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3026Background: In melanoma (mel) patients (pts) treated with immune checkpoint inhibitors, presence of HLA class I (HLA-1) B44 supertype (B44+) correlated with survival (Chowell, Science). B44 pre...

Cancers often arise as the end stage of inflammation in adults, but not in children. As such there is a complex interplay between host immune cells during neoplastic development, with both an ability to promote cancer as well as limit or eliminate it, most often complicit with the host. In humans, defining inflammation and the presence of inflammatory cells within or surrounding the tumor is a critical aspect of modern pathology. Groups defining staging for neoplasms are strongly encouraged to assess and incorporate measures of the presence of apoptosis, autophagy, and necrosis as well as the nature and quality of the immune infiltrate. Both environmental as well as genetic factors enhance the risk of cigarette smoking, H. pylori, hepatitis B/C, human papilloma virus, solar irradiation, asbestos, pancreatitis, or other causes of chronic inflammation. Identifying suitable genetic polymorphisms in cytokines, cytokine receptors, and Toll-like receptors among other immune response genes is also seen as high value as genomic sequencing becomes less expensive. Animal models which incorporate and assess not only the genetic anlagen but also the inflammatory cells and the presence of microbial pathogen [PAMPs] and damage associated molecular pattern molecules [DAMPs] are necessary. Identifying micro-RNAs involved in regulating the response to damage or injury are seen as highly promising. Although no therapeutic strategies to prevent or treat cancers based on insights into inflammatory pathways are currently approved for the common epithelial malignancies, there remains substantial interest in agents targeting COX2 or PPARγ, ethyl pyruvate, as well as steroids and several novel agents on the horizon.

Lung cancer is usually disseminated at diagnosis making prognosis poor. Smokers are at high risk for lung cancer and are targets for prevention and early detection strategies. Sputum is a potential source for lung cancer biomarkers, but no test is currently available with sufficient sensitivity and specificity for clinical screening utility. Chromosomal aneusomy (CA) was measured in sputum samples collected prospectively from 100 incident lung cancer cases and 96 controls matched on age, gender, and date of collection. The CA-FISH assay was performed using a four-target DNA FISH probe including EGFR, MYC, 5p15 and CEP6. Sensitivity for a positive CA-FISH assay (abnormal for ≥ 2 of the 4 markers) was substantially higher for samples collected within 18 months (76%) than >18 months before lung cancer diagnosis (31%). Specificity for a positive FISH by this same definition was 85%. Among subjects providing sputum sample within 18 months before diagnosis, sensitivity was higher for squamous cell cancers (94%) than for other histologic types (69%). The adjusted odds ratios for specimens collected within 18 months of cancer diagnosis were higher using the CA-FISH assay (OR=27.2, 95% CI 7.8 to 94.1) than previous studies assessing cytologic atypia (OR=2.3, CI 0.8 to 6.4) or gene promoter methylation (OR=6.5; CI 1.2 to 35.5). In conclusion, chromosomal aneusomy in sputum is a promising biomarker for prediction of lung cancer risk. Evaluation of the 4-DNA targets was more effective than any single marker and had highest sensitivity for samples collected ≤ 18 months to lung cancer diagnosis and patients diagnosed with squamous cell carcinoma.

Smoking is a major risk factor for lung cancer, which is the leading cause of cancer-related deaths both in the USA and worldwide. Chronic obstructive pulmonary disease and emphysema are comorbid conditions often found in lung cancer patients. The inflammatory pathways that link chronic obstructive pulmonary disease, emphysema, and lung cancer likely involve genetic and epigenetic modulations due to chronic tissue injury and abnormal tumor immunity in susceptible hosts.Chronic airway inflammation contributes to alterations in the bronchial epithelium and lung microenvironment, provoking a milieu conducive to pulmonary carcinogenesis. For example, inflammation-inducible cyclooxygenase-2 is upregulated in nonsmall cell lung cancer and also plays an important role in promoting epithelial-to-mesenchymal transition. Genetic changes in the airway epithelium of smokers may help predict or identify individuals at risk for lung cancer. Finally, radiographic findings of emphysema have been established as independent risk factors for lung cancer.The relationships between inflammation, airflow obstruction, and lung cancer are complex. Deregulated inflammation is complicit in the pathogenesis of chronic obstructive pulmonary disease and lung cancer, but the overlap of signaling events is not yet fully understood. Tobacco exposure is an important risk factor that confers long-term risk of lung disease. Diagnostic sensitivity of detecting lung cancer may improve with the utilization of genetic profiling in combination with pathologic evaluation of airway epithelium. Additional research is required to understand the role of epithelial-to-mesenchymal transition in chronic inflammatory lung diseases and lung carcinogenesis.

We are evaluating the immune enhancing activities of cytokines for their optimum utility in augmenting cellular immune responses against lung cancer. In this study, we evaluated the mechanism of antitumor responses following IL-7 administration to mice bearing established Lewis lung cancer. IL-7 decreased tumor burden with concomitant increases in the frequency of CD4 and CD8 T lymphocyte subsets, T cell activation markers CXCR3, CD69, and CD127low, effector memory T cells, and T cell cytolytic activity against parental tumor cells. Accompanying the antitumor responses were increases in IFN-γ, CXCL9, CXCL10, and IL-12. Individual neutralization of CD4, CD8 T lymphocytes, or the CXCR3 ligands CXCL9 and CXCL10 reversed the antitumor benefit of IL-7, indicating their importance for optimal responses in vivo. Furthermore, IL-7 decreased the tumor-induced apoptosis of T cells with subsequent decrease of the proapoptotic marker Bim. We assessed the impact of IL-7 treatment on regulatory T cells that negatively impact antitumor immune responses. IL-7 decreased regulatory T Foxp3 as well as cell suppressive activity with a reciprocal increase in SMAD7. These results indicate that IL-7 induces CXCR3 ligand-dependent T cell antitumor reactivity in lung cancer.

Green tea polyphenols exhibit multiple anti-tumor activities, and the mechanisms of action are not completely understood. Previously, we reported that green tea extract (GTE)-induced actin remolding is associated with increased cell adhesion and decreased motility in A549 lung cancer cells. To identify the cellular targets responsible for green tea-induced actin remodeling, we performed two-dimensional gel electrophoresis LC-tandem mass spectrometry of A549 cells before and after GTE exposure. We have identified 14 protein spots that changed in expression (≥2 fold) after GTE treatment. These proteins are involved in calcium-binding, cytoskeleton and motility, metabolism, detoxification or gene regulation. In particular we found up-regulation of several genes that modulate actin remodeling and cell migration, including lamin A/C. Our data indicated that GTE-induced lamin A/C up-regulation appears to be at the transcriptional level and the increased expression results in the decrease in the cell motility, as confirmed by siRNA. The result of the study demonstrates that GTE alters the levels of many proteins involved in growth, motility and apoptosis of A549 cells and their identification may explain the multiple anti-tumor activities of GTE.

Worldwide over 1 million people die due to lung cancer each year. It is estimated that cigarette smoking explains almost 90% of lung cancer risk in men and 70 to 80% in women. Clinically evident lung cancers have multiple genetic and epigenetic abnormalities. These abnormalities may result in activation of oncogenes and inactivation of tumor-suppressor genes. Chronic inflammation, which is known to promote cancer, may result both from smoking and from genetic abnormalities. These mediators in turn may be responsible for increased macrophage recruitment, delayed neutrophil clearance, and increase in reactive oxygen species (ROS). Thus, the pulmonary environment presents a unique milieu in which lung carcinogenesis proceeds in complicity with the host cellular network. The pulmonary diseases that are associated with the greatest risk for lung cancer are characterized by abundant and deregulated inflammation. Pulmonary disorders such as chronic obstructive pulmonary disease (COPD)/emphysema are characterized by profound abnormalities in inflammatory and fibrotic pathways. The cytokines and growth factors aberrantly produced in COPD and the developing tumor microenvironment have been found to have deleterious properties that simultaneously pave the way for both epithelial–mesenchymal transition (EMT) and destruction of specific host cell–mediated immune responses. Full definition of these pathways will afford the opportunity to intervene in specific inflammatory events mediating lung tumorigenesis and resistance to therapy.

