Vladimir Ustiyan

Multiple faces of FoxM1 transcription factor: lessons from transgenic mouse models.

FoxM1 transcription factor (previously called HFH-11B, Trident, FoxM1b, Win, and MPP2) is expressed in actively dividing cells and critical for cell cycle progression. FoxM1 expression is induced in a variety of tissues during embryogenesis, and Foxm1-/- mice exhibit embryonic lethal phenotype due to multiple abnormalities in the liver, heart, lung and blood vessels. FoxM1 levels are dramatically decreased in adult tissues, but FoxM1 expression is re-activated during organ injury and numerous cancers. In this review, we discussed the role of FoxM1 in different cell lineages using recent data from transgenic mouse models with conditional “gain-of-function” and “loss-of-function” of FoxM1, as well as tissue samples from human patients. In addition, we provided experimental data showing additional sites of FoxM1 expression in the mouse embryo. Novel cell-autonomous roles of FoxM1 in embryonic development, organ injury and cancer formation in vivo were analyzed. Potential application of these findings for the diagnosis and treatment of human diseases were discussed.

Foxm1 transcription factor is required for lung fibrosis and epithelial‐to‐mesenchymal transition

Alveolar epithelial cells (AECs) participate in the pathogenesis of pulmonary fibrosis, producing pro‐inflammatory mediators and undergoing epithelial‐to‐mesenchymal transition (EMT). Herein, we demonstrated the critical role of Forkhead Box M1 (Foxm1) transcription factor in radiation‐induced pulmonary fibrosis. Foxm1 was induced in AECs following lung irradiation. Transgenic expression of an activated Foxm1 transcript in AECs enhanced radiation‐induced pneumonitis and pulmonary fibrosis, and increased the expression of IL‐1β, Ccl2, Cxcl5, Snail1, Zeb1, Zeb2 and Foxf1. Conditional deletion of Foxm1 from respiratory epithelial cells decreased radiation‐induced pulmonary fibrosis and prevented the increase in EMT‐associated gene expression. siRNA‐mediated inhibition of Foxm1 prevented TGF‐β‐induced EMT in vitro. Foxm1 bound to and increased promoter activity of the Snail1 gene, a critical transcriptional regulator of EMT. Expression of Snail1 restored TGF‐β‐induced loss of E‐cadherin in Foxm1‐deficient cells in vitro. Lineage‐tracing studies demonstrated that Foxm1 increased EMT during radiation‐induced pulmonary fibrosis in vivo. Foxm1 is required for radiation‐induced pulmonary fibrosis by enhancing the expression of genes critical for lung inflammation and EMT.

Forkhead Box M1 Transcriptional Factor is Required for Smooth Muscle Cells during Embryonic Development of Blood Vessels and Esophagus

The forkhead box m1 (Foxm1 or Foxm1b) transcription factor (previously called HFH-11B, Trident, Win, or MPP2) is expressed in a variety of tissues during embryogenesis, including vascular, airway, and intestinal smooth muscle cells (SMCs). Although global deletion of Foxm1 in Foxm1(-/-) mice is lethal in the embryonic period due to multiple abnormalities in the liver, heart, and lung, the specific role of Foxm1 in SMC remains unknown. In the present study, Foxm1 was deleted conditionally in the developing SMC (smFoxm1(-/-) mice). The majority of smFoxm1(-/-) mice died immediately after birth due to severe pulmonary hemorrhage and structural defects in arterial wall and esophagus. Although Foxm1 deletion did not influence SMC differentiation, decreased proliferation of SMC was found in smFoxm1(-/-) blood vessels and esophagus. Depletion of Foxm1 in cultured SMC caused G(2) arrest and decreased numbers of cells undergoing mitosis. Foxm1-deficiency in vitro and in vivo was associated with reduced expression of cell cycle regulatory genes, including cyclin B1, Cdk1-activator Cdc25b phosphatase, Polo-like 1 and JNK1 kinases, and cMyc transcription factor. Foxm1 is critical for proliferation of smooth muscle cells and is required for proper embryonic development of blood vessels and esophagus.

