Role Of FoxOs Research Articles

FOXO1 promotes cancer cell growth through MDM2-mediated p53 degradation

FOXO1 is a transcription factor and potential tumor suppressor that is negatively regulated downstream of PI3K-PKB/AKT signaling. Paradoxically, FOXO also promotes tumor growth, but the detailed mechanisms behind this role of FOXO are not fully understood. In this study, we revealed a molecular cascade by which the Thr24 residue of FOXO1 is phosphorylated by AKT and is dephosphorylated by calcineurin, which is a Ca2+-dependent protein phosphatase. Curiously, single nucleotide somatic mutations of FOXO1 in cancer occur frequently at and near Thr24. Using a calcineurin inhibitor and shRNA directed against calcineurin, we revealed that calcineurin-mediated dephosphorylation of Thr24 regulates FOXO1 protein stability. We also found that FOXO1 binds to the promoter region of MDM2 and activates transcription, which in turn promotes MDM2-mediated ubiquitination and degradation of p53. FOXO3a and FOXO4 are shown to control p53 activity, however, the significance of FOXO1 in p53 regulation remains largely unknown. Supporting this notion, FOXO1 depletion increased p53 and p21 protein levels in association with inhibition of cell proliferation. Taken together, these results indicate that FOXO1 is stabilized by calcineurin-mediated dephosphorylation, and that FOXO1 supports cancer cell proliferation by promoting MDM2 transcription and subsequent p53 degradation.

FoxK1 is Required for Ectodermal Cell Differentiation During Planarian Regeneration.

Forkhead box (Fox) genes belong to the “winged helix” transcription factor superfamily. The function of some Fox genes is well known, such as the role of foxO in controlling metabolism and longevity and foxA in controlling differentiation of endodermal tissues. However, the role of some Fox factors is not yet well characterized. Such is the case of FoxK genes, which are mainly studied in mammals and have been implicated in diverse processes including cell proliferation, tissue differentiation and carcinogenesis. Planarians are free-living flatworms, whose importance in biomedical research lies in their regeneration capacity. Planarians possess a wide population of pluripotent adult stem cells, called neoblasts, which allow them to regenerate any body part after injury. In a recent study, we identified three foxK paralogs in the genome of Schmidtea mediterranea. In this study, we demonstrate that foxK1 inhibition prevents regeneration of the ectodermal tissues, including the nervous system and the epidermis. These results correlate with foxK1 expression in neoblasts and in neural progenitors. Although the triggering of wound genes expression, polarity reestablishment and proliferation was not affected after foxK1 silencing, the apoptotic response was decreased. Altogether, these results suggest that foxK1 would be required for differentiation and maintenance of ectodermal tissues.

Propofol postconditioning ameliorates hypoxia/reoxygenation induced H9c2 cell apoptosis and autophagy via upregulating forkhead transcription factors under hyperglycemia

BackgroundAdministration of propofol, an intravenous anesthetic with antioxidant property, immediately at the onset of post-ischemic reperfusion (propofol postconditioning, P-PostC) has been shown to confer cardioprotection against ischemia–reperfusion injury, while the underlying mechanism remains incompletely understood. The FoxO transcription factors are reported to play critical roles in activating cardiomyocyte survival signaling throughout the process of cellular injuries induced by oxidative stress and are also involved in hypoxic postconditioning mediated neuroprotection, however, the role of FoxO in postconditioning mediated protection in the heart and in particular in high glucose condition is unknown.MethodsRat heart-derived H9c2 cells were exposed to high glucose (HG) for 48 h (h), then subjected to hypoxia/reoxygenation (H/R, composed of 8 h of hypoxia followed by 12 h of reoxygenation) in the absence or presence of postconditioning with various concentrations of propofol (P-PostC) at the onset of reoxygenation. After having identified the optical concentration of propofol, H9c2 cells were subjected to H/R and P-PostC in the absence or presence of FoxO1 or FoxO3a gene silencing to explore their roles in P-PostC mediated protection against apoptotic and autophagic cell deaths under hyperglycemia.ResultsThe results showed that HG with or without H/R decreased cell viability, increased lactate dehydrogenase (LDH) leakage and the production of reactive oxygen species (ROS) in H9c2 cells, all of which were significantly reversed by propofol (P-PostC), especially at the concentration of 25 µmol/L (P25) (all P < 0.05, NC vs. HG; HG vs. HG + HR; HG + HR + P12.5 or HG + HR + P25 or HG + HR + P50 vs. HG + HR). Moreover, we found that propofol (P25) decreased H9c2 cells apoptosis and autophagy that were concomitant with increased FoxO1 and FoxO3a expression (all P < 0.05, HG + HR + P25 vs. HG + HR). The protective effects of propofol (P25) against H/R injury were reversed by silencing FoxO1 or FoxO3a (all P < 0.05, HG + HR + P25 vs. HG + HR + P25 + siRNA-1 or HG + HR + P25 + siRNA-5).ConclusionIt is concluded that propofol postconditioning attenuated H9c2 cardiac cells apoptosis and autophagy induced by H/R injury through upregulating FoxO1 and FoxO3a under hyperglycemia.

