IMMUNE MODULATION OF THE MYCOBACTERIUM TUBERCULOSIS GRANULOMATOUS RESPONSE

Tuberculosis (TB) remains a major public health burden. The immunocompetant host responds to Mycobacterium tuberculosis (MTB) infection by the formation of granulomas, which initially prevent uncontrolled bacterial proliferation and dissemination. However, increasing evidence suggests that granuloma formation promotes persistence of the organism by physically separating infected cells from effector lymphocytes and by inducing a state of non-replicating persistence in the bacilli, making them resistant to the action of antibiotics. Additionally, immune-mediated tissue destruction likely facilitates disease transmission. The granulomatous response is in part due to mycobacterial glycolipid antigens. Therefore, studies were first undertaken to determine the innate mechanisms of mycobacterial cord factor trehalose-6’6-dimycolate (TDM) on granuloma formation. Investigations using knock-out mice suggest that TNF-a is involved in the initiation of the granulomatous response, complement factor C5a generates granuloma cohesiveness, and IL-6 is necessary for maintenance of an established granulomatous responses. Studies were next performed to determine the ability of lactoferrin to modulate the immune response and pathology to mycobacterial cord factor. Lactoferrin is an iron-binding glycoprotein with immunomodulatory properties that decrease tissue damage and promote Th1 responses. Mice challenged with TDM and treated with lactoferrin had decreased size and numbers of granulomas at the peak of the granulomatous response, accompanied by increased IL-10 and TGF-b production. Finally, the ability of lactoferrin to serve as a novel therapeutic for the treatment of TB was performed by aerosol challenging mice with MTB and treating them with lactoferrin added to the drinking water. Mice given tap water had lung log10 CFUs of 7.5 ± 0.3 at week 3 post-infection. Lung CFUs were significantly decreased in mice given lactoferrin starting the day of infection (6.4 ± 0.7) and mice started therapeutically on lactoferrin at day 7 after established infection (6.5 ± 0.4). Total lung inflammation in lactoferrin treated mice was significantly decreased, with fewer areas of macrophages, increased total lymphocytes, and increased numbers of CD4+ and CD8+ cells. The lungs of lactoferrin treated mice had increased CD4+ IFN-g+ cells and IL-17 producing cells on ELISpot analysis. It is hypothesized that lactoferrin decreases bacterial burden during MTB infection by early induction of Th1 responses.

Adenosine induced pulmonary fibrosis and the contribution of the adenosine A2B receptor to the induction of osteopontin in a mouse model of pulmonary fibrosis

Pulmonary fibrosis is a devastating and lethal lung disease with no current cure. Research into cellular signaling pathways able to modulate aspects of pulmonary inflammation and fibrosis will aid in the development of effective therapies for its treatment. Our laboratory has generated a transgenic/knockout mouse with systemic elevations in adenosine due to the partial lack of its metabolic enzyme, adenosine deaminase (ADA). These mice spontaneously develop progressive lung inflammation and severe pulmonary fibrosis suggesting that aberrant adenosine signaling is influencing the development and/or progression of the disease in these animals. These mice also show marked increases in the pro-fibrotic mediator, osteopontin (OPN), which are reversed through ADA therapy that serves to lower lung adenosine levels and ameliorate aspects of the disease. OPN is known to be regulated by intracellular signaling pathways that can be accessed through adenosine receptors, particularly the low affinity A2BR receptor, suggesting that adenosine receptor signaling may be responsible for the induction of OPN in our model. In-vitro, adenosine and the broad spectrum adenosine receptor agonist, NECA, were able to induce a 2.5-fold increase in OPN transcripts in primary alveolar macrophages. This induction was blocked through antagonism of the A2BR receptor pharmacologically, and through the deletion of the receptor subtype in these cells genetically, supporting the hypothesis that the A2BR receptor was responsible for the induction of OPN in our model. These findings demonstrate for the first time that adenosine signaling is an important modulator of pulmonary fibrosis in ADA-deficient mice and that this is in part due to signaling through the A2BR receptor which leads to the induction of the pro-fibrotic molecule, otseopontin. ^

Osteopontin and cadherin 11 are novel mediators and drug targets for chronic lung diseases

