Publications by authors named "Tanya V Kalin"

56 Publications

Therapeutic Potential of Endothelial Progenitor Cells in Pulmonary Diseases.

Am J Respir Cell Mol Biol 2021 Jul 22. Epub 2021 Jul 22.

Cincinnati Children's Hospital Medical Center, Pediatrics, Division of Pulmonary Biology, Cincinnati, Ohio, United States;

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 anti-inflammatory drugs have improved clinical management of preterm infants, still those who suffer with severe vascular complications lack viable treatment options. Paucity of the alveolar capillary network in ACDMPV causes respiratory distress and leads to mortality in a vast majority of ACDMPV infants. 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 which are linked to progenitor properties needed to develop cell-based therapies. Focusing to 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, 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 (iPSCs) in vitro, holds promise for restoring vascular growth and function in the lungs of patients with pediatric pulmonary disorders.
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http://dx.doi.org/10.1165/rcmb.2021-0152TRDOI Listing
July 2021

Nanoparticle Delivery of STAT3 Alleviates Pulmonary Hypertension in a Mouse Model of Alveolar Capillary Dysplasia.

Circulation 2021 Aug 11;144(7):539-555. Epub 2021 Jun 11.

Center for Lung Regenerative Medicine, Perinatal Institute (F.S., G.W., A.P., K.X., J.G.-A., Y.Z., G.T.K., Z.D., A.W.D., V.V.K.), Cincinnati Children's Hospital Medical Center, OH.

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 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 mice carrying the mutation (identified in patients with ACDMPV). The ability of mutant embryonic stem cells to differentiate into respiratory cell lineages in vivo was examined using blastocyst complementation. Intravascular delivery of nanoparticles with a nonintegrating expression vector was used to improve neonatal pulmonary angiogenesis in mice and determine its effects on PH and RV hypertrophy.

Results: mice developed PH and RV hypertrophy after birth. The severity of PH in 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 mutation were used to produce chimeras through blastocyst complementation and to demonstrate that embryonic stem cells have a propensity to differentiate into pulmonary myofibroblasts. Intravascular delivery of nanoparticles carrying cDNA protected 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.
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http://dx.doi.org/10.1161/CIRCULATIONAHA.121.053980DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8373823PMC
August 2021

Generation of Pulmonary Endothelial Progenitor Cells for Cell-based Therapy Using Interspecies Mouse-Rat Chimeras.

Am J Respir Crit Care Med 2021 08;204(3):326-338

Center for Lung Regenerative Medicine.

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 cells (ESCs) or induced pluripotent stem cells remains unknown. To investigate the heterogeneity of pulmonary EPCs and derive functional EPCs from pluripotent ESCs. Single-cell RNA sequencing of neonatal human and mouse lung was used to identify the heterogeneity of pulmonary EPCs. CRISPR/Cas9 gene editing was used to genetically label and purify mouse pulmonary EPCs. Functional properties of the EPCs were assessed after cell transplantation into neonatal mice with mutation, a mouse model of alveolar capillary dysplasia with misalignment of pulmonary veins (ACDMPV). Interspecies mouse-rat chimeras were produced through blastocyst complementation to generate EPCs from pluripotent ESCs for cell therapy in ACDMPV mice. We identified a unique population of EPCs, FOXF1cKIT EPCs, as a subset of recently described general capillary cells (gCAPs) expressing SMAD7, ZBTB20, NFIA, and DLL4 but lacking mature arterial, venous, and lymphatic markers. FOXF1cKIT gCAPs are reduced in ACDMPV, and their transcriptomic signature is conserved in mouse and human lungs. After cell transplantation into the neonatal circulation of ACDMPV mice, FOXF1cKIT gCAPs engraft into the pulmonary vasculature, stimulate angiogenesis, improve oxygenation, and prevent alveolar simplification. FOXF1cKIT gCAPs, produced from ESCs in interspecies chimeras, are fully competent to stimulate neonatal lung angiogenesis and alveolarization in ACDMPV mice. Cell-based therapy using donor or ESC/induced pluripotent stem cell-derived FOXF1cKIT endothelial progenitors may be considered for treatment of human ACDMPV.
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http://dx.doi.org/10.1164/rccm.202003-0758OCDOI Listing
August 2021

Targeting EYA3 in Ewing Sarcoma Retards Tumor Growth and Angiogenesis.

