<p> Chemoresistance is a highly significant problem affecting a diverse array of cancers at all clinical stages. In an attempt to identify molecular mechanisms leading to chemoresistance, we performed a RNAi screen against all known and putative kinases and phosphatases in the human genome. The knockdown of one of these genes, MK-STYX, resulted in potent chemoresistance in response to a diverse array of chemotherapeutic agents. As many of these drugs function through the induction of the apoptotic program, we hypothesized that the RNAi-mediated knockdown of MK-STYX blocks the cellular response to chemotherapeutic-induced apoptosis. </p><p> To investigate this hypothesis, we determined the ability of both control and MK-STYX knockdown cells to undergo apoptosis after exposure to an array of cell death inducing agents with different mechanisms of action. The results of these experiments demonstrated that MK-STYX knockdown protects against intrinsic, but not extrinsic apoptotic stimuli. These data were recapitulated with knockdown of the pro-apoptotic genes caspase-9 and Bax/Bak, suggesting that MK-STYX may modulate the regulation of one of these key apoptotic regulatory nodes. We demonstrated that the loss of MK-STYX blocks cytochrome c release, placing the apoptotic deficiency at the level of Bax/Bak-mediated mitochondrial outer membrane permeabilization, or MOMP. MK-STYX was found to localize to the mitochondria, but is neither released from the mitochondria upon apoptotic stress nor localized proximal to the machinery currently known to control MOMP. These results are summarized in Chapter 2. </p><p> In an effort to more fully define molecular mechanism of MK-STYX, we performed an unbiased TAP-tagging experiment to identify its interaction partners. The most significant and unique protein identified was the mitochondrial phosphatase PTPMT1. Interestingly, MK-STYX is a catalytically inactive dual specificity phosphatase, and catalytically inactive phosphatases have a precedent for regulating the activity and/or localization of active phosphatases. Because of this potential phosphatase regulatory mechanism, as well as similar localization patterns of both genes, we chose to further explore the interaction between PTPMT1 and MK-STYX. </p><p> Due to the robust survival phenotype seen in MK-STYX knockdown cells when treated with chemotherapeutic, we predicted that the knockdown of PTPMT1 may have a similar phenotype. Surprisingly, we found that PTPMT1 knockdown causes a Bax/Bak dependent cell death, suggesting that MK-STYX and PTPMT1 may functionally oppose one another in the mitochondria. Experiments in which both enzymes are downregulated show that PTPMT1 is epistatic to MK-STYX, as cells are resensitized to chemotherapeutic agents and cytochrome c release under these conditions. Interestingly, PTPMT1 was recently shown to be an important enzyme in the cardiolipin biosynthetic pathway, positively regulating the synthesis of this mitochondrial lipid. The genetic interaction provided by the robust changes in viability seen when these enzymes are downregulated suggests that MK-STYX may function to dampen PTPMT1 enzymatic activity. This allows us to hypothesize that the loss of MK-STYX results in increased cardiolipin biosynthesis, leading to altered mitochondrial membrane composition and subsequently, an altered apoptotic response. These results are summarized in Chapters 3 and 4. </p><p> We further hypothesize that the upregulation of cardiolipin levels directly inhibits the ability of Bax/Bak to permeabilize the outer mitochondrial membrane, effectively blocking the induction of mitochondrial apoptosis. These data suggest a novel mechanism by which dysregulated cardiolipin can facilitate chemoresistance, and suggest that this pathway could be exploited by recurrent cancers to evade therapies.</p><p>

<p> The mitochondrion is a complex, double membrane organelle that serves several important cellular functions including ATP synthesis, Ca ²⁺ buffering, and ROS homeostasis. Although classic mitochondrial diagrams depict the mitochondrion as a simple oval or &ldquo;bean&rdquo; shaped organelle, the mitochondria can form extensive tubular networks or numerous small spheres in response to various cellular environments through two opposing processes, mitochondrial fission and fusion. Deregulation of mitochondrial dynamics has been implicated in a wide range of diseases, including Parkinson&rsquo;s disease, heart disease and cancer. While significant emphasis for the last 15 years has been placed on the identification of the protein machinery responsible regulating mitochondrial morphology, it remains less clear how mitochondrial morphology affects various cellular functions and cellular fate outcomes. This thesis summarizes our findings on how mitochondrial morphology regulates cellular fate in the context of mitotic cell division and apoptosis. Using live cell microscopy and image analysis software we characterized mitochondrial dynamics with single cell resolution. We found that loss of key components of the mitochondrial fission machinery promotes a defect in cell cycle progression, characterized by an inability for cells to exit G2/M. Prolonged periods of mitochondrial fusion induced potent cell death, suggesting a novel mechanism to target the replicative potential of cancer cells. We also found that mitochondrial fission and fusion can alter the kinetics of cell death following apoptotic stimuli by inducing mitochondrial fusion prior to the commitment step in apoptosis, mitochondrial membrane permeabilization. This thesis summarizes our work in trying to elucidate how the structure of the mitochondria influences both mitochondrial and cellular fate.</p><p>

