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The sulfone derivative of the non-steroidal anti-inflammatory drug (NSAID), sulindac, has been reported to inhibit mammary and colon tumor formation in rodent models of chemically-induced carcinogenesis. Unlike its parent compound, this metabolite lacks cyclo-oxygenase inhibitory activity. A tumor induction protocol, consisting of NNK administration in the drinking water over several weeks to model chronic human exposure, was used to test whether the sulfone (called FGN-1) could inhibit the formation of primary lung tumors in mice. A total of 150 female, AIN-76A-fed, A/J mice received 9 mg of NNK each. Concentrations of FGN-1 that had been previously determined not to affect body weight gain were added to the food at levels of 0, 250, 500 and 750 mg/kg of diet (30 mice/group) starting 2 weeks before NNK administration and continuing for 22 weeks. At that time pleural surface tumors were counted. Tumor incidence decreased significantly from 96% in the control diet and 93% in the 250 FGN-1 mg/kg diet to 63 and 67% in the 500 and 750 mg FGN-1/kg diet groups, respectively (P < 0.001 by chi-square analysis). Lung tumor multiplicity decreased from 18.1 ± 3 tumors/mouse (mean ± SEM, control diet) to 12.3 ± 3 (250), 5.3 ± 1 (500) and 2.1 ± 1 (750) (P < 0.0005 by post hoc ANOVA). In previous studies using this carcinogenesis protocol, the maximum tolerated dose of sulindac inhibited lung tumor multiplicity by no more than 50% with no effect on incidence. This dose-dependent reduction in tumorigenesis by a non-toxic dose of FGN-1 indicates a strong chemopreventive activity against experimental induction of lung carcinogenesis. The greater potency of the sulfone over sulindac and its lack of toxic side effects because of its inability to affect cyclo-oxygenase activity suggests that clinical testing in individuals at high risk for lung cancer should be considered. [ABSTRACT FROM PUBLISHER]

The N-nitroso-trischloroethylurea (NTCU)-induced mouse model of squamous lung carcinoma recapitulates human disease from premalignant dysplasia through invasive tumors, making it suitable for preclinical chemoprevention drug testing. Pioglitazone is a peroxisome proliferator-activated receptor γ (PPARγ) agonist shown to prevent lung tumors in preclinical models. We investigated pioglitazone's effect on lesion development and markers of potential preventive mechanisms in the NTCU model. Female FVB/N mice were exposed to vehicle, NTCU or NTCU + oral pioglitazone for 32 weeks. NTCU induces the appearance of basal cells in murine airways while decreasing/changing their epithelial cell makeup, resulting in development of bronchial dysplasia. H&E and keratin 5 (KRT5) staining were used to detect and grade squamous lesions in formalin fixed lungs. mRNA expression of epithelial to mesenchymal transition (EMT) markers and basal cell markers were measured by qPCR. Dysplasia persistence markers desmoglein 3 and polo like kinase 1 were measured by immunohistochemistry. Basal cell markers KRT14 and p63, club cell specific protein and ciliated cell marker acetylated tubulin were measured by immunofluorescence. Pioglitazone treatment significantly reduced squamous lesions and the presence of airway basal cells, along with increasing normal epithelial cells in the airways of NTCU-exposed mice. Pioglitazone also significantly influenced EMT gene expression to promote a more epithelial, and less mesenchymal, phenotype. Pioglitazone reduced the presence of squamous dysplasia and maintained normal airway cell composition. This work increases the knowledge of mechanistic pathways in PPARγ agonism for lung cancer interception and provides a basis for further investigation to advance this chemoprevention strategy., (© 2022 UICC.)

The transmembrane receptor Frizzled 9 (FZD9) is important for fetal neurologic and bone development through both canonical and non-canonical WNT/FZD signaling. In the adult lung, however, Fzd9 helps to maintain a normal epithelium by signaling through peroxisome proliferator activated receptor γ (PPARγ). The effect of FZD9 loss on normal lung epithelial cells and regulators of its expression in the lung are unknown. We knocked down FZD9 in human bronchial epithelial cell (HBEC) lines and found that downstream EMT targets and PPARγ activity are altered. We used a FZD9 -/- mouse in the urethane lung adenocarcinoma model and found FZD9 -/- adenomas had more proliferation, increased EMT signaling, decreased activation of PPARγ, increased expression of lung cancer associated genes, increased transformed growth, and increased potential for invasive behavior. We identified PPARγ as a transcriptional regulator of FZD9. We also demonstrated that extended cigarette smoke exposure in HBEC leads to decreased FZD9 expression, decreased activation of PPARγ, and increased transformed growth, and found that higher exposure to cigarette smoke in human lungs leads to decreased FZD9 expression. These results provide evidence for the role of FZD9 in lung epithelial maintenance and in smoking related malignant transformation. We identified the first transcriptional regulator of FZD9 in the lung and found FZD9 negative lesions are more dangerous. Loss of FZD9 creates a permissive environment for development of premalignant lung lesions, making it a potential target for intervention., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Sompel, Dwyer-Nield, Smith, Elango, Vanderlinden, Kopf, Keith and Tennis.)

Prevention of premalignant lesion progression is a promising approach to reducing lung cancer burden in high-risk populations. Substantial preclinical and clinical evidence has demonstrated efficacy of the prostacyclin analogue iloprost for lung cancer chemoprevention. Iloprost activates peroxisome proliferator-activated receptor gamma (PPARG) to initiate chemopreventive signaling and in vitro , which requires the transmembrane receptor Frizzled 9 (FZD 9 ). We hypothesized a Fzd 9 -/- mouse would not be protected by iloprost in a lung cancer model. Fzd 9 -/- mice were treated with inhaled iloprost in a urethane model of lung adenoma. We found that Fzd 9 -/- mice treated with iloprost were not protected from adenoma development compared to wild-type mice nor did they demonstrate increased activation of iloprost signaling pathways. Our results established that iloprost requires FZD 9 in vivo for lung cancer chemoprevention. This work represents a critical advancement in defining iloprost's chemopreventive mechanisms and identifies a potential response marker for future clinical trials., Competing Interests: The authors declare no competing interests., (© 2022 The Author(s).)

Lung cancer chemoprevention with the prostacyclin analogue iloprost is the most promising approach to date for intercepting progression of premalignant lung lesions in former smokers. Previous preclinical studies of iloprost used oral delivery, but a study modeling delivery directly to the target organ was needed. In vivo and in vitro studies have identified gene expression changes following iloprost treatment, including increased e-cadherin and Ppargγ and decreased COX2 and vimentin. We used tumor counts and gene expression to demonstrate the effectiveness of intranasal delivery of iloprost in a murine model of premalignant adenomas. Intranasal delivery of iloprost reduced adenoma multiplicity 14 weeks after urethane exposure in FVB/N mice compared with untreated urethane controls. Intranasal iloprost reversed urethane-induced gene expression changes in tumors and whole lung. These results correspond to previous studies of oral iloprost and in vitro treatment of human bronchial epithelial cells. This study demonstrates that intranasal delivery of iloprost in a mouse model of lung premalignant lesions is effective chemoprevention. This will be an essential tool for exploring mechanisms and outcomes of iloprost chemoprevention, along with supporting ongoing clinical trials of inhaled iloprost chemoprevention. PREVENTION RELEVANCE: Iloprost is a promising chemoprevention agent for lung cancer and this work describes a new delivery approach in vivo., (©2021 American Association for Cancer Research.)

Tobacco smoke-induced squamous cell lung cancer (SCC) develops from endobronchial dysplastic lesions that progress to invasive disease. A reproducible murine model recapitulating histologic progression observed in current and former smokers will advance testing of new preventive and therapeutic strategies. Previous studies show that prolonged topical application of N-nitroso-tris-chloroethylurea (NTCU) generates a range of airway lesions in sensitive mice similar to those induced by chronic tobacco smoke exposure in humans. To improve the current NTCU model and better align it with human disease, NTCU was applied to mice twice weekly for 4-5 weeks followed by a recovery period before cigarette smoke (CS) or ambient air (control) exposure for an additional 3-6 weeks. Despite the short time course, the addition of CS led to significantly more premalignant lesions (PML; 2.6 vs. 0.5; P < 0.02) and resulted in fewer alveolar macrophages (52,000 macrophages/mL BALF vs. 68,000; P < 0.05) compared with control mice. This improved NTCU + CS model is the first murine SCC model to incorporate tobacco smoke and is more amenable to preclinical studies because of the increased number of PML, decreased number of mice required, and reduced time needed for PML development., (©2020 American Association for Cancer Research.)