Mol Cancer Ther 2021 05 1;20(5):803-815. Epub 2021 Mar 1.

Division of Developmental Biology, Cincinnati Children's Hospital Medical Center, Department of Pediatrics, University of Cincinnati College of Medicine, Cincinnati, Ohio.

, 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 and 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.
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http://dx.doi.org/10.1158/1535-7163.MCT-20-0749DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8102334PMC
May 2021

FOXF1 is required for the oncogenic properties of PAX3-FOXO1 in rhabdomyosarcoma.

Oncogene 2021 Mar 24;40(12):2182-2199. Epub 2021 Feb 24.

Division of Pulmonary Biology, The Perinatal Institute of Cincinnati Children's Hospital Medical Center, Cincinnati, OH, USA.

The PAX3-FOXO1 fusion protein is the key oncogenic driver in fusion positive rhabdomyosarcoma (FP-RMS), an aggressive soft tissue malignancy with a particularly poor prognosis. Identifying key downstream targets of PAX3-FOXO1 will provide new therapeutic opportunities for treatment of FP-RMS. Herein, we demonstrate that Forkhead Box F1 (FOXF1) transcription factor is uniquely expressed in FP-RMS and is required for FP-RMS tumorigenesis. The PAX3-FOXO1 directly binds to FOXF1 enhancers and induces FOXF1 gene expression. CRISPR/Cas9 mediated inactivation of either FOXF1 coding sequence or FOXF1 enhancers suppresses FP-RMS tumorigenesis even in the presence of PAX3-FOXO1 oncogene. Knockdown or genetic knockout of FOXF1 induces myogenic differentiation in PAX3-FOXO1-positive FP-RMS. Over-expression of FOXF1 decreases myogenic differentiation in primary human myoblasts. In FP-RMS tumor cells, FOXF1 protein binds chromatin near enhancers associated with FP-RMS gene signature. FOXF1 cooperates with PAX3-FOXO1 and E-box transcription factors MYOD1 and MYOG to regulate FP-RMS-specific gene expression. Altogether, FOXF1 functions downstream of PAX3-FOXO1 to promote FP-RMS tumorigenesis.
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http://dx.doi.org/10.1038/s41388-021-01694-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8005492PMC
March 2021

Blastocyst complementation reveals that NKX2-1 establishes the proximal-peripheral boundary of the airway epithelium.

Dev Dyn 2021 Jul 18;250(7):1001-1020. Epub 2021 Jan 18.

Center for Lung Regenerative Medicine, Perinatal Institute, Cincinnati Children's Research Foundation, Cincinnati, Ohio, USA.

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.
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http://dx.doi.org/10.1002/dvdy.298DOI Listing
July 2021

Genome Editing for Rare Diseases.

Curr Stem Cell Rep 2020 Sep 7;6(3):41-51. Epub 2020 Jul 7.

Center for Lung Regenerative Medicine, Cincinnati Children's Hospital Medical Center, Cincinnati, USA.

Purpose Of The Review: Significant numbers of patients worldwide are affected by various rare diseases, but the effective treatment options to these individuals are limited. Rare diseases remain underfunded compared to more common diseases, leading to significant delays in research progress and ultimately, to finding an effective cure. Here, we review the use of genome-editing tools to understand the pathogenesis of rare diseases and develop additional therapeutic approaches with a high degree of precision.

Recent Findings: Several genome-editing approaches, including CRISPR/Cas9, TALEN and ZFN, have been used to generate animal models of rare diseases, understand the disease pathogenesis, correct pathogenic mutations in patient-derived somatic cells and iPSCs, and develop new therapies for rare diseases. The CRISPR/Cas9 system stands out as the most extensively used method for genome editing due to its relative simplicity and superior efficiency compared to TALEN and ZFN. CRISPR/Cas9 is emerging as a feasible gene-editing option to treat rare monogenic and other genetically defined human diseases.

Summary: Less than 5% of ~7000 known rare diseases have FDA-approved therapies, providing a compelling need for additional research and clinical trials to identify efficient treatment options for patients with rare diseases. Development of efficient genome-editing tools capable to correct or replace dysfunctional genes will lead to novel therapeutic approaches in these diseases.
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http://dx.doi.org/10.1007/s40778-020-00175-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7653884PMC
September 2020

Generation of Lung and Thyroid Tissues from Embryonic Stem Cells Using Blastocyst Complementation.