FOXM1 Promotes Allergen-Induced Goblet Cell Metaplasia and Pulmonary Inflammation

ABSTRACT Chronic airway disorders, including chronic obstructive pulmonary disease (COPD), cystic fibrosis, and asthma, are associated with persistent pulmonary inflammation and goblet cell metaplasia and contribute to significant morbidity and mortality worldwide. While the molecular pathogenesis of these disorders is actively studied, little is known regarding the transcriptional control of goblet cell differentiation and mucus hyperproduction. Herein, we demonstrated that pulmonary allergen sensitization induces expression of FOXM1 transcription factor in airway epithelial and inflammatory cells. Conditional deletion of the Foxm1 gene from either airway epithelium or myeloid inflammatory cells decreased goblet cell metaplasia, reduced lung inflammation, and decreased airway resistance in response to house dust mite allergen (HDM). FOXM1 induced goblet cell metaplasia and Muc5AC expression through the transcriptional activation of Spdef. FOXM1 deletion reduced expression of CCL11, CCL24, and the chemokine receptors CCR2 and CX3CR1, resulting in decreased recruitment of eosinophils and macrophages to the lung. Deletion of FOXM1 from dendritic cells impaired the uptake of HDM antigens and decreased cell surface expression of major histocompatibility complex II (MHC II) and costimulatory molecule CD86, decreasing production of Th2 cytokines by activated T cells. Finally, pharmacological inhibition of FOXM1 by ARF peptide prevented HDM-mediated pulmonary responses. FOXM1 regulates genes critical for allergen-induced lung inflammation and goblet cell metaplasia.

FOXF1 Transcription Factor Is Required for Formation of Embryonic Vasculature by Regulating VEGF Signaling in Endothelial Cells

Rationale: Inactivating mutations in the Forkhead Box transcription factor F1 (FOXF1) gene locus are frequently found in patients with alveolar capillary dysplasia with misalignment of pulmonary veins, a lethal congenital disorder, which is characterized by severe abnormalities in the respiratory, cardiovascular, and gastrointestinal systems. In mice, haploinsufficiency of the Foxf1 gene causes alveolar capillary dysplasia and developmental defects in lung, intestinal, and gall bladder morphogenesis. Objective: Although FOXF1 is expressed in multiple mesenchyme-derived cell types, cellular origins and molecular mechanisms of developmental abnormalities in FOXF1-deficient mice and patients with alveolar capillary dysplasia with misalignment of pulmonary veins remain uncharacterized because of lack of mouse models with cell-restricted inactivation of the Foxf1 gene. In the present study, the role of FOXF1 in endothelial cells was examined using a conditional knockout approach. Methods and Results: A novel mouse line harboring Foxf1-floxed alleles was generated by homologous recombination. Tie2-Cre and Pdgfb-CreER transgenes were used to delete Foxf1 from endothelial cells. FOXF1-deficient embryos exhibited embryonic lethality, growth retardation, polyhydramnios, cardiac ventricular hypoplasia, and vascular abnormalities in the lung, placenta, yolk sac, and retina. Deletion of FOXF1 from endothelial cells reduced endothelial proliferation, increased apoptosis, inhibited vascular endothelial growth factor signaling, and decreased expression of endothelial genes critical for vascular development, including vascular endothelial growth factor receptors Flt1 and Flk1, Pdgfb, Pecam1, CD34, integrin &bgr;3, ephrin B2, Tie2, and the noncoding RNA Fendrr. Chromatin immunoprecipitation assay demonstrated that Flt1, Flk1, Pdgfb, Pecam1, and Tie2 genes are direct transcriptional targets of FOXF1. Conclusions: FOXF1 is required for the formation of embryonic vasculature by regulating endothelial genes critical for vascular development and vascular endothelial growth factor signaling.

