Endocrine-Related Cancer (2008) 15 917–929 REVIEW Forkhead box-O : critical conductors of cancer’s fate

Carl Weidinger, Kerstin Krause, Antje Klagge, Stefan Karger and Dagmar Fuhrer

Division of Endocrinology and Diabetology, Department of Internal Medicine, University of Leipzig, Philipp-Rosenthal-Strasse 27, D-04103 Leipzig, Germany (Correspondence should be addressed to D Fuhrer; Email: [email protected])

Abstract Cells have evolved elaborated mechanisms to coordinate the cellular answer of either survival or apoptosis. Recent concepts of human carcinogenesis have suggested disturbances in these cellular relays as a potential link to cellular dedifferentiation and uncontrolled proliferation. Forkhead box-O transcription factors (FOXOs) play an important role in tumour suppression by regulating the expression of involved in stress resistance, DNA damage repair, cell cycle arrest and apoptosis. The specific regulation of FOXO function is tightly controlled by posttranslational modifications such as phosphorylation, acetylation and ubiquitination. Loss of FOXO function has recently been identified in several human cancers. In this review, we will give an overview about recent progress in the understanding of function and regulation of FOXOs, as well as their role in carcinogenesis. Furthermore, we will discuss a potential clinical use of FOXOs by therapeutically restoring their tumour suppressive properties. Endocrine-Related Cancer (2008) 15 917–929

Introduction containing the FOXO consensus binding sequence Eukaryotic cells, from yeast to mammals, respond and TTGTTTAC (Tsai et al. 2007). To date, four different adapt to environmental challenges by an evolutionarily isoforms of FOXOs are known in humans: FOXO1 conserved endogenous system that balances the (FKHR), FOXO3a (FKHRL1), FOXO4 (AFX1) and decision between cell repair and death by a network FOXO6. of signal transduction pathways. The balance between The regulation of FOXOs depends predominantly on cell survival and death is crucial for cellular integrity. opposing signalling pathways that influence the Forkhead box-O transcription factor (FOXO) tran- subcellular distribution of FOXOs, as well as their scription factors were recently identified as an activity and specificity. For instance, activation of the important family of that modulate the PI3K/Akt pathway results in an active export of expression of genes involved in cell differentiation, FOXOs into the cytoplasm. By contrast, in response resistance to oxidative stress, DNA damage repair, cell to stress, FOXOs are activated by the members of the cycle arrest and apoptosis (Arden 2006, Reagan-Shaw c-Jun-N-terminal kinase (JNK) leading to nuclear & Ahmad 2006, van der Horst & Burgering 2007, FOXO accumulation. Huang & Tindall 2007). FOXO transcription factors It has been suggested that FOXOs may regulate belong to the large family of forkhead proteins expression in both ways, e.g. through activation or (Daitoku & Fukamizu 2007). In the past years, over repression, depending on recruitment and interaction 100 forkhead genes have been identified in a large with either co-factors or -repressors (Boura et al. 2007, variety of species, all sharing an evolutionarily Huang & Tindall 2007). conserved ‘forkhead’ DNA-binding domain. This In this review, we will first describe the present DNA-binding domain consists of three a-helices, knowledge on FOXO function and regulation and then three b-strands and two wing-like loops (Boura discuss the pathophysiological consequences of a loss et al. 2007), and binds to a 13 bp DNA duplex, of FOXO function in human cancers.