Lung cancer is one of the deadliest types of cancer and as such requires disease models that are useful for identification of novel pathways for biomarkers as well as to test therapeutic agents. Adenocarcinoma (ADC), the most prevalent type of lung cancer, is a subtype of non-small cell lung carcinoma (NSCLC) and a disease driven mainly by smoking. However, it is also the most common subtype of lung cancer found in non-smokers with environmental exposures. Chemically driven models of lung cancer, also called primary models of lung cancer, are important because they do not overexpress or delete oncogenes or tumor suppressor genes, respectively, to increase oncogenesis. Instead these models test tumor development without forcing a specific pathway (i.e., Kras). The primary focus of this chapter is to discuss a well-established 2-stage mouse model of lung adenocarcinomas. The initiator (3-methylcholanthrene, MCA) does not elicit many, if any, tumors if not followed by exposure to the tumor promoter (butylated hydroxytoluene, BHT). In sensitive strains, such as A/J, FVB, and BALB, significantly greater numbers of tumors develop following the MCA/BHT protocol compared to MCA alone. BHT does not elicit tumors on its own; it is a non-genotoxic carcinogen and promoter. In these sensitive strains, promotion is also associated with inflammation characterized by infiltrating macrophages, lymphocytes, and neutrophils, and other inflammatory cell types in addition to increases in total protein content reflective of lung hyperpermeability. This 2-stage model is a useful tool to identify unique promotion specific events to then test in future intervention studies., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Lung cancer chemoprevention, especially in high-risk former smokers, has great potential to reduce lung cancer incidence and mortality. Thiazolidinediones prevent lung cancer in preclinical studies, and diabetics receiving thiazolidinediones have lower lung cancer rates which led to our double-blind, randomized, phase II placebo-controlled trial of oral pioglitazone in high-risk current or former smokers with sputum cytologic atypia or known endobronchial dysplasia. Bronchoscopy was performed at study entry and after completing 6 months of treatment. Biopsies were histologically scored, and primary endpoint analysis tested worst biopsy scores (Max) between groups; Dysplasia index (DI) and average score (Avg) changes were secondary endpoints. Biopsies also received an inflammation score. The trial accrued 92 subjects (47 pioglitazone, 45 placebo), and 76 completed both bronchoscopies (39 pioglitazone, 37 placebo). Baseline dysplasia was significantly worse for current smokers, and 64% of subjects had mild or greater dysplasia at study entry. Subjects receiving pioglitazone did not exhibit improvement in bronchial dysplasia. Former smokers treated with pioglitazone exhibited a slight improvement in Max, while current smokers exhibited slight worsening. While statistically significant changes in Avg and DI were not observed in the treatment group, former smokers exhibited a slight decrease in both Avg and DI. Negligible Avg and DI changes occurred in current smokers. A trend toward decreased Ki-67 labeling index occurred in former smokers with baseline dysplasia receiving pioglitazone. While pioglitazone did not improve endobronchial histology in this high-risk cohort, specific lesions showed histologic improvement, and further study is needed to better characterize responsive dysplasia., (©2019 American Association for Cancer Research.)

Polycyclic aromatic hydrocarbons (PAHs), prevalent contaminants in our environment, in many occupations, and in first and second-hand smoke, pose significant adverse health effects. Most research focused on the genotoxic high molecular weight PAHs (e.g., benzo[ a ]pyrene), however, the nongenotoxic low molecular weight (LMW) PAHs are emerging as potential co-carcinogens and tumor promoters known to dysregulate gap junctional intercellular communication (GJIC), activate mitogen activated protein kinase pathways, and induce the release of inflammatory mediators. We hypothesize that inflammatory mediators resulting from LMW PAH exposure in mouse lung epithelial cell lines are involved in the dysregulation of GJIC. We used mouse lung epithelial cell lines and an alveolar macrophage cell line in the presence of a binary PAH mixture (1:1 ratio of fluoranthene and 1-methylanthracene; PAH mixture). Parthenolide, a pan-inflammation inhibitor, reversed the PAH-induced inhibition of GJIC, the decreased CX43 expression, and the induction of KC and TNF. To further determine the direct role of a cytokine in regulating GJIC, recombinant TNF (rTNF) was used to inhibit GJIC and this response was further enhanced in the presence of the PAH mixture. Collectively, these findings support a role for inflammation in regulating GJIC and the potential to target these early stage cancer pathways for therapeutics.

Lung cancer is the leading cause of cancer death worldwide and global burden could be reduced through targeted application of chemoprevention. The development of squamous lung carcinoma has been linked with persistent, high-grade bronchial dysplasia. Bronchial histology improved in former smokers in a chemoprevention trial with the prostacyclin analogue iloprost. Prostacyclin acts through peroxisome proliferator-activated receptor gamma (PPARγ) to reverse epithelial to mesenchymal transition and promote anticancer signaling. We hypothesized that the prostacyclin signaling pathway and EMT could provide response markers for prostacyclin chemoprevention of lung cancer. Human bronchial epithelial cells were treated with cigarette smoke condensate (CSC) or iloprost for 2 weeks, CSC for 16 weeks, or CSC for 4 weeks followed by 4 weeks of CSC and/or iloprost, and RNA was extracted. Wild-type or prostacyclin synthase transgenic mice were exposed to 1 week of cigarette smoke or one injection of urethane, and RNA was extracted from the lungs. We measured potential markers of prostacyclin and iloprost efficacy in these models. We identified a panel of markers altered by tobacco carcinogens and inversely affected by prostacyclin, including PPAR γ, 15PGDH, CES1, COX-2, ECADHERIN, SNAIL, VIMENTIN, CRB3, MIR34c, and MIR221 These data introduce a panel of potential markers for monitoring interception of bronchial dysplasia progression during chemoprevention with prostacyclin. Chemoprevention is a promising approach to reduce lung cancer mortality in a high-risk population. Identifying markers for targeted use is critical for success in future clinical trials of prostacyclin for lung cancer chemoprevention. Cancer Prev Res; 11(10); 643-54. ©2018 AACR ., (©2018 American Association for Cancer Research.)

Persistent bronchial dysplasia is associated with increased risk of developing invasive squamous cell carcinoma (SCC) of the lung. In this study, we hypothesized that differences in gene expression profiles between persistent and regressive bronchial dysplasia would identify cellular processes that underlie progression to SCC. RNA expression arrays comparing baseline biopsies from 32 bronchial sites that persisted/progressed to 31 regressive sites showed 395 differentially expressed genes [ANOVA, FDR ≤ 0.05). Thirty-one pathways showed significantly altered activity between the two groups, many of which were associated with cell-cycle control and proliferation, inflammation, or epithelial differentiation/cell-cell adhesion. Cultured persistent bronchial dysplasia cells exhibited increased expression of Polo-like kinase 1 (PLK1), which was associated with multiple cell-cycle pathways. Treatment with PLK1 inhibitor induced apoptosis and G 2 -M arrest and decreased proliferation compared with untreated cells; these effects were not seen in normal or regressive bronchial dysplasia cultures. Inflammatory pathway activity was decreased in persistent bronchial dysplasia, and the presence of an inflammatory infiltrate was more common in regressive bronchial dysplasia. Regressive bronchial dysplasia was also associated with trends toward overall increases in macrophages and T lymphocytes and altered polarization of these inflammatory cell subsets. Increased desmoglein 3 and plakoglobin expression was associated with higher grade and persistence of bronchial dysplasia. These results identify alterations in the persistent subset of bronchial dysplasia that are associated with high risk for progression to invasive SCC. These alterations may serve as strong markers of risk and as effective targets for lung cancer prevention. Significance: Gene expression profiling of high-risk persistent bronchial dysplasia reveals changes in cell-cycle control, inflammatory activity, and epithelial differentiation/cell-cell adhesion that may underlie progression to invasive SCC. Cancer Res; 78(17); 4971-83. ©2018 AACR ., (©2018 American Association for Cancer Research.)

