Nonsteroidal anti-inflammatory drugs (NSAIDs) are effective in cancer prevention and have been shown to suppress the formation of colorectal tumors in both humans and rodents. The chemopreventive action of NSAIDs is believed to be mediated through induction of apoptosis in preneoplastic cells. However, the precise molecular mechanisms of NSAID-induced apoptosis remain unclear. Previous studies demonstrated that second mitochondria-derived activator of caspase (SMAC) plays an important role in executing NSAID-induced apoptosis in colon cancer cells. SMAC-knockout HCT116 colon cancer cells are resistant to NSAID-induced apoptosis, and are deficient in caspase activation and cytosolic release of cytochrome c and apoptosis inducing factor (AIF). In this study, we tested the hypothesis that SMAC regulates the release of cytochrome c and activation of caspase cascade through a feed-back amplification loop. We found that the N-terminal AVPI domain of SMAC is required for the proapoptotic activity of SMAC. Following NSAID treatment, SMAC promotes dissociation of caspase-3 from inhibitor of apoptosis proteins (IAPs), which in turn leads to mitochondrial dysfunction. We also studied the effects of pharmacological manipulation on NSAID-induced apoptosis by employing small-molecule compounds that functionally mimic the AVPI domain of SMAC. A synergistic action of NSAIDs and SMAC mimetics was observed in inducing a robust apoptotic response in several colon cancer cell lines, as well as in NSAID-resistant BAX-KO and SMAC-KO cell lines. SMAC mimetics appear to potentiate NSAID-induced apoptosis by stimulating the release of cytochrome c from mitochondria and activation of caspases. Together, these results suggest that SMAC may be useful as a target for the development of more effective chemopreventive agents.

Numerous reports correlate nitric oxide (NO) production with stalled S-phase progression, but the molecular mechanism(s) of cell cycle arrest remains elusive. Paradoxically numerous human tumors are exposed to vast quantities of nitric oxide and its reactive byproducts in situ, yet they continue to grow and proliferate. The dual-specificity phosphatase Cdc25A promotes cell cycle progression by dephosphorylating and activating cyclin-dependent kinases. Deregulation of Cdc25A is characteristic of human tumors, accelerates the cell cycle, and confers resistance to apoptosis, highlighting the importance of stringent Cdc25A control. Biochemical and structural analyses of Cdc25A indicate the potential for inhibition by S-nitrosation of the catalytic cysteine, providing a linkage between NO and cytostatic signaling. Thus, the overall hypothesis examined in this dissertation was that Cdc25A is a target and transducer of signaling by NO and NO-derived reactive species. The specific aims were to: 1) probe the susceptibility of Cdc25A to enzymatic regulation by NO-derived reactive species; 2) examine regulation of Cdc25A protein in nitrosatively challenged cells; and 3) determine whether Cdc25A activity was limiting for S-phase progression in nitrosatively-challenged tumor cells. My studies identified novel mechanisms controlling Cdc25A abundance and activity. S-Nitrosothiols rapidly S-nitrosated and inactivated Cdc25A in vitro, and Cdc25A activity was restored by reductants. Generation of nitrosative stress in cells either by iNOS-derived NO or the cell-permeable S-nitrosating agent S-nitrosocysteine ethyl ester (SNCEE) caused translational inhibition of Cdc25A via hyperphosphorylation and inhibition of the eukaryotic translational regulator eIF2á. Although iNOS-derived NO and SNCEE inhibited DNA synthesis coincident with Cdc25A loss, restoration of Cdc25A activity in nitrosatively-challenged cells did not alter DNA synthesis inhibition, distinguishing nitrosative inhibition of DNA synthesis from the canonical intra-S-phase checkpoint. SNCEE decoupled Cdc25A from ASK-1 and sensitized cells to chemotherapeutic-induced apoptosis, suggesting that Cdc25A suppression by nitrosative stress may lower the apoptotic threshold in nitrosatively-challenged cells by priming ASK-1 for activation. In summary, these studies describe novel regulation of Cdc25A translation and activity, and a model wherein selective inhibition of Cdc25A phosphatase-dependent and independent activities can occur under nitrosative stress, and implicate Cdc25A as a regulator of apoptotic threshold following nitrosative insult via priming of ASK-1.

