The “O” Class: Crafting Clinical Care with FoxO Transcription Factors

Abstract

Forkhead Transcription Factors: Vital Elements in Biology and Medicine provides a unique platform for the presentation of novel work and new insights into the vital role that forkhead transcription factors play in both cellular physiology as well as clinical medicine. Internationally recognized investigators provide their insights and perspectives for a number of forkhead genes and proteins that may have the greatest impact for the development of new strategies for a broad array of disorders that can involve aging, cancer, cardiac function, neurovascular integrity, fertility, stem cell differentiation, cellular metabolism, and immune system regulation. Yet, the work clearly sets a precedent for the necessity to understand the cellular and molecular function of forkhead proteins since this family of transcription factors can limit as well as foster disease progression depending upon the cellular environment.

With this in mind, our concluding chapter for Forkhead Transcription Factors: Vital Elements in Biology and Medicine offers to highlight both the diversity and complexity of the forkhead transcription family by focusing upon the mammalian forkhead transcription factors of the O class (FoxOs) that include FoxO1, FoxO3, FoxO4, and FoxO6. FoxO proteins are increasingly considered to represent unique cellular targets that can control numerous processes such as angiogenesis, cardiovascular development, vascular tone, oxidative stress, stem cell proliferation, fertility, and immune surveillance. Furthermore, FoxO transcription factors are exciting considerations for disorders such as cancer in light of their pro-apoptotic and inhibitory cell cycle effects as well as diabetes mellitus given the close association FoxOs hold with cellular metabolism. In addition, these transcription factors are closely integrated with several novel signal transduction pathways, such as erythropoietin and Wnt proteins, that may influence the ability of FoxOs to lead to cell survival or cell injury. Further understanding of both the function and intricate nature of the forkhead transcription factor family, and in particular the FoxO proteins, should allow selective regulation of cellular development or cellular demise for the generation of successful future clinical strategies and patient well-being.

Introduction

Clinical care for many disease entities requires new therapeutic strategies that focus upon a number of pathways and systems in the body to modulate cellular proliferation, metabolism, inflammation, and longevity. In this respect, members of the mammalian forkhead transcription factors of the O class (FoxOs) that include FoxO1, FoxO3, FoxO4, and FoxO6 have been identified as important regulators of cellular proliferation, function, and demise. These transcription factors are increasing considered as potential clinical targets for multiple disorders since they control processes associated with angiogenesis, stem cell proliferation, cardiovascular injury, neurodegeneration, tumorigenesis, and cell longevity. More than 100 forkhead genes and 19 human subgroups that range from FOXA to FOXS are now known to exist since the initial discovery of the fly Drosophila melanogaster gene forkhead.¹ The prior nomenclature for these proteins, such as forkhead in rhabdomyosarcoma (FKHR), the Drosophila gene fork head (fkh), and Forkhead RElated ACtivator (FREAC)-1 and -2, has been replaced. The current nomenclature for human Fox proteins places all letters in uppercase, otherwise only the initial letter is listed as uppercase for the mouse, and for all other chordates the initial and subclass letters are in uppercase.² Initially, the FoxOs were first reported in fusion genes in human soft-tissue tumors and leukemias. FOXO1, termed forkhead in rhabdomyosarcoma (FKHR), and FOXO3a, also known as FKHRL1 (forkhead in rhabdomyosarcoma like protein 1), and their genes were identified through chromosomal translocations in alveolar rhabdomyosarcoma tumors.³ The acute leukemia fusion gene located in chromosome X (AFX), also known as the FOXO4 gene, was described as a gene that fused to MLL transcription factor as a result of the t(X; 11) chromosomal translocation in acute lymphoblastic leukemia.⁴ A fusion between FOXO2 and MLL also occurs in some cases of acute myeloid leukemia that also is believed to be identical to FOXO3a.⁵

FoxO Protein Expression

FoxO proteins are found throughout the body and are expressed in tissues of the reproductive system of males and females, skeletal muscle, the cardiovascular system, lung, liver, pancreas, spleen, thymus, and the nervous system.^6–11 Since FoxO proteins are not equally expressed in all tissues, it is possible that individual FoxO proteins may have specificity in regards to cellular function. For example, FoxO6 expression is found in several regions of the brain that play a significant role in cognitive function and emotion, such as the hippocampus, the amygdala, and the nucleus accumbens.⁹ In contrast, FoxO1 may be more suited for the control of motor function and memory formation, since the expression of this protein is primarily in the striatum and sub-regions of the hippocampus.⁹ In addition, FoxO3 is more diffusely represented in the hippocampus, cortex, and cerebellum, suggesting a complementary role for this FoxO protein to control cognitive and motor function. FoxO expression can be variable in other tissues. Although studies in mice have shown that the mRNA distribution of Foxo1, Foxo3a, and Foxo4 is similar in the embryo and adult,⁷ Foxo1 expression was highest in adipose tissue, Foxo3a expression was greatest in the liver, and Foxo4 expression was strongest in muscle.⁷ Subsequent work in mice has described Foxo1 expression in all tissues with high levels in the ovaries.¹² Foxo3a also was found to be expressed in all tissues and Foxo4 expression was considered to be more tissue specific in skeletal muscle.¹²

FoxO Protein Structure and Function as Transcription Factors

Forkhead proteins function as transcription factors to either inhibit or activate target gene expression.¹³ As a result, these proteins must bind to DNA through the forkhead domain that relies upon fourteen protein-DNA contacts. The forkhead domain in Fox proteins consists of three α-helices, three β-sheets, and two loops that are referred to as the wings,¹⁴ but not all winged helix domains are considered to be Fox proteins.¹⁵ On X-ray crystallography¹⁴ or nuclear magnetic resonance imaging,¹⁶ the forkhead domain is described as a “winged helix” as a result of a butterfly-like appearance. High sequence homology is present in the α-helices and β-sheets with variations described in either absent β-sheets and loops or additional α-helices. Although both the first and second loops make contact with DNA, it is the second loop that can influence the stability of DNA binding. In addition, posttranslational modification of FoxO proteins, such as phosphorylation or acetylation that block FoxO activity, alter the binding of the C-terminal basic region to DNA to prevent transcriptional activity.¹⁷ However, other mechanisms may influence DNA binding of forkhead proteins, such as variations in the N-terminal region of the DNA recognition helix, changes in electrostatic distribution, and the ability of forkhead proteins to be shuttled to the cell nucleus.^10,18

