COPD-like Airway Inflammation Research Articles

Toll-like receptors 2, 4, and 9 modulate promoting effect of COPD-like airway inflammation on K-ras-driven lung cancer through activation of the MyD88/NF-ĸB pathway in the airway epithelium.

Toll-like receptors (TLRs) are an extensive group of proteins involved in host defense processes that express themselves upon the increased production of endogenous damage-associated molecular patterns (DAMPs) and pathogen-associated molecular patterns (PAMPs) due to the constant contact that airway epithelium may have with pathogenic foreign antigens. We have previously shown that COPD-like airway inflammation induced by exposure to an aerosolized lysate of nontypeable Haemophilus influenzae (NTHi) promotes tumorigenesis in a K-ras mutant mouse model of lung cancer, CCSPCre/LSL-K-rasG12D (CC-LR) mouse. In the present study, we have dissected the role of TLRs in this process by knocking out TLR2, 4, and 9 and analyzing how these deletions affect the promoting effect of COPD-like airway inflammation on K-ras-driven lung adenocarcinoma. We found that knockout of TLR 2, 4, or 9 results in a lower tumor burden, reduced angiogenesis, and tumor cell proliferation, accompanied by increased tumor cell apoptosis and reprogramming of the tumor microenvironment to one that is antitumorigenic. Additionally, knocking out of downstream signaling pathways, MyD88/NF-κB in the airway epithelial cells further recapitulated this initial finding. Our study expands the current knowledge of the roles that TLR signaling plays in lung cancer, which we hope, can pave the way for more reliable and efficacious prevention and treatment modalities for lung cancer.

Elastase- and LPS-Exposed Cpa3Cre/+ and ST2-/- Mice Develop Unimpaired Obstructive Pulmonary Disease

IL-33 and its receptor ST2, as well as mast cells and their mediators, have been implicated in the development of chronic obstructive pulmonary disease (COPD). However, whether mast cells and the ST2 receptor play a critical role in COPD pathophysiology remains unclear. Here, we performed repeated intranasal administrations of porcine pancreatic elastase and LPS for four weeks to study COPD-like disease in wildtype, ST2-deficient, and Cpa3Cre/+ mice, which lack mast cells and have a partial reduction in basophils. Alveolar enlargement and changes in spirometry-like parameters, e.g. increased dynamic compliance and decreased expiratory capacity, were evident one day after the final LPS challenge and worsened over time. The elastase/LPS model also induced mild COPD-like airway inflammation, which encompassed a transient increase in lung mast cell progenitors, but not in mature mast cells. While ST2-deficient and Cpa3Cre/+ mice developed reduced pulmonary function uninterruptedly, they had a defective inflammatory response. Importantly, both ST2-deficient and Cpa3Cre/+ mice had fewer alveolar macrophages, known effector cells in COPD. Elastase/LPS instillation in vivo also caused increased bronchiole contraction in precision cut lung slices challenged with methacholine ex vivo, which occurred in a mast cell-independent fashion. Taken together, our data suggest that the ST2 receptor and mast cells play a minor role in COPD pathophysiology by sustaining alveolar macrophages.

Transcriptomic analysis and validation reveal the pathogenesis and a novel biomarker of acute exacerbation of chronic obstructive pulmonary disease

BackgroundAcute exacerbation of chronic obstructive pulmonary disease (AECOPD) is the main factor that leads to the deterioration of the disease. Currently, the diagnosis of AECOPD mainly relies on clinical manifestations, good predictors or biomarkers are lacking. We aim to reveal specific biomarkers and potential pathogenesis of AECOPD and provide a research basis for the diagnosis and treatment.MethodsFour patients with AECOPD, four patients with stable COPD, and five control subjects were enrolled for RNA sequencing and KEGG analysis. The mRNA level of target genes was verified by quantitative real-time PCR (qPCR) with an expanded sample size (30 patients with AECOPD, 27 patients with stable COPD, and 35 control subjects). ELISA and immunofluorescence were used to identify the target proteins. Furthermore, the expression and function of WNT/β-catenin signaling pathway were assessed in animal models of COPD.ResultsRNA sequencing showed that 54 genes were up-regulated and 111 genes were down-regulated in the AECOPD. Differentially expressed genes were mainly enriched in WNT signaling pathway, et al. QPCR revealed that multi-genes of the WNT/β-catenin signaling were significantly down-regulated in AECOPD (P < 0.05), and β-catenin protein was significantly decreased in plasma of AECOPD and stable COPD (P < 0.01), while phosphorylated β-catenin was significantly up-regulated in peripheral blood mononuclear cells of AECOPD (P < 0.05). Similarly, WNT ligands, WNT receptors, and downstream signaling molecules were down-regulated, with an increased phosphorylated β-catenin protein in animal models of COPD. Activation of WNT/β-catenin signaling pathway by lithium chloride reduced the expression of phosphorylated β-catenin and ameliorated the COPD-like airway inflammation in mice.ConclusionWNT/β-catenin signaling pathway is down-regulated in AECOPD patients and in animal models of COPD. Increased expression of phosphorylated β-catenin in the blood might be a potential biomarker of AECOPD. Activation of WNT/β-catenin pathway may also represent a therapeutic target for AECOPD.

