Cyclin D1 Determines Estrogen Signaling in the Mammary Gland In Vivo

The CCND1 gene, which is frequently overexpressed in cancers, encodes the regulatory subunit of a holoenzyme that phosphorylates the retinoblastoma protein. Although it is known that cyclin D1 regulates estrogen receptor (ER)alpha transactivation using heterologous reporter systems, the in vivo biological significance of cyclin D1 to estrogen-dependent signaling, and the molecular mechanisms by which cyclin D1 is involved, are yet to be elucidated. Herein, genome-wide expression profiling conducted of 17 beta-estradiol-treated castrated virgin mice deleted of the Ccnd1 gene demonstrated that cyclin D1 determines estrogen-dependent gene expression for 88% of estrogen-responsive genes in vivo. In addition, expression profiling of 17 beta-estradiol-stimulated cyclin D1 small interfering RNA treated MCF7 cells shows cyclin D1 is required for estrogen-mediated gene expression in vitro. Genome-wide chromatin immunoprecipitation-Seq analysis revealed a cyclin D1-DNA bound form associated with genes that were regulated by estrogen in a cyclin D1-dependent manner. The cyclin D1-dependent estrogen signaling pathways identified in vivo were highly enriched for extracellular membrane-associated growth factor receptors (epidermal growth factor receptor, ErbB3, and EphB3) and their ligands (amphiregulin, encoded by AREG gene), and matrix metalloproteinase. The AREG protein, a pivotal ligand for epidermal growth factor receptors to promote cellular proliferation, was induced by cyclin D1 via the AREG promoter. Chromatin immunoprecipitation analysis demonstrated the recruitment of cyclin D1 to the breast cancer 1 (Brca1)/ER alpha binding site of the Areg gene. Cyclin D1 genetic deletion demonstrated the in vivo requirement for cyclin D1 in assembling the estrogen-dependent amplified in breast cancer 1-associated multiprotein complex. The current studies define a requirement for cyclin D1 in estrogen-dependent signaling modules governing growth factor receptor and ligand expression in vivo and reveal a noncanonical function of cyclin D1 at ER alpha target gene promoters. Cyclin D1 mediates the convergence of ER alpha and growth factor signaling at a common cis-element of growth factor genes.