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Compromised alveolar development and pulmonary vascular remodeling are hallmarks of pediatric lung diseases such as bronchopulmonary dysplasia (BPD) and alveolar capillary dysplasia with misalignment of pulmonary veins (ACDMPV). Although advances in surfactant therapy, corticosteroids, and antiinflammatory drugs have improved clinical management of preterm infants, those who suffer with severe vascular complications still lack viable treatment options. Paucity of the alveolar capillary network in ACDMPV causes respiratory distress and leads to mortality in a vast majority of infants with ACDMPV. The discovery of endothelial progenitor cells (EPCs) in 1997 brought forth the paradigm of postnatal vasculogenesis and hope for promoting vascularization in fragile patient populations, such as those with BPD and ACDMPV. The identification of diverse EPC populations, both hematopoietic and nonhematopoietic in origin, provided a need to identify progenitor cell–selective markers that are linked to progenitor properties needed to develop cell-based therapies. Focusing on the future potential of EPCs for regenerative medicine, this review will discuss various aspects of EPC biology, beginning with the identification of hematopoietic, nonhematopoietic, and tissue-resident EPC populations. We will review knowledge related to cell surface markers, signature gene expression, and key transcriptional regulators and will explore the translational potential of EPCs for cell-based therapy for BPD and ACDMPV. The ability to produce pulmonary EPCs from patient-derived induced pluripotent stem cells in vitro holds promise for restoring vascular growth and function in the lungs of patients with pediatric pulmonary disorders.

Rationale: Although pulmonary endothelial progenitor cells (EPCs) hold promise for cell-based therapies for neonatal pulmonary disorders, whether EPCs can be derived from pluripotent embryonic stem...

Rationale: The regeneration and replacement of lung cells or tissues from induced pluripotent stem cell- or embryonic stem cell-derived cells represent future therapies for life-threatening pulmonary disorders but are limited by technical challenges to produce highly differentiated cells able to maintain lung function. Functional lung tissue-containing airways, alveoli, vasculature, and stroma have never been produced via directed differentiation of embryonic stem cells (ESCs) or induced pluripotent stem cells. We sought to produce all tissue components of the lung from bronchi to alveoli by embryo complementation.Objectives: To determine whether ESCs are capable of generating lung tissue in Nkx2-1-/- mouse embryos with lung agenesis.Methods: Blastocyst complementation was used to produce chimeras from normal mouse ESCs and Nkx2-1-/- embryos, which lack pulmonary tissues. Nkx2-1-/- chimeras were examined using immunostaining, transmission electronic microscopy, fluorescence-activated cell sorter analysis, and single-cell RNA sequencing.Measurements and Main Results: Although peripheral pulmonary and thyroid tissues are entirely lacking in Nkx2-1 gene-deleted embryos, pulmonary and thyroid structures in Nkx2-1-/- chimeras were restored after ESC complementation. Respiratory epithelial cell lineages in restored lungs of Nkx2-1-/- chimeras were derived almost entirely from ESCs, whereas endothelial, immune, and stromal cells were mosaic. ESC-derived cells from multiple respiratory cell lineages were highly differentiated and indistinguishable from endogenous cells based on morphology, ultrastructure, gene expression signatures, and cell surface proteins used to identify cell types by fluorescence-activated cell sorter.Conclusions: Lung and thyroid tissues were generated in vivo from ESCs by blastocyst complementation. Nkx2-1-/- chimeras can be used as "bioreactors" for in vivo differentiation and functional studies of ESC-derived progenitor cells.

Rationale: Alveolar capillary dysplasia with misalignment of pulmonary veins (ACDMPV) is a lethal congenital disorder causing respiratory failure and pulmonary hypertension shortly after birth. There are no effective treatments for ACDMPV other than lung transplant, and new therapeutic approaches are urgently needed. Although ACDMPV is linked to mutations in the FOXF1 gene, molecular mechanisms through which FOXF1 mutations cause ACDMPV are unknown. Objectives: To identify molecular mechanisms by which S52F FOXF1 mutations cause ACDMPV. Methods: We generated a clinically relevant mouse model of ACDMPV by introducing the S52F FOXF1 mutation into the mouse Foxf1 gene locus using CRISPR/Cas9 technology. Immunohistochemistry, whole-lung imaging, and biochemical methods were used to examine vasculature in Foxf1(WT/S52F) lungs and identify molecular mechanisms regulated by FOXF1. Measurements and Main Results: FOXF1 mutations were identified in 28 subjects with ACDMPV. Foxf1(WT/S52F) knock-in mice recapitulated histopathologic findings in ACDMPV infants. The S52F FOXF1 mutation disrupted STAT3–FOXF1 protein–protein interactions and inhibited transcription of Stat3, a critical transcriptional regulator of angiogenesis. STAT3 signaling and endothelial proliferation were reduced in Foxf1(WT/S52F) mice and human ACDMPV lungs. S52F FOXF1 mutant protein did not bind chromatin and was transcriptionally inactive. Furthermore, we have developed a novel formulation of highly efficient nanoparticles and demonstrated that nanoparticle delivery of STAT3 cDNA into the neonatal circulation restored endothelial proliferation and stimulated lung angiogenesis in Foxf1(WT/S52F) mice. Conclusions: FOXF1 acts through STAT3 to stimulate neonatal lung angiogenesis. Nanoparticle delivery of STAT3 is a promising strategy to treat ACDMPV associated with decreased STAT3 signaling.

Background: Pulmonary hypertension (PH) is a common complication in patients with alveolar capillary dysplasia with misalignment of pulmonary veins (ACDMPV), a severe congenital disorder associated with mutations in the FOXF1 gene. Although the loss of alveolar microvasculature causes PH in patients with ACDMPV, it is unknown whether increasing neonatal lung angiogenesis could prevent PH and right ventricular (RV) hypertrophy. Methods: We used echocardiography, RV catheterization, immunostaining, and biochemical methods to examine lung and heart remodeling and RV output in Foxf1 WT/S52F mice carrying the S52F Foxf1 mutation (identified in patients with ACDMPV). The ability of Foxf1 WT/S52F mutant embryonic stem cells to differentiate into respiratory cell lineages in vivo was examined using blastocyst complementation. Intravascular delivery of nanoparticles with a nonintegrating Stat3 expression vector was used to improve neonatal pulmonary angiogenesis in Foxf1 WT/S52F mice and determine its effects on PH and RV hypertrophy. Results: Foxf1 WT/S52F mice developed PH and RV hypertrophy after birth. The severity of PH in Foxf1 WT/S52F mice directly correlated with mortality, low body weight, pulmonary artery muscularization, and increased collagen deposition in the lung tissue. Increased fibrotic remodeling was found in human ACDMPV lungs. Mouse embryonic stem cells carrying the S52F Foxf1 mutation were used to produce chimeras through blastocyst complementation and to demonstrate that Foxf1 WT/S52F embryonic stem cells have a propensity to differentiate into pulmonary myofibroblasts. Intravascular delivery of nanoparticles carrying Stat3 cDNA protected Foxf1 WT/S52F mice from RV hypertrophy and PH, improved survival, and decreased fibrotic lung remodeling. Conclusions: Nanoparticle therapies increasing neonatal pulmonary angiogenesis may be considered to prevent PH in ACDMPV.

