During this reporting period the Laboratory of Genetics and Physiology has made progress and elucidated novel mechanisms by which pregnancy-associated hormones, specifically prolactin, control mammary development through the transcription factor STAT5. We have identified that the gene encoding micro RNA 193b (mir-193b) is under control of cytokines through the transcription factor STAT5 and that this micro RNA modulates mammary gland development through restraining mammary stem cells activity and alveolar differentiation. In addition, we have demonstrated an essential role for histone modifying enzymes in the biology of mammary cells and hepatocytes. LGP scientists have also engaged in collaborative research with scientists from the US, Europe, South America and Asia to investigate functions of the transcription factor STAT5 in neurons, mammary tissue and breast cancer. Lastly, LGP scientists have engaged with intramural and extramural researchers in studying the role of interferon and the transcription factor STAT1 in the hematopoietic system and host versus graft disease. Mammary development Integrating mouse genetics with large-scale genomic analyses led researchers in LGP to the discovery that the sequential activation of genetic programs in mouse mammary epithelium during pregnancy depends on the transcription factors STAT5A and STAT5B that are activated by the pregnancy hormone prolactin (Yamaji, 2013). Genome-wide studies have demonstrated that STAT5 binds to specific micro RNA (miRs) genes suggesting their critical role in controlling these loci. MiRNAs are important post transcriptional regulators of messenger RNAs and that by controlling their stability and translation they modulate the physiology of cells. Scientists in LGP focused on miR-193b, whose gene is activated by cytokines in a wide range of cell types, and most specifically in stem cells. To address the biological functions of this particular micro RNA in mammary epithelium the miR-193b locus, which also encodes miR-365-1, was inactivated in mammary stem cells of the mouse. While the miR-193b locus was characterized by active histone marks in mammary tissue, STAT5 binding and expression during pregnancy, it was silent in most non-mammary cells, suggesting a particular importance in mammary epithelium. 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. Differentiation of mammary epithelium lacking the miR-193b locus was accelerated during puberty and pregnancy, which coincided with the loss of Caveolin 3 and elevated levels of the transcription factor Elf5, two molecules that had been associated with developmental programs in the mammary gland.This study reveals a previously unknown link between the mammary-defining transcriptionfactorSTAT5 and a micro RNA cluster in controlling mammary epithelial differentiation and the activity of mammary stem and progenitor cells. Collaborative research investigated the relative role of tamoxifen and letrazole on mammary preneoplasia in mouse models of cancer that depend on the estrogen receptor and aromatase. An immune-associated gene signature linked to tamoxifen resitance was identified. Response to breast cancer chemoprevention can depend upon host genetic makeup and initiating events leading up to preneoplasia. Increased expression of aromatase and ER are found in conjunction with breast cancer. To investigate response or resistance to endocrine therapy, mice with targeted over-expression of Esr1 or CYP19A1 to mammary epithelial cells were employed, representing two direct pathophysiological interventions in estrogen pathway signaling. Both Esr1 and CYP19A1 over-expressing mice responded to letrozole with reduced HAN prevalence and decreased mammary epithelial cell proliferation. CYP19A1 over-expressing mice were tamoxifen-sensitive but Esr1 over-expressing mice were tamoxifen-resistant. Increased ER expression occurred with tamoxifen resistance but no consistent changes in progesterone receptor, pSTAT3, pSTAT5, cyclin D1 or cyclin E levels in association with response or resistance was found. RNA-seq was employed to seek a transcriptome predictive of tamoxifen resistance using these models and a second tamoxifen-resistant model, BRCA1 deficient/Trp53 haploinsufficient mice. Sixty-eight genes associated with immune system processing were upregulated in tamoxifen-resistant Esr1 and Brca1 deficient mice whereas genes related to aromatic compound metabolic process were upregulated in tamoxifen-sensitive CYP19A1 mice. Interferon Regulatory Factor 7 was identified as a key transcription factor regulating these 68 immune processing genes. Two loci encoding novel transcripts with high homology to human IGLL1 were uniquely upregulated in the tamoxifen-resistant models. Letrozole proved to be a successful alternative to tamoxifen. Further study of transcriptional changes associated with tamoxifen resistance including immune-related genes could expand our mechanistic understanding and lead to biomarkers predictive of escape or response to endocrine therapies.
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