Half of lung cancers are diagnosed in former smokers, leading to a significant treatment burden in this population. Chemoprevention in former smokers using the prostacyclin analogue iloprost reduces endobronchial dysplasia, a premalignant lung lesion. Iloprost requires the presence of the WNT receptor Frizzled 9 (Fzd9) for inhibition of transformed growth in vitro. To investigate the relationship between iloprost, cigarette smoke, and Fzd9 expression, we used human samples, mouse models, and in vitro studies. Fzd9 expression was low in human lung tumors and in progressive dysplasias. In mouse models and in vitro studies, tobacco smoke carcinogens reduced expression of Fzd9 while prostacyclin maintained or increased expression. Expression of miR-31 repressed Fzd9 expression, which was abrogated by prostacyclin. We propose a model where cigarette smoke exposure increases miR-31 expression, which leads to decreased Fzd9 expression and prevents response to iloprost. When smoke is removed miR-31 is reduced, prostacyclin can increase Fzd9 expression, and progression of dysplasia is inhibited. Fzd9 and miR-31 are candidate biomarkers for precision application of iloprost and monitoring of treatment progress. As we continue to investigate the mechanisms of prostacyclin chemoprevention and identify biomarkers for its use, we will facilitate clinical trials and speed implementation of this valuable prevention approach.

Squamous cell lung cancer (SCC) is the second leading cause of lung cancer death in the US and has a 5-year survival rate of only 16%. Histological changes in the bronchial epithelium termed dysplasia are precursors to invasive SCC. However, the cellular mechanisms that cause dysplasia are unknown. To fill this knowledge gap, we used topical application of N-nitroso-tris chloroethylurea (NTCU) for 32 weeks to induce squamous dysplasia and SCC in mice. At 32 weeks the predominant cell type in the dysplastic airways was Keratin (K) 5 and K14 expressing basal cells. Notably, basal cells are extremely rare in the normal mouse bronchial epithelium but are abundant in the trachea. We therefore evaluated time-dependent changes in tracheal and bronchial histopathology after NTCU exposure (4, 8, 12, 16, 25 and 32 weeks). We show that tracheal dysplasia occurs significantly earlier than that of the bronchial epithelium (12 weeks vs. 25 weeks). This was associated with increased numbers of K5+/K14+ tracheal basal cells and a complete loss of secretory (Club cell secretory protein expressing CCSP+) and ciliated cells. TUNEL staining of NTCU treated tissues confirmed that the loss of CCSP+ and ciliated cells was not due to apoptosis. However, mitotic index (measured by bromodeoxyuridine incorporation) showed that NTCU treatment increased proliferation of K5+ basal cells in the trachea, and altered bronchial mitotic population from CCSP+ to K5+ basal cells. Thus, we demonstrate that NTCU-induced lung epithelial dysplasia starts in the tracheal epithelium, and is followed by basal cell metaplasia of the bronchial epithelium. This analysis extends our knowledge of the NTCU-SCC model by defining the early changes in epithelial cell phenotypes in distinct airway locations, and this may assist in identifying new targets for future chemoprevention studies.

Chronic inflammation is a risk factor for lung cancer, and low-dose aspirin intake reduces lung cancer risk. However, the roles that specific inflammatory cells and their products play in lung carcinogenesis have yet to be fully elucidated. In mice, alveolar macrophage numbers increase as lung tumors progress, and pulmonary macrophage programing changes within 2 weeks of carcinogen exposure. To examine how macrophages specifically affect lung tumor progression, they were depleted in mice bearing urethane-induced lung tumors using clodronate-encapsulated liposomes. Alveolar macrophage populations decreased to ≤50% of control levels after 4-6 weeks of liposomal clodronate treatment. Tumor burden decreased by 50% compared to vehicle treated mice, and tumor cell proliferation, as measured by Ki67 staining, was also attenuated. Pulmonary fluid levels of insulin-like growth factor-I, CXCL1, IL-6, and CCL2 diminished with clodronate liposome treatment. Tumor-associated macrophages expressed markers of both M1 and M2 programing in vehicle and clodronate liposome-treated mice. Mice lacking CCR2 (the receptor for macrophage chemotactic factor CCL2) had comparable numbers of alveolar macrophages and showed no difference in tumor growth rates when compared to similarly treated wild-type mice suggesting that while CCL2 may recruit macrophages to lung tumor microenvironments, redundant pathways can compensate when CCL2/CCR2 signaling is inactivated. Depletion of pulmonary macrophages rather than inhibition of their recruitment may be an advantageous strategy for attenuating lung cancer progression.

Silibinin inhibits mouse lung tumorigenesis in part by targeting tumor microenvironment. Tumor necrosis factor-alpha (TNF-α) and interferon-gamma (IFN-γ) can be pro- or anti-tumorigenic, but in lung cancer cell lines they induce pro-inflammatory enzymes cyclooxygenase 2 (COX2) and inducible nitric oxide synthase (iNOS). Accordingly, here we examined mechanism of silibinin action on TNF-α + IFN-γ (hereafter referred as cytokine mixture) elicited signaling in tumor-derived mouse lung epithelial LM2 cells. Both signal transducers and activators of the transcription (STAT)3 (tyr705 and ser727) and STAT1 (tyr701) were activated within 15 min of cytokine mixture exposure, while STAT1 (ser727) activated after 3 h. Cytokine mixture also activated Erk1/2 and caused an increase in both COX2 and iNOS levels. Pretreatment of cells with a MEK, NF-κB, and/or epidermal growth factor receptor (EGFR) inhibitor inhibited cytokine mixture-induced activation of Erk1/2, NF-κB, or EGFR, respectively, and strongly decreased phosphorylation of STAT3 and STAT1 and expression of COX2 and iNOS. Also, janus family kinases (JAK)1 and JAK2 inhibitors specifically decreased cytokine-induced iNOS expression, suggesting possible roles of JAK1, JAK2, Erk1/2, NF-κB, and EGFR in cytokine mixture-caused induction of COX2 and iNOS expression via STAT3/STAT1 activation in LM2 cells. Importantly, silibinin pretreatment inhibited cytokine mixture-induced phosphorylation of STAT3, STAT1, and Erk1/2, NF-κB-DNA binding, and expression of COX2, iNOS, matrix metalloproteinases (MMP)2, and MMP9, which was mediated through impairment of STAT3 and STAT1 nuclear localization. Silibinin also inhibited cytokine mixture-induced migration of LM2 cells. Together, we showed that STAT3 and STAT1 could be valuable chemopreventive and therapeutic targets within the lung tumor microenvironment in addition to being targets within tumor itself, and that silibinin inhibits their activation as a plausible mechanism of its efficacy against lung cancer., (Copyright © 2011 Wiley Periodicals, Inc.)

Background: Worldwide, lung cancer kills more people than breast, colon and prostate cancer combined. Alterations in macrophage number and function during lung tumorigenesis suggest that these immune effector cells stimulate lung cancer growth. Evidence from cancer models in other tissues suggests that cancer cells actively recruit growth factor-producing macrophages through a reciprocal signaling pathway. While the levels of lung macrophages increase during tumor progression in mouse models of lung cancer, and high pulmonary macrophage content correlates with a poor prognosis in human non-small cell lung cancer, the specific role of alveolar macrophages in lung tumorigenesis is not clear., Methods: After culturing either an immortalized lung macrophage cell line or primary murine alveolar macrophages from naïve and lung-tumor bearing mice with primary tumor isolates and immortalized cell lines, the effects on epithelial proliferation and cellular kinase activation were determined. Insulin-like growth factor-1 (IGF-1) was quantified by ELISA, and macrophage conditioned media IGF-1 levels manipulated by IL-4 treatment, immuno-depletion and siRNA transfection., Results: Primary macrophages from both naïve and lung-tumor bearing mice stimulated epithelial cell proliferation. The lungs of tumor-bearing mice contained 3.5-times more IGF-1 than naïve littermates, and media conditioned by freshly isolated tumor-educated macrophages contained more IGF-1 than media conditioned by naïve macrophages; IL-4 stimulated IGF-1 production by both macrophage subsets. The ability of macrophage conditioned media to stimulate neoplastic proliferation correlated with media IGF-1 levels, and recombinant IGF-1 alone was sufficient to induce epithelial proliferation in all cell lines evaluated. Macrophage-conditioned media and IGF-1 stimulated lung tumor cell growth in an additive manner, while EGF had no effect. Macrophage-derived factors increased p-Erk1/2, p-Akt and cyclin D1 levels in neoplastic cells, and the combined inhibition of both MEK and PI3K ablated macrophage-mediated increases in epithelial growth., Conclusions: Macrophages produce IGF-1 which directly stimulates neoplastic proliferation through Erk and Akt activation. This observation suggests that combining macrophage ablation therapy with IGF-1R, MEK and/or PI3K inhibition could improve therapeutic response in human lung cancer. Exploring macrophage-based intervention could be a fruitful avenue for future research.