SPDEF Inhibits Prostate Carcinogenesis by Disrupting a Positive Feedback Loop in Regulation of the Foxm1 Oncogene

SAM-pointed domain-containing ETS transcription factor (SPDEF) is expressed in normal prostate epithelium. While its expression changes during prostate carcinogenesis (PCa), the role of SPDEF in prostate cancer remains controversial due to the lack of genetic mouse models. In present study, we generated transgenic mice with the loss- or gain-of-function of SPDEF in prostate epithelium to demonstrate that SPDEF functions as tumor suppressor in prostate cancer. Loss of SPDEF increased cancer progression and tumor cell proliferation, whereas over-expression of SPDEF in prostate epithelium inhibited carcinogenesis and reduced tumor cell proliferation in vivo and in vitro. Transgenic over-expression of SPDEF inhibited mRNA and protein levels of Foxm1, a transcription factor critical for tumor cell proliferation, and reduced expression of Foxm1 target genes, including Cdc25b, Cyclin B1, Cyclin A2, Plk-1, AuroraB, CKS1 and Topo2alpha. Deletion of SPDEF in transgenic mice and cultures prostate tumor cells increased expression of Foxm1 and its target genes. Furthermore, an inverse correlation between SPDEF and Foxm1 levels was found in human prostate cancers. The two-gene signature of low SPDEF and high FoxM1 predicted poor survival in prostate cancer patients. Mechanistically, SPDEF bound to, and inhibited transcriptional activity of Foxm1 promoter by interfering with the ability of Foxm1 to activate its own promoter through auto-regulatory site located in the −745/−660 bp Foxm1 promoter region. Re-expression of Foxm1 restored cellular proliferation in the SPDEF-positive cancer cells and rescued progression of SPDEF-positive tumors in mouse prostates. Altogether, SPDEF inhibits prostate carcinogenesis by preventing Foxm1-regulated proliferation of prostate tumor cells. The present study identified novel crosstalk between SPDEF tumor suppressor and Foxm1 oncogene and demonstrated that this crosstalk is required for tumor cell proliferation during progression of prostate cancer in vivo.

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.

Foxm1 transcription factor is critical for proliferation and differentiation of Clara cells during development of conducting airways

Respiratory epithelial cells are derived from cell progenitors in the foregut endoderm that subsequently differentiate into the distinct cell types lining the conducting and alveolar regions of the lung. To identify transcriptional mechanisms regulating differentiation and maintenance of respiratory epithelial cells, we conditionally deleted Foxm1 transcription factor from the conducting airways of the developing mouse lung. Conditional deletion of Foxm1 from Clara cells, controlled by the Scgb1a1 promoter, dramatically altered airway structure and caused peribronchial fibrosis, resulting in airway hyperreactivity in adult mice. Deletion of Foxm1 inhibited proliferation of Clara cells and disrupted the normal patterning of epithelial cell differentiation in the bronchioles, causing squamous and goblet cell metaplasia, and the loss of Clara and ciliated cells. Surprisingly, conducting airways of Foxm1-deficient mice contained highly differentiated cuboidal type II epithelial cells that are normally restricted to the alveoli. Lineage tracing studies showed that the ectopic alveolar type II cells in Foxm1-deficient airways were derived from Clara cells. Deletion of Foxm1 inhibited Sox2 and Scgb1a1, both of which are critical for differentiation and function of Clara cells. In co-transfection experiments, Foxm1 directly bound to and induced transcriptional activity of Scgb1a1 and Sox2 promoters. Foxm1 is required for differentiation and maintenance of epithelial cells lining conducting airways.

The transcription factor Foxf1 binds to serum response factor and myocardin to regulate gene transcription in visceral smooth muscle cells.

