ABSTRACT
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ABSTRACT
FoxM1b is a cell cycle-regulated transcription factor, whose over-expression is a marker for poor outcome in cancers. Its transcriptional activation function requires phosphorylation by Cdk1 or Cdk2 that primes FoxM1b for phosphorylation by Plk1, which triggers association with the co-activator CBP. FoxM1b also possesses transcriptional repression function. It represses the mammary differentiation gene GATA3 involving DNMT3b and Rb. We investigated what determines the two distinct functions of FoxM1b: activation and repression. We show that Rb binds to the C-terminal activation domain of FoxM1b. Analyses with phospho-defective and phospho-mimetic mutants of FoxM1b identified a critical role of the Plk1 phosphorylation sites in regulating the binding of FoxM1b to Rb and DNMT3b. That is opposite of what was seen for the interaction of FoxM1b with CBP. We show that, in addition to GATA3, FoxM1b also represses the mammary luminal differentiation marker FoxA1 by promoter-methylation, and that is regulated by the Plk1 phosphorylation sites in FoxM1b. Our results show that the Plk1 phosphorylation sites in FoxM1b serve as a regulator for its repressor function, and they provide insights into how FoxM1b inhibits differentiation genes and activates proliferation genes during cancer progression.
Subject(s)
Cell Cycle Proteins/metabolism , Forkhead Box Protein M1/metabolism , Protein Serine-Threonine Kinases/metabolism , Proto-Oncogene Proteins/metabolism , Repressor Proteins/metabolism , Retinoblastoma Protein/metabolism , Binding Sites , DNA (Cytosine-5-)-Methyltransferases/metabolism , DNA Methylation/genetics , Forkhead Box Protein M1/chemistry , GATA3 Transcription Factor/genetics , Humans , MCF-7 Cells , Mutation/genetics , Peptide Fragments/metabolism , Phosphorylation , Promoter Regions, Genetic , Protein Binding , Protein Domains , Sialoglycoproteins/metabolism , DNA Methyltransferase 3B , Polo-Like Kinase 1ABSTRACT
BACKGROUND & AIMS: Overexpression of FoxM1 correlates with poor prognosis in hepatocellular carcinoma (HCC). Moreover, the Ras-signaling pathway is found to be ubiquitously activated in HCC through epigenetic silencing of the Ras-regulators. We investigated the roles of FoxM1 in Ras-driven HCC, and on HCC cells with stem-like features. METHODS: We employed a transgenic mouse model that expresses the oncogenic Ras in the liver. That strain was crossed with a strain that harbor floxed alleles of FoxM1 and the MxCre gene that allows conditional deletion of FoxM1. FoxM1 alleles were deleted after development of HCC, and the effects on the tumors were analyzed. Also, FoxM1 siRNA was used in human HCC cell lines to determine its role in the survival of the HCC cells with stem cell features. RESULTS: Ras-driven tumors overexpress FoxM1. Deletion of FoxM1 inhibits HCC progression. There was increased accumulation of reactive oxygen species (ROS) in the FoxM1 deleted HCC cells. Moreover, FoxM1 deletion caused a disproportionate loss of the CD44+ and EpCAM+ HCC cells in the tumors. We show that FoxM1 directly activates expression of CD44 in human HCC cells. Moreover, the human HCC cells with stem cell features are addicted to FoxM1 for ROS-regulation and survival. CONCLUSION: Our results provide genetic evidence for an essential role of FoxM1 in the progression of Ras-driven HCC. In addition, FoxM1 is required for the expression of CD44 in HCC cells. Moreover, FoxM1 plays a critical role in the survival of the HCC cells with stem cell features by regulating ROS.
