Foxm1 in Lung Tumors Figure 5. Deletion of Foxm1 influenced the expression of several genes. Microdissected tumors from control Foxm1fl/fl+Dox or experimental epFoxm1fl/fl+Dox mouse lungs (n = 3 mice per group) were used to prepare total RNA for Quantitative real-time RT-PCR. A. Decreased Foxm1, TOPO- 2a, cyclin B1, PPARc and increased PPARa mRNAs were observed in epFoxm12/2 (epFoxm1fl/fl+Dox) lung tumors. B. No changes were found in the expression of the c-Myc, TCF-4, cyclin D1, TTF-1 and VEGF-A genes. b-actin mRNA was used for normalization. A p value,0.05 is shown with asterisk (*). doi:10.1371/journal.pone.0006609.g005 itated (IP) either with antibodies specific to Foxm1 or with IgG control antibodies. Binding of TOPO-2a promoter DNA associated with the IP chromatin was determined by qRT-PCR with primers specific to the potential Foxm1-binding region in mouse TOPO-2a Figure 6. Foxm1 deletion after tumor initiation decreases lung tumor expansion/progression. Experimental epFoxm12/2 (epFoxm1fl/fl+Dox) and control Foxm1fl/fl (Foxm1fl/fl+Dox) mice were given Dox in water to activate Cre expression after the initiation 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). All three groups of mice received six weekly injections of urethane to induce lung tumors. A. Experimental design of conditional deletion of Foxm1fl/fl after 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 after tumor initiation significantly reduced (p,0.05) the total number of lung tumors at both time points. doi:10.1371/journal.pone.0006609.g006 promoter. Foxm1 protein specifically bound to the TOPO-2a promoter region as demonstrated by the ability of siFoxm1 to reduce binding of Foxm1 protein to TOPO-2a promoter DNA (Fig. 9D). To determine whether the Foxm1-binding sites were transcriptionally active, co-transfection experiments were per- formed using CMV-Foxm1b expression vector [29] and luciferase (LUC) reporter construct driven by TOPO-2a promoter region. Co-transfection of the CMV-Foxm1b expression vector signifi- cantly increased expression of the –1.5 Kb TOPO-2a reporter plasmid when compared to CMV-empty vector (Fig. 9E), indicating that Foxm1 is a transcriptional activator of TOPO-2a gene. These results demonstrate that Foxm1 directly binds to and transcriptionally activates the mouse TOPO-2a promoter region, indicating that TOPO-2a is a direct Foxm1 target gene. Discussion Existing treatments for lung cancer have not significantly improved survival, leading to a critical need for new approaches. The important role of Foxm1 in lung cancer has already been established. However, our basic understanding of its role in different cell populations of the tumor is limited. Furthermore, the molecular mechanisms whereby cell autonomous Foxm1 expres- sion regulates lung tumorigenesis remain to be established. The important contribution of the current study is the establishment of the critical role of Foxm1 in respiratory epithelial cells during formation of lung cancer. The conditional deletion of Foxm1 from lung epithelial cells strikingly decreased the development of lung tumors in mice, diminished proliferation of tumor cells and reduced the expression of cyclin B1 and TOPO-2a. Foxm1 deficiency was also associated with increased expression of PPAR-a. Altogether, these observations provide new insight into a cell autonomous role of Foxm1 in the pathogenesis of pulmonary adenocarcinoma, and identify the Foxm1 transcription factor as a potential target for treatment of human lung cancer. Increased Foxm1 levels were found in numerous types of human tumors, including non small cell lung cancer [14,20]. Our previous studies demonstrated that when the Foxm fl/fl allele was deleted in all cell types using Mx-Cre transgene, the numbers and sizes of lung adenomas following urethane exposure were reduced [20]. On the contrary, over-expression of Foxm1 in all cell types using Rosa 26 promoter led to striking increase in the numbers PLoS ONE | www.plosone.org 7 August 2009 | Volume 4 | Issue 8 | e6609