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Foxm1 Transcription Factor is required for Macrophage Migration during Lung Inflammation and Tumor Formation

Macrophages play a key role in tumor-associated pulmonary inflammation that supports proliferation of tumor cells and promotes lung tumor growth. Although increased numbers of tumor-associated macrophages (TAM) are linked to poor prognosis in lung cancer patients, little is known regarding the transcriptional mechanisms controlling recruitment of macrophages during lung tumorigenesis. Forkhead Box m1 (Foxm1) transcription factor is induced in multiple cell types within tumor lesions and its increased expression is associated with poor prognosis in patients with lung adenocarcinomas. To determine the role of Foxm1 in recruitment of TAM, a mouse line with macrophage-specific Foxm1 deletion was generated (macFoxm1−/−). Lung tumorigenesis was induced using a MCA/BHT tumor initiation/promotion protocol. Ablation of Foxm1 in macrophages reduced the number and size of lung tumors in macFoxm1−/− mice. Decreased tumorigenesis was associated with diminished proliferation of tumor cells and decreased recruitment of macrophages during the early stages of tumor formation. Expression levels of pro-inflammatory genes iNOS, Cox-2, IL-1b and IL-6, as well as migration related genes MIP-1α, MIP-2 and MMP-12, were decreased in macrophages isolated from macFoxm1−/− mice. Migration of Foxm1-deficient macrophages was reduced in vitro. The chemokine receptors responsible for monocyte recruitment to the lung, CX3CR1 and CXCR4, were decreased in Foxm1-deficient monocytes. In co-transfection experiments, Foxm1 directly bound to and transcriptionally activated CX3CR1 promoter. Adoptive transfer of wild type monocytes to macFoxm1−/− mice restored BHT-induced pulmonary inflammation to the levels observed in control mice. Expression of Foxm1 in macrophages is required for pulmonary inflammation, recruitment of macrophages into tumor sites and lung tumor growth.


INTRODUCTION
Foxm1 is a member of the Forkhead box (Fox) family of transcription factors, which is expressed in all proliferating cells, including epithelial cells, macrophages and endothelial cells (Korver et al 1997, Yao et al 1997, Ye et al 1997, Zhao et al 2006. Consistent with an important role of Foxm1 in cell cycle progression, increased expression of Foxm1 was found in many human tumors (reviewed in , Myatt and Lam 2007 and ). Increased Foxm1 expression in human lung adenocarcinomas and squamous cell carcinomas was associated with increased proliferation of tumor cells (Kim et al 2006). Transgenic over-expression of Foxm1 using Rosa26-Foxm1 mice significantly increased lung tumorigenesis induced by 3-methylcholanthrene (MCA)/ butylated hydroxytoluene (BHT) . Ubiquitous deletion of Foxm1 in Mx-Cre/Foxm1 fl/fl mice significantly reduced the number and size of urethane-induced lung adenomas (Kim et al 2006). Although these studies demonstrated a critical role of Foxm1 in lung tumorigenesis, specific requirements for Foxm1 in distinct cell populations within the lung remain unclear. Recently, we demonstrated that conditional deletion of Foxm1 in respiratory epithelial cells, the precursors of tumor cells, caused a striking delay in initiation and progression of lung tumors in vivo .
Tumor lesions consist of tumor cells as well as stromal and inflammatory cells which contribute to the tumor promoting microenvironment (de Visser et al 2006). The relationship between inflammation and cancer pathogenesis has been extensively studied Werb 2002, Porta et al 2009). Epithelial neoplasia is initiated by mutations, but cells often remain dormant until promotional events recruit and activate inflammatory cells within the lesion. Activated inflammatory cells stimulate growth and progression of epithelial-derived tumors (Balkwill and Mantovani 2001). Accumulating evidence has shown a clear correlation between increased numbers of infiltrating macrophages and a poor prognosis in various mouse and human malignancies, including colon, breast, lung, and prostate cancers (Baron and Sandler 2000, Bingle et al 2002, Borsig et al 2002, Ernst and Gold 2000.
While recent studies have focused on cytokine/chemokine-dependent signaling mechanisms critical for macrophage recruitment to tumors, the transcriptional regulation of this process is relatively unknown. The present study was designed to determine the cell specific role of Foxm1 in macrophages during formation of lung tumors using a transgenic mouse line with macrophage-specific Foxm1 deletion.