Subject(s)
Carcinogenesis/genetics , Carcinoma, Hepatocellular/genetics , Forkhead Transcription Factors/genetics , Gene Expression Regulation, Neoplastic , Liver Neoplasms/genetics , Stem Cells/pathology , ras Proteins/genetics , Animals , Carcinoma, Hepatocellular/metabolism , Carcinoma, Hepatocellular/pathology , Cell Line, Tumor , Cell Proliferation , Disease Progression , Forkhead Box Protein M1 , Forkhead Transcription Factors/biosynthesis , Humans , Immunohistochemistry , Liver Neoplasms/metabolism , Liver Neoplasms/pathology , Male , Mice , Mice, Inbred C57BL , Prognosis , Signal Transduction , Stem Cells/metabolism , ras Proteins/biosynthesisABSTRACT
The forkhead box transcription factor FOXM1 is considered to be a promising target for cancer therapy. However, the significance of FOXM1 in tumors harboring mutation in p53, which is very common, is unclear. In this study, we investigated the efficacy of FoxM1 targeting in spontaneous p53-null tumors using genetic ablation as well as using a peptide inhibitor of FOXM1. We show that conditional deletion of FoxM1 inhibits growth of the p53-null thymic lymphoma and sarcoma cells. In addition, deletion of FoxM1 induces apoptotic cell death of the p53-null tumors, accompanied by reduced expression of the FOXM1 target genes survivin and Bmi1. An ARF-derived peptide that inhibits the activity of FOXM1, by targeting it to the nucleolus, also induces apoptosis in the p53-null sarcoma and lymphoma, leading to a strong inhibition of their metastatic colonization. Together, our observations suggest that FOXM1 is critical for survival and growth of the p53-null lymphoma and sarcoma and provide proof-of-principle that FOXM1 is an effective therapeutic target for sarcoma and lymphoma carrying loss of function mutation in p53.
Subject(s)
Forkhead Transcription Factors/antagonists & inhibitors , Lymphoma/genetics , Lymphoma/metabolism , Sarcoma/genetics , Sarcoma/metabolism , Tumor Suppressor Protein p53/genetics , Allografts , Animals , Apoptosis/drug effects , Apoptosis/genetics , Cell Survival/drug effects , Cell Survival/genetics , Cyclin-Dependent Kinase Inhibitor p16/chemistry , Disease Models, Animal , Forkhead Box Protein M1 , Forkhead Transcription Factors/genetics , Gene Expression Regulation, Neoplastic , Inhibitor of Apoptosis Proteins/genetics , Inhibitor of Apoptosis Proteins/metabolism , Mice , Mice, Knockout , Peptides/pharmacology , Polycomb Repressive Complex 1/genetics , Polycomb Repressive Complex 1/metabolism , Proto-Oncogene Proteins/genetics , Proto-Oncogene Proteins/metabolism , Repressor Proteins/genetics , Repressor Proteins/metabolism , Survivin , Thymus Neoplasms/genetics , Thymus Neoplasms/metabolismABSTRACT
Elevated expression of FoxM1 in breast cancer correlates with an undifferentiated tumor phenotype and a negative clinical outcome. However, a role for FoxM1 in regulating mammary differentiation was not known. Here, we identify another function of FoxM1, the ability to act as a transcriptional repressor, which plays an important role in regulating the differentiation of luminal epithelial progenitors. Regeneration of mammary glands with elevated levels of FoxM1 leads to aberrant ductal morphology and expansion of the luminal progenitor pool. Conversely, knockdown of FoxM1 results in a shift toward the differentiated state. FoxM1 mediates these effects by repressing the key regulator of luminal differentiation, GATA-3. Through association with DNMT3b, FoxM1 promotes methylation of the GATA-3 promoter in an Rb-dependent manner. This study identifies FoxM1 as a critical regulator of mammary differentiation with significant implications for the development of aggressive breast cancers.
