Forkhead Box Protein M1 Promotes Nasopharyngeal Carcinoma Cell Tumorigenesis Possibly via the Wnt/ß-Catenin Signaling Pathway.

BACKGROUND Forkhead box protein M1 (FoxM1) is an important transcription factor involved in the development and progression of various malignancies. However, its role in nasopharyngeal carcinoma (NPC) remains largely unknown. This study aimed to assess the effect of FoxM1 on NPC cell tumorigenesis as well as the underlying mechanism. MATERIAL AND METHODS NPC cell lines CNE-1 and CNE-2 were treated with vehicle and FoxM1 inhibitor thiostrepton or transfected with small interfering RNA. CCK-8 assay, flow cytometric assay, and Hoechst 33258 staining were performed to assess the viability, apoptosis and nuclear morphological impairment, and cell cycle, respectively. The expression of apoptosis-related caspase-3 and caspase-9 was detected by western blot analysis The tumor growth in the mouse xenograft model of NPC treated with thiostrepton or control was assessed. The expression of Wnt/ß-catenin signaling proteins p27, FoxM1, S phase kinase-associated protein 2 (SKP2), and Cyclin D1 were determined both in cells and xenograft tissues by western blot analysis. RESULTS Inhibition of FoxM1 by thiostrepton significantly suppressed NPC cell viability, induced apoptosis, increased cell cycle arrest, impaired nuclear morphology, and reduced NPC cell-derived tumor xenograft growth. Mechanistically, inhibition or knockdown of FoxM1 inactivated the Wnt/ß-catenin signaling pathway, as demonstrated by altered expression of Wnt/ß-catenin signaling-related genes, including p27, SKP2, and cyclin D1, in both NPC cells and xenograft tissues. CONCLUSIONS We identified FoxM1 as a novel regulator of NPC cell tumorigenesis in vitro and in vivo. Targeting FoxM1 could be a promising therapeutic strategy against NPC.

FOXM1D potentiates PKM2-mediated tumor glycolysis and angiogenesis.

Tumor growth, especially in the late stage, requires adequate nutrients and rich vasculature, in which PKM2 plays a convergent role. It has been reported that PKM2, together with FOXM1D, is upregulated in late-stage colorectal cancer and associated with metastasis; however, their underlying mechanism for promoting tumor progression remains elusive. Herein, we revealed that FOXM1D potentiates PKM2-mediated glycolysis and angiogenesis through multiple protein-protein interactions. In the presence of FBP, FOXM1D binds to tetrameric PKM2 and assembles a heterooctamer, restraining PKM2 metabolic activity by about a half and thereby promoting aerobic glycolysis. Furthermore, FOXM1D interacts with PKM2 and NF-κB and induces their nuclear translocation with the assistance of the nuclear transporter importin 4. Once in the nucleus, PKM2 and NF-κB complexes subsequently augment VEGFA transcription. The increased VEGFA is secreted extracellularly via exosomes, an event potentiated by the interaction of FOXM1 with VPS11, eventually promoting tumor angiogenesis. Based on these findings, our study provides another insight into the role of PKM2 in the regulation of glycolysis and angiogenesis.

LncRNA HOXA11-AS Aggravates Keloid Progression by the Regulation of HOXA11-AS-miR-205-5p-FOXM1 Pathway.