Purpose: Sustained nitric oxide (NO) generation positively correlates with lung cancer development and progression. Herein, we genetically confirmed this role of iNOS and evaluated the chemopreventive efficacy of silibinin in carcinogen-treated B6/129 wild-type (WT) and iNOS(-/-) mice., Experimental Design: Male B6/129-Nos2(tm1Lau) (iNOS(-/-)) and B6/129PF2 WT mice were injected i.p. with 1 mg/g body weight urethane once weekly for 7 consecutive weeks, followed by silibinin gavage (742 mg/kg body weight) for 5 d/wk for 18 weeks., Results: Quantification of micro-CT data in real-time showed that silibinin significantly decreases urethane-induced tumor number and size in WT mice, consistent with measurements made ex vivo at study termination. Genetic ablation of iNOS decreased urethane-induced tumor multiplicity by 87% (P < 0.001) compared to WT mice. Silibinin decreased tumor multiplicity by 71% (P < 0.01) in WT mice, but did not show any such considerable effect in iNOS(-/-) mice. Tumors from WT mice expressed more iNOS (P < 0.01) but almost similar eNOS and nNOS than those in silibinin-treated mice. In these tumors, silibinin moderately (P < 0.01) inhibited cell proliferation but strongly (P < 0.01) reduced the number of newly formed nestin-positive microvessels. Silibinin decreased VEGFR2 level, and STAT3 and NF-κB activation in tumors., Conclusions: The lack of effect of silibinin in iNOS(-/-) mice suggests that silibinin exerts most of its chemopreventive and angiopreventive effects through its inhibition of iNOS expression in lung tumors. Our results support iNOS as a potential target for controlling lung cancer, and demonstrate the value of real-time noninvasive micro-CT imaging modality for evaluating the efficacy of lung cancer chemopreventive agents., (©2010 AACR.)

The inflammatory cytokines tumor necrosis factor alpha (TNFα) and interferon gamma (IFNγ) stimulate production of the inflammatory mediators prostaglandin E₂ (PGEγ), prostacyclin (PGIγ), and nitric oxide (NO) in cultured lung epithelial cells. Pretreatment of these cells with the selective MEK1/2 (mitogen-activated protein kinase/extracellular signal-regulated kinase [ERK] kinase 1/2) inhibitor U0126 blocked ERK1/2 activation and inhibited cytokine-induced production of these inflammatory mediators. Primary bronchiolar epithelial Clara cells treated with TNFα and IFNγ also produced increased PGE₂, PGI₂, and NO, and PG and NO production was decreased by MEK inhibition. U0126 differentially affected cyclooxygenase (COX)-1, COX-2, and inducible NO synthase (iNOS) expression in cell lines, however, suggesting that MEK1/2 regulates prostanoid and NO production by means other than inducing their biosynthetic enzymes. Functionally, inhibition of MEK1/2 caused G1 cell cycle arrest and decreased cyclin D1 expression, but these effects were not related to decreased prostanoid production. These results indicate separate proinflammatory and proliferative roles for ERK1/2 in lung epithelial cells. During lung tumor formation in vivo, ERK1/2 phosphorylation increased as lung tumors progressed. Since tumor-derived cells were more sensitive than nontumorigenic cells to the antiproliferative effects of U0126, MEK1/2 inhibition may serve as an attractive chemotherapeutic target.

Alveolar macrophages and BDMCs undergo sequential biochemical changes during the chronic inflammatory response to chemically induced lung carcinogenesis in mice. Herein, we examine two chronic lung inflammation models-repeated exposure to BHT and infection with Mycobacterium tuberculosis-to establish whether similar macrophage phenotype changes occur in non-neoplastic pulmonary disease. Exposure to BHT or M. tuberculosis results in pulmonary inflammation characterized by an influx of macrophages, followed by systemic effects on the BM and other organs. In both models, pulmonary IFN-gamma and IL-4 production coincided with altered polarization of alveolar macrophages. Soon after BHT administration or M. tuberculosis infection, IFN-gamma content in BALF increased, and BAL macrophages became classically (M1) polarized, as characterized by increased expression of iNOS. As inflammation progressed in both models, the amount of BALF IFN-gamma content and BAL macrophage iNOS expression decreased, and BALF IL-4 content and macrophage arginase I expression rose, indicating alternative/M2 polarization. Macrophages present in M. tuberculosis-induced granulomas remained M1-polarized, implying that these two pulmonary macrophage populations, alveolar and granuloma-associated, are exposed to different activating cytokines. BDMCs from BHT-treated mice displayed polarization profiles similar to alveolar macrophages, but BDMCs in M. tuberculosis-infected mice did not become polarized. Thus, only alveolar macrophages in these two models of chronic lung disease exhibit a similar progression of polarization changes; polarization of BDMCs was specific to BHT-induced pulmonary inflammation, and polarization of granuloma macrophages was specific to the M. tuberculosis infection.

Tumor-associated macrophages (TAMs) encourage and coordinate neoplastic growth. In late stage human lung adenocarcinoma, TAMs exhibited mixed M1 (classical; argI(low)iNOS(high)) and M2 (alternative; argI(high)iNOS(low)) polarization based on arginine metabolism. In several murine cancer models including chemically and genetically-induced primary lung tumors, prostate tumors, colon xenografts, and lung metastases, TAMs expressed argI(high)iNOS(low) early during tumor formation; argI(low)iNOS(high) polarization also occurred during malignancy in some models. In a chemically-induced lung tumor model, macrophages expressed argI(high)iNOS(low) within one week after carcinogen treatment, followed by similar polarization of bone marrow-derived monocytes (BDMCs) a few days later. TAMs surrounding murine prostate tumors also expressed argI(high)iNOS(low) early during tumorigenesis, indicating that this polarization is not unique to neoplastic lungs. In a human colon cancer xenograft model, the primary tumor was surrounded by argI(high)iNOS(low)-expressing TAMs, and BDMCs also expressed argI(high)iNOS(low), but pulmonary macrophages adopted argI(high)iNOS(low) polarization only after tumors metastasized to the lungs. Persistence of tumors is required to maintain TAM polarization. Indeed, in both conditional mutant Kras- and FGF10-driven models of lung cancer, mice expressing the transgene develop lung tumors that regress rapidly when the transgene is silenced. Furthermore, pulmonary macrophages expressed argI(high)iNOS(low) on tumor induction, but then returned to argI(low) iNOS(low) (no polarization) after tumors regressed. Manipulating TAM function or depleting TAMs may provide novel therapeutic strategies for preventing and treating many types of cancer.

Background: Human lung cancer patients exhibit different KRAS mutations depending on smoking status. In a mouse model of human cancer, A/J and BALB/cBy mice treated with the tobacco carcinogen, 3-methylcholanthrene (MCA), followed by butylated hydroxytoluene (BHT)-elicited chronic inflammation develop a high multiplicity of lung tumors., Methods: DNA was isolated from MCA-induced lung tumors in A/J and BALB/cByJ mice. Kras codon 12 sequences from these tumors were compared to those in human lung tumors from smokers and never-smokers., Results: The distribution of Kras codon 12 mutations in MCA-induced A/J lung tumors is strikingly similar to those found in adenocarcinomas from human smokers. In contrast, codon 12 mutations in BALB/cBy mice contain predominantly G --> D mutations, which is the most common mutation in never smokers., Conclusions: A single lung carcinogen induces different tumor initiating mutations in different strains of mice. This may be useful for investigating the role of specific KRAS mutations in adenocarcinoma pathogenesis in smokers versus never smokers, identifying mechanisms that select for certain KRAS mutations and developing new drugs that specifically target cells with different KRAS mutations.

Cancer susceptibility results from interactions between sensitivity and resistance alleles. We employed murine chromosome substitution strains to study how resistance alleles affected sensitive alleles during chemically-induced lung carcinogenesis. The C57BL/6J-Chr#(A/J) strains, constructed by selectively breeding sensitive A/J and resistant C57BL/6J (B6) mice, each contain one pair of A/J chromosomes within an otherwise B6 genome. Pas1, the major locus responsible for this differential strain response to urethane carcinogenesis, resides on Chr 6, but C57BL/6J-Chr6(A/J) mice (hereafter CSS-6) developed few tumors following a single urethane injection, which demonstrates epistatic interactions with other B6 alleles. CSS6 mice developed dozens of lung tumors after chronic urethane exposure, however, indicating that these epistatic interactions could be overcome by repeated carcinogen administration. Unlike A/J, but similar to B6 mice, CSS6 mice were resistant to lung carcinogenesis induced by 3-methylcholanthrene (MCA). Tumor multiplicity increased if BHT administration followed urethane exposure, showing that a Chr 6 gene(s) regulates sensitivity to chemically-induced tumor promotion. Unlike A/J tumors (predominantly codon 61 A-->T transversions), Kras mutations in tumors induced by urethane in CSS-6 mice were similar to B6 tumors (codon 61 A-->G transitions). DNA repair genes not located on Chr 6 may determine the nature of Kras mutations. CSS-6 mice are a valuable resource for testing the ability of candidate genes to modulate lung carcinogenesis.