FoxO Proteins, Posttranslational Modulation, Novel Signal Transduction Pathways, and Cell Cycle Regulation

Posttranslational modulation of FoxO proteins involves pathways associated with phosphorylation, acetylation, and ubiquitylation (Fig. 1).^3,10,19–21 The serine-threonine kinase protein kinase B (Akt) is a primary mediator of phosphorylation of FoxO1, FoxO3a, and FoxO4 that can block activity of these proteins.^{3, 22} Activation of Akt is usually cytoprotective, such as during hyperglycemia,²³ hypoxia,²⁴ β-amyloid (Aβ) toxicity,²⁵ cardiomyopathy,²⁶ cellular aging,²⁷ and oxidative stress.^28–30 Akt can prevent cellular apoptosis through the phosphorylation of FoxO proteins.³¹ Posttranslational phosphorylation of FoxO proteins will maintain FoxO transcription factors in the cytoplasm by association with 14-3-3 proteins and prevent the transcription of pro-apoptotic target genes.^32,33 An exception to these observations involving the subcellular trafficking of FoxO proteins involves FoxO6. This FoxO protein usually resides in the nucleus of cells and is phosphorylated by Akt in the nucleus. FoxO6 does not contain a conserved C-terminal Akt motif which limits nuclear shuttling of this protein, but FoxO6 transcriptional activity can be blocked by growth factors independent of shuttling to the cytosol through a FoxO6 N-terminal Akt site.³⁴

Figure 1.

Posttranslational modulation of FoxO proteins is associated with intricate cellular signal transduction pathways. Posttranslational modulation of FoxO proteins involves pathways associated with phosphorylation, acetylation, and ubiquitylation. Protein kinase B (Akt) can prevent cellular apoptosis through the phosphorylation of FoxO proteins and phosphorylation (p) of FoxO proteins will inhibit FoxO transcription factors through cytoplasmic localization by association with 14-3-3 proteins and prevent the transcription of target genes that lead to apoptosis. If activated, FoxOs can prevent inflammatory cell activation through the inhibition of nuclear factor-κB (NF-κB) and may control inflammatory cell activation through membrane phosphatidylserine (PS) externalization. FoxO proteins can lead to apoptotic death pathways that involve mitochondrial (Mito) release of cytochrome c (Cyto c) and caspase activation through a Fas-mediated ligand (Fas L) death pathway, tumor-necrosis-factor-related apoptosis-inducing ligand (TRAIL), BH3-only proteins Noxa and Bim, or p53. Cell cycle inhibition that blocks tumor growth through FoxO protein activation may require c-myc, p27, and NF-κB.

Modulation of Akt activity also oversees apoptotic pathways of caspases that may offer an alternative mechanism to regulate FoxO proteins (Fig. 1).³⁵ Caspases are a family of cysteine proteases that are synthesized as inactive zymogens that are proteolytically cleaved into subunits at the onset of apoptosis.^36–38 The caspases 1 and 3 have been linked to the apoptotic pathways of genomic DNA cleavage, cellular membrane PS exposure, and activation of inflammatory cells.^39–41 Caspase pathways may be tied to the forkhead transcription factor FoxO3a since increased activity of FoxO3a can result in cytochrome c release and caspase-induced apoptotic death.^32,42–44 Pathways that can inhibit caspase 3 activity appear to offer a unique regulatory mechanism. For example, cell death pathways that rely upon FoxO3a also appear to involve caspase 3 activation. Prior studies suggest that not only does FoxO3a activity promote caspase-induced apoptotic death,^32,42–44 but also demonstrate that inhibition of caspase 3 has been shown to maintain the phosphorylated “inactive” state of FoxO3a to prevent cell injury.^32,42,43 Other work has shown that caspase 3 activity and cleavage is promoted during transfection of a triple mutant FoxO3a expression in which three phosphorylation sites have been altered to prevent inactivation of FoxO3a.⁴⁵ Recent work adds further insight to these studies by illustrating that FoxO3a may control early activation and subsequent apoptotic injury in microglia during amyloid (Aβ) exposure through caspase 3 (Fig. 2).⁴⁶ Since Aβ exposure can facilitate the cellular trafficking of FoxO3a from the cytoplasm to the cell nucleus to potentially lead to “pro-apoptotic” programs by this transcription factor,⁴⁶ one program in particular that may be vital for apoptotic injury appears to involve the activation of caspase 3. Aβ exposure leads to a rapid and significant increases in caspase 3 activity with 6 hours following Aβ administration, but that this induction of caspase 3 activity by Aβ requires FoxO3a, since loss of FoxO3a through gene silencing prevents the induction of caspase 3 activity by Aβ.

Figure 2.

During amyloid (Aβ_1-42) exposure in inflammatory microglial cells, FoxO3a translocates to the cell nucleus to govern an initial activation and proliferation of microglial cells. Microglia were followed at 6 hours after Aβ_1-42 (10 μM) (Aβ) administration with immunofluorescent staining for FoxO3a (Texas-red). Nuclei of microglia were counterstained with DAPI. In merged images, control cells have readily visible nuclei (white in color) that illustrate absence of FoxO3a in the nucleus. In contrast, merged images after Aβ_1-42 (10 μM) exposure are not visible (red in color) and demonstrate translocation of FoxO3a to the nucleus. Control = untreated microglia.