Am J Respir Crit Care Med 2021 02;203(4):471-483

Center for Lung Regenerative Medicine, Perinatal Institute.

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. To determine whether ESCs are capable of generating lung tissue in mouse embryos with lung agenesis. Blastocyst complementation was used to produce chimeras from normal mouse ESCs and embryos, which lack pulmonary tissues. chimeras were examined using immunostaining, transmission electronic microscopy, fluorescence-activated cell sorter analysis, and single-cell RNA sequencing. Although peripheral pulmonary and thyroid tissues are entirely lacking in gene-deleted embryos, pulmonary and thyroid structures in chimeras were restored after ESC complementation. Respiratory epithelial cell lineages in restored lungs of 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. Lung and thyroid tissues were generated from ESCs by blastocyst complementation. chimeras can be used as "bioreactors" for differentiation and functional studies of ESC-derived progenitor cells.
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http://dx.doi.org/10.1164/rccm.201909-1836OCDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7885842PMC
February 2021

FOXM1 nuclear transcription factor translocates into mitochondria and inhibits oxidative phosphorylation.

Mol Biol Cell 2020 06 29;31(13):1411-1424. Epub 2020 Apr 29.

Perinatal Institute and Division of Neonatology, Perinatal and Pulmonary Biology.

Forkhead box M1 (FOXM1), a nuclear transcription factor that activates cell cycle regulatory genes, is highly expressed in a majority of human cancers. The function of FOXM1 independent of nuclear transcription is unknown. In the present study, we found the FOXM1 protein inside the mitochondria. Using site-directed mutagenesis, we generated FOXM1 mutant proteins that localized to distinct cellular compartments, uncoupling the nuclear and mitochondrial functions of FOXM1. Directing FOXM1 into the mitochondria decreased mitochondrial mass, membrane potential, respiration, and electron transport chain (ETC) activity. In mitochondria, the FOXM1 directly bound to and increased the pentatricopeptide repeat domain 1 (PTCD1) protein, a mitochondrial leucine-specific tRNA binding protein that inhibits leucine-rich ETC complexes. Mitochondrial FOXM1 did not change cellular proliferation. Thus, FOXM1 translocates into mitochondria and inhibits mitochondrial respiration by increasing PTCD1. We identify a new paradigm that FOXM1 regulates mitochondrial homeostasis in a process independent of nuclear transcription.
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http://dx.doi.org/10.1091/mbc.E19-07-0413DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7353143PMC
June 2020

Loss of FOXM1 in macrophages promotes pulmonary fibrosis by activating p38 MAPK signaling pathway.

PLoS Genet 2020 04 9;16(4):e1008692. Epub 2020 Apr 9.

Division of Pulmonary Biology, the Perinatal Institute of Cincinnati Children's Hospital Research Foundation, Cincinnati, Ohio, United States of America.

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.
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http://dx.doi.org/10.1371/journal.pgen.1008692DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7173935PMC
April 2020

Nanoparticle Delivery of Proangiogenic Transcription Factors into the Neonatal Circulation Inhibits Alveolar Simplification Caused by Hyperoxia.

Am J Respir Crit Care Med 2020 07;202(1):100-111

Department of Pediatrics, University of Cincinnati and Cincinnati Children's Hospital Medical Center, Cincinnati, Ohio.

: 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.: 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.: Newborn mice were exposed to 75% O 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 or 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.: 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.: 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.
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http://dx.doi.org/10.1164/rccm.201906-1232OCDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7328311PMC
July 2020

IODVA1, a guanidinobenzimidazole derivative, targets Rac activity and Ras-driven cancer models.

PLoS One 2020 12;15(3):e0229801. Epub 2020 Mar 12.

Division of Experimental Hematology and Cancer Biology, Children's Hospital Research Foundation, Cincinnati, Ohio, Unites States of America.

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.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0229801PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7067412PMC
July 2020

Molecular, cellular, and bioengineering approaches to stimulate lung regeneration after injury.

Semin Cell Dev Biol 2020 04 25;100:101-108. Epub 2019 Oct 25.

