Factor FOXO1 Research Articles

FOXO transcription factors in ageing and cancer

Ageing is associated with an increased onset of cancer. Understanding the molecular mechanisms that underlie the age dependency of cancer will have important implications for preventing and treating this pathology. The signalling pathway connecting insulin and FOXO transcription factors provides the most compelling example for a conserved genetic pathway at the interface between ageing and cancer. FOXO transcription factors (FOXO) promote longevity and tumour suppression. FOXO transcription factors are directly phosphorylated in response to insulin/growth factor signalling by the protein kinase Akt, thereby causing their sequestration in the cytoplasm. In the absence of insulin/growth factors, FOXO factors translocate to the nucleus where they trigger a range of cellular responses, including resistance to oxidative stress, a phenotype highly coupled with lifespan extension. FOXO factors integrate stress stimuli via phosphorylation, acetylation and mono-ubiquitination of a series of regulatory sites. Understanding how FOXO proteins integrate environmental conditions to control specific gene expression programmes will be pivotal in identifying ways to slow the onset of cancer in ageing individuals.

An HTS Approach to Screen for Antagonists of the Nuclear Export Machinery Using High Content Cell-Based Assays

Intracellular localization is essential for the regulated activity of many signaling molecules associated with disease-relevant pathways. High content screening is a powerful technology to monitor the impact of small molecules or interfering RNAs on translocation of proteins within intact cells. Several assays have been developed to measure the nucleocytoplasmic shuttling of proteins like nuclear factor kappaB, FoxO, or nuclear factor of activated T-cells involved in distinct signaling networks. However, since all these proteins bear a leucine-rich nuclear export signal (NES), modulators of the NES-dependent export machinery can lead to misinterpretation of the assay readout. Here we report the generation of U2nesRELOC, a cell-based system for the identification of nuclear export inhibitors and specific silencers of the nuclear export machinery, and its adaptation to high throughput screening. The assay is based on mammalian cells stably expressing green fluorescent protein (GFP)-labeled Rev protein, which contains a strong heterologous NES. The fluorescent signal of untreated U2nesRELOC cells localizes exclusively to the cytoplasm. Upon treatment with the nuclear export inhibitor leptomycin B the GFP-labeled reporter protein accumulates rapidly in the cell nucleus. The assay has been adapted to 96-multiwell format and fully automated. Pilot experiments with a panel of 50 test compounds using three different concentrations per compound resulted in very consistent data sets with excellent reproducibility and an average Z' value of 0.76. In summary, U2nesRELOC is a cell-based nuclear export assay suitable for high throughput screening, providing counterscreens for pathway deconvolution.

The emerging role of FOXO transcription factors in pancreatic β cells

FOXO transcription factors critically control fundamental cellular processes, including metabolism, cell differentiation, cell cycle arrest, DNA repair, and other reactions to cellular stress. FOXO factors sense the balance between stimuli promoting growth and differentiation versus stress stimuli signaling damage. Integrated through the FOXO system, these divergent stimuli decide on cell fate, a choice between proliferation, differentiation, or apoptosis. In pancreatic beta cells, most recent evidence highlights complex FOXO-dependent responses to glucose, insulin, or other growth factors, which include regulatory feedback. In the short term, FOXO-dependent mechanisms help beta cells to accomplish their endocrine function, and may increase their resistance to oxidative stress due to transient hyperglycemia. In the long term, FOXO-dependent responses lead to the adaptation of beta cell mass, conditioning the future ability of the organism to produce insulin and cope with changes in fuel abundance. FOXO emerges as a key factor for the maintenance of a functional endocrine pancreas and represents an interesting element in the development of therapeutic approaches to treat diabetes. This review on the role of FOXO transcription factors in pancreatic beta cells has three parts. In Part I, FOXO transcription factors will be presented in general: structure, molecular mechanisms of regulation, cellular functions, and physiological roles. Part II will focus on specific data about FOXO factors in pancreatic beta cells. Lastly in Part III, it will be attempted to combine general and beta cell-specific knowledge with the aim to envisage globally the role of FOXO factors in beta cell-linked physiology and disease.

