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

ErbB2/Neu overexpression is observed in 20--30% of human mammary carcinomas and correlates with poor prognosis. We have demonstrated that four ErbB2/Neu tyrosine autophosphorylation sites (YB, YC, YD and YE) are sufficient to mediate transforming signals in vitro and bind distinct adapter proteins, suggesting that transformation functions through distinct pathways. To study the role of each individual tyrosine autophosphorylation site in mammary tumourigenesis, we derived transgenic mice expressing mutant ErbB2/Neu receptors in the mammary gland. Recently, we showed that YB and YD female transgenic mice developed mammary tumours with differences in tumour latency, morphology, and metastatic potential. To further understand the role of the autophosphorylation sites, I characterized the YC and YE transgenic mouse models and showed that although, they exhibit similar phenotypes, they also differ in their latency, morphology and metastatic rate compared to the YB and YD transgenic mouse models. This suggests that recruitment of specific adaptor proteins has distinct biological effects on ErbB2/Neu-mediated mammary tumourigenesis.

Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal

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.

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.

Licentiatavhandling (sammanfattning) Linköping : Linköpings universitet, 2007. / Härtill 3 uppsatser.

Thesis (Ph.D.) -- McMaster University, 1997. / Includes bibliographical references (leaves 204-216). Also available via World Wide Web.

Diss. (sammanfattning) Uppsala : Sveriges lantbruksuniversitet, 2005. / Härtill 5 uppsatser.