BACKGROUND: Keloid is troublesome for patients' skin appearance and mental health, although it is a benign tumor. Long noncoding RNA (lncRNA) troubling keloid is frequently reported. The purpose of this study was to investigate the role of lncRNA homeobox (HOX) A11 antisense (HOXA11-AS) and related action mechanisms during the development of keloid. METHODS: The expression of HOXA11-AS, miR-205-5p, and forkhead box M1 (FOXM1) was measured by quantitative real-time polymerase chain reaction (qRT-PCR). Cell proliferation or apoptosis was assessed using 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium (MTT) assay or flow cytometry assay. Cell migration and invasion were monitored by transwell assay. The protein levels of extracellular matrix (ECM) proteins (collagen I and collagen III), fibronectin, glucose transporter 1 (GLUT1), lactate dehydrogenase A (LDHA), and FOXM1 were quantified by Western blot. Glycolysis processes were investigated by the glycolysis stress test, glucose consumption, and lactate production. The relationship between miR-205-5p and HOXA11-AS or FOXM1 was predicted by the online tool MIRcode or starBase v2.0 and verified by dual-luciferase reporter assay or RNA immunoprecipitation (RIP). RESULTS: HOXA11-AS and FOXM1 were significantly upregulated in keloid tissues and keloid fibroblasts, while miR-205-5p was downregulated. HOXA11-AS knockdown or miR-205-5p enrichment inhibited proliferation, migration, invasion, ECM accumulation, and glycolysis but accelerated apoptosis of keloid fibroblasts. MiR-205-5p was targeted by HOXA11-AS, and its inhibition overturned the effects of HOXA11-AS knockdown. Moreover, FOXM1 was a target of miR-205-5p, and HOXA11-AS regulated the expression of FOXM1 by adsorbing miR-205-5p. FOXM1 overexpression abolished the role of miR-205-5p enrichment. CONCLUSIONS: The HOXA11-AS-miR-205-5p-FOXM1 pathway may be an active mode in which HOXA11-AS participates in the progression of keloid.

Forkhead box M1 transcription factor: a novel target for pulmonary arterial hypertension therapy.

BACKGROUND: Forkhead box M1 (FoxM1), a member of forkhead family, plays a key role in carcinogenesis, progression, invasion, metastasis and drug resistance. Based on the similarities between cancer and pulmonary arterial hypertension, studies on the roles and mechanisms of FoxM1 in pulmonary arterial hypertension have been increasing. This article aims to review recent advances in the mechanisms of signal transduction associated with FoxM1 in pulmonary arterial hypertension. DATA SOURCES: Articles were retrieved from PubMed and MEDLINE published after 1990, including-but not limited to-FoxM1 and pulmonary arterial hypertension. RESULTS: FoxM1 is overexpressed in pulmonary artery smooth muscle cells in both pulmonary arterial hypertension patients and animal models, and promotes pulmonary artery smooth muscle cell proliferation and inhibits cell apoptosis via regulating cell cycle progression. Multiple signaling molecules and pathways, including hypoxia-inducible factors, transforming growth factor-ß/Smad, SET domain-containing 3/vascular endothelial growth factor, survivin, cell cycle regulatory genes and DNA damage response network, are reported to cross talk with FoxM1 in pulmonary arterial hypertension. Proteasome inhibitors are effective in the prevention and treatment of pulmonary arterial hypertension by inhibiting the expression and transcriptional activity of FoxM1. CONCLUSIONS: FoxM1 has a crucial role in the pathogenesis of pulmonary arterial hypertension and may represent a novel therapeutic target. But more details of interaction between FoxM1 and other signaling pathways need to be clarified in the future.

FOXM1 drives proximal tubule proliferation during repair from acute ischemic kidney injury.

The proximal tubule has a remarkable capacity for repair after acute injury, but the cellular lineage and molecular mechanisms underlying this repair response are incompletely understood. Here, we developed a Kim1-GFPCreERt2 knockin mouse line (Kim1-GCE) in order to perform genetic lineage tracing of dedifferentiated cells while measuring the cellular transcriptome of proximal tubule during repair. Acutely injured genetically labeled clones coexpressed KIM1, VIMENTIN, SOX9, and KI67, indicating a dedifferentiated and proliferative state. Clonal analysis revealed clonal expansion of Kim1+ cells, indicating that acutely injured, dedifferentiated proximal tubule cells, rather than fixed tubular progenitor cells, account for repair. Translational profiling during injury and repair revealed signatures of both successful and unsuccessful maladaptive repair. The transcription factor Foxm1 was induced early in injury, was required for epithelial proliferation in vitro, and was dependent on epidermal growth factor receptor (EGFR) stimulation. In conclusion, dedifferentiated proximal tubule cells effect proximal tubule repair, and we reveal an EGFR/FOXM1-dependent signaling pathway that drives proliferative repair after injury.