Foxm1 deficiency in myeloid cells does not alter the number of macrophages in untreated lungs
To address the macrophages-specific role of Foxm1, we generated conditional knockout mice containing LoxP-flanked exons 4-7 of the Foxm1 gene (Foxm1 fl/fl ) and LysM-Cre transgene (LysM-Cre/Foxm1 fl/fl or macFoxm1 −/− mice). In LysM-Cre mice, Cre is expressed in myeloid cells due to targeted insertion of Cre cDNA into the endogenouse lysozyme M gene locus. Previous lineage-tracing studies demonstrated that Cre-mediated recombination in LysM-Cre mice occurs in 85-98% of macrophages and 100% of neutrophils (Clausen et al 1999). macFoxm1 −/− mice were fertile with no obvious abnormalities. Deletion of Foxm1 from macrophages and granulocytes did not change the morphological structure of the lung, and alveolar macrophages were present both in control Foxm1 fl/fl and macFoxm1 −/− lungs (Fig. 1A). Immunostaining for macrophage-specific Mac-3 antigen demonstrated that the total number of alveolar macrophages in control and macFoxm1 −/− mice were similar ( Fig.  1A-B). In peripheral blood, no differences were found in either total leukocyte counts or differential cell counts (Fig. 1D, left and middle panel). Flow cytometry analysis of the peripheral blood confirmed that the number of monocytes [CD11b + Ly-6C + Ly-6G − MCSFR + cells] and granulocytes [CD11b + Ly-6C + Ly-6G + MCSFR − cells] was similar in the macFoxm1 −/− and control mice (4.0 ± 0.3% vs 3.7 ± 0.6% for monocytes; and 46.4 ± 3.2% vs 49 ± 4.5% for granulocytes). Flow cytometry analysis of bone marrow (BM) cells showed no significant changes in the numbers of monocytes and granulocytes between macFoxm1 −/− and control mice (Fig. 1D, right panel). Furthermore, similar numbers of monocytes and macrophages (Supplemental Fig. 1) were present in the bone marrow, blood and lungs of 7 month old control and macFoxm1 −/− mice, suggesting that Foxm1 deletion did not influence the total number of these cells in older animals.
Broncho-alveolar lavage (BAL)-derived cells consisted primarily of macrophages (>95%). Both viability and cell numbers of BAL macrophages were similar in control and macFoxm1 −/− mice (data not shown). To determine the efficiency of Foxm1 deletion, Foxm1 mRNA in BAL-derived macrophages was examined by qRT-PCR. Compared to control mice, a 90% decrease in Foxm1 mRNA was found in BAL macrophages from macFoxm1 −/− mice (Fig. 1C), confirming the efficient Foxm1 deletion from macrophages.

Foxm1 deletion from macrophages reduced the number and size of lung tumors induced by MCA/BHT
To determine the role of Foxm1 in tumor-associated macrophages, experimental macFoxm1 −/− and control male mice were subjected to a 3-methylcholanthrene (MCA)/ butylated hydroxytoluene (BHT) lung tumor induction/promotion protocol. MCA is a mutagen found in tobacco smoke, which serves as a tumor initiator. BHT is a phenolic antioxidant, which promotes lung tumors by inducing pulmonary inflammation with macrophage infiltration. (Adamson et al 1977, Marino andMitchell 1972). Thirty weeks after MCA injection, mouse lungs were harvested and examined for lung tumors. The total number of lung tumors and tumor size' were significantly decreased in macFoxm1 −/− mice compared to control Foxm1 fl/fl mice ( Fig. 2A). Thus, Foxm1 deletion from macrophages was sufficient to decrease lung tumor formation after MCA/BHT treatment.

