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.

In the United States in 2009, 192,370 women are expected to be diagnosed with invasive breast cancer, and 62,280 with in situ disease. About 70% of these cases are estrogen receptor positive (ER+). There are two isoforms of the ER, α and β, that differ somewhat in structure and action. ERβ expression plays a protective role in breast cancer, and selective targeting of this isoform would have many beneficial effects. Tamoxifen has long been the standard of care for patients with ER+ breast cancer. A major problem with tamoxifen is the development of drug resistance. One of the mechanisms proposed for the development of tamoxifen resistance involves the loss of ERβ expression. The first objective of this study was to screen a library of biphenyl C-cyclopropylalkylamides for their ability to function as ERβ-selective ligands. Two compounds were identified with modest selectivity for ERβ and anti-proliferative effects in breast cancer cells where they inhibited expression of c-Myc. The nuclear matrix (NM), the structural scaffolding of the nucleus, plays a major role in many fundamental processes of the cell. Using the ER+ breast cancer cell line MCF-7 and an antiestrogen resistant derivative, along with subtype selective ER ligands, alterations in the abundance of specific proteins present in the NM were identified using a mass spectrometry (MS)-based relative quantitative methodology. Some of the most interesting proteins with altered abundance are NuMA, serpin H1, hnRNP R, and dynein heavy chain 5. These proteins may represent putative biomarkers to customize treatment. The alterations also provide a mechanistic understanding of tamoxifen resistance. The NM was also investigated by MS in the earliest stage of breast cancer, ductal carcinoma in situ (DCIS), utilizing novel cell lines derived from normal (breast reduction), DCIS, and non-diseased contralateral breast surgical specimens. Two of the interesting proteins found to be altered in DCIS were HSP90 and EEF1D. These studies may provide biomarkers to aid in the diagnosis and treatment of breast cancer. In addition by understanding the mechanism behind the development of breast cancer, prevention becomes a possibility.

Caveolin-3 is a muscle specific scaffolding protein with both structural and signaling roles. Lack of caveolin-3 expression has been implicated in limb-girdle muscular dystrophy, along with distal myopathy and rippling muscle disease. These diseases are characterized by progressive muscle weakness and muscle wasting. Nicotinic acetylcholine receptor (nAChR) clustering and localization are important for efficient nerve to muscle contractile signal transmission. It is hypothesized that muscle weakness could originate through disrupted nAChR clustering, disrupting the efficiency of signaling from the motorneuron to the muscle. While the molecular mechanisms involved in nAChR clustering remain to be fully defined, we hypothesize caveolin-3 is important for nAChR clustering and overall neuromuscular junction function. Caveolin-3 and the nAChR co-localize and associate evidenced by immunofluorescence and immunoprecipitation. These results were replicated in differentiated wildtype myotubes treated with the nAChR clustering agent, neural agrin. In differentiated caveolin-3 null myotubes, agrin treatment yields a 60% reduction in nAChR clusters as compared to agrin treated wildtype myotubes. Agrin induces nAChR clustering, through activation of muscle specific kinase (MuSK) and downstream through Rac-1 activation. In differentiated wildtype myotubes, Rac-1 activation peaks at 1 hour of agrin treatment, while in differentiated caveolin-3 null myotubes there is dramatically reduced Rac-1 activation upon agrin treatment. Immunoprecipitation of MuSK shows that caveolin-3 and MuSK association peaks at 1 hour of agrin treatment in wildtype cells. This corresponds to the peak of MuSK phosphorylation which also occurs at 1 hour. Agrin induced MuSK phosphorylation was decreased more significantly than the overall decrease in MuSK expression in the caveolin-3 null cells as compared to the wildtype results. These results indicate a role for caveolin-3 in efficient nAChR clustering. Electromyography studies in anesthetized mice indicated lengthened latencies of the muscle action potential in the caveolin-3 null mice as compared to wildtype mice. There were also decreased overall electromyography (EMG) amplitude and EMG area under the curve in caveolin-3 null mice. Comparison of contractile strength in wildtype and caveolin-3 null animals indicated tetanic contractions to be less stable in the caveolin-3 null animals, though there was late potentiation in actual contractile strength. Lack of caveolin-3 affects the neuromuscular junction formation and transmission without affecting overall contractile strength. This research opens a novel view, that correct neuromuscular junction formation and neuromuscular transmission is important in the development of muscular dystrophies.

