As a barrier to metastases, cells normally undergo apoptosis after they lose contact with their extra cellular matrix or their neighbouring cells. This cell death process has been termed “anoikis”. Tumour cells that acquire malignant potential have developed mechanisms to resist anoikis and thereby survive after detachment from their primary site and while travelling through the lymphatic and circulatory systems. The understanding of the molecular regulators of anoikis resistance will allow for a better understanding of the metastatic process and the development of novel anti-metastatic therapeutics. To better determine the molecular underpinnings of anoikis resistance, we have used both chemical biology and genetic approaches. Using chemical biological approaches such as small molecule screens, we determined that both FLIP and Na+/K+ ATPase could modulate a cell’s response to anoikis. Through the use of a shRNA genome wide lentiviral screen we determined that ABHD4 was able to inhibit a cell’s response to anoikis. We also showed the importance of anoikis resistance in the ability of malignant cancer cells to survive in circulation. By decreasing a cell’s ability to resist anoikis, one is able to decrease the ability of a cancer cell to survive in circulation and form tumours in distant organs. Taken together, we have identified novel regulators of anoikis resistance and demonstrated the importance of anoikis in metastatic progression, which may lead to the development of novel treatments for metastatic cancers.

Septins are a family of guanine nucleotide-binding proteins that function in eukaryotic cell division, where they form a high-order cortical structure at the site of division, which is essential in most eukaryotes. Expanded roles have evolved for septins in metazoans, where they also have essential functions in terminally-differentiated cell types, such as neurons and spermatozoa. Specific details of septin function are lacking in most roles described, due at least in part to the limited number of characterized binding partners. In this work, yeast two-hybrid screens and pull-downs from tissue homogenate were used to identify novel septin binding partners for subsequent characterization. The neuron-enriched septin, SEPT5, interacted directly with SUMO E3 ligases of the PIAS family. However, I was not able to demonstrate endogenous sumoylation of SEPT5 and SUMO isoforms did not concentrate with the septins during cytokinesis. SEPT5 also interacted with a novel septin, SEPT12, which I further characterized to be testis-specific and localized to the annulus in mature spermatozoa. Further, using SEPT12-specific reagents, I determined that the annulus forms via sequestration and subsequent segregation from the Golgi during spermiogenesis. SEPT9 pull-downs identified another novel testis-specific septin, SEPT14. Reagents specific to SEPT2 and SEPT9 also revealed a septin-rich structure in the seminiferous epithelium in close association with the ectoplasmic specialization. The specific role of septins in this structure awaits further characterization. Several other intriguing candidate septin-interaction partners were identified and the further study of their possible in vivo interaction with septins may provide substantial insight into the mechanisms of septin function in eukaryotes.

Assembly of intestinal chylomicron particles (lipid-protein complexes) is the fundamental mechanism by which we absorb dietary fat. Two intestinal lipid transporters, Cluster of Differentiation 36 (CD36) and fatty acid-binding protein 1 (FABP1), have been shown to play a role in lipid absorption, however, it remains unclear how knockdown of these proteins bleads to aberrant intestinal chylomicron secretion. In an enterocyte-like cell culture model, Caco-2 cells, we hypothesized that knockdown of CD36 or FABP1 using short-hairpin RNA interference techniques would impair triacylglycerol (TG) and apolipoprotein B (apoB) secretion. Surprisingly, knockdown of these lipid transporters lead to an increase in TG and apoB secretion that was associated with an increase in fatty acid synthase and fatty acid transport protein 4 (FATP4) protein levels. De novo fatty acid synthesis was slightly increased in CD36-, but not FABP1-knockdown Caco-2 cells. This study highlights the importance of fatty acid targeting in regulating chylomicron production.

