Foxm1 Expression in Prostate Epithelial Cells Is Essential for Prostate Carcinogenesis*

Background: Foxm1 is up-regulated in prostate adenocarcinomas and its expression correlates with the poor prognosis. Results: Conditional depletion of Foxm1 in prostate epithelial cells inhibits tumor cell proliferation, angiogenesis, and metastasis. Conclusion: Foxm1 expression in prostate epithelial cells is essential for prostate carcinogenesis in mouse models. Significance: Foxm1 may play a key role in the pathogenesis of prostate cancer in human patients. The treatment of advanced prostate cancer (PCa) remains a challenge. Identification of new molecular mechanisms that regulate PCa initiation and progression would provide targets for the development of new cancer treatments. The Foxm1 transcription factor is highly up-regulated in tumor cells, inflammatory cells, and cells of tumor microenvironment. However, its functions in different cell populations of PCa lesions are unknown. To determine the role of Foxm1 in tumor cells during PCa development, we generated two novel transgenic mouse models, one exhibiting Foxm1 gain-of-function and one exhibiting Foxm1 loss-of-function under control of the prostate epithelial-specific Probasin promoter. In the transgenic adenocarcinoma mouse prostate (TRAMP) model of PCa that uses SV40 large T antigen to induce PCa, loss of Foxm1 decreased tumor growth and metastasis. Decreased prostate tumorigenesis was associated with a decrease in tumor cell proliferation and the down-regulation of genes critical for cell proliferation and tumor metastasis, including Cdc25b, Cyclin B1, Plk-1, Lox, and Versican. In addition, tumor-associated angiogenesis was decreased, coinciding with reduced Vegf-A expression. The mRNA and protein levels of 11β-Hsd2, an enzyme playing an important role in tumor cell proliferation, were down-regulated in Foxm1-deficient PCa tumors in vivo and in Foxm1-depleted TRAMP C2 cells in vitro. Foxm1 bound to, and increased transcriptional activity of, the mouse 11β-Hsd2 promoter through the −892/−879 region, indicating that 11β-Hsd2 was a direct transcriptional target of Foxm1. Without TRAMP, overexpression of Foxm1 either alone or in combination with inhibition of a p19ARF tumor suppressor caused a robust epithelial hyperplasia, but was insufficient to induce progression from hyperplasia to PCa. Foxm1 expression in prostate epithelial cells is critical for prostate carcinogenesis, suggesting that inhibition of Foxm1 is a promising therapeutic approach for prostate cancer chemotherapy.


The treatment of advanced prostate cancer (PCa) remains a challenge. Identification of new molecular mechanisms that regulate PCa initiation and progression would provide targets for the development of new cancer treatments. The Foxm1 transcription factor is highly up-regulated in tumor cells, inflammatory cells, and cells of tumor microenvironment. However, its functions in different cell populations of PCa lesions are unknown. To determine the role of Foxm1 in tumor cells during
PCa development, we generated two novel transgenic mouse models, one exhibiting Foxm1 gain-of-function and one exhibiting Foxm1 loss-of-function under control of the prostate epithelial-specific Probasin promoter. In the transgenic adenocarcinoma mouse prostate (TRAMP) model of PCa that uses SV40 large T antigen to induce PCa, loss of Foxm1 decreased tumor growth and metastasis. Decreased prostate tumorigenesis was associated with a decrease in tumor cell proliferation and the down-regulation of genes critical for cell proliferation and tumor metastasis, including Cdc25b, Cyclin B1, Plk-1, Lox, and Versican. In addition, tumor-associated angiogenesis was decreased, coinciding with reduced Vegf-A expression. The mRNA and protein levels of 11␤-Hsd2, an enzyme playing an important role in tumor cell proliferation, were down-regulated in Foxm1-deficient PCa tumors in vivo and in Foxm1-depleted TRAMP C2 cells in vitro. Foxm1 bound to, and increased transcriptional activity of, the mouse 11␤-Hsd2 promoter through the ؊892/؊879 region, indicating that 11␤-Hsd2 was a direct transcriptional target of Foxm1. Without TRAMP, overexpression of Foxm1 either alone or in combination with inhibition of a p19 ARF tumor suppressor caused a robust epithelial hyperplasia, but was insufficient to induce progression from hyperplasia to PCa. Foxm1 expression in prostate epithelial cells is critical for prostate carcinogenesis, suggesting that inhibition of Foxm1 is a promising therapeutic approach for prostate cancer chemotherapy.
