Breast cancer is the most common cancer and the leading cause of cancer-related death among Canadian women. Despite improvements in treatment and early detection, there is still a need to develop novel therapies for breast cancer management. Aberrant Notch signaling is tumorigenic and is associated with poor clinical outcomes in breast cancer, as well as in several other types of cancer. Activation of Notch signaling requires -secretase-mediated Notch receptor cleavage. Thus, strategies to inhibit Notch signaling, including -secretase inhibition, are being evaluated for potential anti-tumor effects. The strongest justification for targeting Notch in breast cancer, and more specifically for using -secretase inhibitors, came from two studies that reported that the -secretase inhibitor (GSI) Z-LLNle-CHO inhibited the growth of breast cancer cells both in vitro and in vivo without causing significant side effects. In Chapter 2, we compared the enzymatic activities and cytotoxicity of Z-LLNle-CHO to those of two other specific GSIs and three proteasome inhibitors and demonstrated that the cytotoxicity of Z-LLNle-CHO in breast cancer cells is mediated by proteasome inhibition, not by -secretase inhibition. In Chapter 3, we characterized the protein complexes formed in breast cancer cells by the intracellular domains (NICD) of the four Notch paralogs. We found that the assembly of NICD protein complexes is dose-dependent and availability of MAML proteins becomes the limiting factor for continuous formation of NICD/RBPj/MAML transactivation complex. This suggests that the formation of some non-canonical NICD complex might occur preferentially at high levels of NICD, conditions under which aberrant Notch signaling induces tumorigenesis in breast cancer. Consequently, these non-canonical interactions might be good targets to specifically block oncogenic, but not physiological, Notch signaling. In addition, we found that the relative affinities of individual NICD paralogs to several known NICD-interacting proteins were different. This may account for the paralog-specific activities of Notch that have been previously reported. Together, these results may be of value for the development of new reagents to block Notch signaling for therapeutic benefit in breast cancer treatment. / Experimental Oncology

Epsin is an endocytic protein that binds Clathrin, the plasma membrane, Ubiquitin, and also a variety of other endocytic proteins through well-characterized motifs. Although Epsin is a general endocytic factor, genetic analysis in Drosophila and mice revealed that Epsin is essential specifically for internalization of ubiquitinated transmembrane ligands of the Notch receptor, a process required for Notch activation. How Epsin promotes ligand endocytosis and thus Notch signaling is unclear. Here, by generating Drosophila lines containing transgenes that express a variety of different Epsin deletion and substitution variants, I tested each of the five protein or lipid interaction modules of Epsin for a role in Notch activation by each of the two Drosophila ligands, Serrate and Delta. here are five main results of this work that impact present thinking about endocytic machinery/Epsin, Epsin/ligand, or ligand/receptor interactions at the plasma membrane. First, I discovered that deletion or mutation of both UIMs destroys Epsin’s function in Notch signaling and has a greater negative effect on Epsin’s ability to function than removal of any other module type. Second, only one of the two UIMs of Epsin is essential. Third, the lipid-binding function of the ENTH domain is required for maximal Epsin activity. Fourth, although the C-terminal Epsin modules that interact with Clathrin, the adapter protein complex AP-2, or endocytic accessory proteins are necessary collectively for Epsin activity, their functions are highly redundant. Finally, I detected no ligand-specific requirements for Epsin modules. Most unexpected was the finding that Epsin’s Clathrin binding motifs were dispensable. All of these observations are consistent with a model where Epsin’s essential function in ligand cells is to link ubiquitinated Notch ligands to Clathrin-coated vesicles through other Clathrin adapter proteins. / text