Introduction Cyclooxygenase-2 overexpression may mediate resistance to epidermal growth factor receptor tyrosine kinase inhibition through prostaglandin E2-dependent promotion of epithelial to mesenchymal transition (EMT). Suppression of epithelial markers, such as E-cadherin, can lead to resistance to erlotinib. Prostaglandin E2 down-regulates E-cadherin expression by up-regulating transcriptional repressors, including ZEB1 and Snail. Furthermore, E-cadherin can be modulated by matrix metalloproteinases (MMPs) and tissue inhibitors of MMPs (TIMPs), promoting tumor invasion and metastasis. Markers of EMT and tumor invasion were evaluated in patient serum from a phase I clinical trial investigating the combination of celecoxib and erlotinib in non-small cell lung cancer (NSCLC) patients. Methods Samples from 22 subjects were evaluated. Soluble E-cadherin (sEC) was evaluated by enzyme linked immunosorbent assay in patient serum at baseline, week 4, and week 8 of treatment. Other markers of EMT and angiogenesis were evaluated by enzyme linked immunosorbent assay, including MMP-9, TIMP-1, and CCL15. Results Serum sEC, MMP-9, TIMP-1, and CCL15 levels were determined at baseline and week 8. Patients with a partial response to therapy had a significant decrease in sEC, TIMP-1, and CCL15 at week 8. In patients who responded to the combination therapy, baseline MMP-9 was significantly lower compared with nonresponders (p = 0.006). Conclusions sEC, MMP-9, TIMP-1, and CCL15 levels correlate with response to combination therapy with erlotinib and celecoxib in patients with NSCLC. A randomized phase II trial is planned comparing erlotinib and celecoxib with erlotinib plus placebo in advanced NSCLC. This study will prospectively assess these and other biomarkers in serum and tumor tissue.

© 2016 The Authors. Monocarboxylate transporter 1 (MCT1) inhibition is thought to block tumor growth through disruption of lactate transport and glycolysis. Here, we show MCT1 inhibition impairs proliferation of glycolytic breast cancer cells co-expressing MCT1 and MCT4 via disruption of pyruvate rather than lactate export. MCT1 expression is elevated in glycolytic breast tumors, and high MCT1 expression predicts poor prognosis in breast and lung cancer patients. Acute MCT1 inhibition reduces pyruvate export but does not consistently alter lactate transport or glycolytic flux in breast cancer cells that co-express MCT1 and MCT4. Despite the lack of glycolysis impairment, MCT1 loss-of-function decreases breast cancer cell proliferation and blocks growth of mammary fat pad xenograft tumors. Our data suggest MCT1 expression is elevated in glycolytic cancers to promote pyruvate export that when inhibited, enhances oxidative metabolism and reduces proliferation. This study presents an alternative molecular consequence of MCT1 inhibitors, further supporting their use as anti-cancer therapeutics. Hong et al. show a lactate-transport-independent role for MCT1 in promoting proliferation of glycolytic breast cancer cells that co-express MCT1 and MCT4. Their results suggest a key role for MCT1 in mediating tumor pyruvate export, which has important implications for use of MCT1 inhibitors in the clinic.

Cyclooxygenase-2 (COX-2) is a key enzyme in the production of prostaglandins and thromboxanes from free arachidonic acid. Increasing evidence suggests that COX-2 plays a role in tumorigenesis. A variety of stimuli induce COX-2 and it is overexpressed in many tumors, including non-small cell lung cancer (NSCLC). We studied the regulation of COX-2 expression in immortalized human bronchial epithelial cells (HBECs) by transforming growth factor-beta1 (TGF-beta1) and epidermal growth factor (EGF) because these two growth factors are present in both the pulmonary milieu of those at risk for lung cancer as well as in the tumor microenvironment. EGF significantly enhanced TGF-beta1-mediated induction of COX-2 and corresponding prostaglandin E2 (PGE2) production. TGF-beta1 and EGF induced COX-2 at the transcriptional and post-transcriptional levels. EGF receptor (EGFR) inhibition, neutralizing antibody against amphiregulin, or mitogen-activated protein kinase kinase (MEK) inhibition blocked TGF-beta1-mediated COX-2 induction. COX-2 induction by TGF-beta1 depended upon Smad3 signaling and required the activity of EGFR or its downstream mediators. Autocrine amphiregulin signaling maintains EGFR in a constitutively active state in HBECs, allowing for COX-2 induction by TGF-beta1. Thus, EGFR ligands, which are abundant in the pulmonary microenvironment of those at risk for lung cancer, potentiate and are required for COX-2 induction by TGF-beta1 in HBEC. These findings emphasize the central role of EGFR signaling in COX-2 induction by TGF-beta1 and suggest that inhibition of EGFR signaling should be investigated further for lung cancer prevention.

Lung carcinogenesis is a complex process requiring the acquisition of genetic mutations that confer the malignant phenotype as well as epigenetic alterations that may be manipulated in the course of therapy. Inflammatory signals in the lung cancer microenvironment can promote apoptosis resistance, proliferation, invasion, metastasis, and secretion of proangiogenic and immunosuppressive factors. Here, we discuss several prototypical inflammatory mediators controlling the malignant phenotype in lung cancer. Investigation into the detailed molecular mechanisms underlying the tumor-promoting effects of inflammation in lung cancer has revealed novel potential drug targets. Cytokines, growth factors and small-molecule inflammatory mediators released in the developing tumor microenvironment pave the way for epithelial-mesenchymal transition, the shift from a polarized, epithelial phenotype to a highly motile mesenchymal phenotype that becomes dysregulated during tumor invasion. Inflammatory mediators within the tumor microenvironment are derived from neoplastic cells as well as stromal and inflammatory cells; thus, lung cancer develops in a host environment in which the deregulated inflammatory response promotes tumor progression. Inflammation-related metabolic and catabolic enzymes (prostaglandin E(2) synthase, prostaglandin I(2) synthase and 15-hydroxyprostaglandin dehydrogenase), cell-surface receptors (E-type prostaglandin receptors) and transcription factors (ZEB1, SNAIL, PPARs, STATs and NF-kappaB) are differentially expressed in lung cancer cells compared with normal lung epithelial cells and, thus, may contribute to tumor initiation and progression. These newly discovered molecular mechanisms in the pathogenesis of lung cancer provide novel opportunities for targeted therapy and prevention in lung cancer.