Autophagy Promotes Cigarette Smoke-Initiated and Elastin-Driven Bronchitis-Like Airway Inflammation in Mice.

Cigarette smoke (CS)-induced macrophage activation and airway epithelial injury are both critical for the development of chronic obstructive pulmonary disease (COPD), while the eventual functions of autophagy in these processes remain controversial. We have recently developed a novel COPD mouse model which is based on the autoimmune response sensitized by CS and facilitated by elastin. In the current study, we therefore utilized this model to investigate the roles of autophagy in different stages of the development of bronchitis-like airway inflammation. Autophagic markers were increased in airway epithelium and lung tissues, and Becn+/- or Lc3b-/ - mice exhibited reduced neutrophilic airway inflammation and mucus hyperproduction in this COPD mouse model. Moreover, treatment of an autophagic inhibitor 3-methyladenine (3-MA) either during CS-initiated sensitization or during elastin provocation significantly inhibited the bronchitis-like phenotypes in mice. Short CS exposure rapidly induced expression of matrix metallopeptidase 12 (MMP12) in alveolar macrophages, and treatment of doxycycline, a pan metalloproteinase inhibitor, during CS exposure effectively attenuated the ensuing elastin-induced airway inflammation in mice. CS extract triggered MMP12 expression in cultured macrophages, which was attenuated by autophagy impairment (Becn+/- or Lc3b-/ -) or inhibition (3-MA or Spautin-1). These data, taken together, demonstrate that autophagy mediates both the CS-initiated MMP12 activation in macrophages and subsequent airway epithelial injury, eventually contributing to development COPD-like airway inflammation. This study reemphasizes that inhibition of autophagy as a novel therapeutic strategy for CS-induced COPD.

COPD-Type lung inflammation promotes K-ras mutant lung cancer through epithelial HIF-1α mediated tumor angiogenesis and proliferation.

Chronic obstructive pulmonary disease (COPD), an inflammatory disease of the lung, is an independent risk factor for lung cancer. Lung tissues obtained from human smokers with COPD and lung cancer demonstrate hypoxia and up-regulated hypoxia inducible factor-1 (HIF-1). HIF-1 activation is the central mechanism for controlling the cellular response to hypoxia during inflammation and tumor development. These facts suggest a link between COPD-related airway inflammation, HIF-1, and lung cancer. We have previously established a mouse model of COPD-like airway inflammation that promotes lung cancer in a K-ras mutant mouse model (CC-LR). Here we show that tumors in the CC-LR model have significantly elevated levels of HIF-1α and HIF-1 activity. To determine the tumor-promoting functions of HIF-1 in CC-LR mice, the gene Hif1a which encodes HIF-1α and is required for HIF-1 activity, was disrupted in the lung epithelium of CC-LR animals. Airway epithelial specific HIF-1α deficient mice demonstrated significant reductions in lung surface tumor numbers, tumor angiogenesis, and tumor cell proliferation in the absence or presence of COPD-like airway inflammation. In addition, when CC-LR mice were bred with transgenic animals that overexpress a constitutively active mutant form of human HIF-1α in the airway epithelium, both COPD- and adenocarcinoma-like phenotypes were observed. HIF-1α overexpressing CC-LR mice had significant emphysema, and they also showed potentiated tumorigenesis, angiogenesis, and cell proliferation accompanied by an invasive metastatic phenotype. Our gain and loss of function studies support a key role for HIF-1α in the promotion of lung cancer by COPD-like inflammation.

