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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. Hamp;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.

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.

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.

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 PPARγ, 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.

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 G2–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.

Prostacyclin (prostaglandin I2, PGI2) overproduction in FVB/N mice prevents the formation of carcinogen and tobacco smoke–induced adenomas, and administration of the oral prostacyclin analogue iloprost to wild-type mice also prevented carcinogen-induced mouse lung adenoma formation. Former smokers taking oral iloprost showed improved bronchial dysplasia histology compared with placebo. Next-generation oral prostacyclin analogues, like treprostinil, were developed for the treatment of pulmonary arterial hypertension (PAH). On the basis of our prior studies with iloprost, we performed preclinical studies examining the ability of treprostinil to chemoprevent urethane-induced murine lung adenocarcinoma. We determined the MTD in chow (prior studies had delivered treprostinil by gavage), and this dose produced serum levels in the experimental animals similar to those found in PAH patients treated with treprostinil. We then examined the chemopreventive efficacy of treprostinil exposure initiated both before (1 week) and after (6 weeks) urethane exposure to better model chemoprevention studies conducted in former smokers. Neither of these dosing strategies prevented murine lung cancer; however, we did detect changes in pulmonary inflammatory cell infiltrate and expression of CXCR4 (a chemokine receptor previously shown to increase in response to treprostinil exposure) in tumor-bearing, treprostinil-treated animals, indicating that the drug was bioavailable. One potential explanation stems from iloprost and treprostinil differentially activating cell surface prostaglandin receptors and intracellular peroxisome proliferator-activated receptors. When murine lung tumor cells were treated with treprostinil, their proliferation rate increased; in contrast, iloprost had no effect on proliferation. Future investigations comparing these two agents will provide insight into iloprost's chemopreventive mechanisms. Cancer Prev Res; 10(11); 671–9. ©2017 AACR.

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.

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. © 2011 Wiley Periodicals, Inc.

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-α (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-1α 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-κB (NF-κB) and signal transducers and activators of transcription (STAT). Silibinin decreased the phosphorylation of p65NF-κB (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-1α, NF-κB, and STAT-3 activation, and up-regulation of the angiogenic inhibitors, Ang-2 and Tie-2.

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.

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. © 2004 Wiley-Liss, Inc.

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.

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.

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 RIα mutant of the PKA I regulatory subunit and was raised in E9 cells by stable transfection with a wild-type Cα 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.

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.

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, AIN76A-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 K 3 tumors/ mouse (mean 6 SEM, control diet) to 12.3 K 3 (250), 5.3 K 1 (500) and 2.1 K 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.

Several dozen lung epithelial cell lines have been established in culture over the past 20 years from normal lung explants and their spontaneous transformants, and from lung tumors that arose spontaneously or were induced with chemicals, viruses, or oncogenic transgenes. To provide information from which to choose appropriate lines for investigating problems in lung cell biology and pulmonary neoplasia, this review describes the origins of these lines and some of their characteristics. These include growth, morphology, tumorigenicity, ability to metastasize, xenobiotic metabolism, mutational status, signal transducing activities, cytogenetics, ability to form domes, and electric conductance. In addition to collecting this information in a single place for the first time, we describe previously unpublished apoptosis features of some of these lines. An increasing number of investigations are beginning to use these lines and this review contains references into 1997.

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-Nos2tm1Lau (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. Clin Cancer Res; 17(4); 1–9. ©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.

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.

One area of intensive investigation is to understand complex cellular and signaling interactions in the tumor microenvironment. Using a novel, although straightforward, microarray approach, we defined a gene expression signature from the lung tumor microenvironment in the murine A/J-urethane model of human lung adenocarcinoma. The tumor microenvironment is reflected by the composition of the cell types present and alterations in mRNA levels, resulting in a “Field Effect” around the tumor. The genes composing the Field Effect expression signature include proteases and their inhibitors, inflammation markers, and immune signaling molecules. By several criteria, the Field Effect expression signature can be attributed to the macrophage lineage, suggesting a qualitative change in the expression pattern of tumor-associated macrophages (TAM) observed in lung tumors. The protein expression levels for a number of Field Effect genes were verified by Western blot analysis of lung homogenates, and for their expression in macrophages and parenchymal cells outside of the tumors by immunohistochemistry. In addition, the Field Effect expression signature was used to classify bronchoalveolar lavage (BAL) cells from tumor-bearing or age-matched control mice. Using a variety of statistical measures, the Field Effect expression signature correctly classified the BAL cells >94% of the time. Finally, the protein levels for several Field Effect genes were higher in cell-free BAL fluid, indicating they may be secreted by the TAMs. This work suggests that TAMs generate a unique gene expression signature within the tumor microenvironment, and this signature could potentially be used for identifying lung cancer from BAL cells and/or fluid. [Cancer Res 2008;68(1):34–43]

