There is currently little known about the role of extracellular matrix (ECM) in the regulation of microRNA (miRs), a family of short, non-coding RNA that repress gene expression at the post-translational level by binding to the 3’-untranslated region (3’UTR) of target mRNA.This thesis uses the mouse mammary gland (MG) to address this question by investigating whether the extracellular matrix regulates miR expression in mammary epithelial cells (MECs).miR expression profiles were generated using MECs cultured in 2D on collagen Ι and in 3D on laminin-rich basement membrane (LrBM). I identified 88 miRs that are more highly expressed in collagen cultured MECs and 8 miRs that have higher expression in MECs cultured on LrBM including miR-146b, a miR known to reduce metastases to the lung in breast cancer. The culture model used compares not only collagen to LrBM but also a stiff environment to a soft environment; raising the question of whether miR-146b is regulated by MEC interaction with ECM proteins or by cellular tension imposed by the microenvironment. Further investigation into miR-146b expression in MECs showed that its expression also increases in response to prolactin stimulation. Expression of the prolactin receptor and subsequently prolactin signalling is reduced in MECs cultured on collagen, but increases in MECs treated with blebbistatin or Y27632, which release cellular tension. However, neither drug had any affect on expression of miR-146. The ECM adhesion receptor β1-intregrin regulates MEC differentiation via cross-talk with prolactin receptor signalling. By using MECs from β1- itgfx/fx;CreER mice I identified a novel mechanism of miR regulation in which β1-intregrin signalling regulates transcription of miR-146b. This study has shows the importance of ECM in the regulation of miR expression and, whilst further investigations are still required, highlights the importance of ECM culture models in studying miR expression and function.

Previous research has shown that dairy cows require a dry period of at least 40 d for maximal milk yield in the subsequent lactation. Reducing the dry period requirement could prove beneficial to animal health and dairy profitability if subsequent milk yield was not reduced. Studies were conducted to evaluate the effects of continuous milking (CM) and hormonal treatments on milk yield, mammary epithelial cell (MEC) turnover during late gestation and early lactation, and mammary gene expression in dairy cows. A commercial trial using primiparous and multiparous cows demonstrated equal milk yields in bST-supplemented, CM and 60-d dry (CTL) multiparous cows, but lower milk yields in bST-supplemented CM, primiparous cows treated with bST. Subsequent experiments evaluated mammary development requirements during the dry period in primiparous cows and methods of rescuing milk yield. MEC growth was lower in CM glands during most of late gestation. Maintenance of lactation in CM glands resulted in a marked reduction in the MEC turnover process that occurs in the early dry period. In the last 20 d of gestation, MEC growth remained reduced in CM glands. By the last week of gestation, MEC growth was 50% less in CM tissue vs. CTL tissue. MEC apoptosis was unaffected by CM during the last 20 d of gestation, but a premature decrease in early lactation apoptosis occurred in CM glands at 7 d postpartum. Mammary gene expression demonstrated bax and insulin-like growth factor binding protein 5 are involved in apoptosis and cyclin D1, CCAT/enhancer binding protein-β, and bcl2 are involved in mammary development. Ultrastructure of CM tissue revealed large populations of resting or involuting alveoli by d 20 postpartum, whereas CTL glands had a homogenous population of secretory alveoli. Collectively, these data suggest that a 40-53% reduction in milk yield in CM glands is caused by reductions in MEC renewal and reduced secretory capacity. Treatments (bST, prostaglandin E2) to stimulate milk synthesis or MEC growth in CM primiparous glands were unsuccessful. In conclusion, primiparous cows continue to require a 60-d dry period, but multiparous cows are good candidates for short dry periods, and potentially no dry period.

