The adapter protein Shc is a ubiquitously expressed Src homology 2 (SH2) domain protein implicated in the transmission of activation signals to Ras. She proteins become phosphorylated on tyrosine in cells stimulated with a variety of growth factors and in v-𝘴𝘳𝘤 transformed cells and are able to transform fibroblasts and differentiate PC12 cells in a Ras-dependent fashion. To assess the transforming ability of Shc in the mouse mammary gland, I generated transgenic mice harbouring the p52ˢʰᶜ cDNA under the transcriptional control of the mouse mammary tumor virus long terminal repeat (MMTV LTR). While p52ˢʰᶜ expression was correlated with multiple enlarged terminal end buds in virgin mouse mammary glands, multiparous mice developed mammary hyperplasias and mammary carcinomas. The frequency, latency and focal nature with which these tumors arose suggests that additional events are necessary to induce malignant conversion of primary mammary epithelial cells. To directly test the role of Shc in established mammary tumor models, I have generated two strains of bigenic mice. When Shc was overexpressed with NDL 1-2, a constitutively activated form of the Neu receptor tyrosine kinase, latency of tumor onset was decreased over that of parental MMTV/NDL 1-2 mice. Polyomavirus middle T antigen (PyV MT) mutants with a functionally inactive Shc binding site (MT Y250F) are debilitated in mammary tumor formation compared to wild-type PyV MT transgenic animals. Concurrent overexpression of Shc with MT Y250F accelerated tumor kinetics and increased the propensity for metastasis to the lungs of bigenic animals. / Thesis / Master of Science (MSc)

No description available.

No description available.

c-Myb/MYB is an established and key player in hematopoietic malignancies but more recently a strong case for c-Myb as an oncogene in breast cancer has emerged. c-Myb and its transcriptional target genes have direct bearing on tumour initiation and progression and thus this has opened new opportunities to the development of therapeutic approaches in a range of cancer types with the aim of treating cancer at its various stages. In this study, the requirement of c-Myb during mammary gland tumourigenesis is being examined. In addition a direct therapeutic approach to targeting c-Myb-driven gene grp78/GRP78 in the context of primary and metastatic breast cancer was assessed. / The first aim of this study is to examine the expression of c-Myb during normal mammary gland development. The expression of c-Myb is extensively characterised in a temporal and spatial fashion. Nuclear staining of c-Myb by immunohistochemisty was found to be most elaborately expressed in the ductal epithelium during early mammary gland development. Mouse mammary gland lacking c-myb showed disorganised ductal structure in virgin mice, but did not affect subsequent pregnancy and lactation. / To extend the view that c-Myb is involved in mammary tumourigenesis c-myb-transduced immortalised mammary epithelial cells and two mammary tumour prone transgenic mouse models were examined. NMuMG cells transduced with c-myb showed enhanced proliferation and reduced Annexin V staining consistent with the protection from apoptosis. This reduced apoptosis is consistent with, and perhaps contingent upon, the elevated expression observed for bcl-2 and grp78. The data assembled by expression studies raised the possibility that c-Myb is essential for the establishment of mammary gland tumor in both MMTV-Neu and MMTV-PyMT spontaneous mammary gland tumor models. Loss of c-Myb expression in these models significantly delayed and in most instances completely abolished the onset of mammary gland tumours in both models. Preliminary evidence also indicated that Stat3 phosphorylation may underpin the elevated c-Myb expression in mouse mammary tumour cells. / The focus of my thesis then shifted to examining ways to exploit elevated c-Myb target gene GRP78 expression on the cell surface of mammary tumour cells. To do this I employed a GRP78 binding pro-apoptotic chimera peptide that specifically binds to GRP78 where I examined its efficacy against primary and metastatic breast cancer models. My results demonstrated the anti-tumour activity of the GRP78-chimera peptide both in vitro and in vivo. More importantly, the peptide is also effective at prolonging disease-free survival in mice with established metastatic disease. / Evidence obtained within these studies suggests that c-Myb plays an important role in mammary gland development and tumourgenesis. Although it may be difficult to directly target c-Myb in malignant disease, alternative anti-tumoural therapy may be developed against c-Myb-regulated target genes that are also implicated in mammary tumours. Collectively my thesis studies have advanced our understanding of c-Myb in mammary cancer initiation, progression and as a direct or indirect therapeutic target.