Both the N-terminal Loop and Wing W2 of the Forkhead Domain of Transcription Factor Foxo4 Are Important for DNA Binding

FoxO4 belongs to the "O" subset of forkhead transcription factors, which participate in various cellular processes. The forkhead DNA binding domain (DBD) consists of three-helix bundle resting on a small antiparallel beta-sheet from which two extended loops protrude and create two wing-like structures. The wing W2 of FoxO factors contains a 14-3-3 protein-binding motif that is phosphorylated by protein kinase B in response to insulin or growth factors. In this report, we investigated the role of the N-terminal loop (portion located upstream of first helix H1) and the C-terminal region (loop known as wing W2) of the forkhead domain of transcription factor FoxO4 in DNA binding. Although the deletion of either portion partly reduces the FoxO4-DBD binding to the DNA, the simultaneous deletion of both regions inhibits DNA binding significantly. Förster resonance energy transfer measurements and molecular dynamics simulations suggest that both studied N- and C-terminal regions of FoxO4-DBD directly interact with DNA. In the presence of the N-terminal loop the protein kinase B-induced phosphorylation of wing W2 by itself has negligible effect on DNA binding. On the other hand, in the absence of this loop the phosphorylation of wing W2 significantly inhibits the FoxO4-DBD binding to the DNA. The binding of the 14-3-3 protein efficiently reduces DNA-binding potential of phosphorylated FoxO4-DBD regardless of the presence of the N-terminal loop. Our results show that both N- and C-terminal regions of forkhead domain are important for stability of the FoxO4-DBD.DNA complex.

FOXO transcription factors

FOXO transcription factors

Foxo Transcription Factors Blunt Cardiac Hypertrophy by Inhibiting Calcineurin Signaling

Cellular hypertrophy requires coordinated regulation of progrowth and antigrowth mechanisms. In cultured neonatal cardiomyocytes, Foxo transcription factors trigger an atrophy-related gene program that counters hypertrophic growth. However, downstream molecular events are not yet well defined. Here, we report that expression of either Foxo1 or Foxo3 in cardiomyocytes attenuates calcineurin phosphatase activity and inhibits agonist-induced hypertrophic growth. Consistent with these results, Foxo proteins decrease calcineurin phosphatase activity and repress both basal and hypertrophic agonist-induced expression of MCIP1.4, a direct downstream target of the calcineurin/NFAT pathway. Furthermore, hearts from Foxo3-null mice exhibit increased MCIP1.4 abundance and a hypertrophic phenotype with normal systolic function at baseline. Together, these results suggest that Foxo proteins repress cardiac growth at least in part through inhibition of the calcineurin/NFAT pathway. Given that hypertrophic growth of the heart occurs in multiple contexts, our findings also suggest that certain hypertrophic signals are capable of overriding the antigrowth program induced by Foxo. Consistent with this, multiple hypertrophic agonists triggered inactivation of Foxo proteins in cardiomyocytes through a mechanism requiring the PI3K/Akt pathway. In addition, both Foxo1 and Foxo3 are phosphorylated and consequently inactivated in hearts undergoing hypertrophic growth induced by hemodynamic stress. This study suggests that inhibition of the calcineurin/NFAT signaling cascade by Foxo and release of this repressive action by the PI3K/Akt pathway are important mechanisms whereby Foxo factors govern cell growth in the heart.

