Abstract 4865: Inhibition of histone methyltransferase G9a as a promising new target for the treatment of liver cancer

Histone methyltransferase G9a is a key enzyme for histone H3 dimethylation at lysine-9 (H3K9me2), an epigenetic mark of gene suppression. Knockdown of G9a markedly inhibits the growth of prostate cancer cells. G9a is highly expressed in human liver cancer cells; however, little is known about its role in the pathology of liver cancer. The purpose of the present study was to elucidate the mechanistic role of G9a in liver carcinogenesis, using the specific G9a inhibitor BIX-01294 (IC 50 2 µM). We found that G9a protein was expressed much higher in mouse hepatoma Hepa-1 cells than in mouse liver. Treatment of Hepa-1 cells with BIX-01294 (4 µM) for 24 h and 72 h decreased the number of live cells (MTT assay) by 50 and >90%, respectively. After 24 h exposure of Hepa-1 cells to BIX-01294, there was an 8-fold increase of caspase-3/7 activity, indicating that the anticancer effect of G9a inhibition is due to induction of apoptosis. Also at 24 h, BIX-01294 abolished mRNA expression of Cyclin D1, but induced mRNA expression of C/EBP homolog protein (CHOP), a transcription factor that promotes apoptosis and inhibits the proliferation of hepatoma cells. Silencing of the tumor-suppressor genes PTEN and p21 in cancer cells has been associated with H3K9me2 of their gene promoters. We found that G9a inhibition of Hepa-1 cells resulted in a 2-fold induction of PTEN mRNA and a marked induction of p21 mRNA, without inducing p53. In contrast, G9a inhibition markedly induced C/EBPβ, a transactivator of p21 and CHOP. BIX-01294 also induced C/EBPβ (8.8 fold), p21 (11 fold), and CHOP (22 fold) in p53-negative Hep3B cells. In HepG2 cells, BIX-01294 also induced p21 mRNA and decreased H3K9me2 of the p21 promoter (ChIP-qPCR analysis). Moreover, exposure of proliferating HepG2, Huh7, and Hep3B cells to BIX-01294 (4 µM) for 72 h resulted in >90% decrease in the numbers of live cells. In contrast, treatment with 8 µM of BIX-01294 for 96 h had no effect on the survival of a differentiated human hepatoma HepaRG cell line. By crossing G9a floxed mice with Alb-cre mice, we have generated mice with adult-hepatocyte-specific knockout of G9a, which appear normal and have normal livers. In conclusion, the present data demonstrate that the G9a specific inhibitor BIX-01294 markedly induces p21 and CHOP in a p53-independent mechanism, probably due to a combined effect of desuppression of C/EBPβ (a transactivator for p21 and CHOP) and/or removal of the suppressive mark H3K9me2 from the p21 promoter. G9a inhibition has potent anticancer effects in both mouse and human hepatoma cells, but has little cytotoxicity in fully differentiated human hepatoma cells, and loss of G9a in mouse liver is well-tolerated under unstressed conditions. Therefore, G9a inhibitors may be a promising new class of epigenetic drugs for the treatment of liver cancer. (Supported by NIH grants RR021940, ES013714, and a KU cancer center grant) Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4865.