Development of cancer is a multistep process involving gain-offunction mutations in oncogenes leading to increased cell proliferation and survival (1). Cancer progression also requires inactivation of tumor suppressor genes that arrest cell proliferation in response to oncogenic stimuli (2). In normal prostate epithelium, the relatively low rate of cell proliferation is balanced by a low rate of apoptosis (3). In contrast, prostatic intraepithelial neoplasia (PIN) 3 and early invasive carcinomas are characterized by an increase in the proliferation rate, whereas advanced and/or metastatic prostate cancers also display a significant decrease in the rate of apoptosis. Altered cell-cycle control is therefore likely to play a role in progression of disease, whereas deregulation of apoptosis may be more important for advanced carcinoma. Published studies have demonstrated significant activation of the PI3K/Akt and ERK mitogen-activated protein kinase (MAPK) signaling pathways in prostate carcinomas (4,5).
The Foxm1 transcription factor is a member of the Forkhead box (Fox) family of transcription factors and broadly expressed in actively dividing cells (6 -9). Activation of the 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 (1,2,10). Activated ERK directly phosphorylates the Foxm1 protein, contributing to its transcriptional activation (11). In proliferating cells, expression of the Foxm1 is induced during the G 1 /S-phase of the cell cycle and its expression continues throughout mitosis. 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 (12). Foxm1 was shown to be essential for diminishing nuclear accumulation of p21 Cip1 and p27 Kip1 , proteins that inhibit Cdk2 activity in G 1 (13)(14)(15). Expression of the alternative reading frame (ARF) tumor suppressor is induced in response to oncogenic stimuli (2). ARF prevents aberrant cell proliferation by targeting Mdm2 to nucleolus and increasing stability of the p53 tumor suppressor (16,17). The ARF protein also targets E2F1, c-Myc, and Foxm1 transcription factors to the nucleolus, thus preventing the transcriptional activation of their target genes (18,19). Expression of the ARF protein is lost in a variety of tumors through DNA methylation and silencing of the ARF promoter region (2,17).
Prostate cancer (PCa) continues to be the most common malignancy diagnosed in American men and the second leading cause of male cancer mortality (20). Major efforts have been directed toward identifying early detection and prognostic markers for PCa. In contrast, significantly less research has been devoted to understanding the molecular mechanisms underlying PCa pathogenesis. Identification of genes regulating the initiation and/or progression of PCa would provide novel targets for diagnosis and treatment of human PCa. The transgenic adenocarcinoma of the mouse prostate (TRAMP) recapitulates multiple stages of human PCa by using the probasin promoter to drive the expression of the SV40 virus large T antigen (Tag) oncoprotein specifically in prostate epithelial cells (21,22). T antigen inactivates the tumor suppressor proteins retinoblastoma (Rb), p53, and PP2A serine/threonine-specific phosphatase (23), effectively inducing prostate tumors in adult mice. At early stages, TRAMP mice develop prostate epithelial cell hyperplasia and PIN, that progress to histopathologically invasive PCa (21,24). Both the reproducibility and progressive nature of PCa development in the TRAMP mouse model has provided a greater understanding of the molecular mechanisms involved in PCa development and progression (25).
Increased expression of Foxm1 is observed in tumor cells, inflammatory cells, and stromal cells of numerous human tumors, implying that Foxm1 plays an important role in tumor progression (reviewed in Refs. 12 and 26 -31). Indeed, overexpression of Foxm1 using the ubiquitous Rosa26 promoter accelerated tumor growth induced by SV40 T large/small t antigens (29). However, given that expression of the Rosa26-Foxm1 transgene was ubiquitous, the specific requirements for Foxm1 in different cell types of PCa remain unknown. The present study was designed to determine the cell autonomous role of Foxm1 in prostate epithelial cells during formation of prostate adenocarcinomas in vivo.

