RORα Binds to E2F1 To Inhibit Cell Proliferation and Regulate Mammary Gland Branching Morphogenesis

Abstract

Retinoic acid receptor-related orphan nuclear receptor alpha (ROR␣) is a potent tumor suppressor that reduces cell proliferation and inhibits tumor growth. However, the molecular mechanism by which it inhibits cell proliferation remains unknown. We demonstrate a noncanonical nuclear receptor pathway in which ROR␣ binds to E2F1 to inhibit cell cycle progression. We showed that ROR␣ bound to the heptad repeat and marked box region of E2F1 and suppressed E2F1-regulated transcription in epithelial cells. Binding of ROR␣ inhibited E2F1 acetylation and its DNA-binding activity by recruiting histone deacetylase 1 (HDAC1) to the protein complexes. Knockdown of HDAC1 or inhibition of HDAC activity at least partially rescued transcription factor activity of E2F1 that was repressed by ROR␣. Importantly, ROR␣ levels were increased in mammary ducts compared to terminal end buds and inversely correlated with expression of E2F1 target genes and cell proliferation. Silencing ROR␣ in mammary epithelial cells significantly enhanced cell proliferation in the ductal epithelial cells and promoted side branching of the mammary ducts. These results reveal a novel link between ROR␣ and E2F1 in regulating cell cycle progression and mammary tissue morphogenesis. N uclear receptors, a family of ligand-dependent transcription factors, regulate gene expression by directly binding to the cis response elements in the regulatory regions (1, 2). Retinoic acid receptor-related orphan nuclear receptor alpha (ROR␣) is an orphan nuclear receptor and plays critical roles in many physiological processes, including cell differentiation, metabolism, inflammation, transformation, and circadian rhythm (3-11). The canonical way that ROR␣ regulates gene expression is through ROR response elements in the regulatory regions of the target genes (12, 13). ROR␣ recruits a number of transcriptional coactivators and corepressors to the target genes, including steroid receptor coactivator 1 (14) and p300 (15), nuclear receptor corepressor, and silencing mediator for retinoic acid and thyroid hormone receptors (16). These transcriptional cofactors modulate the chromatin structure to induce or repress transcription. Unlike the canonical pathway of nuclear receptor to regulate gene expression, a number of recent studies show that ROR␣ acts as a transcriptional cofactor, binding to ␤-catenin or p53 to regulate target gene expression or modulate protein stability (7,17).Spatial and temporal regulation of cell proliferation is crucial for normal tissue development and maintenance of the differentiated state (18). For instance, mammary epithelial cells in the mature ducts remain growth arrested, while cells in terminal end buds are highly proliferative (19,20). The different cell proliferation status in terminal end buds and ducts is important for mammary gland branching morphogenesis and maintenance of the ductal structure (21, 22). Enhancing cell proliferation in mammary epithelial cells in the long run is sufficient to induce tumorigenesis (23, 24). We and others have...

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“…286–290 These antitumor effects of RORs have contributed to decreased cell proliferation as well as inhibition of the EMT. 286,289,291 The inverse correlation between RORα/γ expression and tumorigenesis suggests that agonists might inhibit tumor growth and progression and provide a promising new strategy for anticancer therapy.…”

Lipid-sensors, enigmatic-orphan and orphan nuclear receptors as therapeutic targets in breast-cancer

“…NR1F1 may stimulate ER + /cell-growth via ERα binding/activation or aromatase induction [63]. Besides inhibiting growth, NR1F1 exerts anti-invasive actions in ER − /cells and tumors [64–66]. In ER − /cells and xenografts, transcriptional Semaphorin-3F induction via a ROR-Response-Element contributes to NR1F1 anti-invasive and growth-inhibitory activities [64].…”

“…In ER − /cells and xenografts, transcriptional Semaphorin-3F induction via a ROR-Response-Element contributes to NR1F1 anti-invasive and growth-inhibitory activities [64]. In addition, NR1F1 binds E2F1-transcription-factor , inhibits E2F1 -acetylation and transcriptional activity, reducing ductal epithelial-cell proliferation [66]. No studies on NR1F2 in breast-cancer are available and the number of publications involving NR1F3 is limited [64, 67, 68].…”

Niacin Stimulates Mammary Gland Development in Pubertal Mice through Activation of the AKT/mTOR and ERK1/2 Signaling Pathways

“…Previous studies have shown that cyclin D promotes Rb phosphorylation by binding to cyclin-dependent kinases. Phosphorylation of Rb promotes Rb-E2F1 complex separation and E2F1 target gene transcription, which promotes cell cycle progression and cell proliferation[22, 23]. To test whether niacin increases the release of Rb-bound E2F1 by enhancing the phosphorylation of Rb, an IP assay was carried out.…”