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Functions of FOXO pathway. Furthermore, the transcription of the pro- apoptotic Bim (BCL2L11) is induced in human T-cells Cell cycle control after FOXO3a stimulation (Stahl et al. 2002). Bim is In mammalian cells, proliferation is controlled by believed to promote the mitochondrial release of factors that regulate the transition at the G1/S and the cytochrome c, via the inhibition of Bcl2, thereby G2/M checkpoints, which are responsible for the activating the intrinsic apoptosis pathway (Puthalakath initiation and completion of DNA replication and control et al. 1999, Adams & Cory 2007). Moreover, FOXO3a of cell division respectively. The regulative function of has been demonstrated to induce PUMA (BBC3) FOXOs on cell cycle occurs at different stages. transcription (You et al. 2006) in human lymphoid First, ectopic expression of FOXO1, FOXO3a or cells and mouse embryonic fibroblast cells, conferring FOXO4 in human cells blocks cell cycle transition at G1 increased sensibility for proapoptotic signals. phase by transcriptional upregulation of the cell cycle In addition, FOXOs may repress the transcription of inhibitor p27kip1 (CDKN1B) (Dijkers et al.2000, prosurvival genes of the BCL2 family. For instance, Medema et al. 2000). By subsequent binding of FOXO4 has been shown to inhibit the expression of the p27kip1 to the cyclin E/cdk2 complex, the entry of prosurvival gene Bcl-xL in HeLa cells (Tang et al. cells into S phase is prevented (Reynisdottir et al. 1995). 2002). In addition, FOXO1 and FOXO3a inhibit G1/S phase transition by downregulation of cyclins D1 and D2 DNA repair (Smith et al. 1996, Ramaswamy et al. 2002). FOXOs are emerging as important regulators of genes Secondly, at the G2/M boundary, FOXO3a prevents cell cycle transition by induction of cyclin G2 and involved in DNA damage repair. DNA repair is Gadd45a (growth arrest and DNA damage-inducible initiated by the capability of FOXO3a and FOXO4 to 45), interfering with cyclin/CDK complexes induce the transcription of Gadd45a, which harbours (Furukawa-Hibi et al. 2002, Martinez-Gac et al. 2004). base excision activity and maintains genomic integrity, Thirdly, FOXOs may induce a state of cellular e.g. through DNA demethylation and cell cycle control (Hollander et al. 1999, Smith et al. 2000, Furukawa- quiescence (G0) by transcriptional upregulation of the Rb family member p130 (RBL-2). The FOXO isoforms Hibi et al. 2002, Tran et al. 2002, Kobayashi et al. FOXO3a and FOXO4 directly bind to consensus sites 2005). As a result, FOXO3a-induced DNA repair is in the promoter of the p130 gene, e.g. in murine and severely impaired in Gadd45a-deficient fibroblasts human colon carcinoma cells, thereby activating its (Tran et al. 2002). Recent data have suggested that FOXO3a can also transcription (Kops et al. 2002b). In G0 cells, p130 represses the expression of genes required for re-entry promote DNA repair in a non-transcriptional manner. into the cell cycle through activation of the anti- Tsai et al. reported a direct interaction between proliferative transcription factor (Smith et al. FOXO3a and the ataxia telangiectasia mutated 1996). (ATM) protein in response to DNA damage. They showed that exposure of LNCAP prostate cells to ionizing radiation led to covalent binding of FOXO3a Apoptosis to ATM, resulting in an autophosphorylation of ATM on Ser1981, which in turn was blocked by knock-down Activation of FOXOs can directly promote the of FOXO3a (Tsai et al. 2008). transcription of genes involved in the extrinsic and/or intrinsic apoptotic pathways. In a pioneer study by Brunet et al. it was shown in Detoxification different cell lines that Fas ligand (FASLG), a major FOXO transcription factors play a crucial role in the player in the extrinsic apoptosis pathway, is a down- fine-tuning of the antioxidative system. An increase in stream target of FOXO3a. Thus, the expression of a reactive oxygen species leads to FOXO3a- and constitutively activating FOXO3a mutant in Jurkat FOXO1-mediated upregulation of antioxidative T-lymphocytes induced apoptosis in a FASLG-depen- proteins, such as manganese superoxide dismutase dent manner (Brunet et al. 1999). Modur et al. (2002) (MnSOD) and catalase (Kops et al. 2002a, Adachi showed that restoration of FOXO function (FOXO1 et al. 2007, Alcendor et al. 2007). and FOXO3a) in prostate cancer cells resulted in the Recently, peroxiredoxin III (Prx III) has been upregulation of the tumour necrosis factor-related identified as another FOXO3a target gene induced by apoptosis-inducing ligand (TRAIL; TNFSF10), which oxidative stress in cardiac fibroblasts. Inhibitory is another important mediator of the extrinsic apoptosis phosphorylation of FOXO3a through PI3K/Akt