Chronic lung diseases (CLDs) are a considerable source of morbidity and mortality and are thought to arise from dysregulation of normal wound healing processes. An aggressive, feature of many CLDs is pulmonary fibrosis (PF) and is characterized by excess deposition of extracellular matrix (ECM) proteins from myofibroblasts in airways. However, factors regulating myofibroblast biology are incompletely understood. Proteins in the cadherin family contribute epithelial to mesenchymal transition (EMT), a suggested source of myofibroblasts. Cadherin 11 (CDH11) contributes to developmental and pathologic processes that parallel those seen in PF and EMT. Utilizing Cdh11 knockout (Cdh11 -/-) mice, the goal of this study was to characterize the contribution of CDH11 in the bleomycin model of PF and assess the feasibility of treating established PF. We demonstrate CDH11 in macrophages and airway epithelial cells undergoing EMT in lungs of mice given bleomycin and patients with PF. Endpoints consistent with PF including ECM production and myofibroblast formation are reduced in CDH11-targeted mice given bleomycin. Findings suggesting mechanisms of CDH11-dependent fibrosis include the regulation of the profibrotic mediator TGF-â in alveolar macrophages and CDH11-mediated EMT. The results of this study propose CDH11 as a novel drug target for PF. In addition, another CLD, chronic obstructive pulmonary disease (COPD), is characterized by airway inflammation and destruction. Adenosine, a nucleoside signaling molecule generated in response to cell stress is upregulated in patients with COPD and is suggested to contribute to its pathogenesis. An established model of adenosine-mediated lung injury exhibiting features of COPD is the Ada -/- mouse. Previous studies in our lab suggest features of the Ada -/- phenotype may be secondary to adenosine-dependent expression of osteopontin (OPN). OPN is a protein implicated in a variety of human pathology, but its role in COPD has not been examined. To address this, Ada/Opn -/- mice were generated and endpoints consistent with COPD were examined in parallel with Ada -/- mice. Results demonstrate OPN-mediated pulmonary neutrophilia and airway destruction in Ada -/- mice. Furthermore, patients with COPD exhibit increased OPN in airways which correlate with clinical airway obstruction. These results suggest OPN represents a novel biomarker or therapeutic target for the management of patients with COPD. The importance of findings in this thesis is highlighted by the fact that no pharmacologic interventions have been shown to interfere with disease progression or improve survival rates in patients with COPD or PF.

Proteomic identification of in vivo substrates for matrix metalloproteinases 2 and 9 reveals a mechanism for resolution of inflammation.

Clearance of allergic inflammatory cells from the lung through matrix metalloproteinases (MMPs) is necessary to prevent lethal asphyxiation, but mechanistic insight into this essential homeostatic process is lacking. In this study, we have used a proteomics approach to determine how MMPs promote egression of lung inflammatory cells through the airway. MMP2- and MMP9-dependent cleavage of individual Th2 chemokines modulated their chemotactic activity; however, the net effect of complementing bronchoalveolar lavage fluid of allergen-challenged MMP2(-/-)/MMP9(-/-) mice with active MMP2 and MMP9 was to markedly enhance its overall chemotactic activity. In the bronchoalveolar fluid of MMP2(-/-)/MMP9(-/-) allergic mice, we identified several chemotactic molecules that possessed putative MMP2 and MMP9 cleavage sites and were present as higher molecular mass species. In vitro cleavage assays and mass spectroscopy confirmed that three of the identified proteins, Ym1, S100A8, and S100A9, were substrates of MMP2, MMP9, or both. Function-blocking Abs to S100 proteins significantly altered allergic inflammatory cell migration into the alveolar space. Thus, an important effect of MMPs is to differentially modify chemotactic bioactivity through proteolytic processing of proteins present in the airway. These findings provide a molecular mechanism to explain the enhanced clearance of lung inflammatory cells through the airway and reveal a novel approach to target new therapies for asthma.

Role of A2B adenosine receptor signaling in adenosine-dependent pulmonary inflammation and injury.