Bone marrow-derived progenitor cell populations have been implicated in tissue regeneration and also in human disease pathology. This thesis investigated the hypothesis that Clara Cell Secretory Protein positive (CCSP+) epithelial-like progenitor cells and circulating fibrocyte numbers are altered in human lung disease and injury, and aimed to determine the predictive value of these cell profiles. It was found that cystic fibrosis patients have an increased number of CCSP+ cells in their bone marrow and peripheral blood, while patients with bronchiolitis obliterans syndrome (BOS) have a decreased number. In addition, BOS and pulmonary fibrosis patients have increased circulating fibrocytes. In response to ischemia reperfusion injury, an increase in CCSP+ cells in the peripheral blood was found at 24 hrs following lung transplant. Lastly, in patients studied greater than 1-year from transplant, those diagnosed with BOS had a higher number of fibrocytes and a loss of CCSP+ peripheral blood cells when compared to patients with stable lung function, with increased fibrocytes being associated with time post-transplant. In these patients, the ratio of fibrocytes-to-CCSP+ cells was predictive of lung function. Multiplex protein arrays were used to investigate corresponding patient plasma, aiming to elucidate key mediators of progenitor cell recruitment. While differences in various cytokines were found between end-stage diseases, a specific relationship between Stem Cell Growth Factor- and CCSP+ cells was identified and between Monocyte Chemotactic Protein-1 and fibrocytes. Conversely, response of CCSP+ cells following transplant appears to be mediated by known mobilizing factors SDF-1 and GM-CSF. Interestingly, in patients followed long-term after transplant, MCP-1 was associated with the number of CCSP+ cells, while SDF-1 correlated with fibrocyte numbers. These observations suggest common pathways acting on both populations that may be altered by the microenvironment, and may further suggest a common origin. This work contributes important information regarding changes in lung progenitor cells and their association with human disease and tissue repair, which could ultimately support future directions that directly advance therapy and improve patient care.

Septins are a family of guanine nucleotide-binding proteins that function in eukaryotic cell division, where they form a high-order cortical structure at the site of division, which is essential in most eukaryotes. Expanded roles have evolved for septins in metazoans, where they also have essential functions in terminally-differentiated cell types, such as neurons and spermatozoa. Specific details of septin function are lacking in most roles described, due at least in part to the limited number of characterized binding partners. In this work, yeast two-hybrid screens and pull-downs from tissue homogenate were used to identify novel septin binding partners for subsequent characterization. The neuron-enriched septin, SEPT5, interacted directly with SUMO E3 ligases of the PIAS family. However, I was not able to demonstrate endogenous sumoylation of SEPT5 and SUMO isoforms did not concentrate with the septins during cytokinesis. SEPT5 also interacted with a novel septin, SEPT12, which I further characterized to be testis-specific and localized to the annulus in mature spermatozoa. Further, using SEPT12-specific reagents, I determined that the annulus forms via sequestration and subsequent segregation from the Golgi during spermiogenesis. SEPT9 pull-downs identified another novel testis-specific septin, SEPT14. Reagents specific to SEPT2 and SEPT9 also revealed a septin-rich structure in the seminiferous epithelium in close association with the ectoplasmic specialization. The specific role of septins in this structure awaits further characterization. Several other intriguing candidate septin-interaction partners were identified and the further study of their possible in vivo interaction with septins may provide substantial insight into the mechanisms of septin function in eukaryotes.

Bone marrow-derived progenitor cell populations have been implicated in tissue regeneration and also in human disease pathology. This thesis investigated the hypothesis that Clara Cell Secretory Protein positive (CCSP+) epithelial-like progenitor cells and circulating fibrocyte numbers are altered in human lung disease and injury, and aimed to determine the predictive value of these cell profiles. It was found that cystic fibrosis patients have an increased number of CCSP+ cells in their bone marrow and peripheral blood, while patients with bronchiolitis obliterans syndrome (BOS) have a decreased number. In addition, BOS and pulmonary fibrosis patients have increased circulating fibrocytes. In response to ischemia reperfusion injury, an increase in CCSP+ cells in the peripheral blood was found at 24 hrs following lung transplant. Lastly, in patients studied greater than 1-year from transplant, those diagnosed with BOS had a higher number of fibrocytes and a loss of CCSP+ peripheral blood cells when compared to patients with stable lung function, with increased fibrocytes being associated with time post-transplant. In these patients, the ratio of fibrocytes-to-CCSP+ cells was predictive of lung function. Multiplex protein arrays were used to investigate corresponding patient plasma, aiming to elucidate key mediators of progenitor cell recruitment. While differences in various cytokines were found between end-stage diseases, a specific relationship between Stem Cell Growth Factor- and CCSP+ cells was identified and between Monocyte Chemotactic Protein-1 and fibrocytes. Conversely, response of CCSP+ cells following transplant appears to be mediated by known mobilizing factors SDF-1 and GM-CSF. Interestingly, in patients followed long-term after transplant, MCP-1 was associated with the number of CCSP+ cells, while SDF-1 correlated with fibrocyte numbers. These observations suggest common pathways acting on both populations that may be altered by the microenvironment, and may further suggest a common origin. This work contributes important information regarding changes in lung progenitor cells and their association with human disease and tissue repair, which could ultimately support future directions that directly advance therapy and improve patient care.

