Promotion of lung carcinogenesis by chronic obstructive pulmonary disease-like airway inflammation in a K-ras-induced mouse model

Abstract

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.

Figure 1.

Cre-mediated activation and expression of K-ras^G12D in lung tumors. (A) Schematic of wild-type (WT) (top), floxed (middle), LSL-K-ras^G12D, and Lox–K-ras^G12D (bottom) alleles. K-ras exons 0 and 1 are indicated in boxes. P₁ and P₂ represent the location of the primers used to detect excision of the stop cassette after Cre recombination. (B) Schematic of Clara cell secretory protein (CCSP) targeting event (CCSP with the neomycin cassette excised [CCSP^Cre] or without the neomycin cassette excised [CCSP^Cre-Neo]). (C and D) Identification of Cre editing of the LSL-K-ras^G12D allele in DNA isolated from WT, CCSP^Cre-Neo, LSL–K-ras^G12D, and CCSP^Cre-Neo/LSL–K-ras^G12D (lanes 1–4). The WT, CCSP^Cre-Neo, and LSL-K-ras^G12D allele (lanes 1–3) generates a 285-bp PCR product, whereas the activated mutant allele, Lox–K-ras^G12D, from CCSP^Cre-Neo/LSL–K-ras^G12D (lane 4), generates a product of 315 bp as a result of the presence of the remaining loxP site. (C) CCSP^Cre-Neo. (D) CCSP^Cre. (E and F) RT-PCR analysis of mRNA expression in the lungs of WT, CCSP^Cre-Neo, LSL-K-ras^G12D, and CCSP^Cre-Neo/LSL–K-ras^G12D (lanes1–4). Only in the PCR product from CCSP^Cre-Neo/LSL–K-ras^G12D mouse (lane 4), the WT K-ras band is seen at 448 bp. The mutant K-ras^G12D is identified by the 300- and 148-bp products after HindIII digestion (lane 4). (E) CCSP^Cre-Neo. (F) CCSP^Cre.

Figure 2.

Tumor growth and survival. (A and B) Developmental time course of lung lesions after expression of oncogenic K-ras. The first isolated small lung lesions were observed in 4- and 1-month-old mice in CCSP^Cre-Neo/LSL–K-ras^G12D and CCSP^Cre/LSL–K-ras^G12D mice, respectively, and progress after that (arrows) (10× magnification; scale bars = 200 μm). (C) Survival curves. The CCSP^Cre-Neo/LSL–K-ras^G12D and CCSP^Cre/LSL–K-ras^G12D mice displayed a shortened life span compared with LSL–K-ras^G12D littermate controls.

Figure 3.

Pathology and immunostainning of lung lesions. (A) Pathological diagnosis of lung lesions (panels 1 and 2). Atypical papillary bronchiolar hyperplasia (panels 3 and 4), solid adenoma (panels 5 and 6), papillary adenomas (panels 7 and 8), solid adenoma with atypical cytological features (panels 9 and 10), papillary adenoma with atypical cytological features, and adenocarcinoma (panels 11 and 12). Panels 1, 3, 5, 7, 9, and 11 are 10× magnification; scale bars = 100 μm; panels 2, 4, 6, 8, 10, and 12 are 40× magnification; scale bars = 40 μm. (B) Immunohistochemistry of lungs from CCSP^Cre/LSL–K-ras^G12D using anti-CCSP, and anti-SPC in regions with atypical hyperplasia, adenoma and adenocarcionma (panels 1, 2, 4, and 5 are 20× magnification; scale bars = 50 μm; panels 3 and 6 are 40× magnification; scale bars = 20 μm). (C) LacZ staining of CCSP^cre/R26R mouse lung shows activity (blue staining) in the airways (panels 1 and 2), whereas CCSP^cre/LSL-K-ras^G12D/R26R mouse lung shows staining in airways and alveoli (panels 3 and 4). Dual immunofluorescence staining of CCSP^cre/R26R mouse lung for CCSP (green) and β-galactosidase (red) (panel 5), and SPC (green) and β-galactosidase (red) (panel 6) shows colocalization for CCSP and β-galactosidase (arrows), whereas CCSP^cre/LSL-K-ras^G12D/R26R mouse lung shows colocalization for CCSP and β-galactosidase (arrows) (panel 7) and SPC and β-galactosidase (arrows) (panel 8) (panels 1 and 3: 4× magnification; panels 5 and 6: 40× magnification; panels 2, 4, 7, and 8: 20× magnification; scale bars = 50 μm).

