Foxm1 in Lung Tumors Figure 8. epFoxm12/2 tumors displayed diminished Foxm1 staining and decreased proliferation after MCA/BHT induced tumorigenesis. Lungs from epFoxm12/2 and control Foxm1fl/fl mice, that were given Dox in water starting 4 weeks before MCA/BHT treatment, were dissected and fixed 25 weeks after the first injection. Lung sections were used for immunohistochemistry with either Foxm1 (A–F) or KI-67 antibodies (G–L). Nuclear Foxm1 staining (shown with arrows) was detected in a subset of tumor cells (Tu in A–B) and epithelial cells of peribronchial regions of Foxm1fl/fl lungs (C). Although Foxm1 protein was not detected in epFoxm12/2 tumors, Foxm1-positive epithelial cells were still found in alveolar region of epFoxm12/2 lungs (arrows in D and F). Diminished KI-67 staining was observed in epFoxm12/2 lungs (J–L) when compared to control Foxm1fl/fl lungs (G–I). Magnification is 1006. B, E, H and K are inserts from A, D, G and J. doi:10.1371/journal.pone.0006609.g008 and promotion of tumorigenesis [6]. In the second model, MCA, a carcinogen found in tobacco smoke [26], acts only as an initiator (DNA damage), while BHT, acts as a tumor promoter by causing chronic pulmonary inflammation due to necrosis of type I epithelial cells and macrophage infiltration. Both models showed that Foxm1 is critical for lung tumorigenesis. Consistent with these findings, the expression of Foxm1 in colon epithelial cells [30] and hepatocytes [18] was essential for progression of colon cancer and hepatocellular carcinoma, respectively. Moreover, the present data suggest that the main role of Foxm1 in epithelial cells occurs during tumor progression/expansion. We found a similar decrease in lung tumorigenesis regardless if Foxm1 was deleted prior to the tumor initiation or during tumor progression. Therefore, Foxm1 is critical for the proliferation of tumor cells during the expansion of lung tumors. Since lung tumors were still found in urethane-treated epFoxm12/2 mice, our results suggest that a subset of lung tumor cells can proliferate in the absence of Foxm1. Interestingly, the epFoxm12/2 tumors still maintained normal expression levels of the TTF-1 protein, a lung epithelial-specific transcription factor, implicated in controlling cellular proliferation during embryogen- esis and formation of non-small cell lung cancer [24]. Published studies demonstrated that increased TTF-1 expression and amplification of TTF-1 gene occurred in many cases of NSCLC in human patients [31,32]. We also found that epFoxm12/2 tumors displayed normal expression levels of the cell cycle promoting c- Myc and Cyclin D1. Our results suggest that TTF-1, c-Myc and Cyclin D1 can contribute to maintaining the low proliferation rates in epFoxm12/2 tumors. Alternatively, it is also possible that the formation of lung tumors in epFoxm12/2 mice may result from PLoS ONE | www.plosone.org 9 August 2009 | Volume 4 | Issue 8 | e6609