Transgenic Mice
Loss-of-Function of Foxm1 in Prostate Epithelium-Foxm1 fl/fl mice (32) were bred with Pb-Cre transgenic mice (33) to generate Pb-Cre tg/Ϫ /Foxm1 fl/fl mice. In Pb-Cre tg/Ϫ /Foxm1 fl/fl mice (Fig. 1A), Cre recombinase is specifically expressed in prostate epithelial cells under control of rat probasin (Pb) promoter. Pb-Cre tg/Ϫ /Foxm1 fl/fl mice were crossed with TRAMP transgenic mice containing Pb-driven SV40-T large and small antigens (5,6). Pb-Cre tg/Ϫ /Foxm1 fl/fl /TRAMP mice were fertile with no obvious abnormalities. Pb-Cre tg/Ϫ /Foxm1 fl/fl , Foxm1 fl/fl , and Pb-Cre tg/Ϫ /TRAMP, TRAMP littermates were used as controls. Mouse prostate glands were harvested 8 and 23 weeks after birth and used for isolation of total prostate mRNA or for immunohistochemistry. Animal studies were approved by the Animal Care and Use Committee of Cincinnati Children's Hospital Research Foundation.

ChIP Assay
TRAMP C2 mouse prostate adenocarcinoma cells were transfected with either CMV-Foxm1 or CMV-empty plasmids using the Neon transfection system following the manufacturer's protocol (Invitrogen). 24 h after transfection, cells were cross-linked by addition of formaldehyde, sonicated, and used for immunoprecipitation with a Foxm1 rabbit polyclonal antibody (C-20, Santa Cruz, CA) as described previously (37). DNA fragments were between 500 and 1000 bp in size. Reversed crosslinked ChIP DNA samples were subjected to qPCR using oligonucleotides specific to promoter regions of mouse 11␤-Hsd2: Ϫ706/ Ϫ873 (5Ј-GAG ATG GAA AGG TCA ATG AAG GC-3Јand 5Ј-CAT ACA CAC AGG GAG GGA AAT GC-3Ј) and Ϫ2366/ Ϫ2480 (5Ј-GGA AAA GCA AGA AAG TGG AGC G-3Ј and 5Ј-GGA GCC GAG ACA AAG GAT TCA G-3Ј). Binding of Foxm1 was normalized to DNA of the samples immunoprecipitated with isotype control serum.

Cloning of the Mouse 11␤-Hsd2 Promoter Region and Luciferase Assay
We used PCR of mouse genomic DNA to amplify the Ϫ2757 to Ϫ57 bp region of mouse 11␤-Hsd2 promoter (GenBank number NC_000074.6) using the following primers: 5Ј-ctg agg tac ctg gtg cta gtg agt tac tgg tca c-3Ј and 5Ј-GAC TCG AGG CTA GGA CAC GGA ATG-3Ј. To create the deletion mutants of the Ϫ2757 bp 11␤-Hsd2 promoter region we used the following primers: Ϫ2267 bp, 5Ј-ctg agg tac cgg tct cct gac tta gaa aat ggg g-3Ј and 5Ј-GAC TCG AGG CTA GGA CAC GGA ATG-3Ј; Ϫ1263 bp, 5Ј-ctg agg tac cgc tgt gat aag gaa tct atg acc c-3Ј and 5Ј-GAC TCG AGG CTA GGA CAC GGA ATG-3Ј; Ϫ649 bp, 5Ј-ctg agg tac cta cca ggg acc taa ggc cat gcct-3Ј and 5Ј-GAC TCG AGG CTA GGA CAC GGA ATG-3Ј. To mutate the potential Foxm1 binding site in the Ϫ879 to Ϫ892 bp region (5Ј-aca cac aaacaag-3Ј) of the mouse 11␤-Hsd2 promoter we utilized the GeneArt Site-directed Mutagenesis System (Life Technologies, Grand Island, NY) to completely eliminate the site without affecting neighboring sequences. The following primers were used: 5Ј-TGG CGC AGG GTG CCA GGC CAG GAC CTT CAT ACA CAC AGG GA-3Јand 5Ј-TCC CTG TGT GTA TGA AGG TCC TGG CCT GGC ACC CTG CGC CA-3Ј. The PCR products was cloned into a pGL2-Basic firefly luciferase (LUC) reporter plasmid (Promega, Madison, WI) and verified by DNA sequencing. TRAMP C2 cells were transfected with CMV-Foxm1b or CMV-empty plasmids, as well as with LUC reporter driven by the Ϫ2.7-kb 11␤-Hsd2 promoter region (11␤-Hsd2-LUC) or the deletion mutants. CMV-Renilla was used as an internal control to normalize transfection efficiency. A dual luciferase assay (Promega) was performed 24 h after transfection as described previously (38).