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Luminal epithelial cells in the mammary gland require two types of signals to proliferate: soluble signals (growth factor signals) and signals from the extracellular matrix (ECM). The composition of the ECM is sensed by adhesion receptors such as integrins. Integrins modulate cell behaviour and play a key role in cell cycle entry. Altered integrin expression and signalling has been associated with breast cancer and studies using mouse mammary epithelial cells (MECs) have shown that the absence of β1-integrin induces growth arrest. However, it is not completely understood how integrins transduce the signals from the plasma membrane to the nucleus to induce cell cycle entry. Thus, the first aim of this project was to determine how β1-integrin controls proliferation in MECs. I established a model to study the effects of depleting β1-integrin using the FSK7 mammary epithelial cell line. The proliferation defect observed in this β1-integrin knockdown model was rescued by expressing a constitutively active Rac1 or Pak. Moreover, inhibiting Rac1 or Pak prevented normal proliferation in MECs in a similar fashion as β1-integrin depletion. Furthermore, in this thesis I have identified the complex comprised of Src, paxillin and p130Cas as a potential link between β1-integrin and Rac1. These results provide an insight into the mechanism that regulates proliferation downstream of β1-integrin. During breast cancer initiation, β1-integrin signals are disrupted. This indicates that additional signals must be driving proliferation during tumorigenesis. Therefore, the second aim of this project was to test whether expression of breast oncogenes can overcome the proliferation defect present in β1-integrin null cells. In order to do so, an oncogenic ErbB2, a constitutively active form of Akt (myrAkt) and the Notch1 intracellular domain (NICD) were transfected in the β1-integrin knockdown MECs. The results showed that ErbB2 overcomes the need for β1-integrin by signalling to Pak. NICD does not require β1-integrin to drive proliferation by an unknown mechanism. Expression of myrAkt did not restore normal levels of proliferation in β1-integrin depleted MECs. This finding suggests that Akt is not sufficient to induce cell cycle entry by itself and instead, both Akt and Erk signalling are needed to exert this function. This work has further delineated the specific signals controlling proliferation downstream of β1-integrin, and has provided a model to test the dependence of oncogenes for β1-integrin to drive proliferation in MECs. These studies are important to understand the role of β1-integrin in breast cancer formation and to define the types of breast cancer where β1-integrin can be used as an effective therapeutic target.

Consumer demand for milk fat has declined due to the increased risk of cardiovascular disease associated with consuming a high saturated fat diet. Milk fat synthesis is energetically expensive for the dairy cow, especially during early lactation or periods of poor nutrition. Thus, manipulating milk fat production and composition may promote the synthesis of more market-valuable milk components and improve energy utilization in dairy cows during periods of increased energy demand. Therefore, the objective of the present studies was to identify molecular proteins that regulate fatty acid synthesis in bovine mammary epithelial cells. The regulation of lipogenic genes including acetyl-CoA carboxylase (ACC) and fatty acid synthase (FAS) is controlled by transcription factors including sterol regulatory element binding protein-1 (SREBP1) and liver X receptor (LXR). In vivo, diet-induced milk fat depression or supplementing diets with polyunsaturated fatty acids inhibits milk fat synthesis by regulating SREBP1 expression. Results confirm that polyunsaturated fatty acids inhibit fatty acid synthesis in bovine mammary epithelial cells by regulating the expression of SREBP1. In hepatocytes, LXR can regulate the transcription of SREBP1 in addition to ACC and FAS. Results confirm that LXR activation enhanced synthesis of fatty acids in bovine mammary epithelial cells by promoting the transcription of FAS and SREBP1. Activation of LXR was unable to prevent the inhibitory effect of polyunsaturated fatty acids on fatty acid synthesis. In the lactating mammary gland, LXR may contribute to the synthesis of fatty acids by regulating the expression of SREBP1. In addition to modifying the expression of lipogenic genes, some enzymes can be phosphorylated by AMP-activated protein kinase (AMPK), an energy-sensing protein, inhibiting their activity. Presence of AMPK mRNA was identified in bovine mammary epithelial cells and activation of AMPK dramatically decreased fatty acid synthesis in bovine mammary epithelial cells. In the lactating mammary gland, AMPK may sense energy availability and regulate milk fat synthesis to control energy utilization. Identification of SREBP1, LXR, and AMPK as regulators of fatty acid synthesis in bovine mammary epithelial cells may lead to the development of technologies allowing dairy producers to modify milk fat production and composition to meet consumer demand and maximize profitability. / Ph. D.

