Tatiana Kalin

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Journals/Publications

• Do, J., Verma, G., Maurice, Y., Abdumanobova, M., Deng, Z., Kalin, T. V., & Kalinichenko, V. V. (2025). Protocol for minicircle production for gene therapy without subsequent cleanup steps. STAR Protocols, 6(Issue 3). doi:10.1016/j.xpro.2025.103982
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  Current techniques for producing minicircles have low yields because of the cleanup steps required. Here, we present a protocol that can increase minicircle yield up to 10-fold by eliminating post-induction cleanup steps. We describe steps for bacterial culture, same-day arabinose induction, and DNA harvesting. This protocol is relevant for all minicircles produced from parental plasmids containing attB, attP, and 32 copies of Isce-I endonuclease sites in the minicircle-producing bacteria, ZYCY10PS3T2.
• Bian, F., Goda, C., Wang, G., Lan, Y. W., Deng, Z., Gao, W., Acharya, A., Reza, A. A., Gomez-Arroyo, J., Merjaneh, N., Ren, X., Goveia, J., Carmeliet, P., Kalinichenko, V. V., & Kalin, T. V. (2024). FOXF1 promotes tumor vessel normalization and prevents lung cancer progression through FZD4. EMBO molecular medicine, 16(5), 1063-1090.
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  Cancer cells re-program normal lung endothelial cells (EC) into tumor-associated endothelial cells (TEC) that form leaky vessels supporting carcinogenesis. Transcriptional regulators that control the reprogramming of EC into TEC are poorly understood. We identified Forkhead box F1 (FOXF1) as a critical regulator of EC-to-TEC transition. FOXF1 was highly expressed in normal lung vasculature but was decreased in TEC within non-small cell lung cancers (NSCLC). Low FOXF1 correlated with poor overall survival of NSCLC patients. In mice, endothelial-specific deletion of FOXF1 decreased pericyte coverage, increased vessel permeability and hypoxia, and promoted lung tumor growth and metastasis. Endothelial-specific overexpression of FOXF1 normalized tumor vessels and inhibited the progression of lung cancer. FOXF1 deficiency decreased Wnt/β-catenin signaling in TECs through direct transcriptional activation of Fzd4. Restoring FZD4 expression in FOXF1-deficient TECs through endothelial-specific nanoparticle delivery of Fzd4 cDNA rescued Wnt/β-catenin signaling in TECs, normalized tumor vessels and inhibited the progression of lung cancer. Altogether, FOXF1 increases tumor vessel stability, and inhibits lung cancer progression by stimulating FZD4/Wnt/β-catenin signaling in TECs. Nanoparticle delivery of FZD4 cDNA has promise for future therapies in NSCLC.
• Bian, F., Goda, C., Wang, G., Lan, Y., Deng, Z., Gao, W., Acharya, A., Reza, A., Gomez-Arroyo, J., Merjaneh, N., Ren, X., Goveia, J., Carmeliet, P., Kalinichenko, V., & Kalin, T. (2024). FOXF1 promotes tumor vessel normalization and prevents lung cancer progression through FZD4. EMBO Molecular Medicine, 16(5). doi:10.1038/s44321-024-00064-8
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  Cancer cells re-program normal lung endothelial cells (EC) into tumor-associated endothelial cells (TEC) that form leaky vessels supporting carcinogenesis. Transcriptional regulators that control the reprogramming of EC into TEC are poorly understood. We identified Forkhead box F1 (FOXF1) as a critical regulator of EC-to-TEC transition. FOXF1 was highly expressed in normal lung vasculature but was decreased in TEC within non-small cell lung cancers (NSCLC). Low FOXF1 correlated with poor overall survival of NSCLC patients. In mice, endothelial-specific deletion of FOXF1 decreased pericyte coverage, increased vessel permeability and hypoxia, and promoted lung tumor growth and metastasis. Endothelial-specific overexpression of FOXF1 normalized tumor vessels and inhibited the progression of lung cancer. FOXF1 deficiency decreased Wnt/β-catenin signaling in TECs through direct transcriptional activation of Fzd4. Restoring FZD4 expression in FOXF1-deficient TECs through endothelial-specific nanoparticle delivery of Fzd4 cDNA rescued Wnt/β-catenin signaling in TECs, normalized tumor vessels and inhibited the progression of lung cancer. Altogether, FOXF1 increases tumor vessel stability, and inhibits lung cancer progression by stimulating FZD4/Wnt/β-catenin signaling in TECs. Nanoparticle delivery of FZD4 cDNA has promise for future therapies in NSCLC.
• Deng, Z., Gao, W., Kohram, F., Li, E., Kalin, T. V., Shi, D., & Kalinichenko, V. V. (2024). Fluorinated amphiphilic Poly(β-Amino ester) nanoparticle for highly efficient and specific delivery of nucleic acids to the Lung capillary endothelium. Bioactive materials, 31, 1-17.
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  Endothelial cell dysfunction occurs in a variety of acute and chronic pulmonary diseases including pulmonary hypertension, viral and bacterial pneumonia, bronchopulmonary dysplasia, and congenital lung diseases such as alveolar capillary dysplasia with misalignment of pulmonary veins (ACDMPV). To correct endothelial dysfunction, there is a critical need for the development of nanoparticle systems that can deliver drugs and nucleic acids to endothelial cells with high efficiency and precision. While several nanoparticle delivery systems targeting endothelial cells have been recently developed, none of them are specific to lung endothelial cells without targeting other organs in the body. In the present study, we successfully solved this problem by developing non-toxic poly(β-amino) ester (PBAE) nanoparticles with specific structure design and fluorinated modification for high efficiency and specific delivery of nucleic acids to the pulmonary endothelial cells. After intravenous administration, the PBAE nanoparticles were capable of delivering non-integrating DNA plasmids to lung microvascular endothelial cells but not to other lung cell types. IVIS whole body imaging and flow cytometry demonstrated that DNA plasmid were functional in the lung endothelial cells but not in endothelial cells of other organs. Fluorination of PBAE was required for lung endothelial cell-specific targeting. Hematologic analysis and liver and kidney metabolic panels demonstrated the lack of toxicity in experimental mice. Thus, fluorinated PBAE nanoparticles can be an ideal vehicle for gene therapy targeting lung microvascular endothelium in pulmonary vascular disorders.
• Li, E., Wen, B., Gao, D., Kalin, T. R., Wang, G., Kalin, T. V., & Kalinichenko, V. V. (2024). The bone marrow of mouse-rat chimeras contains progenitors of multiple pulmonary cell lineages. Frontiers in cell and developmental biology, 12, 1394098.
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  Radiation-induced lung injury (RILI) is a common complication of anti-cancer treatments for thoracic and hematologic malignancies. Bone marrow (BM) transplantation restores hematopoietic cell lineages in cancer patients. However, it is ineffective in improving lung repair after RILI due to the paucity of respiratory progenitors in BM transplants. In the present study, we used blastocyst injection to create mouse-rat chimeras, these are artificial animals in which BM is enriched with mouse-derived progenitor cells. FACS-sorted mouse BM cells from mouse-rat chimeras were transplanted into lethally irradiated syngeneic mice, and the contribution of donor cells to the lung tissue was examined using immunostaining and flow cytometry. Donor BM cells provided long-term contributions to all lung-resident hematopoietic cells which includes alveolar macrophages and dendritic cells. Surprisingly, donor BM cells also contributed up to 8% in pulmonary endothelial cells and stromal cells after RILI. To identify respiratory progenitors in donor BM, we performed single-cell RNA sequencing (scRNAseq). Compared to normal mouse BM, increased numbers of hematopoietic progenitors were found in the BM of mouse-rat chimeras. We also identified unique populations of hemangioblast-like progenitor cells expressing , and along with mesenchymal stromal cells expressing , and that were absent or ultra-rare in the normal mouse BM. In summary, by using rats as "bioreactors", we created a unique mouse BM cell transplant that contributes to multiple respiratory cell types after RILI. Interspecies chimeras have promise for future generations of BM transplants enriched in respiratory progenitor cells.
• Li, E., Wen, B., Gao, D., Kalin, T., Wang, G., Kalin, T., & Kalinichenko, V. (2024). The bone marrow of mouse-rat chimeras contains progenitors of multiple pulmonary cell lineages. Frontiers in Cell and Developmental Biology, 12(Issue). doi:10.3389/fcell.2024.1394098
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  Radiation-induced lung injury (RILI) is a common complication of anti-cancer treatments for thoracic and hematologic malignancies. Bone marrow (BM) transplantation restores hematopoietic cell lineages in cancer patients. However, it is ineffective in improving lung repair after RILI due to the paucity of respiratory progenitors in BM transplants. In the present study, we used blastocyst injection to create mouse-rat chimeras, these are artificial animals in which BM is enriched with mouse-derived progenitor cells. FACS-sorted mouse BM cells from mouse-rat chimeras were transplanted into lethally irradiated syngeneic mice, and the contribution of donor cells to the lung tissue was examined using immunostaining and flow cytometry. Donor BM cells provided long-term contributions to all lung-resident hematopoietic cells which includes alveolar macrophages and dendritic cells. Surprisingly, donor BM cells also contributed up to 8% in pulmonary endothelial cells and stromal cells after RILI. To identify respiratory progenitors in donor BM, we performed single-cell RNA sequencing (scRNAseq). Compared to normal mouse BM, increased numbers of hematopoietic progenitors were found in the BM of mouse-rat chimeras. We also identified unique populations of hemangioblast-like progenitor cells expressing Hes1, Dntt and Ebf1, along with mesenchymal stromal cells expressing Cpox, Blvrb and Ermap that were absent or ultra-rare in the normal mouse BM. In summary, by using rats as “bioreactors”, we created a unique mouse BM cell transplant that contributes to multiple respiratory cell types after RILI. Interspecies chimeras have promise for future generations of BM transplants enriched in respiratory progenitor cells.
• Merjaneh, N., Hajjar, M., Lan, Y. W., Kalinichenko, V. V., & Kalin, T. V. (2024). The Promise of Combination Therapies with FOXM1 Inhibitors for Cancer Treatment. Cancers, 16(4).
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  Forkhead box M1 (FOXM1) is a transcription factor in the forkhead (FOX) family, which is required for cellular proliferation in normal and neoplastic cells. FOXM1 is highly expressed in many different cancers, and its expression is associated with a higher tumor stage and worse patient-related outcomes. Abnormally high expression of FOXM1 in cancers compared to normal tissue makes FOXM1 an attractive target for pharmacological inhibition. FOXM1-inhibiting agents and specific FOXM1-targeted small-molecule inhibitors have been developed in the lab and some of them have shown promising efficacy and safety profiles in mouse models. While the future goal is to translate FOXM1 inhibitors to clinical trials, potential synergistic drug combinations can maximize anti-tumor efficacy while minimizing off-target side effects. Hence, we discuss the rationale and efficacy of all previously studied drug combinations with FOXM1 inhibitors for cancer therapies.