The number of macrophages in macFoxm1 −/− tumors was decreased
Histological examination confirmed the reduced size of lung tumors in macFoxm1 −/− mice and showed that these tumors displayed morphological characteristics of lung adenomas (Fig. 2B). The number of Ki-67 positive tumor cells was significantly decreased in the macFoxm1 −/− lungs compared to control Foxm1 fl/fl lungs (Fig. 2C). No differences were found in the number of apoptotic cells in tumors from control and macFoxm1 −/− mice (data not shown). Interestingly, tumors from macFoxm1 −/− mice contained fewer tumorinfiltrating macrophages, as demonstrated by immunostaining for Mac3 (Fig. 2D). No significant differences in the number of neutrophils were found in the macFoxm1 −/− tumors (data not shown), indicating that Foxm1 deletion did not influence neutrophil recruitment into tumor sites. The percentage of peripheral blood monocytes present in control and macFoxm1 −/− mice after MCA/BHT treatment was also not significantly different (Supplemental Fig. 2). The percentage of monocytes in BM of macFoxm1 −/− animals was increased, suggesting that the decreased number of macrophages in macFoxm1 −/− lungs were not the attributed to decreased monocyte generation in the bone marrow. Thus, deletion of Foxm1 from macrophages impaired tumor growth, decreased proliferation of tumor cells and reduced the number of tumor-infiltrating macrophages.

macFoxm1 −/− mice are resistant to BHT-induced lung inflammation
Previous studies demonstarted that BHT promotes lung tumorigenesis by inducing pulmonary inflammation and macrophage infiltration into the lung tissue (Blaine et al 2005, Malkinson et al 1997. To assess the effect of Foxm1 deletion on pulmonary inflammation at early stages of tumor promotion, lungs of macFoxm1 −/− mice and control littermates were collected 2-5 days after the first BHT injection. Lung inflammation was examined by H&E staining. Reduced number of inflammatory cells in the alveolar region, diminished lung congestion and decreased thickening of the alveolar septae were observed in macFoxm1 −/− mice at 2 and 5 days after BHT treatment ( Figure 3A). A decrease in the number of inflammatory cells was also observed in the perivascular and peribronchial areas of macFoxm1 −/− lungs (Fig. 3B). qRT-PCR analysis of total lung RNA demonstrated decreased mRNA levels of several inflammatory mediators, including Cox2, MIP-1α, iNOS, IL-6 and MIP-2 in macFoxm1 −/− lungs (Fig. 3C). However, systemic inflammation after BHT treatment was similar in control and macFoxm1 −/− mice, as indicated by similar protein levels of Cox-2, IL-6, IL-1b, CCL2 and CCL3 in blood serum from these mice (Fig.  3D). These results indicate that macrophage-specific deletion of Foxm1 reduced the severity of pulmonary inflammation at early stages of tumor promotion without changing the systemic inflammatory response to BHT treatment.
A decreased number of inflammatory cells was observed in BAL from BHT-treated macFoxm1 −/− lungs (Fig. 4C). While the number of macrophages was significantly reduced, the number of neutrophils and lymphocytes were not changed (Fig. 4C). These results are consistent with the diminished lung inflammation in macFoxm1 −/− mice found by histological evaluation (Fig. 3A-B) and by gene expression profiling (Fig. 3C). The number of macrophages in lung tissue was determined by immunohistochemistry with macrophagespecific Mac3 antibodies. Two and 5 days after BHT injection, the number of Mac3-positive cells was significantly decreased in macFoxm1 −/− lungs when compared to controls (Fig. 4A and 4B). Thus, deletion of Foxm1 from myeloid cells reduced the number of macrophages in BHT-treated lungs without influencing the number of neutrophils or lymphocytes.