Background Distinct boundaries between the proximal conducting airways and more peripheral-bronchial regions of the lung are established early in foregut embryogenesis, demarcated in part by the distribution of SOX family and NKX2-1 transcription factors along the cephalo-caudal axis of the lung. We used blastocyst complementation to identify the role of NKX2-1 in the formation of the proximal-peripheral boundary of the airways in mouse chimeric embryos. Results While Nkx2-1-/- mouse embryos form primordial tracheal cysts, peripheral pulmonary structures are entirely lacking in Nkx2-1-/- mice. Complementation of Nkx2-1-/- embryos with NKX2-1-sufficient embryonic stem cells (ESCs) enabled the formation of all tissue components of the peripheral lung but did not enhance ESC colonization of the most proximal regions of the airways. In chimeric mice, a precise boundary was formed between NKX2-1-deficient basal cells co-expressing SOX2 and SOX9 in large airways and ESC-derived NKX2-1+ SOX9+ epithelial cells of smaller airways. NKX2-1-sufficient ESCs were able to selectively complement peripheral, rather than most proximal regions of the airways. ESC complementation did not prevent ectopic expression of SOX9 but restored β-catenin signaling in Nkx2-1-/- basal cells of large airways. Conclusions NKX2-1 and β-catenin function in an epithelial cell-autonomous manner to establish the proximal-peripheral boundary along developing airways.

EWSR1/FLI1, the most common fusion gene in Ewing sarcoma, upregulates expression of the Eyes Absent 3 (EYA3) transactivator–phosphatase protein. The purpose of this study was to investigate molecular and cellular mechanisms through which EYA3 might promote Ewing sarcoma tumor growth and to determine whether the EYA3 tyrosine phosphatase activity represents a viable therapeutic target. We used genetic and pharmacologic modulation of EYA3 in cell line–based xenografts to examine how loss of EYA3 tyrosine phosphatase activity affects tumor growth and angiogenesis. Molecular mechanisms were evaluated in vivo and in vitro through analyses of tumor tissue and multicellular tumor spheroids. Our results show that both loss of EYA3 in Ewing sarcoma cells and pharmacologic inhibition of the EYA3 tyrosine phosphatase activity inhibit tumor growth and tumor angiogenesis. EYA3 regulates levels of VEGFA in Ewing tumors, as well as promoting DNA damage repair and survival of Ewing sarcoma tumor cells. Target engagement is demonstrated in tumor tissue through elevated levels of the EYA3 substrate H2AX-pY142 upon loss of EYA3 or with Benzarone treatment. The efficacy of EYA3 tyrosine phosphatase inhibition in attenuating tumor growth and angiogenesis is corroborated in an Ewing sarcoma patient-derived tumor xenograft. Together, the results presented here validate EYA3 as a target for the development of novel Ewing sarcoma therapeutic strategies, and set the stage for evaluating the efficacy of combining the antiangiogenic and anti-cell survival effects of EYA3 inhibition with cytotoxic chemotherapy.

Rationale: Advances in neonatal critical care have greatly improved the survival of preterm infants, but the long-term complications of prematurity, including bronchopulmonary dysplasia (BPD), cause mortality and morbidity later in life. Although VEGF (vascular endothelial growth factor) improves lung structure and function in rodent BPD models, severe side effects of VEGF therapy prevent its use in patients with BPD.Objectives: To test whether nanoparticle delivery of proangiogenic transcription factor FOXM1 (forkhead box M1) or FOXF1 (forkhead box F1), both downstream targets of VEGF, can improve lung structure and function after neonatal hyperoxic injury.Methods: Newborn mice were exposed to 75% O2 for the first 7 days of life before being returned to a room air environment. On Postnatal Day 2, polyethylenimine-(5) myristic acid/polyethylene glycol-oleic acid/cholesterol nanoparticles containing nonintegrating expression plasmids with Foxm1 or Foxf1 cDNAs were injected intravenously. The effects of the nanoparticles on lung structure and function were evaluated using confocal microscopy, flow cytometry, and the flexiVent small-animal ventilator.Measurements and Main Results: The nanoparticles efficiently targeted endothelial cells and myofibroblasts in the alveolar region. Nanoparticle delivery of either FOXM1 or FOXF1 did not protect endothelial cells from apoptosis caused by hyperoxia but increased endothelial proliferation and lung angiogenesis after the injury. FOXM1 and FOXF1 improved elastin fiber organization, decreased alveolar simplification, and preserved lung function in mice reaching adulthood.Conclusions: Nanoparticle delivery of FOXM1 or FOXF1 stimulates lung angiogenesis and alveolarization during recovery from neonatal hyperoxic injury. Delivery of proangiogenic transcription factors has promise as a therapy for BPD in preterm infants.

Idiopathic pulmonary fibrosis (IPF) is a chronic disease with high mortality and is refractory to treatment. Pulmonary macrophages can both promote and repress fibrosis, however molecular mechanisms regulating macrophage functions during fibrosis remain poorly understood. FOXM1 is a transcription factor and is not expressed in quiescent lungs. Herein, we show that FOXM1 is highly expressed in pulmonary macrophages within fibrotic lungs of IPF patients and mouse fibrotic lungs. Macrophage-specific deletion of Foxm1 in mice (myFoxm1-/-) exacerbated pulmonary fibrosis. Inactivation of FOXM1 in vivo and in vitro increased p38 MAPK signaling in macrophages and decreased DUSP1, a negative regulator of p38 MAPK pathway. FOXM1 directly activated Dusp1 promoter. Overexpression of DUSP1 in FOXM1-deficient macrophages prevented activation of p38 MAPK pathway. Adoptive transfer of wild-type monocytes to myFoxm1-/- mice alleviated bleomycin-induced fibrosis. Altogether, contrary to known pro-fibrotic activities in lung epithelium and fibroblasts, FOXM1 has anti-fibrotic function in macrophages by regulating p38 MAPK., Author summary Idiopathic pulmonary fibrosis (IPF) is a severe chronic lung disease, which is refractory to treatment. The environmental, age-related, and genetic factors increase susceptibility of the lung to injury. Repeated injury and deregulated repair result in scarring of the lung tissue and ultimate loss of respiratory function. Macrophages are involved at all stages of lung injury and repair, and can promote as well as repress fibrosis. However, molecular mechanisms regulating macrophage functions during fibrosis remain poorly understood. We have previously identified the FOXM1 protein to be highly increased in fibrotic lungs. High levels of FOXM1 in type II epithelial cells and fibroblasts exacerbated pulmonary fibrosis. In the present study, we found a novel and unexpected role of FOXM1. Contrary to pro-fibrotic functions of FOXM1 in epithelial cells and fibroblasts, FOXM1 has an anti-fibrotic role in macrophages. Mice lacking FOXM1 in macrophages developed severe pulmonary fibrosis following repeated lung injury with bleomycin. FOXM1 inhibited the p38 MAPK signaling pathway in pulmonary macrophages through transcriptional activation of DUSP1, a negative regulator of p38 MAPK pathway. Adoptive transfer of FOXM1-expressing monocytes protected FOXM1-deficient mice from bleomycin-induced pulmonary fibrosis. Altogether, FOXM1 has anti-fibrotic function in macrophages, limiting potential clinical use of FOXM1 inhibitors in IPF.

