The Histone Methyltransferase Gene G9A Is Regulated by Nuclear Receptor 4A1 in Alveolar Rhabdomyosarcoma Cells.

The histone methyltransferase G9A (EHMT2) gene catalyzes methylation of histone 3 lysine 9 (H3K9), and this gene silencing activity contributes to the tumor promoter-like activity of G9A in several tumor types including alveolar rhabdomyosarcoma (ARMS). Previous studies show the orphan nuclear receptor 4A1 (NR4A1, Nur77) is overexpressed in rhabdomyosarcoma and exhibits pro-oncogenic activity. In this study, we show that knockdown of NR4A1 in ARMS cells decreased expression of G9A mRNA and protein. Moreover, treatment of ARMS cells with several bis-indole-derived NR4A1 ligands (antagonists) including 1,1-bis(3'-indolyl)-1-(4-hydroxyphenyl)methane (CDIM8), 3,5-dimethyl (3,5-(CH3)2), and 3-bromo-5-methoxy (3-Br-5-OCH3) analogs also decreased G9A expression. Furthermore, NR4A1 antagonists also decreased G9A expression in breast, lung, liver, and endometrial cancer cells confirming that G9A is an NR4A1-regulated gene in ARMS and other cancer cell lines. Mechanistic studies showed that the NR4A1/Sp1 complex interacted with the GC-rich 511 region of the G9A promoter to regulate G9A gene expression. Moreover, knockdown of NR4A1 or treatment with NR4A1 receptor antagonists decreased overall H3K9me2, H3K9me2 associated with the PTEN promoter, and PTEN-regulated phospho-Akt. In vivo studies showed that the NR4A1 antagonist (3-Br-5-OCH3) inhibited tumor growth in athymic nude mice bearing Rh30 ARMS cells and confirmed that G9A was an NR4A1-regulated gene that can be targeted by NR4A1 receptor antagonists.