Sim2s is a basic helix-loop-helix Per-Arnt-Sim (bHLH-PAS) transcription factor. In Drosophila, the Sim2 homolog, sim, is necessary for cell fate determination during central nervous system (CNS) development. In mammals, both Sim2 isoforms are involved in development of various tissues, including muscle, cartilage, and mammary gland. Loss-of-function studies revealed a role for Sim2s in specifying epithelial cell fate during mammary development and inhibiting growth and invasion of aggressive breast cancer cells. This study determined the role of Sim2s in mammary epithelial cell differentiation. Our hypothesis is that Sim2s is sufficient to promote lactogenic differentiation in vivo, characterized by expression of lactation-specific genes. Two models were used to test this hypothesis: (1) a transgenic mouse, expressing Sim2s under control of the MMTV-LTR, and (2) the mouse mammary epithelial cell line HC11. Together, these models allow analysis of the effect of Sim2s on global mammary gland differentiation and the mechanism through which it accomplishes this in a relatively homogenous population of cells. We determined that precocious expression of Sim2s in vivo is associated with upregulation of a subset of milk protein genes in nulliparous females. During early pregnancy, Sim2s regulation of lactogenic differentiation extended to a larger group of genes. Following pup removal, Sim2s appears to promote survival of alveolar epithelial cells. In vitro, Sim2s expression is necessary for maximal Csn2 expression, as determined by loss-of-function studies. Overexpression of Sim2s is sufficient to enhance prolactin-mediated Csn2 expression. Chromatin immunoprecipitation assays performed in HC11 cells revealed enhanced recruitment of Stat5a and RNA Polymerase II (RNAPII) to the regulatory region of Csn2 in the presence of Sim2s. In addition, Sim2s and RNAPII were found in a complex that was localized to both the promoter and coding region of the Csn2 gene. These studies support the idea that Sim2s is upregulated in a developmental stage-specific manner in the mouse mammary gland to promote the survival and differentiation of alveolar epithelial cells expressing high levels of milk protein genes. Further, Sim2s may regulate the function of a specific subset of alveolar cells by targeting the RNAPII holoenzyme complex to genes expressed during lactogenic differentiation.

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Chronic inflammation stimulates mammary tumourigenesis by disrupting signalling interactions between the epithelial ducts and the surrounding stromal microenvironment. Adipocyte enhancer-binding protein 1 (AEBP1) promotes mammary epithelial cell hyperplasia as a stromal factor that enhances activity of the proinflammatory transcription factor Nuclear Factor-?B (NF-?B) in macrophages. Aberrant NF-?B activity in macrophages elevates production of proinflammatory signals and the ligand sonic hedgehog (Shh), a significant contributor to tumourigenesis. In this study, Shh expression was elevated in macrophages isolated from transgenic mice (AEBP1TG) that overexpress AEBP1. Transient overexpression of AEBP1 in a macrophage cell line resulted in increased Shh expression. Furthermore, hedgehog target genes Gli1 and Bmi1 were up-regulated in mammary epithelium of AEBP1TG mice and HC11 mammary epithelial cells co-cultured with AEBP1TG macrophages. Growth of HC11 cells and mammary tumours was enhanced in response to AEBP1TG macrophages. These findings suggest that macrophage AEBP1 overexpression contributes to mammary hyperplasia through enhanced hedgehog signalling.

Selenium, an essential trace mineral that becomes anticarcinogenic at supranutritional levels, is readily incorporated into milk proteins when cows are fed high levels of selenium. The objective of this study was to investigate the effects of selenized milk protein on human mammary tumor progression. Four isonitrogenous diets with Se levels of 0.16, 0.51, 0.85 and 1.15 ppm were formulated by mixing low- and high-selenium milk protein isolates with a rodent premix. MCF-7 human breast cancer cells were inoculated into the mammary fat pad of female BALB/c nude mice implanted with slow-release 17 β-estradiol pellets. Mice with palpable tumors were randomly assigned to one of four diets for 10 weeks. Increasing Se intake reduced final tumor volume and the number of tumors > 500 mm3 in volume. There was a two-fold higher proportion of apoptotic cells in tumors exposed to the highest Se level. / Financial support was provided by Dairy Farmers of Ontario, Alltech Canada, Inc., and NSERC Canada.

Adult mammopoiesis occurs in close synchronization with reproductive development when the hypothalamic-pituitary-ovarian axis delivers integral systemic hormone cues to propel mammary morphogenesis during puberty, remodeling during reproductive cycles and functional differentiation following pregnancy. While hormones remain the driving force behind normal glandular development, increased life-time hormone exposure is a strong risk factor for breast cancer. Breast cancer heterogeneity has been attributed to different cells of origin and/or different mutation repertoires. Stem/progenitor cells are intensely investigated as cells of origin given their regenerative and self-renewal properties that provide conceivable advantage in cancer. Although hormones have a fundamental influence in breast cancer, their capacity to regulate stem/progenitor cells was unknown, and presents the central directive in this thesis. Employing mouse models, we show that mammary epithelial subpopulations and in particular, stem cells, are highly responsive to ovarian hormones and depend on key molecular events. A progesterone peak during the luteal phase of reproductive cycles results in a significant increase in stem cell-enriched basal cells and an expansion of stem cells measured by in vivo transplantation assays, with rapid development of lobuloalveoli. Progesterone was found to stimulate expression of mitogenic ligands, RANKL and Wnt4, in ER+PR+ luminal epithelial niche cells concomitant with increased expression of their receptors and target genes in the ER-PR- basal stem cell population, suggesting a cross-talk between luminal and basal cells that elicits stem cell expansion within the niche. The requirement of RANKL signaling for hormone-induced mammary stem cell dynamics was further explored utilizing mice deficient for its receptor, RANK, and by pharmacological inhibition of RANKL. Disruption of RANKL/RANK signaling resulted in abrogated activation of the basal stem cell-enriched population and alveolar progenitor cells in response to progesterone. This was accompanied by a marked reduction in cell proliferation, cell cycle regulators, alveolar lineage determinants and notably, in epithelial Wnt responsiveness. Thus, progesterone orchestrates a series of molecular events in the mammary stem cell niche where RANK is effectively positioned to deliver instructive signals to stem cells, culminating in stem cell recruitment and alveolar regeneration, processes which when deregulated have considerable potential to promote breast cancer pathogenesis.