SUMMARY Idiopathic pulmonary fibrosis (IPF) is characterized by aberrant accumulation of collagen-secreting myofibroblasts. Development of effective therapies is limited due to incomplete understanding of molecular mechanisms regulating myofibroblast expansion. FOXF1 transcription factor is expressed in resident lung fibroblasts, but its role in lung fibrosis remains unknown due to the lack of genetic mouse models. Through comprehensive analysis of human IPF genomics data, lung biopsies, and transgenic mice with fibroblast-specific inactivation of FOXF1, we show that FOXF1 inhibits pulmonary fibrosis. FOXF1 deletion increases myofibroblast invasion and collagen secretion and promotes a switch from N-cadherin (CDH2) to Cadherin-11 (CDH11), which is a critical step in the acquisition of the profibrotic phenotype. FOXF1 directly binds to Cdh2 and Cdh11 promoters and differentially regulates transcription of these genes. Re-expression of CDH2 or inhibition of CDH11 in FOXF1-deficient cells reduces myofibroblast invasion in vitro. FOXF1 inhibits pulmonary fibrosis by regulating a switch from CDH2 to CDH11 in lung myofibroblasts., In Brief Black et al. demonstrated that FOXF1 inhibits pulmonary fibrosis by preventing CDH2 to CDH11 cadherin switch in myofibroblasts.

We report the synthesis and preliminary characterization of IODVA1, a potent small molecule that is active in xenograft mouse models of Ras-driven lung and breast cancers. In an effort to inhibit oncogenic Ras signaling, we combined in silico screening with inhibition of proliferation and colony formation of Ras-driven cells. NSC124205 fulfilled all criteria. HPLC analysis revealed that NSC124205 was a mixture of at least three compounds, from which IODVA1 was determined to be the active component. IODVA1 decreased 2D and 3D cell proliferation, cell spreading and ruffle and lamellipodia formation through downregulation of Rac activity. IODVA1 significantly impaired xenograft tumor growth of Ras-driven cancer cells with no observable toxicity. Immuno-histochemistry analysis of tumor sections suggests that cell death occurs by increased apoptosis. Our data suggest that IODVA1 targets Rac signaling to induce death of Ras-transformed cells. Therefore, IODVA1 holds promise as an anti-tumor therapeutic agent.

The lung is susceptible to damage from a variety of sources throughout development and in adulthood. As a result, the lung has great capacities for repair and regeneration, directed by precisely controlled sequences of molecular and signaling pathways. Impairments or alterations in these signaling events can have deleterious effects on lung structure and function, ultimately leading to chronic lung disorders. When lung injury is too severe for the normal pathways to repair, or if those pathways do not function properly, lung regenerative medicine is needed to restore adequate structure and function. Great progress has been made in recent years in the number of regenerative techniques and their efficacy. This review will address recent progress in lung regenerative medicine focusing on pharmacotherapy including the expanding role of nanotechnology, stem cell-based therapies, and bioengineering techniques. The use of these techniques individually and collectively has the potential to significantly improve morbidity and mortality associated with congenital and acquired lung disorders.

Rationale: Disruption of alveologenesis is associated with severe pediatric lung disorders, including bronchopulmonary dysplasia (BPD). Although c-KIT(+) endothelial cell (EC) progenitors are abundant in embryonic and neonatal lungs, their role in alveolar septation and the therapeutic potential of these cells remain unknown. Objectives: To determine whether c-KIT(+) EC progenitors stimulate alveologenesis in the neonatal lung. Methods: We used single-cell RNA sequencing of neonatal human and mouse lung tissues, immunostaining, and FACS analysis to identify transcriptional and signaling networks shared by human and mouse pulmonary c-KIT(+) EC progenitors. A mouse model of perinatal hyperoxia-induced lung injury was used to identify molecular mechanisms that are critical for the survival, proliferation, and engraftment of c-KIT(+) EC progenitors in the neonatal lung. Measurements and Main Results: Pulmonary c-KIT(+) EC progenitors expressing PECAM-1, CD34, VE-Cadherin, FLK1, and TIE2 lacked mature arterial, venal, and lymphatic cell-surface markers. The transcriptomic signature of c-KIT(+) ECs was conserved in mouse and human lungs and enriched in FOXF1-regulated transcriptional targets. Expression of FOXF1 and c-KIT was decreased in the lungs of infants with BPD. In the mouse, neonatal hyperoxia decreased the number of c-KIT(+) EC progenitors. Haploinsufficiency or endothelial-specific deletion of Foxf1 in mice increased apoptosis and decreased proliferation of c-KIT(+) ECs. Inactivation of either Foxf1 or c-Kit caused alveolar simplification. Adoptive transfer of c-KIT(+) ECs into the neonatal circulation increased lung angiogenesis and prevented alveolar simplification in neonatal mice exposed to hyperoxia. Conclusions: Cell therapy involving c-KIT(+) EC progenitors can be beneficial for the treatment of BPD.

The oncogenic transcription factor FOXM1 has been previously shown to play a critical role in carcinogenesis by inducing cellular proliferation in multiple cancer types. A small-molecule compound, Robert Costa Memorial drug-1 (RCM-1), has been recently identified from high-throughput screen as an inhibitor of FOXM1 in vitro and in mouse model of allergen-mediated lung inflammation. In the present study, we examined antitumor activities of RCM-1 using tumor models. Treatment with RCM-1 inhibited tumor cell proliferation as evidenced by increased cell-cycle duration. Confocal imaging of RCM-1–treated tumor cells indicated that delay in cellular proliferation was concordant with inhibition of FOXM1 nuclear localization in these cells. RCM-1 reduced the formation and growth of tumor cell colonies in the colony formation assay. In animal models, RCM-1 treatment inhibited growth of mouse rhabdomyosarcoma Rd76-9, melanoma B16-F10, and human H2122 lung adenocarcinoma. RCM-1 decreased FOXM1 protein in the tumors, reduced tumor cell proliferation, and increased tumor cell apoptosis. RCM-1 decreased protein levels and nuclear localization of β-catenin, and inhibited protein–protein interaction between β-catenin and FOXM1 in cultured tumor cells and in vivo. Altogether, our study provides important evidence of antitumor potential of the small-molecule compound RCM-1, suggesting that RCM-1 can be a promising candidate for anticancer therapy.

FOXM1, a member of the Forkhead box (FOX) family of transcription factors, has previously been shown to play a critical role in carcinogenesis by inducing cellular proliferation in multiple types o...