Background: Cyclooxygenase-2 (COX-2) over-expression and subsequent prostaglandin E2 (PGE2) production are frequently associated with human non-small-cell lung cancer (NSCLC) and are involved in tumor proliferation, invasion, angiogenesis, and resistance to apoptosis. Here, we report that ciglitazone downregulates PGE2 in NSCLC cells. Methods: PGE2 ELISA assay and COX-2 ELISA assay were performed for measuring PGE2 and COX-2, respectively, in NSCLC. The mRNA level of COX-2 was measured by semi-quantitative RT-PCR. The transient transfection experiments were performed to measure COX-2 and peroxisome proliferator-response element (PPRE) promoter activity in NSCLC. Western blots were unitized to measure PGE synthase (PGES) and 15-hydroxyprostaglandin dehydrogenase (15-PGDH) protein expression. Results: COX-2 ELISA assays suggested that ciglitazone-dependent inhibition of PGE2 occurs through the suppression of COX-2. Ciglitazone treatment suppressed COX-2 mRNA expression and COX-2 promoter activity while upregulating PPRE promoter activity. Ciglitazone did not modify the expression of enzymes downstream of COX-2 including PGES and 15-PGDH. Utilization of a dominant-negative PPAR γ showed that the suppression of COX-2 and PGE2 by ciglitazone is mediated via non-PPAR pathways. Conclusion: Taken together, our findings suggest that ciglitazone is a negative modulator of COX-2/PGE2 in NSCLC.

Green tea polyphenols exhibit multiple antitumor activities in various in vitro and in vivo tumor models, and the mechanisms of action are not clear. Previously, we found that green tea extract (GTE) regulates actin remodeling in different cell culture systems. Actin remodeling plays an important role in cancer cell morphology, cell adhesion, motility, and invasion. Using proteomic approaches, we found GTE-induced expression of annexin-I, a multifunctional actin binding protein, in these cell lines. In this study, we aimed to further define the functional role of GTE-induced annexin-I expression in actin remodeling, cell adhesion, and motility in lung adenocarcinoma A549 cells. We found that GTE stimulates the expression of annexin-I in a dose-dependent fashion. The GTE-induced annexin-I expression appears to be at the transcription level, and the increased annexin-I expression mediates actin polymerization, resulting in enhanced cell adhesion and decreased motility. Annexin-I specific interference resulted in loss of GTE-induced actin polymerization and cell adhesion, but not motility. In fact, annexin-I specific interference itself inhibited motility even without GTE. Together, annexin-I plays an important role in GTE-induced actin remodeling, and it may serve as a potential molecular target associated with the anticancer activities of green tea.

Lung cancer cells elaborate the immunosuppressive and antiapoptotic mediator prostaglandin E(2) (PGE(2)), a product of cyclooxygenase-2 (COX-2) enzyme activity. Because peroxisome proliferator-activated receptor (PPAR)gamma ligands, such as thiazolidinediones (TZDs), inhibit lung cancer cell growth, we examined the effect of the TZDs pioglitazone and rosiglitazone on PGE(2) levels in non-small-cell lung cancer (NSCLC) A427 and A549 cells. Both TZDs inhibited PGE(2) production in NSCLC cells via a COX-2 independent pathway. To define the mechanism underlying COX-2 independent suppression of PGE(2) production, we focused on other enzymes responsible for the synthesis and degradation of PGE(2). The expression of none of the three prostaglandin synthases (microsomal PGES1, PGES2 and cystosolic PGES) was down-regulated by the TZDs. It is noteworthy that 15-hydroxyprostaglandin dehydrogenase (15-PGDH), an enzyme that produces biologically inactive 15-ketoprostaglandins from active PGE(2), was induced by TZDs. The TZD-mediated suppression of PGE(2) concentration was significantly inhibited by small interfering RNA to 15-PGDH. Studies using dominant-negative PPARgamma overexpression or 2-chloro-5-nitrobenzanilide (GW9662; a PPARgamma antagonist) revealed that the suppressive effect of the TZDs on PGE(2) is PPARgamma-independent. Together, these findings indicate that it is possible to use a clinically available pharmacological intervention to suppress tumor-derived PGE(2) by enhancing catabolism rather than blocking synthesis.

Cyclooxygenase-2 (COX-2) is frequently overexpressed in human cancers and contributes to the malignant phenotype. Our data indicate unphosphorylated signal transducers and activators of transcription 6 (STAT6) may transcriptionally upregulate COX-2 expression and protect against apoptosis in NSCLC cells. In A427 and H2122, NSCLC cell lines that constitutively express COX-2, only unphosphorylated STAT6 was detectable by western blot, thus, all of the following STAT6-dependent effects are attributed to the unphosphorylated protein. In both cell lines, small-interfering RNA-mediated knockdown of STAT6 or stable expression of dominant-negative STAT6 decreased COX-2 expression. In contrast, transfection with a phosphorylation-deficient mutant STAT6 increased COX-2 levels. Immunofluorescent staining revealed the presence of STAT6 in H2122 nuclei, suggesting a direct role in gene regulation for the unphosphorylated protein. Consistent with this hypothesis, unphosphorylated STAT6 increased luciferase expression from a COX-2 promoter reporter construct. STAT6 co-immunoprecipitated with the transcriptional co-activator, p300, and chromatin immunoprecipitation assays demonstrated that these proteins bind a consensus STAT6 binding site located within the COX-2 promoter. STAT6 DNA-binding specificity was confirmed by electrophoretic mobility shift assay. As COX-2 over-expression has been clearly linked to apoptosis resistance and other hallmarks of malignancy, these findings suggest a novel role of unphosphorylated STAT6 in the pathogenesis of non-small cell lung cancer.

Inactivation of the LKB1 tumor suppressor is a frequent event in non–small cell lung carcinoma (NSCLC) leading to the activation of mTOR complex 1 (mTORC1) and sensitivity to the metabolic stress inducer phenformin. In this study, we explored the combinatorial use of phenformin with the mTOR catalytic kinase inhibitor MLN0128 as a treatment strategy for NSCLC bearing comutations in the LKB1 and KRAS genes. NSCLC is a genetically and pathologically heterogeneous disease, giving rise to lung tumors of varying histologies that include adenocarcinomas and squamous cell carcinomas (SCC). We demonstrate that phenformin in combination with MLN0128 induced a significant therapeutic response in KRAS/LKB1–mutant human cell lines and genetically engineered mouse models of NSCLC that develop both adenocarcinomas and SCCs. Specifically, we found that KRAS/LKB1–mutant lung adenocarcinomas responded strongly to phenformin + MLN0128 treatment, but the response of SCCs to single or combined treatment with MLN0128 was more attenuated due to acquired resistance to mTOR inhibition through modulation of the AKT-GSK signaling axis. Combinatorial use of the mTOR inhibitor and AKT inhibitor MK2206 robustly inhibited the growth and viability of squamous lung tumors, thus providing an effective strategy to overcome resistance. Taken together, our findings define new personalized therapeutic strategies that may be rapidly translated into clinical use for the treatment of KRAS/LKB1–mutant adenocarcinomas and squamous cell tumors. Cancer Res; 75(22); 4910–22. ©2015 AACR.

Cyclooxygenase 2 (COX-2)-dependent signaling represents a potential mechanism of resistance to therapy with epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors. This is mediated in part through an EGFR-independent activation of mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (Erk) by prostaglandin E2 (PGE2). PGE2 promotes downregulation of E cadherin and epithelial to mesenchymal transition. The current study investigated EGFR and COX-2 inhibition in patients with non-small cell lung cancer (NSCLC) and elevated baseline urinary metabolite of PGE2 (PGEM).Patients with stage IIIB/IV (AJCC 6th edition) NSCLC who progressed after at least 1 line of therapy or refused standard chemotherapy were randomized to receive erlotinib and celecoxib versus erlotinib and placebo. The primary endpoint was progression-free survival (PFS) with 80% power to detect a 50% improvement with a 1-sided significance level of .2 in the intent-to-treat and elevated baseline PGEM populations. Secondary endpoints included response rate, overall survival, and evaluation of molecular markers to assess targeting COX-2-related pathways and evaluate EGFR tyrosine kinase inhibitor resistance.A total of 107 patients were enrolled with comparable baseline characteristics. Among the patients treated with celecoxib, those with wild-type EGFR were found to have an increased PFS (3.2 months vs 1.8 months; P = .03). PFS was numerically improved among patients in the intent-to-treat group who received erlotinib and celecoxib compared with those treated with erlotinib and placebo (5.4 months vs 3.5 months; P = .33) and was increased in patients in the erlotinib and celecoxib arm with elevated baseline PGEM (5.4 months vs 2.2 months; P = .15). Adverse events were similar in both treatment arms.The combination of erlotinib and celecoxib did not appear to improve outcomes in an unselected population, but selection by elevated baseline PGEM led to an increase in PFS with this combination. Patients with EGFR wild-type status may benefit from the combination of erlotinib and celecoxib.