[YAP induces chromosomal instability in liver cancer patients]. / YAP induziert chromosomale Instabilität in Leberkrebspatienten.

BACKGROUND: The Hippo/YAP signaling pathway is a central regulator of organ growth and cell proliferation. Activation of the transcriptional co-activator and oncogene YAP (yes-associated protein) supports the development of liver cancer. AIMS: The aim of this work was to analyze the molecular mechanisms which are responsible for YAP-induced hepatocarcinogenesis. METHODS: YAP was silenced using siRNAs in liver cancer cell lines and effects on target gene expression were analyzed via real-time polymerase chain reaction (PCR) and western immunoblotting. Immunoprecipitation and chromatin immunoprecipitation was used to study interacting proteins and binding to target gene promoter regions, respectively. Transgenic mice with liver-specific and inducible YAP expression were used for in vivo analysis. Gene expression data from hepatocellular carcinoma (HCC) patients were used to analyze YAP-dependent gene signatures and to correlate with clinical data. HCC tissue microarrays were analyzed using immunohistochemistry. RESULTS: Together with the transcription factors TEAD4 and FOXM1, YAP induces the expression of genes which are responsible for the development of chromosomal instability (CIN). The overexpression of these CIN genes characterizes liver cancer patients with a poor prognosis. Mechanistically, YAP/TEAD4 and FOXM1 bind to the promoter regions of the CIN genes to directly regulate their expression. The treatment of YAP-transgenic mice with a specific FOXM1 inhibitor reduces the YAP-dependent hepatomegaly, CIN gene expression and CIN. The analysis of human HCC tissue samples confirms the statistical correlation between YAP, FOXM1 and CIN. DISCUSSION: These results reveal a new oncogenic mechanism of the Hippo/YAP signaling pathway and identify YAP and FOXM1 as potential targets for targeted therapies.

Smooth muscle cell-specific FoxM1 controls hypoxia-induced pulmonary hypertension.

RATIONALE: Forkhead box M1 (FoxM1) is a transcription factor that promotes cell proliferation by regulating a broad spectrum of genes that participate in cell cycle regulation, such as Cyclin B, CDC25B, and Aurora B Kinase. We have shown that hypoxia, a well-known stimulus for pulmonary hypertension (PH), induces FoxM1 in pulmonary artery smooth muscle cells (PASMC) in a HIF-dependent pathway, resulting in PASMC proliferation, while the suppression of FoxM1 prevents hypoxia-induced PASMC proliferation. However, the implications of FoxM1 in the development of PH remain less known. METHODS: We determined FoxM1 levels in the lung samples of idiopathic PAH (pulmonary arterial hypertension) (IPAH) patients and hypoxia-induced PH mice. We generated constitutive and inducible smooth muscle cell (SMC)-specific FoxM1 knockdown or knockout mice as well as FoxM1 transgenic mice which overexpress FoxM1, and exposed them to hypoxia (10% O2, 90% N2) or normoxia (Room air, 21% oxygen) for four weeks, and measured PH indices. We also isolated mouse PASMC (mPASMC) and mouse embryonic fibroblasts (MEF) from these mice to examine the cell proliferation and expression levels of SMC contractile proteins. RESULTS: We showed that in hypertensive human lungs or mouse lungs, FoxM1 levels were elevated. Constitutive knockout of FoxM1 in mouse SMC caused early lethality, whereas constitutive knockdown of FoxM1 in mouse SMC prevented hypoxia-induced PH and PASMC proliferation. Inducible knockout of FoxM1 in SMC reversed hypoxia-induced pulmonary artery wall remodeling in existing PH. Overexpression of FoxM1 enhanced hypoxia-induced pulmonary artery wall remodeling and right ventricular hypertrophy in mice. Alteration of FoxM1 status did not affect hypoxia-induced hypoxia-inducible factor (HIF) activity in mice. Knockout of FoxM1 decreased PASMC proliferation and induced expression of SMC contractile proteins and TGF-ß/Smad3 signaling. CONCLUSIONS: Our studies provide clear evidence that altered FoxM1 expression in PASMC contributes to PH and uncover a correlation between Smad3-dependent signaling in FoxM1-mediated proliferation and de-differentiation of PASMC.