Posttranslational modulation of FoxO proteins also requires pathways associated with ubiquitylation and acetylation.^47,48 Akt phosphorylation of FoxO proteins not only retains these transcription factors in the cytoplasm, but also leads to ubiquitination and degradation through the 26S proteasome 19, 48. In the absence of Akt, IκB kinase (IKK) also can directly phosphorylate and block the activity of FoxO proteins, such as FoxO3a.^3,10 This leads to the proteolysis of FoxO3a via the Ub-dependent proteasome pathway.^3,10,19–21 The serum- and glucocorticoid-inducible protein kinase (Sgk), a member of a family of kinases termed AGC (protein kinase A/protein kinase G/protein kinase C) kinases which includes Akt, also can phosphorylate and retain FoxO3a in the cytoplasm.⁴⁹ Knowledge that Sgk and Akt can phosphorylate FoxO3a at different sites may offer new opportunities to more effectively prevent apoptotic cell injury that may be mediated by FoxO3a activity. Yet, phosphorylation of FoxO proteins does not always lead to negative regulation. The protein kinase mammalian sterile 20-like kinase-1 also can phosphorylate FoxO proteins directly and lead to their activation.⁵⁰ The ability of sterile 20-like kinase-1 to activate FoxO proteins may be linked to c-Jun N-terminal kinase ( JNK), since sterile 20-like kinase-1 can increase JNK activation.⁵¹ FoxO proteins also are acetylated by histone acetyltransferases that include p300, the CREB-binding protein (CBP), and the CBP-associated factor and are deacetylated by histone deacetylases, such as Sirt1, a NAD+-dependent deacetylase and the mammalian ortholog of the silent information regulator 2 (Sir2) protein (Fig. 1).¹⁰ Acetylation of FoxO proteins provides another avenue for the control of these proteins. Once acetylated such as by CBP, FoxO proteins may translocate to the cell nucleus but have diminished activity since acetylation of lysine residues on FoxO proteins has been shown to limit the ability of FoxO proteins to bind to DNA.⁵² In addition, acetylation can increase phosphorylation of FoxO proteins by Akt.⁵²

FoxO proteins are also tied to other unique signal transduction pathways that involve proteins derived from the Drosophila Wingless (Wg) and the mouse Int-1 genes.²⁰ The Wnt proteins are secreted cysteine-rich glycosylated proteins that can control cell proliferation, differentiation, survival, and tumorigenesis.^53,54 More than eighty target genes of Wnt signaling pathways have been demonstrated in human, mouse, Drosophila, Xenopus, and zebrafish. These genes are present in several cellular populations, such as neurons, cardiomyocytes, endothelial cells, cancer cells, and pre-adipocytes.⁵⁵ At least nineteen of twenty-four Wnt genes that express Wnt proteins have been identified in the human.^53,54,56

The canonical Wnt pathway controls target gene transcription through β-catenin.^53,54 It is the β-catenin pathway that appears to tie FoxO proteins and Wnt signaling together.⁵⁷ For example, in relation to Alzheimer’s disease, Aβ is toxic to cells,^25,58 and is associated with the phosphorylation of FoxO1 and FoxO3a that can be blocked with ROS scavengers.⁵⁹ A common denominator in the pathways linked to Ab toxicity involves Wnt signaling through β-catenin. β-catenin may increase FoxO transcriptional activity and competitively limit β-catenin interaction with members of the lymphoid enhancer factor/T cell factor family⁶⁰ and β-catenin also has been demonstrated to be necessary for protection against Aβ toxicity in neuronal cells.⁵⁸

Additional shared signal transduction pathways between Wnt and FoxO proteins involve Akt. Processes that involve cellular proliferation, injury, and immune system modulation with FoxO proteins⁶¹ also have parallel cellular pathways with Wnt and Akt. For example, Wnt relies upon Akt for the proliferation and differentiation of cardiomyocytes.⁶² In addition, reduction in tissue injury during pressure overload cardiac hypertrophy and the cytoprotective benefits of cardiac ischemic preconditioning also appear to depend upon Akt.^53,54 Furthermore, Wnt over-expression can independently increase the phosphorylation and the activation of Akt to promote cellular protection and control microglial activation.⁵⁸

Yet, other members of the forkhead family in addition to FoxOs also rely upon Wnt signaling in several scenarios that involve regulated as well as unchecked cell proliferation.^53,54,63 For example, FoxD3 is activated by the Wnt pathway to control neural plate development⁶⁴ and Foxl1 activates the Wnt/β-catenin pathway to increase extracellular proteoglycans, promote gastrointestinal cell proliferation, and possibly foster carcinogenesis.⁶⁵ The Wnt pathway also utilizes forkhead members to modulate endocrine activity and can activate Foxn1 for regulatory control of thymic function.⁶⁶ In other examples of cell development, Wnt signaling has been shown to rely upon Foxf1 and Foxf2 during intestinal maturation in murine models.⁶⁷ In addition, Foxa2 in mice may be a significant component in early anterior-posterior axis polarization.⁶⁸ Deregulation of Wnt alone also promotes activation of β-catenin that has been associated with the proliferation of medulloblastoma tumors.⁶⁹ In addition, reduced expression of inhibitors of the Wnt pathway, such as axin, may foster lung cancer cell invasion.⁷⁰ Multiple other studies also point to the activation of the Wnt pathway during gastric cancer. For example, Wnt5a expression has been correlated with advanced gastric cancer stages and a poor prognosis⁷¹ while experimental activation of the β-catenin pathway leads to the development of gastric tumors.⁷² In conjunction with forkhead proteins, loss of Foxl1 that can regulate the Wnt pathway and prevent β-catenin nuclear accumulation is believed to be a significant etiology for gastrointestinal tumorigenesis.⁶⁵

FoxO proteins also appear to be ideal to cellular proliferation not only through Wnt mediated pathways, but also through the blockade of cell cycle progression. For example, FoxO3a and FoxO4 can promote cell cycle arrest in mouse myoblastic cell lines through modulation of growth-arrest and DNA-damage-response protein 45.^10,73 Treatment of chronic myelogenous leukemia cell lines with the Bcr-Abl tyrosine kinase inhibitor imatinib requires FoxO3a activation to antagonize cell proliferation and promote apoptotic cell death through increased TRAIL production.⁷⁴ In addition, the transcription factor E2F-1 that controls the induction of the cell cycle has been reported in cell lines to increase the endogenous expression of FoxO1 and FoxO3a to lead to cell cycle arrest.⁷⁵ In contrast, the loss of FoxO3a activity in association with c-myc, p27, and nuclear factor-κB (NF-κB) can result in cell cycle induction and malignant transformation of mouse cells in the presence of oncogene activation (Fig. 1).^3,10 Other work suggests that FoxO proteins utilize the p53 upstream regulator p19(Arf ) through myc to block cell cycle induction and lymphoma progression.⁷⁶