Proliferation of macFoxm1 −/− macrophages is reduced
Foxm1 protein was not detected in quiescent lungs by immunochistochemistry, but was dramatically induced after BHT-induced lung injury in both alveolar macrophages and respiratory epithelial cells of control

Foxm1 deficiency reduced macrophage migration in vitro
The observed decrease in the number of macrophages in the macFoxm1 −/− lung tumors could be the result of impaired macrophage migration. Macrophages were isolated from macFoxm1 −/− or control mice, and in vitro migration was assessed using a Transwell invasion chamber assay. The purity of isolated macrophages was similar (91.4% ± 4.1 for control macrophages, and 87.8% ± 5.7 for macFoxm1 −/− macrophages) as demonstrated by flow cytometry. The viability of isolated macrophages was similar for both groups of mice. Reduced Foxm1 mRNA ( Fig. 6A) was associated with a decreased migration of macFoxm1 −/− macrophages through the chamber membrane ( Fig. 6B). When RNA was prepared from control and macFoxm1 −/− macrophages that were able to migrate through the membrane, no differences were found in mRNA levels of Foxm1 or its targets, Cyclin B1, Cyclin D1 and Cdc25b (Supplemental Fig. 3B). These results suggest that macFoxm1 −/− macrophages that migrated through the Transwell membrane had incomplete Foxm1 deletion. Thus, Foxm1 is essentialfor macrophage migration in vitro.

Foxm1 regulates expression of genes critical for pulmonary inflammation and macrophage migration
To identify potential target genes regulated by Foxm1 in macrophages, BAL macrophages from macFoxm1 −/− and control mice were used to prepare total RNA. In control mice, BHT treatment induced Foxm1 expression in BAL macrophages, coinciding with increased expression of pro-inflammatory genes (Fig. 6C). However, in macFoxm1 −/− mice, BHT Balli et al. Page 5 Oncogene. Author manuscript; available in PMC 2013 February 23. treatment failed to induce Foxm1 expression in macrophages to the same levels as in control mice, being consistent with efficient Foxm1 deletion (Fig. 6C). Foxm1 deficiency in BHTtreated macFoxm1 −/− BAL macrophages was associated with decreased mRNA levels of Cdc25b phosphatase and Cox-2, both of which are direct Foxm1 target genes (Wang et al 2005. Reduced expression of Cdc25b and Cox-2 was also observed in macFoxm1 −/− macrophages isolated from lung tissues of BHT-treated mice by flow cytometry-based cell sorting (Fig. 6D).
mRNAs encoding inflammatory mediators, iNOS and IL-6, were significantly reduced in both BAL macrophages (Fig. 6C) and pulmonary macrophages ( Fig. 6D) of BHT-treated macFoxm1 −/− mice ( Fig. 6C-D). Generation of reactive oxygen and nitrogen species by macrophages during chronic inflammation is known to cause genomic alterations in epithelial cells promoting tumorigenesis (Kanwar et al 2009). Thus, the reduced expression of iNOS and other pro-inflammatory genes may contribute to decreased tumorigenesis observed in macFoxm1 −/− mice. However, no differences were found in mRNA levels of the pro-inflammatory IL-1α and IL-1β cytokines ( Fig. 6D), suggesting that Foxm1 does not regulate expression of these genes. Arginase1 and IL-12 mRNAs were not changed (