Background: The role of Foxf1 in smooth muscle development is unknown. Results: Foxf1 binds to serum response factor and myocardin to regulate transcription and affect contractility of visceral smooth muscle cells. Conclusion: Foxf1 is required for normal development of gastrointestinal smooth muscle. Significance: Forkhead proteins interact with the SRF/myocardin axis to control the phenotype of smooth muscle cells. Smooth muscle cells (SMCs) modulate their phenotype from a quiescent contractile state to a dedifferentiated, proliferative and migratory state during the pathogenesis of many diseases, including intestinal pseudoobstruction. Understanding how smooth muscle gene expression is regulated in these different phenotypic states is critical for unraveling the pathogenesis of these diseases. In the current study we examined the specific roles of Foxf1 in visceral SMC differentiation. Data show that Foxf1 is specifically required for expression of several contractile and regulatory proteins such as telokin, smooth muscle γ-actin, and Cav1.2b in visceral SMCs. Mechanistically, Foxf1 directly binds to and activates the telokin promoter. Foxf1 also directly binds to serum response factor (SRF) and myocardin-related transcription factors (MRTFs). Unlike Foxo4 and Foxq1, which bind to MRTFs and block their interaction with SRF, Foxf1 acts synergistically with these proteins to regulate telokin expression. Knock-out of Foxf1 specifically in SMCs results in neonatal lethality, with mice exhibiting GI tract abnormalities. Mice heterozygous for Foxf1 in SMC exhibited impaired colonic contractility and decreased expression of contractile proteins. These studies together with previous studies, suggest that different forkhead proteins can regulate gene expression in SMCs through modulating the activity of the SRF-myocardin axis to either promote or inhibit differentiation and proliferation thereby altering gastrointestinal contractility and development.

The transcription factor FOXF1 promotes prostate cancer by stimulating the mitogen-activated protein kinase ERK5

Prostate cancers positive for FOXF1 depend on the kinase ERK5. Out-FOXing prostate cancer Some types of prostate cancer can be aggressive, and these metastatic forms of the disease have few treatment options. Fulford et al. found that prostate tumors from some patients express the gene encoding the transcription factor forkhead box F1 (FOXF1), which is not expressed in normal prostate epithelium. Overexpressing FOXF1 increased tumor growth and metastatic progression when tumor cells were implanted into mice. FOXF1 bound to and increased the expression of genes encoding the kinase ERK5 and two upstream kinases, MAP3K2 and WNK1. Pharmacologically inhibiting ERK5 or knocking down FOXF1, ERK5, or both MAP3K2 and WNK1 suppressed prostate tumor growth and metastasis. The findings indicate targets for personalizing therapeutic intervention in prostate cancer patients with FOXF1-positive tumors. Forkhead box F1 (FOXF1) is a stromal transcription factor that is not expressed in epithelial cells of normal prostate tissue. The role of FOXF1 in cancer is conflicting; its loss in some cancers suggests a tumor suppressive function, but its abundance in others is associated with protumorigenic and metastatic traits. Extracellular signal–regulated kinase 5 (ERK5) is associated with advanced-stage prostate adenocarcinoma (PCa) in patients. We detected a population of FOXF1-positive tumor cells in aggressive mouse and human PCa. Using two murine orthotopic models of PCa, we found that overexpression of FOXF1 in Myc-CaP and TRAMP prostate tumor cells induced tumor growth in the prostate and progression to peritoneal metastasis. Increased growth of FOXF1-positive prostate tumors was associated with increased phosphorylation of ERK5, a member of the mitogen-activated protein kinase (MAPK) family. FOXF1 transcriptionally induced and directly bound to promoter regions of genes encoding the kinases MAP3K2 and WNK1, which promoted the phosphorylation and activation of ERK5. Knockdown of ERK5 or both MAP3K2 and WNK1 in FOXF1-overexpressing PCa cells reduced cell proliferation in culture and suppressed tumor growth and tumor metastasis when implanted into mice. In human tumors, FOXF1 expression correlated positively with that of MAP3K2 and WNK1. Thus, in contrast to some tumors where FOXF1 may function as a tumor suppressor, FOXF1 promotes prostate tumor growth and progression by activating ERK5 signaling. Our results also indicate that ERK5 may be a new therapeutic target in patients with FOXF1-positive PCa.