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Effect of Manipulation of Notch Signaling Pathway on Neural Stem Cell Proliferation in the Hippocampus Following Traumatic Brain Injury By Seung L. Kim A thesis statement submitted for degree requirement in Mater of Science Virginia Commonwealth University, 2019 Advisor: Dong Sun, MD. PhD. Department of Anatomy & Neurobiology The Notch signaling pathway is known as a core signaling system in maintaining neural stem cells (NSCs) in embryonic development and adulthood including cell proliferation, maturation, and cell fate decision. Proliferation of NSCs persists throughout lifespan in neurogenic niches and is often upregulated following neurological insults including traumatic brain injury (TBI). Therefore, NSCs are viewed as the brain’s endogenous source for repair and regeneration. We speculate Notch signaling pathway is also involved in injury-induced cell proliferation in the neurogenic niche following TBI. TBI, which is a leading cause of death and disability, has been a huge burden to our society. Many efforts have been made in attempt to treat and manage TBI. In this study, we examined the involvement of Notch signaling pathway in injury induced NSC proliferation in the neurogenic niche, by administering exogenous Notch ligands including, Notch agonist or antagonist. Adult rats were intraventricularly infused with Notch1 receptor agonists (anti-Notch1 antibody at the dose of 0.5, 2 or 4μg/ml), Notch1 receptor antagonist (recombinant Jagged1 fusion protein at the dose of 25, 50 or 100μg/ml) or vehicle for 7 days following TBI. 5-bromo-2-deoxyuridine (BrdU) was administered single daily via intraperitoneal injection to label proliferating cells for 7 days post injury. The animals were sacrificed on the 7th day at 2 hours after the last BrdU injection. Sequential vibratome sliced coronal brain sections were processed for proliferation marker BrdU, Ki67 or immature neuronal marker DCX staining. BrdU, Ki67 or DCX-labeled cells in the dentate gyrus of the hippocampus were quantified using unbiased stereological method. We found TBI in the form of moderate lateral fluid percussion injury (LFPI) induced cell proliferation was further augmented by 7-day infusion of Notch agonist (Notch1-2μg/ml) as shown by BrdU and Ki67 labeling. Further, 7-day infusion of Notch antagonist (Jagged1-50μg/ml) post-injury greatly reduced the number of BrdU+ cells. However, ambiguous dose related responses were also observed where 7-day infusion of higher dose of Notch agonist (Notch1-4μg/ml) resulted in reduced cell proliferation. No major changes in the numbers of newly generated neurons were observed across the animals, except a slight reduction in Notch agonist (Notch1-2μg/ml) and Notch antagonist (Jagged1-50μg/ml) infused animals as shown by DCX labeling. Infusion of Notch agonist or antagonist affects NSC proliferation following TBI suggesting the involvement of Notch signaling pathway in regulating post-TBI NSC proliferation in the neurogenic niche. For the unexpected opposite results of higher dosing of Notch 1 agonist, the presence of other Notch receptors regulating NSC in the neurogenic niche should be considered. Future studies involving selective manipulation of these Notch receptors and their downstream effectors would clear some results.

Pax7 is a paired box transcription factor expressed by all satellite cells which are critically required for muscle regeneration and growth. The absolute requirements of Pax7 in the maintenance of the satellite cell pool are widely acknowledged. However the mechanisms by which Pax7 executes muscle regeneration or contributes to satellite cell homeostasis remain elusive. We performed cell and molecular analysis of Pax7 null satellite cells to investigate muscle stem cell function. Through genome wide studies, we found that genes involved in cell cell interactions, regulation of migration, control of lipid metabolism and inhibition of myogenic differentiation were significantly perturbed in Pax7 null satellite cells. Analysis of satellite cells in vitro showed that Pax7 null satellite cells undergo precocious myogenic differentiation and have perturbed expression of genes involved in the Notch signaling pathway. We showed that Notch 1 is a novel Pax7 target gene and by using a genetic approach we demonstrated that ectopic expression of the constitutively active intracellular domain of Notch1 (NICD1) in Pax7 null satellite cells is sufficient to maintain the satellite cell pool as well as to restore their proliferation. Instead of differentiating into myogenic cells and in the absence of a myogenic cue, NICD1 Pax7 null satellite cells become a source of ectopic brown fat within muscles and give rise to brown adipocytes both in vivo and in vitro. In conclusion we showed that Notch 1 partially rescues Pax7 deficient satellite cells loss and proliferation. Additionally we provide the first evidence that Notch signalling contributes to satellite cell fate by inhibiting terminal myogenic differentiation and inducing brown adipogenesis.