• Mohammed, A. N., Kohram, F., Lan, Y. W., Li, E., Kolesnichenko, O. A., Kalin, T. V., & Kalinichenko, V. V. (2024). Transplantation of alveolar macrophages improves the efficacy of endothelial progenitor cell therapy in mouse model of bronchopulmonary dysplasia. American journal of physiology. Lung cellular and molecular physiology, 327(1), L114-L125.
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  Bronchopulmonary dysplasia (BPD) is a severe complication of preterm births, which develops due to exposure to supplemental oxygen and mechanical ventilation. Published studies demonstrated that the number of endothelial progenitor cells (EPC) is decreased in mouse and human BPD lungs and that adoptive transfer of EPC is an effective approach in reversing the hyperoxia-induced lung damage in mouse model of BPD. Recent advancements in macrophage biology identified the specific subtypes of circulating and resident macrophages mediating the developmental and regenerative functions in the lungs. Several studies reported the successful application of macrophage therapy in accelerating the regenerative capacity of damaged tissues and enhancing the therapeutic efficacy of other transplantable progenitor cells. In the present study, we explored the efficacy of combined cell therapy with EPC and resident alveolar macrophages (rAM) in hyperoxia-induced BPD mouse model. rAM and EPC were purified from neonatal mouse lungs and were used for adoptive transfer to the recipient neonatal mice exposed to hyperoxia. Adoptive transfer of rAM alone did not result in engraftment of donor rAM into the lung tissue but increased the mRNA level and protein concentration of proangiogenic CXCL12 chemokine in recipient mouse lungs. Depletion of rAM by chlodronate-liposomes decreased the retention of donor EPC after their transplantation into hyperoxia-injured lungs. Adoptive transfer of rAM in combination with EPC enhanced the therapeutic efficacy of EPC as evidenced by increased retention of EPC, increased capillary density, improved arterial oxygenation, and alveolarization in hyperoxia-injured lungs. Dual therapy with EPC and rAM has promise in human BPD. Recent studies demonstrated that transplantation of lung-resident endothelial progenitor cells (EPC) is an effective therapy in mouse model of bronchopulmonary dysplasia (BPD). However, key factors regulating the efficacy of EPC are unknown. Herein, we demonstrate that transplantation of tissue-resident alveolar macrophages (rAM) increases CXCL12 expression in neonatal mouse lungs. rAM are required for retention of donor EPC in hyperoxia-injured lungs. Co-transplantation of rAM and EPC improves the efficacy of EPC therapy in mouse BPD model.
• Mohammed, A., Kohram, F., Lan, Y., Li, E., Kolesnichenko, O., Kalin, T., & Kalinichenko, V. (2024). Transplantation of alveolar macrophages improves the efficacy of endothelial progenitor cell therapy in mouse model of bronchopulmonary dysplasia. American journal of physiology. Lung cellular and molecular physiology, 327(1). doi:10.1152/ajplung.00274.2023
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  Bronchopulmonary dysplasia (BPD) is a severe complication of preterm births, which develops due to exposure to supplemental oxygen and mechanical ventilation. Published studies demonstrated that the number of endothelial progenitor cells (EPC) is decreased in mouse and human BPD lungs and that adoptive transfer of EPC is an effective approach in reversing the hyperoxia-induced lung damage in mouse model of BPD. Recent advancements in macrophage biology identified the specific subtypes of circulating and resident macrophages mediating the developmental and regenerative functions in the lungs. Several studies reported the successful application of macrophage therapy in accelerating the regenerative capacity of damaged tissues and enhancing the therapeutic efficacy of other transplantable progenitor cells. In the present study, we explored the efficacy of combined cell therapy with EPC and resident alveolar macrophages (rAM) in hyperoxia-induced BPD mouse model. rAM and EPC were purified from neonatal mouse lungs and were used for adoptive transfer to the recipient neonatal mice exposed to hyperoxia. Adoptive transfer of rAM alone did not result in engraftment of donor rAM into the lung tissue but increased the mRNA level and protein concentration of proangiogenic CXCL12 chemokine in recipient mouse lungs. Depletion of rAM by chlodronate-liposomes decreased the retention of donor EPC after their transplantation into hyperoxia-injured lungs. Adoptive transfer of rAM in combination with EPC enhanced the therapeutic efficacy of EPC as evidenced by increased retention of EPC, increased capillary density, improved arterial oxygenation, and alveolarization in hyperoxia-injured lungs. Dual therapy with EPC and rAM has promise in human BPD.NEW & NOTEWORTHY Recent studies demonstrated that transplantation of lung-resident endothelial progenitor cells (EPC) is an effective therapy in mouse model of bronchopulmonary dysplasia (BPD). However, key factors regulating the efficacy of EPC are unknown. Herein, we demonstrate that transplantation of tissue-resident alveolar macrophages (rAM) increases CXCL12 expression in neonatal mouse lungs. rAM are required for retention of donor EPC in hyperoxia-injured lungs. Co-transplantation of rAM and EPC improves the efficacy of EPC therapy in mouse BPD model.
• Wang, G., Wen, B., Guo, M., Li, E., Zhang, Y., Whitsett, J. A., Kalin, T. V., & Kalinichenko, V. V. (2024). Identification of endothelial and mesenchymal FOXF1 enhancers involved in alveolar capillary dysplasia. Nature communications, 15(1), 5233.
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  Mutations in the FOXF1 gene, a key transcriptional regulator of pulmonary vascular development, cause Alveolar Capillary Dysplasia with Misalignment of Pulmonary Veins, a lethal lung disease affecting newborns and infants. Identification of new FOXF1 upstream regulatory elements is critical to explain why frequent non-coding FOXF1 deletions are linked to the disease. Herein, we use multiome single-nuclei RNA and ATAC sequencing of mouse and human patient lungs to identify four conserved endothelial and mesenchymal FOXF1 enhancers. We demonstrate that endothelial FOXF1 enhancers are autoactivated, whereas mesenchymal FOXF1 enhancers are regulated by EBF1 and GLI1. The cell-specificity of FOXF1 enhancers is validated by disrupting these enhancers in mouse embryonic stem cells using CRISPR/Cpf1 genome editing followed by lineage-tracing of mutant embryonic stem cells in mouse embryos using blastocyst complementation. This study resolves an important clinical question why frequent non-coding FOXF1 deletions that interfere with endothelial and mesenchymal enhancers can lead to the disease.
• Wang, G., Wen, B., Guo, M., Li, E., Zhang, Y., Whitsett, J., Kalin, T., & Kalinichenko, V. (2024). Identification of endothelial and mesenchymal FOXF1 enhancers involved in alveolar capillary dysplasia. Nature Communications, 15(1). doi:10.1038/s41467-024-49477-6
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  Mutations in the FOXF1 gene, a key transcriptional regulator of pulmonary vascular development, cause Alveolar Capillary Dysplasia with Misalignment of Pulmonary Veins, a lethal lung disease affecting newborns and infants. Identification of new FOXF1 upstream regulatory elements is critical to explain why frequent non-coding FOXF1 deletions are linked to the disease. Herein, we use multiome single-nuclei RNA and ATAC sequencing of mouse and human patient lungs to identify four conserved endothelial and mesenchymal FOXF1 enhancers. We demonstrate that endothelial FOXF1 enhancers are autoactivated, whereas mesenchymal FOXF1 enhancers are regulated by EBF1 and GLI1. The cell-specificity of FOXF1 enhancers is validated by disrupting these enhancers in mouse embryonic stem cells using CRISPR/Cpf1 genome editing followed by lineage-tracing of mutant embryonic stem cells in mouse embryos using blastocyst complementation. This study resolves an important clinical question why frequent non-coding FOXF1 deletions that interfere with endothelial and mesenchymal enhancers can lead to the disease.
• Wen, B., Li, E., Wang, G., Kalin, T. R., Gao, D., Lu, P., Kalin, T. V., & Kalinichenko, V. V. (2024). CRISPR-Cas9 Genome Editing Allows Generation of the Mouse Lung in a Rat. American journal of respiratory and critical care medicine, 210(2), 167-177.
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  Recent efforts in bioengineering and embryonic stem cell (ESC) technology allowed the generation of ESC-derived mouse lung tissues in transgenic mice that were missing critical morphogenetic genes. Epithelial cell lineages were efficiently generated from ESC, but other cell types were mosaic. A complete contribution of donor ESCs to lung tissue has never been achieved. The mouse lung has never been generated in a rat. We sought to generate the mouse lung in a rat. Clustered regularly interspaced short palindromic repeat (CRISPR)-Cas9 genome editing was used to disrupt the gene in rat one-cell zygotes. Interspecies mouse-rat chimeras were produced by injection of wild-type mouse ESCs into -deficient rat embryos with lung agenesis. The contribution of mouse ESCs to the lung tissue was examined by immunostaining, flow cytometry, and single-cell RNA sequencing. Peripheral pulmonary and thyroid tissues were absent in rat embryos after CRISPR-Cas9-mediated disruption of the gene. Complementation of rat blastocysts with mouse ESCs restored pulmonary and thyroid structures in mouse-rat chimeras, leading to a near-99% contribution of ESCs to all respiratory cell lineages. Epithelial, endothelial, hematopoietic, and stromal cells in ESC-derived lungs were highly differentiated and exhibited lineage-specific gene signatures similar to those of respiratory cells from the normal mouse lung. Analysis of receptor-ligand interactions revealed normal signaling networks between mouse ESC-derived respiratory cells differentiated in a rat. A combination of CRISPR-Cas9 genome editing and blastocyst complementation was used to produce mouse lungs in rats, making an important step toward future generations of human lungs using large animals as "bioreactors."
• Acharya, A., Bian, F., Gomez-Arroyo, J., Wagner, K. A., Kalinichenko, V. V., & Kalin, T. V. (2023). Hypoxia represses FOXF1 in lung endothelial cells through HIF-1α. Frontiers in physiology, 14, 1309155.
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  Forkhead Box F1 (FOXF1) transcription factor plays a critical role in lung angiogenesis during embryonic development and lung repair after injury. FOXF1 expression is decreased in endothelial cells after lung injury; however, molecular mechanisms responsible for the FOXF1 transcript changes in injured lung endothelium remain unknown. We used immunostaining of injured mouse lung tissues, FACS-sorted lung endothelial cells from hypoxia-treated mice, and data from patients diagnosed with hypoxemic respiratory failure to demonstrate that hypoxia is associated with decreased FOXF1 expression. Endothelial cell cultures were used to induce hypoxia and identify the upstream molecular mechanism through which hypoxia inhibits FOXF1 gene expression. Bleomycin-induced lung injury induced hypoxia in the mouse lung tissue which was associated with decreased expression. Human mRNA was decreased in the lungs of patients diagnosed with hypoxemic respiratory failure. Mice exposed to hypoxia exhibited reduced expression in the lung tissue and FACS-sorted lung endothelial cells. , hypoxia (1% of O) or treatment with cobalt (II) chloride increased HIF-1α protein levels but inhibited expression in three endothelial cell lines. Overexpression of HIF-1α in cultured endothelial cells was sufficient to inhibit expression. siRNA-mediated depletion of HIF-1α prevented the downregulation of gene expression after hypoxia or cobalt (II) chloride treatment. Hypoxia inhibits FOXF1 expression in endothelial cells in a HIF-1α dependent manner. Our data suggest that endothelial cell-specific inhibition of HIF-1α via gene therapy can be considered to restore FOXF1 and improve lung repair in patients with severe lung injury.