FoxO Proteins, Apoptosis, and Oxidative Stress

Although genes linked to apoptosis sometimes foster cellular proliferation rather than cell death, cellular apoptosis can become a significant component for pathology in diseases such as neurodegenerative disease, diabetes mellitus (DM), and cardiovascular injury.⁷⁷ More importantly, regulation of apoptotic pathways appears to serve a critical juncture for the control of tumor growth and unregulated cell proliferation.^10,57 Apoptotic cell death is considered to be a dynamic process that involves both early and late events. Membrane phosphatidylserine (PS) externalization is an early event during cell apoptosis that assists microglia to target cells for phagocytosis.^29,78 This process occurs with the expression of the phosphatidylserine receptor (PSR) on microglia during oxidative stress,^79–81 since blockade of PSR function in microglia prevents the activation of microglia.^30,40 As an example, externalization of membrane PS residues occur in cells during periods of oxidative stress that involve anoxia,⁵⁸ reactive oxygen species (ROS) exposure,⁸² and with agents that produce ROS, such as 6-hydroxydopamine.⁸³ In contrast to cells with PS exposure, the cleavage of genomic DNA into fragments is considered to be a later event during apoptotic injury.^77,84 Endonucleases responsible for DNA degradation have been identified and include the acidic, cation independent endonuclease (DNase II), cyclophilins, and the 97 kDa magnesium—dependent endonuclease. In the nervous system, endonucleases include a constitutive acidic cation-independent endonuclease, a constitutive calcium/magnesium-dependent endonuclease, and an inducible magnesium-dependent endonuclease.^77,84

Interestingly, the induction of apoptosis in cells through FoxO proteins may require pathways aligned with oxidative stress. Oxidative stress is a result of the release of reactive oxygen species (ROS) that consist of oxygen free radicals and other chemical entities. Oxygen free radicals and mitochondrial DNA mutations have become associated with tissue injury, aging, and accumulated toxicity for an organism.⁷⁷ ROS include superoxide free radicals, hydrogen peroxide, singlet oxygen, nitric oxide, and peroxynitrite.⁸⁴ Most reactive species are produced at low levels during normal physiological conditions and are scavenged by endogenous antioxidant systems that include superoxide dismutase, glutathione peroxidase, catalase, and small molecules, such as vitamins C, E, D3 and nicotinamide, the amide form of niacin or vitamin B3.^78,85,86 During periods of oxidative stress, FoxO transcription factors can lead to apoptosis,³¹ since forkhead transcription factors such as FoxO1 and FoxO3a must be present for oxidative stress to result in apoptotic cell injury.⁸⁷ Under other conditions of oxidative stress, FoxO3a in conjunction with JNK have been shown to modulate an apoptotic ligand activating a Fas-mediated death pathway in cultured motoneurons,⁸⁸ to lead to apoptosis through tumor-necrosis-factor-related apoptosis-inducing ligand (TRAIL) and BH3-only proteins Noxa and Bim in neuroblastoma cells,⁴⁴ and to promote pro-apoptotic activity of p53.⁸⁹ Additional work shows that loss of FoxO expression during oxidative stress is protective to cells. For example, protein inhibition or gene knockdown of FoxO1 or FoxO3a can lead to reduction in ischemic infarct size in the brain,⁹⁰ mediate protection of metabotropic glutamate receptors during vascular injury,⁴² enhance pancreatic β-cell or neuronal survival through NAD+ precursors during oxidative stress,⁴³ and provide trophic factor protection with erythropoietin (EPO)³² and neurotrophins.⁹¹ Yet, it should be noted that some studies suggest that the loss of FoxO1, FoxO3a, and FoxO4 protein expression may actually lead to an increase in free radical release that can be responsible for oxidative stress.⁹² In addition, FoxO proteins may be protective during aging and exercise, since FoxO3a activity may enhance vascular smooth muscle antioxidant properties in aged animals and be beneficial to the cardiovascular system during physical exertion.⁹³

FoxO Proteins, Metabolism and Cell Longevity

Clinical and experimental studies highlight the role of FoxO proteins during cellular metabolism and cellular longevity. When one considers DM, this disorder is a significant health concern for both young and older populations.^94,95 Approximately 16 million individuals in the United States and more than 165 million individuals worldwide suffer from DM. By the year 2030, it is predicted that more than 360 million individuals will be afflicted with DM and its debilitating conditions. Type 2 DM represents at least 80% of all diabetics and is dramatically increasing in incidence as a result of changes in human behavior and increased body mass index.^85,94 Type 1 insulin-dependent DM is present in 5–10% of all diabetics, but is increasing in adolescent minority groups.^85,94 Furthermore, the incidence of undiagnosed diabetes and impaired glucose tolerance in the population raises additional concerns.

Patients with DM can develop significant neurodegenerative^55,85,96 and cardiovascular disease.^85,97 Interestingly, the development of insulin resistance and the complications of DM can be the result of cellular oxidative stress.^85,94 Hyperglycemia can lead to increased production of ROS in endothelial cells, liver cells, and pancreatic β-cells.^85,94,95 Recent clinical correlates support these experimental studies to show that elevated levels of ceruloplasmin are suggestive of increased ROS.^85,94,95 Furthermore, acute glucose swings in addition to chronic hyperglycemia can trigger oxidative stress mechanisms, illustrating the importance for therapeutic interventions during acute and sustained hyperglycemic episodes.^85,94

Early work with FoxO proteins has shown that metabolic signaling with these transcription factors is conserved among multiple species including Caenorhabditis elegans, Drosophila melanogaster, and mammals. FoxO proteins are homologous to the transcription factor DAuer Formation-16 (DAF-16) in the worm Caenorhabditis elegans that can determine metabolic insulin signaling and lead to lifespan extension,^98,99 suggesting a significant role for FoxO proteins in relation to mammalian cell function.^3,10 In fact, FoxO proteins can stimulate the insulin-like growth factor binding protein-1 (IGFBP1) promoter by binding to the insulin-responsive sequence (IRS).¹⁰⁰ Both insulin and insulin-like growth factor-1 (IGF-1) can suppress this activity through activation of Akt.^100,101

