Anemia occurs in 60% of patients with Shwachman Diamond Syndrome (SDS). Although bi-allelic mutations in SBDS cause SDS, it is unclear whether SBDS is critical for erythropoiesis and what the pathogenesis of anemia is in SDS. I hypothesize that SBDS protects early erythroid progenitors from p53 family member mediated apoptosis by promoting ribosome biosynthesis and translation. SBDS deficiency by vector-based shRNA led to impaired cell expansion of differentiating K562 cells due to accelerated apoptosis and reduced proliferation. Furthermore, the cells showed general reduction of 40S, 60S, 80S ribosomal subunits, loss of polysomes and impaired global translation during differentiation. An upregulation of the pro-apoptotic p53 family member, TAp73, was found in resting SBDS deficient cells; however, not in differentiating cells. These results demonstrate SBDS plays a critical role in erythroid expansion by promoting survival of early erythroid progenitors and in maintaining ribosome biogenesis during erythroid maturation independently of p53 family members.

Constitutive secretion is responsible for the targeting of transmembrane proteins to the plasma membrane, and for the secretion of extracellular matrix proteins, hormones, and other cellular products. The basic steps of secretion are well understood – proteins synthesized in the endoplasmic reticulum are transported in lipid-bound intermediates to the Golgi, and from the Golgi to the plasma membrane or cell exterior. Dysfunction of the secretory pathway – either constitutive or regulated – is involved in many disease states. One such state is diabetic nephropathy (DN). DN is characterized by renal hypertrophy and fibrosis, and is the leading cause of renal failure worldwide. Our lab had previously shown that munc13 is both upregulated and activated in the diabetic kidney, and that munc13 is an effector of rab34. Study of rab34 in HeLa cells revealed that rab34 is localized to the Golgi, and that it is required for the secretion of the Vesicular Stomatitis Virus glycoprotein. Colocalization experiments, as well as the use of Brefeldin A, localized the effect of rab34 to intra-Golgi transport. Further experiments indicated that glucose-induced upregulation of munc13 in rat mesangial cells increased the rate of constitutive secretion to the plasma membrane, and that this effect depended on its interaction with rab34. Finally, munc13 and rab34 were found to be required for the high glucose-mediated stimulation of Transforming Growth Factor-β secretion from mesangial cells, placing these two proteins at a key point in a pathway of physiological significance in the pathology of DN.

In recent years, research in the area of cancer stem cells has spiked tremendously. Numerous investigators have found that several types of cancers contain a subpopulation of tumor cells that display many defining characteristics of normal tissue stem cells, including multipotent differentiation potential, long-term self-renewal capacity, and expression of molecular markers of stemness. Most importantly, these cancer stem cells (CSCs) have very high tumor initiating potential, a finding that has led to the development of the cancer stem cell model for tumor progression. This model suggests that tumors are organized in a developmental hierarchy (similar to healthy tissue), with long-term tumor progression being driven by self-renewing CSCs at the top of the hierarchy. The CSC model represents a significant shift in our understanding of tumor progression, and as such, it may be possible to expand our knowledge of other aspects of tumor biology by re-examining them in the context of the CSC model. My work focuses on investigating interactions between CSCs and the tumor vasculature. Previous work has demonstrated heterogeneity in the proangiogenic potential of cells in a tumor. Considering the possibility that angiogenesis may be driven by specific subsets of tumor cells, I investigated the contribution of the CSC fraction to tumor angiogenesis. Comparing tumors with low or high CSC fractions, I have found that CSCs contribute to tumor vascular development through promotion of endothelial cell activity and recruitment of bone marrow-derived proangiogenic cells, mediated in part by vascular endothelial growth factor (VEGF) and stromal-derived factor 1 (SDF1). Since some tissue stem cells are known to reside in a vascular niche, I investigated the possibility that CSCs may also be supported by blood vessels in the tumor microenvironment, and that consequently CSCs may be targeted by disruption of tumor vasculature with antiangiogenic therapy. By testing multiple antiangiogenic therapeutic strategies, I have found that antiangiogenic therapy sensitizes CSCs to the effects of cytotoxic chemotherapy. Taken together, my work demonstrates a bi-directional relationship in which CSCs promote tumor vascular development, and tumor vasculature supports and protects CSCs. This work has implications for our understanding of CSC biology, tumor angiogenesis and antiangiogenic therapy, and provides insight into strategies for targeting the critical CSC population.