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.

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.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.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, p0.001). The lung tumor burden, an indicator of growth rate, also declined (57%, p0.05). All the mice exhibited similar inflammatory cell infiltration.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 fl avanone 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 infl ammation, proliferation, and apoptosis. All statistical tests were two-sided. Results: Urethane-injected mice exposed to silibinin had statistically signifi cantly 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% confi dence interval [CI] = 13 to 37 tumors/mouse, P = .005). Lung tumors of silibininfed 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/400× fi eld in tumors from control mice versus 6 microvessels/400× fi eld in tumors from urethane + 1% silibinin-treated mice [difference = 50 microvessels/400× fi eld, 95% CI = 46 to 54 microvessels/400× fi eld; P

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.

Human adenocarcinoma (AC) is the most frequently diagnosed human lung cancer, and its absolute incidence is increasing dramatically. Compared to human lung AC, the A/J mouse-urethane model exhibits similar histological appearance and molecular changes. We examined the gene expression profiles of human and murine lung tissues (normal or AC) and compared the two species' datasets after aligning approximately 7500 orthologous genes. A list of 409 gene classifiers (P value0.0001), common to both species (joint classifiers), showed significant, positive correlation in expression levels between the two species. A number of previously reported expression changes were recapitulated in both species, such as changes in glycolytic enzymes and cell-cycle proteins. Unexpectedly, joint classifiers in angiogenesis were uniformly down-regulated in tumor tissues. The eicosanoid pathway enzymes prostacyclin synthase (PGIS) and inducible prostaglandin E(2) synthase (PGES) were joint classifiers that showed opposite effects in lung AC (PGIS down-regulated; PGES up-regulated). Finally, tissue microarrays identified the same protein expression pattern for PGIS and PGES in 108 different non-small cell lung cancer biopsies, and the detection of PGIS had statistically significant prognostic value in patient survival. Thus, the A/J mouse-urethane model reflects significant molecular details of human lung AC, and comparison of changes in orthologous gene expression may provide novel insights into lung carcinogenesis.

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 A 2 (cPLA 2 ) 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 cPLA 2 in mouse lung tumorigenesis by using mice genetically deficient in cPLA 2 . After multiple urethane injections into cPLA 2 null mice and wild-type littermates, the number of lung tumors was determined. cPLA 2 null mice developed 43% fewer tumors (from 16 ± 2 to 9 ± 2 tumors/mouse; P < 0.05) than wild-type littermates. cPLA 2 , COX-1, COX-2 and microsomal prostaglandin E 2 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 cPLA 2 null mice. Although the steady-state levels of prostaglandin E 2 and prostaglandin I 2 in lung tissue extracts prepared from wild-type or cPLA 2 (-/-) mice were not significantly different, both prostaglandins markedly increased in tumors from wild-type mice, an increase that was significantly blunted in tumors from cPLA 2 (-/-) mice. These results demonstrate a role for cPLA 2 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.

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% (P0.0001). 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.

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

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.

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.