Statistical Analysis
We used Microsoft Excel to calculate S.D. and statistically significant differences between samples using the Student's t test. p values Ͻ 0.05 were considered statistically significant.

Conditional Deletion of Foxm1 from Prostate Epithelial Cells-
To investigate the role of Foxm1 in prostate epithelial cells during PCa formation, we generated transgenic mice containing LoxP-flanked exons 4 -7 of the Foxm1 gene (Foxm1 fl/fl ) and the Probasin-Cre transgene (Pb-Cre tg/Ϫ /Foxm1 fl/fl mice). In Pb-Cre tg/Ϫ /Foxm1 fl/fl mice (Fig. 1A), Cre recombinase is specifically expressed in prostate epithelial cells under control of rat probasin (PB) promoter (33). To induce prostate carcinogenesis, Pb-Cre tg/Ϫ /Foxm1 fl/fl mice were crossed with TRAMP mice containing PB-driven SV40-Tag transgene (22,24). Pb-Cre/ Foxm1 fl/fl /TRAMP mice were fertile with no obvious abnormalities. To determine the efficiency of Foxm1 deletion, total prostate RNA from 8-week-old Pb-Cre/Foxm1 fl/fl /TRAMP male mice was examined by qRT-PCR. Compared with control Foxm1 fl/fl /TRAMP tissues, a 90% decrease in Foxm1 mRNA was only observed in prostate tissue, but not in lung tissues from Pb-Cre/Foxm1 fl/fl /TRAMP mice (Fig. 1B), demonstrating that the deletion of Foxm1 was prostate-specific. The number of Foxm1-positive epithelial cells in Pb-Cre/Foxm1 fl/fl /TRAMP prostates was greatly reduced, confirming the efficient Foxm1 deletion by the Cre recombinase ( Foxm1 Deficiency in Prostate Epithelial Cells Prevents Adenocarcinoma Formation Caused by SV40 T-antigen-To determine whether Foxm1 expression in prostate epithelial cells was critical for prostate carcinogenesis, Foxm1 fl/fl /TRAMP and Pb-Cre/Foxm1 fl/fl /TRAMP mouse prostates were analyzed at 23 weeks of age. The weights of the prostate glands from control Foxm1 fl/fl /TRAMP and TRAMP groups of mice were 2.3 Ϯ 1.2 and 2.7 Ϯ 2.3 g, respectively ( Fig. 2A). In contrast, a significant 20-fold decrease in prostate weight was observed in Foxm1deficient Pb-Cre/Foxm1 fl/fl /TRAMP mice (prostate weight 0.12 Ϯ 0.03 g, p Ͻ 0.01) ( Fig. 2A). The mRNA levels of Foxm1 were decreased in Pb-Cre/Foxm1 fl/fl /TRAMP prostates, a finding consistent with efficient Foxm1 deletion (Fig. 2B). Histopathological analysis of the H&E-stained sections demonstrated that only 8% (1 of 12) of Pb-Cre/Foxm1 fl/fl /TRAMP mice developed adenocarcinomas compared with 90% (9 of 10) of TRAMP mice and 80% (8 of 10) of Foxm1 fl/fl /TRAMP mice (Table 1 and Fig. 2C, upper panel). All tumor lesions, including those that developed in Foxm1-deficient Pb-Cre/Foxm1 fl/fl / TRAMP prostates, stained positively for the Foxm1 protein, indicating that tumors developed from non-recombined epithelial cells (Fig. 2C, bottom panel). Furthermore, deletion of Foxm1 prevented the development of mouse PIN at early stages Foxm1 Requires for Prostate Carcinogenesis AUGUST (Fig. 4A). The tumors in control TRAMP mice were well vascularized as demonstrated by immunostaining for endothelial-specific protein Flk-1 (Fig. 4B, middle). In contrast, aberrant angiogenesis did not occur in Foxm1-defecient prostates (Fig. 4B, right). siRNA-mediated depletion of Foxm1 caused a significant reduction in Vegf-a mRNA in cultured TRAMP C2 mouse prostate adenocarcinoma cells in vitro (Fig. 4C), a find-ing consistent with regulation of Vegf-a by Foxm1 in pancreatic tumor cells (42). Thus, reduced Vegf-a levels may inhibit prostate carcinogenesis in Pb-Cre/Foxm1 fl/fl /TRAMP mice.