Abstract 2687: Toll like receptors mediated inflammatory signals mediate promotion of K-ras mutant lung cancer by chronic obstructive pulmonary disease

Abstract Lung cancer is the leading cause of cancer death worldwide, and cigarette smoking is its main cause. However, smoker with chronic obstructive pulmonary disorder (COPD), an inflammatory disease of the lung, have an increased risk of lung cancer (3 to 10 fold) compared to smokers without COPD. Importantly, lung inflammation persists and lung function continues to deteriorate as does the increased risk of lung cancer even after cessation of cigarette smoking among former smokers. These facts suggest a strong link between COPD-related airway inflammation and lung cancer promotion, however, the precise mechanistic link is not known. We have previously developed a COPD-like mouse model of airway inflammation through repeated aerosol challenge to a lysate of nontypeable (i.e., unencapsulated) Haemophilus influenzae (NTHi). NTHi is the most common colonizing bacteria in the lower respiratory tract of patients with COPD and could be a potential cause of perpetuating and promoting airway injury and inflammation in these patients. We then showed that this type of airway inflammation promotes lung cancer in a K-ras mutant mouse model of lung cancer (CC-LR), which was associated with the activation of MyD88/NF-κB pathway and increased expression of its downstream targets in the lung. We have further shown that lack of NF-kB or MyD88 (an adaptor protein upstream to NF-κB) in the airway epithelium of CC-LR mice changes the bronchoalveolar lavage fluid cellular component of CC-LR mice and inhibits the promoting effect of COPD-like airway inflammation on lung tumorigensis. Upstream to MyD88 and NF-κB and downstream to bacterial stimuli is the toll-like receptors family (TLRs), which play critical role in the innate immune response. Among TLRs, TLR-2, TLR4, and TLR9 play critical roles in mediating inflammatory responses in lung and are required for primary epithelial response to inflammatory stimuli and activation of MyD88/NF-kB pathway in the airway epithelium. Accordingly, we further hypothesized that TLR-2, 4, and 9 mediate promoting effect of inflammtion on lung tumorigenesis in an MyD88/NF-kB dependent manner. Therefore, CC-LR mice were separately crossed to TLR-2, TLR-4 and TLR-9 knock out mice in order to test this hypothesis. We found that genetic ablation of these TLRs in CC-LR mice, causes significant reduction in lung surface tumor numbers compared to age and sex matched control CC-LR mice in the presence of COPD-like airway inflammation. This tumor reduction was associated with significant reduction in the numbers of inflammatory cells in bronchoalveolar lavage fluid of mice with lack of these TLRs. Taken these together, we conclude that promoting effect of COPD on lung cancer is mediated through TLR2, 4, 9-mediated activation of epithelial MyD88/NF-kB pathway. Citation Format: Nasim Khosravi, Nelly Torres-Garza, Soudabeh Daliri, Maria Miguelina De La Garza, Amber Cumpian, Evelyn Beltran, Misha Umer, Diana Del Bosque, Saba Akbani, Scott Evans, Seyed Javad Moghaddam. Toll like receptors mediated inflammatory signals mediate promotion of K-ras mutant lung cancer by chronic obstructive pulmonary disease [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2687. doi:10.1158/1538-7445.AM2017-2687

Tumor necrosis factor links chronic obstructive pulmonary disease and K-ras mutant lung cancer through induction of an immunosuppressive pro-tumor microenvironment

ABSTRACTTumor necrosis factor (TNF) is known as an important regulator of tumor microenvironment and inflammation. TNF levels are markedly elevated in the bronchoalveolar lavage fluid (BALF) of patients with chronic obstructive pulmonary disease (COPD), which is an independent risk factor for lung cancer. We have previously shown that COPD-like airway inflammation promotes lung cancer in a K-ras mutant mouse model (CC-LR mouse). This was associated with a significant increase of neutrophils in BALF, accompanied by a marked increase in TNF level, suggesting a link between COPD, TNF, and lung cancer promotion. Therefore, we first overexpressed TNF in the airway epithelium of CC-LR mice, which promoted lung cancer by ∼2-fold. This was associated with increased numbers of Ki67 and CD31 positive cells in lung tumors of CC-LR/TNF-Tg mice. We also found a robust increase in NF-κB activation, and numbers of neutrophils and myeloid-derived suppressor cells (MDSCs) in lung. Accordingly, we depleted MDSCs in CC-LR/TNF-Tg mice, which lead to significant tumor suppression emphasizing on the role of TNF-induced MDSCs in K-ras induced lung tumorigenesis. Finally, we targeted TNF expression by crossing CC-LR mice with TNF knock-out mice (CC-LR/TNF-KO), which resulted in a significant decrease in lung tumor burden in the absence or presence of COPD-like airway inflammation. Interestingly, there were less MDSCs and lower Ki67 and CD31 expression in the lung of the CC-LR/TNF-KO mice. We conclude that TNF links COPD to lung cancer promotion by induction of an immunosuppressive MDSC response, and subsequent amplification of proliferation and angiogenesis in tumors.