• Bian, F., Lan, Y. W., Zhao, S., Deng, Z., Shukla, S., Acharya, A., Donovan, J., Le, T., Milewski, D., Bacchetta, M., Hozain, A. E., Tipograf, Y., Chen, Y. W., Xu, Y., Shi, D., Kalinichenko, V. V., & Kalin, T. V. (2023). Lung endothelial cells regulate pulmonary fibrosis through FOXF1/R-Ras signaling. Nature communications, 14(1), 2560.
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  Pulmonary fibrosis results from dysregulated lung repair and involves multiple cell types. The role of endothelial cells (EC) in lung fibrosis is poorly understood. Using single cell RNA-sequencing we identified endothelial transcription factors involved in lung fibrogenesis, including FOXF1, SMAD6, ETV6 and LEF1. Focusing on FOXF1, we found that FOXF1 is decreased in EC within human idiopathic pulmonary fibrosis (IPF) and mouse bleomycin-injured lungs. Endothelial-specific Foxf1 inhibition in mice increased collagen depositions, promoted lung inflammation, and impaired R-Ras signaling. In vitro, FOXF1-deficient EC increased proliferation, invasion and activation of human lung fibroblasts, and stimulated macrophage migration by secreting IL-6, TNFα, CCL2 and CXCL1. FOXF1 inhibited TNFα and CCL2 through direct transcriptional activation of Rras gene promoter. Transgenic overexpression or endothelial-specific nanoparticle delivery of Foxf1 cDNA decreased pulmonary fibrosis in bleomycin-injured mice. Nanoparticle delivery of FOXF1 cDNA can be considered for future therapies in IPF.
• Donovan, J., Deng, Z., Bian, F., Shukla, S., Gomez-Arroyo, J., Shi, D., Kalinichenko, V. V., & Kalin, T. V. (2023). Corrigendum: Improving anti-tumor efficacy of low-dose Vincristine in rhabdomyosarcoma the combination therapy with FOXM1 inhibitor RCM1. Frontiers in oncology, 13, 1163510.
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  [This corrects the article DOI: 10.3389/fonc.2023.1112859.].
• Donovan, J., Deng, Z., Bian, F., Shukla, S., Gomez-Arroyo, J., Shi, D., Kalinichenko, V. V., & Kalin, T. V. (2023). Improving anti-tumor efficacy of low-dose Vincristine in rhabdomyosarcoma the combination therapy with FOXM1 inhibitor RCM1. Frontiers in oncology, 13, 1112859.
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  Rhabdomyosarcoma (RMS) is a highly metastatic soft-tissue sarcoma that often develops resistance to current therapies, including vincristine. Since the existing treatments have not significantly improved survival, there is a critical need for new therapeutic approaches for RMS patients. FOXM1, a known oncogene, is highly expressed in RMS, and is associated with the worst prognosis in RMS patients. In the present study, we found that the combination treatment with specific FOXM1 inhibitor RCM1 and low doses of vincristine is more effective in increasing apoptosis and decreasing RMS cell proliferation compared to single drugs alone. Since RCM1 is highly hydrophobic, we developed innovative nanoparticle delivery system containing poly-beta-amino-esters and folic acid (NP), which efficiently delivers RCM1 to mouse RMS tumors . The combination of low doses of vincristine together with intravenous administration of NP nanoparticles containing RCM1 effectively reduced RMS tumor volumes, increased tumor cell death and decreased tumor cell proliferation in RMS tumors compared to RCM1 or vincristine alone. The combination therapy was non-toxic as demonstrated by liver metabolic panels using peripheral blood serum. Using RNA-seq of dissected RMS tumors, we identified as a uniquely downregulated gene after the combination treatment. Knockdown of in RMS cells recapitulated the effects of the combination therapy. Altogether, combination treatment with low doses of vincristine and nanoparticle delivery of FOXM1 inhibitor RCM1 in a pre-clinical model of RMS has superior anti-tumor effects and decreases CHAC1 while reducing vincristine toxicity.
• Kolesnichenko, O. A., Flood, H. M., Zhang, Y., Ustiyan, V., Cuervo Jimenez, H. K., Kalin, T. V., & Kalinichenko, V. V. (2023). Endothelial progenitor cells derived from embryonic stem cells prevent alveolar simplification in a murine model of bronchopulmonary dysplasia. Frontiers in cell and developmental biology, 11, 1209518.
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  Vascular remodeling and compromised alveolar development are hallmarks of chronic pulmonary diseases such as bronchopulmonary dysplasia (BPD). Despite advances in neonatal healthcare the number of BPD cases worldwide continues to increase. One approach to overcoming the premature arrest in lung development seen in BPD is to stimulate neonatal angiogenesis via delivery and engraftment of endothelial progenitor cells (EPCs). One such population is resident to the pulmonary microvasculature and expresses both FOXF1 and c-KIT. Previous studies have shown that c-KITFOXF1 EPCs are highly sensitive to elevated levels of oxygen (hyperoxia) and are decreased in premature infants with BPD and hyperoxia-induced BPD mouse models. We hypothesize that restoring EPCs through transplantation of c-KITFOXF1 EPCs derived from pluripotent embryonic stem cells (ESCs), will stimulate neonatal angiogenesis and alveolarization in mice with hyperoxia-induced lung injury. Utilizing a novel ESC line with a FOXF1:GFP reporter, we generated ESC-derived c-KITFOXF1 EPCs . Using a second ESC line which contains FOXF1:GFP and tdTomato transgenes, we differentiated ESCs towards c-KITFOXF1 EPCs and tracked them after injection into the neonatal circulation of hyperoxia-injured mice. After a recovery period in room air conditions, we analyzed c-KITFOXF1 EPC engraftment and quantified the number of resident and circulating endothelial cells, the size of alveolar spaces, and the capillary density after EPC transplantations. Herein, we demonstrate that addition of BMP9 to the directed endothelial differentiation protocol results in very efficient generation of c-KITFOXF1 EPCs from pluripotent ESCs. ESC-derived c-KITFOXF1 EPCs effectively engraft into the pulmonary microvasculature of hyperoxia-injured mice, promote vascular remodeling in alveoli, increase the number of resident and circulating endothelial cells, and improve alveolarization. Altogether, these results provide a proof-of-principle that cell therapy with ESC-derived c-KITFOXF1 EPCs can prevent alveolar simplification in a hyperoxia-induced BPD mouse model.
• Pradhan, A., Che, L., Ustiyan, V., Reza, A. A., Pek, N. M., Zhang, Y., Alber, A. B., Kalin, T. R., Wambach, J. A., Gu, M., Kotton, D. N., Siefert, M. E., Ziady, A. G., Kalin, T. V., & Kalinichenko, V. V. (2023). Novel FOXF1-Stabilizing Compound TanFe Stimulates Lung Angiogenesis in Alveolar Capillary Dysplasia. American journal of respiratory and critical care medicine, 207(8), 1042-1054.
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  Alveolar capillary dysplasia with misalignment of pulmonary veins (ACDMPV) is linked to heterozygous mutations in the (Forkhead Box F1) gene, a key transcriptional regulator of pulmonary vascular development. There are no effective treatments for ACDMPV other than lung transplant, and new pharmacological agents activating FOXF1 signaling are urgently needed. Identify-small molecule compounds that stimulate FOXF1 signaling. We used mass spectrometry, immunoprecipitation, and the ubiquitination assay to identify TanFe (transcellular activator of nuclear FOXF1 expression), a small-molecule compound from the nitrile group, which stabilizes the FOXF1 protein in the cell. The efficacy of TanFe was tested in mouse models of ACDMPV and acute lung injury and in human vascular organoids derived from induced pluripotent stem cells of a patient with ACDMPV. We identified HECTD1 as an E3 ubiquitin ligase involved in ubiquitination and degradation of the FOXF1 protein. The TanFe compound disrupted FOXF1-HECTD1 protein-protein interactions and decreased ubiquitination of the FOXF1 protein in pulmonary endothelial cells . TanFe increased protein concentrations of FOXF1 and its target genes , , and in LPS-injured mouse lungs, decreasing endothelial permeability and inhibiting lung inflammation. Treatment of pregnant mice with TanFe increased FOXF1 protein concentrations in lungs of embryos, stimulated neonatal lung angiogenesis, and completely prevented the mortality of mice after birth. TanFe increased angiogenesis in human vascular organoids derived from induced pluripotent stem cells of a patient with ACDMPV with deletion. TanFe is a novel activator of FOXF1, providing a new therapeutic candidate for treatment of ACDMPV and other neonatal pulmonary vascular diseases.
• Shukla, S., Saha, T., Rama, N., Acharya, A., Le, T., Bian, F., Donovan, J., Tan, L. A., Vatner, R., Kalinichenko, V., Mascia, A., Perentesis, J. P., & Kalin, T. V. (2023). Ultra-high dose-rate proton FLASH improves tumor control. Radiotherapy and oncology : journal of the European Society for Therapeutic Radiology and Oncology, 186, 109741.
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  Proton radiotherapy (PRT) offers potential benefits over other radiation modalities, including photon and electron radiotherapy. Increasing the rate at which proton radiation is delivered may provide a therapeutic advantage. Here, we compared the efficacy of conventional proton therapy (CONV) to ultrahigh dose-rate proton therapy, FLASH, in a mouse model of non-small cell lung cancers (NSCLC).
• Ribeiro, R., Macedo, J., Costa, M., Ustiyan, V., Shindyapina, A., Tyshkovskiy, A., Gomes, R., Castro, J., Kalin, T., Nascimento, D., Dmitriev, S., Gladyshev, V., Kalinichenko, V., Logarinho, E., & Vasques-Nóvoa, F. (2022). In vivo cyclic induction of the FOXM1 transcription factor delays natural and progeroid aging phenotypes and extends healthspan. Nature Aging, 2(5). doi:10.1038/s43587-022-00209-9
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  The FOXM1 transcription factor exhibits pleiotropic C-terminal transcriptional and N-terminal non-transcriptional functions in various biological processes critical for cellular homeostasis. We previously found that FOXM1 repression during cellular aging underlies the senescence phenotypes, which were vastly restored by overexpressing transcriptionally active FOXM1. Yet, it remains unknown whether increased expression of FOXM1 can delay organismal aging. Here, we show that in vivo cyclic induction of an N-terminal truncated FOXM1 transgene on progeroid and naturally aged mice offsets aging-associated repression of full-length endogenous Foxm1, reinstating both transcriptional and non-transcriptional functions. This translated into mitigation of several cellular aging hallmarks, as well as molecular and histopathological progeroid features of the short-lived Hutchison–Gilford progeria mouse model, significantly extending its lifespan. FOXM1 transgene induction also reinstated endogenous Foxm1 levels in naturally aged mice, delaying aging phenotypes while extending their lifespan. Thus, we disclose that FOXM1 genetic rewiring can delay senescence-associated progeroid and natural aging pathologies.
