Despite 35 years of clinical trials, there has been little improvement in one-year survival rates with any chemotherapeutic regimen for the treatment of metastatic melanoma due to resistance to all known agents. Regardless of advances in detection and prevention, diagnosis of metastatic disease remains a death sentence. Resistance mechanisms, including aberrant kinase signaling and drug transport pumps, indicate a need for identification of other therapeutic targets that impinge upon multiple signaling pathways. The Abl family of non-receptor tyrosine kinases (c-Abl, Arg) has been indicted as a causative force in leukemia for more than three decades; however, their role in solid tumors has only recently been described. We first demonstrated that activated Abl family kinases promote breast cancer development and progression, and recently identified them to be novel therapeutic targets in metastatic melanoma cells by demonstrating that they promote proliferation, survival, invasion, and metastasis. We now present evidence that inhibitors of Abl family kinases abrogate resistance to a number of commonly used chemotherapeutics (i.e., 5-fluorouracil, cisplatin, paclitaxel, camptothecin) in a panel of breast cancer cells. We proceed to show that inhibitors of Abl family kinases, likewise, sensitize both breast cancer and melanoma cells to doxorubicin by blocking cell proliferation and dramatically inducing apoptosis. These findings were extended to advanced multi-drug resistant melanoma cells, in which we show for the first time that c- Abl promotes expression of the drug transporter, ABCB1, during acquired resistance, and drugs that inhibit c-Abl/Arg prevent ABCB1 expression and function. Moreover, c-Abl/Arg also promote acquired chemoresistance independent of ABCB1 by modulating multiple survival pathways. We demonstrate that c-Abl/Arg promote chemoresistance by upregulating STAT3, preventing doxorubicin-mediated conversion of NF-κB into a transcriptional repressor, activating an HSP27/p38/Akt survival pathway, and modulating ERK signaling. Therefore, c-Abl/Arg promote chemoresistance in highly resistant melanoma cells by impinging on drug transporter and cell survival pathways. Taken together, these data indicate that c-Abl/Arg inhibitors are likely to reverse acquired resistance in metastatic melanomas harboring activated c-Abl/Arg, and thus, may be effective in a combination regimen.

Acute myocardial infarction (AMI) triggers the mobilization of stem/progenitor cells from bone marrow (BMSPCs) into peripheral blood (PB). The underlying mechanisms orchestrating this mobilization and subsequent homing of BMSPCs to the myocardium are poorly understood. While the role of traditional chemokines in the mobilization and homing of hematopoietic stem cell (HSCs) to BM niches is undisputed, their role in directing BMSPCs to the highly proteolytic environment of the ischemic myocardium is debatable and other redundant mechanism may exist. Based on our observation that bioactive lipids, such as sphingosine-1 phosphate (S1P) and ceramide-1 phosphate (C1P), play an important role in regulating trafficking of HSCs; we explored if they also direct trafficking of BMSPCs in the setting of myocardial ischemia. While BMSPCs expressed S1P receptors regardless of the source, the expression of S1P receptor 1 (S1PR1) and receptor 3 (S1PR3), which are responsible for migration and chemotaxis, was elevated in BMSPCs in naïve BM cells and was reduced following mobilization. This expression correlated to differential response of BMSPCs to S1P in chemotaxis assays. By employing flow cytometry analyses, we observed an increase in circulating PB CD34+, CD133+ and CXCR4+ lineage negative (Lin-)/CD45- cells that are enriched in non-HSCs (P < 0.05 vs. controls). This corroborated our mass spectrometry studies showing a temporal increase in S1P and C1P plasma levels. At the same time, plasma obtained in the early phases following AMI strongly chemoattracted human BM-derived CD34+/Lin- and CXCR4+/Lin- cells in Transwell chemotaxis assays in an S1P dependent fashion. We examined other mechanisms that may contribute to the homing of BMSPCs to the infarcted myocardium due to the reduction of S1PRs upon mobilization. We observed that hypoxia induced higher expression of cathelicidins in cardiac tissues. Indeed, PB cells isolated from patients with AMI migrated more efficiently to low, yet physiological, gradient of SDF-1 in Transwell migration assays compared to SDF-1 alone. Together, these observations suggest that while elevated S1P plasma levels early in the course of AMI may trigger mobilization of non-HSCs into PB, cathelicidins appear to play an important role in their homing to ischemic and damaged myocardium.