The tyrosine kinase receptor Tie-2 is expressed on the surface of endothelial cells and is necessary for angiogenesis and vascular stability. To date, the best characterized ligands for Tie-2 are Angiopoietin-1 (Ang-1) and Angiopoietin-2 (Ang-2). Ang-1 has been identified as the main activating ligand for Tie-2 while the role of Ang-2 has been controversial since its discovery; some studies reported Ang-2 as a Tie-2 antagonist while others described Ang-2 as a Tie-2 agonist. The purpose of this thesis was to understand: (1) how the receptor Tie-2 is regulated by Ang-1 and Ang-2 in endothelial cells, (2) to compare the effects of Ang-1 and Ang-2, and (3) to determine the arrangement and distribution of Tie-2 in endothelial cells. The research presented in this thesis indicates that Tie-2 is arranged in variably sized clusters on the endothelial cell surface. Clusters of Tie-2 were expressed on all surfaces of cells: on the apical plasma membrane, on the tips of microvilli, and on the basolateral plasma membrane. When endothelial cells were stimulated with Ang-1, Tie-2 was rapidly internalized and degraded. Upon Ang-1 stimulation, Tie-2 localized to clathrin-coated pits on all surfaces of endothelial cells indicating that one pathway mediating Tie-2 internalization is through clathrin-coated pits. After activation of Tie-2, Ang-1 dissociates from the endothelial cell surface and accumulates in the surrounding medium. When experiments were repeated with Ang-2, it was discovered that Ang-2 induced all of the same effects on Tie-2 as Ang-1 but at a much reduced level and rate, indicating that Ang-2 likely functions as a partial agonist for Tie-2 in endothelial cells. / PhD

Despite the combination of chemotherapy and surgery in osteosarcoma treatment, the survival of patients remains low. The lack of treatment improvement prompted me to investigate deregulated Hedgehog pathway as a potential target for intervention. During development, the Hedgehog pathway is tightly regulated to control organ and tissue development. Specifically, the Indian Hedgehog pathway (IHH) is important for bone development. Aberrantly activated Hedgehog pathway through ligand-dependent or ligand-independent mechanisms has been reported in numerous cancers. Several small molecule antagonists of the Hedgehog pathway are being explored clinically to improve patient outcome. I examined the expression of IHH pathway components, IHH, SMO, PTCH1 and GLI1, in 43 high-grade primary osteosarcoma tumors and 6 cell lines and found various transcript levels of IHH pathway components in osteosarcoma samples. The high levels of co-expressed IHH and targets, PTCH1 and GLI1, in a subset of osteosarcoma samples are indicative of ligand-dependent activation. Trends toward worse survival for patients with higher IHH (positive regulator) levels and toward better survival for patients with higher PTCH1 (negative regulator) levels were observed. Through genetic analyses, PTCH1 inactivating mutations and GLI1 amplification were found not to be responsible for ligand-independent activation in osteosarcoma. Characterization of Hedgehog signaling in osteosarcoma cell lines showed that cells with high IHH, PTCH1, and GLI1 levels were sensitive to small molecule modulators of both SMO and GLI, which supported the ligand-dependent activation observed in the clinical samples. The inverse correlation of endogenous GLI2 levels and Hedgehog pathway induction levels, and the sensitivity of high-GLI2 cells to GLI inhibition, but not SMO inhibition, in cell lines suggest that GLI2 overexpression may be a mechanism of ligand-independent activation in osteosarcoma. Furthermore, in patient-derived osteosarcoma xenograft models, I observed autocrine and possibly paracrine ligand-dependent Hedgehog signaling in the tumor and stromal compartments. I also showed that a clinically relevant SMO antagonist, IPI-926, was effective at specifically inhibiting all ligand-dependent Hedgehog signaling interactions. A trend toward decreased proliferation and increased apoptosis in treated tumors was observed warranting additional research and demonstrating the potential of Hedgehog pathway inhibitors as novel targeted therapeutics for osteosarcoma treatment.

Large intergenic RNAs (lincRNA) are involved in numerous cellular processes, including many relevant to normal development and cancer progression. In my doctoral research, we hypothesized that lncRNAs are functionally important to human endothelial biology, more specifically, to the process of human blood vessel formation or angiogenesis. To detect lincRNAs that are functionally important to human angiogenesis, a custom microarray was used to profile long noncoding transcripts in human vascular endothelium in two-dimensional versus three-dimensional pro-angiogenic cultures, with or without VEGF-A165. We identified a VEGF-A-responsive lincRNA near the VEGFR1 gene, which we termed lincRNA-VEGFR1 (LIVE1). Unbiased mRNA microarrays defined a number of potential target genes when LIVE1 was functionally disrupted using RNA interference. Importantly, knockdown and over-expression studies indicated that LIVE1 exerts transcriptional control over VEGFR1 as well as other VEGF receptors and direct angiogenesis in vitro. Furthermore, we found that LIVE1 is highly expressed in glioblastoma, and is enriched in glioma stem cell (GSC) fractions and neoplastic endothelial progenitor populations. In vivo knockdown of LIVE1 in a glioblastoma xenograft model decreased microvascular density, vascular perfusion, pericyte coverage, tumor volume and slowed tumour progression. Our results establish LIVE1 as a key mediator of angiogenesis and demonstrate the potential of lincRNA-based therapeutics.