Purpose: The mechanisms underlying the sensitivity of non–small cell lung cancer to epidermal growth factor receptor (EGFR) kinase inhibitors are complex, and there are no established markers to accurately predict treatment outcome in individual patients.Experimental Design: We investigated whether tumors responding to EGFR inhibitors can be identified by measuring treatment-induced changes in glucose utilization by positron emission tomography with the glucose analogue fluorodeoxyglucose (FDG-PET). We studied a panel of cell lines with a spectrum of sensitivity to EGFR kinase inhibitors. After incubation with the EGFR kinase inhibitor gefitinib for various time points, FDG uptake, glucose transport rates, and hexokinase activity were determined. FDG uptake in vivo was assessed by microPET imaging of tumor xenografts in mice.Results: In gefitinib-sensitive cell lines, there was a dramatic decrease in FDG uptake as early as 2 hours after treatment. Immunoblots showed the translocation of glucose transporters (GLUT3) from the plasma membrane to the cytosol; glucose transport rates were reduced 2.6-fold at this time. There was also a modest reduction of hexokinase activity. These metabolic alterations preceded changes in cell cycle distribution, thymidine uptake, and apoptosis. MicroPET studies showed an up to 55% decrease of tumor FDG uptake in sensitive xenografts within 48 hours. In contrast, gefitinib-resistant cells exhibited no measurable changes in FDG uptake, either in cell culture or in vivo.Conclusion: Glucose metabolic activity closely reflects response to gefitinib therapy. FDG-PET may be a valuable clinical predictor, early in the course of treatment, for therapeutic responses to EGFR kinase inhibitors.

Oncogenic RAS expression occurs in up to 40% of multiple myeloma (MM) cases and correlates with aggressive disease. Since activated RAS induces cyclooxygenase-2 (cox-2) expression in other tumor models, we tested a role for cox-2 in mutant RAS–containing MM cells. We used the ANBL-6 isogenic MM cell lines in which the IL-6–dependent parental line becomes cytokine independent following transfection with mutated N-RAS or K-RAS. Both mutated N-RAS– and K-RAS–expressing ANBL-6 cells demonstrated a selective up-regulation of cox-2 expression and enhanced secretion of PGE2, a product of cox-2. Furthermore, in 3 primary marrow specimens, which contained MM cells expressing mutated RAS, 15% to 40% of tumor cells were positive for cox-2 expression by immunohistochemistry. We used cox-2 inhibitors, NS398 and celecoxib, and neutralizing anti-PGE2 antibody to test whether cox-2/PGE2 was involved in the aggressive phenotype of MM ANBL-6 cells containing mutated RAS. Although these interventions had no effect on IL-6–independent growth or adhesion to marrow stromal cells, they significantly inhibited the enhanced binding of mutant RAS– containing MM cells to fibronectin and the enhanced resistance to melphalan. These results indicate a selective induction of cox-2 in MM cells containing RAS mutations, which results in heightened binding to extracellular matrix protein and chemotherapeutic drug resistance.

Elevated tumor cyclooxygenase-2 (COX-2) expression is associated with tumor invasion, metastasis, and poor prognosis in non–small cell lung cancer (NSCLC). Here, we report that COX-2-dependent pathways contribute to the modulation of E-cadherin expression in NSCLC. First, whereas genetically modified COX-2-sense (COX-2-S) NSCLC cells expressed low E-cadherin and showed diminished capacity for cellular aggregation, genetic or pharmacologic inhibition of tumor COX-2 led to increased E-cadherin expression and resulted in augmented homotypic cellular aggregation among NSCLC cells in vitro. An inverse relationship between COX-2 and E-cadherin was shown in situ by double immunohistochemical staining of human lung adenocarcinoma tissue sections. Second, treatment of NSCLC cells with exogenous prostaglandin E2 (PGE2) significantly decreased the expression of E-cadherin, whereas treatment of COX-2-S cells with celecoxib (1 μmol/L) led to increased E-cadherin expression. Third, the transcriptional suppressors of E-cadherin, ZEB1 and Snail, were up-regulated in COX-2-S cells or PGE2-treated NSCLC cells but decreased in COX-2-antisense cells. PGE2 exposure led to enhanced ZEB1 and Snail binding at the chromatin level as determined by chromatin immunoprecipitation assays. Small interfering RNA–mediated knockdown of ZEB1 or Snail interrupted the capacity of PGE2 to down-regulate E-cadherin. Fourth, an inverse relationship between E-cadherin and ZEB1 and a direct relationship between COX-2 and ZEB1 were shown by immunohistochemical staining of human lung adenocarcinoma tissue sections. These findings indicate that PGE2, in autocrine or paracrine fashion, modulates transcriptional repressors of E-cadherin and thereby regulates COX-2-dependent E-cadherin expression in NSCLC. Thus, blocking PGE2 production or activity may contribute to both prevention and treatment of NSCLC. (Cancer Res 2006; 66(10): 5338-45)

IL-4 is a type 2 cytokine that may mediate pleiotropic effects in the NSCLC microenvironment. Here, we investigated whether IL-4 regulates PGE(2) production in NSCLC cells. We found that IL-4 inhibited constitutive COX-2 expression and PGE(2) production in A427 and H2122 NSCLC cell lines, and also suppressed IL-1beta-induced COX-2 expression in A549 and RH2 NSCLC cell lines. COX-2 mRNA was decreased in response to IL-4, and promoter analysis indicated that IL-4 inhibited both constitutive and IL-1beta-induced COX-2 transcription. IL-4 inhibited IL-1beta-stimulated ERK phosphorylation, which may mediate the inhibition of IL-1beta-induced COX-2 by IL-4. IL-4 did not modulate additional arachidonic acid pathway enzymes mPGES-1 and 15-PGDH, which could potentially be responsible for regulating PGE(2) production. Overall, our studies demonstrate that IL-4 has the capacity to inhibit COX-2 mRNA transcription in NSCLC cells and the inhibition of PGE(2) appears to be predominately COX-2 dependent.