CX 3 CR1 promoter is a direct target of Foxm1 transcription factor
Since Foxm1 deficient monocytes exhibited decreased CX 3 CR1 mRNA (Fig. 7B) and the promoter region of mouse CX 3 CR1 gene contained several potential Foxm1-binding sites, we investigated whether Foxm1 directly bound to and transcriptionally activated the promoter region of the CX 3 CR1 gene. Two potential Foxm1 binding sites were found in the −1.0 Kb promoter region of the mouse CX 3 CR1 gene (Fig. 7D). Chromatin Immunoprecipitation (ChIP) assays were used to determine whether Foxm1 protein directly binds to the CX 3 CR1 promoter region in mouse Raw264.7 macrophages. Foxm1 protein specifically bound the −835/−842 bp binding site (but not to the −337/−343 bp site), as demonstrated by the ability of CMV-Foxm1 to induce binding of Foxm1 protein to the CX 3 CR1 promoter DNA (Fig. 7C). To determine whether the binding of Foxm1 results in transcriptional activation of the CX 3 CR1 promoter, co-transfection experiments were performed using a CMV-Foxm1b expression vector (Kim et al 2005) and a Luc reporter driven by the −1.0 Kb CX 3 CR1 promoter region. Co-transfection of the CMV-Foxm1b expression vector significantly increased Luc activity when compared to CMV-empty vector (Fig. 7D). Together, these results demonstrate that Foxm1 binds to and transcriptionally activates the −1.0 Kb CX 3 CR1 promoter region, suggesting that CX 3 CR1 gene is a direct Foxm1 target.
Adoptive transfer of Foxm1 fl/fl monocytes to macFoxm1 −/− mice restored macrophage recruitment after BHT treatment To determine whether Foxm1 plays a cell-autonomous role in macrophages during BHTinduced pulmonary inflammation, the adoptive transfer of wild type monocytes to macFoxm1 −/− mice was performed. Monocytes were isolated from bone marrow of control Foxm1 fl/fl mice using CD115 antibody and labeled with CFSE fluorescent dye. CFSElabeled monocytes were injected into the tail vein of BHT-treated macFoxm1 −/− and control Foxm1 fl/fl mice. Additional controls included macFoxm1 −/− and Foxm1 fl/fl mice without adoptive transfer. Seventy-two hours after adoptive transfer, CFSE-labeled cells were present in macFoxm1 −/− lungs as demonstrated by flow cytometry (Fig. 8A). CFSE-labeled cells expressed markers of mature macrophages: CD11b and F4/80 (Fig. 8A). Thus, transferred Foxm1 fl/fl monocytes were effectively recruited to macFoxm1 −/− lungs and differentiated into mature macrophages. Histological assessment of pulmonary inflammation revealed that adoptive transfer of Foxm1 fl/fl monocytes to macFoxm1 −/− mice restored pulmonary inflammation in response to BHT (Fig. 8B). Altogether, our results demonstrated that cell autonomous expression of Foxm1 in cells of monocyte lineage is required for monocyte proliferation and migration during lung injury and tumor promotion.