Subject(s)
Cell Lineage , Forkhead Transcription Factors/metabolism , Mammary Glands, Animal/metabolism , Mammary Glands, Animal/pathology , Mammary Glands, Human/metabolism , Mammary Glands, Human/pathology , Animals , Base Sequence , Binding Sites , Breast Neoplasms/genetics , Breast Neoplasms/pathology , Cell Differentiation , Cell Line, Tumor , Cell Proliferation , DNA (Cytosine-5-)-Methyltransferases , DNA Methylation/genetics , Female , Forkhead Box Protein M1 , GATA3 Transcription Factor/metabolism , Gene Knockdown Techniques , Humans , Mammary Glands, Animal/growth & development , Mammary Glands, Human/growth & development , Mice , Mice, Inbred C57BL , Molecular Sequence Data , Pregnancy , Promoter Regions, Genetic/genetics , Retinoblastoma Protein/metabolism , Transcription, Genetic , DNA Methyltransferase 3BABSTRACT
Malignant neuroblastomas contain stem-like cells. These tumors also overexpress the Forkhead box transcription factor FoxM1. In this study, we investigated the roles of FoxM1 in the tumorigenicity of neuroblastoma. We showed that depletion of FoxM1 inhibits anchorage-independent growth and tumorigenicity in mouse xenografts. Moreover, knockdown of FoxM1 induces differentiation in neuroblastoma cells, suggesting that FoxM1 plays a role in the maintenance of the undifferentiated progenitor population. We showed that inhibition of FoxM1 in malignant neuroblastoma cells leads to the downregulation of the pluripotency genes sex determining region Y box 2 (Sox2) and Bmi1. We provided evidence that FoxM1 directly activates expression of Sox2 in neuroblastoma cells. By using a conditional deletion system and neurosphere cultures, we showed that FoxM1 is important for expression of Sox2 and Bmi1 in the mouse neural stem/progenitor cells and is critical for its self-renewal. Together, our observations suggested that FoxM1 plays an important role in the tumorigenicity of the aggressive neuroblastoma cells through maintenance of the undifferentiated state.
Subject(s)
Forkhead Transcription Factors/physiology , Neural Stem Cells/physiology , Neuroblastoma/etiology , Animals , Cell Differentiation , Cell Line, Tumor , Forkhead Box Protein M1 , Humans , Mice , Neuroblastoma/pathology , Promoter Regions, Genetic , SOXB1 Transcription Factors/genetics , SOXB1 Transcription Factors/physiologyABSTRACT
The forkhead box M1b (FoxM1b) transcription factor is over-expressed in human cancers, and its expression often correlates with poor prognosis. Previously, using conditional knockout strains, we showed that FoxM1b is essential for hepatocellular carcinoma (HCC) development. However, over-expression of FoxM1b had only marginal effects on HCC progression. Here we investigated the effect of FoxM1b expression in the absence of its inhibitor Arf. We show that transgenic expression of FoxM1b in an Arf-null background drives hepatic fibrosis and metastasis of HCC. We identify novel mechanisms of FoxM1b that are involved in epithelial-mesenchymal transition, cell motility, invasion and a pre-metastatic niche formation. FoxM1b activates the Akt-Snail1 pathway and stimulates expression of Stathmin, lysyl oxidase, lysyl oxidase like-2 and several other genes involved in metastasis. Furthermore, we show that an Arf-derived peptide, which inhibits FoxM1b, impedes metastasis of the FoxM1b-expressing HCC cells. The observations indicate that FoxM1b is a potent activator of tumour metastasis and that the Arf-mediated inhibition of FoxM1b is a critical mechanism for suppression of tumour metastasis.
Subject(s)
Forkhead Transcription Factors/metabolism , Neoplasm Metastasis , ADP-Ribosylation Factors/genetics , ADP-Ribosylation Factors/metabolism , Animals , Carcinoma, Hepatocellular/metabolism , Carcinoma, Hepatocellular/pathology , Carcinoma, Hepatocellular/secondary , Epithelial-Mesenchymal Transition , Forkhead Box Protein M1 , Forkhead Transcription Factors/antagonists & inhibitors , Forkhead Transcription Factors/genetics , Humans , Liver Cirrhosis/metabolism , Liver Cirrhosis/pathology , Liver Neoplasms/metabolism , Liver Neoplasms/pathology , Mice , Mice, Transgenic , Protein-Lysine 6-Oxidase/metabolism , Proto-Oncogene Proteins c-akt/metabolism , Signal Transduction , Snail Family Transcription Factors , Stathmin/metabolism , Transcription Factors/metabolismABSTRACT
Inherent and acquired therapeutic resistance in breast cancer remains a major clinical challenge. In human breast cancer samples, overexpression of the oncogenic transcription factor FoxM1 has been suggested to be a marker of poor prognosis. In this study, we report that FoxM1 overexpression confers resistance to the human epidermal growth factor receptor 2 monoclonal antibody Herceptin and microtubule-stabilizing drug paclitaxel, both as single agents and in combination. FoxM1 altered microtubule dynamics to protect tumor cells from paclitaxel-induced apoptosis. Mechanistic investigations revealed that the tubulin-destabilizing protein Stathmin, whose expression also confers resistance to paclitaxel, is a direct transcriptional target of FoxM1. Significantly, attenuating FoxM1 expression by small interfering RNA or an alternate reading frame (ARF)-derived peptide inhibitor increased therapeutic sensitivity. Our findings indicate that targeting FoxM1 could relieve therapeutic resistance in breast cancer.