Purpose: In neurofibromatosis type 1 (NF1) and in highly aggressive malignant peripheral nerve sheath tumors (MPNSTs), constitutively active RAS-GTP and increased MAPK signaling are important in tumorigenesis. Dual specificity phosphatases (DUSPs) are negative regulators of MAPK signaling that dephosphorylate p38, JNK, and ERK in different settings. Although often acting as tumor suppressors, DUSPs may also act as oncogenes, helping tumor cells adapt to high levels of MAPK signaling. We hypothesized that inhibiting DUSPs might be selectively toxic to cells from NF1-driven tumors. Experimental Design: We examined DUSP gene and protein expression in neurofibroma and MPNSTs. We used small hairpin RNA (shRNA) to knock down DUSP1 and DUSP6 to evaluate cell growth, downstream MAPK signaling, and mechanisms of action. We evaluated the DUSP inhibitor, (E)-2-benzylidene-3-(cyclohexylamino)-2,3-dihydro-1H-inden-1-one (BCI), in MPNST cell lines and in cell-line and patient-derived MPNST xenografts. Results: DUSP1 and DUSP6 are expressed in NF1-deleted tumors. Knockdown of DUSP1 and DUSP6, alone or in combination, reduced MPNST cell growth and led to ERK and JNK hyperactivation increasing downstream TP53 and p-ATM. The DUSP inhibitor, BCI, diminished the survival of NF1-deleted Schwann cells and MPNST cell lines through activation of JNK. In vivo, treatment of an established cell-line xenograft or a novel patient-derived xenograft (PDX) of MPNSTs with BCI increased ERK and JNK activation, caused tumor necrosis and fibrosis, and reduced tumor volume in one model. Conclusions: Targeting DUSP1 and DUSP6 genetically or with BCI effectively inhibits MPNST cell growth and promotes cell death, in vitro and in xenograft models. The data support further investigation of DUSP inhibition in MPNSTs.

BACKGROUND Lung morphogenesis is regulated by interactions between the canonical Wnt/β-catenin and Kras/ERK/Foxm1 signaling pathways that establish proximal-peripheral patterning of lung tubules. How these interactions influence the development of respiratory epithelial progenitors to acquire airway as compared to alveolar epithelial cell fate is unknown. During branching morphogenesis, SOX9 transcription factor is normally restricted from conducting airway epithelial cells and is highly expressed in peripheral, acinar progenitor cells that serve as precursors of alveolar type 2 (AT2) and AT1 cells as the lung matures. RESULTS To identify signaling pathways that determine proximal-peripheral cell fate decisions, we used the SFTPC gene promoter to delete or overexpress key members of Wnt/β-catenin and Kras/ERK/Foxm1 pathways in fetal respiratory epithelial progenitor cells. Activation of β-catenin enhanced SOX9 expression in peripheral epithelial progenitors, whereas deletion of β-catenin inhibited SOX9. Surprisingly, deletion of β-catenin caused accumulation of atypical SOX9-positive basal cells in conducting airways. Inhibition of Wnt/β-catenin signaling by Kras(G12D) or its downstream target Foxm1 stimulated SOX9 expression in basal cells. Genetic inactivation of Foxm1 from Kras(G12D) -expressing epithelial cells prevented the accumulation of SOX9-positive basal cells in developing airways. CONCLUSIONS Interactions between the Wnt/β-catenin and the Kras/ERK/Foxm1 pathways are essential to restrict SOX9 expression in basal cells. Developmental Dynamics 245:590-604, 2016. © 2016 Wiley Periodicals, Inc.

We report the synthesis and preliminary characterization of IODVA1, a potent small molecule that is active in xenograft mouse models of Ras-driven lung and breast cancers. In an effort to inhibit oncogenic Ras signaling, we combined in silico screening with inhibition of proliferation and colony formation of Ras-driven cells. NSC124205 fulfilled all criteria. HPLC analysis revealed that NSC124205 was a mixture of at least three compounds, from which IODVA1 was determined to be the active component. IODVA1 decreased 2D proliferation, 3D spheroid formation, spreading and lamellipodia formation in breast cancer cells. IODVA1 significantly impaired xenograft tumor growth of Ras-driven cancer cells with no observable toxicity. Immuno-histochemistry analysis of tumor sections suggests that cell death occurs by increased apoptosis. Our data suggest that IODVA1 targets a node used by Ras to regulate cell survival, spreading, and movement including lamellipodia formation. Therefore, IODVA1 holds promise as an anti-tumor therapeutic agent.

The unique architecture of the mammalian lung is required for adaptation to air breathing at birth and thereafter. Understanding the cellular and molecular mechanisms controlling its morphogenesis provides the framework for understanding the pathogenesis of acute and chronic lung diseases. Recent single-cell RNA sequencing data and high-resolution imaging identify the remarkable heterogeneity of pulmonary cell types and provides cell selective gene expression underlying lung development. We will address fundamental issues related to the diversity of pulmonary cells, to the formation and function of the mammalian lung, and will review recent advances regarding the cellular and molecular pathways involved in lung organogenesis. What cells form the lung in the early embryo? How are cell proliferation, migration, and differentiation regulated during lung morphogenesis? How do cells interact during lung formation and repair? How do signaling and transcriptional programs determine cell-cell interactions necessary for lung morphogenesis and function?

Organogenesis is regulated by mesenchymal-epithelial signaling events that induce expression of cell-type specific transcription factors critical for cellular proliferation, differentiation and appropriate tissue patterning. While mesenchymal transcription factors play a key role in mesenchymal-epithelial interactions, transcriptional networks in septum transversum and splanchnic mesenchyme remain poorly characterized. Forkhead Box F1 (FOXF1) transcription factor is expressed in mesenchymal cell lineages; however, its role in organogenesis remains uncharacterized due to early embryonic lethality of Foxf1(−/−) mice. In the present study, we generated mesenchyme-specific Foxf1 knockout mice (Dermol-Cre Foxf1(−/−)) and demonstrated that FOXF1 is required for development of respiratory, cardiovascular and gastrointestinal organ systems. Deletion of Foxf1 from mesenchyme caused embryonic lethality in the middle of gestation due to multiple developmental defects in the heart, lung, liver and esophagus. Deletion of Foxf1 inhibited mesenchyme proliferation and delayed branching lung morphogenesis. Gene expression profiling of micro-dissected distal lung mesenchyme and ChIP sequencing of fetal lung tissue identified multiple target genes activated by FOXF1, including Wnt2, Wnt11, Wnt5A and Hoxb7. FOXF1 decreased expression of the Wnt inhibitor Wif1 through direct transcriptional repression. Furthermore, using a global Foxf1 knockout mouse line (Foxf1(−/−)) we demonstrated that FOXF1-deficiency disrupts the formation of the lung bud in foregut tissue explants. Finally, deletion of Foxf1 from smooth muscle cell lineage (smMHC-Cre Foxf1(−/−)) caused hyper-extension of esophagus and trachea, loss of tracheal and esophageal muscle, mispatterning of esophageal epithelium and decreased proliferation of smooth muscle cells. Altogether, FOXF1 promotes lung morphogenesis by regulating mesenchymal-epithelial signaling and stimulating cellular proliferation in fetal lung mesenchyme.

