Deletion of Forkhead Box M1 Transcription Factor from Respiratory Epithelial Cells Inhibits Pulmonary Tumorigenesis I-Ching Wang1, Lucille Meliton 2 , Xiaomeng Ren 1 , Yufang Zhang 1 , David Balli1, Jonathan Snyder1, Jeffrey A. Whitsett1, Vladimir V. Kalinichenko1*, Tanya V. Kalin1* 1 Division of Pulmonary Biology and the Perinatal Institute, Cincinnati Children’s Hospital Research Foundation, Cincinnati, Ohio, United States of America, 2 Department of Medicine, University of Chicago, Chicago, Illinois, United States of America Abstract The Forkhead Box m1 (Foxm1) protein is induced in a majority of human non-small cell lung cancers and its expression is associated with poor prognosis. However, specific requirements for the Foxm1 in each cell type of the cancer lesion remain unknown. The present study provides the first genetic evidence that the Foxm1 expression in respiratory epithelial cells is essential for lung tumorigenesis. Using transgenic mice, we demonstrated that conditional deletion of Foxm1 from lung epithelial cells (epFoxm12/2 mice) prior to tumor initiation caused a striking reduction in the number and size of lung tumors, induced by either urethane or 3-methylcholanthrene (MCA)/butylated hydroxytoluene (BHT). 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 levels. Moreover, Foxm1 directly bound to and induced transcription of the mouse TOPO-2a promoter region, indicating that TOPO-2a is a direct target of Foxm1 in lung tumor cells. Finally, we demonstrated that a conditional deletion of Foxm1 in pre-existing lung tumors dramatically reduced tumor growth in the lung. Expression of Foxm1 in respiratory epithelial cells is critical for lung cancer formation and TOPO- 2a expression in vivo, suggesting that Foxm1 is a promising target for anti-tumor therapy. Citation: Wang I-C, Meliton L, Ren X, Zhang Y, Balli D, et al. (2009) Deletion of Forkhead Box M1 Transcription Factor from Respiratory Epithelial Cells Inhibits Pulmonary Tumorigenesis. PLoS ONE 4(8): e6609. doi:10.1371/journal.pone.0006609 Editor: Mikhail V. Blagosklonny, Roswell Park Cancer Institute, United States of America Received June 8, 2009; Accepted July 10, 2009; Published August 12, 2009 Copyright: 2009 Wang et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by the Research Grant from the American Cancer Society, Ohio Division (TVK), the Grant from Concern Foundation 84794 (TVK), US Public Health Service Grant HL 84151-01 (VVK), Career Development Award from National Lung Cancer Partnership (ICW) and Research Scholar Grant RSG-06-187-01 from the American Cancer Society, National office (VVK). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: kal4ti@cchmc.org (TVK); Vladimir.Kalinichenko@cchmc.org (VVK) Introduction Lung cancer is the leading cause of cancer-related deaths in men and women in the United States [1]. It has a high mortality because it is difficult to detect early and is frequently resistant to available chemotherapy and radiotherapy. Therefore, identification of proteins regulating the proliferation of lung tumor cells will provide novel targets for diagnosis and treatment of human lung cancer. Lung cancer is classified into small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC). Adenocarcinoma, the most common type of NSCLC, is frequently associated with gain of function mutations in the K-Ras oncogene [2,3], activation of c-myc protein, as well as the loss of function mutations in the tumor suppressor gene p53 [4,5]. Activating mutations in K-Ras also occur in the majority of spontaneous and chemically induced lung tumors in mice [6]. The Foxm1 transcription factor is broadly expressed in actively proliferating cells of all origins [7]. Activation of the Ras-MAPK signaling pathway drives cell cycle progression by regulating the temporal expression of Cyclin regulatory subunits, that activate their corresponding Cyclin-dependent kinases (Cdk) through complex formation. Cdk/cyclin complexes phosphorylate and activate a variety of cell cycle regulatory proteins, including Foxm1 [2,5,8]. Activated MAPK (ERK) kinase directly phosphorylates the Foxm1 protein, contributing to its transcriptional activation [9]. Foxm1 directly stimulates the transcription of genes essential for progression into DNA replication and mitosis, including cyclin B1, Cdc25B phosphatase, Aurora B kinase and Polo-like kinase 1 [10]. Foxm12/2 mice exhibit embryonic lethality due to severe proliferation defects in the developing heart, liver, and blood vessels [11]. Consistent with an important role of Foxm1 in cell cycle progression, increased expression of Foxm1 was found in human lung adenocarcinomas and squamous cell carcinomas, prostate adenocarcinomas, basal cell carcinomas, intrahepatic cholangio- carcinomas, anaplastic astrocytomas and glioblastomas, infiltrating ductal breast carcinomas, as well as in many other solid tumors (reviewed in [10,12,13,14]). Inhibition of Foxm1 in cultured tumor cells with either siRNA transfection or pharmacological agents caused a cell cycle arrest [15,16,17,18,19]. Previous studies with Mx-Cre Foxm1fl/fl mice demonstrated that deletion of Foxm1 from all cell types of the body caused a significant reduction in number and size of lung adenomas induced by urethane [20]. Further- more, over-expression of Foxm1 in all cell types in Rosa26-Foxm1 transgenic mice significantly increased the number and size of lung tumors induced by MCA/BHT lung tumor induction/promotion PLoS ONE | www.plosone.org 1 August 2009 | Volume 4 | Issue 8 | e6609