Downloaded from Bioscientifica.com at 10/01/2021 04:22:14AM via free access 918 www.endocrinology-journals.org Endocrine-Related Cancer (2008) 15 917–929 prevented binding of FOXO3a to the Prx III promoter, of FOXO1 and FOXO3a in vitro (Nowak et al. 2007). while FOXO3a overexpression increased Prx III Additionally, regulation of FOXO gene transcription promoter activity (Chiribau et al. 2008). by nutritional and hormonal status was found in rat In addition, FOXOs have been found to interact with hepatocytes in vivo. A diminished nutrient availability a number of other transcription factors, thereby and artificially induced resistance led to modifying their transcriptional activity and specificity, increased mRNA expression of FOXO1, FOXO3a for instance, of SMAD, RUNX3 and , which has and FOXO4 (Imae et al. 2003). been excellently reviewed elsewhere (Gomis et al. The activity of FOXOs is tightly regulated by 2006, Allen & Unterman 2007, van der Horst & posttranslational modifications (PTMs) such as phos- Burgering 2007). Figure 1 shows a summary of known phorylation, acetylation and ubiquitination (Fig. 2; Van functions of FOXO proteins. Note that not all FOXO Der Heide et al. 2004, Vogt et al. 2005, van der Horst isoforms may be expressed in all tissues, and that their & Burgering 2007, Huang & Tindall 2007, Reagan- function may differ in an isoform-specific way. Shaw & Ahmad 2006). PTMs of FOXOs ensure the short-term integration of prosurvival and -apoptotic signals, while transcriptional and translational modifi- Regulation of FOXOs cations predominantly control the long-term adaptation to a changing cellular micromilieu. Forkhead activity is controlled at both the transcrip- In the following section, we will summarize PTMs tional and posttranslational levels (Huang & Tindall that regulate FOXO activity. This will be of substantial 2007). Recent work has suggested that the FOXO value for a better understanding of the role of FOXOs isoforms FOXO1 and FOXO3a are at least partly under in carcinogenesis. transcriptional control of the transcription factor . The proximal gene promoters of FOXO1 and FOXO3a harbour an evolutionarily conserved E2F1-binding site Inhibitory phosphorylation of FOXOs (Nowak et al. 2007). Furthermore, induction of E2F1 One prerequisite for the transcriptional regulatory in human glioblastoma cells promotes the transcription function of FOXOs is their nuclear localization. In

Figure 1 FOXOs regulate a number of genes, involved in tumour suppression. By binding to the promoter regions of their target genes, FOXOs either repress or induce mRNA transcription. For instance, FOXOs upregulate proapoptotic targets such as Bim and PUMA ([), while targets with mitogenic functions such as cyclins D1 and D2 are repressed (Y). Note that this figure represents a simplified model, in which the single isoforms of FOXOs are not distinguished with respect to their target gene specificity.

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Figure 2 FOXO activity is regulated by posttranslational modifications. (A) FOXOs can be excluded from the nucleus in response to growth signalling cascades. Akt and other mitogenic signalling cascades, such as Ikkb, SGK1, CDK1 and CDK2 can phosphorylate FOXOs on isoform-specific phosphorylation sites (1). This phosphorylation enables FOXOs to bind members of the 14-3-3 protein family, leading to the disruption of DNA binding and the nuclear export of FOXOs (2). Once cytoplasmatic, FOXOs can get polyubiquitinated by SKP2 (3), which promotes the proteasomal degradation of FOXOs (4). Note that this simplified model is not valid for FOXO6, which lacks this export mechanism, resulting in its permanent nuclear distribution. (B) Cytoplasmatic FOXOs can translocate into the nucleus in a JNK dependent manner. An increase in oxidative stressors leads to a JNK-mediated phosphorylation of FOXOs on phosphorylation sites that differ from those targeted by mitogenic kinases (1). This phosphorylation triggers the nuclear import of FOXOs, whose acetylation status is relevant for the fine-tuning of target gene transcription (2). various mammalian cells, the FOXO isoforms FOXO1, export sequences enables the shift of FOXOs into the FOXO3a and FOXO4 were demonstrated to underlie cytoplasm (Brunet et al. 2002). Moreover, binding of inhibitory phosphorylation on isoform-specific phos- 14-3-3 proteins suppresses the nuclear re-import of phorylation sites, resulting in their nuclear exclusion FOXOs by masking their nuclear localization sequence and a consecutive loss of their transcriptional function. (Obsilova et al. 2005, Hermeking & Benzinger 2006). Several pathways mostly related to mitogenic signalling However, Yan et al. (2008) recently revealed a can inactivate FOXOs in a phosphorylation-dependent mechanism that allowed FOXO1 to re-enter the manner. In particular, the PI3K/Akt signalling cascade, nucleus after Akt-induced translocation into the which is a downstream target of insulin signalling, cytoplasm: the protein phosphatase 2A (PP2A) disrupts is known to be a major inhibitor of FOXO function the binding of FOXO1 and proteins of the 14-3-3 (Biggs et al. 1999, Brunet et al. 1999, Kops et al. family by dephosphorylating the Akt phosphorylation 2002b). For example, Akt phosphorylates mammalian sites Thr32 and Ser256, leading to nuclear relocation of FOXO1 at sites Thr24, Ser256 and Ser319 (Biggs FOXO1 and reactivation of its proapoptotic transcrip- et al. 1999). FOXO3a can be phosphorylated by Akt tional function in response to growth factor on Thr32, Ser256 and Ser319 (Kashii et al. 2000), withdrawal. whereas inactivation of FOXO4 is associated with Akt- In striking contrast to the other mammalian FOXO mediated phosphorylation on Thr32 and Ser197 isoforms, Akt-mediated phosphorylation of FOXO6 on (Matsuzaki et al. 2005b). Thr26 and Ser184 appears to inhibit its activity without Upon PI3K/Akt-induced phosphorylation of a subsequent translocation into the cytoplasm. Studies FOXO1, FOXO3a and FOXO4, two binding sites for in HEK239T cells revealed that FOXO6 lacks the proteins of the 14-3-3 family are generated, which conserved C-terminal Akt motif, which mediates the results in the disruption of FOXO-DNA binding 14-3-3 protein-dependent shuttling into the cytoplasm (Brunet et al. 2002). Successive to the association (Van Der Heide et al. 2005). Moreover, recent data with 14-3-3 proteins, the binding of exportins, e.g. on the crystal structure of the mammalian forkhead Crm1 and Ran, to isoform-specific FOXO nuclear DNA-binding domain suggest that Akt-mediated