<p> Osteosarcoma (OS) is the most common type of solid bone cancer, mainly arising in children and young adults, and remains the second leading cause of cancer-related death in this age group. Chemotherapy resistance underlying latent recurrence and metastasis represent major contributors to poor outcome for many cancer patients especially those with OS. Tumor hypoxia is an essential element intrinsic to most solid tumor microenvironments and is associated with resistance to therapy and a malignant phenotype, while metastatic dissemination is dependent on a cells ability to resist anoikis, i.e., programmed cell death in the absence of attachment to an extracellular matrix. We sought to better characterize hypoxia and anoikis resistance in human OS using established and novel patient-derived OS cells and OS animal models with the long-term goal of identifying and validating targetable signaling pathways. We show that hypoxia-inducible factors (HIFs), canonical proteins associated with the hypoxic response, are present and can be induced in human OS cells. We demonstrate that the Wnt/&beta;-catenin signaling pathway, a key pathway in OS pathogenesis, is down-regulated in response to hypoxia in OS cells, and that this appears to result from both HIF-dependent and HIF-independent mechanisms. Hypoxia promotes resistance of human OS cells to standard chemotherapy, which is mitigated by treatment with Wnt/&beta;-catenin signaling inhibitors. Using an anchorage-independent growth model, we show that anoikis-resistant OS subpopulations have altered growth rates, increased resistance to standard chemotherapies, and display distinct changes in gene expression and DNA methylation. Finally, we validate the use of two FDA-approved epigenetic therapies predicted by expression profiling in both inhibiting anchorage-independent growth and sensitizing anoikis-resistant OS cells to chemotherapy. In summary, despite the heterogeneity of human OS, our work suggests that unique and effectively targetable signaling pathways underlie the phenotypic consequences in response to hypoxia and anoikis resistance.</p><p>

<p> Group I Paks are serine/threonine kinases that function as major effectors of the small GTPases Rac1 and Cdc42. They regulate many cellular functions, including cell polarity, cytoskeletal dynamics, and transcription. Pak1 and Pak2 are redundantly essential for embryonic skeletal myoblast fusion in <i> Drosophila</i>, with Pak2 playing the more important role. Both are expressed in mammalian skeletal muscle, but little is known as to their function in myogenesis. We find that Pak1 and Pak2 are expressed in mammalian myoblasts and are activated specifically during differentiation. Individual genetic deletions of <i>Pak1</i> and <i>Pak2</i> in mice show no overt defects in muscle development or regeneration. However, young adult mice with muscle-specific deletion of <i>Pak1</i> and <i>Pak2 </i> together (dKO mice) present with reduced muscle mass and a higher proportion of myofibers with smaller cross-sectional area compared to controls. This phenotype is exacerbated after repair to acute injury. Primary myoblasts from dKO animals show delayed differentiation, with lower expression of myogenic markers and inefficient myotube formation. Additionally, with age, dKO mice develop a chronic myopathy. Histological analyses of resting muscle show the presence of central nuclei in the majority of fibers, as well as significant fibrosis, inflammation, necrosis, and hypertrophy with fiber splitting. Ultrastructural analysis revealed grossly elongated and branched intermyofibrillar mitochondria, known as megaconial mitochondria, along with occasional accumulation of subsarcolemmal mitochondria. Moreover, dKO mice show impaired mitochondrial function, with significantly reduced Complex I and II activity. These characteristics are absent in control animals. We conclude that the role of Pak1 and Pak2 in embryonic myoblast fusion, first identified in the fly, is not conserved in mammals. Rather, our data demonstrate that Pak1 and Pak2 function redundantly in regulating myoblast differentiation, thereby impacting overall postnatal muscle size. Furthermore, their major function appears to be in muscle homeostasis. Few protein kinases have been implicated in muscle disease. Group I Paks have wide roles in cell regulation, and the generation of dKO mice provides a genetic system to gain new mechanistic insights into muscle maintenance, as well as to discover the substrates of Paks that regulate this process.</p><p>