A FoxO–Smad synexpression group in human keratinocytes

Transforming growth factor beta (TGF-beta) signals through activation of Smad transcription factors. Activated Smad proteins associate with different DNA-binding cofactors for the recognition and regulation of specific target genes. Members of the forkhead box O family (FoxO1, FoxO3, and FoxO4) play such a role in the induction of the cyclin-dependent kinase inhibitors p15Ink4b and p21Cip1. To delineate the organization of the TGF-beta response in human keratinocytes, we defined the set of genes whose activation by TGF-beta requires both FoxO and Smad functions. FoxO factors are shown to be essential for 11 of the 115 immediate gene activation responses to TGF-beta in these cells. FoxO1, FoxO3, and FoxO4 act redundantly as mediators of these effects. Smad4, which functions as a partner of receptor-phosphorylated Smad2/3, is required for all of these responses. These results define a FoxO-Smad synexpression group or group of genes that are jointly induced by a common mechanism in response to TGF-beta. In addition to p15INK4b and p21CIP1, these genes include mediators of stress responses (GADD45A, GADD45B, and IER1) and adaptive cell signaling responses (CTGF, JAG1, LEMD3, SGK, CDC42EP3, and OVOL1). Bioinformatic analysis of the promoter region of these genes reveals diverse configurations of Smad and FoxO binding elements, implying differences in the regulatory properties of this group of genes. Indeed, a subset of FoxO/Smad-dependent TGF-beta gene responses additionally require the transcription factor CCAAT/enhancer-binding protein beta. The composition of the FoxO-Smad synexpression group suggests that stress reactions and adaptive functions accompany the cytostatic response of keratinocytes to TGF-beta.

Foxo Factors: A Matter of Life and Death

Forkhead box O-class (FOXO) transcription factors, including FOXO1, FOXO3a and FOXO4, function as tumor-suppressor proteins by inhibiting cell proliferation, promoting apoptotic cell death and protecting cells from DNA damage and oxidative stress. The potency of these functions is regulated tightly by phosphorylation, acetylation and ubiquitination. Emerging evidence indicates that protein levels of FOXO1 are under dual regulation by Ak-mediated phosphorylation and Skp2-mediated ubiquitination. Given that Akt and Skp2 proteins are highly activated in human cancers due to the loss of phosphatase and tensin homolog (PTEN), deregulation of the FOXO1 protein appears to be a promising target for future drug discovery and cancer therapy.

Spatial and temporal expression of FoxO transcription factors in the developing and adult murine brain

In order to obtain leads to molecular mechanisms of signal transduction pathways and controled gene expression in neuronal development we have screened the adult mouse brain for expressed forkhead transcription factors using a degenerate RT-PCR approach. Here, we focus on three FoxO genes found to be expressed in the brain: FoxO1, FoxO3 and FoxO6. The FoxO subfamily of forkhead transcription family is emerging as a central keypoint in an array of cellular functions, such as metabolism, differentiation and transformation. In situ hybridization experiments on adult and embryonic mouse brain showed differential expression patterns for three FoxO members. FoxO1 was strongly expressed in the striatum and neuronal subsets of the hippocampus (dentate gyrus and the ventral/posterior part of the CA regions), whereas FoxO3 was more diffusely expressed throughout the brain including all hippocampal areas, cortex and cerebellum. FoxO6 expression was eminent in various parts of the adult mouse brain, including the entire hippocampus, the amygdalohippocampal area and the shell of the nucleus accumbens. Remarkably, all three FoxO transcription factors were expressed relatively late in the developing murine brain, starting between E12.5 and E14. In summary, the presented data show FoxO factors to be expressed in the adult and developing mouse brain, in a spatially end temporally restricted manner.

FOXO transcription factors at the interface between longevity and tumor suppression

A wide range of human diseases, including cancer, has a striking age-dependent onset. However, the molecular mechanisms that connect aging and cancer are just beginning to be unraveled. FOXO transcription factors are promising candidates to serve as molecular links between longevity and tumor suppression. These factors are major substrates of the protein kinase Akt. In the presence of insulin and growth factors, FOXO proteins are relocalized from the nucleus to the cytoplasm and degraded via the ubiquitin-proteasome pathway. In the absence of growth factors, FOXO proteins translocate to the nucleus and upregulate a series of target genes, thereby promoting cell cycle arrest, stress resistance, or apoptosis. Stress stimuli also trigger the relocalization of FOXO factors into the nucleus, thus allowing an adaptive response to stress stimuli. Consistent with the notion that stress resistance is highly coupled with lifespan extension, activation of FOXO transcription factors in worms and flies increases longevity. Emerging evidence also suggests that FOXO factors play a tumor suppressor role in a variety of cancers. Thus, FOXO proteins translate environmental stimuli into changes in gene expression programs that may coordinate organismal longevity and tumor suppression.