Figure 4.

Analysis of lung inflammation after repetitive exposure to the aerosolized NTHi lysate. Control and CCSP^Cre/LSL–K-ras^G12D (K-ras^G12D) mice were exposed weekly for 8 weeks to an NTHi aerosol, and then killed the first day after the last exposure for analysis. (A) Total and lineage-specific leukocyte numbers in bronchoalveolar lavage fluid (BALF) after the eighth NTHi aerosol exposure are shown (mean ± SE; *P < 0.05 for control with NTHi exposure or CCSP^Cre/LSL–K-ras^G12D without NTHi exposure, or CCSP^Cre/LSL–K-ras^G12D with NTHi exposure versus WT without NTHi exposure; ^#P < 0.05 for CCSP^Cre/LSL–K-ras^G12D with NTHi exposure versus CCSP^Cre/LSL–K-ras^G12D without NTHi exposure). (B) hematoxylin and eosin–stained sections of lungs after the eighth NTHi aerosol exposure are shown (scale bar = 20 μm).

Figure 5.

Immunohistochemical identification of NF-κB activation and leukocytes infiltrating the lungs after repetitive NTHi lysate aerosol exposure. CCSP^Cre/LSL–K-ras^G12D mice were exposed weekly for 8 weeks to the aerosolized NTHi lysate, then killed 1 day after the last exposure and their lungs processed for light microscopy by immunohistochemical labeling with specific antibodies and hematoxylin counterstaining. (A) Labeling with α-p65 (antibody against the p65 subunit of NF-κB) showing intense nuclear staining of tumoral, and inflammatory cells. (B) Labeling with α-p40 to detect neutrophils. (C) Labeling with α-F4/80 to detect macrophages. (D) Labeling with α-CD20 to detect B cells. (E) Labeling with α-CD4 to detect helper T cells. (F) Labeling with α-CD8 to detect cytolytic T cells. Scale bar = 50 μm (applicable to all panels).

Figure 6.

Lung tumor burden and CCSP expression after repetitive exposure to the aerosolized NTHi lysate. WT, CCSP^Cre/LSL–K-ras^G12D (K-ras^G12D), and CCSP–T antigen (TAg) mice were exposed weekly for 8 weeks to an NTHi aerosol, and killed at various time points to assess lung surface tumor number or CCSP expression. (A) Lung surface tumor numbers in CCSP^Cre/LSL–K-ras^G12D mice before aerosol exposure (Week 6), after four and eight weekly exposures to the aerosolized NTHi lysate (Weeks 10 and 14, open circles), or after 4 and 8 weeks without aerosol exposure (Weeks 10 and 14, closed circles) are shown (mean ± SE; *P < 0.05 for NTHi exposed versus unexposed). (B) Quantitative RT-PCR analysis of CCSP transcripts in whole-lung lysate from WT C57BL/6 mice 1 day after a single exposure to the aerosolized NTHi lysate (W1-D1) and at multiple time points after eight weekly exposures are shown (mean ± SE; *P < 0.05 for NTHi exposed versus unexposed). (C) Western blot analysis of CCSP protein in BALF from WT C57BL/6 mice 1 day after a single exposure to the aerosolized NTHi lysate (W1-D1) and at multiple time points after eight weekly exposures are shown (mean ± SE; *P < 0.05 for NTHi exposed versus unexposed). (D) Lung surface tumor number in CCSP-TAg mice before aerosol exposure (Week 14) after four and eight weekly exposures to the aerosolized NTHi lysate (Weeks 18 and 22, open circles), or after 4 and 8 weeks without aerosol exposure (Weeks 18 and 22, closed circles) are shown (mean ± SE; *P < 0.05 for NTHi exposed versus unexposed).