• Sun, X., Perl, A., Li, R., Bell, S., Sajti, E., Kalinichenko, V., Kalin, T., Misra, R., Deshmukh, H., Clair, G., Kyle, J., Crotty Alexander, L., Masso-Silva, J., Kitzmiller, J., Wikenheiser-Brokamp, K., Deutsch, G., Guo, M., Du, Y., Morley, M., , Valdez, M., et al. (2022). A census of the lung: CellCards from LungMAP. Developmental Cell, 57(1). doi:10.1016/j.devcel.2021.11.007
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  The human lung plays vital roles in respiration, host defense, and basic physiology. Recent technological advancements such as single-cell RNA sequencing and genetic lineage tracing have revealed novel cell types and enriched functional properties of existing cell types in lung. The time has come to take a new census. Initiated by members of the NHLBI-funded LungMAP Consortium and aided by experts in the lung biology community, we synthesized current data into a comprehensive and practical cellular census of the lung. Identities of cell types in the normal lung are captured in individual cell cards with delineation of function, markers, developmental lineages, heterogeneity, regenerative potential, disease links, and key experimental tools. This publication will serve as the starting point of a live, up-to-date guide for lung research at https://www.lungmap.net/cell-cards/. We hope that Lung CellCards will promote the community-wide effort to establish, maintain, and restore respiratory health.
• Wang, G., Wen, B., Deng, Z., Zhang, Y., Kolesnichenko, O., Ustiyan, V., Pradhan, A., Kalin, T., & Kalinichenko, V. (2022). Endothelial progenitor cells stimulate neonatal lung angiogenesis through FOXF1-mediated activation of BMP9/ACVRL1 signaling. Nature Communications, 13(1). doi:10.1038/s41467-022-29746-y
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  Pulmonary endothelial progenitor cells (EPCs) are critical for neonatal lung angiogenesis and represent a subset of general capillary cells (gCAPs). Molecular mechanisms through which EPCs stimulate lung angiogenesis are unknown. Herein, we used single-cell RNA sequencing to identify the BMP9/ACVRL1/SMAD1 pathway signature in pulmonary EPCs. BMP9 receptor, ACVRL1, and its downstream target genes were inhibited in EPCs from Foxf1WT/S52F mutant mice, a model of alveolar capillary dysplasia with misalignment of pulmonary veins (ACDMPV). Expression of ACVRL1 and its targets were reduced in lungs of ACDMPV subjects. Inhibition of FOXF1 transcription factor reduced BMP9/ACVRL1 signaling and decreased angiogenesis in vitro. FOXF1 synergized with ETS transcription factor FLI1 to activate ACVRL1 promoter. Nanoparticle-mediated silencing of ACVRL1 in newborn mice decreased neonatal lung angiogenesis and alveolarization. Treatment with BMP9 restored lung angiogenesis and alveolarization in ACVRL1-deficient and Foxf1WT/S52F mice. Altogether, EPCs promote neonatal lung angiogenesis and alveolarization through FOXF1-mediated activation of BMP9/ACVRL1 signaling.
• Wen, B., Wang, G., Li, E., Kolesnichenko, O., Tu, Z., Divanovic, S., Kalin, T., & Kalinichenko, V. (2022). In Vivo Generation of Bone Marrow from Embryonic Stem Cells in Interspecies Chimeras. eLife, 11(Issue). doi:10.7554/elife.74018
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  Generation of bone marrow (BM) from embryonic stem cells (ESCs) promises to accelerate the development of future cell therapies for life-threatening disorders. However, such approach is limited by technical challenges to produce a mixture of functional BM progenitor cells able to replace all hematopoietic cell lineages. Herein, we used blastocyst complementation to simultaneously produce BM cell lineages from mouse ESCs in a rat. Based on FACS analysis and single-cell RNA sequencing, mouse ESCs differentiated into multiple hematopoietic and stromal cell types that were indistinguishable from normal mouse BM cells based on gene expression signatures and cell surface markers. Receptor-ligand interactions identified Cxcl12- Cxcr4, Lama2-Itga6, App-Itga6, Comp-Cd47, Col1a1-Cd44 and App-Il18rap as major signaling pathways between hematopoietic progenitors and stromal cells. Multiple hematopoietic progenitors, including hematopoietic stem cells (HSCs) in mouse-rat chimeras derived more efficiently from mouse ESCs, whereas chondrocytes predominantly derived from rat cells. In the dorsal aorta and fetal liver of mouse-rat chimeras, mouse HSCs emerged and expanded faster compared to endogenous rat cells. Sequential BM transplantation of ESC-derived cells from mouse-rat chimeras rescued lethally-irradiated syngeneic mice and demonstrated long-term reconstitution potential of donor HSCs. Altogether, a fully functional bone marrow was generated from mouse ESCs using rat embryos as “bioreactors”.
• Deng, Z., Lin, J., Webster, B., Kalin, T., Kalinichenko, V., Shi, D., & Bud’ko, S. (2021). Dual targeting with cell surface electrical charge and folic acid via superparamagnetic fe3o4@cu2–xs for photothermal cancer cell killing. Cancers, 13(21). doi:10.3390/cancers13215275
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  A major challenge in cancer therapy is to achieve high cell targeting specificity for the highest therapeutic efficacy. Two major approaches have been shown to be quite effective, namely, (1) bio-marker mediated cell targeting, and (2) electrical charge driven cell binding. The former utilizes the tumor-specific moieties on nano carrier surfaces for active targeting, while the latter relies on nanoparticles binding onto the cancer cell surfaces due to differences in electrical charge. Cancer cells are known for their hallmark metabolic pattern: high rates of glycolysis that lead to negatively charged cell surfaces. In this study, the nanoparticles of Fe3O4@Cu2–xS were rendered positively charged by conjugating their surfaces with different functional groups for strong electrostatic binding onto the negatively-charged cancer cells. In addition to the positively charged surfaces, the Fe3O4@Cu2–xS nanoparticles were also modified with folic acid (FA) for biomarker-based cell targeting. The dual-targeting approach synergistically utilizes the effectiveness of both charge-and biomarker-based cell binding for enhanced cell targeting. Further, these superparamagnetic Fe3O4@Cu2–xS nanoparticles exhibit much stronger IR absorptions compared to Fe3O4, therefore much more effective in photothermal therapy.
• Li, E., Ustiyan, V., Wen, B., Kalin, G., Whitsett, J., Kalin, T., & Kalinichenko, V. (2021). Blastocyst complementation reveals that NKX2-1 establishes the proximal-peripheral boundary of the airway epithelium. Developmental Dynamics, 250(7). doi:10.1002/dvdy.298
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  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.
• Milewski, D., Shukla, S., Gryder, B., Pradhan, A., Donovan, J., Sudha, P., Vallabh, S., Pyros, A., Xu, Y., Barski, A., Szabo, S., Turpin, B., Pressey, J., Millay, D., Khan, J., Kalinichenko, V., & Kalin, T. (2021). FOXF1 is required for the oncogenic properties of PAX3-FOXO1 in rhabdomyosarcoma. Oncogene, 40(12). doi:10.1038/s41388-021-01694-9
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  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.
• Sun, F., Wang, G., Pradhan, A., Xu, K., Gomez-Arroyo, J., Zhang, Y., Kalin, G., Deng, Z., Vagnozzi, R., He, H., Dunn, A., Wang, Y., York, A., Hegde, R., Woods, J., Kalin, T., Molkentin, J., & Kalinichenko, V. (2021). Nanoparticle Delivery of STAT3 Alleviates Pulmonary Hypertension in a Mouse Model of Alveolar Capillary Dysplasia. Circulation, 144(7). doi:10.1161/circulationaha.121.053980
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  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 Foxf1WT/S52Fmice carrying the S52F Foxf1 mutation (identified in patients with ACDMPV). The ability of Foxf1WT/S52Fmutant 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 Foxf1WT/S52Fmice and determine its effects on PH and RV hypertrophy. Results: Foxf1WT/S52Fmice developed PH and RV hypertrophy after birth. The severity of PH in Foxf1WT/S52Fmice 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 Foxf1WT/S52Fembryonic stem cells have a propensity to differentiate into pulmonary myofibroblasts. Intravascular delivery of nanoparticles carrying Stat3 cDNA protected Foxf1WT/S52Fmice 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.
• Wang, G., Wen, B., Ren, X., Li, E., Zhang, Y., Guo, M., Xu, Y., Whitsett, J., Kalin, T., & Kalinichenko, V. (2021). Generation of pulmonary endothelial progenitor cells for cell-based therapy using interspecies mouse-rat chimeras. American Journal of Respiratory and Critical Care Medicine, 204(3). doi:10.1164/rccm.202003-0758oc
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  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 cells (ESCs) or induced pluripotent stem cells remains unknown. Objectives: To investigate the heterogeneity of pulmonary EPCs and derive functional EPCs from pluripotent ESCs. Methods: 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 S52F Foxf1 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. Measurements and Main Results: We identified a unique population of EPCs, FOXF11cKIT1 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. FOXF11cKIT1 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, FOXF11cKIT1 gCAPs engraft into the pulmonary vasculature, stimulate angiogenesis, improve oxygenation, and prevent alveolar simplification. FOXF11cKIT1 gCAPs, produced from ESCs in interspecies chimeras, are fully competent to stimulate neonatal lung angiogenesis and alveolarization in ACDMPV mice. Conclusions: Cell-based therapy using donor or ESC/induced pluripotent stem cell-derived FOXF11cKIT1 endothelial progenitors may be considered for treatment of human ACDMPV.
• Wang, Y., Pandey, R., Roychoudhury, K., Milewski, D., Kalin, T., Szabo, S., Pressey, J., & Hegde, R. (2021). Targeting EYA3 in ewing sarcoma retards tumor growth and angiogenesis. Molecular Cancer Therapeutics, 20(5). doi:10.1158/1535-7163.mct-20-0749
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  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.
• Wen, B., Li, E., Ustiyan, V., Wang, G., Guo, M., Na, C., Kalin, G., Galvan, V., Xu, Y., Weaver, T., Kalin, T., Whitsett, J., & Kalinichenko, V. (2021). In vivo generation of lung and thyroid tissues from embryonic stem cells using blastocyst complementation. American Journal of Respiratory and Critical Care Medicine, 203(4). doi:10.1164/rccm.201909-1836oc
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  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 Nkx2-12/2 mouse embryos with lung agenesis. Methods: Blastocyst complementation was used to produce chimeras from normal mouse ESCs and Nkx2-12/2 embryos, which lack pulmonary tissues. Nkx2-12/2 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 Nkx2-12/2 chimeras were restored after ESC complementation. Respiratory epithelial cell lineages in restored lungs of Nkx2-12/2 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. Nkx2-12/2 chimeras can be used as “bioreactors” for in vivo differentiation and functional studies of ESC-derived progenitor cells.