DISCUSSION
Using a novel macFoxm1 −/− conditional knockout mouse line in which Foxm1 was selectively deleted from macrophages, we found diminished lung tumorigenesis and a decreased number of tumor-associated macrophages after MCA/BHT treatment. Foxm1deficient monocytes failed to migrate into the lungs at the early stages of tumor promotion. Our data is consistent with previous studies showing that macrophage infiltration promoted tumor growth . In the present study, we established that Foxm1 is a critical transcriptional regulator of various macrophage functions, such as migration to the lung, secretion of pro-inflammatory cytokines and macrophage proliferation in response to MCA/BHT.
The MCA/BHT tumor induction/promotion protocol closely resembles the initial stages of lung carcinogenesis in human smokers. MCA is a tobacco-derived mutagen and BHT is a tumor promoting agent. BHT undergoes pulmonary metabolism in mice, causing pulmonary inflammation, macrophage infiltration and necrosis of alveolar type I epithelial cells with compensatory hyperplasia of alveolar type II cells from which lung adenomas originate (Thompson et al 1987). Chronic BHT treatment induced persistent chronic pulmonary inflammation with increased numbers of infiltrating macrophages and induced expression of macrophage-derived cytokines and chemokines (Bauer et al 2001). The presence of macrophages was previously found to be critical for pulmonary inflammation and tumorigenesis . In this study, we found that macrophage-specific loss of Foxm1 decreased the number of pulmonary macrophages after BHT treatment. This observation could be the result of decreased migration of monocytes from circulating blood into lung tissue and decreased proliferation of resident macrophages. Since Foxm1 is a known cell cycle regulator (Costa et al 2005, Myatt and Lam 2007), we were not surprised to find that Foxm1 was critical for proliferation of pulmonary macrophages in response to BHT treatment. Interestingly, Foxm1 was dispensable for proliferation of hepatic macrophages in response to carbon tetracloride treatment (Ren et al 2010). Our data suggest that Foxm1 plays distinct roles in different macrophage populations and/or different tissue environments . In support of this hypothesis, different mechanisms of cell migration were found in hepatic and pulmonary monocytes ( (Ren et al 2010) and this manuscript). In both injury models, the recruitment of monocytes to injured tissues was significantly reduced by Foxm1 deletion. However, during liver injury the down-regulation of the chemokine receptor CCR2 was responsible for reduced migration of Foxm1-deficient monocytes and CCR2 was shown to be a direct target of Foxm1 (Ren et al 2010 Previous studies demonstrated that MIP-1α, and/or MIP-2 mediate BHT-induced monocyte recruitment (Murdoch et al 2004). Moreover, MIP-1α levels were elevated in non-small cell lung cancer patients, correlating with increased infiltration of macrophages into tumor regions (Arenberg et al 2000, Meyer et al 2006. In the present study, both MIP-1α and MIP-2 were decreased in BHT-treated macFoxm1 −/− macrophages, suggesting that decreased expression of these genes may contribute to decreased migration of Foxm1deficient macrophages (Fig. 9). Furthermore, MMP-12 mRNA levels were reduced in macFoxm1 −/− macrophages. MMP-12 is up-regulated in lung cancer compared to normal tissues and has been associated with decreased survival (Heist et al 2006, Kleiner andStetler-Stevenson 1999). Studies using MMP-12 −/− mice demonstrated that macrophage recruitment to the lung was MMP-12-dependent after long-term exposure to cigarette smoke (Hautamaki et al 1997). The macrophage recruitment observed in response to cigarette smoke was linked to the elastolytic properties of MMP-12 (Nenan et al 2005). Thus, MMP-12 reduction in macFoxm1 −/− macrophages may contribute to decreased recruitment of macrophages into MCA/BHT-treated lungs. Since potential Foxm1-binding sites are present within the promoter regions of the MIP-1α, MIP-2 and MMP-12 genes, Foxm1 may

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Author Manuscript play a role in transcriptional activation of these macrophage genes during pulmonary inflammation and tumorigenesis.
Decreased inflammation in macFoxm1 −/− mice was associated with reduced expression of pro-inflammatory cytokines, including iNOS, Cox-2, IL-1β and IL-6. During chronic inflammation, macrophage-generated reactive oxygen species (ROS) induce DNA damage in proliferating cells by forming peroxynitrite, a mutagenic agent (Lala and Chakraborty 2001). Repeated tissue damage and regeneration of tissue in the presence of ROS leads to genomic alterations including point mutations, deletions or rearrangements (Faux et al 2009). Cox-2 and ROS stimulate tumor growth and metastasis by promoting migration, invasion, and angiogenesis (Lala and Chakraborty 2001). The cytokines IL-1β and IL-6, when persistently produced at chronic inflammatory sites stimulate the proliferation of DNA-damaged tumor cells and promote tumor formation. In this study, we found that Foxm1-deficient macrophages produced significantly lower levels of IL-1β and IL-6. In addition, the total number of macrophages was decreased in macFoxm1 −/− tumors, likely causing even greater decrease in the secretion of these stimulatory factors. Therefore, reduced production of mitogenic cytokines by tumor-associated macrophages can contribute to decreased proliferation of tumor cells in macFoxm1 −/− mice. Altogether, our results demonstrated that Foxm1 influenced various macrophage functions critical for pulmonary inflammation and tumor promotion (Fig. 9).
In summary, Foxm1 ablation in macrophages reduced the number and size of lung tumors induced by MCA/BHT. Decreased tumorigenesis in macFoxm1 −/− mice was associated with a diminished number of proliferating tumor cells, impaired recruitment of TAMs and decreased expression of pro-inflammatory genes. Macrophage-specific ablation of Foxm1 decreased lung inflammation during the early stages of tumor promotion, leading to decreased lung tumorigenesis (Fig. 9). Thus, Foxm1 may represent a promising target for the development of new therapeutic approaches to reduce tumor-promoting pulmonary inflammation.