There is an urgent need for novel effective drug regimens for the treatment of cancer. Current chemotherapy suffers from a slim therapeutic index, with significant toxicity from effective drug doses or tumor recurrence at low drug doses. Identifying synergistic interactions between drugs is a difficult process. To accelerate the discovery of potential drug combinations, I have developed a druggable genome siRNA synthetic lethal screen capable of rapidly identifying novel drug targets that would sensitize cancer cells to sublethal concentrations of microtubule destabilizing agents. I employed a high-throughput cell-based 16,560-siRNA screen to isolate a high-confidence list of genes that, when silenced, enhanced glioblastoma multiforme cancer cell chemosensitivity. Two gene products that were the major focus of my work were midline2 and the neurokinin receptor NK1R. Silencing of midline2, a PP2A-microtubule tether, sensitized cells to two microtubule destabilizing agents, vinblastine and disorazole C1, suggesting a mechanistic dependency of the phosphatidylinositol 3-kinase pathway on microtubule functionality. Combinations of phosphatidylinositol 3-kinase inhibitors with disorazole C1 and several vinca alkaloids confirmed this hypothesis. To verify microtubule destabilizing agent sensitization by NK1R silencing, I demonstrated a significant collaboration of neurokinin receptor NK1R antagonists with low concentrations of vinca alkaloids. These assay results and subsequent novel combination strategies demonstrate the tremendous ability of this synthetic lethal screen to predict potent collaborations between different classes of drugs, as well as identifying molecular constituents mediating those interactions.

Alzheimers disease (AD) is a neurodegenerative disorder that results from a loss of synaptic transmission and ultimately results in cell death. However, the mechanisms that induce neuronal cell death remain elusive. Amyloid plaques composed of amyloid fibrils (Ab) and neurofibrillary tangles (NFTs) composed of hyperphosphorylated tau (pTau) are the main pathogenic hallmarks of AD. Ab and NFT generation is influenced by reactive oxygen species and altered signaling pathways. Focal adhesion proteins assemble into intracellular complexes involved in integrin-mediated communication between the extracellular matrix (ECM) and the actin cytoskeleton, regulating many cell physiological processes. Interestingly, recent studies report that integrins bind to Ab fibrils, mediating Ab signal transmission from extracellular sites of Ab deposits into the cell and ultimately to the nucleus. Hydrogen peroxide-inducible-clone 5 (Hic-5) and paxillin are members of the group III LIM domain protein family that localize to both the nucleus and focal adhesions. Hic-5 and paxillin are expressed in numerous regions of the rat brain including cerebellum, striatum, prefrontal cortex, hippocampus, hypothalamus, thalamus, and spinal cord. While little is known about the specific roles of paxillin and Hic-5 in regulating focal adhesion signaling and gene expression within brain, non-genomic roles for both paxillin and Hic-5 in brain have been described. For example, in cultured neurons, paxillin is rapidly phosphorylated in the presence of fibrillar b-amyloid and colocalized with pTau, leading to altered focal adhesion turnover and loss of synaptic integrity. A functional role for Hic-5 in the brain was revealed by its ability to (1) decrease surface levels of dopamine transporter (DAT) in rat midbrain neuronal cultures and to (2) negatively affect dopamine uptake. A direct interaction between Hic-5 and DAT may be responsible for this effect. While these reports suggest biologic functions of Hic-5 and paxillin in brain, a detailed analysis of paxillin and Hic-5 expression and distribution in normal or AD brain has not been performed. Given the in vitro association between paxillin and b-amyloid-induced toxicity and Hic-5 response to oxidative stress, a blinded retrospective cross-sectional study of the human hippocampus for Hic-5 and paxillin was performed. The expression and subcellular distribution of Hic-5 and paxillin in AD and control hippocampus were determined by immunohistochemistry (IHC) from early and late-stage AD and age-matched control subjects. IHC was also used to examine the subcellular distribution of specific phosphorylated isoforms of paxillin. Laser scanning confocal microscopy (LSCM) was used to visualize or demonstrate colocalization of Hic-5, paxillin and phosphorylated isoforms of paxillin. Observations demonstrate changes in the subcellular distribution of Hic-5, paxillin and specific phosphorylated isoforms of paxillin within particular regions of the hippocampus in AD brain. Hic-5 and phosphorylated isoforms of paxillin colocalize with NFTs, while paxillin is predominantly found in reactive astrocytes (stellate-shaped) in the hippocampus of AD brains. Thus, important scaffolding proteins that link various intracellular signaling pathways to the ECM are modified and exhibit altered subcellular distribution in hippocampus during AD.

