Histone lysine methyltransferase G9a is a novel epigenetic target for the treatment of hepatocellular carcinoma
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Masayuki Yokoyama1,*, Tetsuhiro Chiba1,*, Yoh Zen2, Motohiko Oshima3, Yuko Kusakabe1, Yoshiko Noguchi1, Kaori Yuki1,3, Shuhei Koide3, Shiro Tara3, Atsunori Saraya3, Kazumasa Aoyama3, Naoya Mimura3, Satoru Miyagi3, Masanori Inoue1, Toru Wakamatsu1, Tomoko Saito1, Sadahisa Ogasawara1, Eiichiro Suzuki1, Yoshihiko Ooka1, Akinobu Tawada1, Masayuki Otsuka4, Masaru Miyazaki4, Osamu Yokosuka1, Atsushi Iwama3
1Department of Gastroenterology and Nephrology, Graduate School of Medicine, Chiba University, Chiba, Japan
2Department of Diagnostic Pathology, Kobe University Graduate School of Medicine, Kobe, Japan
3Department of Cellular and Molecular Medicine, Graduate School of Medicine, Chiba University, Chiba, Japan
4Department of General Surgery, Graduate School of Medicine, Chiba University, Chiba, Japan
*These authors contributed equally to this work
Tetsuhiro Chiba, email: [email protected]
Keywords: epigenetics, hepatocellular carcinoma, G9a, BIX-01294, epithelial mesenchymal transition
Received: May 22, 2016 Accepted: January 27, 2017 Published: February 20, 2017
Histone H3 lysine 9 dimethylation (H3K9me2) is mainly regulated by the histone lysine methyltransferase G9a and is associated with the repression of transcription. However, both the role of G9a and the significance of H3K9me2 in hepatocellular carcinoma (HCC) cells remain unclear. In this study, we conducted loss-of-function assay of G9a using short-hairpin RNA and pharmacological interference. Knockdown of G9a reduced H3K9me2 levels and impaired both HCC cell growth and sphere formation. However, transforming growth factor β1-induced epithelial mesenchymal transition (EMT) was not suppressed by G9a knockdown. Combined analyses of chromatin immunoprecipitation followed by sequencing and RNA-sequencing led to successful identification of 96 candidate epigenetic targets of G9a. Pharmacological inhibition of G9a by BIX-01294 resulted in both cell growth inhibition and induction of apoptosis in HCC cells. Intraperitoneal administration of BIX-01294 suppressed the growth of xenograft tumors generated by implantation of HCC cells in non-obese diabetic/severe combined immunodeficient mice. Immunohistochemical analyses revealed high levels of G9a and H3K9me2 in 36 (66.7%) and 35 (64.8%) primary HCC tissues, respectively. G9a expression levels were significantly positively correlated with H3K9me2 levels in tumor tissues. In contrast, in non-tumor tissues, G9a and H3K9me2 were only observed in biliary epithelial cells and periportal hepatocytes. In conclusion, G9a inhibition impairs anchorage-dependent and -independent cell growth, but not EMT in HCC cells. Our data indicate that pharmacological interference of G9a might be a novel epigenetic approach for the treatment of HCC.
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