Subject(s)
Antibodies, Monoclonal/therapeutic use , Antineoplastic Agents, Phytogenic/therapeutic use , Breast Neoplasms/drug therapy , Drug Resistance, Neoplasm , Forkhead Transcription Factors/metabolism , Paclitaxel/therapeutic use , Antibodies, Monoclonal, Humanized , Apoptosis , Blotting, Western , Breast Neoplasms/metabolism , Breast Neoplasms/pathology , Cell Proliferation , Female , Flow Cytometry , Forkhead Box Protein M1 , Forkhead Transcription Factors/genetics , Gene Expression Regulation, Neoplastic , Humans , Peptide Fragments/pharmacology , RNA, Messenger/genetics , RNA, Messenger/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Trastuzumab , Tubulin/metabolism , Tumor Cells, CulturedABSTRACT
Oxygen is a key modulator of many cellular pathways, but current devices permitting in vitro oxygen modulation fail to meet the needs of biomedical research. A microfabricated insert for multiwell plates has been developed to more effectively control the temporal and spatial oxygen concentration to better model physiological phenomena found in vivo. The platform consists of a polydimethylsiloxane insert that nests into a standard multiwell plate and serves as a passive microfluidic gas network with a gas-permeable membrane aimed to modulate oxygen delivery to adherent cells. Equilibration time is on the order of minutes and a wide variety of oxygen profiles can be attained based on the device design, such as the cyclic profile achieved in this study, and even oxygen gradients to mimic those found in vivo. The proper biological consequences of the device's oxygen delivery were confirmed in cellular models via a proliferation assay and western analysis of the upregulation of hypoxia inducible transcription factor-1alpha. These experiments serve as a demonstration for the platform as a viable tool to increase experimental throughput and permit novel experimental possibilities in any biomedical research lab.
Subject(s)
Cell Culture Techniques/instrumentation , Oxygen/metabolism , Biosensing Techniques , Cell Adhesion , Cell Culture Techniques/methods , Cell Line, Tumor , Cell Proliferation , Dimethylpolysiloxanes/chemistry , Equipment Design , Gases , Humans , Hypoxia-Inducible Factor 1, alpha Subunit/metabolism , Microfluidics , Models, Biological , Time FactorsABSTRACT
The transcription factor FoxM1 is over-expressed in most human malignancies. Although it is evident that FoxM1 has critical functions in tumour development and progression, the mechanisms by which FoxM1 participates in those processes are not understood. Here, we describe an essential role of FoxM1 in the regulation of oxidative stress that contributes to malignant transformation and tumour cell survival. We identify a negative feedback loop involving FoxM1 that regulates reactive oxygen species (ROS) in proliferating cells. We show that induction of FoxM1 by oncogenic Ras requires ROS. Elevated FoxM1, in turn, downregulates ROS levels by stimulating expression of ROS scavenger genes, such as MnSOD, catalase and PRDX3. FoxM1 depletion sensitizes cells to oxidative stress and increases oncogene-induced premature senescence. Moreover, tumour cells expressing activated AKT1 are 'addicted' to FoxM1, as they require continuous presence of FoxM1 for survival. Together, our results identify FoxM1 as a key regulator of ROS in dividing cells, and provide insights into the mechanism how tumour cells use FoxM1 to control oxidative stress to escape premature senescence and apoptosis.