Background: Alternative (M2) macrophage activation is associated with tumor development in many tumor types, including those in the lung. Herein the biological consequences of forcing classical (M1) or alternative (M2) macrophage activation on lung tumor development are examined., Materials and Methods: Urethane-induced lung tumor multiplicity and size were compared in IFN-gamma(-/-) mice which lack M1 macrophage activation, IL-4Ralpha(-/-) mice which lack M2 macrophage activation, and wild-type BALB/cJ (background strain of the IFN-gamma(-/-) and IL-4Ralpha(-/-) mice) mice. Tumor-associated macrophage (TAM) and bone marrow-derived monocyte (BDMC) activation were each examined., Results: The TAMs and BDMCs in the IFN-gamma(-/-) mice exhibited M2 activation, and their lung tumors were significantly larger than those in the wild-type mice. In contrast, urethane-treated IL-4Ralpha(-/-) mice, whose TAMs and BDMCs were M1 activated, developed smaller tumors than the wild-type mice., Conclusion: Altered innate immunity can diminish or accelerate lung tumor progression in response to defective cytokine signaling.

The latency period for lung tumor progression offers a window of opportunity for therapeutic intervention. Herein, we studied the effect of oral silibinin (742 mg/kg body weight, 5 d/wk for 10 weeks) on the growth and progression of established lung adenocarcinomas in A/J mice. Silibinin strongly decreased both tumor number and tumor size, an antitumor effect that correlates with reduced antiangiogenic activity. Silibinin reduced microvessel size (50%, P < 0.01) with no change in the number of tumor microvessels and reduced (by 30%, P < 0.05) the formation of nestin-positive microvessels in tumors. Analysis of several proteins involved in new blood vessel formation showed that silibinin decreased the tumor expression of interleukin-13 (47%) and tumor necrosis factor-alpha (47%), and increased tissue inhibitor of metalloproteinase-1 (2-fold) and tissue inhibitor of metalloproteinase-2 (7-fold) expression, without significant changes in vascular endothelial growth factor levels. Hypoxia- inducible factor-1 alpha expression and nuclear localization were also decreased by silibinin treatment. Cytokines secreted by tumor cells and tumor-associated macrophages regulate angiogenesis by activating nuclear factor-kappaB (NF-kappaB) and signal transducers and activators of transcription (STAT). Silibinin decreased the phosphorylation of p65NF-kappaB (ser276, 38%; P < 0.01) and STAT-3 (ser727, 16%; P < 0.01) in tumor cells and decreased the lung macrophage population. Angiopoietin-2 (Ang-2) and Ang-receptor tyrosine kinase (Tie-2) expression were increased by silibinin. Therapeutic efficacy of silibinin in lung tumor growth inhibition and regression by antiangiogenic mechanisms seem to be mediated by decreased tumor-associated macrophages and cytokines, inhibition of hypoxia-inducible factor-1 alpha, NF-kappaB, and STAT-3 activation, and up-regulation of the angiogenic inhibitors, Ang-2 and Tie-2.

Purpose: Erlotinib, a small molecule inhibitor of the tyrosine kinase (TK) domain of epidermal growth factor receptor (EGFR), increases survival of advanced non-small cell lung cancer patients who failed standard chemotherapy (Phase III study). We evaluated whether erlotinib is also effective at an early stage of primary lung tumorigenesis in a carcinogen-induced lung tumor model in mice., Methods: Sixteen weeks after carcinogen (urethane) injection, when small self-contained adenomas are evident, male and female A/J mice were treated IP with 10 mg/kg erlotinib or Captisol vehicle daily over 3.5 weeks (15 mice per group). The efficacy, metabolism and mechanism of action of erlotinib were evaluated., Results: Erlotinib reduced tumor burden in males by twofold compared to vehicle (12.7 +/- 1.2 vs 26.2 +/- 2.5 mg, respectively; p < 0.0001), while tumor burden in erlotinib-treated females slightly increased compared to vehicle by 21% (15.1 +/- 1.2 vs 11.9 +/- 0.9 mg, respectively; p < 0.05). Tumor multiplicity, in contrast, was unaffected by erlotinib. The levels of erlotinib that accumulated in plasma, lung tumor tissue and adjacent uninvolved (UI) lung were comparable in males and females. Males, however, accumulated more OSI-420, an active and pharmacologically equipotent metabolite of erlotinib, than females in plasma, lung tumors, and UI lung. In both genders, 80% of tumors contained Kras mutations at codon 61, but no EGFR mutations were detected. The cellular distribution and concentration of EGFR were also similar between genders. In control mice, however, phosphorylated EGFR (pEGFR) levels were nearly 2.5-fold higher in males compared to females in UI lungs and sevenfold higher in lung tumors. Further, erlotinib decreased the contents of pEGFR in UI lungs and lung tumors, particularly in males., Conclusions: Adenomas from male mice in this early lung cancer model are responsive to erlotinib treatment, possibly because of a greater dependence of male tumor growth on the EGFR pathway compared to females. Importantly, these results indicate that small lung adenomas from male mice that utilize EGFR signaling but also harbor Kras mutations shrink in response to erlotinib, suggesting that erlotinib may be beneficial for some patients very early during lung cancer progression.

Our previous proteomic investigation of lung neoplasia in vitro demonstrated a high concentration of the lung cancer biomarker and splicing factor, hnRNP A2/B1, in the transformed mouse lung epithelial cell line, E9. Since changes in pre-mRNA splicing profoundly affect neoplastic progression, we examined hnRNP A2/B1 expression in chemically induced primary mouse lung tumors, an in vivo model of pulmonary adencocarcinoma. Tumor hnRNP A2/B1 content and spatial distribution assessed by immunohistochemistry varied with stage of progression, genetic background, and whether tumors were induced by a single agent (urethane) or by 2-stage initiation/promotion (3-methylcholanthrene/butylated hydroxytoluene) carcinogenesis. To address mechanisms governing hnRNP A2/B1 expression changes, we utilized in vitro models. hnRNP A2/B1 protein was overexpressed in E9, the spontaneous tranformant of immortalized but non-neoplastic E10 cells, but expression was not strictly a function of enhanced proliferative rate in neoplastic cells. Elevated mRNA content was positively associated with cell division in both E10 and E9, but hnRNP A2/B1 protein levels decreased in proliferating E10 cells. The increased mRNA reflected enhanced mRNA stability, as shown by measuring time-dependent mRNA decay after inhibiting transcription. Dysregulation of hnRNP A2/B1 expression during lung neoplasia in vivo thus depends on complex gene-environmental interactions that affect cell type-specific changes in mRNA processing and, most probably, the rates of translation and/or protein degradation., (Copyright (c) 2007 Wiley-Liss, Inc.)

Inbred mice vary in their susceptibility to develop macroscopic, chemically-induced, pulmonary neoplasias. It is not known, however, whether microscopic lesions appear in resistant strains but do not grow or if no early lesions arise at all. We show herein that resistant C57BL/6J (B6) and intermediately resistant BALB/cByJ (BALB) mice form very few urethane-induced early microadenomas (i.e. adenomas larger than hyperplasic foci, but detectable only by light microscopy). Additionally, while all urethane-induced microadenomas in sensitive A/J mice gave rise to adenomas, most microscopic tumors induced in BALB mice by 2-stage, 3-methylcholanthrene/butylated hydroxytoluene carcinogenesis spontaneously regressed. The formation of microscopic lesions is thus genetically dependent, but whether they continue to grow or regress depends on how they were induced.