Center for Lung Regenerative Medicine, Perinatal Institute, Cincinnati Children's Research Foundation, Cincinnati, OH 45229, United States; Division of Pulmonary Biology, Perinatal Institute, Cincinnati Children's Research Foundation, Cincinnati, OH 45229, United States; Division of Developmental Biology, Perinatal Institute, Cincinnati Children's Research Foundation, Cincinnati, OH 45229, United States; Department of Pediatrics, College of Medicine, University of Cincinnati, Cincinnati, OH 45229, United States. Electronic address:

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.
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http://dx.doi.org/10.1016/j.semcdb.2019.10.006DOI Listing
April 2020

Postnatal Alveologenesis Depends on FOXF1 Signaling in c-KIT Endothelial Progenitor Cells.

Am J Respir Crit Care Med 2019 11;200(9):1164-1176

Center for Lung Regenerative Medicine.

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. To determine whether c-KIT EC progenitors stimulate alveologenesis in the neonatal lung. 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. 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 in mice increased apoptosis and decreased proliferation of c-KIT ECs. Inactivation of either or 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. Cell therapy involving c-KIT EC progenitors can be beneficial for the treatment of BPD.
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http://dx.doi.org/10.1164/rccm.201812-2312OCDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6888649PMC
November 2019

The S52F FOXF1 Mutation Inhibits STAT3 Signaling and Causes Alveolar Capillary Dysplasia.

Am J Respir Crit Care Med 2019 10;200(8):1045-1056

Department of Pediatrics.

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 gene, molecular mechanisms through which FOXF1 mutations cause ACDMPV are unknown. To identify molecular mechanisms by which S52F FOXF1 mutations cause ACDMPV. We generated a clinically relevant mouse model of ACDMPV by introducing the S52F FOXF1 mutation into the mouse gene locus using CRISPR/Cas9 technology. Immunohistochemistry, whole-lung imaging, and biochemical methods were used to examine vasculature in lungs and identify molecular mechanisms regulated by FOXF1. FOXF1 mutations were identified in 28 subjects with ACDMPV. knock-in mice recapitulated histopathologic findings in ACDMPV infants. The S52F FOXF1 mutation disrupted STAT3-FOXF1 protein-protein interactions and inhibited transcription of , a critical transcriptional regulator of angiogenesis. STAT3 signaling and endothelial proliferation were reduced in 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 mice. 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.
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http://dx.doi.org/10.1164/rccm.201810-1897OCDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6794119PMC
October 2019

The FOXM1 Inhibitor RCM-1 Decreases Carcinogenesis and Nuclear β-Catenin.

Mol Cancer Ther 2019 07 30;18(7):1217-1229. Epub 2019 Apr 30.

Perinatal Institute, Division of Pulmonary Biology, Cincinnati Children's Hospital Medical Center, Cincinnati, Ohio.

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 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 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.
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http://dx.doi.org/10.1158/1535-7163.MCT-18-0709DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7341442PMC
July 2019

Targeted Inhibition of the Dual Specificity Phosphatases DUSP1 and DUSP6 Suppress MPNST Growth via JNK.

Clin Cancer Res 2019 07 1;25(13):4117-4127. Epub 2019 Apr 1.

Cincinnati Children's Hospital Medical Center, Cincinnati, OH.

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. , 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, and in xenograft models. The data support further investigation of DUSP inhibition in MPNSTs.
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http://dx.doi.org/10.1158/1078-0432.CCR-18-3224DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6606396PMC
July 2019

The Forkhead box F1 transcription factor inhibits collagen deposition and accumulation of myofibroblasts during liver fibrosis.

Biol Open 2019 Feb 11;8(2). Epub 2019 Feb 11.