Adenosine has been implicated in the pathogenesis of chronic lung diseases such as asthma and chronic obstructive pulmonary disease. In vitro studies suggest that activation of the A2B adenosine receptor (A2BAR) results in proinflammatory and profibrotic effects relevant to the progression of lung diseases; however, in vivo data supporting these observations are lacking. Adenosine deaminase-deficient (ADA-deficient) mice develop pulmonary inflammation and injury that are dependent on increased lung adenosine levels. To investigate the role of the A2BAR in vivo, ADA-deficient mice were treated with the selective A2BAR antagonist CVT-6883, and pulmonary inflammation, fibrosis, and airspace integrity were assessed. Untreated and vehicle-treated ADA-deficient mice developed pulmonary inflammation, fibrosis, and enlargement of alveolar airspaces; conversely, CVT-6883-treated ADA-deficient mice showed less pulmonary inflammation, fibrosis, and alveolar airspace enlargement. A2BAR antagonism significantly reduced elevations in proinflammatory cytokines and chemokines as well as mediators of fibrosis and airway destruction. In addition, treatment with CVT-6883 attenuated pulmonary inflammation and fibrosis in wild-type mice subjected to bleomycin-induced lung injury. These findings suggest that A2BAR signaling influences pathways critical for pulmonary inflammation and injury in vivo. Thus in chronic lung diseases associated with increased adenosine, antagonism of A2BAR-mediated responses may prove to be a beneficial therapy.

Mechanisms of apoptosis in bleomycin-induced lung injury: Implications for pulmonary fibrosis

Chronic inflammation leading to pulmonary fibrosis develops in response to environmental pollutants, radiotherapy, or certain cancer chemotherapeutic agents. Studies have shown that several cell types accumulate during the inflammatory process, but little information is known about what actually triggers and stimulates persistent inflammation culminating in fibrosis. As a first step in defining the events that precipitate inflammation in the lung, the biological mechanism(s) mediating apoptosis and cellular targets must be identified. The purpose of this study was to determine the molecular mechanism(s) of bleomycin-induced apoptosis in the lung using mice deficient in genes that we hypothesized to play a key role in apoptosis. Intratracheal administration of bleomycin led to caspase-mediated DNA fragmentation characteristic of apoptosis. The effects of bleomycin were associated with translocation of p53 from the cytosol to the nucleus only in alveolar macrophages that had been exposed to the drug in vivo, suggesting that the lung microenvironment regulated p53 activation. Experiments with a thiol antioxidant (N-acetylcysteine) in vivo and nitric oxide donors in vitro confirmed that reactive oxygen species were required for p53 activation. A specific role for NO was demonstrated in experiments with iNOS−/− macrophages, which failed to demonstrate nuclear p53 localization after in vivo bleomycin exposure. Strikingly, rates of bleomycin-induced apoptosis were at least two-fold higher in iNOS−/− and p53−/− C57BL/6 mice compared to wild-type controls. Laser Scanning Cytometry (LSC) analysis revealed that bleomycin exposure resulted in a 2-fold induction in Fas and FasL expression in wild-type mice but not iNOS−/− or p53−/− mice. Experiments using gld mice confirmed that the Fas/FasL pathway was the primary mechanism of bleomycin-induced apoptosis in the lung. LSC-mediated analysis indicated that bleomycin exposure resulted in a 2-fold induction in Bax expression in iNOS−/− and P53−/− mice but not wild-type mice. Furthermore, LSC analysis revealed that bleomycin exposure induced a 3-fold increase in thrombospondin expression in wild-type mice. However, thrombospondin was not expressed in either the iNOS−/− or p53−/− mice, implicating a thrombospondin-mediated apoptotic cell clearance mechanism in the lung. Together, these results demonstrate that iNOS and p53 positively regulate apoptosis via the Fas/FasL pathway and mediate a novel apoptosis-suppressing pathway in the lung. ^