• Black, M., Arumugam, P., Shukla, S., Pradhan, A., Ustiyan, V., Milewski, D., Kalinichenko, V., & Kalin, T. (2020). FOXM1 nuclear transcription factor translocates into mitochondria and inhibits oxidative phosphorylation. Molecular Biology of the Cell, 31(13). doi:10.1091/mbc.e19-07-0413
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  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.
• Bolte, C., Ustiyan, V., Ren, X., Dunn, A., Pradhan, A., Wang, G., Kolesnichenko, O., Deng, Z., Zhang, Y., Shi, D., Greenberg, J., Jobe, A., Kalin, T., & Kalinichenko, V. (2020). Nanoparticle delivery of proangiogenic transcription factors into the neonatal circulation inhibits alveolar simplification caused by hyperoxia. American Journal of Respiratory and Critical Care Medicine, 202(1). doi:10.1164/rccm.201906-1232oc
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  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.
• Gasilina, A., Premnauth, G., Gurjar, P., Biesiada, J., Hegde, S., Milewski, D., Ma, G., Kalin, T., Merino, E., Meller, J., Seibel, W., Cancelas, J., Vinnedge, L., & Nassar, N. (2020). IODVA1, a guanidinobenzimidazole derivative, targets Rac activity and Ras-driven cancer models. PLoS ONE, 15(3). doi:10.1371/journal.pone.0229801
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  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.
• Goda, C., Balli, D., Black, M., Milewski, D., Le, T., Ustiyan, V., Ren, X., Kalinichenko, V., & Kalin, T. (2020). Loss of FOXM1 in macrophages promotes pulmonary fibrosis by activating p38 MAPK signaling pathway. PLoS Genetics, 16(4). doi:10.1371/journal.pgen.1008692
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  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.
• Flood, H., Bolte, C., Dasgupta, N., Sharma, A., Zhang, Y., Gandhi, C., Kalin, T., & Kalinichenko, V. (2019). The Forkhead box F1 transcription factor inhibits collagen deposition and accumulation of myofibroblasts during liver fibrosis. Biology Open, 8(2). doi:10.1242/bio.039800
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  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 αSMACreER;Foxf1 fl/fl mice to conditionally inactivate Foxf1 in myofibroblasts during carbon tetrachloride-mediated liver fibrosis. Foxf1 deletion increased collagen depositions and disrupted liver architecture. Timp2 expression was significantly increased in Foxf1-deficient mice while MMP9 activity was reduced. RNA sequencing of purified liver myofibroblasts demonstrated that FOXF1 inhibits expression of pro-fibrotic genes, Col1α2, Col5α2, and Mmp2 in fibrotic livers and binds to active repressors located in promotors and introns of these genes. Overexpression of FOXF1 inhibits Col1a2, Col5a2, and MMP2 in primary murine HSCs in vitro. Altogether, FOXF1 prevents aberrant ECM depositions during hepatic fibrosis by repressing pro-fibrotic gene transcription in myofibroblasts and HSCs.
• Pradhan, A., Dunn, A., Ustiyan, V., Bolte, C., Wang, G., Whitsett, J., Zhang, Y., Porollo, A., Hu, Y., Xiao, R., Szafranski, P., Shi, D., Stankiewicz, P., Kalin, T., & Kalinichenko, V. (2019). The S52F FOXF1 mutation inhibits STAT3 signaling and causes alveolar capillary dysplasia. American Journal of Respiratory and Critical Care Medicine, 200(8). doi:10.1164/rccm.201810-1897oc
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  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 Foxf1WT/S52F lungs and identify molecular mechanisms regulated by FOXF1. Measurements and Main Results: FOXF1 mutations were identified in 28 subjects with ACDMPV. Foxf1WT/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 Foxf1WT/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 Foxf1WT/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.
• Ramkissoon, A., Chaney, K., Milewski, D., Williams, K., Williams, R., Choi, K., Miller, A., Kalin, T., Pressey, J., Szabo, S., Azam, M., Largaespada, D., & Ratner, N. (2019). Targeted inhibition of the dual specificity phosphatases DUSP1 and DUSP6 suppress MPNST growth via JNK. Clinical Cancer Research, 25(13). doi:10.1158/1078-0432.ccr-18-3224
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  Purpose: In neurofibromatosis type 1 (NF1) and in highly aggressive malignant peripheral nerve sheath tumors (MPNSTs), constitutively active RAS-GTP and increasedMAPK 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 cellline 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.
• Ren, X., Ustiyan, V., Guo, M., Wang, G., Bolte, C., Zhang, Y., Xu, Y., Whitsett, J., Kalin, T., & Kalinichenko, V. (2019). Postnatal alveologenesis depends on FOXF1 signaling in c-KIT1 endothelial progenitor cells. American Journal of Respiratory and Critical Care Medicine, 200(9). doi:10.1164/rccm.201812-2312oc
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  Rationale: Disruption of alveologenesis is associated with severe pediatric lung disorders, including bronchopulmonary dysplasia (BPD). Although c-KIT1 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-KIT1 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-KIT1 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-KIT1 EC progenitors in the neonatal lung. Measurements and Main Results: Pulmonary c-KIT1 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-KIT1 ECswas 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-KIT1 EC progenitors. Haploinsufficiency or endothelial-specific deletion of Foxf1 in mice increased apoptosis and decreased proliferation of c-KIT1 ECs. Inactivation of either Foxf1 or c-Kit caused alveolar simplification. Adoptive transfer of c-KIT1 ECs into the neonatal circulation increased lung angiogenesis and prevented alveolar simplification in neonatal mice exposed to hyperoxia. Conclusions: Cell therapy involving c-KIT1 EC progenitors can be beneficial for the treatment of BPD.
• Shukla, S., Milewski, D., Pradhan, A., Rama, N., Rice, K., Le, T., Flick, M., Vaz, S., Zhao, X., Setchell, K., Logarinho, E., Kalinichenko, V., & Kalin, T. (2019). The FOXM1 inhibitor RCM-1 decreases carcinogenesis and nuclear b-catenin. Molecular Cancer Therapeutics, 18(7). doi:10.1158/1535-7163.mct-18-0709
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  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 b-catenin, and inhibited protein–protein interaction between b-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.
• Whitsett, J., Kalin, T., Xu, Y., & Kalinichenko, V. (2019). Building and regenerating the lung cell by cell. Physiological Reviews, 99(1). doi:10.1152/physrev.00001.2018
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  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?.
• Black, M., Milewski, D., Le, T., Ren, X., Xu, Y., Kalinichenko, V., & Kalin, T. (2018). FOXF1 Inhibits Pulmonary Fibrosis by Preventing CDH2-CDH11 Cadherin Switch in Myofibroblasts. Cell Reports, 23(2). doi:10.1016/j.celrep.2018.03.067
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  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. Black et al. demonstrated that FOXF1 inhibits pulmonary fibrosis by preventing CDH2 to CDH11 cadherin switch in myofibroblasts.
• Ustiyan, V., Bolte, C., Zhang, Y., Han, L., Xu, Y., Yutzey, K., Zorn, A., Kalin, T., Shannon, J., & Kalinichenko, V. (2018). FOXF1 transcription factor promotes lung morphogenesis by inducing cellular proliferation in fetal lung mesenchyme. Developmental Biology, 443(1). doi:10.1016/j.ydbio.2018.08.011
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  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.
• Bolte, C., Flood, H., Ren, X., Jagannathan, S., Barski, A., Kalin, T., & Kalinichenko, V. (2017). FOXF1 transcription factor promotes lung regeneration after partial pneumonectomy. Scientific Reports, 7(1). doi:10.1038/s41598-017-11175-3
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  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.
• Milewski, D., Balli, D., Ustiyan, V., Le, T., Dienemann, H., Warth, A., Breuhahn, K., Whitsett, J., Kalinichenko, V., & Kalin, T. (2017). FOXM1 activates AGR2 and causes progression of lung adenomas into invasive mucinous adenocarcinomas. PLoS Genetics, 13(12). doi:10.1371/journal.pgen.1007097
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  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 KrasG12Dtransgenic 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.
• Milewski, D., Pradhan, A., Wang, X., Cai, Y., Le, T., Turpin, B., Kalinichenko, V., & Kalin, T. (2017). FoxF1 and FoxF2 transcription factors synergistically promote rhabdomyosarcoma carcinogenesis by repressing transcription of p21 Cip1 CDK inhibitor. Oncogene, 36(6). doi:10.1038/onc.2016.254
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  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 Cip1 and p27 Kip1. 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 Cip1 promoter through-556/-545 bp region, but did not affect p27 Kip1 transcription. Knockdown of p21 Cip1 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 Cip1 gene promoter. Because of the robust oncogenic activity in RMS tumors, FoxF1 and FoxF2 may represent promising targets for anti-Tumor therapy.
• Sun, L., Ren, X., Wang, I., Pradhan, A., Zhang, Y., Flood, H., Han, B., Whitsett, J., Kalin, T., & Kalinichenko, V. (2017). The FOXM1 inhibitor RCM-1 suppresses goblet cell metaplasia and prevents IL-13 and STAT6 signaling in allergen-exposed mice. Science Signaling, 10(475). doi:10.1126/scisignal.aai8583
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  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. 2017
• Weiler, S., Pinna, F., Wolf, T., Lutz, T., Geldiyev, A., Sticht, C., Knaub, M., Thomann, S., Bissinger, M., Wan, S., Becker, D., Gretz, N., Lang, H., Bergmann, F., Ustiyan, V., Kalin, T., Singer, S., Lee, J., Marquardt, J., , Schirmacher, P., et al. (2017). Induction of Chromosome Instability by Activation of Yes-Associated Protein and Forkhead Box M1 in Liver Cancer. Gastroenterology, 152(8). doi:10.1053/j.gastro.2017.02.018
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  Background & Aims Many different types of cancer cells have chromosome instability. The hippo pathway leads to phosphorylation of the transcriptional activator yes-associated protein 1 (YAP1, YAP), which regulates proliferation and has been associated with the development of liver cancer. We investigated the effects of hippo signaling via YAP on chromosome stability and hepatocarcinogenesis in humans and mice. Methods We analyzed transcriptome data from 242 patients with hepatocellular carcinoma (HCC) to search for gene signatures associated with chromosomal instability (CIN); we investigated associations with overall survival time and cancer recurrence using Kaplan–Meier curves. We analyzed changes in expression of these signature genes, at mRNA and protein levels, after small interfering RNA–mediated silencing of YAP in Sk-Hep1, SNU182, HepG2, or pancreatic cancer cells, as well as incubation with thiostrepton (an inhibitor of forkhead box M1 [FOXM1]) or verteporfin (inhibitor of the interaction between YAP and TEA domain transcription factor 4 [TEAD4]). We performed co-immunoprecipitation and chromatin immunoprecipitation experiments. We collected liver tissues from mice that express a constitutively active form of YAP (YAPS127A) and analyzed gene expression signatures and histomorphologic parameters associated with chromosomal instability. Mice were given injections of thiostrepton and livers were collected and analyzed by immunoblotting, immunohistochemistry, histology, and real-time polymerase chain reaction. We performed immunohistochemical analyses on tissue microarrays of 105 HCCs and 7 nontumor liver tissues. Results Gene expression patterns associated with chromosome instability, called CIN25 and CIN70, were detected in HCCs from patients with shorter survival time or early cancer recurrence. TEAD4 and YAP were required for CIN25 and CIN70 signature expression via induction and binding of FOXM1. Disrupting the interaction between YAP and TEAD4 with verteporfin, or inhibiting FOXM1 with thiostrepton, reduced the chromosome instability gene expression patterns. Hyperplastic livers and tumors from YAPS127A mice had increased CIN25 and CIN70 gene expression patterns, aneuploidy, and defects in mitosis. Injection of YAPS127A mice with thiostrepton reduced liver overgrowth and signs of chromosomal instability. In human HCC tissues, high levels of nuclear YAP correlated with increased chromosome instability gene expression patterns and aneuploidy. Conclusions By analyzing cell lines, genetically modified mice, and HCC tissues, we found that YAP cooperates with FOXM1 to contribute to chromosome instability. Agents that disrupt this pathway might be developed as treatments for liver cancer. Transcriptome data are available in the Gene Expression Omnibus public database (accession numbers: GSE32597 and GSE73396).