Abstract 413: Targeting IL-6 as a preventive and therapeutic strategy for K-ras mutant lung cancer

Abstract Worldwide, lung cancer (LC) leads in cancer mortality and cigarette smoking (CS) is its principal cause. Activating mutations of K-ras are one of the most common alterations in LC and are associated with CS exposure. However, several studies have shown that smokers with chronic obstructive pulmonary disease (COPD), an inflammatory disease of the lung, have a higher risk of developing LC when compared to smokers without COPD. These facts suggest a strong link between COPD-type inflammatory lung microenvironment and K-ras mutant lung cancer. Unfortunately, attempts to directly target K-ras have thus far failed, clearly stating the need for new approaches to bring clinical benefits to patients with this undrugabble profile. Using a conditional K-ras induced lung cancer mouse model (CC-LR) we showed that K-ras mutant lung tumors have intrinsic inflammatory characteristics, and a bacterial lysate-induced COPD-like airway inflammation promotes lung cancer in this model. This was associated with release of the cytokine IL-6 and activation of the IL-6-responsive transcription factor STAT3. We showed that genetic ablation of IL-6 in this model results in a significant tumor reduction indicating an essential role for IL-6/STAT3 pathway in lung cancer promotion and introducing IL-6 as a druggable target for K-ras mutant lung tumor. Therefore, we blocked IL-6 using a monoclonal anti-IL-6 immunotherapy in our K-ras mutant model. Six week old CC-LR mice were injected intraperitoneally with 20 mg/kg dose of an anti-IL-6 monoclonal IgG1 antibody twice a week for eight weeks, while been exposed to the aerosolized bacterial lysate once weekly for 8 weeks. Anti-IL-6 therapy not only inhibited intrinsic lung cancer development by ∼78%, but also inhibited the tumor promoting effect of the COPD-like airway inflammation. It also decreased the expression of P-STAT3, proliferation marker, Ki67, and pro-angiogenic factors: VEGF, MMP-9 and CD31 in lung tissue. qPCR analysis of lung tissues from anti-IL-6 treated mice showed a decrease in Arginase1, FIZZ1, FOXP3, and IL-17 expression, with increased expression of Th1 markers, IFNγ and TBx21. Flow cytometry analysis of total lung inflammatory cells in CC-LR mouse showed a significant increase in the population of tumor associated macrophages (TAMs) during tumor progression from age 6 to 14 weeks, and COPD-like inflammation induced an increase in myeloid derived suppressor cell (MDSCs) population. Interestingly, we found a significant decrease in TAMs and MDSCs population after anti-IL-6 treatment. Taken together, we conclude that IL-6 blockade not only has cell intrinsic effect in K-ras mutant tumors but also edit the lung microenvironment toward an anti-tumor phenotype. Therefore, we propose pharmacological targeting of IL-6 alone or in combination with conventional cytotoxic therapy, or other targeted therapies for prevention and treatment of lung cancer patients with K-ras mutation. Citation Format: Mauricio S. Caetano, Amber M. Cumpian, Lei Gong, Seon H. Chang, Huiyuan Zhang, Humam N. Kadara, Cinthya Sternberg, Carlos G. Ferreira, Stephanie S. Watowich, Seyed J. Moghaddam. Targeting IL-6 as a preventive and therapeutic strategy for K-ras mutant lung cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 413. doi:10.1158/1538-7445.AM2015-413

Promoting effect of neutrophils on lung tumorigenesis is mediated by CXCR2 and neutrophil elastase.