Elucidating the Possible Role of FoxO in Depression.

Forkhead box-O (FoxO) transcriptional factors perform essential functions in several physiological and biological processes. Recent studies have shown that FoxO is implicated in the pathophysiology of depression. Changes in the upstream mediators of FoxOs including brain-derived neurotrophic factor (BDNF) and protein kinase B have been associated with depressive disorder and the antidepressant agents are known to alter the phosphorylation of FoxOs. Moreover, FoxOs might be regulated by serotonin or noradrenaline signaling and the hypothalamic-pituitary-adrenal (HPA)-axis,both of them are associated with the development of the depressive disorder. FoxO also regulates neural morphology, synaptogenesis, and neurogenesis in the hippocampus, which accounts for the pathogenesis of the depressive disorder. The current article underlined the potential functions of FoxOs in the etiology of depressive disorder and formulate few essential proposals for further investigation. The review also proposes that FoxO and its signal pathway might establish possible therapeutic mediators for the management of depressive disorder.

FOXO Regulates Neuromuscular Junction Homeostasis During Drosophila Aging.

The transcription factor foxo is a known regulator of lifespan extension and tissue homeostasis. It has been linked to the maintenance of neuronal processes across many species and has been shown to promote youthful characteristics by regulating cytoskeletal flexibility and synaptic plasticity at the neuromuscular junction (NMJ). However, the role of foxo in aging neuromuscular junction function has yet to be determined. We profiled adult Drosophila foxo- null mutant abdominal ventral longitudinal muscles and found that young mutants exhibited morphological profiles similar to those of aged wild-type flies, such as larger bouton areas and shorter terminal branches. We also observed changes to the axonal cytoskeleton and an accumulation of late endosomes in foxo null mutants and motor neuron-specific foxo knockdown flies, similar to those of aged wild-types. Motor neuron-specific overexpression of foxo can delay age-dependent changes to NMJ morphology, suggesting foxo is responsible for maintaining NMJ integrity during aging. Through genetic screening, we identify several downstream factors mediated through foxo-regulated NMJ homeostasis, including genes involved in the MAPK pathway. Interestingly, the phosphorylation of p38 was increased in the motor neuron-specific foxo knockdown flies, suggesting foxo acts as a suppressor of p38/MAPK activation. Our work reveals that foxo is a key regulator for NMJ homeostasis, and it may maintain NMJ integrity by repressing MAPK signaling.

FOXO1-Mediated NOX4 Expression is Required for Host to Defense in the Model of Ocular Toxoplasmosis

Previous study illustrated that PI3K-AKT pathway is an essential target for Toxoplasma gondii to neutralize host defense system by inhibiting intracellular host ROS level. Here, we demonstrate that FOXO transcription factor as a direct controller of the ROS generating enzyme, NOX4, transcription. The interaction between Nox4 promoter and the FOXO showed that NOX4 expression and host ROS level are strictly controlled by FOXO, and host susceptibility to T. gondii is highly dependent on it. Our findings provide biological insights into the role of FOXO in controlling NOX4 and ROS production and how T. gondii takes advantage of the system for their survival and growth during infection.

1722-P: Loss of Foxos in Muscle Maintains Strength and Mitochondrial Function during Aging, but Does Not Alter Glucose or Insulin Tolerance

