis, especially in hepatitis C virus (HCV)-related cases [9]. Our goal was to determine whether DNA hypomethylation is linked to CIN and influenced by background
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