BackgroundTumor cells produce various cytokines and chemokines that attract leukocytes. Leukocytes can amplify parenchymal innate immune responses, and have been shown to contribute to tumor promotion. Neutrophils are among the first cells to arrive at sites of inflammation, and the increased number of tumor-associated neutrophils is linked to poorer outcome in patients with lung cancer.ResultsWe have previously shown that COPD-like airway inflammation promotes lung cancer in a K-ras mutant mouse model of lung cancer (CC-LR). This was associated with severe lung neutrophilic influx due to the increased level of neutrophil chemoattractant, KC. To further study the role of neutrophils in lung tumorigenesis, we depleted neutrophils in CC-LR mice using an anti-neutrophil antibody. This resulted in a significant reduction in lung tumor number. We further selectively inhibited the main receptor for neutrophil chemo-attractant KC, CXCR2. Similarly, this resulted in suppression of neutrophil recruitment into the lung of CC-LR mice followed by significant tumor reduction. Neutrophil elastase (NE) is a potent elastolytic enzyme produced by neutrophils at the site of inflammation. We crossed the CC-LR mice with NE knock-out mice, and found that lack of NE significantly inhibits lung cancer development. These were associated with significant reduction in tumor cell proliferation and angiogenesis.ConclusionWe conclude that lung cancer promotion by inflammation is partly mediated by activation of the IL-8/CXCR2 pathway and subsequent recruitment of neutrophils and release of neutrophil elastase. This provides a baseline for future clinical trials using the IL-8/CXCR2 pathway or NE inhibitors in patients with lung cancer.

Abstract 4977: An essential role for neutrophils in lung cancer promotion .

Abstract Lung cancer is the leading cause of cancer death worldwide, and cigarette smoking is the principal cause of it. Cigarette smoke-induced lung cancer is characterized by a deregulated inflammatory tumor microenvironment. Tumor cells produce various cytokines and chemokines that attract leukocytes. Leukocytes (innate and adaptive immune cells) can amplify parenchymal innate immune responses, and have been shown to contribute to tumor promotion. In addition, smokers with chronic obstructive pulmonary disease (COPD), an inflammatory disease of the lung, have an increased risk of lung cancer compared to smokers without COPD. These facts suggest a strong link between COPD-type inflammation and lung cancer promotion. In histopathologic specimens of lung and in bronchoalveolar lavage fluid (BALF) from COPD patients, neutrophils and macrophages are prominent. Neutrophils are among the first cells to arrive at sites of inflammation, and the increased number of tumor-associated neutrophils is linked to poorer outcome in patients with lung cancer. We have previously shown that COPD-like airway inflammation promotes lung cancer in a K-ras mutant mouse model of lung cancer (CC-LR). This was associated with severe neutrophilic influx due to increased level of neutrophil chemoattractant, KC, which was partially inhibited by using a natural non-specific anti-inflammatory agent, curcumin 1% in diet, and resulted in significant tumor suppression. To further study the role of neutrophil in lung tumorigenesis, we depleted neutrophils in CC-LR mice using an anti-neutrophil antibody, mLy6G, 5mg/kg twice a week by intraperitoneal injection. This resulted in complete lung neutrophil depletion and 68% (3.2-fold) reduction in lung surface tumor number. Neutrophil elastase (NE) is a potent elastolytic enzyme produced by neutrophils at the site of inflammation. It participates actively in COPD development and might contribute to tumor progression by activating proteolytic cascades. The concentration of NE in tumor extracts was also found to be linked to the invasiveness of lung cancer. Therefore, we crossed the CC-LR mice with NE knock out (KO) mice. We have found that lack of NE significantly inhibited lung cancer development by 43% (1.7-fold) without changing the BALF inflammatory cell component of the CC-LR mice. We further inhibited the main receptor for neutrophil chemo-attractant KC, CXCR2, using a selective inhibitor, SB332235Z, 50 mg/kg orally twice daily by gavage. Similarly, this also resulted in significant suppression of neutrophil recruitment into the lung of CC-LR mice followed by significant (∼64%, 2.8-fold) tumor reduction. We conclude that lung cancer promotion by inflammation partly mediated by activation of IL-8/CXCR2 pathway and subsequent recruitment of neutrophils and release of neutrophil elastase. This provides a baseline for future clinical trails using IL-8/CXCR2 pathway or NE inhibitors in patients with lung cancer. Citation Format: Lei Gong, Amber C. Cumpian, Cesar E. Ochoa, Daniel J. Lapid, Seyedeh Golsar Mirabolfathinejad, Maria Miguelina De la Garza, Seyed Javad Moghaddam. An essential role for neutrophils in lung cancer promotion . [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4977. doi:10.1158/1538-7445.AM2013-4977