In clinical studies, analysis of the genetic variance in FOXO1a and FOXO3a on metabolic profiles, age-related diseases, fertility, fecundity, and mortality have observed higher HbA^1c levels and increased mortality risk associated with specific haplotypes of FOXO1a.¹⁰² These clinical observations may coincide with the demonstration in human endothelial progenitor cells that elevated glucose levels can reduce posttranslational phosphorylation of FOXO1, FOXO3a, and FOXO4 and allow for the nuclear translocation of these proteins to initiate an apoptotic program in endothelial progenitor cells.¹⁰³ In experimental models, FoxO proteins may prevent the toxic effects of high serum glucose levels. Interferon-gamma driven expression of tryptophan catabolism by cytotoxic T lymphocyte antigen 4 may activate Foxo3a to protect dendritic cells from injury in nonobese diabetic mice.¹⁰⁴ Additional studies have demonstrated that adipose tissue-specific expression of Foxo1 in mice improved glucose tolerance and sensitivity to insulin during an elevated fat diet.¹⁰⁵ FoxO proteins also may protect against diminished mitochondrial energy levels known to occur during insulin resistance such as in the elderly populations.^85,94,95 In caloric restricted mice that have decreased energy reserves, Foxo1, Foxo3a, and Foxo4 mRNA levels were noted to progressively increase over a two-year course.⁸ These observations complement studies in Drosophila and mammalian cells that demonstrate an increase in insulin signaling to regulate cellular metabolism during the up-regulation of FoxO1 expression.¹⁰⁶

However, the ability for FoxO proteins to maintain proper physiologic controls over cellular metabolism may be limited and occur only during specific circumstances. For example, mice with a constitutively active Foxo1 transgene have increased microsomal triglyceride transfer protein and elevated plasma triglyceride levels.¹⁰⁷ Studies in cardiomyocytes also suggest detrimental results with enhanced FoxO activity. Increased transcriptional activity of FoxO1, such as by the Sirt1 activator resveratrol, can diminish insulin mediated glucose uptake and result in insulin resistance.¹⁰⁸ In addition, over-expression of Foxo1 in skeletal muscles of mice can lead to reduced skeletal muscle mass and poor glycemic control,¹⁰⁹ illustrating that activation of FoxO proteins also may impair cellular energy reserves. Additional investigations that block the expression of Foxo1 in normal and cachectic mice¹¹⁰ or reduce FoxO3 expression¹¹¹ show the reverse with an increase in skeletal muscle mass or resistance to muscle atrophy. These results become especially relevant in patients with cancer and cachexia, since FoxO protein expression may further muscle wasting for these individuals. Given these concerns, one potential agent to consider for the maintenance of cellular metabolism in cancer patients is nicotinamide,^36,78 an agent that also can inhibit FoxO protein activity.⁴³ In patients with DM, oral nicotinamide protects β-cell function, prevents clinical disease in islet-cell antibody-positive first-degree relatives of Type-1 DM, and can reduce HbA1c levels.^36,78,94 Nicotinamide, which is closely linked to cell longevity pathways,^112,113 may derive its protective capacity through two separate mechanisms of posttranslational modification of FoxO3a. Nicotinamide not only can maintain phosphorylation of FoxO3a and inhibit its activity, but also can preserve the integrity of the FoxO3a protein to block FoxO3a proteolysis that can yield pro-apoptotic amino-terminal fragments.⁴³

As an extension to the work with cellular metabolism, FoxO proteins also have been linked to cell longevity and aging as shown by early studies linking DAF-16 in Caenorhabditis elegans to increased longevity.^3,19,21,114 However, the relationship between FoxO transcription factors and proteins that increased cellular lifespan has been met with controversy. Sirt1 is a NAD⁺-dependent deacetylase and the mammalian ortholog of the silent information regulator 2 (Sir2) protein associated with increased lifespan in yeast. Some studies suggest that stimulation of Sirt1 during starvation is dependent upon FoxO3a activity as well as p53.¹¹⁵ In contrast, other work has shown in cell culture that Sirt1 may repress the activity of FoxO1, FoxO3a, and FoxO4, suggesting that cellular longevity may benefit from reduction in FoxO protein generated apoptosis.¹¹⁶ Additional studies offer alternative views to illustrate that Sirt1 binds to FoxO proteins, such as FoxO4, to catalyze its deacetylation and enhance FoxO4 activity while acetylation of FoxO4 by cyclic-AMP responsive element binding (CREB)-binding protein serves to inhibit FoxO4 transcriptional activity.^3,19,21,114

FoxO proteins also may be protective during aging, cell senescence, and exercise. In cultured human dermal fibroblasts, gene silencing of FoxO3a protein results in cell morphology consistent with cell senescence, cell population doubling times, and the generation of ROS, suggesting that FoxO protein activity may be required to extend cell longevity and limit oxidative stress.¹¹⁷ Additional work in animal models of aging demonstrates a reduction in Sirt1 in the heart, but no significant change in FoxO3a expression with advanced age. However, during exercise training, an up-regulation of FoxO3a and Sirt1 activity is observed in the heart,⁹³ suggesting that the benefits of physical activity for the cardiovascular system may be associated with FoxO proteins. Interestingly, increased levels of Sirt1 less than 7.5-fold can be associated with expression of catalase, an anti-oxidant that is controlled by FoxO1a to possibly reduce cell injury during oxidative stress. Yet, elevated levels of Sirt1 at 12.5-fold can result in cardiomyocyte apoptosis and decreased cardiac function.¹¹⁸ In addition, FoxO proteins may be protective during aging, since loss of FoxO3a activity in explanted vascular smooth muscle of aged animals may limit tissue antioxidant properties through decreased manganese-superoxide dismutase and lead to enhanced cell injury with aging.¹¹⁹ Extension of cellular lifespan that depends upon the prevention of cell senescence at least in primary human cultured vascular cells also may require the negative regulation of Akt to allow for the activation of FoxO3a.¹²⁰

