secondary mutations that allowed tumor formation, bypassing proliferation defects in Foxm1-deficient lung tumor cells. The present study demonstrated that TOPO-2a was decreased after deletion of Foxm1 during pulmonary tumorigenesis and it was shown that Foxm1 directly induces the TOPO-2a promoter activity. Type II topoisomerases are ubiquitous enzymes that play essential roles in regulating DNA under- and over-winding, and resolving knots and tangles in the genetic material through transient double-stranded breaks [33]. Because TOPO-2a gener- ates DNA strand breaks, it has the potential to fragment the genome every time it functions and to cause significant genotoxic damage. TOPO-2a levels increase during the cell cycle, peaking in G2/M [34], in close correlation with the expression levels of Foxm1 transcription factor [7]. In the present study, we established that TOPO-2a expression was significantly decreased in Foxm1 deficient lung tumors as well as in A549 human lung adenocarcinoma cells transfected in vitro with Foxm1-specific siRNA. Chromatin immunoprecipitation assay demonstrated that Foxm1 protein directly binds to the mouse TOPO-2a promoter, suggesting that Foxm1 is a direct transcriptional activator of TOPO-2a gene. This is an important finding that can be used for designing the new therapeutic strategies to treat lung cancer. Deletion of Foxm1 in lung epithelial cells increased PPARa expression during tumorigenesis in vivo. PPARa mRNA was also increased in Foxm1-depleted A549 lung adenocarcinoma cells in vitro. PPARa is the member of the nuclear hormone receptor superfamily that acts as a ligand-activated transcription factor. Recent studies indicate that PPARa expression is increased in mouse or human medullablastoma cells, leading to the gradual accumulation of cells in G1 and G2/M phases of the cell cycle and inhibition of cell proliferation [35]. Since accumulation of cells in G1 and G2/M and inhibition of cell proliferation are main characteristics of Foxm1 deficiency [25], this recent data is consistent with our findings that increased PPARa mRNA in Foxm1 deficient tumors correlated with inhibition of cell prolifetartion. The other study showed that incidence of DEHP- induced hepatocellular carcinomas in PPARa-null mice was significantly higher than in wild type mice [36], suggesting negative relationship between PPARa expression and cell proliferation. Therefore, increased PPARa expression in Foxm1- deficient epithelial cells may contribute to reduced tumorigenesis in epFoxm12/2 mice. In summary, deletion of Foxm1 in respiratory epithelial cells prior to or even after tumor initiation caused a striking decrease in the number and size of lung tumors. Decreased tumor formation in epFoxm12/2 lungs was associated with diminished proliferation of tumor cells. Micro-dissected tumors from epFoxm12/2 lungs showed significant decrease in TOPO-2a mRNA expression. Foxm1 induced TOPO-2a expression in A549 lung adenocarcino- ma cells and directly bound to the TOPO-2a promoter region. The present data demonstrates that Foxm1 expression in respiratory epithelial cells is required for progression/expansion of chemically- induced lung cancer in vivo and provides support for the concept that Foxm1 functions in a cell autonomous manner during pulmonary carcinogenesis. Our studies suggest that Foxm1 represents a potential therapeutic target in treatment of NSCLC lung cancers. Materials and Methods Transgenic mice The generation of Foxm1flox/flox (Foxm1fl/fl) mice was described previously [11]. The Foxm1fl/fl mice were bred with SPC–rtTAtg/2/ TetO-Cretg/tg mice [23] to generate the SPC–rtTAtg/2/TetO-Cretg/2/ Foxm1 in Lung Tumors Foxm1fl/fl triple transgenic mice (epFoxm1fl/fl). Only male mice were used for tumor studies. To induce Cre expression in respiratory epithelium and produce epFoxm12/2 mice, doxycycline (Dox; 1% in drinking water) was given to 8 week old mice. Four weeks later, lung tumors were induced by urethane or MCA/BHT. Dox treatment was continued through the whole experiment (tumor induction and progression studies). In another series of experiments Dox was given starting 1 week after the last urethane or BHT injections until the day of tissue harvest (tumor progression studies). Dox-treated Foxm1fl/fl male littermates lacking either the SPC–rtTA, the TetO-Cre or both transgenes were used as controls. Further controls included Dox-treated SPC–rtTAtg/2/TetO-Cretg/2/Foxm1fl/+ male mice and SPC–rtTAtg/2/TetO-Cretg/2/Foxm1fl/fl male mice without Dox treatment (epFoxm1fl/fl – Dox group). Animal studies were reviewed and approved by the Animal Care and Use Committee of Cincinnati Children’s Hospital Research Foundation. Tumor induction protocols Urethane protocol. Control and epFoxm12/2 male mice were injected i.p. with 1 mg/g of body weight of urethane (Sigma, St. Louis, MO; diluted in saline) once a week for 6 consecutive weeks and tumors were counted and examined 20 and 28 weeks after the initial injection using a dissecting microscope. Individually microdissected lung tumors were used to prepare total RNA with RNA-STAT-60 (Tel-Test ‘‘B’’ Inc. Friendswood, TX). Lung tissues were fixed and embedded in paraffin blocks. MCA/BHT protocol. MCA (Sigma, St. Louis, MO), a polycyclic aromatic hydrocarbon found in tobacco smoke which serves as a tumor initiator, was given as a single i.p. dose of 15 mg/ g of body weight, followed by six weekly i.p. injections with BHT, a known tumor promoter, (200 mg/g of body weight; Sigma, St. Louis, MO). MCA and BHT were solubilized in corn oil. Mice were sacrificed at 25 weeks following MCA injection and examined for lung tumors using a dissecting microscope. Immunohistochemical staining and in situ hybridization Paraffin (5 mm) sections were stained with hematoxylin and eosin (H&E) for morphological examination or immunostained with antibodies against KI-67 (1:500; clone Tec-3, Dako), Cre (1:30,000; no. 69050–3, Novagen), SPC (1;1,500; AB-3428, Chemicon International), CCSP (1:1,000; a gift from B. Stripp, Duke University), TTF-1 (1:3,000, gift of Dr. DiLauro, Stazione Zoologica, Naples, Italy) or Foxm1 (1:2000; clone K-19; Santa Cruze Biotechnology, Santa Cruze, CA). Antibody-antigen com- plexes were detected using biotinylated secondary antibody followed by avidin-horseradish peroxidase (HRP) complex, and DAB substrate (all from Vector Lab, Burlingame, CA) as described [21]. Sections were counterstained with nuclear fast red (Vector Labs, Burlingame, CA). Lung sections were also used for in situ hybridization with 35S-labeled antisense riboprobe specific to 1649–1947 bp region of the mouse Foxm1 mRNA as described [7]. Quantitative real-time RT-PCR (qRT-PCR) Total RNA was prepared from microdissected lung tumors of epFoxm12/2 or control Foxm1fl/fl mice, or from A549 human lung adenocarcinoma cells (purchased from ATCC, Manassas, VA) and analyzed by qRT-PCR using the StepOnePlus Real-Time PCR system (Applied Biosystems, Foster City, CA). Samples were amplified with Taqman Gene Expression Master Mix (Applied Biosystems) combined with inventoried Taqman gene expression assays: mouse Foxm1, Mm00514924_m1; mouse cyclinB1, Mm00838401_g1; mouse PPARa, Mm00440939_m1; mouse PPARc, Mm00440945_m1; mouse TOPO-2a, Mm00495703_m1; mouse b-Actin, Mm00607939_s1; mouse c-Myc, Mm00487804_m1; PLoS ONE | www.plosone.org 11 August 2009 | Volume 4 | Issue 8 | e6609