The antitumor efficiency of dendritic cells transduced with an adenovirus vector expressing secondary lymphoid chemokine (CCL21) was evaluated in a murine model of spontaneous bronchoalveolar cell carcinoma. The transgenic mice (CC-10 TAg) express the SV40 large T antigen (TAg) under the Clara cell promoter, develop bilateral, multifocal, and pulmonary adenocarcinomas, and die at 4 months as a result of progressive pulmonary tumor burden. A single intratracheal administration of CCL21 gene-modified dendritic cells (DC-AdCCL21) led to a marked reduction in tumor burden with extensive mononuclear cell infiltration of the tumors. The reduction in tumor burden was accompanied by the enhanced elaboration of type 1 cytokines [IFN-γ, interleukin (IL)-12, and granulocyte macrophage colony-stimulating factor] and antiangiogenic chemokines (CXCL9 and CXCL10) but a concomitant decrease in the immunosuppressive molecules (IL-10, transforming growth factor-β, prostaglandin E2) in the tumor microenvironment. The DC-AdCCL21 therapy group revealed a significantly greater frequency of tumor-specific T cells releasing IFN-γ compared with the controls. Continuous therapy with weekly intranasal delivery of DC-AdCCL21 significantly prolonged median survival by >7 weeks in CC-10 TAg mice. Both innate natural killer and specific T-cell antitumor responses significantly increased following DC-AdCCL21 therapy. Significant reduction in tumor burden in a model in which tumors develop in an organ-specific manner provides a strong rationale for further evaluation of intrapulmonary-administered DC-AdCCL21 in regulation of tumor immunity and genetic immunotherapy for lung cancer.(Cancer Res 2006; 66(6): 3205-13)

Purpose: This study evaluated the feasibility of cyclooxygenase-2 (COX-2) inhibition for lung cancer chemoprevention. We hypothesized that treatment with oral Celecoxib, a selective COX-2 inhibitor, would favorably alter the biomarkers of lung cancer risk as measured by the Ki-67 proliferative labeling index (Ki-67 LI). Experimental Design: Twenty active heavy smokers were enrolled into a pilot study and treated with Celecoxib for 6 months. Bronchoscopies with bronchial biopsies were done before and after 6 months of Celecoxib treatment. H&E stain for histologic grading and immunohistochemical examination for Ki-67 LI, COX-2, and survivin were carried out on serially matched biopsy samples to determine responses to treatment. Results: Treatment with Celecoxib significantly reduced Ki-67 LI in smokers by 35% (P = 0.016), and increased the expression of nuclear survivin by 23% (P = 0.036) without significantly changing that of cytoplasmic survivin. Conclusions: Our findings suggest that oral Celecoxib may be capable of modulating the proliferation indices and apoptotic balance in bronchial tissue of active smokers.

Lung cancer is the most common cancer in the world. It is a highly lethal disease in women and men, and new treatments are urgently needed. Previous studies implicated a role of estrogens and estrogen receptors in lung cancer progression, and this steroidal growth-stimulatory pathway may be promoted by tumor expression and activity of aromatase, an estrogen synthase. We found expression of aromatase transcripts and protein in human non–small cell lung cancer (NSCLC) cells using reverse transcription-PCR and Western immunoblots, respectively. Aromatase staining by immunohistochemistry was detected in 86% of archival NSCLC tumor specimens from the clinic. Further, biological activity of aromatase was determined in NSCLC tumors using radiolabeled substrate assays as well as measure of estradiol product using ELISA. Significant activity of aromatase occurred in human NSCLC tumors, with enhanced levels in tumor cells compared with that in nearby normal cells. Lung tumor aromatase activity was inhibited by anastrozole, an aromatase inhibitor, and treatment of tumor cells in vitro with anastrozole led to significant suppression of tumor cell growth. Similarly, among ovariectomized nude mice with A549 lung tumor xenografts, administration of anastrozole by p.o. gavage for 21 days elicited pronounced inhibition of tumor growth in vivo. These findings show that aromatase is present and biologically active in human NSCLCs and that tumor growth can be down-regulated by specific inhibition of aromatase. This work may lead to development of new treatment options for patients afflicted with NSCLC. (Cancer Res 2005; 65(24): 11287-91)

Purpose: Cyclooxygenase-2 (COX-2), phosphorylated signal transducers and activators of transcription 3 (STAT3), and interleukin-6 (IL-6) are elevated in non–small cell lung cancer (NSCLC). These molecules affect numerous cellular pathways, including angiogenesis and apoptosis resistance, and, therefore, may act in concert in NSCLC. Experimental Design: We examined IL-6 and phosphorylated STAT3 in COX-2-overexpressing [COX-2 sense-oriented (COX-2-S)] NSCLC cells and control cells. The effect of IL-6, STAT3, phosphatidylinositol 3-kinase, and mitogen-activated protein/extracellular signal-regulated kinase kinase on vascular endothelial growth factor (VEGF) production and apoptosis resistance was assessed in COX-2-overexpresing cells. Results: We report that NSCLC cells overexpressing COX-2 (COX-2-S) have increased IL-6 and phosphorylated STAT3 expression compared with control cells. IL-6 induced expression of VEGF in NSCLC cells. Moreover, blocking IL-6, mitogen-activated protein/extracellular signal-regulated kinase kinase, or phosphatidylinositol 3-kinase decreased VEGF production in COX-2-S cells. The addition of IL-6 to NSCLC cells resulted in increased apoptosis resistance. Furthermore, the inhibition of STAT3 or IL-6 induced apoptosis and reduced survivin expression, a member of the inhibitor of apoptosis protein family in COX-2-S cells. Conclusions: Overall, these findings suggest a novel pathway in which COX-2 activates STAT3 by inducing IL-6 expression. This pathway could contribute to tumor formation by promoting survivin-dependent apoptosis resistance and VEGF production. These findings provide a rationale for the future development of STAT3, IL-6, and/or COX-2-targeted therapies for the treatment of lung cancer.

Cyclooxygenase 2 (COX-2) overexpression is found in a wide variety of human cancers and is linked to all stages of tumorigenesis. Elevated tumor COX-2 expression is associated with increased angiogenesis, tumor invasion, suppression of host immunity and promotes tumor cell resistance to apoptosis. Previous reports have linked the COX-2 product prostaglandin E2 (PGE2) to the abnormal activation of the mitogen-activated protein kinase/Erk kinase pathway. Here we show that PGE2 is able to rapidly stimulate Erk phosphorylation in a subset of non–small cell lung cancer (NSCLC) cell lines. This effect is not evident in bronchial epithelial cells. In contrast to previous reports in colon cancer, we found that Erk activation as well as cellular proliferation induced by PGE2 was not inhibited by pretreatment of the cells with epidermal growth factor receptor (EGFR) inhibitors. Activation of the Erk pathway by PGE2 was also resistant to src kinase inhibitors but sensitive to the protein kinase C inhibition. PGE2 effects are mediated through four G protein–coupled receptors. Selective inhibition of EP receptors revealed the possible involvement of Ca2+-dependent signaling in PGE2-mediated activation of Erk. Our data indicate the presence of an EGFR-independent activation of the mitogen-activated protein kinase/Erk pathway by PGE2 in NSCLC cells. These findings provide evidence for the possible link between tumor COX-2 overexpression and elevated Erk-mediated cancer cell proliferation and migration. Importantly, these findings suggest that COX-2 overexpression may contribute to EGFR inhibitor resistance in NSCLC.

Clinical lung cancer is the ultimate event resulting from a series of genetic and epigenetic alterations in the respiratory epithelium at risk. According to the "field carcinogenesis" theory, these alterations can occur throughout the entire lung. In individuals with a genetic predisposition combined with a sufficient amount of procarcinogenic environmental influences, a few of these sites may eventually progress to malignancies. Recent advances in the understanding of tumor biology have identified new therapeutic targets for lung cancer chemoprevention, among which is cyclooxgygenase (COX)-2. Ample preclinical data suggest that the COX-2/prostaglandin E2 (PGE2) signaling pathway plays a pivotal role in conferring the malignant phenotype. Produced primarily by the action of COX on the free arachidonic acid liberated from membrane phospholipids, overproduction of PGE2, which is predominantly generated by upregulation of COX-2, is associated with a variety of mechanisms known to facilitate tumorigenesis. These mechanisms include abnormal expression of epithelial growth factors, epithelial and microvascular proliferation, resistance to apoptosis, and suppression of antitumor immunity. The lung is one of the major sites of PGE2 production, and previous studies have shown elevated PGE2 levels in bronchoalveolar lavage fluid of patients with bronchogenic carcinoma. In animal models, inhibition of COX-2 and PGE2 synthesis suppresses lung tumorigenesis. These preclinical data suggesting the antineoplastic effect of COX-2 inhibitors provide the basis for several ongoing pilot clinical trials to determine the feasibility of COX-2 inhibition in chemoprevention of bronchogenic carcinoma.