MATERIALS AND METHODS
Further details and additional methods are provided as Supplementary Information.

MCA/BHT lung tumor induction/promotion protocol
MCA (Sigma, St Louis, MO) was given as a single I.P. injection (15 μg/g of body weight) followed by six weekly i.p. injections with BHT (300 μg/g of body weight). BHT (3,5-di-tbutyl-4-hydroxytoluene; Sigma, St Louis, MO) was dissolved in corn oil at a concentration 30mg/ml. Mice were sacrificed 30 weeks following MCA injection and lung tumors were counted using a dissecting microscope . To study the role of Foxm1 during tumor induction, mice were sacrificed one, two, three, and five days after the first Balli et al. Page 9 Oncogene. Author manuscript; available in PMC 2013 February 23.
BHT injection. Lungs were used to prepare total RNA or were fixed and embedded into paraffin blocks .

Quantitative real-time RT-PCR (qRT-PCR)
Total RNA was prepared from lungs, BAL cells or peritoneal macrophages of macFoxm1 −/− or control Foxm1 fl/fl mice, and analyzed by qRT-PCR using the StepOnePlus Real-Time PCR system (Applied Biosystems, Foster City, CA). Further description is provided in Supplementary Materials and Methods.

Cloning of the mouse CX 3 CR1 promoter region and Luciferase assay
We used PCR of mouse genomic DNA to amplify the −970 bp to +87 bp region of mouse CX 3 CR1 promoter (Gene Bank Number NC_000075.5) using the following primers: 5′-TCC CCC GGG AGT TTA GCA GAA ATG AAT AAG GAC ACG G -3′ and 5′-TCC CCC GGG AGT TTA GCA GAA ATG AAT AAG GAC ACG G -3′. The PCR product was cloned into a pGL2-Basic firefly luciferase (LUC) reporter plasmid (Promega, Madison, WI) and verified by DNA sequencing. U2OS cells were transfected with CMV-Foxm1b or CMV-empty plasmids, as well as with LUC reporter driven by -1kb CX 3 CR1 promoter region (CX 3 CR1-LUC). CMV-Renilla was used as an internal control to normalize transfection efficiency. A dual luciferase assay (Promega) was performed 24 hours after transfection as described previously .

Isolation and adoptive transfer of monocytes during BHT lung injury
Monocyte-enriched cells were isolated from the bone marrow of Foxm1 fl/fl mice as described (Ren et al 2010). Erythrocytes and neutrophils were removed by Ficoll gradient centrifugation. Cells were incubated with a biotin-conjugated anti-CD115 (M-CSF receptor) antibody (eBioscience) followed by anti-biotin MACS beads (Miltenyi Biotech). A positive cell fraction was obtained through magnetic separation, and labeled with carboxyfluorescein succinimidyl ester (CFSE) as described (Westcott et al 2009). The percentage of monocytes in the positive fraction was 85% as determined by flow cytometry defining the monocytic phenotype as CD115 + /CD11b + /Ly-6G − /Ly-6C hi /F4/80 lo cells. Twenty-four hours after BHT injection, 1×10 6 labeled cells were injected into tail veins of macFoxm1 −/− or control Foxm1 fl/fl mice. Recipient mice were harvested 72 hours after adoptive transfer.

Statistical analysis
We used Microsoft Excel to calculate SD and statistically significant differences between samples using the Student T-Test. P values <0.05 were considered statistically significant.

