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

Abstract

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.

Flavonoids Kaempferol and Quercetin are Nuclear Receptor 4A1 (NR4A1, Nur77) Ligands and inhibit Rhabdomyosarcoma Cell and Tumor Growth

“…A recent study reported that the avonoid kaempferol decreased G9a expression in gastric cancer cells (30) and this was accompanied by growth inhibition, induction of markers of apoptosis and inhibition of mTOR signaling by induced phosphorylation of AMPK. This pattern of responses observed for kaempferol in gastric cancer cells has previously been observed for CDIM/NR4A1 antagonists or NR4A1 silencing in RMS and other cancer cell lines (21)(22)(23)(24)(25)(26)(27). Therefore, we hypothesized that kaempferol is an NR4A1 ligand and this study shows for the rst time that both kaempferol and quercetin bind NR4A1 and act as NR4A1 antagonists in RMS cells.…”

“…2F) cells. NR4A1 knockdown or treatment with NR4A1 antagonists also inhibits RMS cell migration and invasion (21,25) (Supplemental Figures) and kaempferol and quercetin inhibit migration of Rh30 (Fig. 3A) and Rh41 (Fig.…”

“…Rh30 and Rh41 cell lines were treated with the desired concentrations of avonoids for 24 and 48 hours respectively. Knockdown studies by RNA interference (siNR4A1) were carried out essentially as described (21,22).…”

“…Previous studies show that NR4A1 regulates RMS cell growth, survival and invasion, and related genes including the PAX3-FOX01 fusion oncogene and G9a (21,25) and we therefore further investigated kaempferol and quercetin as inhibitors of these NR4A1-dependent pathways/genes. Treatment of Rh30 cells with 10-100 µM kaempferol and quercetin decreased growth (Fig.…”

“…and other cancer cell lines and the anticancer activities of bis-indole derived (CDIMs) which are NR4A1 ligands that act as receptor antagonists (21)(22)(23)(24)(25)(26)(27). The fusion oncogene PAX3-FOX01 and G9a have been characterized as highly pro-oncogenic factors in RMS (28, 29) and NR4A1 regulated expression of both genes and also β1-integrin and treatment of RMS cells with CDIM/NR4A1 antagonists decreased expression of these genes (21,25). A recent study reported that the avonoid kaempferol decreased G9a expression in gastric cancer cells (30) and this was accompanied by growth inhibition, induction of markers of apoptosis and inhibition of mTOR signaling by induced phosphorylation of AMPK.…”