Department of Pediatrics, Cincinnati Children's Research Foundation, Cincinnati, Ohio 45229, USA

Hepatic fibrosis is the common end stage to a variety of chronic liver injuries and is characterized by an excessive deposition of extracellular matrix (ECM), which disrupts the liver architecture and impairs liver function. The fibrous lesions are produced by myofibroblasts, which differentiate from hepatic stellate cells (HSC). The myofibroblast's transcriptional networks remain poorly characterized. Previous studies have shown that the Forkhead box F1 (FOXF1) transcription factor is expressed in HSCs and stimulates their activation during acute liver injury; however, the role of FOXF1 in the progression of hepatic fibrosis is unknown. In the present study, we generated mice to conditionally inactivate in myofibroblasts during carbon tetrachloride-mediated liver fibrosis. deletion increased collagen depositions and disrupted liver architecture. expression was significantly increased in -deficient mice while MMP9 activity was reduced. RNA sequencing of purified liver myofibroblasts demonstrated that FOXF1 inhibits expression of pro-fibrotic genes, , , and in fibrotic livers and binds to active repressors located in promotors and introns of these genes. Overexpression of FOXF1 inhibits , , and in primary murine HSCs Altogether, FOXF1 prevents aberrant ECM depositions during hepatic fibrosis by repressing pro-fibrotic gene transcription in myofibroblasts and HSCs.
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http://dx.doi.org/10.1242/bio.039800DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6398469PMC
February 2019

Building and Regenerating the Lung Cell by Cell.

Physiol Rev 2019 01;99(1):513-554

Perinatal Institute, Cincinnati Children's Hospital Medical Center, Department of Pediatrics, Division of Neonatology, Perinatal and Pulmonary Biology, Cincinnati, Ohio.

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?
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http://dx.doi.org/10.1152/physrev.00001.2018DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6442926PMC
January 2019

FOXF1 transcription factor promotes lung morphogenesis by inducing cellular proliferation in fetal lung mesenchyme.

Dev Biol 2018 11 25;443(1):50-63. Epub 2018 Aug 25.

Center for Lung Regenerative Medicine, Divisions of Cincinnati Children's Research Foundation, 3333 Burnet Ave., Cincinnati, OH 45229, United States; Pulmonary Biology, Cincinnati Children's Research Foundation, 3333 Burnet Ave., Cincinnati, OH 45229, United States; Developmental Biology and Cincinnati Children's Research Foundation, 3333 Burnet Ave., Cincinnati, OH 45229, United States. Electronic address:

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 (Dermo1-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.
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http://dx.doi.org/10.1016/j.ydbio.2018.08.011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6191344PMC
November 2018

FOXF1 Inhibits Pulmonary Fibrosis by Preventing CDH2-CDH11 Cadherin Switch in Myofibroblasts.

Cell Rep 2018 Apr;23(2):442-458

Divisions of Pulmonary Biology, Perinatal Institute of Cincinnati Children's Hospital Research Foundation, 3333 Burnet Ave., Cincinnati, OH 45229, USA. Electronic address:

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 pro-fibrotic 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.
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http://dx.doi.org/10.1016/j.celrep.2018.03.067DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5947867PMC
April 2018

Transcription Factors Regulating Embryonic Development of Pulmonary Vasculature.

Adv Anat Embryol Cell Biol 2018 ;228:1-20

Center for Lung Regenerative Medicine, Perinatal Institute, Cincinnati Children's Research Foundation, Cincinnati, OH, USA.

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.
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http://dx.doi.org/10.1007/978-3-319-68483-3_1DOI Listing
November 2019

FOXM1 activates AGR2 and causes progression of lung adenomas into invasive mucinous adenocarcinomas.

PLoS Genet 2017 12 21;13(12):e1007097. Epub 2017 Dec 21.

Division of Pulmonary Biology, the Perinatal Institute of Cincinnati Children's Research Foundation, Cincinnati, Ohio, United States of America.

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.
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http://dx.doi.org/10.1371/journal.pgen.1007097DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5755924PMC
December 2017

FOXF1 transcription factor promotes lung regeneration after partial pneumonectomy.

Sci Rep 2017 09 6;7(1):10690. Epub 2017 Sep 6.

Center for Lung Regenerative Medicine, Cincinnati Children's Research Foundation, Cincinnati, Ohio, USA.

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 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 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.
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http://dx.doi.org/10.1038/s41598-017-11175-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5587533PMC
September 2017

The FOXM1 inhibitor RCM-1 suppresses goblet cell metaplasia and prevents IL-13 and STAT6 signaling in allergen-exposed mice.

Sci Signal 2017 Apr 18;10(475). Epub 2017 Apr 18.

Center for Lung Regenerative Medicine, Cincinnati Children's Hospital Medical Center, Cincinnati, OH 45229, USA.