FoxO6 transcriptional activity is regulated by Thr26 and Ser184, independent of nucleo-cytoplasmic shuttling.

Forkhead members of the 'O' class (FoxO) are transcription factors crucial for the regulation of metabolism, cell cycle, cell death and cell survival. FoxO factors are regulated by insulin-mediated activation of PI3K (phosphoinositide 3-kinase)-PKB (protein kinase B) signalling. Activation of PI3K-PKB signalling results in the phosphorylation of FoxO factors on three conserved phosphorylation motifs, which are essential for the translocation of FoxO factors from the nucleus to the cytosol. FoxO6, however, remains mostly nuclear due to the fact that its shuttling ability is dramatically impaired. FoxO1, FoxO3 and FoxO4 all contain an N- and C-terminal PKB motif and a motif located in the forkhead domain. FoxO6 lacks the conserved C-terminal PKB motif, which is the cause of the shuttling impairment. Since FoxO6 can be considered constitutively nuclear, we investigated whether it is also a constitutively active transcription factor. Our results show that FoxO6 transcriptional activity is inhibited by growth factors, independent of shuttling, indicating that it is not constitutively active. The PKB site in the forkhead domain (Ser184) regulated the DNA binding characteristics and the N-terminal PKB site acted as a growth factor sensor. In summary, FoxO6 is not a constitutively active transcription factor and can be regulated by growth factors in a Thr26- and Ser184-dependent manner, independent of shuttling to the cytosol.

14-3-3 Protein Interacts with Nuclear Localization Sequence of Forkhead Transcription Factor FoxO4

The 14-3-3 proteins are a family of regulatory signaling molecules that interact with other proteins in a phosphorylation-dependent manner. 14-3-3 proteins are thought to play a direct role in the regulation of subcellular localization of FoxO forkhead transcription factors. It has been suggested that the interaction with the 14-3-3 protein affects FoxO binding to the target DNA and interferes with the function of nuclear localization sequence (NLS). Masking or obscuring of NLS could inhibit interaction between FoxO factors and nuclear importing machinery and thus shift the equilibrium of FoxO localization toward the cytoplasm. According to our best knowledge, there is no experimental evidence showing a direct interaction between the 14-3-3 protein and NLS of FoxO. Therefore, the main goal of this work was to investigate whether the phosphorylation by protein kinase B, the 14-3-3 protein, and DNA binding affect the structure of FoxO4 NLS. We have used site-directed labeling of FoxO4 NLS with the extrinsic fluorophore 1,5-IAEDANS in conjunction with steady-state and time-resolved fluorescence spectroscopy to study conformational changes of FoxO4 NLS in vitro. Our data show that the 14-3-3 protein binding significantly changes the environment around AEDANS-labeled NLS and reduces its flexibility. On the other hand, the phosphorylation itself and the binding of double-stranded DNA have a small effect on the structure of this region. Our results also suggest that the DNA-binding domain of FoxO4 remains relatively mobile while bound to the 14-3-3 protein.

Skp2 inhibits FOXO1 in tumor suppression through ubiquitin-mediated degradation

Forkhead transcription factors FOXO1 (FKHR), FOXO3a (FKHRL1), and FOXO4 (AFX) play a pivotal role in tumor suppression by inducing growth arrest and apoptosis. Loss of function of these factors due to phosphorylation and proteasomal degradation has been implicated in cell transformation and malignancy. However, the ubiquitin ligase necessary for the ubiquitination of the FOXO factors and the relevance of this regulation to tumorigenesis have not been characterized. Here we demonstrate that Skp2, an oncogenic subunit of the Skp1/Cul1/F-box protein ubiquitin complex, interacts with, ubiquitinates, and promotes the degradation of FOXO1. This effect of Skp2 requires Akt-specific phosphorylation of FOXO1 at Ser-256. Moreover, expression of Skp2 inhibits transactivation of FOXO1 and abolishes the inhibitory effect of FOXO1 on cell proliferation and survival. Furthermore, expression of the FOXO1 protein is lost in a mouse lymphoma model, where Skp2 is overexpressed. These data suggest that the Skp2-promoted proteolysis of FOXO1 plays a key role in tumorigenesis.