The factors controlling desensitization to ATP stimulation were investigated in PC12 cells. Reducing the concentration of ATP to produce half maximal response reduced the degree to which cells desensitize to ATP. Increasing external Me concentration, which produced a comparable decrease in the secretory response of the cells, had no effect on the degree of desensitization. Neither did decreasing external Ca2+ concentration, which produced a similar decrease in secretory response. Desensitizing cells in 0.5 mM Ca2+ did not result in a corresponding decrease in response when cells were subsequently tested in 2.2 mM Ca2+. PC12 cells desensitized in 2.2 mM Ca2+ were found to show the same degree of desensitization when tested in 0.5 mM Ca2+. A similar pattern was found when desensitization to 30 and 300 uM ATP was examined. The role of the ATP receptor subtypes, P2x and P2y, was studied using the ATP agonists 2-MeS ATP and UTP, respectively. 60 uM 2-MeS ATP was found to cause desensitization to the same degree as 30 uM ATP. As with ATP, the initial response to 2-MeS ATP was found to be sensitive to changes in external Mg2+. Unlike ATP, the desensitization to 2-MeS ATP was sensitive to changes in external Mg2+. When cells were co-stimulated with 2-MeS ATP and UTP, the sensitivity of 2-MeS ATP desensitization to Mg2+ remained. UTP in the background solution, however, increased desensitization to ATP and 2-MeS ATP. The effect of voltage-operated Ca2+ channel (VOCC) blockers on the response and desensitization to ATP and 2-MeS ATP was examined. Cd2+ produced a significant increase in the secretory response to both stimulants. Both nicardipine and Cd2+ increased the rate of desensitization to ATP. Only nicardipine was able to increase the rate of desensitization to 2-MeS ATP. These results were integrated to provide insights into the relationship between ATP receptors and VOCCs.

Polychlorinated biphenyls are one type of organic pollutant. Although they are no longer produced due to their resistance to degradation they have persisted in the environment and have become globally distributed. They have been found within the tissues of nearly every organism that has been tested for their presence. Because they are capable of interacting with biological systems their effect on environmental and human health have become a topic of interest and research. It is known that PCBs alter the function and levels of thyroid hormone. This is significant because of the importance of properly regulated thyroid hormone levels during development. To gain a better understanding of the specific effects of PCBs on thyroid hormone function and to better assess the risks associated with PCB exposure I set out to identify mechanisms by which PCBs alter thyroid hormone-directed brain development. I used a number of approaches to identify impacts of PCBs on thyroid hormone-directed brain development. I first looked at gene expression and identified new targets of thyroid hormone, the cofactors N-CoR and SRC-1. I next looked at the effects of PCB exposure on these genes. I found that PCBs did not affect the expression of these genes. I next went on to look at cell and death and proliferation in the cerebellum and found that PCBs did not alter these processes. When I tried an in vitro approach to look at the impact of PCB exposure on receptor-independent effects of thyroid hormone on actin polymerization I found that my cultures did not respond to thyroid hormone as reported. In the last experiment I used a differential display screen to identify targets of PCB exposure and identified number of genes whose expression is putatively affected by PCB exposure. These genes included Stathmin, BESH, and Zic-1 all of which are associated with the cytoskeleton. It has also been shown that the effects of thyroid hormone can alter neuronal migration. These data suggest that PCBs may be altering neuronal migration and that the genes identified in this screen are downstream consequences of the thyroid hormone receptor -independent effects of thyroid hormone.