Large intergenic RNAs (lincRNA) are involved in numerous cellular processes, including many relevant to normal development and cancer progression. In my doctoral research, we hypothesized that lncRNAs are functionally important to human endothelial biology, more specifically, to the process of human blood vessel formation or angiogenesis. To detect lincRNAs that are functionally important to human angiogenesis, a custom microarray was used to profile long noncoding transcripts in human vascular endothelium in two-dimensional versus three-dimensional pro-angiogenic cultures, with or without VEGF-A165. We identified a VEGF-A-responsive lincRNA near the VEGFR1 gene, which we termed lincRNA-VEGFR1 (LIVE1). Unbiased mRNA microarrays defined a number of potential target genes when LIVE1 was functionally disrupted using RNA interference. Importantly, knockdown and over-expression studies indicated that LIVE1 exerts transcriptional control over VEGFR1 as well as other VEGF receptors and direct angiogenesis in vitro. Furthermore, we found that LIVE1 is highly expressed in glioblastoma, and is enriched in glioma stem cell (GSC) fractions and neoplastic endothelial progenitor populations. In vivo knockdown of LIVE1 in a glioblastoma xenograft model decreased microvascular density, vascular perfusion, pericyte coverage, tumor volume and slowed tumour progression. Our results establish LIVE1 as a key mediator of angiogenesis and demonstrate the potential of lincRNA-based therapeutics.

Despite the combination of chemotherapy and surgery in osteosarcoma treatment, the survival of patients remains low. The lack of treatment improvement prompted me to investigate deregulated Hedgehog pathway as a potential target for intervention. During development, the Hedgehog pathway is tightly regulated to control organ and tissue development. Specifically, the Indian Hedgehog pathway (IHH) is important for bone development. Aberrantly activated Hedgehog pathway through ligand-dependent or ligand-independent mechanisms has been reported in numerous cancers. Several small molecule antagonists of the Hedgehog pathway are being explored clinically to improve patient outcome. I examined the expression of IHH pathway components, IHH, SMO, PTCH1 and GLI1, in 43 high-grade primary osteosarcoma tumors and 6 cell lines and found various transcript levels of IHH pathway components in osteosarcoma samples. The high levels of co-expressed IHH and targets, PTCH1 and GLI1, in a subset of osteosarcoma samples are indicative of ligand-dependent activation. Trends toward worse survival for patients with higher IHH (positive regulator) levels and toward better survival for patients with higher PTCH1 (negative regulator) levels were observed. Through genetic analyses, PTCH1 inactivating mutations and GLI1 amplification were found not to be responsible for ligand-independent activation in osteosarcoma. Characterization of Hedgehog signaling in osteosarcoma cell lines showed that cells with high IHH, PTCH1, and GLI1 levels were sensitive to small molecule modulators of both SMO and GLI, which supported the ligand-dependent activation observed in the clinical samples. The inverse correlation of endogenous GLI2 levels and Hedgehog pathway induction levels, and the sensitivity of high-GLI2 cells to GLI inhibition, but not SMO inhibition, in cell lines suggest that GLI2 overexpression may be a mechanism of ligand-independent activation in osteosarcoma. Furthermore, in patient-derived osteosarcoma xenograft models, I observed autocrine and possibly paracrine ligand-dependent Hedgehog signaling in the tumor and stromal compartments. I also showed that a clinically relevant SMO antagonist, IPI-926, was effective at specifically inhibiting all ligand-dependent Hedgehog signaling interactions. A trend toward decreased proliferation and increased apoptosis in treated tumors was observed warranting additional research and demonstrating the potential of Hedgehog pathway inhibitors as novel targeted therapeutics for osteosarcoma treatment.

The presence of microglia at sites of CNS injury can potentially shift the balance between neuronal survival and death; however, the mechanisms regulating their mobilization to these sites are still poorly understood. Here I report that microglia express podosomes, short-lived, punctate organelles which adhere to and degrade extracellular matrix (ECM). Podosomes and related invadopodia of cancer cells have recently been the focus of much interest for their roles in migration and invasion. Microglial podosomes degraded ECM, providing impetus for further study of their function in microglia. Further, I report the Ca2+-activated SK3 channel as a novel component of the podosome core. While SK3 and SK4 channels are reported to play redundant roles in activated microglia (Kaushal et al., 2007; Schlichter et al., 2010), immunostaining work suggests that they are differentially regulated during microglial activation. Together, these results suggest unique functions for these channel subtypes in microglia.

The presence of microglia at sites of CNS injury can potentially shift the balance between neuronal survival and death; however, the mechanisms regulating their mobilization to these sites are still poorly understood. Here I report that microglia express podosomes, short-lived, punctate organelles which adhere to and degrade extracellular matrix (ECM). Podosomes and related invadopodia of cancer cells have recently been the focus of much interest for their roles in migration and invasion. Microglial podosomes degraded ECM, providing impetus for further study of their function in microglia. Further, I report the Ca2+-activated SK3 channel as a novel component of the podosome core. While SK3 and SK4 channels are reported to play redundant roles in activated microglia (Kaushal et al., 2007; Schlichter et al., 2010), immunostaining work suggests that they are differentially regulated during microglial activation. Together, these results suggest unique functions for these channel subtypes in microglia.