A common bipotent thymocyte precursor gives rise to both lineages of T cells, αβ and γδ. This thesis addresses how the interplay between intrinsic T cell receptor (TCR) signals and cell extrinsic signals provided by Notch and TCR ligands help to assign and support a final lineage fate decision. Emerging data supports a model in which differential TCR signaling capacity plays an instructional role in specifying lineage fate, particularly through induction of the ERK - early growth response gene (Egr) - inhibitor of DNA binding 3 (Id3) pathway. In particular, Id3 expression serves to regulate adoption of the γδ fate. Moreover, Id3 is both necessary and sufficient to enable γδ-lineage cells to differentiate independently of Notch signaling and become competent interferon (IFN)-γ-producing effectors. These findings identify Id3 as a central player that controls both adoption of the γδ fate and their maturation in the thymus. While loss of Notch signaling in γδTCR-expressing CD4-CD8- (DN)3 cells does not affect development, Notch signals are critical for pre-TCR-bearing cells to transition to the CD4+CD8+ (DP) stage of αβ T cell development. Notch signals affect the activation of the PI3K/Akt pathway, which is required for pTα/TCRβ (pre-TCR)-induced survival, differentiation and proliferation of developing αβ-lineage thymocytes. Here, I identify the key molecular players responsible for the interaction between the Notch and PI3K pathways at this critical developmental stage. Notch induction of Hes1 expression is necessary to repress the expression of the PI3K/Akt pathway inhibitor, PTEN, which in turn facilitates pre-TCR-induced differentiation. c-Myc, another critical target of Notch, is required for proliferation during β-selection. Lastly, I find that the majority of DN3 cells expressing both pre-TCR and γδTCR follow the signal strength model for lineage development, and commit and mature along the γδ-lineage. However, manipulation of signal strength, through γδTCR ligand availability or Id3 expression, can skew this development outcome. Taken together, the results from this thesis provide a detailed examination of the molecular mechanisms that are instrumental in determining lineage fate, survival, and proliferation of differentiating thymocytes. Central to these outcomes is the interplay between the Notch, TCR, PI3K, and MAPK signaling pathways.

Glioblastoma is the commonest form of brain neoplasm and among the most malignant forms of cancer. The identification of a subpopulation of self-renewing and multipotent cancer stem cells within glioblastoma has revealed a novel cellular target for the treatment of this disease. The role of developmental cell signaling pathways in these cell populations remains poorly understood. Herein, we examine the role of the Notch signaling pathway in glioblastoma stem cells. In this thesis we have demonstrated that the canonical Notch pathway is active in glioblastoma stem cells and functions to inhibit neuronal lineage commitment in a subset of patient derived glioblastoma stem cells in vitro. Gamma secretase (γ-secretase) small molecule inhibitors or dominant-negative co-activators inhibit glioblastoma stem cell proliferation and induce neuronal lineage commitment in a fashion that synergizes with Wingless pathway activation via GSK-3β blockade. Our data suggest that subsets of patient samples show a Notch gene expression profile that predicts their abilities to undergo neuronal lineage differentiation in response to γ-secretase small molecule inhibitors. Additionally, the data suggests that Notch may perturb the relative fractions of cells undergoing symmetric division, in favour of asymmetric division, limiting clonal expansion from single cells. These data may have important implications for treating human glioblastoma, and suggest that in addition to inhibition of proliferation, influencing lineage choice of the tumor stem cells may be a mechanism by which these tumors may be pharmacologically inhibited.

Glioblastoma is the commonest form of brain neoplasm and among the most malignant forms of cancer. The identification of a subpopulation of self-renewing and multipotent cancer stem cells within glioblastoma has revealed a novel cellular target for the treatment of this disease. The role of developmental cell signaling pathways in these cell populations remains poorly understood. Herein, we examine the role of the Notch signaling pathway in glioblastoma stem cells. In this thesis we have demonstrated that the canonical Notch pathway is active in glioblastoma stem cells and functions to inhibit neuronal lineage commitment in a subset of patient derived glioblastoma stem cells in vitro. Gamma secretase (γ-secretase) small molecule inhibitors or dominant-negative co-activators inhibit glioblastoma stem cell proliferation and induce neuronal lineage commitment in a fashion that synergizes with Wingless pathway activation via GSK-3β blockade. Our data suggest that subsets of patient samples show a Notch gene expression profile that predicts their abilities to undergo neuronal lineage differentiation in response to γ-secretase small molecule inhibitors. Additionally, the data suggests that Notch may perturb the relative fractions of cells undergoing symmetric division, in favour of asymmetric division, limiting clonal expansion from single cells. These data may have important implications for treating human glioblastoma, and suggest that in addition to inhibition of proliferation, influencing lineage choice of the tumor stem cells may be a mechanism by which these tumors may be pharmacologically inhibited.

