Abstract
BackgroundTherapeutic strategies exist for human pulmonary neoplasia, however due to the heterogeneity of the disease, most are not very effective. The innate immunity gene, toll-like receptor 4 (TLR4), protects against chronic pulmonary inflammation and tumorigenesis in mice, but the mechanism is unclear. This study was designed to identify TLR4-mediated gene expression pathways that may be used as prognostic indicators of susceptibility to lung tumorigenesis in mice and provide insight into the mechanism.MethodsWhole lung mRNA was isolated from C.C3H-Tlr4Lps-d (BALBLps-d; Tlr4 mutant) and BALB/c (Tlr4 normal) mice following butylated hydroxytoluene (BHT)-treatment (four weekly ip. injections; 150-200 mg/kg/each; "promotion"). mRNA from micro-dissected tumors (adenomas) and adjacent uninvolved tissue from both strains were also compared 27 wks after a single carcinogen injection (3-methylcholanthrene (MCA), 10 μg/g; "control") or followed by BHT (6 weekly ip. injections; 125-200 mg/kg/each; "progression"). Bronchoalveolar lavage fluid was analyzed for inflammatory cell content and total protein determination, a marker of lung hyperpermeability; inflammation was also assessed using immunohistochemical staining for macrophages (F4/80) and lymphocytes (CD3) in mice bearing tumors (progression).ResultsDuring promotion, the majority of genes identified in the BALBLps-d compared to BALB/c mice (P < 0.05) were involved in epithelial growth factor receptor (EGFR) signaling (e.g. epiregulin (Ereg)), secreted phosphoprotein 1(Spp1)), which can lead to cell growth and eventual tumor development. Inflammation was significantly higher in BALBLps-d compared to BALB/c mice during progression, similar to the observed response during tumor promotion in these strains. Increases in genes involved in signaling through the EGFR pathway (e.g. Ereg, Spp1) were also observed during progression in addition to continued inflammation, chemotactic, and immune response gene expression in the BALBLps-d versus BALB/c mice (P < 0.05), which appears to provide more favorable conditions for cell growth and tumor development. In support of these findings, the BALB/c mice also had significantly reduced expression of many immune response and inflammatory genes in both the tumors and uninvolved tissue.ConclusionThis transcriptomic study determined the protective effect of TLR4 in lung carcinogenesis inhibition of multiple pathways including EGFR (e.g. Ereg), inflammatory response genes (e.g. Cxcl5), chemotaxis (e.g. Ccr1) and other cell proliferation genes (e.g. Arg1, Pthlh). Future studies will determine the utility of these pathways as indicators of immune system deficiencies and tumorigenesis.
Highlights
Adenocarcinoma (AC), a non-small cell lung carcinoma (NSCLC) [1], remains the leading type of lung cancer among smokers and nonsmokers
This transcriptomic study determined the protective effect of toll-like receptor 4 (TLR4) in lung carcinogenesis inhibition of multiple pathways including epidermal growth factor receptor (EGFR) (e.g. Ereg), inflammatory response genes (e.g. Cxcl5), chemotaxis (e.g. Ccr1) and other cell proliferation genes (e.g. Arg1, Pthlh)
butylated hydroxytoluene (BHT)-induced transcriptome changes between BALB vs. BALBLps-d mice during tumor promotion (Protocol 1, "Promotion") Three different gene expression profiles were identified
Summary
Adenocarcinoma (AC), a non-small cell lung carcinoma (NSCLC) [1], remains the leading type of lung cancer among smokers and nonsmokers. Inflammatory mediators and effector pathways might provide a source of early biomarkers and further insight into mechanisms of lung cancer. Multiple exposures to butylated hydroxytoluene (BHT) elicit lung injury and inflammation during tumor promotion that are significantly correlated with tumor multiplicity in mice [7,8] This 2-stage carcinogenesis model uses a low dose of a tobacco smokexs carcinogen, 3-methlycholanthrene (MCA), followed by multiple doses of BHT as the tumor promoter [9]. It is not BHT per se, but oxidative metabolites of BHT produced in high concentrations in mouse lung that are responsible for these pneumotoxicities and inflammatory activities [10]. This study was designed to identify TLR4-mediated gene expression pathways that may be used as prognostic indicators of susceptibility to lung tumorigenesis in mice and provide insight into the mechanism
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