The porcine mammary gland can be used for the production of recombinant proteins by directing a transgene to the mammary gland with a milk protein gene promoter. In order to determine whether or not the protein will be expressed, the animals must be maintained at least through their first lactation. An experiment was performed to determine if hormonal induction of lactogenesis in transgenic virgin pigs could be used as a method for identifying those gilts that are likely to express the recombinant protein during a natural lactation. Mammary development and lactogenesis were induced by administration of subcutaneous implants designed to release 7.1 mg of estradiol-17 beta and 18 mg of progesterone daily for 21 d. Histological analysis of tissue samples before and after the treatment period indicated that mammary secretory tissue underwent dramatic proliferation resulting in a greater degree of alveolar and individual epithelial cell differentiation. The presence of beta-lactoglobulin mRNA was detected in high levels in post-implant tissue samples, and minimally detected in samples cultured in media supplemented with insulin, hydrocortisone, and prolactin. However, protein expression was only detected in the post-implant samples, indicating that beta-lactoglobulin was not maintained well by in vitro culture. The transgene mRNA, recombinant human fibrinogen (A-alpha chain), was detected in all analyzed samples at varying levels. However, the corresponding protein was not detected in any sample, under either reduced or nonreduced conditions. These results indicate that lactogenesis was successfully induced using the hormonal implants. Also, the transgene was activated by the hormonal induction in vivo and in vitro, but the corresponding protein could not be detected. This study indicates that induction of lactogenesis can be used to detect the presence of transgene mRNA in mammary tissue of gilts. However, we cannot conclusively demonstrate that this procedure can be used to identify those gilts that are likely to express the recombinant protein during a natural lactation. / Master of Science

Dairy herds are major contributors to N pollution because 70% of the N intake is lost to the environment and 30% or less is retained in milk protein. Plasma amino acids (AA) that are not used for protein synthesis in mammary glands (MG) are catabolized in post splanchnic tissues (liver plus gastrointestinal tract, pancreas, spleen, portal system, and associated adipose tissue) and two thirds of the net supply of essential AA (EAA) are cleared in splanchnic tissues. Thus, increasing AA capture in MG would be expected to reduce AA catabolism and thereby increase efficiency of AA utilization. The objectives of the work presented in this dissertation were to test the effect of energy and N intake on cell regulatory mechanisms, nutrient kinetics, milk, milk protein yield, and N efficiency in dairy cows. The aim of the first study was to test whether metabolizable protein (MP) and dietary energy exerted independent effects on milk protein synthesis and postabsorptive N efficiency. Forty mid-lactation cows (32 multiparous Holstein and 8 primiparous Holstein x Jersey cross-breds) were used in a complete randomized design with a 2 x 2 factorial arrangement of diets. Cows were assigned to one of four dietary treatments: high-energy, high-protein (HE/HP); high-energy, low-protein (HE/LP); low-energy, high-protein (LE/HP); and low-energy, low-protein (LE/LP). Energy concentrations were 1.55 (HE/HP and HE/LP) or 1.44 (LE/HP and LE/LP) Mcal NEL/kg DM according to the NRC model. Changes in predicted MP were achieved by feeding diets with 6.6 (HE/HP and LE/HP) or 4.6% (HE/LP and LE/LP) ruminally undegradable protein (DM basis). Ruminally degradable protein was held constant at 10.1% of DM. All cows were fed HE/HP diet from day 1 to 21 followed by the respective treatments from day 22 to 43 (n=10). Milk protein yield was reduced as dietary energy was reduced. There were no interactions between dietary energy and protein for either milk or protein yield. Milk urea N was significantly affected by energy and protein with an interaction (HE/HP=17.2, HE/LP=12.2, LE/HP=21.0, LE/LP=12.2 mg/dl). Nitrogen efficiency was affected by energy and protein supplies with no interaction and ranged from a low of 31% (LE/HP) to a high of 43% (HE/LP). Although energy and protein independently affected milk and protein yield the tissue and cellular mechanisms that regulate milk production were not studied. The second experiment studied cellular mechanisms in MG that contributed to the regulation of protein synthesis in the presence of energy or protein supply. We hypothesized that metabolism of AA in the MG is controlled by systemic and local tissue adaptations and when combined with altered mammary cell function controlled milk protein yield. Six primiparous mid-lactation Holstein cows with rumen cannulas were randomly assigned to abomasal infusions of casein and starch using a 2 x 2 factorial arrangement. The design was a replicated incomplete 4 x 4 Latin-square. All animals received the same basal diet (17.6% CP and 1.58 Mcal NEL/kg DM) throughout the study. Cows were restricted to 70% of ad libitum intake and infused abomasally for 36 h with water, starch (2 kg/d), casein (0.86 kg/d), or the combination (2 kg/d starch + 0.86 kg/d casein) using peristaltic pumps. Milk weights, milk samples, and arterial and venous blood samples were collected during the last 8 h of infusions. Mammary biopsy samples were collected and tissue protein prepared to evaluate cell signaling. Animals infused with casein had increased arterial concentrations of NEAA and EAA, as well as net uptake and clearance; however, milk protein yield did not increase. Animals infused with starch however, exhibited reduced arterial concentrations of NEAA and EAA but increased clearance and net uptake of most AA. Additionally, infusions of starch increased circulating concentration of insulin, IGF-I, and glucose as well as the rate of mammary plasma flow. Abomasal infusions of starch activated mammary activity of ribosomal protein S6 irrespective of other treatments. However, mammary tissue mTOR increased activity in response to casein only when starch was present during the infusions. These results suggest that cell signaling activation responded to different nutritional stimuli. Milk and protein yield increased in animals infused with starch. Therefore, MG positively responded to energy supply and engaged local and intracellular regulatory mechanisms to achieve that response. Understanding these adaptations could be beneficial in the development of mathematical representations for nutrients utilization in lactating animals. These two studies supported our hypotheses that regulatory mechanism are activated during limiting supply of AA to sustain protein synthesis in MG. The accuracy of mathematical models for lactating animals would increase if effects of energy on AA metabolism and cell signaling related to protein synthesis were included in the representation of milk protein synthesis. / Ph. D.