Downloaded from Bioscientifica.com at 10/01/2021 04:22:14AM via free access 920 www.endocrinology-journals.org Endocrine-Related Cancer (2008) 15 917–929 phosphorylation itself diminishes the affinity of Acetylation FOXOs for DNA binding through modulation of van The transcriptional activity and specificity of FOXOs der Waals forces (Tsai et al. 2007). have been shown to be tightly controlled by acetylation Besides PIK3K/Akt, further kinases involved in and deacetylation in the nucleus. Nuclear FOXOs can mitogenic signalling have been identified to mimic bind to various proteins, influencing FOXOs’ acetylation PI3K/Akt-mediated phosphorylation and inhibition of status on conserved lysine residues in vitro and in vivo FOXO3a and FOXO4. For example, Ikkb,an (Daitoku et al.2004, van der Horst et al.2004, Frescas important mediator of NF-kB signalling, phosphory- et al.2005, Perrot & Rechler 2005, Arden 2006): for lates FOXO3a on Ser644, resulting in the nuclear example, in response to oxidative stress, nuclear exclusion of FOXO3a (Hu et al. 2004, Hu & Hung FOXO3a associates with the histone acetylase CREB- 2005). A similar effect has been demonstrated for the binding protein (CREBBP) and P300 (Daitoku et al. serum- and glucocorticoid-inducible kinase SGK1 2004, Perrot & Rechler 2005). This association is (Arteaga et al. 2007). Moreover, it has been shown required for the assembly of an active initiation complex, that FOXO1 can be inactivated by the cyclin- responsible for FOXOs’ transcriptional function. dependent kinases CDK2 and CDK1 via increased Furthermore, CREBBP acetylates FOXO3a and simul- phosphorylation at Ser249 in prostate cancer cells taneously recruits nuclear proteins possessing deacety- (Huang et al. 2006, Liu et al.2008). lating functions (Giannakou & Partridge 2004). Thus, the ‘acetylation signature’ of FOXO3a can be modified by Activating phosphorylation of FOXOs deacetylating partners, enabling the fine-tuning of target gene expression (Greer & Brunet 2008). For instance, In response to oxidative and genomic damage, FOXO subsequent to H2O2 exposure, pre-acetylated FOXO3a is transcription factors are activated by the Ral/JNK and increasingly deacetylated by mammalian sirtuin proteins ERK pathways. This involves FOXO phosphorylation (SIRTs). This in turn triggers the FOXO-mediated at sites that are distinct from those targeted by expression of antioxidative target genes and inhibits PI3K/Akt and other mitogenic signalling cascades the expression of proapoptotic targets, e.g. Bim and (Asada et al. 2007). Stress-induced phosphorylation of FASL6 (Brunet et al.2004). Furthermore, it has been FOXOs results in a nuclear retention of FOXOs and shown that deacetylation of FOXO1 by SIRTs alters the activation of their transcriptional regulatory functions susceptibility of FOXOs to PI3K/Akt signalling in vitro (Vogt et al. 2005, van der Horst & Burgering 2007, by sensitizing FOXO1 for phosphorylation by Akt Huang & Tindall 2007). (Matsuzaki et al.2005a). For example, studies by Essers et al. (2004) provide However, future studies are needed to elucidate the evidence in murine cells, that H O -dependent acti- 2 2 importance of FOXO deacetylation as a prerequisite vation of JNK increases phosphorylation of FOXO4 on Thr447 and Thr451, resulting in nuclear accumu- for phosphorylation by Akt, and to decipher in which lation of FOXO4 and increased transcription of the way this is mandatory for PI3K/Akt-FOXO signalling. FOXO targets MnSOD and catalase. Additionally, the stress-induced protein kinase MST1 was shown to Ubiquitination activate FOXO3a in a phosphorylation-dependent Two functionally opposing types of FOXO ubiqui- manner. Using an in vitro model of mammalian tination have been discovered and involve polyubi- neurons, Lehtinen et al. (2006) found that MST1 quitination by SKP2 (Huang et al.2005)and phosphorylates FOXO3a upon oxidative stress on monoubiquitination by yet unidentified binding part- Ser207, thereby inhibiting the interaction between ners (van der Horst et al. 2004, Vogt et al. 2005). FOXO3a and 14-3-3 proteins. Huang et al. demonstrated in murine cells that Furthermore, Greer et al. (2007) demonstrated in overexpression of SKP2, an oncogenic subunit of the human kidney cells that glucose deprivation increases Skp1/Cul1/F-box protein ubiquitin complex, signi- the phosphorylation of FOXO3a on six specific ficantly decreased the cellular content of FOXO1. phosphorylation sites in an AMP kinase-dependent Thereby SKP2 induced the polyubiquitination of manner. When phosphorylated at these specific FOXO1, leading to its proteasomal degradation. Inter- residues, FOXO3a does not change its subcellular estingly, an Akt-mediated phosphorylation at FOXO1 distribution but changes the expression of its target site 256 is required for SKP2 action (Huang et al.2005, genes. For instance, AMP kinase-mediated phos- Arden 2006, Reagan-Shaw & Ahmad 2006). phorylation of FOXO3a significantly induced the By contrast, monoubiquitination of unphosphory- transcription of Gadd45a in mice. lated cytoplasmic FOXOs is thought to trigger the