Background: Manipulating prostaglandin (PG) production modulates tumor development. Elevated PGI2 production prevents murine lung cancer, while decreasing PGE2 content protects against colon cancer. PGE2 receptor subtype 2 (EP2) -deficient mice were hypothesized to be resistant to lung tumorigenesis., Materials and Methods: EP2 null BALB/c mice and their wild-type littermates were exposed to an initiation-promotion carcinogenesis protocol and lung tumorigenesis was examined. Chronic lung inflammation was induced to determine whether EP2 ablation influenced inflammatory cell infiltration., Results: Tumor multiplicity in EP2 null mice was 34% lower than in their wild-type littermates (21.9+/-3.0 vs. 14.5+/-2.9 tumors/mouse, p<0.001). The lung tumor burden, an indicator of growth rate, also declined (57%, p<0.05). All the mice exhibited similar inflammatory cell infiltration., Conclusion: PGE2, acting through EP2, enhanced lung tumorigenesis through a mechanism that may be distinct from its proinflammatory activity. Thus, EP2 is a potential target for novel chemoprevention strategies.

Background: Silibinin, a flavanone from milk thistle, inhibits the growth of tumors in several rodent models. We examined the effects of dietary silibinin on the growth, progression, and angiogenesis of urethane-induced lung tumors in mice., Methods: A/J mice (15 per group) were injected with urethane (1 mg/g body weight) or saline alone and fed normal diets for 2 weeks, after which they were fed diets containing different doses of silibinin (0%-1% [wt/wt] silibinin) for 18 or 27 weeks. Immunohistochemistry and Western blot analysis were used to examine angiogenesis and enzymatic markers of inflammation, proliferation, and apoptosis. All statistical tests were two-sided., Results: Urethane-injected mice exposed to silibinin had statistically significantly lower lung tumor multiplicities than urethane-injected mice fed the control diet lacking silibinin (i.e., control mice). Mice that received urethane and 1% (wt/wt) dietary silibinin for 18 weeks had 93% fewer large (i.e., 1.5-2.5-mm-diameter) lung tumors than control mice (mean number of tumors/mouse: 27 in the urethane group versus 2 in the urethane + 1% silibinin group, difference = 25 tumors/mouse, 95% confidence interval [CI] = 13 to 37 tumors/mouse, P = .005). Lung tumors of silibinin-fed mice had 41%-74% fewer cells positive for the cell proliferation markers proliferating cell nuclear antigen and cyclin D1 than lung tumors of control mice. Tumor microvessel density was reduced by up to 89% with silibinin treatment (e.g., 56 microvessels/400x field in tumors from control mice versus 6 microvessels/400x field in tumors from urethane + 1% silibinin-treated mice [difference = 50 microvessels/400x field, 95% CI = 46 to 54 microvessels/400x field; P<.001]). Silibinin decreased lung tumor expression of vascular endothelial growth factor (VEGF) and of inducible nitric oxide synthase and cyclooxygenase-2, two enzymes that promote lung tumor growth and progression by inducing VEGF expression., Conclusions: Silibinin inhibits lung tumor angiogenesis in an animal model and merits investigation as a chemopreventive agent for suppressing lung cancer progression.

Administration of butylated hydroxytoluene (BHT) to mice causes lung damage characterized by the death of alveolar type I pneumocytes and the proliferation and subsequent differentiation of type II cells to replace them. Herein, we demonstrate this injury elicits an inflammatory response marked by chemokine secretion, alveolar macrophage recruitment, and elevated expression of enzymes in the eicosanoid pathway. Cytosolic phospholipase A(2) (cPLA(2)) catalyzes release of arachidonic acid from membrane phospholipids to initiate the synthesis of prostaglandins and other inflammatory mediators. A role for cPLA(2) in this response was examined by determining cPLA(2) expression and enzymatic activity in distal respiratory epithelia and macrophages and by assessing the consequences of cPLA(2) genetic ablation. BHT-induced lung inflammation, particularly monocyte infiltration, was depressed in cPLA(2) null mice. Monocyte chemotactic protein-1 (MCP-1) content in bronchoalveolar lavage fluid increases after BHT treatment but before monocyte influx, suggesting a causative role. Bronchiolar Clara cells isolated from cPLA(2) null mice secrete less MCP-1 than Clara cells from wild-type mice, consistent with the hypothesis that cPLA(2) is required to secrete sufficient MCP-1 to induce an inflammatory monocytic response.

Studies using transgenic and knockout mice have demonstrated that particular cytokines influence lung tumor growth and identified prostaglandin E2 (PGE2), prostacyclin (PGI2) and nitric oxide (NO) as critical mediators of this process. PGE2 and NO were pro-tumorigenic while PGI2 was antitumorigenic. We describe herein an in vitro experimental approach to examine interactions among cytokines, prostaglandins (PGs) and NO. PGE2, PGI2, and NO levels were assayed in culture media from non-tumorigenic mouse lung epithelial cell lines, their spontaneous transformants and mouse lung tumor-derived cell lines, before or after exposure to the cytokines TNFalpha, IFNgamma and IL1beta, alone and in combination. More PGE2 than PGI2 was produced by neoplastic cells, while the opposite was observed in non-tumorigenic lines. Cytokine exposure magnified the extent of these differential concentrations. The PGE2 to PGI2 ratio was also greater in chemically-induced mouse lung tumors than in adjacent tissue or control lungs, supporting the physiological relevance of this in vitro model. Expression of PG biosynthetic enzymes in these cell lines correlated with production of the corresponding PGs. Cytokine treatment enhanced NO production by inducing the inflammation-associated biosynthetic enzyme, inducible NO synthase (iNOS), but this did not correlate with the neoplastic status of cells. Inhibition of iNOS or cyclooxygenase 2 activity using aminoguanidine or NS-398 respectively, demonstrated that NO did not affect PG production nor did PGs influence NO production. Since lack of iNOS inhibits mouse lung tumor formation, we propose that this is independent of any modulation of PG synthesis in epithelial cells. The similar normal/neoplastic trends in PGE2 to PGI2 ratios both in vitro and in vivo, together with an amplification of this difference upon cytokine exposure, are consistent with the hypothesis that cytokines released during inflammation exacerbate differences in the behavior of neoplastic and normal lung cells.

Pre-mRNA processing is an important mechanism for globally modifying cellular protein composition during tumorigenesis. To understand this process during lung cancer, expression of two key pre-mRNA alternative splicing factors was compared in a mouse model of early lung carcinogenesis and during regenerative growth following reversible lung injury. Heterogeneous nuclear ribonucleoprotein (hnRNP) A1 and alternative splicing factor/splicing factor 2 (ASF/SF2) act antagonistically to modulate splice site selection. Both hnRNP A1 and ASF/SF2 contents rose in adenomas and during injury-induced hyperplasia compared to control lungs, as measured by immunoblotting. While both proteins increased similarly during compensatory hyperplasia, hnRNP A1 increased to a much greater extent than ASF/SF2 in tumors, resulting in a 6-fold increase of the hnRNP A1 to ASF/SF2 ratio. Immunohistochemical analysis showed that hnRNP A1 localized exclusively within tumor nuclei, while ASF/SF2 appeared in cytoplasm and/or nuclei, depending on the growth pattern of the tumor cells. We also demonstrated cancer-associated changes in the pre-mRNA alternative splicing of CD44, a membrane glycoprotein involved in cell-cell and cell-extracellular matrix interactions. hnRNP A1 and ASF/SF2 expression is thus differentially altered in neoplastic lung cells by mechanisms that do not strictly arise from increased cell division. These changes are influenced by tumor histology and may be associated with production of variant CD44 mRNA isoforms., ((c) 2004 Wiley-Liss, Inc.)

Activating mutations in Ras oncoproteins represent attractive targets for cancer immunotherapy, but few vectors capable of generating immune responses required for tumor killing without vector neutralization have been described. Whole recombinant yeast heterologously expressing mammalian mutant Ras proteins were used to immunize mice in a carcinogen-induced lung tumor model. Therapeutic immunization with the whole recombinant yeast caused complete regression of established Ras mutation-bearing lung tumors in a dose-dependent, antigen-specific manner. In combination with the genomic sequencing of tumors in patients, the yeast-based immunotherapeutic approach could be applied to treat Ras mutation-bearing human cancers.