Abstract A32: Mechanistic Dissection of Lung Cancer Promotion by Airway Inflammation

Abstract Cigarette smoking is the principal cause of lung carcinogenesis, however, smokers with chronic obstructive pulmonary disease (COPD) have an increased risk of lung cancer (3 to 10 fold) compared to smokers with comparable cigarette exposure but without COPD. Histopathologic studies have clearly demonstrated lung inflammation in COPD, and it persists even after cessation of cigarette smoking. These facts suggest a strong link between COPD-related airway inflammation and lung cancer promotion independent of smoking but the precise mechanistic link is not known. We developed a COPD-like mouse model of airway inflammation through repeated aerosol challenge to a lysate of non-typeable Haemophilus influenzae (NTHi) which commonly colonizes the airways of COPD patients. Then, we showed that this type of airway inflammation, but not asthma-like airway inflammation, promotes lung cancer in a K-ras mutant mouse model of lung cancer (CC-LR). This was associated with NF-kB pathway activation, and up-regulation of its downstream target genes, including IL-6 and T helper 17 (Th17) response. Therefore, we studied the role of NF-kB in lung cancer promotion by targeting its expression in airway epithelium. CC-LR mice were crossed with IKK-βf/f mice to develop a mouse with lack of NF-kB activity in airway secretory cells (CC-LR-IKKβ mice). NF-kB deficiency in the airway epithelium changed the bronchoalveolar lavage fluid (BALF) cellular component (mostly reduction in macrophage number) of the CC-LR mice, and resulted in a ∼70% (3.4-fold) reduction in lung surface tumor number compared to age and sex matched control CC-LR mice. It also significantly reduced the number of visible tumors on the lung surface of the CC-LR mice by &amp;gt;60% (2.6-fold) after inducing COPD-like airway inflammation using weekly NTHi exposure for 8 weeks. We then genetically ablated IL-6 in the CC-LR mice. Lack of IL-6 did not change the BALF inflammatory cell component of the CC-LR mice. However, it not only inhibited intrinsic lung cancer development (1.7-fold, 41%), but also inhibited the promoting effect of extrinsic NTHi-induced COPD-like airway inflammation (2.6-fold, 62%) on lung carcinogenesis, suggesting a role for IL-6 mediated epithelial cell signaling in tumor promotion by COPDlike airway inflammation. This is under further investigation by our group. IL-6 is required for differentiation of Th17 cells from naive T cells. Th17 cells mostly produce IL-17A, and IL-17F. These both bind to the IL-17 receptor (IL-17R), and IL-17R signaling is required for lung CXC chemokine expression and neutrophil recruitment, however, we found that only the IL-17R KO and IL-17A KO but not IL-17F KO mice showed lower levels of neutrophilic influx into the BALF and less inflammatory cell infiltration in lung tissue in response to NTHi. Furthermore, IL-17A induces more production of IL-6 by epithelial cells. So, CC-LR mice were crossed with IL-17A KO mice. Lack of IL-17A resulted in a ∼70% (3.4-fold) reduction in lung surface tumor number compared to age and sex matched control CC-LR mice. It also significantly reduced the number of visible tumors on the lung surface of the CCLR mice by ∼54% (2.2-fold) after inducing COPD-like airway inflammation using weekly NTHi exposure for 4 weeks from the age of 10 weeks. We conclude that airway inflammation through NF-kB mediated activation of the IL-6/Th17 signaling pathway promotes lung carcinogenesis.

Interleukin 6, but not T helper 2 cytokines, promotes lung carcinogenesis.

Several epidemiologic studies have found that smokers with chronic obstructive pulmonary disease (COPD), an inflammatory disease of the lung, have an increased risk of lung cancer compared with smokers without COPD. We have shown a causal role for COPD-like airway inflammation in lung cancer promotion in the CCSP(Cre)/LSL-K-ras(G12D) mouse model (CC-LR). In contrast, existing epidemiologic data do not suggest any definite association between allergic airway inflammation and lung cancer. To test this, CC-LR mice were sensitized to ovalbumin (OVA) and then challenged with an OVA aerosol weekly for 8 weeks. This resulted in eosinophilic lung inflammation associated with increased levels of T helper 2 cytokines and mucous metaplasia of airway epithelium, similar to what is seen in asthmatic patients. However, this type of inflammation did not result in a significant difference in lung surface tumor number (49 ± 9 in OVA vs. 52 ± 5 in control) in contrast to a 3.2-fold increase with COPD-like inflammation. Gene expression analysis of nontypeable Haemophilus influenzae (NTHi)-treated lungs showed upregulation of a different profile of inflammatory genes, including interleukin 6 (IL-6), compared with OVA-treated lungs. Therefore, to determine the causal role of cytokines that mediate COPD-like inflammation in lung carcinogenesis, we genetically ablated IL-6 in CC-LR mice. This not only inhibited intrinsic lung cancer development (1.7-fold) but also inhibited the promoting effect of extrinsic COPD-like airway inflammation (2.6-fold). We conclude that there is a clear specificity for the nature of inflammation in lung cancer promotion, and IL-6 has an essential role in lung cancer promotion.