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 and , 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.
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http://dx.doi.org/10.1126/scisignal.aai8583DOI Listing
April 2017

FoxF1 and FoxF2 transcription factors synergistically promote rhabdomyosarcoma carcinogenesis by repressing transcription of p21 CDK inhibitor.

Oncogene 2017 02 18;36(6):850-862. Epub 2016 Jul 18.

Division of Pulmonary Biology, the Perinatal Institute of Cincinnati Children's Hospital Research Foundation, 3333 Burnet Ave., Cincinnati, OH 45229.

The role of Forkhead Box F1 (FoxF1) transcription factor in carcinogenesis is not well characterized. Depending on tissue and histological type of cancer, FoxF1 has been shown to be either an oncogene or a tumor suppressor. Alveolar rhabdomyosarcoma (RMS) is the most aggressive pediatric soft-tissue sarcoma. Although FoxF1 is highly expressed in alveolar RMS, the functional role of FoxF1 in RMS is unknown. The present study demonstrates that expression of FoxF1 and its closely related transcription factor FoxF2 are essential for RMS tumor growth. Depletion of FoxF1 or FoxF2 in RMS cells decreased tumor growth in orthotopic mouse models of RMS. The decreased tumorigenesis was associated with reduced tumor cell proliferation. Cell cycle regulatory proteins Cdk2, Cdk4/6, Cyclin D1 and Cyclin E2 were decreased in FoxF1- and FoxF2-deficient RMS tumors. Depletion of either FoxF1 or FoxF2 delayed G1-S cell cycle progression, decreased levels of phosphorylated retinoblastoma protein (Rb) and increased protein levels of the CDK inhibitors, p21 and p27. Depletion of both FoxF1 and FoxF2 in tumor cells completely abrogated RMS tumor growth in mice. Overexpression of either FoxF1 or FoxF2 in tumor cells was sufficient to increase tumor growth in orthotopic RMS mouse model. FoxF1 and FoxF2 directly bound to and repressed transcriptional activity of p21 promoter through -556/-545 bp region, but did not affect p27 transcription. Knockdown of p21 restored cell cycle progression in the FoxF1- or FoxF2-deficient tumor cells. Altogether, FoxF1 and FoxF2 promoted RMS tumorigenesis by inducing tumor cell proliferation via transcriptional repression of p21 gene promoter. Because of the robust oncogenic activity in RMS tumors, FoxF1 and FoxF2 may represent promising targets for anti-tumor therapy.
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http://dx.doi.org/10.1038/onc.2016.254DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5243941PMC
February 2017

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

Sci Signal 2016 05 10;9(427):ra48. Epub 2016 May 10.

Division of Pulmonary Biology, Perinatal Institute of Cincinnati Children's Research Foundation, 3333 Burnet Avenue, Cincinnati, OH 45229, USA.

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.
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http://dx.doi.org/10.1126/scisignal.aad5582DOI Listing
May 2016

FOXF1 maintains endothelial barrier function and prevents edema after lung injury.

Sci Signal 2016 Apr 19;9(424):ra40. Epub 2016 Apr 19.

Division of Pulmonary Biology, Cincinnati Children's Research Foundation, 3333 Burnet Avenue, Cincinnati, OH 45229, USA. The Perinatal Institute, Cincinnati Children's Research Foundation, Cincinnati, OH 45229, USA.

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.
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http://dx.doi.org/10.1126/scisignal.aad1899DOI Listing
April 2016

β-catenin and Kras/Foxm1 signaling pathway are critical to restrict Sox9 in basal cells during pulmonary branching morphogenesis.

Dev Dyn 2016 05 8;245(5):590-604. Epub 2016 Mar 8.

Division of Pulmonary Biology, Perinatal Institute of Cincinnati Children's Research Foundation, Cincinnati, Ohio.

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.
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http://dx.doi.org/10.1002/dvdy.24393DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4844868PMC
May 2016

Forkhead transcription factor FoxF1 interacts with Fanconi anemia protein complexes to promote DNA damage response.

Oncotarget 2016 Jan;7(2):1912-26

Division of Pulmonary Biology, Perinatal Institute of Cincinnati Children's Research Foundation, Cincinnati, OH 45229, USA.

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.
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http://dx.doi.org/10.18632/oncotarget.6422DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4811506PMC
January 2016
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