Adenosine mediated lung mast cell degranulation in the mouse

Adenosine has been implicated to play a role in inflammatory processes associated with asthma. Most notable is adenosine's ability to potentiate mediator release from mast cells. Mast cells are bone marrow derived inflammatory cells that can release mediators that have both immediate and chronic effects on airway constriction and inflammation. Most physiological roles of adenosine are mediated through adenosine receptors. Four subtypes of adenosine receptors have been identified, A1, A2A, A2B and A 3. The mechanisms by which adenosine can influence the release of mediators from lung tissue mast cells is not understood due to lack of in vivo models. Mice deficient in the enzyme adenosine deaminase (ADA) have been generated. ADA controls the levels of adenosine in tissues and cells, and consequently, adenosine accumulates in the lungs of ADA-deficient mice. ADA-deficient mice develop features seen in asthmatics, including lung eosinophilia and mucus hypersecretion. In addition, lung tissue mast cell degranulation was associated with elevated adenosine in ADA-deficient lungs and can be prevented by ADA enzyme therapy. We established primary murine lung mast cell cultures, and used real time RT-PCR and immunofluorescence to demonstrate that A 2A, A2B and A3 receptors are expressed on murine lung mast cells. Studies using selective adenosine receptor agonists and antagonists and A3 receptor deficient (A3−/−) mast cells suggested that activation of A3 receptors could induce mast cell mediator release in vitro. Furthermore, this mediator release was associated with increases in intracellular Ca++ that appeared to be mediated through a Gi and PI3K pathway. In addition, nebulized A3 receptor agonist directly induced lung mast cell degranulation in wild type mice while having no effect in A3−/− mice. These results demonstrate that the A3 receptor plays an important role in adenosine mediated murine lung mast cell degranulation. Therefore, the A3 adenosine receptor and its signaling pathways may represent novel therapeutic targets for the treatment and prevention of asthma. ^

Characterization of cytochrome P450 4F subfamily: Functional role and response in inflammation

CYP4F subfamily comprises a group of enzymes that metabolize LTB4 to biologically less active metabolites. These inactive hydroxy products are incapable of chemotaxis and recruitment of inflammatory cells. This has led to a hypothesis that CYP4Fs may modulate inflammatory conditions serving as a signal of resolution. ^ We investigated the regulation of rat CYP4F gene expression under various inflammatory prompts including a bacterial lipopolysaccharide (LPS) treated model system, controlled traumatic brain injury (TBI) model as well as using direct cytokine challenges. CYP4Fs showed an isoform specific response to LPS. The pro-inflammatory cytokines IL-1β, IL-6 and TNF-α produced an overall inductive CYP4F response whereas IL-10, an anti-inflammatory cytokine, suppressed CYP4F gene expression in primary hepatocytes. The molecular mechanism behind IL-6 mediated CYP4F induction was partially STAT3 dependent. ^ An alternate avenue of triggering the inflammatory cascade is TBI, which is known to cause several secondary effects leading to multiorgan dysfunction syndrome. The results from this study elicited that trauma to the brain can produce acute inflammatory changes in organs distant from the injury site. Local production of LTB4 after CNS injury caused mobilization of inflammatory cells such as neutrophils to the lung. In the resolution phase, CYP4F expression increased with time along with the associated activity causing a decline in LTB4 concentration. This marked a significant reduction in neutrophil recruitment to the lung which led to subsequent recovery and repair. In addition, we showed that CYP4Fs are localized primarily in pulmonary endothelium. We speculate that the temporally regulated LTB4 clearance in the endothelium may be a novel target for treatment of pulmonary inflammation following injury. ^ In humans, several CYP4F isoforms have been identified and shown to metabolize LTB4 and other endogenous eicosanoids. However, the specific activity of the recently cloned human CYP4F11 is unknown. In the final part of this thesis, CYP4F11 protein was expressed in yeast in parallel to CYP4F3A. To our surprise, CYP4F11 displayed a different substrate profile than CYP4F3A. CYP4F3A metabolized eicosanoids while CYP4F11 was a better catalyst for therapeutic drugs. Thus, besides their endogenous function in clearing inflammation, CYP4Fs also may play a part in drug metabolism. ^

The contribution of A3 adenosine receptor signaling to pulmonary inflammation and mucus secretion in the mouse