Classically, the epithelial lining of the urinary bladder, also called the urothelium, has been thought of as a passive barrier against toxins present in urine. However, recent studies are beginning to emerge that demonstrate an active role for the urothelium in the sensory functions of the bladder. For example, the urothelium expresses a number of the same receptors as sensory nerves and can respond to and release transmitters. One such transmitter, acetylcholine, has been shown to be released from the urothelium in response to physical stimuli, and is thought to act back on the urothelium in an autocrine/paracrine manner to effect urothelial signaling. This study was undertaken to determine if the urothelium expresses the proper receptors to respond to acetylcholine, specifically nicotinic acetylcholine receptors, and if these receptors play a role in influencing bladder physiology. Our research indicates that the urothelium expresses the proper nicotinic receptor subunits to form two classes of receptor: 1) α3 heteromeric receptors and 2) α7 homomeric receptors. Both of these classes of urothelial receptor are functional and can alter bladder reflexes in the anesthetized rat. Specifically, α7 receptors mediate an inhibitory pathway as measured by a bladder cystometrogram, while α3 receptors mediate an excitatory pathway. Finally, we examined intracellular and extracellular pathways that may mediate these physiological effects in vivo. These experiments suggest that nicotinic receptors in the urothelium mediate their effects through intracellular calcium signaling, resulting in the modulation of the release of the excitatory transmitter ATP. Specifically, our research indicates that α3 stimulation can potentiate the release of ATP from urothelial cells, while α7 stimulation inhibits it. This effect may be due to the fact that each receptor subtype modulates [Ca+2]i through distinct pathways: α3 receptors through influx of extracellular Ca+2 and α7 receptors through release from intracellular stores. Additionally, our research indicates that α7 receptors can inhibit signaling through α3 receptors, indicating another possible mechanism for the inhibitory effects α7 receptors exhibit in vivo. This research, which is the first to indicate an interaction between two types of nicotinic receptor, suggests that urothelial nicotinic receptors could play a significant role in bladder physiology and may represent a viable target for treatments into bladder pathology.

Caveolin-1 (Cav-1) is a membrane associated scaffolding protein that regulates a myriad of signaling molecules. It has been implicated as both a tumor suppressor and promoter. Here, we examine the proteins link to senescence and cancer, and identify a novel pathway through which Cav-1 mediates stress induced premature senescence (SIPS) through p53 activation. Oxidative stress triggers p38MAPK , which activates the transcription factor Sp1. Sp1 binds to two GC-rich regions in the caveolin-1 promoter up-regulating the protein. Cav-1 binds to p53s negative regulator, MDM2, sequestering the E3 ligase to allow p53 to become active. p53 activates its downstream targets, such as p21WAF/CIP1, which initiates SIPS. This pathway is dysfunctional in many cancers that have a downregulated Cav-1 gene. The effects of oxidative stress in Cav-1 null backgrounds were examined. Breast cancer cells that do not express Cav-1 cannot undergo oxidatively induced SIPS. However, upon re-expression of Cav-1, the SIPS phenotype is restored. Utilization of Cav-1 knockout mouse embryonic fibroblasts show that without Cav-1 to sequester MDM2, allowing for the upregulation of p53 leading to SIPS, cells continued to proliferate. These results distinguish Cav-1 as a molecular senescence switch, because in its absence oxidative SIPS does not occur, but in its presence it does. This effect is also not specific to a particular cell type; data supports Cav-1 as a molecular switch in epithelial and fibroblast cell lines. Finally, senescence is known to have antagonistic pleiotropic effects on an organism. That is, cell senescence is beneficial for younger organisms, as it prevents the proliferation of mutated genomes through growth arrest. However, an accumulation of senescent cells can lead to aging and become detrimental. Cav-1s role in the antagonistic pleiotropic effects of senescent fibroblasts on neoplastic epithelial cells is also explored. Data shows that senescence of fibroblasts depends upon Cav-1 sequestering MDM2, which activates p53 and induces SIPS. These fibroblasts can secrete factors that make it advantageous for NIH 3T3 RasG12V transformed fibroblasts and MDA-MB-231 breast cancer epithelial cells to proliferate in vitro and in vivo. Hence, we propose that the Cav-1 gene functions with antagonistic pleiotropy.