FoxO Proteins, Stem Cells and Cardiovascular Development

FoxO proteins represent important targets for several disorders, but high on the list may be therapies to block cancer growth since FoxO proteins can modulate stem cell proliferation and new vessel growth. The initial identification of FoxO proteins in soft-tissue tumors and leukemias, neoplasms now believed to harbor cancer stem cells for tumor self-renewal,⁶⁹ suggests that FoxO proteins may be closely associated with the oversight of stem cell proliferation and differentiation. For example, either simultaneous deletion of Foxo1, Foxo3a, and Foxo4 or single deletion of Foxo3a in mice prevents the repopulation of hematopoietic stem cells and leads to apoptosis in these stem cell populations.^92,121 Furthermore, vascular cytoprotective agents, such as the growth factor EPO,^33,122,123 also may be required to modulate FoxO protein activity such as during erythroid progenitor cell development,^73,124 suggesting that current clinical use of agents such as EPO during anemia or cancer may have less defined treatment implications for patients than originally anticipated.^33,124 In cell culture and animal studies, EPO is cytoprotective in vascular cells and can stimulate postnatal neovascularization by increasing endothelial progenitor cell mobilization from the bone marrow.^73,124,125 Interestingly, the ability of EPO to foster eythroid progenitor cell development is dependent upon the inhibition of FoxO3a activity,^33,124 but also may require regulation of specific gene expression through an EPO-FoxO3a association to promote erythropoiesis in cultured cells.¹²⁶ In relation to the reproductive potential of an organism, deletion of the FoxO3a gene results in the depletion of oocytes and subsequent infertility.¹²⁷ Other work using a mouse model of FoxO3a over-expression in oocytes further suggests that FoxO3a retards oocyte growth and follicular development and leads to anovulation and luteinization of unruptured follicles.¹²⁸ These studies may suggest a role for FoxO proteins, and specifically FoxO3a, in relation to not only the development of cancer stem cell niches, but also in regards to oocyte and follicular cell maturation. For example, in a small percentage of women who suffer from premature ovarian failure mutations in FOXO3a and FOXO1a have been observed.¹²⁹

In addition to the modulation of stem cell development, FoxO proteins play a significant role to govern new vessel growth that can impact upon tumor cell growth and dispersion. New capillary formation from pre-existing vessels into an avascular area is a process known as angiogenesis that is present during embryogenesis, during menstruation, and during pathological processes that involve wound healing, chronic inflammation, and tumor growth.^54,124 FoxO proteins are intimately involved in endothelial cell development and angiogenesis. For example, Foxo3a −/minus; and Foxo4 minus;/minus; mice develop without incidence and are indistinguishable from control littermates. However, mice that are singly deficient in Foxo1 die by embryonic day eleven and lack development of the vascular system.¹³⁰ Additional studies illustrate that endothelial cell colonies in Foxo1-deficient mice fail to respond to vascular endothelial growth factor in a manner similar to wild-type endothelial cells,¹³¹ suggesting that FoxOs are necessary for the development of vascular cells as well as for the biological response to cellular mediators.

During cardiac development, FoxO proteins also appear to be necessary to modulate cardiomyocyte proliferation. Both FoxO1 and FoxO3 are expressed during embryonic through prenatal stages in the developing myocardium. The expression of these FoxO proteins is believed to negatively regulate cardiomyocyte growth, since overexpression of FoxO1 blocks cardiomyocyte proliferation but expression of dominant negative FoxO1 leads to enhanced cardiomyocyte growth.¹³² These observations may provide clues into the roles of FoxO proteins during cardiac hypertrophy. Atrogin-1, a protein that can block cardiac hypertrophy, may rely upon the up-regulation of Foxo1 and Foxo3a to disrupt cardiac hypertrophy, since mice lacking atrogin-1 are susceptible to cardiac hypertrophy and do not yield increased expression of Foxo1 and Foxo3a.¹³³

In regards to smooth muscle cell growth, Foxo3a has been demonstrated to block vascular smooth muscle proliferation and may lessen the effects from disorders such as atherosclerosis and hypertension. In a rat balloon carotid arterial injury model, gene transfer of FoxO3a can inhibit neointimal hyperplasia through the prevention of vascular smooth muscle growth.¹³⁴ However, not all FoxO proteins may exert an inhibitory effect upon vascular smooth muscle cells. FoxO4 may inhibit smooth muscle cell differentiation through the repression of the transcriptional coactivator of smooth muscle genes myocardin,¹³⁵ but other work suggests that FoxO4 also can increase matrix metalloproteinase 9 expression to promote vascular smooth muscle migration and foster neointimal hyperplasia.¹³⁶

In light of the ability of FoxO proteins to regulate vascular smooth muscle cell proliferation, these transcription factors may have a significant clinical role in regards to disorders that involve hypertension and cardiac failure. Vascular smooth muscle cells are vital for the regulation of vascular tone and systemic arterial blood pressure. For example, high flow states in vessels can reduce FoxO1 activity, resulting in the potential proliferation of vascular smooth muscle cells, vascular neointimal hyperplasia, and subsequent pathological states such as hypertension.¹³⁷ In fact, α1-adrenergic agonists that increase systemic blood pressure can have the reverse effect and stimulate the expression of FoxO1 and its nuclear translocation that ultimately may lead to apoptotic endothelial cell injury.¹³⁸ In addition, more than moderate levels of vessel cyclic stretch that can occur during hypertension may lead to the phosphorylation and inhibition of Foxo1 and Foxo3a in smooth muscle cells to further contribute to pathological smooth muscle cell proliferation.¹³⁹ Furthermore, in human as well as murine models of cardiac failure, increased expression of Fox transcription factors, such as FoxO1a, have been observed to suggest a potential association of FoxO proteins with imminent cardiac failure.¹⁴⁰