Forkhead box F1 (Foxf1) transcription factor is an important regulator of embryonic development but its role in tumor cells remains incompletely understood. While 16 proteins were characterized in Fanconi anemia (FA) core complex, its interactions with cellular transcriptional machinery remain poorly characterized. Here, we identified FoxF1 protein as a novel interacting partner of the FA complex proteins. Using multiple human and mouse tumor cell lines and Foxf1+/− mice we demonstrated that FoxF1 physically binds to and increases stability of FA proteins. FoxF1 co-localizes with FANCD2 in DNA repair foci in cultured cells and tumor tissues obtained from cisplatin-treated mice. In response to DNA damage, FoxF1-deficient tumor cells showed significantly reduced FANCD2 monoubiquitination and FANCM phosphorylation, resulting in impaired formation of DNA repair foci. FoxF1 knockdown caused chromosomal instability, nuclear abnormalities, and increased tumor cell death in response to DNA-damaging agents. Overexpression of FoxF1 in DNA-damaged cells improved stability of FA proteins, decreased chromosomal and nuclear aberrations, restored formation of DNA repair foci and prevented cell death after DNA damage. These findings demonstrate that FoxF1 is a key component of FA complexes and a critical mediator of DNA damage response in tumor cells.

Alterations in the forkhead box F2 gene expression have been reported in numerous pathologies, and Foxf2(-/-) mice are perinatal lethal with multiple malformations; however, molecular mechanisms pertaining to Foxf2 signaling are severely lacking. In this study, Foxf2 requirements in murine smooth muscle cells were examined using a conditional knock-out approach. We generated novel Foxf2-floxed mice, which we bred to smMHC-Cre-eGFP mice to generate a mouse line with Foxf2 deleted specifically from smooth muscle. These mice exhibited growth retardation due to reduced intestinal length as well as inflammation and remodeling of the small intestine. Colons of Tg(smMHC-Cre-eGFP(+/-));Foxf2(-/-) mice had expansion of the myenteric nerve plexus and increased proliferation of smooth muscle cells leading to thickening of the longitudinal smooth muscle layer. Foxf2 deficiency in colonic smooth muscle was associated with increased expression of Foxf1, PDGFa, PDGFb, PDGF receptor α, and myocardin. FOXF2 bound to promoter regions of these genes indicating direct transcriptional regulation. Foxf2 repressed Foxf1 promoter activity in co-transfection experiments. We also show that knockdown of Foxf2 in colonic smooth muscle cells in vitro and in transgenic mice increased myocardin/serum response factor signaling and increased expression of contractile proteins. Foxf2 attenuated myocardin/serum response factor signaling in smooth muscle cells through direct binding to the N-terminal region of myocardin. Our results indicate that Foxf2 signaling in smooth muscle cells is essential for intestinal development and serum response factor signaling.

Lung morphogenesis is a highly orchestrated process beginning with the appearance of lung buds on approximately embryonic day 9.5 in the mouse. Endodermally derived epithelial cells of the primitive lung buds undergo branching morphogenesis to generate the tree-like network of epithelial-lined tubules. The pulmonary vasculature develops in close proximity to epithelial progenitor cells in a process that is regulated by interactions between the developing epithelium and underlying mesenchyme. Studies in transgenic and knockout mouse models demonstrate that normal lung morphogenesis requires coordinated interactions between cells lining the tubules, which end in peripheral saccules, juxtaposed to an extensive network of capillaries. Multiple growth factors, microRNAs, transcription factors, and their associated signaling cascades regulate cellular proliferation, migration, survival, and differentiation during formation of the peripheral lung. Dysregulation of signaling events caused by gene mutations, teratogens, or premature birth causes severe congenital and acquired lung diseases in which normal alveolar architecture and the pulmonary capillary network are disrupted. Herein, we review scientific progress regarding signaling and transcriptional mechanisms regulating the development of pulmonary vasculature during lung morphogenesis.

Lung cancer remains one of the most prominent public health challenges, accounting for the highest incidence and mortality among all human cancers. While pulmonary invasive mucinous adenocarcinoma (PIMA) is one of the most aggressive types of non-small cell lung cancer, transcriptional drivers of PIMA remain poorly understood. In the present study, we found that Forkhead box M1 transcription factor (FOXM1) is highly expressed in human PIMAs and associated with increased extracellular mucin deposition and the loss of NKX2.1. To examine consequences of FOXM1 expression in tumor cells in vivo, we employed an inducible, transgenic mouse model to express an activated FOXM1 transcript in urethane-induced benign lung adenomas. FOXM1 accelerated tumor growth, induced progression from benign adenomas to invasive, metastatic adenocarcinomas, and induced SOX2, a marker of poorly differentiated tumor cells. Adenocarcinomas in FOXM1 transgenic mice expressed increased MUC5B and MUC5AC, and reduced NKX2.1, which are characteristics of mucinous adenocarcinomas. Expression of FOXM1 in KrasG12D transgenic mice increased the mucinous phenotype in KrasG12D-driven lung tumors. Anterior Gradient 2 (AGR2), an oncogene critical for intracellular processing and packaging of mucins, was increased in mouse and human PIMAs and was associated with FOXM1. FOXM1 directly bound to and transcriptionally activated human AGR2 gene promoter via the -257/-247 bp region. Finally, using orthotopic xenografts we demonstrated that inhibition of either FOXM1 or AGR2 in human PIMAs inhibited mucinous characteristics, and reduced tumor growth and invasion. Altogether, FOXM1 is necessary and sufficient to induce mucinous phenotypes in lung tumor cells in vivo., Author summary Lung cancer is the leading cause of cancer-related deaths in men and women in the United States. It has a high rate of mortality due to late detection and aggressive metastasis. While multiple genes were implicated in the development of lung cancer, the molecular mechanisms that trigger the progression of benign lung adenomas to aggressive adenocarcinomas are still unclear. FOXM1 is a transcription factor, which is induced in lung cancers and is correlated with poor prognosis. In the present study, we used genetically-altered mice to express an activated form of FOXM1 protein in pre-existing benign lung adenomas. FOXM1 induced progression of lung adenomas into invasive, metastatic adenocarcinomas with a mucinous phenotype. In an orthotopic xenograft model of human mucinous lung adenocarcinoma, inhibition of FOXM1 suppressed mucinous characteristics and reduced the tumor invasion and metastasis. FOXM1 directly activated the AGR2 gene, a key regulator of mucinous phenotype in lung cancer cells. We have demonstrated that FOXM1 is sufficient to drive progression of adenomas to adenocarcinomas and is nessesary to maintain a mucinous phenotype. Therapeutic targeting of FOXM1 can be beneficial for treatment of PIMA patients.