The hallmark of chronic myelogenous leukemia (CML) is a chromosomal translocation between the c-abl gene (chromosome 9) and the bcr gene (chromosome 22). This event gives rise to BcrAbl, a chimeric protein with constitutive tyrosine kinase activity that drives the pathogenesis of the disease. Imatinib, a Bcr-Abl kinase inhibitor is the frontline therapy in CML. Although imatinib is very effective in the chronic phase of CML, patients in advanced stages develop resistance. An increased understanding of the signaling pathways implicated in CML pathogenesis and imatinib resistance is critical to the development of improved therapies. Previous work in our laboratory found that A-419259, a broad-spectrum Src family kinase (SFK) inhibitor induces growth arrest and apoptosis in CML cells, suggesting that SFKs are required for Bcr-Abl transformation of myeloid progenitors. Additionally, Hck couples BcrAbl to Stat5 activation in myeloid cells, which may contribute to survival. Furthermore, studies on samples from some imatinib-resistant patients found increased expression and activity of Hck and Lyn. In this dissertation, using two chemical genetic methods, I addressed the contribution of Hck to Bcr-Abl signaling and imatinib resistance. To explore the individual contribution of Hck to Bcr-Abl signaling, I developed an A419259-resistant mutant of Hck (Hck-T338M). Expression of Hck-T338M fully protected K562 CML cells from A-419259-induced apoptosis, an effect that correlated with sustained Stat5 activation. In addition, the Hck-T338M partially protected CML cells against the growth inhibition induced by A-419259. These studies suggest that Hck plays a non-redundant role as a key downstream survival partner for Bcr-Abl. I also tested whether Hck overexpression was sufficient to induce imatinib resistance in CML cells. For this study, I developed a mutant of Hck (Hck-T338A) that is uniquely sensitive to NaPP1, an analog of the generic SFK inhibitor pyrrazolo-pyrimidine 1. Overexpression of Hck or Hck-T338A in K562 cells induced resistance to imatinib-dependent apoptosis and growth arrest. Furthermore, NaPP1 reversed imatinib resistance in K562-Hck-T338A cells, suggesting that Hck-induced imatinib resistance requires Hck kinase activity. Taken together, my work validates Hck as a target for the development of apoptosis-inducing drugs and that are likely to be effective in imatinib-resistant patients.

The mammalian lung supports the transport and diffusion of inspired and expired gasses that are critical for aerobic life. With every inspiration the lung is exposed to environmental agents including microbes, virus, and environmental pollutants. In the event that injury occurs the epithelium is repaired by an abundant facultative progenitor pool and a sequestered population of adult tissue stem cells. Chronic lung diseases, such as asthma, chronic obstructive pulmonary disease, and bronchopulmonary dysplasia, are characterized by extensive epithelial remodeling resulting in a reduction to the number of non-ciliated bronchiolar Clara cells. Given the established role for Clara cells as abundant facultative progenitors, these data suggest that epithelial repair has been compromised. In addition to affects on the epithelium, these diseases are also accompanied by extensive subepithelial fibroproliferation, mesenchymal remodeling, and elevated extracellular matrix deposition as well as a profound increase to lung inflammation. It has been postulated, but never tested in vivo that mesenchymal remodeling and uncontrolled deposition of extracellular matrix may be a result of impaired airway epithelial reparative capacity. Moreover, the finding that airway epithelial cells are essential for modulation of innate immunity suggests that the enhanced inflammatory response described in chronic lung disease may be a result of attenuated airway epithelial cell function. Therefore, this dissertation tests the hypothesis that airway epithelial reparative capacity moderates extracellular matrix deposition and innate immunity. Through the use of in vivo models of injury, inflammation, and attenuated Clara cell function, this dissertation research work identifies a previously uncharacterized process in which extracellular matrix is dynamically and reversibly regulated during productive epithelial repair and severely disrupted by blocking stem cell mediated repair. In addition, the use of mouse models of decreased Clara cell abundance and secretion demonstrate airway epithelium modulates pulmonary innate immunity through regulation of macrophage behavior and inhibition of pulmonary inflammation. This work defines two phenotypes that are the result of attenuated epithelial repair and supports the paradigm that epithelial reparative capacity may be a principal determinant of lung disease.