Regulation of FoxO activity by CBP/p300-mediated acetylation

Forkhead box, class O (FoxO) transcription factors are inhibited by insulin-induced FoxO phosphorylation. Recently, acetylation of FoxO factors by calcium response element-binding (CREB)-binding protein (CBP) and/or p300 has been identified as a novel regulatory pathway, although the exact consequences of acetylation remain unclear. We propose that binding of CBP/p300 to FoxO factors is essential for FoxO-mediated transcription. CBP and p300 act as FoxO cofactors by weakening histone-DNA interactions. Acetylation of FoxO factors, however, attenuates FoxO-mediated transcriptional activity by disrupting the interaction between FoxO factors and target DNA. Therefore, acetylation shifts the function of FoxO from cell-cycle arrest and protection against oxidative stress towards cell death.

Myc-induced proliferation and transformation require Akt-mediated phosphorylation of FoxO proteins.

Myc synergizes with Ras and PI3-kinase in cell transformation, yet the molecular basis for this behavior is poorly understood. We now show that Myc recruits TFIIH, P-TEFb and Mediator to the cyclin D2 and other target promoters, while the PI3-kinase pathway controls formation of the pre-initiation complex and loading of RNA polymerase II. The PI3-kinase pathway involves Akt-mediated phosphorylation of FoxO transcription factors. In a nonphosphorylated state, FoxO factors inhibit induction of multiple Myc target genes, Myc-induced cell proliferation and transformation by Myc and Ras. Abrogation of FoxO function enables Myc to activate target genes in the absence of PI3-kinase activity and to induce foci formation in primary cells in the absence of oncogenic Ras. We suggest that the cooperativity between Myc and Ras is at least in part due to the fact that Myc and FoxO proteins control distinct steps in the activation of an overlapping set of critical target genes.

FOXO4 Is Acetylated upon Peroxide Stress and Deacetylated by the Longevity Protein hSir2SIRT1

FOXO transcription factors have important roles in metabolism, cellular proliferation, stress tolerance, and aging. FOXOs are negatively regulated by protein kinase B/c-Akt-mediated phosphorylation. Here we show that FOXO factors are also subject to regulation by reversible acetylation. We provide evidence that the acetyltransferase CREB-binding protein (CBP) binds FOXO resulting in acetylation of FOXO. This acetylation inhibits FOXO transcriptional activity. Binding of CBP and acetylation are induced after treatment of cells with peroxide stress. Deacetylation of FOXOs involves binding of the NAD-dependent deacetylase hSir2(SIRT1). Accordingly, hSir2(SIRT1)-mediated deacetylation precludes FOXO inhibition through acetylation and thereby prolongs FOXO-dependent transcription of stress-regulating genes. These data demonstrate that acetylation functions in a second pathway of negative control for FOXO factors and provides a novel mechanism whereby hSir2(SIRT1) can promote cellular survival and increase lifespan.

The ins and outs of FoxO shuttling: mechanisms of FoxO translocation and transcriptional regulation.

FoxO (forkhead box O; forkhead members of the O class) are transcription factors that function under the control of insulin/insulin-like signalling. FoxO factors have been associated with a multitude of biological processes, including cell-cycle, cell death, DNA repair, metabolism and protection from oxidative stress. Central to the regulation of FoxO factors is a shuttling system, which confines FoxO factors to either the nucleus or the cytosol. Shuttling of FoxO requires protein phosphorylation within several domains, and association with 14-3-3 proteins and the nuclear transport machinery. Description of the FoxO-shuttling mechanism contributes to the understanding of FoxO function in relation to signalling and gene regulation.