Adenosine has been implicated in chronic lung diseases such as asthma and COPD. Most physiological actions of adenosine are mediated through G-protein coupled adenosine receptors. Four subtypes of adenosine receptors have been identified, A1, A2A, A2B, and A 3. However, the specific roles of the various adenosine receptors in processes central to asthma and COPD are not well understood in part due to the lack of adequate animal models that examine the effect of adenosine on the development of lung disease. In this study we have investigated the expression and function of the A3 adenosine receptor in pulmonary eosinophilia and mucus production/secretion in adenosine deaminase (ADA)-deficient mice in which adenosine levels are elevated. ADA-deficient mice develop features of asthma and COPD, including lung eosinophilia and mucus hyperplasia in association with elevated lung adenosine levels. The A3 receptor was found to be expressed in eosinophils and mucus producing cells in the airways of ADA-deficient. Disruption of A3 receptor signaling in ADA-deficient mice by genetic removal of the receptor or treatment with MRS 1523, a selective A3 adenosine receptor antagonist, prevented airway eosinophilia and mucus production. Although eosinophils were decreased in the airways of ADA-deficient mice with disrupted A3 receptor signaling, elevations in circulating and lung interstitial eosinophils persisted, suggesting signaling through the A3 receptor is needed for the migration of eosinophils into the airways. Further examination of the role of the A3 receptor in mucus biology demonstrated that the A3 receptor is neither required nor is overexpression of the receptor in clara cells sufficient for mucus production in naive mice. Transgenic overexpression of the A3 receptor did elucidate a role for the A3 receptor in the secretion of mucus into the airways of ovalbumin challenged mice. These findings identify an important role for the A3 adenosine receptor in regulating lung eosinophilia and mucus secretion in inflammatory lung diseases. Therefore, the A3 adenosine receptor may represent a novel therapeutic target for the treatment and prevention of asthma. ^

Evaluation of the C5a anaphylatoxin and its receptor (CD88) in allergic lung disease

Allergic asthma is characterized by airflow obstruction, airway hyperresponsiveness (AHR) and chronic airway inflammation. We and others have reported that complement component C3 and the anaphylatoxin C3a receptor promote while C5 protects against the development of the biological and physiological hallmarks of allergic lung disease in mice. In this study, we assessed if the protective responses could be mediated by C5a, an activation-induced C5 cleavage product. Mice with ablation of the C5a receptor (C5aR) either by genetic deletion or by pharmacological blockade exhibited significantly exacerbated AHR compared to allergen-challenged wild-type (WT) mice. However, there were no significant differences in many of the other hallmarks of asthma such as airway infiltration by eosinophils or lymphocytes, pulmonary IL-4-producing cell numbers, goblet cell metaplasia, mucus secretion or total serum IgE levels. In contrast to elevated AHR, numbers of IL-5 and IL-13 producing pulmonary cells, and IL-5 and IL-13 protein levels, were significantly reduced in allergen-challenged C5aR-/- mice compared to allergen-challenged WT mice. Administration of a specific cysteinyl leukotriene receptor 1 (cysLT1R) antagonist before each allergen-challenge abolished AHR in C5aR-/- as well as in WT mice. Pretreatment with a C3aR antagonist dose-dependently reduced AHR in allergen-challenged WT and C5aR-/- mice. Additionally, allergen-induced upregulation of pulmonary C3aR expression was exaggerated in C5aR-/- mice compared to WT mice. In summary, deficiency or antagonism of C5aR in a mouse model of pulmonary allergy increased AHR, which was reversed or reduced by blockade of the cysLT1R and C3aR, respectively. In conclusion, this study suggests that C5a and C5aR mediate protection against AHR by suppressing cysLT and C3aR signaling pathways, which are known to promote AHR. This also supports important and opposing roles of complement components C3a/C3aR and C5a/C5aR in AHR. ^

Transcriptional regulation of lung tumor suppressor GPRC5A in malignant and normal cells