A common bipotent thymocyte precursor gives rise to both lineages of T cells, αβ and γδ. This thesis addresses how the interplay between intrinsic T cell receptor (TCR) signals and cell extrinsic signals provided by Notch and TCR ligands help to assign and support a final lineage fate decision. Emerging data supports a model in which differential TCR signaling capacity plays an instructional role in specifying lineage fate, particularly through induction of the ERK - early growth response gene (Egr) - inhibitor of DNA binding 3 (Id3) pathway. In particular, Id3 expression serves to regulate adoption of the γδ fate. Moreover, Id3 is both necessary and sufficient to enable γδ-lineage cells to differentiate independently of Notch signaling and become competent interferon (IFN)-γ-producing effectors. These findings identify Id3 as a central player that controls both adoption of the γδ fate and their maturation in the thymus. While loss of Notch signaling in γδTCR-expressing CD4-CD8- (DN)3 cells does not affect development, Notch signals are critical for pre-TCR-bearing cells to transition to the CD4+CD8+ (DP) stage of αβ T cell development. Notch signals affect the activation of the PI3K/Akt pathway, which is required for pTα/TCRβ (pre-TCR)-induced survival, differentiation and proliferation of developing αβ-lineage thymocytes. Here, I identify the key molecular players responsible for the interaction between the Notch and PI3K pathways at this critical developmental stage. Notch induction of Hes1 expression is necessary to repress the expression of the PI3K/Akt pathway inhibitor, PTEN, which in turn facilitates pre-TCR-induced differentiation. c-Myc, another critical target of Notch, is required for proliferation during β-selection. Lastly, I find that the majority of DN3 cells expressing both pre-TCR and γδTCR follow the signal strength model for lineage development, and commit and mature along the γδ-lineage. However, manipulation of signal strength, through γδTCR ligand availability or Id3 expression, can skew this development outcome. Taken together, the results from this thesis provide a detailed examination of the molecular mechanisms that are instrumental in determining lineage fate, survival, and proliferation of differentiating thymocytes. Central to these outcomes is the interplay between the Notch, TCR, PI3K, and MAPK signaling pathways.

Notch signals are required to promote T lineage commitment and development and suppress alternative cell fates in the thymus. Although the Notch activating ligand(s) in the thymus is(are) not known, studies have shown that hematopoietic progenitors are sensitive to Delta-like (DL), but not Jagged (Jag)-type ligands. In Chapter 3, I show that DL-expressing bone marrow stromal cell lines exhibit Notch ligand-independent functional heterogeneity in their capacity to support T cell development in vitro. These findings thus suggest the existence of stromal cell-derived signals that work with Notch to support T cell development. In Chapters 4 and 5, I investigated the ability of Fringe proteins to modulate Notch ligand-receptor interactions and the developmental consequences of these interactions for hematopoetic progenitors. Fringe proteins are glycosyl-transferases that enhance Notch activation by DL ligands and inhibit Notch activation by Jag ligands. In Chapter 4 I show that Lunatic Fringe (Lfng) enhances the strength of DL-mediated Notch activation to drive proliferation and expansion of early thymocytes and that DL4 and DL1 display different potencies to induce Notch-dependent outcomes. In Chapter 5, I demonstrate for the first time in a mammalian system that Lfng and Manic Fringe (Mfng) co-operate to enhance DL-Notch interactions and inhibit Jag-Notch interactions in hematopoietic stem cells. Thus, Lfng and Mfng function together to induce T cell development and inhibit B cell, myeloid and NK cell development. Collectively, these data highlight the importance of Fringe proteins in modulating the strength and specificity of Notch signaling levels during hematopoieisis.