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.

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.

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.

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.

The Wilms' Tumour Suppressor gene 1, WT1, encodes for a complex protein which is essential in mammals throughout life. Its roles vary with the developmental stages: in the embryo, it regulates the epithelial-mesenchymal balance required for a correct organogenesis and acts as a tumour suppressor; in the adult, it is involved in the maintenance of tissue homeostasis and has been controversially considered as an oncogene. Breast cancer is one of the adult tumours in which WT1 oncogenic function was first hypothesised. This malignancy is the most common in women, with more than one million cases being diagnosed worldwide every year, and represents the leading cause of cancer related deaths. Because of its major health burden, this disease has been extensively studied and special attention has also been paid to normal mammary gland biology: several works have shown that breast cancer can be divided into many molecular subtypes, which may reflect the cell of origin of the tumour; moreover, many genes involved in the normal development of the mammary gland have been proven to also play a role in breast tumorigenesis. WT1 expression has been previously reported in both healthy mammary glands and breast cancer samples, however, its function in this context is not well understood and the evidence gathered so far is extremely contradictory. This thesis aimed to investigate the exact role played by WT1 in both mammary gland and breast cancer biology, using a combination of in vivo and in vitro techniques. Following flow cytometry isolation, Wt1 mRNA expression was detected in the myoepithelial and stem cell subpopulations of the healthy gland. To investigate the effects of WT1 loss, Wt1 conditional mice were crossed with two different mammary specific Cre lines: the knockout animals developed, bred and lactated normally, however, they showed a significant increase of ductular branches during pregnancy, suggesting that WT1 may be involved in the regulation of branching morphogenesis. In order to study WT1 role in mammary tumours, the gene was knocked out in a breast cancer mouse model and knocked down in several breast cancer cell lines, using both constitutive and inducible lentivirus-based systems. WT1 loss did not seem to affect cell proliferation, but resulted in a significant increase in cell migration in vitro and in the upregulation of mesenchymal markers. Furthermore, bioinformatics analysis showed that the WT1-positive tumours mainly belong to the luminal/ER-positive subtypes and express lower levels of mesenchymal markers than the WT1-negative tumours. As a whole, the findings of this thesis characterise WT1 expression in the healthy mammary gland and provide the first evidence of its possible function in this organ; moreover, this work seems to rule out an oncogenic role for WT1 in breast cancer, while suggesting that it could be an upstream regulator of cell migration. Additional experiments are required to confirm this result in vivo and verify whether it could lead to any clinical application.

The mammary gland of lactating transgenic animals has several advantages for production of heterologous proteins including a high cell density that results in high concentrations of secreted protein and the ability to perform several types of post-translational modifications. Transgenes were constructed from the 4.1 kbp murine Whey Acidic Protein promoter (mWAP) and the three cDNAs coding for the Aα, Bβ and γ fibrinogen chains to evaluate the requirements of the transgenic murine mammary gland for high level secretion of fully assembled human fibrinogen. After introducing the constructs into the murine zygotes by microinjection, secretion of fully assembled fibrinogen into milk was measured at concentrations between 10 ug/ml to 200 ug/ml. In one line of mice the total secretion of fibrinogen and unassembled subunits approached 700 ug/ml in milk. The level of assembled fibrinogen was proportional to the lowest amount of subunit produced where both the Bβ and γ chains were rate limiting. Also, the subunit complexes γ₂, Aαγ₂ and the individual subunits Aα, Bβ and γ were found as secretion products. This is the first time that secretion of individual Bβ-subunits by any cell type has been reported and suggests the organization of the secretion pathway in mammary epithelia is different from that in liver. Glycosylated forms of individual Bβ-chain contained a complex saccharide with low mannose. Glycosylation of the γ-chain was also observed. These results suggest the 4.1 mWAP promoter can drive expression of fibrinogen cDNAs to high levels and that the amount of fully assembled fibrinogen secreted is equal to the level of the lowest expressing chain. / Master of Science

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<p> PEA3 is a member of the ets family of transcription factors. It is expressed throughout embryonic development and in mouse mammary adenocarcinomas induced by expression of the receptor tyrosine kinase Neu. Mice lacking PEA3 due to a targeted disruption of the gene, develop normally, however, male mice fail to mate for yet undetermined reasons. To further understand the role of PEA3 in mammary gland development and tumorigenesis, the effects of loss of function of PEA3 were examined in tumor formation and in mammary gland development. </p> <p> Analysis of tumor formation in PEA3 +I+ and PEA3 -/-animals failed to show a statistically significant difference in tumor onset. Loss of PEA3 did not affect the tumor morphology, nor did it inhibit metastasis of these tumors to the lung. These data indicate that PEA3 is not required for tumor formation or metastasis. </p> <p> PEA3 deficient animals displayed defects in branching morphogenesis in the mammary gland. Decreased ductal branching was observed in virgin and pregnant females. Mice with decreased levels of PEA3 expression also exhibited defects in branching morphogenesis, indicating a dosage effect. PEA3 is expressed in the myoepithelial cells during puberty and pregnancy. It is also express in the highly proliferative cap cell layer of the terminal end bud. In the embryonic mammary gland, PEA3 is expressed as early as 10.5 days in the mammary epithelium and continues late in embryogenesis. Expression in the male mammary gland is lost at approximately embryonic day 16. </p> / Thesis / Candidate in Philosophy

Aristaless-like homeobox 4 is a homeodomain transcription factor that has important functions during mouse development. A recent report demonstrated that Alx4 expression is required in periductal stromal cells in the mouse mammary gland for normal mammary morphogenesis. To test the hypothesis that ALX4 is expressed in the normal human breast, and this expression is altered in breast cancer, immunohistochemistry was performed on normal and breast cancer tissue and breast tissue microarrays. In the normal breast, ALX4 was expressed in stromal fibroblasts and luminal epithelial cells, but not in myoepithelial cells. Expression was lost in breast cancer in both cell compartments. Upon global demethylation induced by 5-aza-2’-deoxycytidine, normal and breast cancer cell lines expressed ALX4, suggesting that hypermethylation may repress expression of ALX4 during malignant transformation of the breast. These results demonstrate that ALX4 may be used as a biomarker for breast cancer, and may act as a tumour suppressor.