Malignant mesothelioma (MM) is a rare tumor associated with occupational exposure to asbestos with no effective treatment regime. Evaluation of mitochondrial function in human MM cell lines revealed a common tumor phenotype: in comparison to immortalized or primary human mesothelial cells, MM tumor cells displayed a more oxidized mitochondrial environment, increased expression of mitochondrial antioxidant enzymes, and altered mitochondrial metabolism. Earlier work by our laboratory indicated that increases in mitochondrial reactive oxygen species (mROS) in MM cell lines supports expression of FOXM1, an oncogenic transcription factor that contributes to increased cell proliferation and chemoresistance. These studies sought to investigate targeting of mitochondrial structure and function as a therapeutic avenue in MM. MM cells have reduced mitochondrial reserve capacity, a redox vulnerability exploitable by pro-oxidant therapeutics. Targeting of the mitochondrial peroxidase peroxiredoxin 3 (PRX3) with the anti-cancer compound thiostrepton (TS) induces irreversible modifications to PRX3 protein, increased mROS, and selective MM cell death. Mass spectrometry showed TS targets conserved cysteine residues in PRX3. In vitro and in MM cells, TS failed to modify human PRX3 harboring mutations to Cys108, Cys127 or Cys229. Pre-incubation of MM cells with dimedone blocked cysteine adduction of PRX3 by TS, suggesting adduction requires an active PRX3 catalytic cycle. Studies with immortalized and primary human mesothelial cells showed adduction of PRX3 by TS occurred at a much lower rate in normal cells than MM cells, and this difference correlated with markedly decreased cytotoxicity. Moreover, MM cells transduced with shRNA to PRX3 grew more slowly and were less sensitive to TS than their wild type counterparts, indicating PRX3 is a major target of TS in MM cells. Studies with a xenoplant mouse model of MM showed TS alone or in combination with the TRX2 inhibitor gentian violet significantly reduced tumor volume. Tumor cell mitochondria have an increased mitochondrial membrane potential, therefore numerous drugs have been developed that selectively accumulatte into energized mitochondria to enhance drug efficacy and specificity. Here two mitochondrial-targeted nitroxides, Mito-carboxy-proxyl (MCP) and Mito-TEMPOL (MT), were investigated for their anti-cancer effects. Treatment of MM cells with MCP or MT led to rapid disruption of the mitochondrial reticulum, increased oxidant levels, and reduced FOXM1 and PRX3 protein expression. Immunostaining revealed a pool of cytoplasmic FOXM1 associated with PRX3 in mitochondria, suggesting PRX3 participates in regulating FOXM1 expression. Combination of MCP or MT with TS led to synergistic effects on MM cell viability. Upregulation of mitochondrial antioxidant enzymes is an adaptive response that ameliorates mitochondrial oxidative stress and supports tumor cell survival. Studies here indicate that enhanced dependency on the PRX3 catalytic cycle in tumor cells promotes inactivation of PRX3 by TS, providing a therapeutic window dependent on a mitochondrial phenotype common to many human tumor types. Therefore TS, alone or in combination with other agents, may prove useful in the management of intractable tumors such as MM.

Ovarian cancer is the deadliest of all the gynecologic cancers and is known for its clinically occult and asymptomatic dissemination. Most ovarian malignancies are diagnosed in the late stages of the disease and the high rate of morbidity is thought to be due, in part, to the highly metastatic nature of ovarian carcinomas. Cancer metastasis relies on the ability of cells to migrate away from primary tumors and invade into target tissues. Though the processes are distinct, cancer cell invasion relies on the underlying migration machinery to invade target tissues. Cell migration requires the coordinated effort of numerous spatially-regulated signaling pathways to extend protrusions, create new adhesion to the extracellular matrix (ECM), translocate the cell body, and retract the cell rear. Our lab established that the cyclic-AMP dependent protein kinase (PKA) subunits and enzymatic activity are localized to the leading edge of migrating cells and are required for cell movement. Despite the importance for localized PKA activity during migration, neither its role in regulating ovarian cancer cell migration and invasion nor the mechanism regulating leading edge PKA activity have been determined. Therefore, the objective of the enclosed work is to establish the importance of PKA for ovarian cancer cell migration and invasion and elucidate the molecular mechanism governing leading edge PKA. We demonstrate, for the first time, that PKA activity and spatial distribution through A-Kinase Anchoring Proteins (AKAPs) is required for efficient ovarian cancer cell migration and invasion. Additionally, we establish a link between leading edge PKA activity in migrating cells, ECM stiffness sensing, and the regulation of both PKA activity and ovarian cancer cell migration by the mechanical properties of the ECM. Finally, we delineate the hierarchy of cell signaling events that regulate leading edge PKA activity and, ultimately, the migration of ovarian cancer cells. Specifically, we elucidate a mechanism where leading edge protrusions elicit leading edge calcium currents through the stretch-activated calcium channel (SACC) of the transient receptor potential family melastatin 7 (TrpM7) to activate actomyosin contractility. ECM substrate stiffness is sensed by the actin cytoskeleton and actomyosin contractility, which, in turn, regulates the activity of leading edge PKA activity. These studies have provided important insights into the regulation of cell migration and have established the mechanistic details governing leading edge PKA activity during cell migration.

