With more than 150,000 deaths per year in the US alone, lung cancer has the highest number of deaths for any cancer. These poor outcomes reflect a lack of treatment for the most common form of lung cancer, non-small cell lung carcinoma (NSCLC). Lung adenocarcinoma (ADC) is the most prevalent subtype of NSCLC, with the main oncogenic drivers being KRAS and epidermal growth factor receptor (EGFR). Whereas EGFR blockade has led to some success in lung ADC, effective KRAS inhibition is lacking. KRAS-mutant ADCs are characterized by high levels of gel-forming mucin expression, with the highest mucin levels corresponding to worse prognoses. Despite these well-recognized associations, little is known about roles for individual gel-forming mucins in ADC development causatively. We hypothesized that MUC5AC/Muc5ac, a mucin gene known to be commonly expressed in NSCLC, is crucial in KRAS/Kras-driven lung ADC. We found that MUC5AC was a significant determinant of poor prognosis, especially in patients with KRAS-mutant tumors. In addition, by using mice with lung ADC induced chemically with urethane or transgenically by mutant-Kras expression, we observed significantly reduced tumor development in animals lacking Muc5ac compared with controls. Collectively, these results provide strong support for MUC5AC as a potential therapeutic target for lung ADC, a disease with few effective treatments.

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.

IntroductionToll-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.MethodsIn 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.ResultsWe 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.DiscussionOur 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.

Lung cancer, particularly K-ras mutant lung cancer, is the leading cause of cancer death worldwide, and cigarette smoking (CS) is its main cause. Epidemiologic studies had consistently revealed a strong association (3 to 9 fold increase) between lung cancer and COPD (chronic obstructive pulmonary disease), after controlling for CS exposure. COPD is an inflammatory disease of the airways with smoking being the main cause of it. 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 with COPD. These facts suggest a strong link between COPD-related airway inflammation and lung cancer promotion independent of smoking. We have previously shown that weekly exposure to an aerosolized bacterial lysate of nontypeable Haemophilus influenzae (NTHi), induces lung inflammation with a profile of mediators and inflammatory cells similar to that observed in COPD patients excluding mucous metaplasia. We further showed that NTHi-induced COPD type airway inflammation promotes lung cancer 3.2-fold in a K-ras mutant mouse model remarkably similar to that in the epidemiologic literature. 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 persistent airway inflammation after CS exposure in these patients. Therefore, we further studied the effect of combined CS and NTHi exposure in the induction of COPD phenotype and promotion of lung cancer. Briefly, 6-week old K-ras mutant mice were exposed to NTHi lysate once a week for 8 weeks and to cigarette smoke (CS) daily for 2 hours/day, 5 days/week for 8 weeks and studied at the age of 14 weeks. The CS exposure was conducted by burning 3R4F reference cigarettes (University of Kentucky, Tobacco Research Institute), using an InExpose System (SCIREQ Scientific Respiratory Equipment Inc). We found that CS exposure alone caused a mild macrophage dominant airway inflammation, induced airway epithelial mucous metaplasia, and led to a 2.3 fold increase in lung tumor burden. Notably, combined NTHi and CS exposure resulted in a robust neutrophilic lung inflammation and mucous metaplasia and promoted K-ras mutant lung cancer by 4.3 folds (2 times more than CS alone). Our experimental results suggest that CS exposure and colonization of smoke-injured airways with NTHi induce an inflammatory and structural COPD phenotype fully recapitulating human COPD and provide a microenvironment that has a significant promoting effect on K-ras mutant lung cancer. Citation Format: Marco A. Ramos, Misha Umer, Susana Castro, Berenice Adriana Gutierrez, Nassim Khosravi, Seyed Javad Moghaddam. Synergistic effect of cigarette smoke and bacterial-induced chronic obstructive pulmonary disease type airway inflammation on promotion of K-ras mutant lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5731.

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