Downloaded from Bioscientifica.com at 10/01/2021 04:22:14AM via free access www.endocrinology-journals.org 921 C Weidinger et al.: Function and regulation of FOXOs nuclear import and activation of FOXOs by the transcriptional activity of TCF/LEF and vice versa mechanisms that are still incompletely understood (Hoogeboom et al.2008). An imbalance consequential to (Arden 2006, van der Horst et al. 2006). oxidative stress tipping target gene expression towards the FOXO axis has been recently proposed, but further b-catenin binding studies are required to fully elucidate the physiological Recently, FOXOs have been reported to associate and pathophysiological consequences of a limited directly with cytoplasmatic b-catenin upon exposure to b-catenin pool for FOXO- and TCF/LEF-dependent oxidative stress in HEK239T cells (Essers et al.2004). target gene expression (Almeida et al.2007, Manolagas b-catenin is a prominent member of the canonical Wnt & Almeida 2007, Hoogeboom et al.2008). pathway and has been described to act as a coactivator of several transcription factors such as TCF/LEF, thereby promoting proliferation and dedifferentiation The role of FOXOs in cancer (Hoppler & Kavanagh 2007). In turn, binding of b-catenin to FOXOs was found to augment their The ability of FOXOs to control cell survival and cell transcriptional activity, resulting in the upregulation of death suggests that FOXOs may function as tumour anti-proliferative genes, such as p27kip1 and proapoptotic suppressors. Indeed, loss of FOXO function has been genes, such as Bim (Essers et al.2005, Almeida et al. observed in a number of human cancers and may 2007). Interestingly, there is evidence that the pool of represent a common feature of carcinogenesis. cytoplasmatic b-catenin is limited, resulting in a Generally, FOXOs can be deregulated through genetic competitive binding of FOXOs and other transcription defects and altered PTMs (Fig. 3). It is conceivable that factors (TCF/LEF) to b-catenin (Manolagas & Almeida each type of cancer may harbour an individual 2007). Thus, the binding of b-catenin to FOXOs signature of FOXO deregulation determining its augments their activation, while in parallel opposing unique phenotype.