Chronic exposure of the airways to cigarette smoke induces inflammatory response and genomic instability that play important roles in lung cancer development. Nuclear factor kappa B (NF-κB), the major intracellular mediator of inflammatory signals, is frequently activated in preneoplastic and malignant lung lesions. ^ Previously, we had shown that a lung tumor suppressor GPRC5A is frequently repressed in human non-small cell lung cancers (NSCLC) cells and lung tumor specimens. Recently, other groups have shown that human GPRC5A transcript levels are higher in bronchial samples of former than of current smokers. These results suggested that smoking represses GPRC5A expression and thus promotes the occurrence of lung cancer. We hypothesized that cigarette smoking or associated inflammatory response repressed GPRC5A expression through NF-κB signaling. ^ To determine the effect of inflammation, we examined GPRC5A protein expression in several lung cell lines following by TNF-α treatment. TNF-α significantly suppressed GPRC5A expression in normal small airway epithelial cells (SAEC) as well as in Calu-1 cells. Real-time PCR analysis indicated that TNF-α inhibits GPRC5A expression at the transcriptional level. NF-κB, the major downstream effectors of TNF-α signaling, mediates TNF-α-induced repression of GPRC5A because over-expression of NF-κB suppressed GPRC5A. To determine the region in the GPRC5A promoter through which NF-κB acts, we examined the ability of TNF-α to inhibit a series of reporter constructs with different deletions of GPRC5A promoter. The luciferase assay showed that the potential NF-κB binding sites containing region are irresponsible for TNF-α-induced suppression. Further analysis using constructs with different deletions in p65 revealed that NF-κB-mediated repression of GPRC5A is transcription-independent. Co-immunoprecipitation assays revealed that NF-κB could form a complex with RAR/RXR heterodimer. Moreover, the inhibitory effect of NF-κB has been found to be proportional to NF-κB/RAR ratio in luciferase assay. Finally, Chromatin IP demonstrated that NF-κB/p65 bound to GPRC5A promoter as well as RAR/RXR and suppressed transcription. Taken together, we propose that inflammation-induced NF-κB activation disrupts the RA signaling and suppresses GPRC5A expression and thus contributes to the oncogenesis of lung cancer. Our studies shed new light on the pathogenesis of lung cancer and potentially provide novel interventions for preventing and treating this disease. ^