The flow of information through neuronal circuits relies on the ability of neurons to form synaptic connections with specific temporal and spatial properties. These properties are not static but have the ability to change, allowing synaptic connections to be strengthened or weakened. It is this plastic nature of synapses that is central to higher order processes such as learning and memory. However, major gaps remain in our understanding of this process. Throughout my dissertation work I have examined mechanisms of a form of structural synaptic plasticity by analyzing the roles of a variety of proteins that we have found serve to regulate the formation and maintenance of glutamatergic synapses at the Drosophila NMJ. These proteins include APPL, the Drosophila homolog of Alzheimer's disease-associated β-Amyloid Precursor Protein (APP), the tumor suppressor protein Scribble (Scrib), the secreted signaling molecule Wingless (Wg), and the cell adhesion molecule Fasciclin II (FasII). In this work, in collaboration with members of the labs of Dr. Vivian Budnik, Dr. Kalpana White, and Dr. Susan Cumberledge, I have demonstrated that Wingless (Wg) provides a secreted signal that is required to initiate the formation of pre- and postsynaptic structures. Further, I have also demonstrated that once synapse formation is initiated, presumably by Wg signaling, APPL regulates synaptic bouton proliferation. This process also involves signaling by FasII, a protein required for synapse maintenance, and growth. Moreover, I have also demonstrated that Scrib is a scaffolding protein that plays a key role at these synapses in influencing neurotransmitter release.

It is widely accepted that activating mutations of genes encoding the Notch family of transmembrane receptors, specifically Notch1, are associated with oncogenic transformation. Previous data from our lab has shown that an active form of Notch1 (Nic) provides a protective effect against apoptosis in D011.10 T cells, and that this effect may be attributed to Nic binding the pro-apoptotic protein Nur77. Nur77 is an immediate early gene that is upregulated during negative selection of thymocytes and activation-induced apoptosis in D011.10 T cells. Nur77 upregulation is tightly regulated and requires MEF2D, NFAT, and the co-activator, p300, to effectively respond to apoptotic stimuli. In this report we show that Nic has the ability to interfere with the induction of transcription of Nur77, and that this interference is directly related to the inability of p300 to bind the Nur77 promoter in the presence of Nic. We also show that blocking Notch activation through gamma secretase-inhibitors or siRNA directed against Notch1 in T cell acute lymphoblastic leukemia (T-ALL) cell lines restores Nur77 upregulation in response stimuli. These observations support a model in which during thymocytes negative selection activating mutations of Notch1 inhibit the upregulation of a crucial proapoptotic molecule. Studies to determine the mechanism by which Nur77 induces apoptosis have indentified a unique translocation of Nur77 from the nucleus to the cytosol. It has been determined that once in the cytosol Nur77 interacts with members of the Bcl-2 family of proteins at the mitochondrial membrane. This interaction induces a conformational change of Bcl-2 so that is becomes pro-apoptotic instead of protective. Of similar interest is the role that Nur77 itself plays during the induction of activation-induced apoptosis which may be independent of Bcl-2 conformational change. In an effort to describe possible functions of Nur77, DO11.10 cells that have Nur77 under a tet-inducible promoter were observed for changes IP3R. Initial results from our lab suggest that Nur77 alone has the ability to induce cell death in DO11.10 cells using this tet-inducible system. Interestingly we have been able to identify distinct changes in IP3R isoforms during stimulation induced apoptosis and Nur77-dependent apoptosis. Current experiments are focused on a mechanism beyond the known function of the Nur77/Bcl-2 interaction; that Nur77 may also be acting as a physical barrier between the known anti-apoptotic interaction of IP3R and Bcl-2, leading to sustained calcium flux.