FoxO Proteins and the Immune System

Forkhead transcription factors have a vital role in maintaining immune system function. For example, the forkhead family member FoxP3 can control the development and function of thymic-derived CD4(+)CD25(+) regulatory T cells (Treg) that impart autoimmunity. Loss of FoxP3 can result in autoimmune disorders.¹⁴¹ In addition, recent work identifies the expression of FoxP3 in tumor cells, such as melanoma,¹⁴² as well as in Tregs which may significantly affect patient mortality since the increased presence of Tregs in cancer patients combined with FoxP3 expression in tumors may impair antitumor autoimmune responses and lead to high mortality.¹⁴³

In regards to FoxO proteins, these forkhead transcription factors also may impact upon neoplastic progression since they lead to the induction of apoptotic pathways and may influence early apoptotic membrane PS externalization (Fig. 1). The ability to regulate early apoptotic membrane PS exposure⁴⁰ and inflammatory cell activity²⁹ can ultimately impact upon cell survival since activated immune cells can lead to the phagocytic removal of tumor cells.^79,84 Inflammatory cells, such as macrophages or microglia, require the activation of intracellular cytoprotective pathways to proliferate and remove injured cells.^80,144 At times, this can be a beneficial process and form a barrier for the removal of foreign micro-organisms and promote tissue repair during cell injury.^73,85 However, inflammatory cells also may lead to cellular damage through the generation of ROS and through the production of cytokines.⁷³ Interestingly, in mice deficient for Foxo3a, lymphoproliferation, organ inflammation of the salivary glands, lung, and kidney, and increased activity of helper T cells results, supporting an important role for FoxO3a in preventing T cell hyperactivity.¹⁴⁵ FoxO3a also appears to be necessary for neutrophil activity, since Foxo3a null mice are resistant to models of neutrophilic inflammation that involve immune complex-mediated inflammatory arthritis.¹⁴⁶

In clinical studies, patients with rheumatoid arthritis and osteoarthritis show phosphorylation of FOXO3a in T lymphocytes as well as FOXO1 and FOXO4 in synovial macrophages, suggesting that loss of functional FOXO family members may lead to inflammatory cell activation in these disorders.¹⁴⁷ FOXO1 gene transcript levels also are down-regulated in peripheral blood mononuclear cells of patients with systemic lupus erythematosus and rheumatoid arthritis,¹⁴⁸ illustrating a potential etiology through the loss of functional FOXO proteins for these disorders and possibly providing a biomarker of disease activity. Other work has demonstrated that FOXO1 protein regulates L-selectin expression that can regulate human T lymphocyte trafficking.¹⁴⁹ More importantly, studies suggest a relationship between the regulation of immune system activity and the induction of apoptotic pathways that are dependent upon FoxO proteins. Prevention of inflammatory activation and apoptosis in the nervous system such as in systemic lupus erythematosus in animal models may require the up-regulation of different Fox proteins, such as FoxJ1 and FoxO3a, that can block NF-κB activation and interferon-gamma secretion.¹⁵⁰ FoxO proteins also may work in concert with Fas signaling to clear activated T cells following a decrease in cytokine stimulation in patients with autoimmune lymphoproliferative syndromes,¹⁵¹ suggesting that activation of specific FoxO proteins may be beneficial for autoimmune disorders but may impair treatments designed to target tumor cells through immune mediated pathways.

FoxO Proteins and Cancer

As previously mentioned, one of the most important treatment possibilities for FoxO proteins involves strategies designed to control human cancer progression in light of the ability of FoxO proteins to lead to apoptosis and block cell cycle progression. For example, studies with prostate cancer have shown that the tumor suppressor phosphatase and tensin homolog deleted on chromosome 10 (PTEN) is mutated in approximately 80% of tumors with the loss of FOXO1 and FOXO3a activity. In cell cultures, over-expression of FoxO1 and FoxO3a in prostrate tumor cell lines also leads to apoptosis, suggesting that FoxO1 and FoxO3a are necessary for limiting prostate cell tumor growth.¹¹ In addition, it has been shown that inhibition of FoxO3a activity can result in enhanced prostate tumor cell growth¹⁵² while agents that increase FoxO3a activity in both androgen sensitive and androgen insensitive prostate cell lines prevent prostate cancer cell progression.¹⁵³ Furthermore, therapeutic strategies that rely upon the over-expression of a nonphosphorylatable form of FoxO3a that cannot be inactivated can sensitize prostate cancer cells to androgen-withdrawal-induced apoptosis.¹⁵⁴ Yet, it should be noted that in prostate cell lines FoxO3a can be a positive regulator of androgen receptor expression and therefore may play a complex role in prostate cancer cell proliferation and growth inhibition.¹⁵⁵ Other factors that control FoxO protein function also may play a role during prostate tumor progression. In prostate cancer cells, cyclin-dependent kinase 1 (CDK1) can become over-expressed and subsequently phosphorylate FOXO1 to block its transcriptional activity and contribute to prostate tumorigenesis.¹⁵⁶ In a similar manner, it has been shown that astrocyte-elevated gene-1 (AEG-1) can be upregulated in clinical prostate cancer,¹⁵⁷ possibly lead to activation of Akt that suppresses FOXO3a¹⁵⁸ and apoptosis in prostate tumor cells.

Initial investigations of FOXO3a in clinical breast cancer suggested that activation of FOXO3a was associated with lymph nodal metastasis and a poor prognosis.¹⁵⁹ In contrast to these observations, other studies reported that FOXO3a was inactivated by IKK and that inactivation of FOXO3a was associated with a poor prognosis in breast cancer,¹⁶⁰ suggesting that FOXO3a sub-cellular localization and pathways that enhance its activity could be used not only as prognostic assays but also as therapeutic targets. Other work in breast cancer cells demonstrate the tumor repressive ability of FoxOs by illustrating that increased activity of FoxO3a in association with JNK in breast cancer cell lines¹⁶¹ or in association with cyclin-dependent kinase inhibitor p27 in isolated human breast cancer cells can prevent breast cancer growth.¹⁶² In addition, FoxO proteins may be able to modulate estrogen function and indirectly block breast cancer growth. Over-expression of FoxO3a in breast cancer cell lines can decrease the expression of estrogen receptor regulated genes and inhibits 17beta-estradiol (E2)-dependent breast cancer growth.¹⁶³