COX-2 overexpression and subsequent PGE2 production are frequently associated with non-small cell lung cancer and are implicated in tumor-mediated angiogenesis. Here, we report for the first time that IL-20 downregulates COX-2 and PGE2 in human bronchial epithelial and endothelial cells. Flow cytometry analysis suggests that IL-20-dependent inhibition of COX-2/PGE2 occurs through the IL-22R1/IL-20R2 dimers. In addition, we report that IL-20 exerts anti-angiogenic effects, inhibiting experimental angiogenesis. IL-20-mediated inhibition of PMA-induced angiogenesis occurs through the COX-2 regulatory pathway. Altogether our findings revealed that IL-20 is a negative modulator of COX-2/PGE2 and inhibits angiogenesis.

Cyclooxygenase (COX)-2 and its product prostaglandin (PG) E2 underlie an immunosuppressive network that is important in the pathogenesis of non–small cell lung cancer. CD4+CD25+ T regulatory (Treg) cells play an important role in maintenance of immunologic self-tolerance. CD4+CD25+ Treg cell activities increase in lung cancer and appear to play a role in suppressing antitumor immune responses. Definition of the pathways controlling Treg cell activities will enhance our understanding of limitation of the host antitumor immune responses. Tumor-derived COX-2/PGE2 induced expression of the Treg cell-specific transcription factor, Foxp3, and increased Treg cell activity. Assessment of E-prostanoid (EP) receptor requirements revealed that PGE2-mediated induction of Treg cell Foxp3 gene expression was significantly reduced in the absence of the EP4 receptor and ablated in the absence of the EP2 receptor expression. In vivo, COX-2 inhibition reduced Treg cell frequency and activity, attenuated Foxp3 expression in tumor-infiltrating lymphocytes, and decreased tumor burden. Transfer of Treg cells or administration of PGE2 to mice receiving COX-2 inhibitors reversed these effects. We conclude that inhibition of COX-2/PGE2 suppresses Treg cell activity and enhances antitumor responses.

Although tumor growth leads to inflammatory responses, the immune system develops tolerance to cancer. One way to break host tolerance to tumors is to activate key immune effector activities. Toward this end, various adjuvants are under investigation in an effort to harness the immune system to overcome tolerance to tumor associated self-antigens. There is enthusiasm for the use of specific ligands for toll-like 3 receptors (TLR3) that play a key role in the innate immune system. TLR3 agonists serve as immune adjuvants because they potently induce innate immune responses by activating dendritic cell (DC) maturation and inflammatory cytokine secretion. These activities facilitate the bridge between the innate and adaptive immune systems promoting the expansion of cytotoxic T lymphocytes (CTL) that destroy cancer cells. TLR3 agonists either alone or in combination with tumor antigens have shown success in terms of enhancing immune responses and eliciting antitumor activity in preclinical models. However, TLR3 agonists can also impact regulatory cells that dampen immune responses. Thus, immune strategies that utilize TLR3 agonists should consider the relative induction of suppressive as well as beneficial anti tumor immune activities. Herein, we summarize the TLR3 agonists that will hopefully come to clinical fruition.

CXC chemokines have been found to be important in the regulation of angiogenesis and tumor-related immunity, and in promoting organ-specific metastases. This review highlights the importance of CXC chemokine ligands and receptors in mediating non-small cell lung cancer tumor-associated angiogenesis, "immunoangiostasis," and organ-specific metastases. These findings may ultimately lead to clinical strategies to target CXC chemokines in non-small cell lung cancer.

Monitoring the spontaneous antibody (Ab) response against a panel of relevant tumor-associated antigens (TAA) in cancer patients may provide useful information regarding the clinical status of cancer. However, current Ab detection approaches require the purification of recombinant proteins, which is often difficult to achieve. In order to bypass the purification of recombinant proteins, we identified a dominant B-cell epitope from a shared tumor antigen NY-ESO-1. A synthetic peptide of the epitope, ESO:1-40, was as sensitive as the recombinant protein for detecting Ab against NY-ESO-1 in most patients. NY-ESO-1 specific Ab present in the sera of patients with melanoma, prostate cancer, nonsmall cell lung cancer, esophageal cancer, gastric cancer and hepatocellular carcinoma reacted with the dominant peptide at a similar frequency as the recombinant protein. To our knowledge, ESO:1-40 is the first peptide epitope recognized by sera from a wide spectrum of cancer patients but not healthy donors. This simple and straightforward approach may allow the investigation of the clinical significance of spontaneous Ab responses against multiple TAA and their correlation with the clinical course of malignant diseases in the future.

Dendritic cell (DC) migration is crucial for the initiation of immune responses. The balance between metalloproteinases (MMP) and tissue inhibitors of metalloproteinases (TIMP) has been shown to modulate DC migration. PGE2, which is overproduced in a wide variety of human malignancies, has been implicated in MMP and TIMP regulation in various cells, including monocytes. In the present study, we hypothesized that tumor-derived PGE2 would affect DC migratory capacity through the extracellular matrix (ECM) by altering MMP and TIMP balance. Treatment of monocyte-derived immature DC with exogenous PGE2 induced TIMP-1 secretion but not MMP-9 production and was correlated with reduced DC migration through ECM. Because recombinant TIMP-1 replicated PGE2 inhibition of DC migration while anti-TIMP-1 neutralizing Ab reversed it, we conclude that PGE2-mediated induction of TIMP-1 was responsible for the reduced migration of PGE2-treated DC. Similarly, DC cultured for 48 h in supernatants from cyclooxygenase-2 overexpressing lung cancer cells that secrete high levels of PGE2, exhibited decreased migration through ECM. Finally, analysis of E prostanoid receptor expression and their selective inhibition revealed that the enhanced TIMP-1 secretion in PGE2-treated DC was mediated predominantly by the E prostanoid receptor 2. These findings indicate that PGE2-dependent enhancement of TIMP-1 production causes reduced migration of DC through ECM.

Purpose: Emerging data continue to link carcinogenesis to inflammatory events involving the eicosanoid metabolic pathways. We therefore evaluated the effects of cyclooxygenase (COX)-2 inhibition on leukotriene (LT) B4 synthesis in the lungs of active smokers, as part of a pilot lung cancer chemoprevention study with celecoxib (Celebrex), an oral COX-2 inhibitor. Experimental Design: Bronchoalveolar lavage was performed before celecoxib treatment and after 1 month of celecoxib treatment to recover alveolar macrophages (AMs) and lining fluid for study. After harvest, AMs were immediately stimulated in vitro with the calcium ionophore A23187. AMs obtained from smokers before treatment and from ex-smoker control subjects were also cultured overnight with SC58236, a selective COX-2 inhibitor, with or without lipopolysaccharide stimulation. Results: Treatment with oral celecoxib only modestly increased LTB4 levels in bronchoalveolar lavage, without increasing the mRNA transcription of 5-lipoxygenase (5-LOX) or 5-LOX-activating protein in AMs, whereas the acute calcium ionophore-stimulated LTB4 production from smokers’ AMs was markedly increased by 10.6-fold. In addition, smokers’ AMs were twice as responsive in producing LTB4 when exposed to lipopolysaccharide compared with ex-smokers’ AMs. Concomitant COX-2 inhibition with SC58236, however, did not significantly impact these changes, whereas the 5-LOX inhibitor Zileuton blocked the generation of LTB4 in a dose-responsive manner. Finally, cycloheximide increased the production of LTB4 under all conditions, suggesting a shunting phenomenon and/or the presence of pathway inhibitors. Conclusions: Our findings suggest that whereas oral celecoxib is capable of modulating LTB4 production in the lung microenvironment, under physiologic conditions, this effect is probably not functionally significant.