Foxm1 Deficiency in Prostate Epithelial Cells Alters Expression of Genes Critical for Prostate Carcinogenesis-To identify additional Foxm1-regulated target genes, we examined the expression of several genes critical for prostate cancer progression and metastasis. Total RNA was isolated from excised prostate glands and used for real time RT-PCR analysis. In Pb-Cre/ Foxm1 fl/fl /TRAMP prostates, mRNAs encoding the 11␤-Hsd2, Fzd1, Igf1, AR, Cxcl12, and Cxcr3 genes known to mediate carcinogenesis (43)(44)(45) were all decreased, whereas Hif-1a, Spdef, and Foxf1 mRNA levels did not change (Fig. 5A). Consistent with the in vivo data (Fig. 1B), knockdown of Foxm1 in cultured TRAMP C2 tumor cells decreased mRNA levels of the cell-

Foxm1 Requires for Prostate Carcinogenesis
cycle regulatory genes Cyclin B1, Cyclin D1, Cdc25b, and Plk-1 in addition to 11␤-Hsd2, Igf1, AR, and Hif-1␣ mediators of prostate carcinogenesis (Fig. 5B). Because all these genes are critical for proliferation of prostate tumor cells and formation of prostate cancer, reduced expression of these genes may contribute to reduced prostate carcinogenesis in Pb-Cre/ Foxm1 fl/fl /TRAMP mice. Moreover, depletion of Foxm1 was associated with decreased mRNA expression of Lox and Versican (Fig. 5C), genes critical for tumor metastasis (46,47). Consistent with these results, the percentage of Pb-Cre/Foxm1 fl/fl / TRAMP mice with metastasis to the lymph nodes and the lung was dramatically decreased compared with control Foxm1 fl/fl / TRAMP mice. Taken together, Foxm1 not only regulates expression of genes critical for PCa cell proliferation and TRAMP tumor formation, but also those that promote tumor progression and metastatic disease.
To demonstrate that the role of Foxm1 is not limited to the TRAMP model of prostate cancer, we used Myc-CaP prostate adenocarcinoma cells for the orthotopic injection into mouse prostates. Foxm1 mRNA and protein were increased in Myc-CaP prostate tumors compared with normal prostates (Fig. 6, A  and B). Depletion of Foxm1 in Myc-CaP cells by shRNA reduced expression of cell cycle regulatory genes (Fig. 6C) and decreased proliferation of Myc-CaP cells in vitro (Fig. 6D).

JOURNAL OF BIOLOGICAL CHEMISTRY 22533
Foxm1-⌬N transgenic protein at 24 weeks of Dox treatment (Fig. 7A, middle panels). Foxm1 overexpression on a p19 ARF ϩ/Ϫ genetic background did not promote PCa development, but caused atrophy of prostate epithelial tubules as evidenced by smaller size of prostate tubules embedded into fibrotic tissue (Fig. 7A, right panels). Altogether, our data demonstrated that overexpression of activated Foxm1-⌬N in adult prostates induced epithelial hyperplasia, but was insufficient to stimulate progression of epithelial hyperplasia to prostate adenocarcinoma. In contrast to hepatocellular car-

Foxm1 Requires for Prostate Carcinogenesis
cinomas (50), simultaneous overexpression of Foxm1 and inhibition of the p19 ARF tumor suppressor did not induce prostate carcinogenesis in vivo. Foxm1 Directly Regulates Hsd11b2 Gene Transcription-Foxm1 induced 11␤-Hsd2 mRNA and protein in mouse prostates in vivo and in cultured TRAMP C2 cells as shown by qRT-PCR (Fig. 5, A and B). Consistent with these observations, 11␤-Hsd2 protein levels were reduced in Foxm1-defecient TRAMP C2 cells (Fig. 8C) and Pb-Cre/Foxm1 fl/fl /TRAMP prostates (Fig. 8D). We next investigated whether 11␤-Hsd2 was a direct transcriptional target of Foxm1 in prostate tumor cells. Two potential Foxm1 binding sites were identified within the Ϫ2.69-kb promoter