No description available.

The mammary gland is a branched epithelial organ comprised of myoepithelial, ductal and alveolar cells that are derived from resident stem and progenitor cells. The progression from mammary gland stem cell(s) to the differentiated mammary gland cell types is poorly understood. Here, I describe the identification and characterization of two luminal progenitor cell populations in the mouse mammary gland, and investigate the role of the transcription factor C/EBPβ in their development. In Chapter 2, I describe the isolation of two luminal progenitor cell populations (Sca1+ and Sca1- luminal cells) and show that they are differentially primed in their gene expression towards ductal and alveolar cell fates, respectively. Furthermore, I show that in vivo genetic priming affects the in vitro differentiation potential of Sca1+ and Sca1- luminal cells. In Chapter 3, I show that C/EBPβ is required for the appropriate specification of ductal and alveolar lineages, and in its absence, alveolar lineage priming is lost, and ductal lineage priming is up-regulated in both Sca1+ and Sca1- cells. Preliminary data also shows that in addition to severe proliferation defects, the changes in in vivo lineage priming in Cebpb-/- mice also affect the in vitro differentiation potential of Cebpb-/- Sca1+ and Sca1- luminal progenitors. Lastly, in Chapter 4, I describe the genome-wide binding characteristics of C/EBPβ in Sca1+, Sca1- and P16.5 alveolar cells. These experiments reveal that genome-wide C/EBPβ occupancy is correlated with alveolar cells fate, and that C/EBPβ target genes perform distinct cellular functions in alveolar cells (Sca1- cells and P16.5). Furthermore, I show that Elf5 is directly regulated by C/EBPβ, and posit that direct regulation of Elf5 by C/EBPβ may be one mechanism through which C/EBPβ exerts its alveolar cell fate programming.

The role that each of the Notch receptors play in controlling alveolar development and cell fate determination in the mouse mammary gland has remained unclear. By utilizing a cre-conditional constitutively active intracellular Notch1 knock-in I define, in vivo, that ectopic Notch1 activation is sufficient to inhibit ductal outgrowth, cause the formation of alveolar-like cell accumulations, and promote Elf5+/ER- cell fate, at the expense of ER+ cell fate, in the mammary gland of pubescent mice. Furthermore, ectopic Notch1 in the pregnant mammary gland is sufficient to promote the formation of pregnancy/lactation-dependent lactating adenomas. These lactating adenomas consist of differentiated secretory cells and normally regress during involution but progress into non-regressing tumours after multiple pregnancies. These lactating adenomas exhibit decapitation secretions characteristic of apocrine differentiation. Together these results suggest that Notch1 may function to promote Elf5+/ER- cell fate and may be misregulated in pregnancy-associated masses and apocrine-carcinoma of the breast in humans.

The role that each of the Notch receptors play in controlling alveolar development and cell fate determination in the mouse mammary gland has remained unclear. By utilizing a cre-conditional constitutively active intracellular Notch1 knock-in I define, in vivo, that ectopic Notch1 activation is sufficient to inhibit ductal outgrowth, cause the formation of alveolar-like cell accumulations, and promote Elf5+/ER- cell fate, at the expense of ER+ cell fate, in the mammary gland of pubescent mice. Furthermore, ectopic Notch1 in the pregnant mammary gland is sufficient to promote the formation of pregnancy/lactation-dependent lactating adenomas. These lactating adenomas consist of differentiated secretory cells and normally regress during involution but progress into non-regressing tumours after multiple pregnancies. These lactating adenomas exhibit decapitation secretions characteristic of apocrine differentiation. Together these results suggest that Notch1 may function to promote Elf5+/ER- cell fate and may be misregulated in pregnancy-associated masses and apocrine-carcinoma of the breast in humans.

No description available.