Assembly of intestinal chylomicron particles (lipid-protein complexes) is the fundamental mechanism by which we absorb dietary fat. Two intestinal lipid transporters, Cluster of Differentiation 36 (CD36) and fatty acid-binding protein 1 (FABP1), have been shown to play a role in lipid absorption, however, it remains unclear how knockdown of these proteins bleads to aberrant intestinal chylomicron secretion. In an enterocyte-like cell culture model, Caco-2 cells, we hypothesized that knockdown of CD36 or FABP1 using short-hairpin RNA interference techniques would impair triacylglycerol (TG) and apolipoprotein B (apoB) secretion. Surprisingly, knockdown of these lipid transporters lead to an increase in TG and apoB secretion that was associated with an increase in fatty acid synthase and fatty acid transport protein 4 (FATP4) protein levels. De novo fatty acid synthesis was slightly increased in CD36-, but not FABP1-knockdown Caco-2 cells. This study highlights the importance of fatty acid targeting in regulating chylomicron production.

Kawasaki disease (KD) is an acute inflammatory disease characterized by persistent inflammation of the coronary arteries. KD is characterized by the release of cytokines such as tumor necrosis factor alpha (TNFα) and is thought to be initiated by a superantigen (SAg). The Lactobacillus casei cell wall extract model of KD demonstrates a critical requirement for TNFα and its receptor during pathogenesis, although the precise effect of TNFα is unknown. A persistent T cell infiltrate in the coronary artery disagrees with established fates of SAg activated cells, which undergo apoptosis. In this work, TNFα was found to promote the survival of SAg-reactive T cells. The results demonstrate that TNFα regulates B7.2 molecule expression on antigen presenting cells, and that TNFα indirectly promotes the survival of SEB-stimulated T cells by driving costimulation. These observations demonstrate how TNFα prevents T cell apoptosis and lend support to KD therapies which target TNFα and B7.

Robust bipolar spindle formation and faithful transmission of genetic material are vital to the maintenance of genome integrity and cellular homeostasis. Chromosome segregation errors can result in aneuploidy, a hallmark of human solid tumors. The assembly of a microtubule-based mitotic spindle relies on the concerted action of centrosomes, spindle microtubules, molecular motors and nonmotor spindle proteins. Before mitosis, centrosomes need to duplicate and increase in size in order to gain sufficient microtubule nucleation activity during bipolar spindle formation. This process is called centrosome maturation and coincides with a dramatic change of centrosome structure. However, the architecture of centrosomes and the organization of centrosome components in both interphase and mitosis have long remained elusive. In this thesis, I describe the identification and characterization of novel regulators that are essential for centrosome and mitotic spindle organization in human cells. One such regulator is human Augmin, an evolutionarily conserved eight-subunit protein complex that has essential functions for centrosome and spindle integrity. I present evidence that human Augmin promotes microtubule-dependent nucleation of microtubules by targeting microtubule-nucleating complexes to the mitotic spindle. This function of Augmin is important for generation and/or stabilization of kinetochore microtubules within the mitotic spindle, and its loss results in destabilization of kinetochore microtubules and spindle assembly errors. These errors culminate in cells displaying multipolar spindles with fragmented centrosomes and mitotic arrest. A second regulator of centrosome and spindle organization described in this thesis is CEP192. I show that CEP192 is critical for recruitment of microtubule-nucleating complexes to centrosomes and, consequently, for centrosome maturation, mitotic spindle formation, and centriole duplication. Finally, I describe novel organizational features of the centrosome using a subdiffraction microscopy approach. Because of a lack of higher-order structural information, centrosomes have traditionally been described as amorphous clouds. My results now reveal that centrosome components instead occupy separable spatial domains throughout the cell cycle and highlight the role of higher-order protein organization in the regulation of centrosome assembly and function. Collectively, this work has significantly expanded our current knowledge of centrosome architecture and biogenesis and of the mechanisms that underlie robust bipolar spindle assembly.

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.