SETD3 negatively regulates VEGF expression during hypoxic pulmonary hypertension in rats.

Angiogenesis and activation of vascular endothelial growth factor (VEGF) signaling are tightly regulated under the condition of hypoxic pulmonary hypertension (HPH); therefore, deciphering the regulatory mechanisms associated with VEGF is important. SET domain-containing 3 (SETD3) and VEGF expression in lung tissue during hypoxia exposure and lentivirus. SETD3 treatments were detected by real-time PCR and Western blot analysis. Remodeling of pulmonary vasculature and hypertrophy of the RV were evaluated. The effects of SETD3 over-expression on the interaction between SETD3 and forkhead box protein M1 (FoxM1) at the VEGF promoter and downstream of the VEGF signal pathway during chronic hypoxia were detected. SETD3 lentiviral vector treatment not only inhibited the increase in VEGF expression but also significantly relieved pulmonary vasculature remodeling and hypertrophy of the RV during HPH. The functional interplay between SETD3 and FoxM1 on chromatin may negatively regulate VEGF expression under HPH through the VEGF receptor-extracellular signal-regulated kinase-hypoxia-induced factor-1 signal pathway. SETD3-mediated transcriptional modification of VEGF may be a potential target to inhibit the development of HPH.

FOXM1 contributes to taxane resistance by regulating UHRF1-controlled cancer cell stemness.

Therapy-induced expansion of cancer stem cells (CSCs) has been identified as one of the most critical factors contributing to therapeutic resistance, but the mechanisms of this adaptation are not fully understood. UHRF1 is a key epigenetic regulator responsible for therapeutic resistance, and controls the self-renewal of stem cells. In the present study, taxane-resistant cancer cells were established and stem-like cancer cells were expanded. UHRF1 was overexpressed in the taxane-resistant cancer cells, which maintained CSC characteristics. UHRF1 depletion overcame taxane resistance in vitro and in vivo. Additionally, FOXM1 has been reported to play a role in therapeutic resistance and the self-renewal of CSCs. FOXM1 and UHRF1 are highly correlated in prostate cancer tissues and cells, FOXM1 regulates CSCs by regulating uhrf1 gene transcription in an E2F-independent manner, and FOXM1 protein directly binds to the FKH motifs at the uhrf1 gene promoter. This present study clarified a novel mechanism by which FOXM1 controls CSCs and taxane resistance through a UHRF1-mediated signaling pathway, and validated FOXM1 and UHRF1 as two potential therapeutic targets to overcome taxane resistance.

Endothelial and Smooth Muscle Cell Interaction via FoxM1 Signaling Mediates Vascular Remodeling and Pulmonary Hypertension.