No description available.

Metastasis is the leading cause of death in patients with cancer, and the extracellular matrix is critical to cancer dissemination. The adhesion receptor, CD44, mediates cellular communication with the extracellular matrix by binding to the glycosaminoglycan, hyaluronan (HA). CD44 and HA play critical roles in cancer progression and development. HA is deposited in extracellular and pericellular matrices where it directs intracellular signaling through interactions with cell-surface CD44. CD44-HA interactions, in turn, direct signaling that is relevant to cancer progression. Importantly, these molecules can both promote and inhibit the oncogenic cascade, although the mechanism by which they promote dual and contrasting functions is unknown.Here we show that HA can both activate and suppress EGFR, a critical regulator of oncogenic signaling, in a context-dependent fashion. Using a 3D collagen system in which HA is either polymerized in collagen matrix or provided soluble in the media (sHA) we report that collagen-embedded HA (eHA) inhibits EGFR activation, filopodia formation, and cell spreading on a collagen matrix. Additionally, we show that CD44 is subject to cell-type changes during cancer progression. We have found that CD44 is expressed in the myoepithelium of the developing mammary gland and regulates the normal function of this cell type. The myoepithelial function of CD44 is also relevant to its role in cancer progression as CD44 is expressed in the basal cells of early-stage breast and prostate cancer but undergoes a basal to luminal epithelial switch with increasing tumorigenicity and is strongly expressed by tumor epithelium. These findings demonstrate a novel role for eHA as a protective molecule when encountered in the collagen matrix during cancer progression and highlight the importance of understanding cell-type specific contributions during cancer progression. Taken together, the findings reported in this dissertation point to a mechanism by which CD44 and HA can function in tumor suppression and promotion, depending on cell-type specific expression and modulation of the extracellular matrix.

Worldwide, more than 1,000,000 women develop breast cancer each year, and more than 400,000 die because of it. Basal-like breast cancers, which account for 8% to 20% of all breast cancer cases, represent the most aggressive of breast cancers with the majority resistant to existing targeted therapies. Accumulating lines of evidence implicate the Notch pathway in the aetiology of these basal-like breast cancers; while current work in our lab supports the notion that signalling through the Ectodermal Dysplasin Receptor (EDAR) pathway is also important. Notch signalling functions in normal development to control cell fate decisions and is mediated primarily, although not exclusively, through the CBF1 / RBP-Jĸ transcription factor. Aberrant Notch signalling leads to mammary tumourigenesis in mice; however at the outset of this work it was unclear whether signalling through RBP-Jĸ and / or through alternative pathways is required. This thesis presents novel data showing that elevated Notch signalling through the RBP-Jĸ-dependent pathway alone in murine mammary glands causes a number of developmental defects, including reduced ductal outgrowth, increased ductal side branching at puberty and, most significantly, is sufficient to induce mammary tumourigenesis. The data presented also provide supporting evidence that Notch signalling through RBP-Jĸ likely contributes to tumourigenesis, at least in part, via the suppression of apoptosis. At the outset of this thesis far less was known regarding the role(s) of the EDAR pathway within the mammary gland. Despite its recognised function in the development of ectodermal appendages it has been predominantly studied in the context of hair and tooth development. We show here that elevated Edar signalling affects the morphology of numerous ectodermally-derived glands, including the mammary gland, where in general, it results in glands that are enlarged or more elaborately branched. Most significantly, we show that elevated Edar signalling causes mammary tumourigenesis in mice, and provide data to support the hypothesis that elevated EDAR signalling might also be important in a subset of basal-like breast cancers in humans. The murine mammary gland phenotypes seen in response to elevated Edar signalling, including the squamous metaplasia within Edar-induced tumours, are very similar to those observed when Wnt signalling is increased. We provide data to support a positive correlation between activation of the EDAR and Wnt pathways in murine mammary tumourigenesis and provide data to support a comparable interaction in the aetiology of basal-like breast cancer showing squamous differentiation in humans. In summary, this thesis identifies the EDAR pathway as a novel potential therapeutic target in the treatment of a subset of basal-like breast cancers, and provides evidence that signalling through the RBP-Jĸ-dependent Notch pathway is sufficient to induce mammary tumourigenesis, most likely through the suppression of apoptosis.

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.