Curcumin inhibits COPD-like airway inflammation and lung cancer progression in mice

Recent studies have demonstrated that K-ras mutations in lung epithelial cells elicit inflammation that promotes carcinogenesis in mice (intrinsic inflammation). The finding that patients with chronic obstructive pulmonary disease (COPD), an inflammatory disease of the lung, have an increased risk of lung cancer after controlling for smoking suggests a further link between lung cancer and extrinsic inflammation. Besides exposure to cigarette smoke, it is thought that airway inflammation in COPD is caused by bacterial colonization, particularly with non-typeable Hemophilus influenzae (NTHi). Previously, we have shown that NTHi-induced COPD-like airway inflammation promotes lung cancer in an airway conditional K-ras-induced mouse model. To further test the role of inflammation in cancer promotion, we administered the natural anti-inflammatory agent, curcumin, 1% in diet before and during weekly NTHi exposure. This significantly reduced the number of visible lung tumors in the absence of NTHi exposure by 85% and in the presence of NTHi exposures by 53%. Mechanistically, curcumin markedly suppressed NTHi-induced increased levels of the neutrophil chemoattractant keratinocyte-derived chemokine by 80% and neutrophils by 87% in bronchoalveolar lavage fluid. In vitro studies of murine K-ras-induced lung adenocarcinoma cell lines (LKR-10 and LKR-13) indicated direct anti-tumoral effects of curcumin by reducing cell viability, colony formation and inducing apoptosis. We conclude that curcumin suppresses the progression of K-ras-induced lung cancer in mice by inhibiting intrinsic and extrinsic inflammation and by direct anti-tumoral effects. These findings suggest that curcumin could be used to protract the premalignant phase and inhibit lung cancer progression in high-risk COPD patients.

Promotion of Lung Carcinogenesis by Chronic Obstructive Pulmonary Disease–Like Airway Inflammation in a K-ras–Induced Mouse Model

Lung cancer is the leading cause of cancer deaths in the United States. In addition to genetic abnormalities induced by cigarette smoke, several epidemiologic studies have found that smokers with chronic obstructive pulmonary disease (COPD), an inflammatory disease of the lungs, have an increased risk of lung cancer (1.3- to 4.9-fold) compared to smokers without COPD. This suggests a link between chronic airway inflammation and lung carcinogenesis, independent of tobacco smoke exposure. We studied this association by assaying the inflammatory impact of products of nontypeable Haemophilus influenzae, which colonizes the airways of patients with COPD, on lung cancer promotion in mice with an activated K-ras mutation in their airway epithelium. Two new mouse models of lung cancer were generated by crossing mice harboring the LSL-K-ras(G12D) allele with mice containing Cre recombinase inserted into the Clara cell secretory protein (CCSP) locus, with or without the neomycin cassette excised (CCSP(Cre) and CCSP(Cre-Neo), respectively). Lung lesions in CCSP(Cre-Neo)/LSL-K-ras(G12D) and CCSP(Cre)/LSL-K-ras(G12D) mice appeared at 4 and 1 month of age, respectively, and were classified as epithelial hyperplasia of the bronchioles, adenoma, and adenocarcinoma. Weekly exposure of CCSP(Cre)/LSL-K-ras(G12D) mice to aerosolized nontypeable Haemophilus influenzae lysate from age 6-14 weeks resulted in neutrophil/macrophage/CD8 T-cell-associated COPD-like airway inflammation, a 3.2-fold increase in lung surface tumor number (156 +/- 9 versus 45 +/- 7), and an increase in total lung tumor burden. We conclude that COPD-like airway inflammation promotes lung carcinogenesis in a background of a G12D-activated K-ras allele in airway secretory cells.