Muscle mitochondrial decline plays an important role in aging-related muscle weakness and is associated with insulin resistance. FoxO transcription factors are targets in insulin action and are determinants of longevity across species. However, the role of FoxOs in age-related strength and muscle mitochondrial decline is unknown. We measured strength, glucose/insulin tolerance, and muscle mitochondrial bioenergetics in 4.5- month (Young) and 24-month-old (Aged) muscle specific FoxO1/3/4 triple KO (TKO) and littermate control mice. Lean mass and muscle weights were increased 22-48% in young and aged TKO mice compared to age-matched controls. Ad libitum glucose levels were ∼12 mg/dL higher in aged groups compared to young, without differences between TKO and controls. Similarly, glucose and insulin tolerance were unchanged between TKO and control mice in both young and aged groups. While treadmill tests were not different between TKO and controls at either age, the aged control mice show a 20% decrease in max force of ankle dorsi-flexor muscles compared to young controls which was prevented in aged TKOs. Reductions in muscle mitochondrial respiration using glutamate/malate/succinate substrates were also prevented in aged TKO mice compared to aged controls. Mitochondrial Complex I activity was higher in both young and aged TKO compared to their respective controls. We did not see any differences in activity of citrate synthase, and protein expression of subunits from complex I and III, but not other complexes, showed an age-related decline in both TKO and control muscle. These data indicate that loss of FoxOs in muscle prevents age-related mitochondrial dysfunction and muscle weakness, without altering glucose or insulin tolerance, and may be a therapeutic target to prevent muscle atrophy. Disclosure C.M. Penniman: None. G. Bhardwaj: None. T. Junck: None. C. Boyer: None. J. Jena: None. J. Fuqua: None. V.A. Lira: None. B.T. ONeill: None.

Other Molecular Mechanisms Regulating Autophagy.

Autophagy is a catabolic process in eukaryotic cells that delivers cytoplasmic components and organelles to the lysosomes for digestion. It is thought that various environmental signaling pathways are somehow integrated with autophagy signaling, such as the lack of nutrients (amino acids or glucose or others), changes in pH or osmotic pressure. Autophagy has been seen as an adaptive response to stress. In the extreme cases of starvation, the breakdown of cellular components promotes survival by maintaining cellular energy levels. A series of studies have found that the signaling pathway regulating autophagy is very complex. In addition to 40 autophagy-related genes (ATG) involved in the formation of autophagosomes, there are many other transcription factors that participated in the regulation of autophagy. This chapter focuses on the role of FoxO, NFκB, E2F and TFEB in autophagy.

Hydra as Model to Determine the Role of FOXO in Longevity.

In non-senescent Hydra, continuously high activity of transcription factor FOXO contributes to continuous stem cell proliferation. Here, we describe how genetic manipulation of Hydra polyps using embryo-microinjection allows uncovering the role of FOXO in coordinating both stem cell proliferation and antimicrobial peptide0073 , effector molecules of the innate immune system, and regulators of the microbiome.

The Role of FoxOs in Bone Health and Disease.

Recent studies with murine models of cell-specific loss- or gain-of-function of FoxOs have provided novel insights into the function and signaling of these transcription factors on the skeleton. They have revealed that FoxO actions in chondrocytes are critical for normal skeletal development, and FoxO actions in cells of the osteoclast or osteoblast lineage greatly influence bone resorption and formation and, consequently, bone mass. FoxOs also act in osteoblast progenitors to inhibit Wnt signaling and bone formation. Additionally, FoxOs decrease bone resorption via direct antioxidant effects on osteoclasts and upregulation of the antiosteoclastogenic cytokine OPG in cells of the osteoblast lineage. Deacetylation of FoxOs by the NAD-dependent histone deacetylase Sirt1 in both osteoblasts and osteoclasts stimulates bone formation and inhibits bone resorption, making Sirt1 activators promising therapeutic agents for diseases of low bone mass. In this chapter, we review these advances and discuss their implications for the pathogenesis and treatment of estrogen deficiency-, Type 1 diabetes-, and age-related osteoporosis.

FOXO transcription factors protect against the diet-induced fatty liver disease

Forkhead O transcription factors (FOXOs) have been implicated in glucose and lipid homeostasis; however, the role of FOXOs in the development of nonalcoholic fatty liver disease (NAFLD) is not well understood. In this study, we designed experiments to examine the effects of two different diets—very high fat diet (HFD) and moderately high fat plus cholesterol diet (HFC)—on wildtype (WT) and liver-specific Foxo1/3/4 triple knockout mice (LTKO). Both diets induced severe hepatic steatosis in the LTKO mice as compared to WT controls. However, the HFC diet led to more severe liver injury and fibrosis compared to the HFD diet. At the molecular levels, hepatic Foxo1/3/4 deficiency triggered a significant increase in the expression of inflammatory and fibrotic genes including Emr1, Ccl2, Col1a1, Tgfb, Pdgfrb, and Timp1. Thus, our data suggest that FOXO transcription factors play a salutary role in the protection against the diet-induced fatty liver disease.

The non-genomic loss of function of tumor suppressors: an essential role in the pathogenesis of chronic myeloid leukemia chronic phase.