Despite their widespread use, the precise molecular actions of anesthetics and alcohol are unknown. Although anesthetics have made surgical intervention palatable, anesthetics are not free of alarming side effects such as anesthetic awareness and post-operative cognitive deficits. While alcohol is consumed for positive effects such as anti-anxiety, euphoria, and relaxation, alcohol consumption also results in adverse behavioral effects such as sedation, motor incoordination, and cognitive impairment. Chronic alcohol consumption results in alcohol withdrawal syndrome, tolerance, and dependence. Understanding the mechanisms of action of anesthetics and alcohol are critical for preventing anesthetic side effects and for developing effective treatments for alcohol abuse and alcoholism. Many putative targets of anesthetics and alcohol have been identified in brain including sodium channels, potassium channels, glutamate receptors, glycine receptors, and especially GABA receptors. Phasic and tonic inhibitory currents mediated by GABA type A receptors are sensitive to modulation by anesthetics and alcohol. α4 subunit-containing GABA A receptors mediate tonic inhibitory currents, are highly sensitive to GABA, and are strongly implicated in the effects of volatile, intravenous, and neurosteroid anesthetics. Global α4 knockout mice showed reduced tonic current that was not potentiated by the volatile anesthetic isoflurane or the neurosteroid anesthetic, alphaxalone. Specific Aim 1 tested the hypothesis that volatile and intravenous anesthetic effects are mediated via a4-containing receptors by comparing behavioral responses to these drugs in wild type and α4 KO mice. Results obtained indicate that while α4-containing receptors are required for the amnestic effects of isoflurane, they are not required for mediating the effects of volatile and intravenous anesthetics on other behavioral endpoints. Interestingly, α4-containing receptors are required for low dose alphaxalone-induced locomotor stimulation, but not high dose effects. α4-containing receptors, when paired with the δ subunit, have been proposed to possess a common binding pocket for ethanol and pharmacologic antagonists of ethanol action. Previous studies in the Homanics lab indicated that the ethanol-reversing effects of Ro15-4513, an imidazobenzodiazepine ethanol-antagonist, were dramatically reduced in α4 KO mice both at the cellular and the behavioral level. Specific Aim 2 tested the hypothesis that a4-containing receptors are required for the ethanol antagonistic effects of RY023, a derivative of Ro15-4513. α4 KO mice showed differential sensitivity to the effects of RY023 in the presence and absence of ethanol on loss of righting reflex and locomotor behavior. We conclude that α4 containing receptors are involved in some intrinsic effects of RY023 but not in the ethanol-antagonistic effects of RY023. This study of α4-containing receptors has advanced our understanding of anesthetic action and eliminated the theory of a unitary target for ethanol antagonism.

Electrophilic lipids are emerging as critical mediators of anti-inflammatory signaling pathways, although many biologically relevant electrophiles may still remain unknown. Nitro derivatives (NO2-FA) and á,â-unsaturated carbonyl derivatives of unsaturated fatty acids are naturally occurring electrophilic products of redox reactions, and can modulate a variety of cellular signaling processes including the transcriptional activity of the peroxisome proliferator-activated receptor-ã (PPARã). PPARã binds diverse ligands to regulate the expression of genes involved in metabolism and inflammation. Activators of PPARã include anti-hyperglycemic drugs such as thiazolidinediones (TZDs) and intermediates of lipid metabolism and oxidation that bind PPARã with very low affinity. Recently TZDs have raised concern after being linked with increased risk of peripheral edema, weight gain, and adverse cardiovascular events. In contrast, NO2-FA act as partial agonists of PPARã at nM concentrations and covalently bind PPARã via Michael addition. NO2-FA show selective PPARã modulator characteristics by inducing coregulator protein interactions distinctively different from those induced by the TZD Rosiglitazone. In further exploring the electrophilic lipidome, a new subclass of electrophilic lipid has been revealed. Using a recently developed â-mercaptoethanol (BME) alkylation reaction, followed by HPLC-MS/MS-based screening, we report six novel electrophilic fatty acid derivatives (EFADs) specifically formed during macrophage activation (RAW264.7 and THP-1 cell lines and primary macrophages treated with IFNã and LPS). The major EFADs are á,â-unsaturated oxo-derivatives of omega-3 fatty acids as confirmed by cell culture and in vitro studies and by MS/MS structural analysis. The isomers of two major EFADs were identified as 13- and 17-keto derivatives of docosapentaenoic acid (DPA) and docosahexaenoic acid (DHA). Purified cyclooxygenase-2 (COX-2) product profiles and treatment of activated macrophages with COX-2 inhibitors confirmed EFAD synthesis to be catalyzed by inducible COX-2, followed by hydroxy-dehydrogenase activity. EFAD production was increased 2.5 fold in activated macrophages treated with acetylsalicylic acid (ASA; aspirin). Internal standard-based quantification showed that EFADs are highly abundant electrophiles in activated macrophages, reaching intracellular concentrations as high as 350 nM. Importantly, EFADs form reversibly reactive covalent adducts with both proteins and small molecule thiols in activated macrophages, supporting a potential for post-translational protein modification-mediated cell signaling. Furthermore, synthetic isomers of EFAD-1 and -2 (17-oxo-DHA and 17-oxo-DPA, respectively) act as partial agonists of PPARã, activate Nrf2 (nuclear factor-erythroid 2-related factor 2)-dependent gene expression, and inhibit pro-inflammatory cytokine production and iNOS expression in IFNã and LPS-activated RAW264.7 cells and in primary macrophages. In conclusion, it has been demonstrated that upon activation macrophages generate omega-3 derived electrophilic signaling molecules at biologically relevant concentrations that act as autocrine mediators.