FOXF1, a member of the forkhead box family of transcription factors, has been previously shown to be critical for lung development, homeostasis, and injury responses. However, the role of FOXF1 in lung regeneration is unknown. Herein, we performed partial pneumonectomy, a model of lung regeneration, in mice lacking one Foxf1 allele in endothelial cells (PDGFb-iCre/Foxf1 fl/+ mice). Endothelial cell proliferation was significantly reduced in regenerating lungs from mice deficient for endothelial Foxf1. Decreased endothelial proliferation was associated with delayed lung regeneration as shown by reduced respiratory volume in Foxf1-deficient lungs. FACS-sorted endothelial cells isolated from regenerating PDGFb-iCre/Foxf1 fl/+ and control lungs were used for RNAseq analysis to identify FOXF1 target genes. Foxf1 deficiency altered expression of numerous genes including those regulating extracellular matrix remodeling (Timp3, Adamts9) and cell cycle progression (Cdkn1a, Cdkn2b, Cenpj, Tubb4a), which are critical for lung regeneration. Deletion of Foxf1 increased Timp3 mRNA and protein, decreasing MMP14 activity in regenerating lungs. ChIPseq analysis for FOXF1 and histone methylation marks identified DNA regulatory regions within the Cd44, Cdkn1a, and Cdkn2b genes, indicating they are direct FOXF1 targets. Thus FOXF1 stimulates lung regeneration following partial pneumonectomy via direct transcriptional regulation of genes critical for extracellular matrix remodeling and cell cycle progression.

Goblet cell metaplasia and excessive mucus secretion associated with asthma, cystic fibrosis, and chronic obstructive pulmonary disease contribute to morbidity and mortality worldwide. We performed a high-throughput screen to identify small molecules targeting a transcriptional network critical for the differentiation of goblet cells in response to allergens. We identified RCM-1, a nontoxic small molecule that inhibited goblet cell metaplasia and excessive mucus production in mice after exposure to allergens. RCM-1 blocked the nuclear localization and increased the proteasomal degradation of Forkhead box M1 (FOXM1), a transcription factor critical for the differentiation of goblet cells from airway progenitor cells. RCM-1 reduced airway resistance, increased lung compliance, and decreased proinflammatory cytokine production in mice exposed to the house dust mite and interleukin-13 (IL-13), which triggers goblet cell metaplasia. In cultured airway epithelial cells and in mice, RCM-1 reduced IL-13 and STAT6 (signal transducer and activator of transcription 6) signaling and prevented the expression of the STAT6 target genes Spdef and Foxa3, which are key transcriptional regulators of goblet cell differentiation. These results suggest that RCM-1 is an inhibitor of goblet cell metaplasia and IL-13 signaling, providing a new therapeutic candidate to treat patients with asthma and other chronic airway diseases.

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 β3, 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.

Published studies with transgenic mice convincingly showed that Forkhead Box transcription factor M1 (FOXM1) transcription factor is an important component of the KRAS/ERK signaling pathway in respiratory epithelial cells. FOXM1 is required for oncogenic KRAS signaling in mouse lung cancer models and therefore, clear potential exists to target FOXM1 in human NSCLC driven by activated KRAS mutations. To date, several approaches to inhibit FOXM1 in cancer cells have been explored. These include siRNA/shRNA-mediated inhibition of Foxm1 mRNA, sequestration of FOXM1 protein in nucleoli using ARF peptide, inhibition of FOXM1 binding to its target promoter DNAs by the FDI-6 small-molecule compound and inhibition of proteasomes by thiazole antibiotics. Additional studies are needed to determine if inhibition of FOXM1 is beneficial for treatment of KRAS mutant NSCLCs in human patients and to develop effective delivery systems for FOXM1 inhibitors. If successful, additional strategies can be explored to screen for novel FOXM1 inhibitors, such as targeting FOXM1 nuclear localization, nuclear export or protein-protein interactions with activating kinases and co-activator proteins. Altogether, inhibition of FOXM1, either alone or in combination with other anticancer drugs, could be beneficial for treatment of KRAS mutant NSCLCs that are resistant to conventional chemotherapy.

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.

Multiple signaling pathways, structural proteins, and transcription factors are involved in the regulation of endothelial barrier function. The forkhead protein FOXF1 is a key transcriptional regulator of embryonic lung development, and we used a conditional knockout approach to examine the role of FOXF1 in adult lung homeostasis, injury, and repair. Tamoxifen-regulated deletion of both Foxf1 alleles in endothelial cells of adult mice (Pdgfb-iCreER/Foxf1(-/-)) caused lung inflammation and edema, leading to respiratory insufficiency and death. Deletion of a single Foxf1 allele made heterozygous Pdgfb-iCreER/Foxf1(+/-)mice more susceptible to acute lung injury. FOXF1 abundance was decreased in pulmonary endothelial cells of human patients with acute lung injury. Gene expression analysis of pulmonary endothelial cells with homozygous FOXF1 deletion indicated reduced expression of genes critical for maintenance and regulation of adherens junctions. FOXF1 knockdown in vitro and in vivo disrupted adherens junctions, enhanced lung endothelial permeability, and increased the abundance of the mRNA and protein for sphingosine 1-phosphate receptor 1 (S1PR1), a key regulator of endothelial barrier function. Chromatin immunoprecipitation and luciferase reporter assays demonstrated that FOXF1 directly bound to and induced the transcriptional activity of the S1pr1 promoter. Pharmacological administration of S1P to injured Pdgfb-iCreER/Foxf1(+/-)mice restored endothelial barrier function, decreased lung edema, and improved survival. Thus, FOXF1 promotes normal lung homeostasis and repair, in part, by enhancing endothelial barrier function through activation of the S1P/S1PR1 signaling pathway.

Macrophages have a key role in tumor-associated pulmonary inflammation that supports the proliferation of tumor cells and promotes lung tumor growth. Although increased numbers of tumor-associated macrophages 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 tumor-associated macrophages, a mouse line with macrophage-specific Foxm1 deletion was generated (macFoxm1(-/-)). Lung tumorigenesis was induced using a 3-methylcholanthrene/butylated hydroxytoluene (BHT; 3,5-di-t-butyl-4-hydroxytoluene) 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. The expression levels of the pro-inflammatory genes iNOS, Cox-2, interleukin-1b (IL-1b) and IL-6, as well as the migration-related genes macrophage inflammatory protein-1 (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, CX(3)CR1 and CXCR4, were decreased in Foxm1-deficient monocytes. In co-transfection experiments, Foxm1 directly bound to and transcriptionally activated the CX(3)CR1 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.