Subject(s)
Cell Transformation, Neoplastic/metabolism , Forkhead Transcription Factors/metabolism , Oxidative Stress , Animals , Cell Line, Tumor , Cell Transformation, Neoplastic/genetics , Forkhead Box Protein M1 , Forkhead Transcription Factors/genetics , Gene Expression Regulation, Neoplastic , Genes, ras , Humans , Mice , Mice, Inbred C57BL , NIH 3T3 Cells , Osteosarcoma/metabolism , Proto-Oncogene Proteins c-akt/genetics , Proto-Oncogene Proteins c-akt/metabolism , Reactive Oxygen Species/metabolismABSTRACT
We discovered intronic mutations in two episodic ataxia type 2 (EA2) families: a four-nucleotide GAGT deletion at IVS41+(3-6) and a single nucleotide insertion (insT) at IVS24+3. We expressed minigenes harboring the mutations in cell lines to demonstrate exon skipping from the deletion mutation and the activation of a cryptic splice donor site from the insertion mutation. The identification of these disease-causing mutations expands the spectrum of EA2 mutations and emphasizes the importance of intronic sequences in regulating gene expression.
Subject(s)
Ataxia/genetics , Introns , Mutation , Nerve Tissue Proteins/genetics , Adolescent , Adult , Animals , COS Cells , Calcium Channels, L-Type , Child , Chlorocebus aethiops , DNA Mutational Analysis/methods , Exons , Family Health , Female , Genetic Predisposition to Disease , Humans , Male , RNA Splicing/physiology , RNA, Small Nuclear/physiology , Reverse Transcriptase Polymerase Chain Reaction/methods , Transfection/methodsABSTRACT
The mechanisms controlling axon guidance are of fundamental importance in understanding brain development. Growing corticospinal and somatosensory axons cross the midline in the medulla to reach their targets and thus form the basis of contralateral motor control and sensory input. The motor and sensory projections appeared uncrossed in patients with horizontal gaze palsy with progressive scoliosis (HGPPS). In patients affected with HGPPS, we identified mutations in the ROBO3 gene, which shares homology with roundabout genes important in axon guidance in developing Drosophila, zebrafish, and mouse. Like its murine homolog Rig1/Robo3, but unlike other Robo proteins, ROBO3 is required for hindbrain axon midline crossing.
Subject(s)
Axons/physiology , Ophthalmoplegia/genetics , Receptors, Immunologic/genetics , Receptors, Immunologic/metabolism , Rhombencephalon/growth & development , Scoliosis/genetics , Adult , Alternative Splicing , Amino Acid Motifs , Amino Acid Sequence , Evoked Potentials, Motor , Evoked Potentials, Somatosensory , Female , Functional Laterality , Genetic Linkage , Humans , In Situ Hybridization , Magnetic Resonance Imaging , Male , Medulla Oblongata/growth & development , Medulla Oblongata/pathology , Microsatellite Repeats , Molecular Sequence Data , Morphogenesis , Mutation , Neural Pathways , Ophthalmoplegia/pathology , Ophthalmoplegia/physiopathology , Pedigree , Protein Structure, Tertiary , Receptors, Cell Surface , Receptors, Immunologic/chemistry , Reverse Transcriptase Polymerase Chain Reaction , Rhombencephalon/pathology , Scoliosis/pathology , Scoliosis/physiopathology , Sequence Analysis, DNA , SyndromeABSTRACT
We investigated the effects of methamphetamine (METH) on core body temperature (Tb) and motor activity (MA) with or without exposure to a peripheral immune challenge. Mice were exposed to an escalating METH treatment and then to a METH treatment known to cause neurotoxicity (binge METH treatment). This was followed by a challenge with lipopolysaccharide (LPS). Three days later, METH and saline-treated control groups were challenged with an acute test dose of METH (METH test). Animals exposed to the escalating METH treatment exhibited a significant increase in Tb only after the initial exposure to METH (Day 1) and following the METH test (Day 7). The hyperthermic effect produced by the METH test (Day 7) was reduced in mice previously exposed to combined exposure to binge METH and LPS treatments. The escalating METH treatment produced MA sensitization to the METH test. Animals treated with the binge METH, LPS injection or both treatments combined prevented MA sensitization to the METH test. These findings suggest that induction of peripheral endotoxemia in animals with a history of METH reduced the hyperthermic response to a subsequent challenge with METH.