• Cai, Y., Bolte, C., Le, T., Goda, C., Xu, Y., Kalin, T., & Kalinichenko, V. (2016). FOXF1 maintains endothelial barrier function and prevents edema after lung injury. Science Signaling, 9(424). doi:10.1126/scisignal.aad1899
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  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.
• Fulford, L., Milewski, D., Ustiyan, V., Ravishankar, N., Cai, Y., Le, T., Masineni, S., Kasper, S., Aronow, B., Kalinichenko, V., & Kalin, T. (2016). The transcription factor FOXF1 promotes prostate cancer by stimulating the mitogen-activated protein kinase ERK5. Science Signaling, 9(427). doi:10.1126/scisignal.aad5582
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  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.
• Pradhan, A., Ustiyan, V., Zhang, Y., Kalin, T., & Kalinichenko, V. (2016). Forkhead transcription factor FoxF1 interacts with Fanconi anemia protein complexes to promote DNA damage response. Oncotarget, 7(2). doi:10.18632/oncotarget.6422
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  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.
• Ustiyan, V., Zhang, Y., Perl, A., Whitsett, J., Kalin, T., & Kalinichenko, V. (2016). β-catenin and Kras/Foxm1 signaling pathway are critical to restrict Sox9 in basal cells during pulmonary branching morphogenesis. Developmental Dynamics, 245(5). doi:10.1002/dvdy.24393
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  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 KrasG12D or its downstream target Foxm1 stimulated SOX9 expression in basal cells. Genetic inactivation of Foxm1 from KrasG12D-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.
• Bolte, C., Ren, X., Tomley, T., Ustiyan, V., Pradhan, A., Hoggatt, A., Kalin, T., Herring, B., & Kalinichenko, V. (2015). Forkhead box F2 regulation of platelet-derived growth factor and myocardin/serum response factor signaling is essential for intestinal development. Journal of Biological Chemistry, 290(12). doi:10.1074/jbc.m114.609487
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  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.
• Xia, H., Ren, X., Bolte, C., Ustiyan, V., Zhang, Y., Shah, T., Kalin, T., Whitsett, J., & Kalinichenko, V. (2015). Foxm1 regulates resolution of hyperoxic lung injury in newborns. American Journal of Respiratory Cell and Molecular Biology, 52(5). doi:10.1165/rcmb.2014-0091oc
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  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.
• Cheng, X., Black, M., Ustiyan, V., Le, T., Fulford, L., Sridharan, A., Medvedovic, M., Kalinichenko, V., Whitsett, J., & Kalin, T. (2014). SPDEF Inhibits Prostate Carcinogenesis by Disrupting a Positive Feedback Loop in Regulation of the Foxm1 Oncogene. PLoS Genetics, 10(9). doi:10.1371/journal.pgen.1004656
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  SAM-pointed domain-containing ETS transcription factor (SPDEF) is expressed in normal prostate epithelium. While its expression changes during prostate carcinogenesis (PCa), the role of SPDEF in prostate cancer remains controversial due to the lack of genetic mouse models. In present study, we generated transgenic mice with the loss- or gain-of-function of SPDEF in prostate epithelium to demonstrate that SPDEF functions as tumor suppressor in prostate cancer. Loss of SPDEF increased cancer progression and tumor cell proliferation, whereas over-expression of SPDEF in prostate epithelium inhibited carcinogenesis and reduced tumor cell proliferation in vivo and in vitro. Transgenic over-expression of SPDEF inhibited mRNA and protein levels of Foxm1, a transcription factor critical for tumor cell proliferation, and reduced expression of Foxm1 target genes, including Cdc25b, Cyclin B1, Cyclin A2, Plk-1, AuroraB, CKS1 and Topo2alpha. Deletion of SPDEF in transgenic mice and cultures prostate tumor cells increased expression of Foxm1 and its target genes. Furthermore, an inverse correlation between SPDEF and Foxm1 levels was found in human prostate cancers. The two-gene signature of low SPDEF and high FoxM1 predicted poor survival in prostate cancer patients. Mechanistically, SPDEF bound to, and inhibited transcriptional activity of Foxm1 promoter by interfering with the ability of Foxm1 to activate its own promoter through auto-regulatory site located in the −745/−660 bp Foxm1 promoter region. Re-expression of Foxm1 restored cellular proliferation in the SPDEF-positive cancer cells and rescued progression of SPDEF-positive tumors in mouse prostates. Altogether, SPDEF inhibits prostate carcinogenesis by preventing Foxm1-regulated proliferation of prostate tumor cells. The present study identified novel crosstalk between SPDEF tumor suppressor and Foxm1 oncogene and demonstrated that this crosstalk is required for tumor cell proliferation during progression of prostate cancer in vivo.
• Ren, X., Ustiyan, V., Pradhan, A., Cai, Y., Havrilak, J., Bolte, C., Shannon, J., Kalin, T., & Kalinichenko, V. (2014). FOXF1 transcription factor is required for formation of embryonic vasculature by regulating VEGF signaling in endothelial cells. Circulation Research, 115(8). doi:10.1161/circresaha.115.304382
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  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 '3, ephrin B2, Tie2, and the noncoding RNA Fendrr. Chromatin immunoprecipitation assay demonstrated that Flt1, Flk1, Pdgfb, Pecam1, and Tie2 genes are direct transcriptional targets of FOXF1. Conclusions: FOXF1 is required for the formation of embryonic vasculature by regulating endothelial genes critical for vascular development and vascular endothelial growth factor signaling.
• Sen, P., Dharmadhikari, A., Majewski, T., Mohammad, M., Kalin, T., Zabielska, J., Ren, X., Bray, M., Brown, H., Welty, S., Thevananther, S., Langston, C., Szafranski, P., Justice, M., Kalinichenko, V., Gambin, A., Belmont, J., & Stankiewicz, P. (2014). Comparative analyses of lung transcriptomes in patients with alveolar capillary dysplasia with misalignment of pulmonary veins and in Foxf1 heterozygous knockout mice. PLoS ONE, 9(4). doi:10.1371/journal.pone.0094390
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  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. © 2014 Sen et al.
• Varma, S., Mahavadi, P., Sasikumar, S., Cushing, L., Hyland, T., Rosser, A., Riccardi, D., Lu, J., Kalin, T., Kalinichenko, V., Guenther, A., Ramirez, M., Pardo, A., Selman, M., & Warburton, D. (2014). Grainyhead-like 2 (GRHL2) distribution reveals novel pathophysiological differences between human idiopathic pulmonary fibrosis and mouse models of pulmonary fibrosis. American Journal of Physiology - Lung Cellular and Molecular Physiology, 306(5). doi:10.1152/ajplung.00143.2013
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  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. © 2014 the American Physiological Society.
• Wang, I., Ustiyan, V., Zhang, Y., Cai, Y., Kalin, T., & Kalinichenko, V. (2014). Foxm1 transcription factor is required for the initiation of lung tumorigenesis by oncogenic KrasG12D. Oncogene, 33(46). doi:10.1038/onc.2013.475
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  Lung cancer is the leading cause of deaths in cancer patients in the United States. Identification of new molecular targets is clearly needed to improve therapeutic outcomes of this devastating human disease. Activating mutations in K-Ras oncogene and increased expression of FOXM1 protein are associated with poor prognosis in patients with non-small-cell lung cancer. Transgenic expression of activated KrasG12D in mouse respiratory epithelium is sufficient to induce lung adenocarcinomas; however, transcriptional mechanisms regulated by K-Ras during the initiation of lung cancer remain poorly understood. Foxm1 transcription factor, a downstream target of K-Ras, stimulates cellular proliferation during embryogenesis, organ repair and tumor growth, but its role in tumor initiation is unknown. In the present study, we used transgenic mice expressing KrasG12D under control of Sftpc promoter to demonstrate that Foxm1 was induced in type II epithelial cells before the formation of lung tumors. Conditional deletion of Foxm1 from KrasG12D-expressing respiratory epithelium prevented the initiation of lung tumors in vivo. The loss of Foxm1 inhibited expression of K-Ras target genes critical for the nuclear factor-κB (NF-κB) and c-Jun N-terminal kinase (JNK) pathways, including Ikbkb, Nfkb1, Nfkb2, Rela, Jnk1, N-Myc, Pttg1 and Cdkn2a. Transgenic overexpression of activated FOXM1 mutant was sufficient to induce expression of these genes in alveolar type II cells. FOXM1 directly bound to promoter regions of Ikbkb, Nfkb2, N-Myc, Pttg1 and Cdkn2a, indicating that these genes are direct FOXM1 targets. FOXM1 is required for K-Ras-mediated lung tumorigenesis by activating genes critical for the NF-κB and JNK pathways.
• Balli, D., Ustiyan, V., Zhang, Y., Wang, I., Masino, A., Ren, X., Whitsett, J., Kalinichenko, V., & Kalin, T. (2013). Foxm1 transcription factor is required for lung fibrosis and epithelial-to-mesenchymal transition. EMBO Journal, 32(2). doi:10.1038/emboj.2012.336
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  Alveolar epithelial cells (AECs) participate in the pathogenesis of pulmonary fibrosis, producing pro-inflammatory mediators and undergoing epithelial-to-mesenchymal transition (EMT). Herein, we demonstrated the critical role of Forkhead Box M1 (Foxm1) transcription factor in radiation-induced pulmonary fibrosis. Foxm1 was induced in AECs following lung irradiation. Transgenic expression of an activated Foxm1 transcript in AECs enhanced radiation-induced pneumonitis and pulmonary fibrosis, and increased the expression of IL-1β, Ccl2, Cxcl5, Snail1, Zeb1, Zeb2 and Foxf1. Conditional deletion of Foxm1 from respiratory epithelial cells decreased radiation-induced pulmonary fibrosis and prevented the increase in EMT-associated gene expression. siRNA-mediated inhibition of Foxm1 prevented TGF-β-induced EMT in vitro. Foxm1 bound to and increased promoter activity of the Snail1 gene, a critical transcriptional regulator of EMT. Expression of Snail1 restored TGF-β-induced loss of E-cadherin in Foxm1-deficient cells in vitro. Lineage-tracing studies demonstrated that Foxm1 increased EMT during radiation-induced pulmonary fibrosis in vivo. Foxm1 is required for radiation-induced pulmonary fibrosis by enhancing the expression of genes critical for lung inflammation and EMT. © 2013 European Molecular Biology Organization.
