protocol [21]. Although these studies demonstrated a critical role of Foxm1 in lung tumorigenesis, specific requirements for this transcription factor in different populations of respiratory cells remain unknown. Since lung cancer lesions contain a heteroge- neous population of cells, that includes tumor cells, originated from genetically modified epithelial cells, and different inflamma- tory and stromal cells [22], it is important to know the cell autonomous role of Foxm1 during lung tumorigenesis. In the present study, transgenic mice were generated in which the Foxm1 gene was conditionally deleted in lung epithelial cells (epFoxm12/2 mice) either prior to the initiation of chemically-induced lung cancer, or during cancer progression/expansion. Deletion of Foxm1 from epithelial cells caused a striking decrease in the number and size of lung adenomas. Decreased lung tumorigenesis in epFoxm12/2 mice was associated with diminished proliferation of tumor cells and reduced expression of Topoisomerase-2a (TOPO- 2a), a critical regulator of tumor cell proliferation. Depletion of Foxm1 mRNA in cultured lung adenocarcinoma cells significantly decreased TOPO-2a mRNA and protein. Foxm1 directly bound to and induced transcription of the mouse TOPO-2a promoter region, indicating that TOPO-2a is a direct transcriptional target of Foxm1. Taken together, our data demonstrate that Foxm1 expression in respiratory epithelial cells is critical for expansion of lung cancer and TOPO-2a expression in lung tumor cells. Results Conditional deletion of Foxm1 transcription factor in lung epithelial cells To determine the role of Foxm1 in epithelial cells during formation of lung tumors, we generated triple-transgenic mice containing LoxP-flanked exons 4–7 of the Foxm1 gene (Foxm1fl/fl), the SP-C–rtTAtg/2 and the TetO-Cretg/2 transgenes (SP-C–rtTAtg/2/ TetO-Cretg/2/Foxm1fl/fl or epFoxm1fl/fl mice). To induce Cre expression in lung epithelial cells, Dox was given to the adult mice in water for 4 weeks. Previous studies demonstrated that using this protocol, Cre-mediated recombination occurs in type II epithelial cells and Clara cells of the adult lung [23]. In the presence of Dox, the reverse tetracycline transactivator (rtTA) binds to the TetO promoter and induces expression of Cre recombinase, deleting exons 4–7, which encode DNA binding and transcriptional activation domains of the Foxm1 protein (Fig. 1A); thus, lung epithelial-specific Foxm1 knockout mice (epFoxm12/2) are generated. Consistent with previous studies, Cre protein was detected in epithelial type II (Fig. 1B) and Clara cells (Fig. 1C) of Dox-treated epFoxm12/2 mice but not in control Foxm1fl/fl mice (Fig. 1B–C). No morphological changes were observed in either epFoxm12/2 or control Foxm1fl/fl lungs after 4 weeks of Dox treatment (Fig. 1D). Thus, the Foxm1 deletion from adult lungs does not alter lung structure. Foxm1 deletion from respiratory epithelial cells reduces numbers and sizes of lung tumors To determine the role of Foxm1 in epithelial cells during formation of lung tumors, experimental epFoxm1fl/fl and control male mice were given Dox for 4 weeks. Control groups included Dox-treated Foxm1fl/fl male mice and epFoxm1fl/+mice, as well as epFoxm1fl/fl transgenic mice without Dox treatment. Mice were subjected to 6 weekly urethane injections to induce lung tumors (Fig. 2A). Dox treatment was continued for additional 20 or 28 weeks after the first urethane injection. In this model, deletion of Foxm1 from lung epithelial cells was induced prior to the initiation of lung tumorigenesis. Foxm1 in Lung Tumors The total number of urethane-induced lung tumors was decreased approximately 5-fold in epFoxm12/2 (epFoxm1fl/fl+Dox) mice compared to all control groups (Fig. 2B and data not shown). Numbers of large (.2 mm), medium (1–2 mm) and small-sized (,1 mm) tumors were significantly decreased in epFoxm12/2 mice at both 20 and 28 weeks after the first urethane injection (Fig. 2C– D and Fig. 3A). Histological examination of H&E-stained sections confirmed the reduced size of lung tumors from epFoxm12/2 mice and showed that these tumors displayed morphological charac- teristics of lung adenomas (Fig. 3B). To identify the origin of the lung tumor cells, the immunohistochemical staining was per- formed using antibodies against either surfactant protein C (SPC), a type II lung alveolar epithelial cell marker, or Clara cell specific protein (CCSP), Clara cells marker. All tumors in control and epFoxm12/2 mice were SPC- positive (Fig. 3C), indicating that they developed from alveolar type II epithelial cells. No CCSP- positive tumors were found in either epFoxm12/2 or control epFoxm1fl/fl lungs (Fig. 3D). Interestingly, epFoxm12/2 tumors maintained normal expression levels of TTF-1, a lung epithelial- specific transcription factor, implicated in controlling cellular proliferation during embryogenesis and formation of non-small cell lung cancer [24]. Foxm1 deletion from respiratory epithelial cells diminishes tumor cell proliferation To address the efficiency of deletion of the Foxm1fl/fl allele from lung tumors, Foxm1 mRNA was examined by in situ hybridization of lung paraffin sections. Consistent with efficient Cre-mediated recombination, Foxm1 mRNA was decreased in lung tumors from epFoxm12/2 mice compared to Foxm1fl/fl mice (Fig. 4A and 4B). Furthermore, numbers of KI-67 positive cells were significantly reduced in epFoxm12/2 tumors (Fig. 4C–D and 4E). We did not observe any differences in the number of apoptotic cells in the tumors from control and epFoxm12/2 mice (data not shown). Altogether, these results indicate that Foxm1 deletion from type II lung epithelial cells is sufficient to reduce proliferation of tumor cells in vivo and decrease urethane-mediated lung tumorigenesis. Tumors from epFoxm12/2 mice demonstrate altered expression of genes important for cellular proliferation and lung tumorigenesis To identify new molecular targets of Foxm1 in respiratory epithelial cells, lung tumors were dissected from control and urethane-treated mice and then used to prepare total RNA. Even with the contribution of non-tumor cell RNA (endothelial cells, macrophages, fibroblasts), the Real-time RT-PCR analysis revealed decreased Foxm1 mRNA levels in epFoxm12/2 tumors compared to control Foxm1fl/fl tumors (Fig. 5A). Foxm1 deficiency in epFoxm12/2 tumors was associated with decreased mRNA levels of cyclin B1 (Fig. 5A), a known transcriptional target for Foxm1 [25]. Expression of several genes important for cellular prolifer- ation and lung carcinogenesis was also studied, including c-Myc, cyclin D1, TOPO-2a, TCF4, VEGF-A, TTF-1, PPARc and PPARa. While TOPO-2a and PPARc mRNA were decreased in lung tumors from epFoxm12/2 mice, PPARa mRNAs was significantly increased (Fig. 5A). No changes were found in the mRNA expression of c-Myc, cyclin D1, TCF4, TTF-1 and VEGF-A genes (Fig. 5B). Foxm1 deletion after tumor initiation decreases lung tumor expansion/progression Having established that deletion of Foxm1 from lung epithelial cells prior to tumor initiation significantly reduced lung tumori- PLoS ONE | www.plosone.org 2 August 2009 | Volume 4 | Issue 8 | e6609