region of the mouse 11␤-Hsd2 gene (Fig.  8A). Chromatin immunoprecipitation (ChIP) assay was performed to demonstrate that the Foxm1 protein physically bound to the Ϫ706/Ϫ873 bp 11␤-Hsd2 promoter region, whereas no specific binding of Foxm1 to the Ϫ2366/Ϫ2480 bp region was observed (Fig. 8A). To identify functional Foxm1binding sites in the 11␤-Hsd2 promoter region, we performed luciferase reporter assays using the constructs as outlined in Fig. 7B. CMV-Foxm1b significantly increased transcriptional activity of the Ϫ2.69 kb 11␤-Hsd2 promoter region (Fig. 8B). Analysis of deletion mutants revealed that the Ϫ1263/Ϫ649-bp region of the 11␤-Hsd2 promoter was required for full transcriptional activation of 11␤-Hsd2 by Foxm1 (Fig. 8B). Sitedirected mutagenesis of Foxm1-binding sequences in the Ϫ892/Ϫ879 region decreased transcriptional activity of the 11␤-Hsd2 promoter, indicating that the Ϫ892/Ϫ879 region is important for transcriptional regulation of the 11␤-Hsd2 gene by Foxm1 (Fig. 8B). Thus, Foxm1 directly bound to and induced transcriptional activity of the 11␤-Hsd2 promoter, indicating that 11␤-Hsd2 is a direct Foxm1 target.
Finally, to determine whether 11␤-Hsd2 is important for cellular proliferation in Foxm1-expressing cells, TRAMP C2 tumor cells were transduced with sh11␤-Hsd2 lentivirus. Depletion of 11␤-Hsd2 was sufficient to decrease cellular proliferation as demonstrated by the reduced number of TRAMP C2 cells in two separate proliferation assays (Fig. 9, A-D). Depletion of 11␤-Hsd2 decreased mRNA levels of cell cycle regulatory genes, such as Cyclin D1 and Cdc25b (Fig. 9B), sup-  AUGUST 2, 2013 • VOLUME 288 • NUMBER 31 porting our conclusion that 11␤-Hsd2 is important for cellular proliferation in prostate cancer cells.

DISCUSSION
Our previous studies demonstrated that aberrant overexpression of Foxm1 in all cell types using ubiquitous Rosa26 promoter accelerated prostate carcinogenesis in TRAMP and LADY transgenic mice (31). Prostate cancer lesions contain a heterogeneous population of cells, which includes epithelial, inflammatory (macrophages, granulocytes), and stromal cells, all of each up-regulate Foxm1 expression during cancer formation (51). Recent studies highlighted the important role of Foxm1 in regulation of cancer-associated inflammation and production of inflammatory cytokines and chemokines (28), suggesting that Foxm1 may indirectly influence prostate carcinogenesis in Rosa26-Foxm1 mice by inducing tumor-associ-   Hsd2). Location of two potential Foxm1 DNA binding sites are indicated (boxes). The ChIP assay demonstrated that Foxm1 protein directly binds to the Ϫ706/Ϫ873-bp 11␤-Hsd2 promoter region, but not to the Ϫ2366/Ϫ2480-bp region. Cross-linked chromatin from mock-transfected TRAMP C2 mouse prostate adenocarcinoma cells or TRAMP C2 cells transfected with Foxm1-specific siRNA was immunoprecipitated (IP) with either Foxm1 antibodies or IgG control. Genomic DNA in the IP fraction was analyzed for the amount of 11␤-Hsd2 promoter DNA using qPCR. Foxm1 binding to genomic DNA was normalized to IgG control. Diminished binding of Foxm1 to the Ϫ892/Ϫ879-bp 11␤-Hsd2 promoter region was observed after siFoxm1 transfection. B, Foxm1 induces transcriptional activity of the mouse 11␤-Hsd2 promoter. Schematically shown the luciferase (LUC) reporter constructs that include either the Ϫ2.69-kb mouse 11␤-Hsd2 promoter region (I, includes both Foxm1 binding sites) or one of its deletion mutants (II-IV). TRAMP C2 cells were transfected with CMV-Foxm1b expression vector and one