The Forkhead Box f1 (Foxf1) transcriptional factor (previously known as HFH-8 or Freac-1) is expressed in endothelial and smooth muscle cells in the embryonic and adult lung. To assess effects of Foxf1 during lung injury, we used CCl(4) and butylated hydroxytoluene (BHT) injury models. Foxf1(+/-) mice developed severe airway obstruction and bronchial edema, associated with increased numbers of pulmonary mast cells and increased mast cell degranulation after injury. Pulmonary inflammation in Foxf1(+/-) mice was associated with diminished expression of Foxf1, increased mast cell tryptase, and increased expression of CXCL12, the latter being essential for mast cell migration and chemotaxis. After ovalbumin (OVA) sensitization and OVA challenge, increased lung inflammation, airway hyperresponsiveness to methacholine, and elevated expression of CXCL12 were observed in Foxf1(+/-) mice. During lung development, Foxf1(+/-) embryos displayed a marked increase in pulmonary mast cells before birth, and this was associated with increased CXCL12 levels in the lung. Expression of a doxycycline-inducible Foxf1 dominant-negative transgene in primary cultures of lung endothelial cells increased CXCL12 expression in vitro. Foxf1 haploinsufficiency caused pulmonary mastocytosis and enhanced pulmonary inflammation after chemically induced or allergen-mediated lung injury, indicating an important role for Foxf1 in the pathogenesis of pulmonary inflammatory responses.

The proliferation-specific Forkhead Box m1 (Foxm1 or Foxm1b) transcription factor (previously called HFH-11B, Trident, Win, or MPP2) regulates expression of cell cycle genes essential for progression into DNA replication and mitosis. Expression of Foxm1 is found in a variety of distinct human cancers including hepatocellular carcinomas, intrahepatic cholangiocarcinomas, basal cell carcinomas, ductal breast carcinomas, and anaplastic astrocytomas and glioblastomas. In this study, we show that human Foxm1 protein is abundantly expressed in highly proliferative human non–small cell lung cancers (NSCLC) as well as in mouse lung tumors induced by urethane. To determine the role of Foxm1 during the development of mouse lung tumors, we used IFN-inducible Mx-Cre recombinase transgene to delete mouse Foxm1 fl/fl–targeted allele before inducing lung tumors with urethane. We show that Mx-Cre Foxm1−/− mice exhibit diminished proliferation of lung tumor cells causing a significant reduction in number and size of lung adenomas. Transient transfection experiments with A549 lung adenocarcinoma cells show that depletion of Foxm1 levels by short interfering RNA caused diminished DNA replication and mitosis and reduced anchorage-independent growth of cell colonies on soft agar. Foxm1-depleted A549 cells exhibit reduced expression of cell cycle–promoting cyclin A2 and cyclin B1 genes. These data show that Foxm1 stimulates the proliferation of tumor cells during progression of NSCLC. (Cancer Res 2006; 66(4): 2153-61)

The proliferation-specific Forkhead Box M1 (FoxM1 or FoxM1b) transcription factor is overexpressed in a number of aggressive human carcinomas. Mouse hepatocytes deficient in FoxM1 fail to proliferate and are highly resistant to developing carcinogen-induced liver tumors. We previously developed a transgenic (TG) mouse line in which the ubiquitous Rosa26 promoter was used to drive expression of the human FoxM1b cDNA transgene in all mouse cell types. To investigate the role of FoxM1b in prostate cancer progression, we bred Rosa26-FoxM1b mice with both TRAMP and LADY TG mouse models of prostate cancer. We show that increased expression of FoxM1b accelerated development, proliferation, and growth of prostatic tumors in both TRAMP and LADY double TG mice. Furthermore, development of prostate carcinomas in TRAMP/Rosa26-FoxM1b double TG mice required high levels of FoxM1 protein to overcome sustained expression of the alternative reading frame tumor suppressor, a potent inhibitor of FoxM1 transcriptional activity. Depletion of FoxM1 levels in prostate cancer cell lines PC-3, LNCaP, or DU-145 by small interfering RNA transfection caused significant reduction in proliferation and anchorage-independent growth on soft agar. This phenotype was associated with increased nuclear levels of the cyclin-dependent kinase inhibitor protein p27Kip1 and diminished expression of S-phase promoting cyclin A2 and M-phase promoting cyclin B1 proteins. Finally, we show that elevated levels of FoxM1 protein correlate with high proliferation rates in human prostate adenocarcinomas. Our results suggest that the FoxM1 transcription factor regulates development and proliferation of prostate tumors, and that FoxM1 is a novel target for prostate cancer treatment. Cancer Res 2006; 66(3): 1712-20

Current treatments for inflammation associated with bronchopulmonary dysplasia (BPD) fail to show clinical efficacy. Foxm1, a transcription factor of the Forkhead box family, is a critical mediator of lung development and carcinogenesis, but its role in BPD-associated pulmonary inflammation is unknown. Immunohistochemistry and RNA analysis were used to assess Foxm1 in lung tissue from hyperoxia-treated mice and patients with BPD. LysM-Cre/Foxm1(-/-) mice, in which Foxm1 was deleted from myeloid-derived inflammatory cells, including macrophages, monocytes, and neutrophils, were exposed to neonatal hyperoxia, causing lung injury and remodeling. Measurements of lung function and flow cytometry were used to evaluate the effects of Foxm1 deletion on pulmonary inflammation and repair. Increased Foxm1 expression was observed in pulmonary macrophages of hyperoxia-exposed mice and in lung tissue from patients with BPD. After hyperoxia, deletion of Foxm1 from the myeloid cell lineage decreased numbers of interstitial macrophages (CD45(+)CD11b(+)Ly6C(-)Ly6G(-)F4/80(+)CD68(-)) and impaired alveologenesis and lung function. The exaggerated BPD-like phenotype observed in hyperoxia-exposed LysM-Cre/Foxm1(-/-) mice was associated with increased expression of neutrophil-derived myeloperoxidase, proteinase 3, and cathepsin g, all of which are critical for lung remodeling and inflammation. Our data demonstrate that Foxm1 influences pulmonary inflammatory responses to hyperoxia, inhibiting neutrophil-derived enzymes and enhancing monocytic responses that limit alveolar injury and remodeling in neonatal lungs.

Alveolar Capillary Dysplasia with Misalignment of Pulmonary Veins (ACDMPV) is a developmental disorder of the lungs, primarily affecting their vasculature. FOXF1 haploinsufficiency due to heterozygous genomic deletions and point mutations have been reported in most patients with ACDMPV. The majority of mice with heterozygous loss-of-function of Foxf1 exhibit neonatal lethality with evidence of pulmonary hemorrhage in some of them. By comparing transcriptomes of human ACDMPV lungs with control lungs using expression arrays, we found that several genes and pathways involved in lung development, angiogenesis, and in pulmonary hypertension development, were deregulated. Similar transcriptional changes were found in lungs of the postnatal day 0.5 Foxf1 +/− mice when compared to their wildtype littermate controls; 14 genes, COL15A1, COL18A1, COL6A2, ESM1, FSCN1, GRINA, IGFBP3, IL1B, MALL, NOS3, RASL11B, MATN2, PRKCDBP, and SIRPA, were found common to both ACDMPV and Foxf1 heterozygous lungs. Our results advance knowledge toward understanding of the molecular mechanism of ACDMPV, lung development, and its vasculature pathology. These data may also be useful for understanding etiologies of other lung disorders, e.g. pulmonary hypertension, bronchopulmonary dysplasia, or cancer.