BackgroundChronic Myeloid Leukemia was always referred as a unique cancer due to the apparent independence from tumor suppressors’ deletions/mutations in the early stages of the disease. However, it is now well documented that even genetically wild-type tumor suppressors can be involved in tumorigenesis, when functionally inactivated. In particular, tumor suppressors’ functions can be impaired by subtle variations of protein levels, changes in cellular compartmentalization and post-transcriptional/post-translational modifications, such as phosphorylation, acetylation, ubiquitination and sumoylation. Notably, tumor suppressors inactivation offers challenging therapeutic opportunities. The reactivation of an inactive and genetically wild-type tumor suppressor could indeed promote selective apoptosis of cancer cells without affecting normal cells.Main bodyChronic Myeloid Leukemia (CML) could be considered as the paradigm for non-genomic loss of function of tumor suppressors due to the ability of BCR-ABL to directly promote functionally inactivation of several tumor suppressors.Short conclusionIn this review we will describe new insights on the role of FoxO, PP2A, p27, BLK, PTEN and other tumor suppressors in CML pathogenesis. Finally, we will describe strategies to promote tumor suppressors reactivation in CML.

Abstract B32: FOXOs support the metabolic requirements of normal and tumor cells by regulating IDH1 expression

Abstract FOXO transcription factors are considered bona fide tumor suppressors, however recent studies showed FOXOs are also required for tumor survival. Here we identify FOXOs as transcriptional regulators of IDH1. FOXOs regulate IDH1 expression and thereby maintain the cytosolic levels of α-ketoglutarate and NADPH. In cancer cells carrying mutant IDH1, FOXOs likewise regulate mutant IDH1 expression and maintain the levels of the oncometabolite 2-hydroxyglutarate, which stimulates cancer cell proliferation likely by repressing differentiation. Combined, our data provide a new paradigm for the paradoxical role of FOXOs in both tumor suppression and promotion. Furthermore these data suggest a role for FOXOs in maintaining the de-differentiated state by regulating α-ketoglutarate and/or 2-hydroxyglutarate levels. Indeed, in models of differentiation (e.g. 3T3-L1 differentiation towards adipocytes) loss of FOXO, as well as loss of IDH1 expression repress differentiation and we are currently further exploring the molecular underpinnings. Note: This abstract was not presented at the conference. Citation Format: Paraskevi Charitou, Maria Rodriguez-Colman, Johan Gerrits, Nanda Verhoeven-Duif, Boudewijn Burgering. FOXOs support the metabolic requirements of normal and tumor cells by regulating IDH1 expression. [abstract]. In: Proceedings of the AACR Special Conference: Metabolism and Cancer; Jun 7-10, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(1_Suppl):Abstract nr B32.

Suppression of FoxO6 by lipopolysaccharide in aged rat liver.

The beneficial role of FoxO during aging has been proposed for its promotion of resistance to oxidative stress and inhibition of pro-inflammatory mediators. On the other hand, NF-κB is a pro-inflammatory transcription factor which is a key mediator of inflammatory cytokine generation. However, the correlation between FoxO6 and NF-κB during aging has not fully been explored.The main purpose of the present study was to elucidate mechanisms underlying the protective role of FoxO6 in the maintenance of cellular homeostasis under potent pro-inflammatory conditions induced by LPS. Initial experimentation revealed that reduced FoxO6 activity during aging was caused by its phosphorylation, which suppressed its transcriptional activity in aged livers. Transfection with FoxO6-wt virus and FoxO6-siRNA in HepG2 cells revealed that FoxO6 phosphorylation by LPS leads to NF-κB activation via Akt and Pak1 pathways. Furthermore, Pak1 activity was increased in a phosphatidylinositol 3-kinase independent manner, and LPS-induced FoxO6 phosphorylation and FoxO6 inactivation were Pak1-dependent in nuclear fractions of cells. Further revealed Pak1 phosphorylation by LPS permitted interaction between FoxO6 and Akt.Current study suggests FoxO6 phosphorylation facilitates the nuclear translocation of NF-κB via Akt and Pak1 pathways induced by LPS in aged rats.

