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.

This study was designed to determine the distribution of fatty acids and cholesterol in total tumor and mammary tissues. Fatty acid profiles of phospholipid fractions from tumors and mammary glands were also determined. Fatty acids and cholesterols were analyzed by gas liquid chromatography. Methodology was developed for phospholipid separation by high performance liquid chromatography.Tumors derived from mammary glands in Strain A/ST mice were found to contain two to three times the amount of cholesterol compared to normal mammary glands. Mammary glands from safflower fed mice contained significantly higher percentages of linoleic acid. Linoleic acid content in stearicacid (SA-1) fed mice was sharply reduced. linoleic acid in mammary glands of animals fed a high fat Stearic acid (SA-4) , corn oil and stock diet fed animals displayed similar fatty acid profiles. Fatty acid analysis of tumors excised from mice fed the experimental diets showed similar patterns in comparison to normal mammary glands. The similar distributions were in the 18 carbon fatty acids. Distributions of phospholipid fatty acids in tumors and mammary glands were similar. Mammary gland phospholipids displayed increased percentages of short chain (14 carbons and under) fatty acids. Results of these studies demonstrate an increased availability of diet rich in polyunsaturated fatty acids.

The extracellular matrix (ECM) plays an important role in mammary gland development and function. Osteopontin (OPN), a secreted glycophosphoprotein, with several functional and structural properties of ECM proteins, is expressed at elevated levels during normal and pathologic development of the mammary gland and is present in milk. However, whether it plays any developmental role in the mammary gland is unknown. To investigate this possibility, transgenic AS-OPN mice were generated using a transgene expressing OPN antisense RNA under the control of the MMTV-LTR promoter/enhancer. The mammary glands of AS-OPN mice express low levels of OPN, compared to control littermates, show excessive branching of the ductal epithelium at the virgin stage, impaired proliferation of the mammary epithelium, and abnormal alveolar structure formation during pregnancy, and mild to severe lactation deficiency. To further examine the role of OPN at the cellular level, an established murine mammary epithelial cell line (NMuMG), which is capable of differentiation in culture, was transfected with the same DNA construct used to generate AS-OPN mice. Several antisense OPN cDNA transfected clones, which secrete decreased amounts of OPN compared to mock-transfected cells were obtained. These cells were found to have lost their ability to form branched duct-like structures, as judged by branching morphogenesis assays, by growth in collagen gels and stimulation with hepatocyte growth factor. They also failed to spread on type I collagen, although their binding to type IV collagen was unaffected. The antisense transfectants also assumed a mesenchymal phenotype, characterized by fibroblast-like morphology, an apparent loss of cell-cell contacts and spontaneous cell scattering. Transmigration assays and wounding experiments indicated that these cells also have a higher migratory activity than control cells. Northern blot and immunofluorescence analyses showed that migrating cells downregulate OP

Mammary gland development is controlled by hormones such as progesterone and prolactin, which activate a genomic regulatory network. Identification of the components and regulatory links that comprise this network will provide the basis for defining the network's dynamic response during normal development and its perturbation during breast carcinogenesis. This thesis investigates two molecules in detail, Elf5 and KIBRA, which were identified as potential prolactin targets in a transcript profiling screen for key members in this genetic program of mammary morphogenesis. We examined the effect of expression of Elf5, a transcription factor critical in alveolar differentiation, in a 3D culture model of non-transformed mammary epithelial MCF-10A cells. We discovered that Elf5 expression was selectively repressed over time in these cells when cultured on a basement membrane, and that Elf5 overexpression disrupted the architecture of acini resulting in luminal filling. This occurred due to an increase in the expression of epidermal growth factor receptor (EGFR) with repressed the induction of the pro-apoptotic molecule, Bim. We also observed that Elf5 is up-regulated with progesterone treatment, and that suppression of Elf5 expression in T47D breast cancer cells inhibits proliferation. Data obtained from the suppression of Elf5 expression in the presence of progesterone suggested that the role played by Elf5 in the Pg signalling pathway in T47D cells is relatively minor, and that rather than being a major downstream factor, the induction of Elf5 expression is utilised more to influence and potentiate other signalling pathways, such as the Prl pathway. We characterised expression of KIBRA in the mammary gland and breast cancer cell lines, and observed that KIBRA was also up-regulated with progesterone treatment. Using a bioinformatic approach, we identified the tyrosine kinase receptor DDR1 as a binding partner of KIBRA. We have demonstrated that the WW domains of KIBRA bind to a PPxY motif in DDR1, and that these molecules dissociate upon treatment with the DDR1 ligand, collagen. Finally, overexpression and knockdown studies demonstrate that KIBRA promotes the collagen-stimulated activation of the MAPK cascade. Thus KIBRA may play a role in how the reproductive state influences the mammary epithelial cell to respond to changing cell-context information, such as experienced during the tissue remodelling events of mammary gland development. Overall, the data presented in this thesis contributes to our growing knowledge of the genetic program responsible for mammary development and carcinogenesis.

Thesis (Ph. D.)--University of Texas at Austin, 2003. / Supervisor: Jaquelin P. Dudley. Vita. Includes bibliographical references. Also available from UMI.

Thesis (Ph. D.)--University of Texas at Austin, 2001. / Vita. Includes bibliographical references. Available also in a digital version from UMI/Dissertation Abstracts International.

Thesis (Ph. D.)--University of Texas at Austin, 2005. / Supervisor: Jaquelin P. Dudley. Vita. Includes bibliographical references.

Thesis (Ph. D.)--University of Texas at Austin, 2005. / Supervisor: Martin Poenie. Vita. Includes bibliographical references.

Thesis (Ph. D.)--University of Texas at Austin, 2005. / Supervisor: Jaquelin P. Dudley. Vita. Includes bibliographical references.

Thesis (Ph. D.)--University of Wisconsin--Madison, 1969. / Typescript. Vita. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references.