Chronic injury of alveolar lung epithelium leads to epithelial disintegrity in idiopathic pulmonary fibrosis (IPF). We had reported earlier that Grhl2, a transcriptional factor, maintains alveolar epithelial cell integrity by directly regulating components of adherens and tight junctions and thus hypothesized an important role of GRHL2 in pathogenesis of IPF. Comparison of GRHL2 distribution at different stages of human lung development showed its abundance in developing lung epithelium and in adult lung epithelium. However, GRHL2 is detected in normal human lung mesenchyme only at early fetal stage (week 9). Similar mesenchymal reexpression of GRHL2 was also observed in IPF. Immunofluorescence analysis in serial sections from three IPF patients revealed at least two subsets of alveolar epithelial cells (AEC), based on differential GRHL2 expression and the converse fluorescence intensities for epithelial vs. mesenchymal markers. Grhl2 was not detected in mesenchyme in intraperitoneal bleomycin-induced injury as well as in spontaneously occurring fibrosis in double-mutant HPS1 and HPS2 mice, whereas in contrast in a radiation-induced fibrosis model, with forced Forkhead box M1 (Foxm1) expression, an overlap of Grhl2 with a mesenchymal marker was observed in fibrotic regions. Grhl2's role in alveolar epithelial cell plasticity was confirmed by altered Grhl2 gene expression analysis in IPF and further validated by in vitro manipulation of its expression in alveolar epithelial cell lines. Our findings reveal important pathophysiological differences between human IPF and specific mouse models of fibrosis and support a crucial role of GRHL2 in epithelial activation in lung fibrosis and perhaps also in epithelial plasticity.

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. 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.

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‐1 β, 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.

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.

The ability to form a metastatic niche is a unique feature of tumor cells. The niche provides signals for tumor cells extravasation, survival, dormancy or proliferation, ultimately leading to the formation of metastasis. Endothelial cells of metastatic niche become activated and play a key role in tumor cells extravasation and establishing inflammatory microenvironment that supports metastatic outgrowth. Although multiple signaling pathways have been implicated in formation of metastatic niche, the transcriptional network which controls reprogramming of normal endothelial cells (EC) into activated endothelial cells of the metastatic niche (aEC) are poorly understood. Screening for multiple regulators of the endothelial reprogramming, we identified Forkhead Box F1 (Foxf1) transcription factor, which is highly expressed in normal pulmonary ECs and is down-regulated in aECs within mouse and human lung metastases. Tamoxifen-regulated inactivation of both Foxf1 alleles in EC of adult Pdgfb-CreER/Foxf1fl/fl mice caused lung inflammation and edema, leading to respiratory insufficiency and uniform mortality. Deletion of a single Foxf1 allele from mouse ECs was sufficient to increase the number and size of pulmonary metastases in the spontaneous orthotopic mouse model of breast cancer. Increased lung metastasis in Foxf1-deficient mice was associated with increased recruitment of macrophages to the metastatic niche. The genomic signature of Foxf1 deletion from purified lung ECs indicated increased expression of cell adhesion molecules and pro-inflammatory chemokines, as well as reduced expression of genes critical for maintenance of adherence junctions in ECs. Foxf1 knockdown in vitro and in vivo disrupted adherence junctions, increased endothelial permeability and decreased mRNA and protein levels of VE-cadherin, a key regulator of endothelial barrier function. Foxf1 directly bound to and induced transcriptional activity of the VE-cadherin (Cdh5) promoter through -788/-775 bp Cdh5 promoter region. Altogether, Foxf1 transcription factor controls reprogramming of normal pulmonary ECs into aECs of the metastatic niche by regulating expression of endothelial genes critical for recruitment of macrophages and extravasation of tumor cells through endothelial barrier. Citation Format: Yuqi Cai, Tien Le, David Malewski, Craig Bolte, Chinmayee Goda, Tanya V. Kalin. Foxf1 transcription factor reprograms pulmonary endothelial cells to promote metastasis. [abstract]. In: Proceedings of the AACR Special Conference: Function of Tumor Microenvironment in Cancer Progression; 2016 Jan 7–10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2016;76(15 Suppl):Abstract nr C14.

Heart disease remains a leading cause of morbidity and mortality in the industrialized world. Hypertrophic cardiomyopathy is the most common genetic cardiovascular disorder and the most common cause of sudden cardiac death. Foxm1 transcription factor (also known as HFH-11B, Trident, Win or MPP2) plays an important role in the pathogenesis of various cancers and is a critical mediator of post-injury repair in multiple organs. Foxm1 has been previously shown to be essential for heart development and proliferation of embryonic cardiomyocytes. However, the role of Foxm1 in postnatal heart development and in cardiac injury has not been evaluated. To delete Foxm1 in postnatal cardiomyocytes, αMHC-Cre/Foxm1(fl/fl) mice were generated. Surprisingly, αMHC-Cre/Foxm1(fl/fl) mice exhibited normal cardiomyocyte proliferation at postnatal day seven and had no defects in cardiac structure or function but developed cardiac hypertrophy and fibrosis late in life. The development of cardiomyocyte hypertrophy and cardiac fibrosis in aged Foxm1-deficient mice was associated with reduced expression of Hey2, an important regulator of cardiac homeostasis, and increased expression of genes critical for cardiac remodeling, including MMP9, αSMA, fibronectin and vimentin. We also found that following aortic constriction Foxm1 mRNA and protein were induced in cardiomyocytes. However, Foxm1 deletion did not exacerbate cardiac hypertrophy or fibrosis following chronic pressure overload. Our results demonstrate that Foxm1 regulates genes critical for age-induced cardiomyocyte hypertrophy and cardiac fibrosis.

While Kras/mitogen-activated protein kinase (MAPK) and canonical Wnt/β-catenin are critical for lung morphogenesis, mechanisms integrating these important signaling pathways during lung development are unknown. Herein, we demonstrate that the Foxm1 transcription factor is a key downstream target of activated Kras(G12D). Deletion of Foxm1 from respiratory epithelial cells during lung formation prevented structural abnormalities caused by activated Kras(G12D). Kras/Foxm1 signaling inhibited the activity of canonical Wnt signaling in the developing lung in vivo. Foxm1 decreased T-cell factor (TCF) transcriptional activity induced by activated β-catenin in vitro. Depletion of Foxm1 by short interfering RNA (siRNA) increased nuclear localization of β-catenin, increased expression of β-catenin target genes, and decreased mRNA and protein levels of the β-catenin inhibitor Axin2. Axin2 mRNA was reduced in distal lung epithelium of Foxm1-deficient mice. Foxm1 directly bound to and increased transcriptional activity of the Axin2 promoter region. Foxm1 is required for Kras signaling in distal lung epithelium and provides a mechanism integrating Kras and canonical Wnt/β-catenin signaling during lung development.

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.