Gap junctional intercellular communication (GJIC) and connexin expression are frequently decreased in neoplasia and may contribute to defective growth control and loss of differentiated functions. GJIC, in E9 mouse lung carcinoma cells and WB-aB1 neoplastic rat liver epithelial cells, was elevated by forced expression of the gap junction proteins, connexin43 (Cx43) and connexin32 (Cx32), respectively. Transfection of Cx43 into E9 cells increased fluorescent dye-coupling in the transfected clones, E9-2 and E9-3, to levels comparable to the nontransformed sibling cell line, E10, from which E9 cells originated. Transduction of Cx32 into WB-aB1 cells also increased dye-coupling in the clone, WB-a/32-10, to a level that was comparable to the nontransformed sibling cell line, WB-F344. The cell cycle distribution was also affected as a result of forced connexin expression. The percentage of cells in G(1)-phase increased and the percentage in S-phase decreased in E9-2 and WB-a/32-10 cells as compared to E9 and WB-aB1 cells. Concomitantly, these cells exhibited changes in G(1)-phase cell cycle regulators. E9-2 and WB-a/32-10 cells expressed significantly less cyclin D1 and more p27(kip-1) protein than E9 and WB-aB1 cells. Other growth-related properties (expression of platelet-derived growth factor receptor-beta, epidermal growth factor receptor, protein kinase C-alpha, protein kinase A regulatory subunit-Ialpha, and production of nitric oxide in response to a cocktail of pro-inflammatory cytokines) were minimally altered or unaffected. Thus, enhancement of connexin expression and GJIC in neoplastic mouse lung and rat liver epithelial cells restored G(1) growth control. This was associated with decreased expression of cyclin D1 and increased expression of p27(kip-1), but not with changes in other growth-related functions.

Modulation of gene expression at the level of mRNA stability has emerged as an important regulatory paradigm. In this context, differential expression of numerous mRNAs in normal versus neoplastic tissues has been described. Altered expression of these genes, at least in part, has been demonstrated to be at the level of mRNA stability. Two ubiquitously expressed mRNA binding proteins have recently been implicated in the stabilization (Hu antigen R/HuR) or destabilization (AU-rich element mRNA binding protein [AUF1]/heterogeneous nuclear ribonucleoprotein D) of target mRNAs. Further, their functional activity appears to require cytoplasmic localization. In the present study, we demonstrate a strong correlation between increased cytoplasmic expression of both AUF1 and HuR with urethane-induced neoplasia and with butylated hydroxytoluene-induced compensatory hyperplasia in mouse lung tissue. In addition, when compared with slower growing cells, rapidly growing neoplastic lung epithelial cell lines expressed a consistently higher abundance of both AUF1 and HuR proteins. Moreover, in nontumorigenic cell lines, both AUF1 and HuR protein abundance decreased with confluence and growth arrest. In contrast, in spontaneous transformants, AUF1 and HuR abundance was unaffected by changes in cell density. We suggest that growth-regulated alterations in AUF1 and HuR abundance may have pleiotropic effects on the expression of a number of highly regulated mRNAs and that this significantly impacts the onset, maintenance, and progression of the neoplastic phenotype. Mol. Carcinog. 28:76-83, 2000.

Mouse lung tumorigenesis is a convenient model for examining all stages of lung adenocarcinoma (AC) progression. Because enhanced cyclooxygenase 2 (COX-2) expression has been observed in advanced human AC, we investigated the intracellular concentrations of the two cyclooxygenases, cyclooxygenase 1 (COX-1) and COX-2, at different times after carcinogen administration to A/J mice. The concentrations of both proteins were much higher in urethane-induced adenomas and carcinomas compared with control A/J mouse lung tissue (P < 0.03 and P < 0.01 in adenomas and AC, respectively, for COX-1; P < 0.003 and P < 0.004 in adenomas and AC, respectively, for COX-2). Small benign tumors that arose spontaneously in 13-month-old mice also stained for COX-1 and COX-2, showing that this elevated enzyme content does not depend on chemical induction. COX-1 and COX-2 immunostaining was observed in normal bronchiolar and alveolar epithelia, alveolar macrophages and bronchiolar smooth muscle. This is the first report of the cellular distribution of COX-1 and COX-2 in murine lungs and the first in any species to demonstrate their co-localization. COX content in isolated bronchiolar Clara cells, a putative cell of tumor origin, was equal to that found in tumors, suggesting that the high enzyme content in neoplasms is due to their proportionally high concentration of these tumor precursor cells. Different patterns of COX-1 and COX-2 expression were observed in tumors of different growth patterns; only occasional small foci stained in solid adenomas, while most cells in papillary adenomas were immunoreactive. This staining pattern was also seen in adenocarcinomas, but some of the papillary portions also included focally stained and unstained regions. The continued expression during neoplastic progression of these specialized enzymes present in normal cells of tumor origin suggests their function in maintenance of the neoplastic state.