Figure 3 Oncogenic signalling cascades and molecular events contributing to loss of FOXO function. FOXOs can be deregulated through genetic defects on the one hand and posttranslational modifications on the other hand. Thus, each type of cancer might harbour an individual signature of FOXO deregulation determining its unique phenotype. For example, inhibitory phosphorylation of FOXOs by mitogenic signalling cascades, e.g. PI3K/Akt, has been observed in various human cancers. Furthermore, genetic defects can disrupt FOXO function. In addition, proteasomal degradation has been shown to impair FOXO expression in human cancers.

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Genetic defects of FOXOs Generally, three different molecular mechanisms Several FOXO gene alterations have been identified in were found responsible for a constitutive activation of human cancers: for instance, a chromosomal fusion of the PI3K/Akt signalling and loss of FOXO function in C-terminal domain of FOXO1 and the N-terminal malignancies. domain of the transcription factors PAX3 or PAX7 was First, physiological inhibitors of the PI3K/Akt cascade found in human alveolar rhabdomyosarcoma (ARMS; such as the tumour suppressor phosphatase PTEN can be Bois & Grosveld 2003, Bois et al.2007). These chimera lost due to genetic mutations or decreased expression constitutively promote the transcription of PAX3 and leading to consecutive Akt activation. Consequently, PAX7 target genes respectively, which are associated FOXO1 and FOXO3a are phosphorylated and trans- with increased proliferation and cell motility (Kikuchi located into the cytosol (Modur et al.2002). Consistent et al.2008). In addition, the transcription of tumour with these findings, Emerling et al.(2008)reported an K/K suppressive FOXO target genes is thought to be impaired overactivation of PI3K/Akt signalling in PTEN (Arden 2006). In line with this concept, studies revealed LNCAP prostate cancer cells, resulting in the nuclear that PAX3 upregulation itself was not sufficient to induce exclusion and inhibition of FOXO3a activity. Using ARMS in mice, but required FOXO1 downregulation for murine astrocytes, de la Iglesia and coworkers revealed a malignant transformation (Anderson et al.2001). direct interrelationship between loss of PTEN- and Akt- Accordingly, Bois et al.(2005)demonstrated that loss mediated cytoplasmatic translocation of FOXO3a (De la of FOXO1 expression is a common feature of ARMS, Iglesias et al.2008). while re-expression of FOXO1 in ARMS-derived cells Secondly, some human tumours harbour transgenes, induced apoptosis and cell cycle arrest. such as RET/PTC, ZNF198-FGFR1 or BCR-ABL, that Moreover, Dong et al. (2006) have demonstrated confer constitutive activation of PI3K/Akt signalling. For that human prostate cancers harbour a loss of example, Jung et al.(2005)could demonstrate in human heterozygosity on 13q14, which is the embryonic kidney (HEK) cells that the overexpression of known gene locus of FOXO1. Thereby, loss of FOXO1 the RET/PTC chimera led to increased activation of Akt gene expression was directly correlated with increased signalling and subsequently inhibited the transcriptional cell proliferation in cancer cells. activity of FOXO1. Moreover, the ZNF198-FGFR1 Similar to PAX/FOXO1 transgenes, chromosomal fusion protein has been shown to stimulate the PI3K/Akt breakpoints occurring in human acute myeloid leukae- cascade in acute myeloid leukaemia and lymphoma, mia were found to involve FOXO3a and FOXO4. For resulting in FOXO3a Thr32 phosphorylation and a example the mixed lineage leukaemia gene (MLL) can 14-3-3 protein-dependent nuclear exclusion of fuse with FOXO3a and FOXO4, thereby putatively FOXO3a (Dong et al.2007). Likewise, in chronic disturbing their physiological tumour suppressive myeloid leukaemia (CML), the chimeric BCR-ABL function (Parry et al. 1994, So & Cleary 2003). fusion protein has been found to confer FOXO3a inactivation through Akt-mediated phosphorylation on the phosphorylation sites Thr32 and Ser253 Alteration of posttranslational FOXO protein (Birkenkamp et al.2007). modification Third, PI3K copy gains or overexpression of the As outlined above, FOXOs are modified by a multitude catalytic subunit p110a are frequently found in human of posttranslational events, which directly influence their malignancies (Kikuno et al. 2007) and are directly transcriptional activity and specificity. With regard to the associated with increased phosphorylation of FOXO1 importance of PTMs for FOXO activity, it can be and FOXO3a as shown in human colon cancers assumed that disruption of this sophisticated network (Samuels et al. 2005). results in loss of FOXO function and may trigger Furthermore, the astrocyte-elevated gene-1, AEG-1, carcinogenesis. has been shown to confer PI3K/Akt activation in several tumours. For instance, human prostate cancers PI3K/Akt pathway cells display elevated levels of AEG-1 resulting in Akt The PI3K/Akt signalling pathway has emerged as a activation and aberrant cell proliferation. Interestingly, central mitogenic pathway in various types of knock-down of AEG-1 in prostate cancer cell lines, mammalian cancer (Zbuk & Eng 2007) and there is such as LNCAP and PC-3, decreased the level of amble evidence for a direct correlation between phospho-Akt and simultaneously increased the fraction PI3K/Akt activation and FOXO inactivation in cancers of nuclear FOXO3a, as well as FOXO p27kip1 target (Kim et al. 2007, Yip et al. 2008). gene transcription (Kikuno et al. 2007).