Breast cancer tops the list of new cancer cases and is predicted to be the second leading cause of cancer deaths in women in 2012. The primary objective of the present study was to provide insights into the molecular mechanisms underlying the aggressive growth and metastasis of triple-negative and basal-like breast cancers. To study increased growth and invasive behavior in triple-negative and basal-like breast cancers we utilize both an interesting and relevant cell culture model and examination of human tissue. S-phase kinase-associated protein 2 (SKP2) plays an important role in cell cycle regulation by targeting p27 for degradation. The cyclin-dependent kinase (CDK) inhibitor p27 regulates G1/S transition by binding cyclin/CDK complexes and abrogating its activity. By targeting p27 for degradation, SKP2 frees the complexes needed to progress into the S phase of the cell cycle. Evaluation of SKP2 expression in TMX2-28 revealed significantly higher levels than in other breast cancer cell lines. Despite the high levels of SKP2 expression, p27 protein was not reduced. However, levels of the Serine 10 phosphorylated form of p27 (pSer10p27), which has been associated with increased proliferation rates, was found to be increased. Furthermore, suppression of SKP2 completely eliminated the pSer10p27 and slowed cycle progression confirming the role of SKP2 in the aggressive growth of TMX2-28 cells. Assessment of mRNA from 30 frozen human breast cancers demonstrated that SKP2 is more highly expressed in ER&agr;-negative and basal-like breast cancers. Immunohistochemical analysis of 188 breast cancers and 50 benign reduction mammoplasty tissues confirmed that SKP2 is more highly expressed in ER&agr;-negative breast cancers and for the first time demonstrated that triple-negative breast cancers are more likely to overexpress SKP2 than are non-triple-negative, but still ER&agr;-negative, tumors. In contrast to some previous reports, we did not observe an inverse relationship between SKP2 and p27 expression. Only 11% of tumors expressed high SKP2 and low p27, while 32% of tumors had high SKP2 and high p27. Although no significant relationship between SKP2 and p27 expression was observed in human breast cancers, a significant positive relationship was discovered between SKP2 and pSer10p27. Furthermore, high levels of SKP2 and pSer10p27 were observed significantly more often in ER&agr;-negative and triple negative breast tumors than in ER&agr;-positive breast cancers. Based on these results and those of the cell culture experiments showing complete elimination of pSer10p27 after suppression of SKP2 it appears that levels of pSer10p27 may be a better indicator of SKP2-dependent p27 degradation than are levels of p27. Therefore, that inhibiting SKP2 in triple-negative breast cancers expressing high levels of both SKP2 and pSer10p27 regardless of p27 levels may be a valid therapeutic approach. We determined that TMX2-28 lack MMP-1 mRNA, and MMP-2/MMP-9 protein expression; each of which is important in protease-dependent invasion. Furthermore, TMX2-28 cells have low expression of other genes key to protease-dependent invasion including Slug, Zeb 1, Zeb 2, Vimentin, Fibronectin and N-cadherin. RhoA is a member of the Rho superfamily of GTPases that acts as a molecular switch to control signal transduction and is critical to the amoeboid invasion mechanism. TMX2-28 cells have high expression of protease-independent invasion genes such as RhoA, ROCK 1, ROCK 2, and E-cadherin. Finally, treating TMX2-28 cells with a RhoA pathway inhibitor or an shRNA targeting RhoA significantly reduces their invasiveness. These data suggest that TMX2-28 cells use a RhoA-dependent, proteolytic-independent invasion mechanism. Collectively, the data presented here demonstrate the roles of SKP2 and RhoA in triple-negative and basal-like breast cancers, making both genes, as well as their pathways, desirable therapeutic targets. (Abstract shortened by UMI.)

Glioblastoma Multiforme (GBM) is the most common brain cancer and one of the most fatal forms of cancer overall. The average survival time is 10-14 months, and less than 10% of patients survive more than 5 years after diagnosis. It is characterized by extreme vasculature, chemo/radioresistance, and invasiveness into the normal brain. The current standard of care, which includes surgical removal of tumor, radiation, and the chemotherapeutic agent temozolomide, initially stunt tumor growth. Nevertheless, the tumor invariably rebounds and the patient succumbs to the disease. Therefore, there is an urgent need to develop new therapies for this devastating disease. YKL-40 is one of the most over-expressed proteins by GBM cells, and is elevated in the serum of patients with GBM. YKL-40 is implicated in a host of inflammatory diseases and has been shown to play a major role in the maturation of some cells of the immune system, especially macrophages. Thus, it has been suggested that YKL-40 may act as a prognostic biomarker for cancer and inflammatory disease. However, little is known about the role of YKL-40 in relation to cancer development and progression, and more work needs to be done to validate it as a biomarker or as a therapeutic target. It was the goal of the work described herein to uncover some of the key molecular mechanisms of GBM development and progression in the hopes of offering new therapeutic targets. Using a wide variety of in vitro and in vivo techniques, the role of a secreted glycoprotein YKL-40 in GBM was probed. It was demonstrated that YKL-40 enhanced angiogenesis, radioresistance, and progression of GBM cells. Moreover, inhibition of YKL-40 in mouse models markedly arrested tumor growth and vascularization, lending support to the idea of YKL-40 as a therapeutic target. Finally, YKL-40 drove GBM cells into a mesenchymal phenotype, where the tumor cells act as mural-like cells, supporting tumor vasculature networks. Hopefully, the results from these studies will offer the rationale to develop drugs against YKL-40 and potentially extend the lives of patients with this terrible disease.