• Cai, Y., Balli, D., Ustiyan, V., Fulford, L., Hiller, A., Misetic, V., Zhang, Y., Paluch, A., Waltz, S., Kasper, S., & Kalin, T. (2013). Foxm1 expression in prostate epithelial cells is essential for prostate carcinogenesis. Journal of Biological Chemistry, 288(31). doi:10.1074/jbc.m113.455089
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  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. © 2013 by The American Society for Biochemistry and Molecular Biology, Inc.
• Hoggatt, A., Kim, J., Ustiyan, V., Ren, X., Kalin, T., Kalinichenko, V., & Herring, B. (2013). The transcription factor Foxf1 binds to serum response factor and myocardin to regulate gene transcription in visceral smooth muscle cells. Journal of Biological Chemistry, 288(40). doi:10.1074/jbc.m113.478974
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  Background:The role of Foxf1 in smooth muscle development is unknown. Results:Foxf1 binds to serum response factor and myocardin to regulate transcription and affect contractility of visceral smooth muscle cells. Conclusion:Foxf1 is required for normal development of gastrointestinal smooth muscle. Significance:Forkhead proteins interact with the SRF/myocardin axis to control the phenotype of smooth muscle cells. © 2013 by The American Society for Biochemistry and Molecular Biology, Inc.
• Ren, X., Shah, T., Ustiyan, V., Zhang, Y., Shinn, J., Chen, G., Whitsett, J., Kalin, T., & Kalinichenko, V. (2013). FOXM1 promotes allergen-induced goblet cell metaplasia and pulmonary inflammation. Molecular and Cellular Biology, 33(2). doi:10.1128/mcb.00934-12
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  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. © 2013, American Society for Microbiology.
• Balli, D., Ren, X., Chou, F., Cross, E., Zhang, Y., Kalinichenko, V., & Kalin, T. (2012). Foxm1 transcription factor is required for macrophage migration during lung inflammation and tumor formation. Oncogene, 31(34). doi:10.1038/onc.2011.549
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  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. © 2012 Macmillan Publishers Limited. All rights reserved.
• Bolte, C., Zhang, Y., York, A., Kalin, T., Schultz, J., Molkentin, J., & Kalinichenko, V. (2012). Postnatal Ablation of Foxm1 from Cardiomyocytes Causes Late Onset Cardiac Hypertrophy and Fibrosis without Exacerbating Pressure Overload-Induced Cardiac Remodeling. PLoS ONE, 7(11). doi:10.1371/journal.pone.0048713
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  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/Foxm1fl/fl mice were generated. Surprisingly, αMHC-Cre/Foxm1fl/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. © 2012 Bolte et al.
• Ustiyan, V., Wert, S., Ikegami, M., Wang, I., Kalin, T., Whitsett, J., & Kalinichenko, V. (2012). Foxm1 transcription factor is critical for proliferation and differentiation of Clara cells during development of conducting airways. Developmental Biology, 370(2). doi:10.1016/j.ydbio.2012.07.028
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  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. © 2012 Elsevier Inc.
• Wang, I., Snyder, J., Zhang, Y., Lander, J., Nakafuku, Y., Lin, J., Chen, G., Kalin, T., Whitsett, J., & Kalinichenko, V. (2012). Foxm1 mediates cross talk between kras/mitogen-activated protein kinase and canonical wnt pathways during development of respiratory epithelium. Molecular and Cellular Biology, 32(19). doi:10.1128/mcb.00355-12
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  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 KrasG12D. Deletion of Foxm1 from respiratory epithelial cells during lung formation prevented structural abnormalities caused by activated KrasG12D. 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. © 2012, American Society for Microbiology.
• Balli, D., Zhang, Y., Snyder, J., Kalinichenko, V., & Kalin, T. (2011). Endothelial cell-specific deletion of transcription factor foxM1 increases urethane-induced lung carcinogenesis. Cancer Research, 71(1). doi:10.1158/0008-5472.can-10-2004
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  Vascular endothelial cells provide essential support to the tumor microenvironment, but little is known about the transcriptional control of endothelial functions during tumorigenesis. Here we define a critical role for the Forkhead transcription factor FoxM1 in modulating the development of tumor-associated endothelial cells. Pulmonary tumorigenesis induced by urethane administration was compared in mice genetically deleted for FoxM1 in endothelial cells (enFoxm1-/- mice). Notably, lung tumor number and size were increased in enFoxm1-/- mice. Increased tumorigenesis was associated with increased proliferation of tumor cells and increased expression of c-Myc and cyclin D1. Furthermore, perivascular infiltration by inflammatory cells was elevated and inflammatory cells in BAL fluid were increased. Expression of Flk-1 (vascular endothelial growth factor receptor 2) and FoxF1, known regulators of pulmonary inflammation, was decreased in enFoxm1-/- mice. siRNA-mediated knockdown of FoxM1 in endothelial cells reduced Flk-1 and FoxF1 expression, which was driven by direct transcriptional induction by FoxM1 as target genes. Endothelial specific deletion of FoxM1 in vivo or in vitro also decreased expression of Sfrp1 (secreted frizzled-related protein 1), a known inhibitor of canonical Wnt signaling, in a manner that was associated with increased Wnt signaling. Taken together, our results suggest that endothelial-specific expression of FoxM1 limits lung inflammation and canonical Wnt signaling in lung epithelial cells, thereby restricting lung tumorigenesis. ©2011 AACR.
• Bolte, C., Zhang, Y., Wang, I., Kalin, T., Molkentin, J., & Kalinichenko, V. (2011). Expression of Foxm1 transcription factor in cardiomyocytes is required for myocardial development. PLoS ONE, 6(7). doi:10.1371/journal.pone.0022217
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  Forkhead Box M1 (Foxm1) is a transcription factor essential for organ morphogenesis and development of various cancers. Although complete deletion of Foxm1 in Foxm1 -/- mice caused embryonic lethality due to severe abnormalities in multiple organ systems, requirements for Foxm1 in cardiomyocytes remain to be determined. This study was designed to elucidate the cardiomyocyte-autonomous role of Foxm1 signaling in heart development. We generated a new mouse model in which Foxm1 was specifically deleted from cardiomyocytes (Nkx2.5-Cre/Foxm1 fl/f mice). Deletion of Foxm1 from cardiomyocytes was sufficient to disrupt heart morphogenesis and induce embryonic lethality in late gestation. Nkx2.5-Cre/Foxm1 fl/fl hearts were dilated with thinning of the ventricular walls and interventricular septum, as well as disorganization of the myocardium which culminated in cardiac fibrosis and decreased capillary density. Cardiomyocyte proliferation was diminished in Nkx2.5-Cre/Foxm1 fl/fl hearts owing to altered expression of multiple cell cycle regulatory genes, such as Cdc25B, Cyclin B 1, Plk-1, nMyc and p21 cip1. In addition, Foxm1 deficient hearts displayed reduced expression of CaMKIIδ, Hey2 and myocardin, which are critical mediators of cardiac function and myocardial growth. Our results indicate that Foxm1 expression in cardiomyocytes is critical for proper heart development and required for cardiomyocyte proliferation and myocardial growth. © 2011 Bolte et al.
• Ren, X., Zhang, Y., Snyder, J., Cross, E., Shah, T., Kalin, T., & Kalinichenko, V. (2010). Forkhead box M1 transcription factor is required for macrophage recruitment during liver repair. Molecular and Cellular Biology, 30(22). doi:10.1128/mcb.00876-10
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  Acute liver injury results from exposure to toxins, pharmacological agents, or viral infections, contributing to significant morbidity and mortality worldwide. While hepatic inflammation is critical for liver repair, the transcriptional mechanisms required for the recruitment of inflammatory cells to the liver are not understood. Forkhead box M1 (Foxm1) transcription factor is a master regulator of hepatocyte proliferation, but its role in inflammatory cells remains unknown. In this study, we generated transgenic mice in which Foxm1 was deleted from myeloid-derived cells, including macrophages, monocytes, and neutrophils. Carbon tetrachloride liver injury was used to demonstrate that myeloid-specific Foxm1 deletion caused a delay in liver repair. Although Foxm1 deficiency did not influence neutrophil infiltration into injured livers, the total numbers of mature macrophages were dramatically reduced. Surprisingly, Foxm1 deficiency did not influence the proliferation of macrophages or their monocytic precursors but impaired monocyte recruitment during liver repair. Expression of L-selectin and the CCR2 chemokine receptor, both critical for monocyte recruitment to injured tissues, was decreased. Foxm1 induced transcriptional activity of the mouse CCR2 promoter in cotransfection experiments. Adoptive transfer of monocytes to Foxm1-deficient mice restored liver repair and rescued liver function. Foxm1 is critical for liver repair and is required for the recruitment of monocytes to the injured liver. Copyright © 2010, American Society for Microbiology. All Rights Reserved.
• Ustiyan, V., Wang, I., Ren, X., Zhang, Y., Snyder, J., Xu, Y., Wert, S., Lessard, J., Kalin, T., & Kalinichenko, V. (2009). Forkhead box M1 transcriptional factor is required for smooth muscle cells during embryonic development of blood vessels and esophagus. Developmental Biology, 336(2). doi:10.1016/j.ydbio.2009.10.007
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  The forkhead box m1 (Foxm1 or Foxm1b) transcription factor (previously called HFH-11B, Trident, Win, or MPP2) is expressed in a variety of tissues during embryogenesis, including vascular, airway, and intestinal smooth muscle cells (SMCs). Although global deletion of Foxm1 in Foxm1-/- mice is lethal in the embryonic period due to multiple abnormalities in the liver, heart, and lung, the specific role of Foxm1 in SMC remains unknown. In the present study, Foxm1 was deleted conditionally in the developing SMC (smFoxm1-/- mice). The majority of smFoxm1-/- mice died immediately after birth due to severe pulmonary hemorrhage and structural defects in arterial wall and esophagus. Although Foxm1 deletion did not influence SMC differentiation, decreased proliferation of SMC was found in smFoxm1-/- blood vessels and esophagus. Depletion of Foxm1 in cultured SMC caused G2 arrest and decreased numbers of cells undergoing mitosis. Foxm1-deficiency in vitro and in vivo was associated with reduced expression of cell cycle regulatory genes, including cyclin B1, Cdk1-activator Cdc25b phosphatase, Polo-like 1 and JNK1 kinases, and cMyc transcription factor. Foxm1 is critical for proliferation of smooth muscle cells and is required for proper embryonic development of blood vessels and esophagus. © 2009 Elsevier Inc. All rights reserved.