Clostridium difficile is a Gram-positive bacterium that causes a toxin-mediated disease, typically in individuals whose normal intestinal flora has been compromised by antibiotic therapy. C. difficile is naturally resistant to many antibiotics and produces spores that can withstand harsh environmental conditions and many disinfectants, making the infection difficult to clear and easy to spread. The infection begins when spores from the environment are ingested and germinate upon exposure to taurocholate and glycine in the digestive tract. This germination process is required to initiate infection and thus represents a good target for the development of novel therapeutics. Although spore germination is necessary for disease transmission, the molecular mechanisms regulating this process are poorly understood. Germination relies on sensing a germinant and triggering degradation of the cortex layer of the spore, which is important for spore resistance. Once the cortex is degraded, the spore can undergo outgrowth to a vegetative cell and secrete toxins to cause disease symptoms. There are several discrete steps to the proteolytic cascade that ultimately lead to cortex hydrolysis. First, the pseudoprotease CspC acts as a germinant receptor for the bile salt taurocholate; CspC then relays this signal to the subtilisin-like serine protease, CspB. CspB is required for efficient cleavage and activation of the cortex hydrolase. SleC. Upon proteolytic activation of SleC, cortex hydrolysis can proceed, which allows subsequent outgrowth. To better understand the mechanistic basis of the germination process, we solved the 1.6 Å structure of the required germination protease, CspB, from C. perfringens (a related pathogen). This structure revealed that CspB is comprised of three domains: an associated prodomain, a subtilase domain, and a jellyroll domain. Our work significantly advanced our understanding of the proteolytic cascade that leads to germination; in particular the structure and function of the CspB protease, and the role of its three domains. We have described the four domains of the cortex hydrolase, SleC, and how they contribute to the activity of SleC. We have recently obtained diffraction-quality crystals of the pseudoprotease, CspC, from an organism more closely related to C. difficile, C. bifermentans. Our latest work, focusing on the germination receptor, CspC, has brought us closer to a three-dimensional structure of this protein, which will likely reveal how it binds ligands and functions in germination.

The most common malignant glioma, glioblastoma multiforme (GBM), remains an intractable tumor despite advances in therapy. Its proclivity to infiltrate surrounding brain tissue contributes greatly to its treatment failure and the grim prognosis of patients. Radiation is a staple in modern therapeutic regimens, though cells surviving radiation become more aggressive and invasive. Consequently, it is imperative to define further the cellular mechanisms that control GBM invasion and identify promising novel therapeutic targets. Melanoma differentiation associated gene-9 (MDA-9/Syntenin) is a highly conserved PDZ domain-containing scaffolding protein that promotes invasion and metastasis in human melanoma models. We show that MDA-9/Syntenin is robustly expressed in GBM cell lines and patient samples, and expression increases by tumor grade. These findings are confirmed through database analysis, which revealed MDA-9/Syntenin expression correlates with shorter survival times and patient tumors high in MDA-9/Syntenin have a worse prognosis when undergoing radiotherapy. Modulating MDA-9/Syntenin levels produced changes in invasion, angiogenesis, and signaling, indicating MDA-9/Syntenin enhances glioma pathogenesis. Overexpression of MDA-9/Syntenin enhances invasion, while knockdown inhibits invasion, migration, and anchorage-independent growth in soft agar. MDA-9/Syntenin increases activation of c-Src, P38MAPK, and NF-kB, leading to elevated MMP2 expression and IL-8 secretion. Through an orthotopic tumor model, we show that shmda-9 tumor cells formed smaller tumors and had a less invasive phenotype in vivo. Knockdown of MDA-9/Syntenin radiosensitizes GBM cells and significantly reduces post-radiation invasion gains through abrogation of radiation-induced Src and EphA2 activity. In efforts to pharmacologically inhibit MDA-9/Syntenin, we describe the effects of a novel small molecule, PDZ1i, which targets the PDZ1 domain of MDA-9/Syntenin and successfully reduces invasion gains in GBM cells following radiation. While it does not effect astrocyte radiosensitivity, PDZ1i radiosensitizes GBM cells. PDZ1i inhibits crucial GBM signaling including FAK and mutant EGFR, EGFRvIII, and can negate gains in secreted proteases, such as MMP2 and MMP9, following radiation. In a model of glioma, PDZ1i treatment combined with radiation results in less invasive tumors and extends survival. Our findings indicate that MDA-9/Syntenin is a novel and important mediator of GBM pathogenesis, and further identify it as a targetable protein that enhances radiotherapy for treatment in glioma.