Epidemiological investigations suggest that chronic lung inflammation increases lung cancer risk. Pharmacologic and genetic evidence in mouse models indicates that lipid mediators released during pulmonary inflammation enhance lung tumor formation. Cytosolic phospholipase A2 (cPLA2) catalyzes arachidonic acid (AA) release from membrane phospholipids. AA can then lead to the synthesis of several classes of lipid mediators, including prostaglandin (PG) biosynthesis through the cyclooxygenase (COX) pathway. We investigated a role for cPLA2 in mouse lung tumorigenesis by using mice genetically deficient in cPLA2. After multiple urethane injections into cPLA2 null mice and wild-type littermates, the number of lung tumors was determined. cPLA2 null mice developed 43% fewer tumors (from 16 +/- 2 to 9 +/- 2 tumors/mouse; P < 0.05) than wild-type littermates. cPLA2, COX-1, COX-2 and microsomal prostaglandin E2 synthase (mPGES), examined by immunohistochemistry, are present in alveolar and bronchiolar epithelia and in alveolar macrophages in lungs from naive mice and tumor-bearing mice. Tumors express higher levels of each of these four enzymes than control lungs, as determined by immunoblotting. No differences were detected in the contents of COX-1, COX-2 and mPGES between wild-type and cPLA2 null mice. Although the steady-state levels of prostaglandin E2 and prostaglandin I2 in lung tissue extracts prepared from wild-type or cPLA2 (-/-) mice were not significantly different, both prostaglandins markedly increased in tumors from wild-type mice, an increase that was significantly blunted in tumors from cPLA2 (-/-) mice. These results demonstrate a role for cPLA2 in mouse lung tumorigenesis that may be mediated by decreased prostaglandin synthesis.

Most murine lung tumors are composed of differentiated epithelial cells. We have reported previously that surfactant protein (SP)-D is expressed in urethane-induced tumors. Serum levels of SP-D are increased in patients with interstitial lung disease and acute respiratory distress syndrome and in rats with acute lung injury but have not been measured in mice. In this study, we sought to determine whether SP-D could be detected in murine serum and discovered that it was increased in mice bearing lung tumors. Serum SP-D concentration was 5.0 +/- 0.2 ng/ml in normal C57BL/6 mice, essentially absent in SP-D nulls, and 63.6 +/- 9.0 ng/ml in SP-D-overexpressing mice. SP-D in serum was verified by immunoblotting. Serum SP-D was increased in mice bearing tumors induced by three different protocols, and the SP-D level correlated with tumor volume. However, in mice with a single adenoma or a few adenomas, SP-D levels were usually within the normal range. SP-D was expressed by the tumor cells, and there was also a field effect whereby type II cells near the tumor expressed more SP-D than type II cells in the remainder of the lung. Serum SP-D was also increased by lung inflammation. In airway inflammation induced by aerosolized ovalbumin in sensitized BALB/c mice, the serum levels were elevated after challenge. In conclusion, serum SP-D concentration is increased in mice bearing lung tumors and generally reflects the tumor burden but is also elevated during lung inflammation.

Murine pulmonary adenomas progress to malignancy with many similarities to human pulmonary adenocarcinoma, the most common form of lung cancer. Inbred mice vary in their susceptibility to lung tumor development, and induced genetic modifications are a powerful tool for understanding this susceptibility. Many transgenic and null mutations relevant to lung cancer pathogenesis were derived on the highly resistant C57BL/B6 (B6) background. Since the inability to reliably induce lung tumors in B6 mice limits these studies, we systematically examined several carcinogenesis protocols in B6 mice. Ten weekly ethyl carbamate (EC) doses caused a nearly 100% lung tumor incidence with a tumor multiplicity >2; multiple EC dosing is thus an alternative to the time-consuming transfer of transgenes and null mutations to susceptible backgrounds.

A model system to investigate the promotion phase of pulmonary carcinogenesis involves chronic exposure of carcinogen-initiated mice to the food additive, butylated hydroxytoluene (BHT). Previous studies strongly suggested that this activity is due to the cytochrome p450-catalyzed formation of quinone methides 2,6-di-tert-butyl-4-methylenecyclohexa-2,5-dienone (BHT-QM) and 6-tert-butyl-2-(1',1'-dimethyl-2'-hydroxy)ethyl-4-methylenecyclohexa-2,5-dienone (BHTOH-QM). The effects of these electrophiles on non-tumorigenic C10 and E10 epithelial cell lines derived from a normal mouse lung explant were compared with effects on their corresponding neoplastic siblings, the A5 and E9 spontaneous transformants, respectively. The tumorigenic cells were more resistant to cell killing, with LC(50) values of 165-180 microM for BHT-QM and 12-22 microM for BHTOH-QM, versus LC(50) values in the non-tumorigenic cells of 105-118 microM and 5.0-6.0 microM, respectively. Constitutive glutathione (GSH) concentrations were 12-20 nmol/10(6) cells, and BHT-QM toxicity was enhanced >2-fold by depleting GSH with buthionine sulfoximine (BSO). Formation of the GSH conjugate of BHT-QM accounted for a substantial fraction of the cellular GSH lost by quinone methide exposure. Enhanced lipid peroxidation and superoxide formation occurred in all cell lines treated with BHT-QM, but both tumorigenic lines contained higher levels of GSH S-transferase and superoxide dismutase (SOD) activities. These data suggest the possibility that BHT-derived quinone methides may exert their promoting effects by inducing oxidative stress; such stress is better tolerated by tumorigenic cells, which have higher levels of antioxidant enzymes. Normal cells are destroyed more readily which allows neoplastic cells to expand their proliferation.

Inducible nitric oxide synthase (iNOS) content is elevated in human lung adenocarcinomas, and lung cancer patients exhale more nitric oxide (NO) than healthy individuals. The mechanism of this association of chronically elevated NO with tumorigenesis has not been defined. We investigated the role of iNOS in murine lung tumorigenesis, a model of human lung adenocarcinoma, using wild-type (+/+) and iNOS (-/-) mice. Genetic disruption of iNOS decreased urethane-induced lung tumor multiplicity by 80% (P < 0.0001). iNOS protein was expressed in lung tumors growing in wild-type mice and bronchiolar Clara cells isolated from normal mouse lungs, but was undetectable in whole lung extracts by immunoblotting. Because NO regulates vascular endothelial growth factor (VEGF) expression in other systems, we examined the effect of iNOS deficiency on VEGF protein concentration in mouse lung tumors. VEGF concentration was 54% lower in lung tumors isolated from iNOS (-/-) mice versus controls, implying that NO modulates angiogenesis in these tumors. Lung tumors also have elevated levels of cyclooxygenase (COX) -1 and COX-2 contents relative to normal lungs, but iNOS deficiency did not change COX expression in the tumors. Chronic inflammation predisposes mice to lung tumorigenesis; accordingly, we examined whether butylated hydroxytoluene-induced chronic lung inflammation was influenced by iNOS deficiency. Butylated hydroxytoluene-induced alveolar macrophage infiltration was unaffected by iNOS (-/-) status, suggesting that although NO is a critical mediator of mouse lung tumorigenesis, it is not essential in this model of lung inflammation. The substantial (80%) reduction in lung tumor multiplicity in iNOS (-/-) mice strongly supports examining iNOS-specific inhibitors as potential lung cancer chemopreventive agents.

Cyclooxygenase (COX) enzyme expression is elevated in human and rodent lung tumors, and non-steroidal anti-inflammatory drugs (NSAIDs) such as indomethacin reduce lung tumor formation in mice. These observations, along with the well-characterized protection that NSAID treatment engenders for colon cancer, have prompted clinical trials testing whether celecoxib, a COX-2-specific inhibitor, can prevent lung cancer development in populations at high risk. Protection by celecoxib in murine models of pulmonary inflammation and lung tumorigenesis has not yet been evaluated, however, and we now report such studies. Chronic administration of butylated hydroxytoluene (BHT) to mice stimulates pulmonary inflammation characterized by vascular leakage and macrophage infiltration into the air spaces, increased PGE2 production, and translocation of 5-lipoxygenase (5-LO) from the cytosol to the particulate fraction. Dietary celecoxib limited macrophage infiltration, abrogated PGE2 production and reduced particulate 5-LO content. Celecoxib and aspirin were ineffective at preventing lung tumorigenesis in a two-stage carcinogenesis protocol in which 3-methylcholanthrene administration is followed by chronic BHT. Celecoxib also did not reduce the multiplicity of lung tumors after induction by urethane; lung tumors in celecoxib-treated mice were larger than those in mice that did not receive celecoxib. Tumors induced in celecoxib-fed mice contained 60% less PGE2 than tumors in mice fed control diets, so reducing lung PGE2 levels was insufficient to prevent lung tumor formation. As the production of eicosanoids in addition to PGE2 is also inhibited by celecoxib, and as celecoxib has COX-independent interactions, its effects on tumor formation may vary in different organ systems.