The majority of bovine mammary gland research focuses on the main cell types - mammary epithelial cells and fibroblasts. However, the extracellular matrix (ECM) within the mammary gland is also of importance for its ability to regulate cell shape, proliferation, polarity, differentiation, gene transcription, protein synthesis, and secretion. Decorin is an ECM proteoglycan known to impact mammary cell proliferation in humans and rodents. Prior to this work, very little was known about decorin in bovine mammary biology. A series of bovine mammary cell culture experiments was conducted. The first experiment demonstrated existence of decorin pathway molecules in immortalized bovine mammary cells, but stopped short of demonstrating mature decorin proteoglycan deposition into the extracellular space. During the investigation it was noted that when cultured under basal conditions, intracellular decorin core protein (DCP) localization patterns appeared to be coordinated with specific phases of the cell cycle. Therefore, the objective of the second set of experiments was to characterize DCP localization patterns in bovine mammary epithelial cells (BME) at known phases of the cell cycle. The work was carried out in two sequential experiments. The hypothesis of the first experiment was that DCP accumulates in BME during S-phase of the cell cycle; the research rejected this hypothesis. The hypothesis of the second experiment, formulated after completion of the first experiment for this objective, was that DCP accumulates in BME during metaphase of the cell cycle. However, the experiment was unable to confirm of reject this hypothesis. Major findings were that both BME and mammary fibroblasts produce DCP and known decorin pathway molecules. BME produce intracellular DCP, but it is not accumulated during the S-phase of the cell cycle. However, it is still unknown if DCP is accumulated in BME during metaphase. Future research should focus on further characterization of decorin and its associated pathway molecules to learn if decorin induces proliferation or apoptosis of bovine mammary epithelial cells. This is important because number and activity of mammary epithelial cells ultimately determine milk yield in dairy cows. Fundamental knowledge gained in this research area may one day be applied at the animal-level and lead to gains in milk production efficiency by altering the cellular composition of mammary glands. / Ph. D. / The mammary gland is a heterogeneous tissue with two main cell types - mammary epithelial cells and fibroblasts. These two cells types produce the majority of the extracellular matrix (ECM) within the mammary gland. The ECM is important for regulation of cell shape, proliferation, polarity, differentiation, gene transcription, protein synthesis, and secretion. Decorin is an ECM proteoglycan known to impact mammary cell proliferation in humans and rodents. Prior to this work, very little was known about decorin in bovine mammary biology. A series of bovine mammary cell culture experiments was conducted. The first experiment demonstrated existence of decorin pathway molecules in bovine mammary cells, but stopped short of demonstrating mature decorin proteoglycan deposition into the extracellular space. During the investigation it was noted that when cultured under basal conditions, intracellular decorin core protein (DCP) localization patterns appeared to be coordinated with specific phases of the cell cycle. Therefore, the objective of the second set of experiments was to characterize DCP localization patterns in bovine mammary epithelial cells (BME) at known phases of the cell cycle. The work was carried out in two sequential experiments. The hypothesis of the first experiment was that DCP accumulates in BME during S-phase of the cell cycle; the research rejected this hypothesis. The hypothesis of the second experiment, formulated after completion of the first experiment for this objective, was that DCP accumulates in BME during metaphase of the cell cycle. Major findings were that both BME and mammary fibroblasts produce DCP and known decorin pathway molecules. BME produce intracellular DCP, but it is not accumulated during the S-phase of the cell cycle. However, it is still unknown if DCP is accumulated in BME during metaphase. Future research should focus on further characterization of decorin and its associated pathway molecules to learn if decorin induces proliferation or apoptosis of bovine mammary cells. This is important because number and activity of mammary epithelial cells ultimately determine milk yield in dairy cows. Fundamental knowledge gained in this research area may one day be applied at the animal-level and lead to gains in milk production efficiency by altering the cellular composition of mammary glands.

The goal of this dissertation was to evaluate genomic regulation during bovine mammary epithelial cell (BMEC) growth and differentiation. To accomplish this goal, a collagen gel cell culture system was developed that was capable of mimicking the prepartum stages of epithelial development and differentiation. In addition, a 4,600-cDNA bovine microarray was developed in order to profile gene expression. Analysis of BMEC in collagen cultures using various lactogenic conditions highlighted the critical importance of both hormonal and structural signals. The objective of the first study utilizing the microarray was to evaluate the contribution of the two prominent lactogenic factors in vitro, 1) prolactin and 2) gel release. Collectively, lactogenic stimulation appears to turn off genes associated with structural progression and morphogenesis, and turn on genes involved in alveolar MEC differentiation such as cell polarization, milk protein synthesis and ER/Golgi transport. The objective of the second study utilizing these resources was to evaluate the direct effects of thermal stress on BMEC growth and development. The structural response to thermal stress was characterized by morphogenic inhibition and dramatic regression of the ductal branches. Microarray analysis revealed an overall up-regulation of genes associated with stress response, DNA repair, protein degradation and cell death. In contrast, genes associated with cellular and MEC-specific biosynthesis, metabolism, and morphogenesis, were generally down-regulated. Subsequent to the analysis of BMEC differentiation was a targeted effort focusing on two small molecules hypothesized to be involved in regulating the BMEC secretory response: serotonin and prostaglandin E2. A pilot study suggested that serotonin is produced by bovine MEC and a model was proposed that describes serotonin's role as a feedback inhibitor during milk synthesis and secretion. A second pilot study demonstrated that PGE2 had a consistently positive influence on lumen diameter of alveolar structures in vitro. Overall, this dissertation provides new resources for studying bovine functional genomics, particularly within the mammary gland, and it provides a strong foundation for understanding genomic regulation of mammary epithelial structure and function. Furthermore, it establishes potential roles for local regulation of milk production by serotonin and PGE2.