Constitutive overexpression of cyclooxygenase-2 (COX-2) occurs frequently in several different malignancies, including lung, colon, breast, and prostate cancer. Clinical studies have established elevated serum insulin-like growth factor (IGF-I) content and IGF-I:IGF-binding protein 3 (IGFBP-3) ratio as risk factors for these same malignancies. Therefore, we sought to determine the link between COX-2 expression and the IGF axis in COX-2 gene-modified human non–small-cell lung cancer (NSCLC) cells. Overexpression of COX-2 in NSCLC cells enhanced the antiapoptotic and mitogenic effects of IGF-I and IGF-II, facilitated the autophosphorylation of the type 1 IGF receptor, increased class IA phosphatidylinositol 3′-kinase activity, and decreased expression of IGFBP-3. Thus, these findings show that COX-2 augments the stimulatory arm of the IGF axis.

Elevated tumor cyclooxygenase 2 (COX-2) expression is associated with increased angiogenesis, tumor invasion, and promotion of tumor cell resistance to apoptosis. In our previous studies using non-small cell lung cancer (NSCLC) cell lines constitutively expressing COX-2 cDNA in sense and antisense orientations, we demonstrated that constitutive overexpression of COX-2 leads to stabilization of the inhibitor of apoptosis protein survivin resulting in the elevated apoptosis resistance of COX-2–overexpressing cells. Genetic or pharmacologic suppression of COX-2 activity increased proteasomal degradation of survivin and cellular response to apoptosis induction. Our data show that expression of survivin in non-small cell lung cancer cells can be significantly down-regulated by RNA interference. Whereas COX-2–overexpressing NSCLC cells have significantly higher apoptosis resistance than the parental cells, inhibition of survivin expression by small interfering RNA decreases apoptosis resistance to the level of the parental non-small cell lung cancer. We conclude that COX-2-dependent expression of survivin is critical for apoptosis resistance in non-small cell lung cancer.

Cyclooxygenase-2 (COX-2) overexpression is seen in many malignancies including lung cancer. In non-small cell lung cancer (NSCLC), COX-2 is overexpressed in most adenocarcinomas and squamous cell carcinomas. Elevated tumor COX-2 prostaglandin E(2) (PGE(2)) levels have been implicated in angiogenesis, tumor invasion, resistance to apoptosis, and suppression of antitumor immunity. Preclinical animal model studies show tumor reduction when animals are treated with either nonspecific or specific inhibitors of COX-2. These studies suggest nonsteroidal anti-inflammatory drugs may act on multiple tumor-progression targets via both COX-2-dependent and-independent pathways. Consistent with these findings, epidemiologic evidence has shown a decreased incidence of lung cancer in patients who use nonsteroidal anti-inflammatory drugs. Based on these observations, celecoxib, a selective COX-2 inhibitor, has been evaluated in combination with chemotherapy for the management of metastatic NSCLC in patients who have failed prior chemotherapy. Several clinical trials are ongoing that evaluate celecoxib in combination with chemoradiation for unresectable, locally advanced NSCLC. Another trial evaluating celecoxib in a preoperative combination with paclitaxel and carboplatin has generated overall clinical response rates at least comparable to those reported in the Bimodality Lung Oncology Team trial. Ongoing clinical trials are also evaluating the combination of celecoxib with chemotherapy and/or radiation or celecoxib in combination with epidermal growth factor receptor inhibitors of NSCLC. This article reviews preclinical information on COX-2 inhibitors in lung cancer and presents updated data from several ongoing clinical trials that are evaluating celecoxib in NSCLC.

The antitumor efficacy of EBV-induced molecule 1 ligand CC chemokine (ELC/CCL19) was evaluated in a murine lung cancer model. The ability of ELC/CCL19 to chemoattract both dendritic cells and T lymphocytes formed the rationale for this study. Compared with diluent-treated tumor-bearing mice, intratumoral injection of recombinant ELC/CCL19 led to significant systemic reduction in tumor volumes (p < 0.01). ELC/CCL19-treated mice exhibited an increased influx of CD4 and CD8 T cell subsets as well as dendritic cells at the tumor sites. These cell infiltrates were accompanied by increases in IFN-γ, MIG/CXCL9, IP-10/CXCL10, GM-CSF, and IL-12 but a concomitant decrease in the immunosuppressive molecules PGE2 and TGFβ. Transfer of T lymphocytes from ELC/CCL19 treated tumor-bearing mice conferred the antitumor therapeutic efficacy of ELC/CCL19 to naive mice. ELC/CCL19 treated tumor-bearing mice showed enhanced frequency of tumor specific T lymphocytes secreting IFN-γ. In vivo depletion of IFN-γ, MIG/CXCL9, or IP-10/CXCL10 significantly reduced the antitumor efficacy of ELC/CCL19. These findings provide a strong rationale for further evaluation of ELC/CCL19 in tumor immunity and its use in cancer immunotherapy.

The antitumor efficiency of dendritic cells transduced with an adenovirus vector expressing interleukin (IL)-7 (DC-AdIL-7) was evaluated in a murine model of spontaneous bronchoalveolar cell carcinoma. These transgenic mice (CC-10 TAg), expressing the SV40 large T antigen under the Clara cell promoter, develop bilateral multifocal pulmonary adenocarcinomas and die at 4 months as a result of progressive pulmonary tumor burden. Injection of DC-AdIL-7 in the axillary lymph node region (ALNR) weekly for 3 weeks led to a marked reduction in tumor burden with extensive lymphocytic infiltration of the tumors and enhanced survival. The antitumor responses were accompanied by the enhanced elaboration of interferon (IFN)-gamma and IL-12 as well as an increase in the antiangiogenic chemokines, IFN-gamma-inducible protein 10 (IP-10/CXCL10) and monokine induced by IFN-gamma (MIG/CXCL9). In contrast, production of the immunosuppressive mediators IL-10, transforming growth factor (TGF)-beta, prostaglandin E(2) (PGE(2)), and the proangiogenic modulator vascular endothelial growth factor (VEGF) decreased in response to DC-AdIL-7 treatment. Significant reduction in tumor burden in a model in which tumors develop in an organ-specific manner provides a strong rationale for further evaluation of DC-AdIL-7 in regulation of tumor immunity and its use in lung cancer genetic immunotherapy.