• Wang, I., Meliton, L., Ren, X., Zhang, Y., Balli, D., Snyder, J., Whitsett, J., Kalinichenko, V., & Kalin, T. (2009). Deletion of forkhead box M1 transcription factor from respiratory epithelial cells inhibits pulmonary tumorigenesis. PLoS ONE, 4(8). doi:10.1371/journal.pone.0006609
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  The Forkhead Box m1 (Foxm1) protein is induced in a majority of human non-small cell lung cancers and its expression is associated with poor prognosis. However, specific requirements for the Foxm1 in each cell type of the cancer lesion remain unknown. The present study provides the first genetic evidence that the Foxm1 expression in respiratory epithelial cells is essential for lung tumorigenesis. Using transgenic mice, we demonstrated that conditional deletion of Foxm1 from lung epithelial cells (epFoxm1-/- mice) prior to tumor initiation caused a striking reduction in the number and size of lung tumors, induced by either urethane or 3-methylcholanthrene (MCA)/butylated hydroxytoluene (BHT). Decreased lung tumorigenesis in epFoxm1-/- mice was associated with diminished proliferation of tumor cells and reduced expression of Topoisomerase-2α (TOPO-2α), a critical regulator of tumor cell proliferation. Depletion of Foxm1 mRNA in cultured lung adenocarcinoma cells significantly decreased TOPO-2α mRNA and protein levels. Moreover, Foxm1 directly bound to and induced transcription of the mouse TOPO-2α promoter region, indicating that TOPO-2α is a direct target of Foxm1 in lung tumor cells. Finally, we demonstrated that a conditional deletion of Foxm1 in pre-existing lung tumors dramatically reduced tumor growth in the lung. Expression of Foxm1 in respiratory epithelial cells is critical for lung cancer formation and TOPO-2α expression in vivo, suggesting that Foxm1 is a promising target for anti-tumor therapy. © 2009 Wang et al.
• Kalin, T., Meliton, L., Meliton, A., Zhu, X., Whitsett, J., & Kalinichenko, V. (2008). Pulmonary mastocytosis and enhanced lung inflammation in mice heterozygous null for the Foxf1 gene. American Journal of Respiratory Cell and Molecular Biology, 39(4). doi:10.1165/rcmb.2008-0044oc
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  The Forkhead Box f1 (Foxf1) transcriptional factor (previously known as HFH-8 or Freac-1) is expressed in endothelial and smooth muscle cells intheembryonic andadult lung. To assess effectsof Foxf1 during lung injury, we used CCl4 and butylated hydroxytoluene (BHT) injury models. Foxf +/- 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 Foxf+/- 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 Foxf +/- mice. During lung development, Foxf+/- embryos displayed a marked increase in pulmonary mast cells before birth, and this was associated with increased CXCL12 levels in the lung. Expression of adoxycycline-inducible Foxf1dominant-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.
• Kalin, T., Wang, I., Meliton, L., Zhang, Y., Wert, S., Ren, X., Snyder, J., Bell, S., Graf, L., Whitsett, J., & Kalinichenko, V. (2008). Forkhead Box m1 transcription factor is required for perinatal lung function. Proceedings of the National Academy of Sciences of the United States of America, 105(49). doi:10.1073/pnas.0806748105
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  The Forkhead Box m1 (Foxm1 or Foxm1b) transcription factor (previously called HFH-11B, Trident, Win, or MPP2) is an important positive regulator of DNA replication and mitosis in a variety of cell types. Global deletion of Foxm1 in Foxm1-/- mice is lethal in the embryonic period, causing multiple abnormalities in the liver, heart, lung, and blood vessels. In the present study, Foxm1 was deleted conditionally in the respiratory epithelium (epFoxm1-/-). Surprisingly, deletion of Foxm1 did not alter lung growth, branching morphogenesis, or epithelial proliferation but inhibited lung maturation and caused respiratory failure after birth. Maturation defects in epFoxm1-/- lungs were associated with decreased expression of T1-α and aquaporin 5, consistent with a delay of type I cell differentiation. Expression of surfactant-associated proteins A, B, C, and D was decreased by deletion of Foxm1. Foxm1 directly bound and induced transcriptional activity of the mouse surfactant protein B and A (Sftpb and Sftpa) promoters in vitro, indicating that Foxm1 is a direct transcriptional activator of these genes. Foxm1 is critical for surfactant homeostasis and lung maturation before birth and is required for adaptation to air breathing. © 2008 by The National Academy of Sciences of the USA.
• Wang, I., Meliton, L., Tretiakova, M., Costa, R., Kalinichenko, V., & Kalin, T. (2008). Transgenic expression of the forkhead box M1 transcription factor induces formation of lung tumors. Oncogene, 27(30). doi:10.1038/onc.2008.60
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  The forkhead box m1 (Foxm1 or Foxm1b) protein (previously called HFH-11B, Trident, Win or MPP2) is abundantly expressed in human non-small cell lung cancers where it transcriptionally induces expression of genes essential for proliferation of tumor cells. In this study, we used Rosa26-Foxm1 transgenic mice, in which the Rosa26 promoter drives ubiquitous expression of Foxm1 transgene, to identify new signaling pathways regulated by Foxm1. Lung tumors were induced in Rosa26-Foxm1 mice using the 3-methylcholanthrene (MCA)/butylated hydroxytoluene (BHT) lung tumor initiation/promotion protocol. Tumors from MCA/BHT-treated Rosa26-Foxm1 mice displayed a significant increase in the number, size and DNA replication compared to wild-type mice. Elevated tumor formation in Rosa26-Foxm1 transgenic lungs was associated with persistent pulmonary inflammation, macrophage infiltration and increased expression of cyclooxygenase-2 (Cox-2), Cdc25C phosphatase, cyclin E2, chemokine ligands CXCL5, CXCL1 and CCL3, cathepsins and matrix metalloprotease-12. Cell culture experiments with A549 human lung adenocarcinoma cells demonstrated that depletion of Foxm1 by either short interfering RNA transfection or treatment with Foxm1-inhibiting ARF 26-44 peptide significantly reduced Cox-2 expression. In co-transfection experiments, Foxm1 protein-induced Cox-2 promoter activity and directly bound to the -2566/-2580 bp region of human Cox-2 promoter. © 2008 Macmillan Publishers Limited All rights reserved.
• Ksmiakrishna, S., Kim, I., Petrovic, V., Malin, D., Wang, I., Kalin, T., Meliton, L., Zhao, Y., Ackerson, T., Qin, Y., Malik, A., Costa, R., & Kalinichenko, V. (2007). Myocardium defects and ventricular hypoplasia in mice homozygous null for the Forkhead Box M1 transcription factor. Developmental Dynamics, 236(4). doi:10.1002/dvdy.21113
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  The Forkhead Box m1 (Foxm1) transcription factor is expressed in cardiomyocytes and cardiac endothelial cells during heart development. In this study, we used a novel Foxm1 -/- mouse line to demonstrate that Foxm1-deletion causes ventricular hypoplasia and diminished DNA replication and mitosis in developing cardioniyocytes. Proliferation defects in Foxm1 -/- hearts were associated with a reduced expression of Cdk1-activator Cdc25B phosphatase and NFATc3 transcription factor, and with abnormal nuclear accumulation of the Cdk-inhibitor p21Cip1 protein. Depletion of Foxm1 levels by siRNA caused altered expression of these genes in cultured HL-1 cardiomyocytes. Endothelial-specific deletion of the Foxm1 fl/fl allele in Tie2-Cre Foxm1 fl/fl embryos did not influence heart development and cardiomyocyte proliferation. Foxm1 protein binds to the -9,259/-9,288-bp region of the endogenous mouse NFATc3 promoter, indicating that Foxm1 is a transcriptional activator of the NFATc3 gene. Foxinl regulates expression of genes essential for the proliferation of cardioniyocytes during heart development. © 2007 Wiley-Liss, Inc.
• Malin, D., Kim, I., Boetticher, E., Kalin, T., Ramakrishna, S., Meliton, L., Ustiyan, V., Zhu, X., & Kalinichenko, V. (2007). Forkhead box f1 is essential for migration of mesenchymal cells and directly induces integrin-beta3 expression. Molecular and Cellular Biology, 27(7). doi:10.1128/mcb.01736-06
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  The Forkhead box f1 (Fox1) transcription factor is expressed in mesenchymal cells of the lung, liver, and gallbladder. Although Foxf1 deficiency causes severe abnormalities in the development of these organs, the molecular mechanisms underlying Foxf1 function remain uncharacterized. In this study we inactivated Foxf1 function in lung mesenchymal cells and mouse embryonic fibroblasts (MEFs) by use of either short interfering RNA duplexes or a membrane-transducing Foxf1 dominant negative (DN) mutant protein (Foxf1 DN), the latter of which is fused to the human immunodeficiency virus TAT protein transduction domain. Although Foxf1 did not influence DNA replication or cell survival, Foxf1 depletion severely diminished mesenchyme migration. Foxf1 deficiency in mesenchymal cells was associated with reduced expression of the integrin-beta3 (Itgβ3) subunit. Furthermore, we generated transgenic mice containing a tetracycline-inducible Foxf1 DN transgene. Adenovirus-mediated activation of Foxf1 DN in transgenic MEFs caused diminished cell migration and reduced Itgβ3 expression. A chromatin immunoprecipitation assay demonstrated that Foxf1 protein binds to the bp -871 to -815 region of the mouse Itgβ3 promoter. Deletion of the -871 to -815 Itgβ3 promoter region completely abolished the ability of Foxf1 to activate transcription of the Itgβ3 promoter in cotransfection experiments, indicating that the mouse Itgβ3 is a direct transcriptional target of Foxf1 protein. Foxf1 plays an essential role in mesenchyme migration by transcriptionally regulating Itgβ3. Copyright © 2007, American Society for Microbiology. All Rights Reserved.
• Kalin, T., Wang, I., Ackerson, T., Major, M., Detrisac, C., Kalinichenko, V., Lyubimov, A., & Costa, R. (2006). Increased levels of the FoxM1 transcription factor accelerate development and progression of prostate carcinomas in both TRAMP and LADY transgenic mice. Cancer Research, 66(3). doi:10.1158/0008-5472.can-05-3138
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  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. ©2006 American Association for Cancer Research.
• Kim, I., Ackerson, T., Ramakrishna, S., Tretiakova, M., Wang, I., Kalin, T., Major, M., Gusarova, G., Yoder, H., Costa, R., & Kalinichenko, V. (2006). The Forkhead Box m1 transcription factor stimulates the proliferation of tumor cells during development of lung cancer. Cancer Research, 66(4). doi:10.1158/0008-5472.can-05-3003
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  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 f1/f1-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. ©2006 American Association for Cancer Research.