Erbin belongs to the LAP (leucine-rich repeat and PDZ domain) family of scaffolding proteins that play important roles in orchestrating cell signaling. Here, we show that Erbin functions as a tumor suppressor in colon cancer. Analysis of Erbin expression in patient specimens reveals that Erbin is downregulated at both mRNA and protein levels in tumor tissues. Functionally, knockdown of Erbin disrupts epithelial cell polarity and increases cell proliferation in 3D culture. In addition, silencing Erbin results in an increase in the amplitude and duration of signaling through Akt and RAS/RAF pathways. Moreover, Erbin-loss induces epithelial-mesenchymal transition (EMT), which coincides with a significant increase in cell migration and invasion. Erbin interacts with KSR1 and displaces it from the RAF/MEK/ERK complex to prevent signaling propagation. Furthermore, genetic deletion of Erbin in Apc knockout mice promotes tumorigenesis and significantly reduces survival. Tumor organoids derived from Erbin/Apc double knockout mice have increased tumor initiation potential along with increased Wnt target gene expression as seen by qPCR. Collectively, the studies within this dissertation identify Erbin as a negative regulator of EMT and tumor progression by directly suppressing Akt and RAS/RAF signaling in vivo.

Arenaviruses and hantaviruses are human pathogens that cause significant morbidity and mortality. The current lack of vaccines and treatment options for these viruses is a global concern. Despite producing only 4 proteins, these viruses are able to maintain a persistent and asymptomatic infection in wild rodents while being continuously shed into the environment. In humans, these viruses cause a spectrum of diseases ranging from aseptic meningitis to severe hemorrhagic fever syndromes. Little is known about how arenavirus and hantavirus proteins engage and interact with the human proteome during the complex process of viral biogenesis, or how the interactions with human proteins contribute to viral propagation as well as the onset and progression of disease. This dissertation provides a road map of the protein interactions formed between a prototypic envelope glycoprotein encoded by either an arenavirus or hantavirus, and the human proteome. The viral envelope glycoprotein (GP) decorates the surface of the virion. The primary function of the GP is to mediate attachment of the virus to specific cellular receptors, and after internalization of the virion, fuse the viral membrane with an internal endosomal membrane. In order to carry out these specific tasks, the viral GPs must first co-opt the extensive machinery found within the cellular secretory pathway to coordinate the proper glycosylation, folding, proteolytic maturation, and targeting of the GP during its biosynthesis. We identified a human protein with a conserved interaction amongst these two groups of viral GPs termed the Endoplasmic Reticulum (ER)-Golgi Intermediate Compartment Protein of 53 kiloDaltons (ERGIC-53). ERGIC-53 is an intracellular cargo receptor that normally cycles within the early secretory pathway of cells, where it is responsible for ferrying a small subset of cellular glycoproteins, most notably the coagulation factors FV and FVIII, from the ER to the Golgi apparatus. Herein we describe a novel role for ERGIC-53 in the propagation of not only arenaviruses, but also coronaviruses and filoviruses. Following infection with an arenavirus, ERGIC-53 leaves the early secretory pathway and becomes incorporated into the virus as it pinches off from the cell surface. Newly formed viruses lacking ERGIC-53 are no longer infectious due, in part, to a defect in their ability to attach to host cells. We suggest that ERGIC-53 represents a promising broad-spectrum antiviral target because of its association with the GPs from many families of pathogenic viruses, as well as its ability to exert control over their infectivity; and finally, because ERGIC-53 itself is not required for human health. The discovery of ERGIC-53 outside of its normal location inside of cells suggests that it may have additional unknown functions. Lastly, by revealing the importance of the cellular protein in controlling viral infectivity, we provide insight into the ongoing co-evolution of virus and host.