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Ikkb/NF-kB pathway instance, the E3-ubiquitin ligase Skp2 has been found Besides the PI3K/Akt pathway, additional mitogenic overexpressed in lymphomas and breast, colorectal, cascades have been found to impair FOXO function in thyroid and lung cancers (Dehan & Pagano 2005). cancer. Inactivation of FOXOs due to overexpressed Recently, Goto et al. reported a correlation between Skp2 Ikkb was reported in breast cancers lacking activation expression and FOXO1 downregulation in endometrial- of the PI3K/Akt cascade. Hu et al. (2004) and Hu & derived cancer cells. Furthermore, the authors observed a Hung (2005) reported that the level of Ikkb/NF-kB decreased protein expression of FOXO1 (and Gadd45a activation was directly correlated with the amount of target gene expression) in the biopsies of human phosphorylated FOXO3a in the cytoplasm. Interest- endometrial cancers and suggested that Skp2 might ingly, Ikkb-mediated phosphorylation of FOXO3a did represent an important factor for tumorigenesis by not only induced cytosolic dislocation of FOXO3a, but accelerating the proteasomal degradation of FOXOs also triggered its proteasomal degradation in (Goto et al.2008). HEK293T cells (Hu et al. 2004). Perspective: FOXOs as drug targets for cancer RAS/ERK pathway treatment Furthermore, both the transcriptional activity and protein Recent approaches to suppress tumour growth by expression of FOXO3a have been shown to be impaired inducing cell cycle arrest and apoptosis in a FOXO- after ERK activation. Overexpression of ERK in the dependent manner have shown promising results human hepatoma cell line Hep-3BX significantly (Sunters et al.2003, Hussain et al.2006, Dong et al. reduced the nuclear content of FOXO3a, as well as its 2007, Jin et al.2007a,b). Interventions on different total cellular expression. ERK phosphorylates FOXO3a regulatory stages of FOXO function resulting in either on Ser294, Ser344 and Ser425, which resulted in a restoration or augmentation of FOXO function seem FOXO3a translocation into the cytosol and MDM2- especially useful for drug-targeting: mediated ubiquitination. When treated with the ERK First, disruption of constitutive PI3K/Akt signalling inhibitor UO126, the cells showed a restored expression by specific kinase inhibitors can save FOXOs from of FOXO3a and an increased transcription of the FOXO inactivating phosphorylation. The possibly best-esta- kip1 targets p27 and Bim. Moreover, induction of a blished example is the treatment of CML with the FOXO3a mutant harbouring constitutive phosphoryl- tyrosine kinase inhibitor STI571 (gleevec), which ation on ERK phosphorylation sites (i.e. causing FOXO inhibits PI3K/Akt and thus FOXO3a phosphorylation inactivation) in the human breast cancer cell line 435 led and restores its transcriptional activity as reflected by to increased cellular proliferation when compared with Bim upregulation (Fernandez et al. 2004, Essafi et al. the cells expressing the phosphorylation resistant 2005). Similarly, it was shown that treatment of human FOXO3a mutant. Transplantation of these cells into osteosarcoma cells with the PI3K/Akt inhibitor grifolin nude mice showed that the phosphorylated FOXO3a reduces the fraction of phosphorylated and inactivated protein did not suppress tumour growth, whereas the non- FOXOs, thereby increasing the rate of apoptosis phosphorylated FOXO3a protein inhibited uncontrolled (Alexia et al. 2006, Jin et al. 2007a). proliferation (Yang et al.2008). Secondly, inhibition of the nuclear export of FOXOs by inhibitors of the 14-3-3 protein family and related Skp2 polyubiquitination exportins, such as the peptide R18, can increase FOXO Increased proteasomal degradation of FOXOs may transcriptional activity. Using a model of human constitute another mechanism contributing to loss of leukaemia cells, Dong et al. (2007) demonstrated that FOXO function in cancers. In fact, the proteasomal treatment of cells with R18 downregulates the machineries run out of control in many malignancies. For expression of 14-3-3 proteins, which in turn leads to