G-protein coupled receptors (GPCRs) are the largest family of transmembrane proteins, constituting 2% of the human genome. They mediate signaling from a diverse set of ligands, ranging from photons to large peptides. Their intracellular signaling cascades are complex and highly malleable depending on cellular context. Yet, GPCR signaling in vivo is highly specific. Cells maintain this tight control over GPCR signaling through the expression of adaptor proteins. These adaptors regulate GPCR function and activation on many levels - they localize receptors to specific subcellular domains, assemble functional signaling complexes, alter the specificity of G-proteins coupling to the receptor, or regulate receptor traffic to and from the plasma membrane. By balancing the expression of these adaptor proteins, cells control where, when and how long GPCRs signal. Na+/H+ Exchanger Regulatory Factor 1 (NHERF1), also known as Ezrin binding phosphoprotein 50kDa (EBP50), is the prototypical PDZ adaptor protein. Expression of NHERF1 clusters parathyroid hormone type 1 receptor (PTH1R) and frizzled (Fzd) along actin stress fibers in non-polarized cells at the apical actin cap in polarized cells. In addition to proper localization, interaction with NHERF1 has several signaling manifestations. For PTH1R, interaction with NHERF1 can cause either a G-protein switch or can scaffold a PTH1R-PKA-calcium channel signaling complex. For Fzd, interaction with NHERF1 blocks Wnt-induced -catenin activation. NHERF1 knockout mice exhibit PTH-resistant phosphate excretion and enhanced PTH-induced vitamin D synthesis, as well as increased mammary duct density secondary to heightened Wnt-Fzd signaling. NHERF1 is a prime example of how PDZ adaptor proteins regulate GPCR localization and diversify GPCR signaling in physiologically significant ways.

Background Lung cancer is currently the second most prevalent form of cancer in the United States and is the leading cause of cancer-related deaths. Currently, there are no effective therapies for those diagnosed in the later stages of lung cancer. The c-Met receptor is a potential therapeutic target for NSCLC along with its ligand, hepatocyte growth factor (HGF). Signaling interactions between c-Met and the mutant Epidermal Growth Factor Receptor (EGFR) have been studied extensively, but the biological importance of lateral signaling to c-Met in EGFR wild-type tumors is minimally understood. Principal Findings Our observations indicate that wild-type EGFR, the receptor most often found in NSCLC tumors, can initiate delayed c-Met activation in NSCLC cell lines. EGFR ligands induce accumulation of activated c-Met which begins at 8 h and continues for 48 h. This effect is accompanied by phosphorylation of critical c-Met tyrosine residues. Gene transcription is required for delayed c-Met activation; however, phosphorylation of c-Met by EGFR occurs without production of HGF or secretion of other factors, supporting an internal mechanism that is independent of c-Met ligand. Lateral signaling is blocked by two selective c-Met tyrosine kinase inhibitors (TKIs), PF2341066 and SU11274, or with gefitinib, an EGFR TKI, suggesting kinase activities of both receptors are required for this effect. The c-Src pathway is essential for EGFR to c-Met communication. This appears to be up- and downstream of delayed c-Met activation. Pre-treatment with pan-SFK inhibitors, PP2 and dasatinib, abolishes delayed c-Met phosphorylation. A c-Src dominant-negative construct reduces EGF-induced c-Met phosphorylation compared to control, further confirming a c-Src requirement. Additionally, delayed c-Src association with c-Met and prolonged c-Src activation are observed following EGF addition. Inhibition of c-Met with PF2341066 and siRNA decreases the EGF-induced phenotypes of invasion by ~86% and motility by ~81%, suggesting that delayed c-Met activation is utilized by EGFR to potentiate its full biological effects possibly through STAT3. Combined targeting of c-Met and EGFR pathways lead to increased NSCLC xenograft anti-tumor activity. Conclusions and Significance Collectively, these data provide an alternative working model of prolonged EGFR signaling, whereby c-Met activation in NSCLC cell lines initiated by wild-type, non-amplified EGFR with wild-type, non-amplified c-Met maximizes EGFR-induced cell motility and invasion. With the identification of this novel pathway, the studies presented here demonstrate that inhibition of both EGFR downstream signaling and EGFR lateral signaling through the EGFR-c-Src-c-Met axis might be effective in treatment of NSCLC. Taken together, these findings will aid in the future development of combination EGFR and c-Met TKI treatments in clinical trials for NSCLC tumors that are wild-type for both EGFR and c-Met.