The likelihood of developing lung cancer within 10 years is 3-fold greater in patients with mild to moderate COPD and 10-fold greater in patients with severe COPD compared to smokers with normal lung function . COPD is thought to be caused by the lung paranchymal response to inflammation from cigarette smoke and bacterial colonization of smoke-injured airways [10, 20]. These epidemiologic data suggest that chronic airway inflammation caused by tobacco smoke and microbial infection promote lung carcinogenesis [21, 22], and that lung cancer risk is positively associated with the severity and duration of inflammation . However, while these data underlie the relationships, they do not establish causal links which are addressed in this study using a novel mouse model of lung carcinogenesis.
Tumor cells themselves produce various cytokines and chemokines that attract leukocytes (intrinsic inflammation). The inflammatory component of a developing neoplasm may include a diverse leukocyte population including neutrophils, dendritic cells, macrophages, and lymphocytes, all of which are capable of producing an array of cytokines, cytotoxic mediators including reactive oxygen species, serine and cysteine proteases, and matrix metalloproteinases (MMPs) [24, 25]. Sustained cell proliferation in this environment rich in inflammatory cells, growth factors, activated stroma, and DNA-damage-promoting agents potentiates neoplastic risk [24, 26]. We have previously shown that the loss of the tumor suppressor gene Gprc5a in mouse lung epithelial cells results in expression of various cytokines and chemokines, followed by recruitment of inflammatory cells into the lung tumor microenvironment . Furthermore, increased activation of NF-κB in the Gprc5a-/- mouse lungs has been implicated in the creation of an inflammatory and proliferative microenvironment . More recently, we have demonstrated that exposure of the Gprc5a knockout mice to the tobacco-specific carcinogen NNK enhances lung tumorigenesis . In the present study, we have shown that extrinsic inflammation caused by bacterial infection and carcinogen exposure emulating COPD lung microenvironment, can further promote tumorigenesis in this mouse model. We previously showed the role of bacterial-induced COPD-like inflammation in promotion of lung tumorigenesis in an oncogenic K-ras induced mouse model of lung cancer . Here we showed the role of bacterial-induced COPD-like inflammation in a background of tobacco carcinogen exposure in lung cancer promotion and progression in a mouse model with lack of Gprc5a activity. Deletion of Gprc5a, which is expressed preferentially in lung tissue, predisposes mice to develop spontaneous lung tumors , but the tumorigenesis process in the Gprc5a-/- mouse takes 1 to 2 years with low multiplicity. Even with exposure to NNK , the time to tumor development is slow (12 months). This is in contrast with the rapid development and high multiplicity of tumors in K-ras mutant mouse model . Therefore, the Gprc5a knockout mouse model emulates better the typical course of lung cancer development in the setting of COPD in humans who have smoked cigarette and became chronically colonized with bacteria.
Cigarette smoke contains ~4,000 chemicals, of which ~60 have been identified as carcinogens . Of these NNK is the most potent carcinogen in laboratory animals and has therefore been implicated as a significant cause of tobacco-associated cancers including lung cancer . Activating mutations of K-ras, which are found in 30%-50% of lung adenocarcinoma (AC), are one of the most common genetic alterations associated with tobacco exposure . Within days after NNK administration, K-ras becomes mutated and activated in alveolar type II pneumocytes and bronchiolar Clara cells, the putative cells from which lung AC originate [31, 32]. However, in the present study, we showed that NNK exposure of Gprc5a knockout mice also results in increased levels of inflammatory mediators and recruitment of inflammatory cells into the lungs, which leads to lung tumor promotion. This is in agreement with the recent finding of a lung cancer promoting effect for smoke-induced inflammation independent of its direct mutagenesis effect in a K-ras mutant mouse model . Chronic NTHi exposure in Gprc5a-/- mice resulted in increased levels of inflammatory mediators followed by recruitment of inflammatory cells into the lungs (COPD-like inflammation) and lung cancer promotion, similar to what we have previously described in our K-ras induced mouse model . The highest levels of inflammation and tumor promotion were seen in Gprc5a-/- mice exposed to both NNK and NTHi, indicating an additive role for smoke- and bacterial-induced inflammation in progression from COPD to lung cancer.
Sustained angiogenesis is one of the hallmarks of cancer , and it is required for tumor promotion. We have also found increased microvessel density in response to NTHi-induced inflammation. This was associated with increased expression of HIF-1α which controls inflammation , and activates the transcription of genes involved in crucial aspects of cancer biology, including angiogenesis . NF-κB is a critical transcriptional activator of HIF-1α activity and is required for HIF-1α protein accumulation during hypoxia [35–37]. Activation of NF-κB, a hallmark of inflammatory responses, is frequently detected in COPD patients , smokers  and tumors . NF-κB is essential for promoting inflammation-associated cancers, and its inactivation decreases tumor multiplicity and delays cancer progression [41–43]. In the lung there are also a limited number of studies which show that NF-κB inhibition suppresses lung cancer in mouse [44, 45]. We have previously found NF-κB activation in the lungs of wild type (WT) mice , Gprc5a-/- mice , and in mice with mutant K-ras exposed to NTHi [13, 46]. We have also shown increased level of HIF-1α transcript in gene expression analysis of whole lung from WT, and mutant K-ras mice after NTHi exposure . In this study, we found that a single exposure to aerosolized NTHi was sufficient to increase NF-κB activation five-fold in the lung of WT and Gprc5a-/- mice (data not shown), along with increased expression of HIF-1α (Figure 5). All together these data suggest a role for NF-κB in lung cancer promotion in the Gprc5a-/- mice in response to bacterial induced inflammation through activation of the HIF-1α pathway and its downstream angiogenic signals.
One of the events downstream of NF-κB activation is production of various cytokines and chemokines (e.g. IL-6, and IL-17) that attract leukocytes , which results in enhanced tumor progression, cancer cell growth and spread, angiogenesis, invasion and tumor immunosuppression [24, 25]. We have found significantly increased level of IL-6 and IL-17 in BALF after NTHi exposure alone or in combination with NNK treatment (table 1). We have previously shown an essential role for IL-6 in promotion of lung cancer by airway inflammation . IL-6 is required for differentiation of Th17 cells from naïve T cells, which mainly produce IL-17 [47, 48]. IL-17 binds to the IL-17 receptor (IL-17R), and IL-17R signaling is required for lung CXC chemokine expression and neutrophil recruitment . In addition to the traditional T helper 1 (Th1) response (IFN-γ) in COPD, recent developments in cytokine biology imply that COPD might be better explained by the Th17 phenotype [50, 51]. These are consistent with our finding after inducing COPD-like inflammation by NTHi exposure (table 1 and Figure 2A). NNK exposure may induce a type of immune response (T regulatory response) which suppresses the anti-tumoral immune response (CD8 T cell, and Th1 responses) and meanwhile balances the Th17 response toward an effective pro-tumoral response.
During immune responses, neutrophils are among the first cells to arrive at sites of inflammation. This is similar to our finding of significant neutrophil recruitment after NTHi exposure in Gprc5a-/- mice (Figure 2A). An increased number of tumor-associated neutrophils (TANs) was linked to poorer outcome in patients with bronchioloalveolar carcinoma . Neutrophils were present within the alveolar airspaces and within the tumor parenchyma during neoplastic development in a urethane-induced mouse model of lung cancer . Using a K-ras induced mouse model, study has shown that TANs were involved in lung tumorigenesis by the production of elastase . We have also shown an indirect anti-tumoral effect for curcumin through inhibition of neutrophil recruitment secondary to suppression of neutrophil chemoattractant (KC) in our K-ras induced lung cancer model .