The STAT5-regulated miR-193b locus restrains mammary stem and progenitor cell activity and alveolar differentiation

Abstract

The transcription factor STAT5 mediates prolactin signaling and controls functional development of mammary tissue during pregnancy. This study has identified the miR-193b locus, also encoding miRNAs 365-1 and 6365, as a STAT5 target in mammary epithelium. While the locus was characterized by active histone marks in mammary tissue, STAT5 binding and expression during pregnancy, it was silent in most non-mammary cells. Inactivation of the miR-193b locus in mice resulted in elevated mammary stem/progenitor cell activity as judged by limiting dilution transplantation experiments of primary mammary epithelial cells. Colonies formed by mutant cells were larger and contained more Ki-67 positive cells. Differentiation of mammary epithelium lacking the miR-193b locus was accelerated during puberty and pregnancy, which coincided with the loss of Cav3 and elevated levels of Elf5. Normal colony development was partially obtained upon ectopically expressing Cav3 or upon siRNA-mediated reduction of Elf5 in miR-193b-null primary mammary epithelial cells. This study reveals a previously unknown link between the mammary-defining transcription factor STAT5 and a microRNA cluster in controlling mammary epithelial differentiation and the activity of mammary stem and progenitor cells.

Loss of EZH2 results in precocious mammary gland development and activation of STAT5-dependent genes

“…For EZH2, total protein was extracted in RIPA buffer (50mM Tris-HCl (PH7.5), 150mM NaCl, 10% Glycerol, 50mM NaF, 0.5mM EDTA and 1% NP-40) and 20 μg of protein was loaded on 7% Tris-Acetate gel (Invitrogen) and probed with anti-EZH2 (Cell Signaling, 1:1000) and β-actin (Millipore, 1:10 000). For H3K27me3 detection, mammary epithelial cells (MECs) were prepared as described previously ( 50 ). Histones were extracted from MECs using 0.2M HCl and 0.5 μg of histones were loaded on 10–20% Tris-Glycine gradient gels (Invitrogen) and reacted with anti-H3K27me3 (Millipore, 1:2000) and anti-Histone H3 (abcam, 1:2000).…”

Histone Demethylase KDM6A Controls the Mammary Luminal Lineage through Enzyme-Independent Mechanisms

“…MECs were stained with biotinylated anti-CD24 -fluorescein isothiocyanate (FITC) (BD Biosciences; 553261), anti-CD49f-R-phycoerythrin (R-PE) (BD Biosciences; 555736), and 7-amino-actinomycin D (7AAD) (BD Biosciences; 51-68981E) and were sorted into basal (CD24 ϩ CD49f hi ) and luminal (CD24 hi CD49f lo ) cell populations after FACS analysis was performed, as previously described (35).…”

“…354230) with EGF medium (Dulbecco's modified Eagle's medium [DMEM]-F-12, 1% penicillin-streptomycin, 1ϫ ITS-X, 10 ng/ml EGF, and 0.0004% heparin) for 6 days. The medium was changed every 48 h. Colonies were fixed and stained for immunofluorescence as described previously (35).…”

Integration of microRNA signatures of distinct mammary epithelial cell types with their gene expression and epigenetic portraits

“…Interestingly, miR-200c targets the mRNA encoding BMI1, a key regulator of the self-renewal of stem cells in multiple tissues. MiR-193b also has been implicated in regulating mammary stem cell activity in vivo and may serve an additional function in controlling the alveolar differentiation during pregnancy [ 21 ]. In the context of breast cancer, many miRNAs have been reported to undergo deregulation, inferring an important role in controlling proliferation versus differentiation decisions.…”