Decreased interaction between FoxO3a and Akt correlates with seizure-induced neuronal death

Status epilepticus (SE) leads to neurodegeneration which likely contributes to the development of chronic temporal lobe epilepsy (TLE). Therefore, neuroprotection following SE is considered as a promising strategy for preventing chronic TLE, but molecular changes that occur following SE still remain unclear. The Forkhead homeobox type O (FoxO) family of Forkhead transcription factors mediates cell death in several pathological conditions, but the role of FoxO in the excitotoxic effects of kainic acid (KA) remains largely unknown. The present study examined how FoxO3a and its interaction with other proteins changed in response to excitotoxic stimuli in the mouse hippocampus after SE. Mice were given intraperitoneal injection of kainate and seizure behavior was monitored for 2h to ensure SE. Western blot analyses, co-immunoprecipitation experiments, sub-cellular fractionation and double immunofluorescence analyses were used to determine changes in levels of FoxO3a, Akt, Bim, cleaved caspase-3 and phospho-FoxO3a or phospho-Akt, and their interactions at 6 or 24h after KA treatment. We found that SE activated FoxO3a and increased levels of Bim or cleaved caspase-3, and decreased levels of phospho-FoxO3a or phospho-Akt in the hippocampus. In addition, we noted extensive hippocampal cell death at 24h after KA treatment, evidenced by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL), fluoro-jade B or anti-active caspase-3 staining. Furthermore, co-immunoprecipitation experiments revealed that phospho-Akt interaction with FoxO3a was significantly lowered in the hippocampus at 24h after KA treatment, paralleling enhanced Bim levels and Bim interaction with Bcl-xL. Moreover, double immunofluorescence analyses showed increased co-localization of FoxO3a or Bim and TUNEL in the hippocampi at 24h after KA treatment. Identifying molecular mechanism underlying SE-induced neuronal death can provide a novel strategy to protect against seizure-induced neuronal injury. We found that Akt-FoxO3a signaling relates to seizure-induced neuronal death, providing insight into neuroprotection following SE.

Correction: FoxO3a Modulates Hypoxia Stress Induced Oxidative Stress and Apoptosis in Cardiac Microvascular Endothelial Cells

[This corrects the article DOI: 10.1371/journal.pone.0080342.].

Role of Forkhead Transcription Factors in Myocardial Ischemic Reperfusion Injury in Diabetes

Diabetes, characterized by hyperglycemia resulting from either insulin deficiency or insulin resistance, is a chronic metabolic disorder. Hearts of subjects with diabetes are more sensitive to myocardial ischemia/reperfusion injury but the underlying mechanism is largely unclear. Recent evidence suggests that alteration in cardiac metabolism is a key contributor to the increased vulnerability of diabetic heart to ischemia/reperfusion injury. The FoxO family of forkhead transcription factors including FoxO1, 3 and 4 play important roles in the regulation of many cellular and biological processes and are critical regulators of cardiac metabolism and cellular oxidative stress in the heart. This brief review focuses on the role of FoxO in regulating cardiac metabolism and its association with myocardial ischemia/reperfusion injury, especially in diabetes.

Role of PI3K-AKT-mTOR and Wnt Signaling Pathways in Transition of G1-S Phase of Cell Cycle in Cancer Cells

The PI3K-Akt pathway together with one of its downstream targets, the mechanistic target of rapamycin (mTOR; also known as the mammalian target of rapamycin) is a highly deregulated pathway in cancers. mTOR exists in two complexes, mTORC1 and mTORC2. Akt phosphorylated at T308 inhibits TSC1/2 complex to activate mTORC1; mTORC2 is recognized as the kinase phosphorylating Akt at S473. Inhibition of autophagy by mTORC1 was shown to rescue disheveled (Dvl) leading to activation of Wnt pathway. Cyclin D1 and the c-Myc are activated by the Wnt signaling. Cyclin D1 is a key player in initiation of cell cycle. c-Myc triggers metabolic reprograming in G1 phase of cell cycle, which also activates the transcription factors like FoxO and p53 that play key roles in promoting the progression of cell cycle. While the role of p53 in cancer cell metabolism in arresting glycolysis and inhibition of pentose phosphate pathway has come to be recognized, there are confusions in the literature on the role of FoxO and that of rictor. FoxO was shown to be the transcription factor of rictor, in addition to the cell cycle inhibitors like p21. Rictor has dual roles; inhibition of c-Myc and constitution of mTORC2, both of which are key factors in the exit of G1-S phase and entry into G2 phase of cell cycle. A model is presented in this article, which suggests that the PI3K-Akt-mTOR and Wnt pathways converge and regulate the progression of cell cycle through G0-G1-S-phases and reprogram the metabolism in cancer cells. This model is different from the conventional method of looking at individual pathways triggering the cell cycle.

Evolution of human longevity: lessons from Hydra

Comment on: Boehm AM et al. FoxO is a critical regulator of stem cell maintenance in immortal Hydra. Proc Natl Acad Sci USA. 2012; 109:19697-19702.

FoxO1 breaks diabetic heart.

FoxO1 breaks diabetic heart.