Genetic Predictors of Clinical Outcomes in Non-Small Cell Lung Cancer Patients

Lung cancer is the leading cause of cancer-related mortality in the US. Emerging evidence has shown that host genetic factors can interact with environmental exposures to influence patient susceptibility to the diseases as well as clinical outcomes, such as survival and recurrence. We aimed to identify genetic prognostic markers for non-small cell lung cancer (NSCLC), a major (85%) subtype of lung cancer, and also in other subgroups. With the fast evolution of genotyping technology, genetic association studies have went through candidate gene approach, to pathway-based approach, to the genome wide association study (GWAS). Even in the era of GWAS, pathway-based approach has its own advantages on studying cancer clinical outcomes: it is cost-effective, requiring a smaller sample size than GWAS easier to identify a validation population and explore gene-gene interactions. In the current study, we adopted pathway-based approach focusing on two critical pathways - miRNA and inflammation pathways. MicroRNAs (miRNA) post-transcriptionally regulate around 30% of human genes. Polymorphisms within miRNA processing pathways and binding sites may influence patients’ prognosis through altered gene regulation. Inflammation plays an important role in cancer initiation and progression, and also has shown to impact patients’ clinical outcomes. We first evaluated 240 single nucleotide polymorphisms (SNPs) in miRNA biogenesis genes and predicted binding sites in NSCLC patients to determine associations with clinical outcomes in early-stage (stage I and II) and late-stage (stage III and IV) lung cancer patients, respectively. First, in 535 early-stage patients, after correcting multiple comparisons, FZD4:rs713065 (hazard ratio [HR]:0.46, 95% confidence interval [CI]:0.32-0.65) showed a significant inverse association with survival in early stage surgery-only patients. SP1:rs17695156 (HR:2.22, 95% CI:1.44-3.41) and DROSHA:rs6886834 (HR:6.38, 95% CI:2.49-16.31) conferred increased risk of progression in the all patients and surgery-only populations, respectively. FAS:rs2234978 was significantly associated with improved survival in all patients (HR:0.59, 95% CI:0.44-0.77) and in the surgery plus chemotherapy populations (HR:0.19, 95% CI:0.07-0.46).. Functional genomics analysis demonstrated that this variant creates a miR-651 binding site resulting in altered miRNA regulation of FAS, providing biological plausibility for the observed association. We then analyzed these associations in 598 late-stage patients. After multiple comparison corrections, no SNPs remained significant in the late stage group, while the top SNP NAT1:rs15561 (HR=1.98, 96%CI=1.32-2.94) conferred a significantly increased risk of death in the chemotherapy subgroup. To test the hypothesis that genetic variants in the inflammation-related pathways may be associated with survival in NSCLC patients, we first conducted a three-stage study. In the discovery phase, we investigated a comprehensive panel of 11,930 inflammation-related SNPs in three independent lung cancer populations. A missense SNP (rs2071554) in HLA-DOB was significantly associated with poor survival in the discovery population (HR: 1.46, 95% CI: 1.02-2.09), internal validation population (HR: 1.51, 95% CI: 1.02-2.25), and external validation (HR: 1.52, 95% CI: 1.01-2.29) population. Rs2900420 in KLRK1 was significantly associated with a reduced risk for death in the discovery (HR: 0.76, 95% CI: 0.60-0.96) and internal validation (HR: 0.77, 95% CI: 0.61-0.99) populations, and the association reached borderline significance in the external validation population (HR: 0.80, 95% CI: 0.63-1.02). We also evaluated these inflammation-related SNPs in NSCLC patients in never smokers. Lung cancer in never smokers has been increasingly recognized as distinct disease from that in ever-smokers. A two-stage study was performed using a discovery population from MD Anderson (411 patients) and a validation population from Mayo Clinic (311 patients). Three SNPs (IL17RA:rs879576, BMP8A:rs698141, and STK:rs290229) that were significantly associated with survival were validated (pCD74:rs1056400 and CD38:rs10805347) were borderline significant (p=0.08) in the Mayo Clinic population. In the combined analysis, IL17RA:rs879576 resulted in a 40% reduction in the risk for death (p=4.1 × 10-5 [p=0.61, heterogeneity test]). We also validated a survival tree created in MD Anderson population in the Mayo Clinic population. In conclusion, our results provided strong evidence that genetic variations in specific pathways that examined (miRNA and inflammation pathways) influenced clinical outcomes in NSCLC patients, and with further functional studies, the novel loci have potential to be translated into clinical use.

Cellular Uptake of Neutrohpil Elastase Links Inflammation to Adaptive Immunity

Many tumors arise from sites of inflammation providing evidence that innate immunity is a critical component in the development and progression of cancer. Neutrophils are primary mediators of the innate immune response. Upon activation, an important function of neutrophils is release of an assortment of proteins from their granules including the serine protease neutrophil elastase (NE). The effect of NE on cancer has been attributed primarily to its ability to degrade the extracellular matrix thereby promoting invasion and metastasis. Recently, it was shown that NE could be taken up by lung cancer cells leading to degradation of insulin receptor substrate-1 thereby promoting hyperactivity of the phosphatidylinositol-3 kinase (PI3K) pathway and tumor cell proliferation. To our knowledge, nobody has investigated uptake of NE by other tumor types. In addition, NE has broad substrate specificity suggesting that uptake of NE by tumor cells could impact processes regulating tumorigenensis other than activation of the PI3K pathway. Neutrophil elastase has been identified in breast cancer specimens where high levels of NE have prognostic significance. These studies have assessed NE levels in whole tumor lysates. Because the major source of NE is from activated neutrophils, we hypothesized that breast cancer cells do not have endogenous NE but may take up NE released by tumor associated neutrophils in the tumor microenvironment and that this could provide a link between the innate immune response to tumors and specific adaptive immune responses. In this thesis, we show that breast cancer cells lack endogenous NE expression and that they are able to take up NE resulting in increased generation of low molecular weight cyclin E (CCNE) and enhanced susceptibility to lysis by CCNE-specific cytotoxic T lymphocytes. We also show that after taking up NE and proteinase 3 (PR3), a second primary granule protease with significant homology to NE, breast cancer cells cross-present the NE- and PR3-derived peptide PR1 rendering them susceptible to PR1-targeted therapies. Taken together, our data support a role for NE uptake in modulating adaptive immune responses against breast cancer.