Histone lysine methyltransferase G9a is a novel epigenetic target for the treatment of hepatocellular carcinoma.

Masayuki Yokoyama,Motohiko Oshima,Naoya Mimura,Atsunori Saraya,Yuko Kusakabe,Akinobu Tawada,Masaru Miyazaki,Kazumasa Aoyama,Yoshiko Noguchi,Osamu Yokosuka,Kaori Yuki,Toru Wakamatsu,Tetsuhiro Chiba,Satoru Miyagi,Shiro Tara,Shuhei Koide,Yoh Zen,Masayuki Otsuka,Eiichiro Suzuki,Atsushi Iwama,Tomoko Saito,Yoshihiko Ooka,Masatoshi Inoue ,Satoshi Ogasawara

doi:10.18632/oncotarget.15528

Masayuki Yokoyama, Motohiko Oshima + Show 22 more

Open Access

https://doi.org/10.18632/oncotarget.15528

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Journal: Oncotarget	Publication Date: Feb 20, 2017
Citations: 38	License type: cc-by

Affiliation: Chiba University, Kobe University

Abstract

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.

Highlights

Epigenetics is defined as a mechanism that regulates gene expression without changes in the underlying DNA sequence [1]
These results suggest that G9a and H3K9me2 levels are closely associated in hepatocellular carcinoma (HCC) cells
We achieved the stable knockdown of G9a in Huh1 and Huh7 cells using lentivirus-mediated short hairpin RNA (shRNA) directed against G9a by enhanced green fluorescent protein (EGFP)positive cell sorting

Summary

Introduction

Epigenetics is defined as a mechanism that regulates gene expression without changes in the underlying DNA sequence [1]. Epigenetic regulation of chromatin structure is achieved through various approaches, including histone modifications, DNA methylation, small and non-coding. RNAs, and chromatin remodeling [2] Among these epigenetic changes, methylation of histone H3 at lysine 4 (H3K4) and histone H3 at lysine 36 (H3K36) is associated with transcriptional activation, while methylation of histone H3 at lysine 9 (H3K9) and histone H3 at lysine 27 (H3K27) is involved in transcriptional repression [3]. SUV39H1, ESET, G9a and GLP function as H3K9-specific HKMTs and the methylation of H3K9 has been shown to play an important role in both gene repression and heterochromatin formation [5]. G9a is a member of the SUV39H group and is responsible for H3K9 mono- and dimethylation (H3K9me2) [6]

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