RATIONALE: Angioproliferative vasculopathy is a hallmark of pulmonary arterial hypertension (PAH). However, little is known about how endothelial cell (EC) and smooth muscle cell (SMC) crosstalk regulates the angioproliferative vascular remodeling. OBJECTIVES: To investigate the role of EC and SMC interaction and underlying signaling pathways in pulmonary hypertension (PH) development. METHODS: SMC-specific Foxm1 (forkhead box M1) or Cxcr4 knockout mice, EC-specific Foxm1 or Egln1 knockout mice, and EC-specific Egln1/Cxcl12 double knockout mice were used to assess the role of FoxM1 on SMC proliferation and PH. Lung tissues and cells from patients with PAH were used to validate clinical relevance. FoxM1 inhibitor thiostrepton was used in Sugen 5416/hypoxia- and monocrotaline-challenged rats. MEASUREMENTS AND MAIN RESULTS: FoxM1 expression was markedly upregulated in lungs and pulmonary arterial SMCs of patients with idiopathic PAH and four discrete PH rodent models. Mice with SMC- (but not EC-) specific deletion of Foxm1 were protected from hypoxia- or Sugen 5416/hypoxia-induced PH. The upregulation of FoxM1 in SMCs induced by multiple EC-derived factors (PDGF-B, CXCL12, ET-1, and MIF) mediated SMC proliferation. Genetic deletion of endothelial Cxcl12 in Egln1Tie2Cre mice or loss of its cognate receptor Cxcr4 in SMCs in hypoxia-treated mice inhibited FoxM1 expression, SMC proliferation, and PH. Accordingly, pharmacologic inhibition of FoxM1 inhibited severe PH in both Sugen 5416/hypoxia and monocrotaline-challenged rats. CONCLUSIONS: Multiple factors derived from dysfunctional ECs induced FoxM1 expression in SMCs and activated FoxM1-dependent SMC proliferation, which contributes to pulmonary vascular remodeling and PH. Thus, targeting FoxM1 signaling represents a novel strategy for treatment of idiopathic PAH.

FOXM1 promotes pulmonary artery smooth muscle cell expansion in pulmonary arterial hypertension.

Pulmonary arterial hypertension (PAH) is a progressive vascular remodeling disease characterized by a persistent elevation of pulmonary artery pressure, leading to right heart failure and premature death. Exaggerated proliferation and resistance to apoptosis of pulmonary artery smooth muscle cells (PASMCs) is a key component of vascular remodeling. Despite major advances in the field, current therapies for PAH remain poorly effective in reversing the disease or significantly improving long-term survival. Because the transcription factor FOXM1 is necessary for PASMC proliferation during lung morphogenesis and its overexpression stimulates proliferation and evasion of apoptosis in cancer cells, we thus hypothesized that upregulation of FOXM1 in PAH-PASMCs promotes cell expansion and vascular remodeling. Our results showed that FOXM1 was markedly increased in distal pulmonary arteries and isolated PASMCs from PAH patients compared to controls as well as in two preclinical models. In vitro, we showed that miR-204 expression regulates FOXM1 levels and that inhibition of FOXM1 reduced cell proliferation and resistance to apoptosis through diminished DNA repair mechanisms and decreased expression of the pro-remodeling factor survivin. Accordingly, inhibition of FOXM1 with thiostrepton significantly improved established PAH in two rat models. Thus, we show for the first time that FOXM1 is implicated in PAH development and represents a new promising target. KEY MESSAGES: FOXM1 is overexpressed in human PAH-PASMCs and PAH animal models. FOXM1 promotes PAH-PASMC proliferation and resistance to apoptosis. Pharmacological inhibition of FOXM1 improves established PAH in the MCT and Su/Hx rat models. FOXM1 may be a novel therapeutic target in PAH.

FOXM1 in Cancer: Interactions and Vulnerabilities.

FOXM1 is a transcription factor of the Forkhead family that is required for cell proliferation of normal cells. However, FOXM1 is repeatedly overexpressed in a variety of human cancers, and it has been implicated in all major hallmarks of cancer delineated by Hanahan and Weinberg. It has been postulated that the oncogenic potential of FOXM1 is determined by its capacity to transactivate target genes that are implicated in different phases of cancer development. However, FOXM1 may also play an oncogenic role by interacting with other proteins, such as ß-catenin or SMAD3 to induce oncogenic WNT and TGFß signaling pathways, respectively. In this review, I will discuss the protein-protein interactions of FOXM1 that are critical for cancer development and may represent novel targets for anticancer drugs. Cancer Res; 77(12); 3135-9. ©2017 AACR.