Increased COX-2 expression and elevated PGE2 have been associated with a poor prognosis in lung cancer. Cannabinoids have been known to exert some of their biological effects via modulation of prostaglandin production. We evaluated the impact of methanandamide on COX-2 expression, PGE2 production, and tumor growth in murine lung cancer. Methanandamide administration (5 mg/kg, four times/wk i.p.) resulted in an increased rate of tumor growth (P0.01 compared with diluent treated controls). The CB1 and CB2 receptor antagonists, SR141716 and SR144528, did not block the methanandamide-mediated increase in tumor growth. In vivo, methanandamide treatment increased the production of PGE2 at the tumor site as well as in splenocytes. Consistent with these results, methanandamide increased PGE2 and COX-2 levels in murine lung cancer cells in vitro via a cannabinoid receptor-independent mechanism. The COX-2-specific inhibitor, SC58236, abrogated methanandamide induction of PGE2 production in vitro and blocked methanandamide-enhanced tumor growth in vivo. Furthermore, the p38/MAPK inhibitor, SB203528, and the p42/44 inhibitor, PD98059, blocked methanandamide-mediated induction of PGE2 and COX-2. These results suggest that methanandamide augments tumor growth by a cannabinoid receptor-independent pathway that is associated with the up-regulation of COX-2.

Metastatic disease is responsible for 90% of death from solid tumors. However, only a minority of metastasis-specific targets has been exploited therapeutically, and effective prevention and suppression of metastatic disease is still an elusive goal. In this review, we will first summarize the current state of knowledge about the molecular features of the disease, with particular focus on steps and targets potentially amenable to therapeutic intervention. We will then discuss the reasons underlying the paucity of metastatic drugs in the current oncological arsenal and potential ways to overcome this therapeutic gap. We reason that the discovery of novel promising targets, an increased understanding of the molecular features of the disease, the effect of disruptive technologies, and a shift in the current preclinical and clinical settings have the potential to create more successful drug development endeavors.

New ground-breaking discoveries in cancer immunotherapeutics are now applicable for lung cancer therapy, exemplified by the recent FDA approval of nivolumab for advanced squamous cell carcinoma of the lung as a second line therapy, harnessing the immune system to fight lung cancer. This opens the door for lung cancer to enter the mainstream of immunotherapeutics along with other malignancies that have been traditionally viewed as immune unresponsive. The durable responses that result in continued separation of the survival curves suggest a new emerging paradigm that may add significantly to the benefit of existing conventional therapies. This chapter reviews immunotherapeutic approaches for lung cancer that have shown promise in early clinical trials and/or have advanced to late-phase development and FDA approval.

Epithelial–mesenchymal transition (EMT) is a type of cellular plasticity by which epithelial cells acquire the form and function of mesenchymal cells. Physiologic EMT is an essential part of normal embryonic development and an adult organism’s ability to overcome acute injury. However, chronic injury and inflammation can yield dysregulated or pathologic EMT that drives organ fibrosis and cancer development. In this chapter, we review seminal work and recent findings regarding the molecular, cellular, microenvironmental, and environmental factors that drive inflammation-induced EMT-dependent lung carcinogenesis. We also discuss potential approaches for treating or perhaps preventing lung cancer by targeting the inflammation-EMT-cancer axis.

Lung cancer is the leading cause of cancer death in the US and in the world. Over the past 30 years, the five-year survival rate for lung cancer has increased by only 5%. With the widespread implementation of screening programs, detection of premalignant and early stage disease is increasing. A better understanding of genomic alterations and the microenvironment along the spectrum of early disease could lead to identification of progression-associated mutations (PAMs), defined as those shared between premalignant lesions and invasive cancer, and their neoepitopes. Unleashing the immune response against pulmonary premalignancy could transform therapy and outcomes. FFPE tissue blocks from patients with resected lung adenocarcinoma (ADC) were obtained from the UCLA Lung Cancer Tissue Repository. For each patient, the following regions were dissected from distal airways utilizing Laser Capture Microdissection: a) normal airway epithelial cells (1-3 regions), b) premalignant atypical adenomatous hyperplasia (AAH, 2-4 regions), c) adenocarcinoma in situ (AIS, when present) and d) ADC, 1-3 regions followed by whole exome sequencing. Forty-one complete cases have been sequenced to date. Our data suggest that premalignant lesions from the same patient may a) have different mutational profiles and b) bear progression-associated mutations, common with the primary lung tumor. This inter-lesion heterogeneity suggests that a progression-associated mutational landscape could be defined in longitudinal studies of pulmonary premalignancy which will be the focus of future investigations. Next, utilizing the mutational data, we performed in silico neoantigen analysis to identify potential neoepitopes among the genes mutated in premalignant lesions. The neoantigen analysis demonstrated that among the top 11 peptides with high binding avidity for autologous MHC, 9 were derived from PAMs. This suggests that neoepitopes exist in premalignancy that could serve as targets for development of future vaccines. Finally, we performed the quantitative immunohistochemical (IHC) and immunofluorescence (IF) staining for Granzyme B, PD-1, PDL-1, CD4+, CD8+ and FOXP3 to evaluate cell-mediated immunity on the same samples that were utilized for WES. We found both infiltration of T effector cells as well as upregulation of checkpoints in premalignancy and detected a significant inter-lesional heterogeneity. Our studies lay the ground work for identification of neoepitopes that can be targeted before the development of invasive lung cancer, thus shifting the approach to disease interception through immunoprevention and treatment of the very earliest phase of the disease. Citation Format: Kostyantyn Krysan, Linh M. Tran, Brandon S. Grimes, Tonya C. Walser, William D. Wallace, Steven M. Dubinett. Evaluation of progression associated neoepitopes and immune contexture in pulmonary premalignancy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1016. doi:10.1158/1538-7445.AM2017-1016

Background: Lung squamous cell carcinoma (SqCC) arises in the epithelial layer of the bronchial airway and is often preceded by the development of premalignant lesions. However, not all premalignant lesions progress to lung SqCC and many regress without therapeutic intervention. Understanding the somatic alterations that contribute to progression of premalignant lesions in the airway will allow us to identify biomarkers for early detection and develop therapeutic strategies for early intervention. Methods: Airway biopsies were obtained from high-risk smokers undergoing lung cancer screening by auto-fluorescence bronchoscopy and chest CT at the Roswell Park Cancer Institute. For each subject (n=30), multiple premalignant lesions were sampled repeatedly over time (n=144 samples). One biopsy from each region was sent for pathological review while another biopsy was taken for molecular studies. DNA was also isolated from the blood or cytologically normal bronchial brushings to serve as a matched normal control. Exome capture was performed using the Illumina TruSeq Rapid Exome kit and sequenced to a mean depth of coverage of 120x at Uniform Services University and Walter Reed National Military Medical Center. Results: The median number of somatic mutations across all premalignant lesions was 0.73 per megabase (range: 0.10 - 9.8 per Mb) and displayed a modest association with histological grade (p=0.07). The most frequently mutated lung cancer genes included KMT2C (12%), NOTCH1 (11%), FAT1 (6%), TP53 (5%), and CDKN2A (7/Mb) were taken from adjacent sites over two time points in the same individual with a prior history of lung squamous cell carcinoma. These lesions had a significantly overlapping set of mutations including FAT1 indicating a common evolutionary ancestor. Conclusions: The somatic alterations observed in known cancer genes such as TP53, KMT2C, NOTCH1, and FAT1 may be among the earliest driver events in lung SqCC development and may be useful as biomarkers for early detection as well as targets for lung cancer interception. Citation Format: Joshua Campbell, Xijun Zhang, Samjot S. Dhillon, Catalina Perdomo, Sarah Mazzilli, Yaron Geshalter, Gang Liu, Sherry Zhang, Hanqiao Lin, Jessica Vick, Christopher Moy, Stefano Monti, Evan Johnson, Matthew Meyerson, Steven Dubinett, Suso Platero, Matthew Wilkerson, Clifton Dalgard, Marc Lenburg, Mary Reid, Jennifer Beane, Avrum Spira. The genomic landscape of premalignant lung squamous cell carcinoma lesions [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3259. doi:10.1158/1538-7445.AM2017-3259