of the 11␤-Hsd2 LUC reporter plasmids. CMV-empty plasmid was used as a control. Cells were harvested 24 h after transfection. Dual LUC assays were used to determine LUC activity. Deletion of both Foxm1 binding sites (construct IV) prevented activation of the 11␤-Hsd2 promoter region by the CMV-Foxm1b plasmid. Site-directed mutagenesis of Foxm1-binding sequences in the Ϫ892/Ϫ879 region (construct V) decreased transcriptional activity of the 11␤-Hsd2 promoter. Transcriptional induction is shown as a fold-change relative to CMV-empty vector (ϮS.D.). A p value Ͻ 0.05 is shown with asterisk (*). C, Foxm1 knockdown by siRNA decreased 11␤-Hsd2 protein levels. TRAMP C2 cells were transfected either with control siRNA (Mock) or with siRNA specific for Foxm1 (siFoxm1). Forty-eight hours after siRNA transfection, protein extracts were analyzed by Western blot. D, decreased 11␤-Hsd2 protein expression was observed in Pb-Cre/Foxm1 fl/fl /TRAMP prostates by immunohistochemistry. Prostate tissue sections of 24-week-old mice were stained with antibodies specific to 11␤-Hsd2. Magnification is ϫ200. AUGUST 2, 2013 • VOLUME 288 • NUMBER 31 at the same time as the initiation of prostate cancer through the Pb-driven SV40 T-antigen (TRAMP model). Therefore, it is possible that Foxm1 is important for both cancer initiation and cancer progression. Because the aberrant expression of Foxm1 is found in human prostate adenocarcinomas and its expression correlates with the severity of the disease (29), our findings provide foundation for the development of new therapeutic approaches based on inhibition of Foxm1.

Foxm1 Requires for Prostate Carcinogenesis
Glucocorticoids (GC) are widely used in combination with other chemotherapeutic drugs to treat prostate cancer (52,53). GC inhibit multiple steps in prostaglandin cascade, including enzymatic activity of cytosolic phospholipase A 2 , which releases the Cox substrate arachidonic acid. GC also inhibit the expression of both Cox-2 and microsomal PGE synthase, the terminal enzyme of Cox-2-mediated prostaglandin E 2 biosynthesis (54,55). However, the adverse side effects of immune suppression limit the use of GC in clinic. 11␤-Hsd2 is an enzyme involved in metabolism of GC, particularly in the conversion of the active ligand cortisol to the inactive agonist cortisone (44). Cortisol binds to the intracellular glucocorticoid receptor, which then translocate into the nucleus and activates anti-proliferative and anti-inflammatory genes. In the presence of agonist cortisone, the glucocorticoid receptor is not activated and is not providing the anti-proliferative/anti-inflam-matory response. Increased expression of 11␤-Hsd2 was associated with increased tumor cell proliferation due to increased conversion of cortisol to agonist cortisone (56,57). In the current study, we demonstrated that 11␤-Hsd2 mRNA and protein were increased in the prostate tumors of TRAMP mice, implicating 11␤-Hsd2 in proliferation of prostate tumor cells in vivo. Conditional deletion of Foxm1 from tumor cells prevented the up-regulation of 11␤-Hsd2, coinciding with decreased carcinogenesis and reduced proliferation of prostate tumor cells. 11␤-Hsd2 is a direct transcriptional target of Foxm1, because Foxm1 directly bound to and increased promoter activity of the mouse 11␤-Hsd2 gene through the Ϫ706/Ϫ873 region. Thus, decreased expression of 11␤-Hsd2 in Foxm1-deficient prostates may contribute to decreased tumor cell proliferation and reduced prostate carcinogenesis in Pb-Cre/Foxm1 fl/fl /TRAMP mice.