Foxo Transcription Factors Induce the Atrophy-Related Ubiquitin Ligase Atrogin-1 and Cause Skeletal Muscle Atrophy

Skeletal muscle atrophy is a debilitating response to fasting, disuse, cancer, and other systemic diseases. In atrophying muscles, the ubiquitin ligase, atrogin-1 (MAFbx), is dramatically induced, and this response is necessary for rapid atrophy. Here, we show that in cultured myotubes undergoing atrophy, the activity of the PI3K/AKT pathway decreases, leading to activation of Foxo transcription factors and atrogin-1 induction. IGF-1 treatment or AKT overexpression inhibits Foxo and atrogin-1 expression. Moreover, constitutively active Foxo3 acts on the atrogin-1 promoter to cause atrogin-1 transcription and dramatic atrophy of myotubes and muscle fibers. When Foxo activation is blocked by a dominant-negative construct in myotubes or by RNAi in mouse muscles in vivo, atrogin-1 induction during starvation and atrophy of myotubes induced by glucocorticoids are prevented. Thus, forkhead factor(s) play a critical role in the development of muscle atrophy, and inhibition of Foxo factors is an attractive approach to combat muscle wasting.

IκB Kinase Promotes Tumorigenesis through Inhibition of Forkhead FOXO3a

Nuclear exclusion of the forkhead transcription factor FOXO3a by protein kinase Akt contributes to cell survival. We investigated the pathological relationship between phosphoylated-Akt (Akt-p) and FOXO3a in primary tumors. Surprisingly, FOXO3a was found to be excluded from the nuclei of some tumors lacking Akt-p, suggesting an Akt-independent mechanism of regulating FOXO3a localization. We provide evidence for such a mechanism by showing that IκB kinase (IKK) physically interacts with, phosphorylates, and inhibits FOXO3a independent of Akt and causes proteolysis of FOXO3a via the Ub-dependent proteasome pathway. Cytoplasmic FOXO3a correlates with expression of IKKβ or Akt-p in many tumors and associates with poor survival in breast cancer. Further, constitutive expression of IKKβ promotes cell proliferation and tumorigenesis that can be overridden by FOXO3a. These results suggest the negative regulation of FOXO factors by IKK as a key mechanism for promoting cell growth and tumorigenesis.

Protein Kinase B-α Inhibits Human Pyruvate Dehydrogenase Kinase-4 Gene Induction by Dexamethasone Through Inactivation of FOXO Transcription Factors

Starvation and diabetes increase pyruvate dehydrogenase kinase-4 (PDK4) expression, which conserves gluconeogenic substrates by inactivating the pyruvate dehydrogenase complex. Mechanisms that regulate PDK4 gene expression, previously established to be increased by glucocorticoids and decreased by insulin, were studied. Treatment of HepG2 cells with dexamethasone increases the relative abundance of PDK4 mRNA, and insulin blocks this effect. Dexamethasone also increases human PDK4 (hPDK4) promoter activity in HepG2 cells, and insulin partially inhibits this effect. Expression of constitutively active PKB alpha abrogates dexamethasone stimulation of hPDK4 promoter activity, while coexpression of constitutively active FOXO1a or FOXO3a, which are mutated to alanine at the three phosphorylation sites for protein kinase B (PKB), disrupts the ability of PKB alpha to inhibit promoter activity. A glucocorticoid response element for glucocorticoid receptor (GR) binding and three insulin response sequences (IRSs) that bind FOXO1a and FOXO3a are identified in the hPDK4 promoter. Mutation of the IRSs reduces the ability of glucocorticoids to stimulate PDK4 transcription. Transfection studies with E1A, which binds to and inactivates p300/CBP, suggest that interactions between p300/CBP and GR as well as FOXO factors are important for glucocorticoid-stimulated hPDK4 expression. Insulin suppresses the hPDK4 induction by glucocorticoids through inactivation of the FOXO factors.