Table 1 Forkhead box-O transcription factor (FOXO) activity can be increased by targeting distinct regulatory mechanisms

Inhibition of nuclear Disruption of persisting Induction of FOXO Influencing FOXO FOXO export FOXO inhibition expression acetylation

Competitive inhibition of Inhibition of PI3K (LY294002, grifolin) Paclitaxel Activation of SIRT1 (resveratrol) 14-3-3 proteins (R18) Inhibition of PI3K/AKT1 (wortmannin) Inhibition of SIRT1 (nicotinamid) Inhibition of tyrosine kinases (imatinib; mesylat)

Downloaded from Bioscientifica.com at 10/01/2021 04:22:14AM via free access 924 www.endocrinology-journals.org Endocrine-Related Cancer (2008) 15 917–929 increased nuclear accumulation of FOXO3a and References restored transcription of its anti-proliferative targets Adachi M, Osawa Y, Uchinami H, Kitamura T, Accili D & p27kip1 and Bim. Brenner DA 2007 The forkhead transcription factor Thirdly, inhibition of the proteasomal degradation of FoxO1 regulates proliferation and transdifferentiation of FOXOs could replenish the cellular content of FOXOs, hepatic stellate cells. Gastroenterology 132 1434–1446. and might thus represent an interesting mode to Adams JM & Cory S 2007 The Bcl-2 apoptotic switch in improve the chemo- and radiation-inducible rates of cancer development and therapy. Oncogene 26 apoptosis. However, so far there are no studies on this 1324–1337. topic available in the literature. Alcendor RR, Gao S, Zhai P, Zablocki D, Holle E, Yu X, Fourthly, induction of FOXO expression potentially Tian B, Wagner T, Vatner SF & Sadoshima J 2007 Sirt1 decreases cellular proliferation and dedifferentiation. regulates aging and resistance to oxidative stress in the Treatment of human breast cancer cells with paclitaxel heart. Circulation Research 100 1512–1521. has been shown to increase FOXO3a expression Alexia C, Bras M, Fallot G, Vadrot N, Daniel F, Lasfer M, 0 and transcription of the FOXO target gene Bim, Tamouza H & Groyer A 2006 Pleiotropic effects of PI-3 which confers apoptosis in breast cancer cells (Sunters kinase/Akt signaling in human hepatoma cell prolifer- et al. 2003). ation and drug-induced apoptosis. Annals of the New York Academy of Sciences 1090 1–17. 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By contrast, the 27298–27305. sirtuin inhibitor nicotinamid prevented SIRT1 binding Anderson MJ, Shelton GD, Cavenee WK & Arden KC 2001 (Frescas et al. 2005). Table 1 summarizes several Embryonic expression of the tumor-associated PAX3- putative strategies to augment FOXOs function as FKHR fusion protein interferes with the developmental tumour suppressor. functions of Pax3. PNAS 98 1589–1594. Arden KC 2006 Multiple roles of FOXO transcription factors In conclusion, FOXO transcription factors have in mammalian cells point to multiple roles in cancer. emerged as key regulators of cellular fate. Loss of Experimental Gerontology 41 709–717. FOXO function occurs at different levels of FOXO Arteaga MF, Alvarez DLR, Alvarez JA & Canessa CM 2007 regulation and is a novel molecular feature of cancer. Multiple translational isoforms give functional specificity In vitro and in vivo data suggest that restoration of FOXO to serum- and glucocorticoid-induced kinase 1. 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Hui RC, Holstege F, Koenderman L, Lam EW & Coffer PJ 2007 FOXO3a induces differentiation of Bcr-Abl- transformed cells through transcriptional down- regulation of Id1. Journal of Biological Chemistry 282 Funding 2211–2220. This work was supported by the DFG (FU 356/3-1) and IZKF Bois PR & Grosveld GC 2003 FKHR (FOXO1a) is required grants (B26) and by the Medical Faculty of the University of for myotube fusion of primary mouse myoblasts. EMBO Leipzig (F1 934000-363 and NBL-Formel. 1–100). Journal 22 1147–1157.

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