Acute pulmonary toxicity and tumor promotion by the food additive 2,6-di-tert-butyl-4-methylphenol (BHT) in mice are well documented. These effects have been attributed to either of two quinone methides, 2,6-di-tert-butyl-4-methylenecyclohexa-2,5-dienone (BHT-QM) formed through direct oxidation of BHT by pulmonary cytochrome P450 or a quinone methide formed by hydroxylation of a tert-butyl group of BHT (to form BHTOH) followed by oxidation of this metabolite to BHTOH-QM. BHTOH-QM is a more reactive electrophile compared to BHT-QM due to intramolecular interactions of the side-chain hydroxyl with the carbonyl oxygen. To further examine this bioactivation pathway, an analogue of BHTOH was prepared, 2-tert-butyl-6-(1'-hydroxy-1'-methyl)ethyl-4-methylphenol (BPPOH), that is structurally very similar to BHTOH but forms a quinone methide (BPPOH-QM) capable of more efficient intramolecular hydrogen bonding and, therefore, higher electrophilicity than BHTOH-QM. BPPOH-QM was synthesized and its reactivity with water, methanol, and glutathione determined to be >10-fold higher than that of BHTOH-QM. The conversions of BPPOH and BHTOH to quinone methides in lung microsomes from male BALB/cByJ mice were quantitatively similar, but in vivo the former was pneumotoxic at one-half of the dose required for the latter and one-eighth of the dose required for BHT, as determined by increased lung weight:body weight ratios following a single i.p. injection. Similar differences were found in the doses of BHT, BHTOH, or BPPOH required for tumor promotion after a single initiating dose of 3-methylcholanthrene followed by three weekly injections of the phenol. The downregulaton of calpain II, previously shown to accompany lung tumor promotion by BHT and BHTOH, also occurred with BPPOH. The correlation between biologic activities of these phenols and the reactivities of their corresponding quinone methides provides additional support for the role of BHTOH-QM as the principal metabolite responsible for the effects of BHT on mouse lung.

An inflammatory response accompanies the reversible pneumotoxicity caused by butylated hydroxytoluene (BHT) administration to mice. Lung tumor formation is promoted by BHT administration following an initiating agent in BALB/cByJ mice, but not in CXB4 mice. To assess the contribution of inflammation to this differential susceptibility, we quantitatively characterized inflammation after one 150 mg/kg body weight, followed by three weekly 200 mg/kg ip injections of BHT into male mice of both strains. This examination included inflammatory cell infiltrate and protein contents in bronchoalveolar lavage (BAL) fluid, cyclooxygenase (COX)-1 and COX-2 expression in lung extracts, and PGE(2) and PGI(2) production by isolated bronchiolar Clara cells. BAL macrophage and lymphocyte numbers increased in BALB mice (P<0.0007 and 0.02, respectively), as did BAL protein content (P<0.05), COX-1 and COX-2 expression (P<0.05 for each), and PGI(2) production (P<0.05); conversely, these indices were not perturbed by BHT in CXB4 mice. BALB mice fed aspirin (400 mg/kg of chow) for two weeks prior to BHT treatment had reduced inflammatory cell infiltration. Our results support a hypothesis that resistance to BHT-induced inflammation in CXB4 mice accounts, at least in part, for the lack of effect of BHT on lung tumor multiplicity in this strain.

Elevations in cyclin D1 content increase the phosphorylation status of retinoblastoma (Rb) protein to encourage cell cycle transit. We sought to determine if cyclin D1 content could be used as an index of cell proliferation in mouse lung epithelia following growth manipulations in vitro and in vivo. Rb protein concentration was high in 82-132 and LM2, two fast-growing neoplastic mouse lung epithelial cell lines. The hyperphosphorylated form of Rb predominated in these two cell lines, while Rb in slower-growing cell lines was predominantly hypophosphorylated. Consistent with this, more cyclin D1 protein was expressed in the fast-growing cell lines than in slower-growing cells. We therefore tested whether cyclin D1 content varied with growth status. The amount of cyclin D1 decreased upon serum removal coincident with growth inhibition and then increased upon serum re-addition which stimulated resumption of proliferation. This correlation between cyclin D1 content and growth status also occurred in vivo. Cyclin D1 content increased when lungs underwent compensatory hyperplasia following damage caused by butylated hydroxytoluene administration to mice and in lung tumor extracts as compared with extracts prepared from uninvolved tissue or control lungs. We conclude that elevated cyclin D1 levels account, at least in part, for the hyperphosphorylation of Rb in neoplastic lung cells, and are associated with enhanced lung growth in vitro and in vivo.

Cell shape is mediated in part by the actin cytoskeleton and the actin-binding protein vinculin. These proteins in turn are regulated by protein phosphorylation. We assessed the contribution of cAMP-dependent protein kinase A isozyme I (PKA I) to lung epithelial morphology using the E10/E9 sibling cell lines. PKA I concentration is high in flattened, nontumorigenic E10 cells but low in their round E9 transformants. PKA I activity was lowered in E10 cells by stable transfection with a dominant negative RIalpha mutant of the PKA I regulatory subunit and was raised in E9 cells by stable transfection with a wild-type Calpha catalytic subunit construct. Reciprocal changes in morphology ensued. E10 cells became rounder and grew in colonies, their actin microfilaments were disrupted, and vinculin localization at cell-cell junctions was diminished. The converse occurred in E9 cells on elevating their PKA I content. Demonstration that PKA I is responsible for the dichotomy in these cellular behaviors suggests that manipulating PKA I concentrations in lung cancer would provide useful adjuvant therapy.

Chronic pulmonary inflammatory diseases predispose towards lung cancer by unknown mechanisms. Butylated hydroxytoluene (BHT) administration to mice causes lung injury and a subsequent inflammatory response, and when administered chronically to certain inbred strains following carcinogen treatment, increases lung tumor multiplicity. We hypothesize that inflammation promotes lung tumor growth in this model system and have begun to examine this hypothesis by assessing inflammatory parameters in inbred strains that vary in their susceptibility to promotion. Positive correlations were found between susceptibilities to tumor promotion and BHT induction of alveolar macrophage and lymphocyte infiltration into alveolar airspaces, and increased vascular permeability (P < .03, P < .04, and P < .005, respectively). The amounts of pulmonary cyclooxygenase (COX)-1 and COX-2 did not strongly correlate with promotion. Because persistent elevation of macrophage content is the hallmark of a chronic inflammatory response, the alveolar macrophage population was depleted by adding chlorine to the drinking water prior to carcinogenesis. This treatment reduced lung tumor multiplicity following 2-stage carcinogenesis (P < .05). These correlations between inflammatory and tumorigenic responses to BHT, along with decreased tumorigenesis after macrophage depletion, are consistent with a role of inflammation in promotion. Inflammatory mediators may provide targets for early diagnosis and chemoprevention.

Gap junctions provide direct pathways for the exchange of molecules and ions between neighboring cells, a process known as gap junctional intercellular communication (GJIC). This GJIC is important for homeostasis and regulation of mitosis, differentiation, and apoptosis. Gap junctions are present in lung airway and alveolar epithelial cells and, in addition to the above roles, might coordinate ciliary beating and surfactant secretion. GJIC is decreased in human and mouse lung carcinoma cells because of reduced expression of the gap junction protein, connexin43 (Cx43), and defects in signal transduction pathways that mediate Cx43 function. This reduced GJIC is important in the behavior of lung carcinoma cells because forced expression of Cx43 in lung carcinoma cells inhibits their growth and tumorigenicity. In this report, we summarize our studies on the role of GJIC in lung neoplasia.

Chronic treatment of BALB and GRS mice with BHT (2,6-di-tert-butyl-4-methylphenol) following a single urethane injection increases lung tumor multiplicity, but this does not occur in CXB4 mice. Previous data suggest that promotion requires the conversion of BHT to a tert-butyl-hydroxylated metabolite (BHTOH) in lung and the subsequent oxidation of this species to an electrophilic quinone methide. To obtain additional evidence for the importance of quinone methide formation, structural analogs that form less reactive quinone methides were tested and found to lack promoting activity in BHT-responsive mice. The possibility that promotion-unresponsive strains are unable to form BHTOH was tested by substituting this compound for BHT in the promotion protocol using CXB4 mice. No promotion occurred, and in-vitro work demonstrated that CXB4 mice are, in fact, capable of producing BHTOH and its quinone methide, albeit in smaller quantities. Incubations with BALB lung microsomes and radiolabeled substrates confirmed that more covalent binding to protein occurs with BHTOH than with BHT and, in addition, BHTOH quinone methide is considerably more toxic to mouse lung epithelial cells than BHT quinone methide. These data are consistent with the hypothesis that a two-step oxidation process, i.e. hydroxylation and quinone methide formation, is required for the promotion of mouse lung tumors by BHT.