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 expression 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-2α. Foxm1 deficiency was also associated with increased expression of PPAR-α. 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 
. 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 
. On the contrary, over-expression of Foxm1 in all cell types using Rosa 26 promoter led to striking increase in the numbers and sizes of lung tumors induced with MCA/BHT in transgenic mice 
. Thus, previous gain-of-function or loss-of-function studies with ubiquitous changes in Foxm1 expression had identified Foxm1 as an important transcription factor during lung tumorigenesis. However, lung cancer lesions contain a heterogeneous population of cells, that includes epithelial, inflammatory (macrophages, granulocytes) and stromal cells, that express increased levels of Foxm1 
and may influence tumorigenesis. Although our previous studies emphasized the essential role of Foxm1 in lung tumorigenesis, its specific role in lung epithelial cells, the precursors of lung adenoma or adenocarcinoma cells, was not addressed. In the present study, deletion of Foxm1 from respiratory epithelial cells was sufficient to significantly decrease lung tumor formation, demonstrating the cell autonomous role of Foxm1 in the progression of pulmonary tumors.
Two different models of lung cancer in mice were used. In the first model, urethane acts as a complete carcinogen leading to DNA damage with K-ras mutations and providing both initiation and promotion of tumorigenesis 
. In the second model, MCA, a carcinogen found in tobacco smoke 
, 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 
and hepatocytes 
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 epFoxm1−/−
mice, our results suggest that a subset of lung tumor cells can proliferate in the absence of Foxm1. Interestingly, the epFoxm1−/−
tumors still maintained normal expression levels of the TTF-1 protein, a lung epithelial-specific transcription factor, implicated in controlling cellular proliferation during embryogenesis and formation of non-small cell lung cancer 
. Published studies demonstrated that increased TTF-1 expression and amplification of TTF-1
gene occurred in many cases of NSCLC in human patients 
. We also found that epFoxm1−/−
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 epFoxm1−/−
tumors. Alternatively, it is also possible that the formation of lung tumors in epFoxm1−/−
mice may result from secondary mutations that allowed tumor formation, bypassing proliferation defects in Foxm1-deficient lung tumor cells.
The present study demonstrated that TOPO-2α
was decreased after deletion of Foxm1 during pulmonary tumorigenesis and it was shown that Foxm1 directly induces the TOPO-2α
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 
. Because TOPO-2α generates DNA strand breaks, it has the potential to fragment the genome every time it functions and to cause significant genotoxic damage. TOPO-2α levels increase during the cell cycle, peaking in G2/M 
, in close correlation with the expression levels of Foxm1 transcription factor 
. In the present study, we established that TOPO-2α
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-2α promoter, suggesting that Foxm1 is a direct transcriptional activator of TOPO-2α
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 PPARα
expression during tumorigenesis in vivo
mRNA was also increased in Foxm1-depleted A549 lung adenocarcinoma cells in vitro
. PPARα is the member of the nuclear hormone receptor superfamily that acts as a ligand-activated transcription factor. Recent studies indicate that PPARα
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 
. Since accumulation of cells in G1 and G2/M and inhibition of cell proliferation are main characteristics of Foxm1 deficiency 
, this recent data is consistent with our findings that increased PPARα
mRNA in Foxm1 deficient tumors correlated with inhibition of cell prolifetartion. The other study showed that incidence of DEHP-induced hepatocellular carcinomas in PPARα-null mice was significantly higher than in wild type mice 
, suggesting negative relationship between PPARα expression and cell proliferation. Therefore, increased PPARα
expression in Foxm1-deficient epithelial cells may contribute to reduced tumorigenesis in epFoxm1−/−
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 epFoxm1−/− lungs was associated with diminished proliferation of tumor cells. Micro-dissected tumors from epFoxm1−/− lungs showed significant decrease in TOPO-2α mRNA expression. Foxm1 induced TOPO-2α expression in A549 lung adenocarcinoma cells and directly bound to the TOPO-2α 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.