In addition to the ability to inhibit prostate and breast tumor growth, FoxO proteins may represent a viable option to control tumor progression in other tissues. FoxO proteins can function as redundant repressors of tumor growth. For example, somatic deletion in mice of Foxo1, Foxo3a, and Foxo4 results in the growth of thymic lymphomas and hemangiomas.¹⁶⁴ Other work illustrates that FoxO3a activation in colon carcinoma cell lines prevents tumor proliferation through Myc target genes that involve the Mad/Mxd family of transcriptional repressors.¹⁶⁵ In addition, the loss of FoxO3a activity may participate in oncogenic transformation in B-chronic lymphocytic leukemia¹⁶⁶ and in the progression of chronic myelogenous leukemia cell lines.⁷⁴ Furthermore, studies suggest that some proteins, such as the Kaposi’s sarcoma-associated herpes virus latent protein LANA2, may specifically block the transcriptional activity of FoxO3a to lead to tumor growth.¹⁶⁷ In cell models of endometrial cancer, presensitization of cells to block Akt activation and foster transcription activity of FoxO1 enhances the effect of chemotherapy to limit tumor growth.¹⁶⁸

Conclusions and Future Perspectives for Clinical Care

The potential translation of FoxO proteins and their signal transduction pathways into viable therapeutic strategies offer exciting prospects for the future. FoxO proteins control several vital cellular pathways in relation to cell proliferation, metabolism, inflammation, and survival. For example, the known mutations in FoxO proteins that exist in several disease entities may provide novel insights for therapeutic strategies that can address a broad range of disorders. Further analysis in larger populations of patients with premature ovarian failure, diabetes, or stroke could enhance our understanding of the role of FoxO proteins in these disorders. When one considers the role of FoxO proteins at the cellular level such as in cardiac and endothelial cells, targeting the activity of FoxO1, FoxO3a, or FoxO4 may prevent the onset of pathological cardiac hypertrophy and neointimal hyperplasia that may result in atherosclerosis. Interestingly, new work suggests that the utilization and combination of multiple biomarkers may improve risk assessment for patients suffering from cardiovascular disorders.¹⁶⁹ These studies illustrate that FoxO proteins may serve as biomarkers of disease activity such as in individuals with imminent cardiac failure.¹⁴⁰

In regards to potential treatments directed against cancer, the ability of FoxO proteins to control cell cycle progression and promote apoptosis highlights the potential of FoxOs to become an important component for new strategies directed against tumorigenesis. For example, use of triple mutant FoxO1 or FoxO3a expression in which three phosphorylation sites have been altered to prevent inactivation of this protein has been proposed as a potential therapeutic agent against melanoma tumors⁴⁵ and endometrial cancer.¹⁷⁰ Other work also offers additional support for the use of FoxO proteins as biomarkers of cancer progression. As an example, down regulation of the phosphatidylinositol 3 kinase and Akt pathways have been associated with increased transcript levels for FOXO1a and FOXO3a in clinical prostate cancer samples and may indicate the onset of precancerous changes or the progression of on-going tumor growth.¹⁷¹ Although loss of Akt activity in prostate cancer cells can result in enhanced FoxO3a activity and subsequent apoptosis of tumor cells,¹⁵⁷ it is conceivable that early stages of cancer may lead to reduced Akt activity with insufficient levels of active forkhead transcription factors to limit tumor progression. In addition, the early and persistent expression of phosphorylated FOXO1a in gastric tumors may not only indicate the onset of cancer, but also suggest an improved prognosis for patients.¹⁷²

Despite the presently known attributes of FoxO proteins to potentially treat a number of disorders, FoxO transcription factors also may limit clinical utility. Further investigations are required since FoxO protein inhibition of cell cycle progression may not consistently lead to apoptotic cell death. Some investigations suggest that during oxidative stress, FoxO3a activation in association with Sirt1 can lead to cell cycle arrest, but not result in apoptotic cell injury.¹⁷³ Furthermore, during hypoxic stress, forkhead transcription factors, such as FOXO3a, may potentiate anti-apoptotic pathways in breast cancer cells to further tumor growth.¹⁷⁴ FoxO proteins also have been linked to potential chemotherapy drug resistance. Increased expression of MDR1 (P-glycoprotein) has been associated with chemotherapy drug resistance in breast cancer cells and recent work shows that FoxO1 can stimulate the transcriptional activity of MDR1 that may promote increased tolerance of tumor cells.¹⁷⁵ In addition, the common pathways shared between Wnt and forkhead proteins may have another side that impacts upon the ability to control tumor growth.^53,63 FoxO proteins may assist with β-catenin activation in the Wnt pathway and lead to tumor cell proliferation.⁵⁴ In the presence of Wnt deregulation and increased β-catenin activity, tumorigenesis may ensue, such as with the proliferation of medulloblastoma tumors.⁶⁹ Therefore, prediction of biological outcomes during FoxO protein involvement may be uncertain and may be influenced by a host of factors such as tissue characteristics, cellular metabolic state, and the age of an individual. Given these circumstances, further basic and clinical investigations will be required to continue to elucidate the immense potential of FoxO proteins as well as to understand the potential limits of these transcription factors.

Abbreviations

Aβ

β-amyloid

Akt

protein kinase B

AFX

acute leukemia fusion gene located in chromosome X

AGC

protein kinase A/protein kinase G/protein kinase C

CBP

CREB-binding protein

DAF-16

DAuer Formation-16

DM

diabetes mellitus

EPO

erythropoietin

FKHR

forkhead in rhabdomyosarcoma

FKHRL1

forkhead in rhabdomyosarcoma like protein 1

IKK

IκB kinase

IGF-1

insulin-like growth factor-1

IGFBP1

insulin-like growth factor binding protein-1

IRS

insulin-responsive sequence

JNK

Jun N-terminal kinase

NF-κB

nuclear factor-κB

PCP

planar cell polarity

PS

phosphatidylserine

PTEN

tumor suppressor phosphatase and tensin homolog deleted on chromosome ten

PSR

phosphatidylserine receptor

ROS

reactive oxygen species

Wg

Drosophila Wingless