<p>L1 syndrome is an X-linked recessive disorder, characterised by congenital hydrocephalus, adducted thumbs, spastic paraplegia, agenesis of the corpus callosum and mental retardation. The disease is caused by mutations in the L1CAM gene, encoding a protein predominantly expressed in the nervous system. This protein has been implicated in a variety of processes including neuronal migration, neurite outgrowth and fasciculation, myelination, and long-term memory formation.</p><p>L1 syndrome was suspected at genetic counselling of a family with a boy suffering from congenital hydrocephalus and mental retardation. Complete sequencing of L1CAM, performed by an external laboratory, revealed a novel mutation in the family, including a boy, affected with L1 syndrome, his sister, his mother and his maternal grandmother.</p><p>To verify this mutation and to be able to detect mutations in the L1CAM gene locally in the future, a method for mutational analysis of this gene was set up using PCR optimisation and cycle sequencing.</p><p>Sequencing of L1CAM was then performed on DNA samples from the four family members and the mutation was verified. A point mutation (c.3458-1G>C) in the 5’ splice site of exon 26 was detected in all of them. This new, not previously described, mutation is supposed to cause a deletion of the 26th exon and a frameshift in the part of the protein encoded by exons 27 and 28. This means that almost the entire cytoplasmic domain of the protein would have a loss of function, explaining the symptoms affecting the boy.</p>

<p>The Notch signalling pathway is an evolutionary conserved pathway, named after the Notch receptors, Notch1-4 in mammals, which upon cell-cell contact and ligand binding releases the intracellular domain (NICD). NICD translocates into the nucleus where it binds the transcriptional repressor RBP-Jk, which together with co-activators belonging to the Mastermind-like family of proteins form a transcriptional activation complex. This complex activates genes controlling cell fate decision, embryonic development, proliferation, differentiation, adult homeostasis and stem cell maintenance. On the other hand, disrupted Notch signalling may result in pathological conditions like cancer, although the mechanisms behind the disruption are often complex and in many cases largely unknown.</p><p>Notch1 drives the lymphocyte differentiation towards a T-cell fate and activating mutations in the gene have been suggested to be involved in T-cell lymphoma. In <em>paper I, </em>genetic alterations in <em>Notch1 </em>and the Notch1 regulating gene <em>CDC4 </em>were investigated in tumours from murine T-cell lymphoma induced with phenolphthalein, 1,3-butadiene or 2’,3’-dideoxycytidine. We identified activating <em>Notch1</em> mutations in 39% of the lymphomas, suggesting that <em>Notch1 </em>is<em> </em>an important target gene for mutations in chemically induced lymphomas.<em></em></p><p>While it is known that constitutively activated Notch signalling has a clear oncogenic function in several solid malignancies as well, the molecular mechanisms are less known in this context. Unpublished data of our lab, together with other recent studies, suggest that mutations of Notch and Notch-related genes <em>per se</em> are uncommon in solid malignancies including colorectal cancer, while a growing body of evidence indicates that aberrant Wnt/b-catenin signalling may result in pro-tumoural Notch activation in these contexts. In <em>paper II</em>, we therefore investigated potential transcriptional interactions between the Notch and Wnt signalling pathways in colorectal cancer cell lines. The proximal Notch and Wnt pathway gene promoters were bioinformatically identified and screened for putative TCF/LEF1 and RBP-Jk sites. In canonical Wnt signalling, Apc negatively regulates b-catenin leading to repression of TCF/LEF1 target genes. Upon repression of the Wnt pathway we observed that several genes in the Notch pathway, including <em>Notch2</em>, were transcriptionally downregulated. We also confirmed binding of Lef1 to <em>Notch2</em> as well as other Notch pathway gene promoters and luciferase assays showed an increased activity for at least one LEF1/TCF-site in the <em>Notch2</em> promoter upon co-transfection of HT29 or HCT116 cells with mutated b-catenin. HT29 cell lines were also treated with the g-secretase inhibitor DAPT, leading to inactivation of the Notch pathway by preventing release of NICD. However, results showed no effects on Apc, b-catenin or their target <em>cyclin D1</em>. Taken together, these results indicate that the Wnt pathway may function as a regulator of the Notch pathway through the TCF/LEF1 target gene program in colon cancer cell lines.</p><p>In summary, Notch pathway deregulation is of importance in both murine T-cell lymphoma and human colorectal cancer, although the mechanisms differ. The current results give new insights in Notch pathway alterations as well as the signalling networks in which the Notch pathway interacts, and thus increase the understanding of Notch’s involvement in malignant diseases.</p> / Studies on molecular genetic alterations in colorectal cancer