Ductal carcinoma in situ (DCIS) has been shown to be a precursor to invasive ductal cancer (IDC). Though the progression of DCIS to IDC is believed to be an important aspect of tumor aggressiveness, prognosis and molecular markers that predict progression are poorly understood. Therefore, determining the mechanisms by which some DCIS progress is critical for future breast cancer diagnostics and treatment. Singleminded-2s (SIM2s) is a member of the bHLH/PAS family of transcription factors and a key regulator of differentiation. SIM2s is highly expressed in mammary epithelial cells and lost in breast cancer. Loss of Sim2s causes aberrant mouse mammary development with features suggestive of malignant transformation, whereas over-expression of Sim2s promotes precocious alveolar differentiation, suggesting that Sim2s is required for establishing and enhancing mammary gland differentiation. We hypothesize that SIM2s expression must be lost in premalignant lesions for breast cancer to develop. We first analyzed Sim2s in the involuting mammary gland, which is a highly tumorpromoting environment. Sim2s is down-regulated during involution, and forced expression delays involution. We then analyzed SIM2s expression in human breast cancer samples and found that SIM2s is lost with progression from DCIS to IDC, and this loss correlates with metastasis. SIM2s expression in DCIS promoted a differentiated phenotype and suppressed genes associated with de-differentiation. Furthermore, loss of SIM2s expression in DCIS xenografts increased metastasis likely due to an increase in hedgehog signaling and matrix metalloproteinase expression. Interestingly, we found metabolic shifts with gain and loss of SIM2s in not only DCIS cells, but also MCF7 and SUM159 cells. SIM2s expression decreased aerobic glycolysis and promoted oxidative phosphorylation through direct upregulation of CDKN1a and senescence. Loss of SIM2s, conversely, promotes mitochondrial dysfunction and induction of the Warburg effect. This is the first time CDKN1a and cellular senescence have been indicated as causative to metabolic shifts within cancer cells. These studies show a new role for SIM2s in metabolic homeostasis, and this regulation is lost during tumorigenesis. These data indicate SIM2s is at the apex where aging, metabolism, and disease meet – regulating the delicate relationship between the three.

The fps proto-oncogene encodes a 92 kDa cytoplasmic tyrosine kinase. Previous studies have shown that Fps expression in the mammary gland changes with development, and Fps has a suppressor function in mammary tumorigenesis. The aim of my thesis was to elucidate the role of the Fps tyrosine kinase in regulating mammary gland development and function. We have shown that the expression of the Fps kinase in the mammary gland increased during pregnancy and reached its maximum during lactation. The level of Fps tyrosine phosphorylation paralleled the expression pattern. Pups reared by fps-null females gained weight more slowly than those reared by wild-type females. Epithelial cells were the primary source of Fps expression. Milk protein and fat content were not affected by the absence of Fps. Similarly, no differences in mammary gland structure were observed with whole mount or histological analysis. Fps was shown to be in a multi-protein complex with E-cadherin, β-catenin and p120-catenin. A strong co-localization signal was observed for Fps and E-cadherin. Immunofluorescence analysis indicated that the localization of E-cadherin and β-catenin was disorganized and less concentrated at sites of cell-cell contacts in the fps-null glands. The interactions between the different adherens junction components were altered in the fps-null tissue. Specifically, less E-cadherin and β-catenin was associated with p120-catenin in the fps-null glands. Suprisingly, no phosphotyrosine differences were detected for the adherens junction components. Conditions were established to grow primary murine epithelial cell cultures that could be used to investigate the function of Fps. Fps expression was up-regulated in these cells in response to lactogenic hormones. A lentiviral system encoding a murine p53 shRNA sequence was used to increase the growth potential of the primary cells. Continual growth of the infected and uninfected primary epithelial cell mixture resulted in the establishment of an immortalized cell line. Immunofluorescent and immunoblot analyses revealed that the cells have undergone an epithelial-to-mesenchymal transition. With the transduction of a myc-epitope tagged Fps into the cells, we have generated cell lines with the appropriate genetic backgrounds to study the function of the Fps kinase in the mammary gland, specifically as it relates to tumorigenesis. / Thesis (Ph.D, Pathology & Molecular Medicine) -- Queen's University, 2008-07-03 11:53:01.135

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.