Butylated hydroxytoluene (BHT) causes lung injury in mice and promotes tumor formation. Hydroxylation of a tert-butyl group on BHT to yield the metabolite, 6-tert-butyl-2-[2′-(2′-hydroxymethyl)-propyl]-4-methylphenol (BHTOH), may be required. BHTOH is more potent than BHT on an equimolar basis in causing lung damage, enhancing lung tumor development, killing isolated bronchiolar non-ciliated Clara cells, and inhibiting lung epithelial gap junctional intercellular communication. One mechanism proposed for tumor promoting agents is selective cytotoxicity; killing normal cells allows uninhibited clonal expansion of neighboring initiated cells. We compared the abilities of BHT, BHTOH, and other BHT metabolites to kill non-tumorigenic and tumorigenic mouse and human lung cell lines, and examined the contribution of apoptosis to this cytotoxicity. These cells lack the cytochrome P450 2B isozyme necessary for converting BHT to BHTOH. BHTOH and 4-hydroperoxy-4-methyl-2,6-di-tert-butyl-2,5-cyclohex-adienone (BHTOOH) were most toxic, BHT and 2,6-di-tert-butyl-1,4-benzoquinone (BHTQu) were less potent, and 4-methyl BHT metabolites that are not pneumotoxic were ineffective. BHTOH most strongly induced apoptosis, based on nuclear condensation and transmission electron microscopy. Non-tumorigenic cells were as susceptible to cell death as the neoplastic cell lines when apoptosis and necrosis are not distinguished, but more sensitive to BHTOH-induced apoptosis. An apoptotic mechanism may underlie the lung tumor promoting actions of BHTOH.

Cytomix, a mixture of interferon-γ, tumor necrosis factor-α, and interleukin-1β, induces nitric oxide (NO) production in lung epithelial cell lines. It is not known whether neoplastic transformation alters a cell’s ability to form NO in response to cytokines. The present study investigated NO formation in two murine lines of immortalized “normal” (nontumorigenic) lung epithelial cells of alveolar type II origin, E10 and C10, and their sibling spontaneous transformants, E9 and A5. Nontumorigenic cells elaborated much more NO after cytomix exposure than did their tumorigenic counterparts. NO production was prevented by inhibiting protein synthesis and NO synthase and attenuated by dexamethasone. Northern and Western blot analyses of inducible NO synthase (iNOS) demonstrated cytomix-induced induction of iNOS only in nontumorigenic cells. The deficiency in NO production in tumorigenic cells was not associated with reduced iNOS mRNA stability or with differences in cytomix-induced nuclear factor-κB activation. Although cytomix caused a greater production of NO in E10 cells than in E9 cells, the same treatment induced equivalent proliferation in both cell lines. These results indicate a specific deficiency in cytokine-induced NO synthesis in transformed murine lung epithelial cells relative to their normal progenitor cells and provide a model for investigating iNOS regulation.

2002;121;82S Chest Kleeberger Al Malkinson, Richard Radcliffe, Alison Bauer, Lori Dwyer-Nield and Steven * CXB Recombinant Inbred Mice Inflammation and Lung Tumor Promotion in Hydroxytoluene-Induced Pulmonary Susceptibility to Both Butylated Quantitative Trait Loci That Regulate http://chestjournal.chestpubs.org/content/121/3_suppl/82S.1.full.html can be found online on the World Wide Web at: The online version of this article, along with updated information and services ) ISSN:0012-3692 http://chestjournal.chestpubs.org/site/misc/reprints.xhtml ( distributed without the prior written permission of the copyright holder. All rights reserved. No part of this article or PDF may be reproduced or College of Chest Physicians, 3300 Dundee Road, Northbrook, IL 60062. has been published monthly since 1935. Copyright2002by the American is the official journal of the American College of Chest Physicians. It Chest