Overexpression of the histone methyltransferase enhancer of zeste homolog 2 was involved in multistep hepatocarcinogenesis and metastasis of hepatocellular carcinoma

Abstract

Background: Post-translational modifications on histone N-terminal tails are tightly associated with gene transcription regulation. Accumulating evidence has suggested that aberrant histone modifications are implicated in malignancy development. However, the roles and mechanisms of aberrant histone modifications in the development of hepatocellular carcinoma (HCC) are still warranted. In a pilot study, we observed that Enhancer of zeste homolog 2 (EZH2), a histone methyltransferase specific for H3K27, was frequently deregulated in human HCC. The purpose of this present study is to investigate the role of EZH2 in hepatocarcinogenesis. Experimental Design: The expression of EZH2 mRNA and protein in primary HCC and corresponding non-tumorous livers were determined by real-time quantitative RT-PCR and immunohistochemistry, respectively. Clinicopathological analysis was also done to correlate EZH2 overexpression with clinicopathological parameters of HCC patients. Ectopic overexpression and RNA interference knockdown approaches were employed to study the functional implication of EZH2 in HCC cell lines. Results: Real-time quantitative RT-PCR was performed in 59 pairs of primary HCC samples and overexpression of EZH2 mRNA was found in 70% of primary HCC. Immunohistochemistry study of tissue microarrays consisted of 108 pairs of primary HCC also revealed that both number of positive cells and the intensity of EZH2 nuclear staining were significantly higher in primary HCC than their corresponding non-tumorous livers. Ectopic overexpression of EZH2 significantly increased the H3K27 trimethylation level in HCC cell lines. We also found that the expression level of EZH2 exhibited a stepwise increase along the multistep hepatocarcinogenesis, suggesting that overexpression of EZH2 may contribute to HCC progression. Furthermore, overexpression of EZH2 in HCC was significantly associated with aggressive tumor behaviors in HCC patients including the presence of venous invasion (p=0.043) and liver invasion (p=0.014), and the absence of tumor encapsulation (p=0.043). Consistently, knockdown of EZH2 by small interfering RNA suppressed migration of HCC cell lines, suggesting that upregulated EZH2 may contribute to the HCC metastasis. Conclusions: Our findings indicated that EZH2 was frequently upregulated in human HCC and was associated with HCC progression as well as the metastatic phenotypes.