Based on our studies with Pb-Foxm1-⌬N mice, it appears that Foxm1 alone is not sufficient to initiate prostate tumorigenesis. The Pb-Foxm1-⌬N mice developed PINs, but the PINs did not progress into prostate adenocarcinomas. These data are consistent with previous studies demonstrating that overexpression of Foxm1 in pulmonary type II cells did not induce the formation of lung tumors (49). One of the explanations of resistance to form tumors in Pb-Foxm1-⌬N prostates is that FIGURE 9. Depletion of 11␤-Hsd2 decreased proliferation of prostate adenocarcinoma cells. TRAMP C2 cells were transduced with sh11␤-Hsd2 lentivirus. A, decreased 11␤-Hsd2 mRNA was found in sh11␤-Hsd2 transduced cells by qRT-PCR. ␤-Actin mRNA was used for normalization. B, depletion of 11␤-Hsd2 decreased Cyclin D1 and Cdc25b mRNAs. C, depletion of 11␤-Hsd2 decreased cellular proliferation. Control and 11␤-Hsd2-depleted TRAMP C2 cells were seeded in triplicates and counted at different time points using hemocytometer. D, decreased proliferation of 11␤-Hsd2-depleted cells is shown by WST1 cell proliferation assay. Control and 11␤-Hsd2-depleted TRAMP C2 cells were seeded in triplicates and processed at different time points. A p value Ͻ 0.05 is shown with an asterisk (*). p19 ARF tumor suppressor, a known inhibitor of Foxm1 (14,48), prevents tumor-promoting properties of Foxm1. In fact, we found that expression of p19 ARF was increased in Pb-Foxm1-⌬N prostates. Loss of p19 ARF function is a critical event for tumor promotion as evidenced by extinguished expression of the p19 ARF protein in a variety of tumors through DNA methylation and silencing of the p19 ARF promoter region (2). Expression of the ARF tumor suppressor is induced in response to oncogenic stimuli and prevents abnormal cell proliferation through stabilization of p53 tumor suppressor (59). p19 ARF also mediates p53-independent cell cycle arrest through the targeting of FoxM1, E2F1, and c-Myc transcription factors to the nucleolus, thus preventing their transcriptional activation of cell cycle regulatory genes (12,14,18,40,58). The p19 ARF protein inhibited Foxm1 transcriptional activity during development of hepatocellular carcinoma (14).
To overcome inhibition of Foxm1 by the p19 ARF tumor suppressor, we created transgenic mice overexpressing Foxm1-⌬N in prostate epithelial cells in p19 ARFϪ/Ϫ background. However, those mice succumb to lymphomas earlier than they developed prostate cancer. Genetic inactivation of one p19 ARF copy in Pb-Foxm1-⌬N/p19 ARFϩ/Ϫ prostates was insufficient to induce prostate carcinogenesis. We also found that overexpression of Foxm1-⌬N alone or in the absence of one copy of tumor suppressor p19 ARF induced epithelial hyperplasia, but was insufficient to stimulate progression of epithelial hyperplasia to prostate adenocarcinomas. These data suggest that either a complete inactivation of p19 ARF , as was achieved in liver tumors (50), or deregulation of other tumorigenic pathways is necessary to promote oncogenic properties of Foxm1 in prostate epithelium. Consistent with this hypothesis, Foxm1 synergized with K-Ras (49) and ␤-catenin (39) to induce progression of lung and brain cancer, respectively. Interestingly, Foxm1overexpressing prostate tubules in Pb-Foxm1-⌬N/p19 ARFϩ/Ϫ mice underwent a complete atrophy with obliteration of lumen. These results indicate that Foxm1/p19 ARF signaling is critical for normal homeostasis in prostate epithelium.
In summary, Foxm1 expression in prostate epithelial cells is required for prostate carcinogenesis induced by SV40 T antigens. Decreased prostate carcinogenesis in Foxm1-deficient mice was associated with decreased proliferation of tumor cells, diminished angiogenesis, and reduced expression of Cdc25b, Cyclin B1, Plk-1, Vegf-a, Lox, and Versican, all of which are critical for cellular proliferation and tumor metastasis. mRNA and protein levels of 11␤-Hsd2, an enzyme playing an important role in tumor cell proliferation, were reduced in Foxm1deficient PCa tumors in vivo and in Foxm1-deficient prostate cancer cells in vitro. Foxm1 bound to and increased promoter activity of the mouse 11␤-Hsd2 gene, indicating that the 11␤-Hsd2 is a direct transcriptional target of Foxm1. Our results suggest that inhibition of Foxm1 is a promising therapeutic approach for prostate cancer chemotherapy in human patients.