Gli1 promotes colorectal cancer metastasis in a Foxm1-dependent manner by activating EMT and PI3K-AKT signaling.

Colorectal cancer(CRC) is one of the most commonly diagnosed cancers in human beings and metastasis is the main death reason. Recently, Gli1 has been reported to be a key regulator of various cancer biologies and genes expressions. However, the detailed molecular mechanism of Gli1 in CRC metastasis remains largely unknown. In this study, we aimed to investigate the role of Gli1 in CRC metastasis. We used qRT-PCR, Immunohistochemistry and Western blot to test the expression levels of Gli1, Foxm1 and other target genes in the tissues and cells; Lentivirus stable transfection to change the expression levels of Gli1 and Foxm1; Wound-healing, cell invasion, migration assays and tail vein metastatic assay to test the role of Gli1 in CRC metastasis in vitro and vivo. We demonstrated that Gli1 was significantly overexpressed in colorectal cancer tissues and cells. Foxm1 level had a positive correlation with Gli1. Furthermore, we found that Gli1 promotes colorectal cancer cells metastasis in a Foxm1-dependent manner by activating EMT and PI3K-AKT signaling. Thus, we proved that Gli1 plays important role in CRC metastasis and provided a new visual field on the therapy of CRC metastasis.

Cadherin-6 type 2, K-cadherin (CDH6) is regulated by mutant p53 in the fallopian tube but is not expressed in the ovarian surface.

High-grade serous ovarian cancer (HGSOC) is the most lethal gynecological malignancy and may arise in either the fallopian tube epithelium (FTE) or ovarian surface epithelium (OSE). A mutation in p53 is reported in 96% of HGSOC, most frequently at R273 and R248. The goal of this study was to identify specific gene targets in the FTE that are altered by mutant p53, but not in the OSE. Gene analysis revealed that both R273 and R248 mutant p53 reduces CDH6 expression in the oviduct, but CDH6 was not detected in murine OSE cells. p53R273H induced SLUG and FOXM1 while p53R248W did not induce SLUG and only modestly increased FOXM1, which correlated with less migration as compared to p53R273H. An oviduct specific PAX8Cre/+/p53R270H/+ mouse model was created and confirmed that in vivo mutant p53 repressed CDH6 but was not sufficient to stabilize p53 expression alone. Overexpression of mutant p53 in the p53 null OVCAR5 cells decreased CDH6 levels indicating this was a gain-of-function. SLUG knockdown in murine oviductal cells with p53R273H restored CDH6 repression and a ChIP analysis revealed direct binding of mutant p53 on the CDH6 promoter. NSC59984, a small molecule that degrades mutant p53R273H, rescued CDH6 expression. In summary, CDH6 is expressed in the oviduct, but not the ovary, and is repressed by mutant p53. CDH6 expression with further validations may aide in establishing markers that inform upon the cell of origin of high grade serous tumors.

Epithelial-mesenchymal transition-related factors in solid tumor and hematological malignancy.

The epithelial-mesenchymal transition (EMT) process plays pivotal roles in regulatory mechanisms of embryogenesis and wound healing physiologically, and organ fibrosis, cancer progression, and metastasis pathologically. EMT is classified as primary, secondary, and tertiary during embryonic development. EMT contributes to repair of tissue injury and fibrogenesis by re-epithelialization and regeneration of fibroblasts, respectively. The hallmarks of EMT include loss of contact inhibition, remodeling of extracellular matrix, and reorganization of cytoskeleton, along with expression of mesenchymal markers and reduction of epithelial markers. Cancer cells acquire stemness, migration and invasive capability, evade apoptosis, and initiate metastasis to distant organs. Several EMT regulators including Snail, Zeb1, Zeb2, and Twist in solid tumor and Sox4, distal-less homeobox gene 4 (DLX4), Prdm14, Bmi1, and the forkhead box family in hematological malignancy are reviewed with regard to their signaling pathways, regulatory mechanisms, and clinical interactions.