Hypoxia represents an important element of the solid tumor microenvironment and contributes to tumorigenesis and resistance to chemo- and radiation therapy. Hypoxia can modulate cell cycle progression although the mechanisms involved remain unclear. The Cdc25A dual specificity phosphatase promotes cell cycle progression by dephosphorylating and activating cyclin-dependent kinases. Cdc25A is the master regulator of the cell cycle and its disruption induces cell cycle arrest in cancer cells. The recent observation that under hypoxic conditions, levels of Cdc25A protein and mRNA are decreased led to the hypothesis that hypoxia-mediated reduction in Cdc25A may represent a novel mechanism in the hypoxic regulation of the cell cycle. Given the prominent role of Cdc25A in regulating the cell cycle and the proposed changes in Cdc25A protein and mRNA under hypoxic conditions, it was hypothesized that Cdc25A plays an essential role in hypoxia-mediated cell cycle arrest in human tumor cells. The specific aims were to: 1) examine the mechanism of Cdc25A downregulation in response to hypoxia, 2) determine the role HIF-1α in Cdc25A regulation and cell cycle arrest, and 3) determine if Cdc25A downregulation is required for hypoxia-induced cell cycle arrest. Under hypoxic conditions, Cdc25A protein levels were specifically and reversibly suppressed. It was found that Cdc25A mRNA levels are significantly decreased by a p21-dependent mechanism. In addition, suppression of Cdc25A was independent of p53. Loss of Cdc25A protein occurred in the absence of checkpoint activation. Recent evidence has linked the microRNA miR-21 to Cdc25A and hypoxia. It is shown here that miR-21 was required for Cdc25A mRNA suppression in hypoxic colon cancer cells and miR-21 levels were increased under hypoxic conditions. The HIF-1α transcription factor was not required for suppression of Cdc25A but must be present for hypoxia-induced cell cycle arrest. Under hypoxic conditions, cells undergo p21- and miR-21- S-phase cell cycle arrest. This study proposes a novel mechanism of transient regulation of Cdc25A via the p21- and miR-21-dependent regulation of mRNA levels in hypoxic cells leading to cell cycle arrest. This previously unknown mechanism may confer protection from hypoxic conditions, contributing to cell survival and the observed resistance to chemo- and radiation therapy.

Head and neck squamous cell carcinoma (HNSCC) is characterized by the overexpression of the epidermal growth factor receptor. However, molecular targeting strategies against EGFR have not improved the 5-year survival rates of HNSCC patients. EGFR tyrosine kinase inhibitors displayed limited clinical responses in Phase II trials and the FDA-approved monoclonal antibody cetuximab (C225) did not prevent the occurrence of secondary tumors and distant metastases. G-protein-coupled receptor ligands; gastrin-releasing peptide (GRP), prostaglandin E2 (PGE2) and bradykinin (BK) have all been reported to activate EGFR in HNSCC via extracellular release of EGFR ligands TGF-á and AR. To improve the efficacy of EGFR inhibition in HNSCC, we investigated the efficacy of targeting common signaling intermediates involved in GPCR-EGFR crosstalk. We previously reported that GRP mediated release of EGFR ligands via phosphoinositide-dependent kinase 1 (PDK1) dependent phosphorylation of a disintegrin and metalloprotease 17 (ADAM17). We subsequently investigated whether PDK1 mediates EGFR activation downstream of PGE2, BK and LPA pathways and the efficacy of different PDK1 targeting strategies in HNSCC. PGE2, BK and LPA-mediated EGFR phosphorylation was abrogated in PDK1 siRNA-transfected HNSCC cells. PDK1 siRNA also decreased PGE2 and BK-mediated HNSCC growth in vitro. Expression of kinase-dead PDK1 (PDK1M) decreased PGE2 -mediated HNSCC growth. PDK1M HNSCC cells demonstrated reduced proliferation compared to control HNSCC cells. HNSCC cells displayed nanomolar sensitivity to the PDK1 inhibitor OSU-03012 compared to normal mucosal cells. Combined treatment with the EGFR TKIs erlotinib or AG1478, plus OSU-03012 enhanced anti-proliferative effects. We have reported that PGE2 and BK mediated MAPK phosphorylation in the presence of EGFR inhibition, and combined GPCR and EGFR demonstrated additive to synergistic anti-tumor effects. To elucidate the EGFR-independent signaling mediated by GPCRs, we used a forward phase phosphoprotein array to identify potential molecular targets that can potentiate EGFR inhibition. We observed that p70S6K phosphorylation was induced in EGFR siRNA-transfected cells and sustained in cetuximab (C225)-treated cells following PGE2 or BK stimulation. Further investigation showed that p70S6K phosphorylation mediated by EGFR downmodulation was dependent on PDK1 and PKCä expression. Combined targeting of EGFR with cetuximab and p70S6K with the mTOR inhibitor RAD001 decreased GPCR-mediated growth in vitro and in vivo. The results from this study have indicated that targeting the GPCR signaling intermediates PDK1 and p70S6K in conjunction with EGFR may be beneficial therapeutic strategies for the subset of HNSCC patients that respond poorly to cetuximab treatment.