Supplementary Material
Refer to Web version on PubMed Central for supplementary material.    Mac-3-positive and negative cells were counted in ten random x400 microscope fields from n=5 mouse lungs per group. C. The total number of BAL cells (left panel) and specific number of BAL macrophages (middle panel) were decreased in macFoxm1 −/− lungs after BHT injury. No change in the number of neutrophils and lymphocytes were observed. Cells were counted in ten random microscope fields from n=5 mouse lungs per group and presented as mean ±SD. Magnification: A panels, 10x. A p value < 0.05 is shown with asterisk (*).   A. qRT-PCR shows diminished Foxm1 mRNA in peritoneal macrophages from macFoxm1 −/− mice. B. Deletion of Foxm1 from macrophages reduced their migration in transwell invasion chamber assays. C. Diminished expression of pro-inflammatory genes in Foxm1-deficient BAL macrophages in response to BHT injury. BAL cells from macFoxm1 −/− or control mice (n=3 mice per group) were collected 5 days after BHT injection and were used to prepare total RNA for qRT-PCR. β-actin mRNA was used for normalization. D. Decreased expression of pro-inflammatory genes in pulmonary macrophages purified from BHT-treated macFoxm1 −/− lungs. Using flow-cytometry based sorting, pulmonary macrophages were isolated from macFoxm1 −/− or control mice (n=3 mice per group) 5 days after BHT injection and were used to prepare total RNA for qRT-PCR. β-actin mRNA was used for normalization. Expression below detection levels were marked as N.D. (Not Detected). A p value < 0.05 is shown with asterisk (*).

Figure 7. Decreased expression of chemokine receptors in macFoxm1 −/− monocytes and macrophages
Using flow-cytometry based sorting, pulmonary macrophages were isolated from macFoxm1 −/− or control mice (n=3 mice per group) 5 days after BHT injection and were used to prepare total RNA for qRT-PCR. A. Reduced expression of CSF-R1 and IL6-Rβ in Foxm1 deficient pulmonary macrophages after BHT treatment. β-actin mRNA was used for normalization. B. Reduced expression of CX 3 CR1 and CXCR4 in monocytes isolated from BHT treated macFoxm1 −/− lungs. C. Foxm1 directly binds to CX 3 CR1 promoter. A schematic drawing of promoter region of the mouse CX 3 CR1 gene. Locations of potential Foxm1 DNA binding sites are indicated (boxes). ChIP assay demonstrated that Foxm1 directly binds to the −835/−842 bp CX3CR1 promoter region, but not to the −337/−343 bp region. Raw264.7 mouse macrophage cells were transfected via electroporation with either CMV-Foxm1 or CMV-empty vector. Foxm1 binding was normalized to DNA samples immunoprecipitated with isotype control serum. D. Luciferase assay demonstrated that Foxm1 induced the transcriptional activity of CX 3 CR1 promoter in U2OS cells. Transcriptional activity of the mouse CX 3 CR1 promoter was increased by CMV-Foxm1b transfection. A p value < 0.05 is shown with asterisk (*).  were restored to control levels after adoptive transfer (bar histogram). Statistically significant differences with p value < 0.01 are indicated by ***.

Figure 9. Role of Foxm1 in monocytes and macrophages during lung tumorigenesis
After Foxm1 deletion from monocytes, expression of CX 3 CR1 and CXCR4 is decreased, causing reduced recruitment of monocytes to the lung. Foxm1 deficiency in pulmonary macrophages results in decreased expression of IL-6R, MIP1a, MIP2 and MMP12, inhibiting macrophage migration. Foxm1-deficient macrophages showed diminished levels of Cox-2, iNOS and IL-6 mRNA, resulting in decreased pulmonary inflammation in macFoxm1 −/− mice. The cell cycle regulatory gene Cdc25b, a known Foxm1 target, was decreased in macFoxm1 −/− macrophages, which is consistent with decreased proliferation of these cells. Impaired migration, proliferation and expression of pro-inflammatory cytokines in cells of macrophage lineage inhibited BHT-induced pulmonary inflammation, decreasing lung tumorigenesis in macFoxm1 −/− mice. Balli et al. Page 25 Oncogene. Author manuscript; available in PMC 2013 February 23.