Luminal STAT5 mediates H2AX promoter activity in distinct population of basal mammary epithelial cells

• Published in: Oncotarget 2016-07, Vol.7 (27), p.41781-41797
• Main Authors: Reichenstein, Moshe, Rauner, Gat, Kfir, Shenhav, Kisliouk, Tatiana, Barash, Itamar
• Format: Article
• Language: English
• Subjects: Animals; Azacitidine - pharmacology; Cell Line; Cells, Cultured; DNA Methylation - drug effects; Enzyme Inhibitors - pharmacology; Epithelial Cells - metabolism; Female; Gene Expression Regulation - drug effects; Histones - genetics; Histones - metabolism; Mammary Glands, Animal - cytology; Mammary Glands, Animal - metabolism; Mice, Knockout; Mice, Transgenic; Pregnancy; Promoter Regions, Genetic; Research Paper; STAT5 Transcription Factor - genetics; STAT5 Transcription Factor - metabolism
• ISSN: 1949-2553; 1949-2553
• DOI: 10.18632/oncotarget.9718

Deregulated STAT5 activity in the mammary gland causes parity-dependent tumorigenesis. Epithelial cell cultures transfected with constitutively active STAT5 express higher levels of the histone H2AX than their non-transfected counterparts. Higher H2AX expression may be involved in tumorigenesis. Here, we aimed to link high STAT5 activity to H2AX-GFP expression by looking for distinct types of mammary cells that express these proteins. In vitro and in transgenic mice, only 0.2 and 0.02%, respectively, of the cells expressed the H2AX-GFP hybrid gene. Its expression correlated with that of the endogenous H2AX gene, suggesting that detectable H2AX-GFP expression marks high levels of H2AX transcript. Methylation of the H2AX promoter characterized non-GFP-expressing H2AX-GFP cells and was inversely correlated with promoter activity. Administration of 5-azacytidine increased H2AX promoter activity in an activated STAT5-dependent manner. In transgenic mice, H2AX-GFP expression peaked at pregnancy. The number of H2AX-GFP-expressing cells and GFP expression decreased in a Stat5a-null background and increased in mice expressing the hyperactivated STAT5. Importantly, H2AX-GFP activity was allocated to basal mammary cells lacking stem-cell properties, whereas STAT5 hyperactivity was detected in the adjacent luminal cells. Knockdown of RANKL by siRNA suggested its involvement in signaling between the two layers. These results suggest paracrine activation of H2AX via promoter demethylation in specific populations of basal mammary cells that is induced by a signal from neighboring luminal cells with hyper STAT5 activity. This pathway provides an alternative route for the luminally confined STAT5 to affect basal mammary cell activity.