Calcium is the major mineral component of milk and is essential for neonatal development. To enrich the milk, calcium must pass from the maternal bloodstream, through mammary epithelial cells, into the alveolar lumen. While calcium extrusion from the epithelial cells is well characterized, no calcium channel or transporter has been identified as the major conduit for calcium to enter the mammary epithelial cell from the bloodstream. A major aim of this thesis was to identify a calcium channel or channels responsible for calcium influx into mammary epithelial cells during lactation. Real time reverse transcription-polymerase chain reaction was used to investigate in vivo expression of calcium channels in the murine mammary gland at the four main stages of mammary gland development. The store-operated calcium channel Orai1 was upregulated during lactation relative to its expression in the nulliparous gland. The classic ORAI1 regulator Stim1 was not similarly overexpressed during lactation, however, its isoform Stim2 was modestly upregulated. HC11 murine mammary cells were used as a model to further investigate the role of STIM2 on calcium handling during lactation. siRNA knockdown of Stim2 reduced both basal and agonist-induced peak cytosolic calcium levels, indicative of its role in calcium regulation. In addition to investigating the role of calcium channels in normal mammary development, their role in breast cancer was examined. Real time reverse transcription-polymerase chain reaction was used to identify calcium channels upregulated in human breast cancer cell lines, relative to non-tumorigenic mammary cell lines. TRPV1, TRPV6, and ORAI1 were upregulated in the breast cancer cell lines. Pharmacological modulation of ORAI1 resulted in modest changes in proliferation, but as there was no specific ORAI1 inhibitor, this effect could not be conclusively attributed to ORAI1 inhibition. siRNA was used to specifically target ORAI1 in three human breast cancer cell lines: MCF-7, MDA-MB-231, and T-47D. siRNA knockdown of ORAI1 was specific and potent, and reduced cell viability and altered calcium handing in all three cell lines. The alterations caused by ORAI1 knock down were not related to the expression of the genes CDK2 and FOS, as these did not change upon ORAI1 knockdown. Data mining was performed using the National Center for Biotechnology Information’s (NCBI’s) expressed sequence tag (EST) database, dbEST, and the Oncomine database. ORAI1 was elevated in estrogen receptor negative breast cancers and in the basal breast cancer molecular subtype, a subtype that has a poor prognosis. Other data suggested that breast cancer cells with high STIM1 and low STIM2 expression also correlated with the basal breast cancer subtype. These data indicate that ORAI and STIM proteins have a role in the physiological process of lactation as well as in the regulation of tumorigenic pathways in the breast, and particular gene expression profiles may be predictors of disease prognosis.

Calcium is the major mineral component of milk and is essential for neonatal development. To enrich the milk, calcium must pass from the maternal bloodstream, through mammary epithelial cells, into the alveolar lumen. While calcium extrusion from the epithelial cells is well characterized, no calcium channel or transporter has been identified as the major conduit for calcium to enter the mammary epithelial cell from the bloodstream. A major aim of this thesis was to identify a calcium channel or channels responsible for calcium influx into mammary epithelial cells during lactation. Real time reverse transcription-polymerase chain reaction was used to investigate in vivo expression of calcium channels in the murine mammary gland at the four main stages of mammary gland development. The store-operated calcium channel Orai1 was upregulated during lactation relative to its expression in the nulliparous gland. The classic ORAI1 regulator Stim1 was not similarly overexpressed during lactation, however, its isoform Stim2 was modestly upregulated. HC11 murine mammary cells were used as a model to further investigate the role of STIM2 on calcium handling during lactation. siRNA knockdown of Stim2 reduced both basal and agonist-induced peak cytosolic calcium levels, indicative of its role in calcium regulation. In addition to investigating the role of calcium channels in normal mammary development, their role in breast cancer was examined. Real time reverse transcription-polymerase chain reaction was used to identify calcium channels upregulated in human breast cancer cell lines, relative to non-tumorigenic mammary cell lines. TRPV1, TRPV6, and ORAI1 were upregulated in the breast cancer cell lines. Pharmacological modulation of ORAI1 resulted in modest changes in proliferation, but as there was no specific ORAI1 inhibitor, this effect could not be conclusively attributed to ORAI1 inhibition. siRNA was used to specifically target ORAI1 in three human breast cancer cell lines: MCF-7, MDA-MB-231, and T-47D. siRNA knockdown of ORAI1 was specific and potent, and reduced cell viability and altered calcium handing in all three cell lines. The alterations caused by ORAI1 knock down were not related to the expression of the genes CDK2 and FOS, as these did not change upon ORAI1 knockdown. Data mining was performed using the National Center for Biotechnology Information’s (NCBI’s) expressed sequence tag (EST) database, dbEST, and the Oncomine database. ORAI1 was elevated in estrogen receptor negative breast cancers and in the basal breast cancer molecular subtype, a subtype that has a poor prognosis. Other data suggested that breast cancer cells with high STIM1 and low STIM2 expression also correlated with the basal breast cancer subtype. These data indicate that ORAI and STIM proteins have a role in the physiological process of lactation as well as in the regulation of tumorigenic pathways in the breast, and particular gene expression profiles may be predictors of disease prognosis.