Foxm1 in Lung Tumors Figure 2. Foxm1 deletion from lung epithelial cells prior to tumor initiation reduced the number and size of urethane-induced lung tumors. Experimental epFoxm12/2 (epFoxm1fl/fl+Dox) and control Foxm1fl/fl (Foxm1fl/fl+Dox) mice were given Dox in water to activate Cre expression prior to induction of lung tumors by multiple urethane injections as described in Materials and Methods. The second control group of epFoxm1fl/fl mice was given regular water (epFoxm1fl/fl – Dox; this mouse group was used as a control for Dox-independent recombination). All three groups of mice received six weekly injections of urethane to induce lung tumors. A. Experimental design of conditional deletion of Foxm1fl/fl prior to tumor induction. B. Lung tumors from experimental epFoxm12/2 (epFoxm1fl/fl+Dox) mice and control mice were counted and measured using a dissecting microscope at 20 weeks and 28 weeks after the first urethane injection. Deletion of Foxm1 prior to tumor induction significantly reduced (p,0.05) the total number of lung tumors at both time points. C–D. The tumor diameters were significantly reduced (p,0.05) at 20 weeks (C) and 28 weeks (D) after tumor induction. Mean number of tumors per lung (6SD) and tumor sizes were calculated from n = 12 mouse lungs per group. doi:10.1371/journal.pone.0006609.g002 genesis, the next series of experiments were designed to determine whether Foxm1 was required during progression/expansion of lung tumors in vivo. Lung tumors in epFoxm1fl/fl and control Foxm1fl/fl mice were induced by six urethane injections. One week after the last urethane injection, Dox-treatment was initiated and continued until the completion of the experiment at 20 or 28 weeks (Fig. 6A). Thus, Foxm1 deletion occurs after initiation of lung tumors. A significant decrease in the number of lung tumors was observed in Dox-treated epFoxm12/2 mice compared to either Dox-treated Foxm1fl/fl mice or epFoxm1fl/fl mice without Dox treatment (Fig. 6B). These results demonstrate that Foxm1 expression in the pulmonary epithelium is required for the progression/expansion of urethane-induced lung cancer. Foxm1 is essential for lung tumorigenesis induced by MCA/BHT To demonstrate that the established critical role of Foxm1 in lung epithelial cells is not limited only to urethane mediated tumorigenesis, we used another experimental mouse model of lung cancer: two-step lung tumor initiation/promotion using 3- methylcholanthrene (MCA)/butylated hydroxytoluene (BHT). Lung tumors in epFoxm12/2 and control male mice were induced by a single injection of MCA, a carcinogen found in tobacco smoke [26,27]. Starting one week after MCA injection, mice were subjected to weekly injections of BHT for 6 consecutive weeks to promote formation of lung tumors by causing chronic pulmonary inflammation and remodeling of the lung [26]. To induce Foxm1 deletion prior to tumor initiation, Dox was administered for 4 weeks prior to MCA injection (Fig. 7A). To avoid negative effects of Dox on lung inflammation, we did not administer Dox to mice during MCA/BHT injections. In another series of experiments, Dox was given one week after BHT treatment, causing Foxm1 deletion after tumor initiation (Fig. 7B). Both types of experiments demonstrated that epFoxm12/2 mice were resistant to lung tumorigenesis as demonstarated by comparison of tumor numbers in Foxm1-deficient mice to either control Foxm1fl/fl mice, or triple transgenic epFoxm1fl/fl mice without Dox treatment (Fig. 7A and 7B). The epFoxm12/2 lungs displayed only few microscopic tumor lesions. Immunohistochemistry demonstrated that decreased numbers and size of lung tumors in epFoxm12/2 lungs were associated with reduced Foxm1 expression (Fig. 8A–F) and decreased proliferation in tumor regions (Fig. 8G–L). Consistent with urethane experiments, MCA/BHT treated epFoxm12/2 lungs displayed decreased TOPO-2a, cyclin B1 and PPARc mRNAs, confirming a critical role of Foxm1 in the regulation of these genes in lung tumor cells in vivo (Fig. 7C). Altogether, our results demonstrate that Foxm1 in epithelial cells plays a critical role during chemically-induced and inflammation-mediated lung tumorigenesis. TOPO-2a is a direct target of Foxm1 transcriptional factor in lung epithelial cells Our in vivo experiments suggested that Foxm1 regulated lung epithelial genes essential for lung cancer formation. To determine whether Foxm1 regulates expression of these genes in vitro, A549 human lung adenocarcinoma cells were transfected with short PLoS ONE | www.plosone.org 4 August 2009 | Volume 4 | Issue 8 | e6609