p16INK4a (p16) traps the cell at the restriction point of the cell cycle by binding to cyclin-dependent kinase 4/6 thus preventing the phosphorylation of the retinoblastoma protein (pRB). As p16 accumulates the cell stops dividing and becomes senescent. This study investigates the modulation of p16 function by the putative membrane protein ABCB5 and a group of five putative oncogenic microRNAs (oncomiRs). ABCB5 is a poorly characterised member of the B-subfamily of human ATP Binding Cassette transporters. ABCB5 is reportedly transcribed into four transcripts, one of which could potentially encode a full-length transporter (ABCB5fl) whilst a second could encode a half-transporter (ABCB5β). The other two transcripts (ABCB5α and ABCB5γ) could only encode short polypeptides. Exogenous expression of ABCB5fl and ABCB5β was achieved in HEK293T cells, but the recombinant protein expressed poorly and localised to the endoplasmic reticulum. Point mutations introduced into the ATP catalytic domain failed to improve expression levels suggesting that protein function was not deleterious to the cell. Exogenous expression in HEK293T cells also allowed commercial antibodies purportedly raised against ABCB5 isoforms to be tested. Several were found not to recognise ABCB5 necessitating re-interpretation of published data. However, one antibody recognised both ABCB5fl and ABCB5β, and was subsequently used to evaluate protein expression levels in other cell types.siRNA knockdown of ABCB5 in human mammary epithelial cells (HMECs) caused a concomitant reduction in p16 expression and an increase in cellular proliferation. Differential siRNAs and RT-qPCR analyses demonstrated ABCB5β to be the relevant transcript with respect to the reduction in p16 expression; however, no native ABCB5β protein was detected in HMECs. Together these data lead to the hypothesis that the ABCB5β transcript may act as a long noncoding RNA to regulate p16. Exogenous expression of each of five distinct putative oncomiRs in HMECs was found to increase cellular proliferation and, surprisingly, increase p16 expression. These results mirror a phenotype commonly observed in p16-positive basal-like breast cancer (BLBC), an aggressive form of breast cancer with poor prognosis and few treatment options. Bioinformatic analysis of the predicted target genes for these oncomiRs identified multiple transcriptional regulators of pRB. These predictions, together with the work performed in a cellular model of p16-positive BLBC, suggest that the oncomiRs may cause unrestricted cell proliferation by indirectly reducing transcription of the pRB gene, RB1. In the absence of pRB, p16 expression is induced via a previously reported oncogeneinduced senescence-like positive feedback loop. These data, and previously published observations, suggest that a similar mechanism may explain the basis of p16-positive BLBC.

Changes in the epigenetic landscape are widespread in neoplasia, with de novo methylation and histone repressive marks commonly occurring in association with gene silencing. However, understanding the dynamics of epigenetic changes is often hindered due to the absence of adequate in vitro model systems that accurately reflect events occurring in vivo. Human mammary epithelial cells (HMECs) grown under standard culture conditions enter a growth arrest termed selection, but a subpopulation is able to escape from arrest and continue to proliferate. These cells, called post-selection cells, have many of the hallmarks seen in the earliest lesions of breast cancer, including transcriptional silencing and hypermethylation of the p16INK4A tumour suppressor gene. The overall aim of my thesis was to use post-selection HMECs as model system to identify and dissect the mechanism involved in early epigenetic aberrations. Firstly, using a microarray approach, I found that multiple members of the TGF-β signalling pathway were concordantly suppressed in post-selection cells, and this was associated with functional disruption of the TGF-β pathway. Interestingly, concordant gene suppression was not associated with aberrant DNA methylation, but with repressive chromatin remodelling. Secondly, to further understand the mechanism underpinning epigenetic silencing, I demonstrated using laser capture technology, that p16INK4A silencing is a precursor to DNA methylation and histone remodelling. Thirdly, I found that individual post-selection HMEC strains during the early passages shared a common 'wave' pattern of regional-specific methylation within the p16INK4A CpG island. Interestingly, the 'wave' pattern of early de novo methylation correlated with the apparent footprint of nucleosomes within the p16INK4A CpG island. Lastly, to further characterise the properties of the HMEC culture system, I demonstrated that post-selection cells do not possess a natural tumour-inducing property when transplanted into the mammary fat pad of immunocompromised mice. However, post-selection HMECs were associated with high expression of a variety of stem/progenitor markers, as well as stem/progenitor associated polycomb genes, thus demonstrating that these cells share some common features of stem/progenitor cells. The research presented in this thesis demonstrate that epigenetic changes occur early in the growth of post-selection HMECs and many of these changes are common in breast cancer.