Hormone and transcription factor regulation of cytokines in the mammary gland

Abstract

Increased number of menstrual cycles is associated with an increased lifetime risk for breast cancer, however the biological basis for this increased risk is not well understood. Previous research in mouse models suggest the immune microenvironment is critically regulated by fluctuations in circulating estrogen and progesterone across the menstrual cycle, which may affect breast cancer susceptibility. The work in this thesis aims to investigate hormonal regulation of transcription factors and cytokines that affect cells of the immune system in the mammary gland, using an array of approaches including primary human mammary epithelial organoid structures (n=6), human mammary epithelial cell lines (MCF7, T47D and ZR751), and mouse mammary gland tissues. Firstly, primary mammary epithelial organoid cultures were treated with combinations of 17-beta estradiol and progesterone for 72 hours, and the abundance of messenger RNA encoding cytokines transforming growth factor beta 1 (TGFB1), tumour necrosis factor alpha (TNFA), signal transducer and activator of transcription 3 (STAT3), STAT5, interleukin-12 (IL12), E74-like factor 5 (ELF5), C-X-C motif chemokine ligand 12 (CXCL12), S100 calcium binding protein A8 (S100A8), S100A9, Forkhead box P3 (FOXP3), and Zinc Finger E-Box Binding Homeobox 1 (ZEB1) were analysed using real-time PCR. Moreover, lentiviral vectors were used to investigate the effects of FOXP3 overexpression on downstream cytokines in the human mammary epithelial organoids. In the second approach, human mammary epithelial cell lines were treated with combinations of 17-beta estradiol and progesterone at different time courses and the abundance of mRNA encoding the cytokines of interest was analysed using real-time PCR. Further, the expression of ELF5, a mammary epithelial morphogenesis transcription factor, was transiently silenced by small interfering RNA oligos in T47D mammary epithelial cell lines to investigate the role of ELF5 in progesterone-mediated cytokine expression. Lastly, to investigate the effects of Foxp3 heterozygosity on mammary ductal morphogenesis, C57BL6 wildtype and Foxp3 heterozygous female mice were tracked over a period of 28 days by histological analysis of vaginal smears. The 4th pair of the mammary glands from each mouse was collected at each of the four stages of the cycle for cytokine expression studies and whole-mount analysis. In primary organoid cultures, there was high variability in cytokine expression between patients; the only consistent result was that combined estradiol and progesterone treatment significantly attenuated TGFB1 mRNA expression (p<0.05). Also, overexpression of FOXP3 in these cells resulted in an insignificant increase in the mRNA expression of ZEB1. Hormone treatments of cell lines at different time courses resulted in different expression of mRNA encoding the cytokines of interest. However, silencing of the ELF5 gene in T47D cells resulted in induced mRNA expression of S100A9 and CXCL12 by 50% compared to non-silenced cells (p<0.05). Finally, analysis of whole-mount images revealed that mammary ductal morphogenesis in Foxp3 heterozygous mice was similar to that of wildtype mice. These results suggest that estrogen and progesterone variably regulate the cytokine synthesis by mammary epithelial cells, depending on the hormone receptor profile of the cells. In this case, ELF5 transcription factor might moderate the effects of progesterone on pro-inflammatory cytokines. Moreover, it seems that Foxp3 heterozygosity does not have any significant effect on mammary glands morphogenesis in mice.