Oncogene (2008) 27, 2300–2311 & 2008 Nature Publishing Group All rights reserved 0950-9232/08 $30.00 www.nature.com/onc REVIEW Many forks in the path: cycling with FoxO

KK Ho, SS Myatt and EW-F Lam

Department of Oncology, Cancer Research UK Labs, Imperial College London, London, UK

FoxO transcription factors are an evolutionary conserved Despite the diversification of FoxO family, a evolu- subfamily of the forkhead transcription factors, charac- tionary conserved feature of FoxO transcription factors terized by the forkhead DNA-binding domain. FoxO is that they function as downstream effectors of the factors regulate a number of cellular processes involved in phosphoinositide 3-kinase (PI3K)– kinase B cell-fate decisions in a cell-type- and environment-specific (PKB also called Akt) pathway (the Age-1-Akt analo- manner, including metabolism, differentiation, apoptosis gues in C. elegans) (Burgering and Kops, 2002). The and proliferation. A key mechanism by which FoxO PI3K–PKB signalling cascade is a key pathway by determines cell fate is through regulation of the cell cycle which cells may respond to a wide range of stimuli, machinery, and as such the cellular consequence of FoxO which may be generated intrinsically (for example, deregulation is often manifested through perturbation of growth factors, cytokines and cell–cell contact) or from the cell cycle. Consequently, the deregulation of FoxO an external source (for example, irradiation, and factors is implicated in the development of numerous physical or genotoxic stress) (Leevers et al., 1999; proliferative diseases, in particular cancer. Hennessy et al., 2005; Wymann and Marone, 2005; Oncogene (2008) 27, 2300–2311; doi:10.1038/onc.2008.23 Samuels and Ericson, 2006). At the molecular level, many of the PI3K–PKB-mediated mitogenic responses Keywords: FoxO; cell cycle; cancer; senescence; are achieved through the direct repression of FoxO development; apoptosis transcription factors by PKB-mediated phosphoryla- tion, promoting nuclear export, proteosomal degrada- tion and a decrease in transactivation activity (Brunet et al., 1999, 2001; Kops et al., 2002b; Jacobs et al., 2003). FoxO is also a target of many other kinases, Introduction including serum and glucocorticoid-regulated kinase, c-Jun N-terminal kinase, a dual specificity tyrosine- The development of multicellular organisms demands phosphorylated and regulated kinase (DYRK1A), an increase in cell type and complexity, often leading to CDK2 and IkB kinase (Brunet et al., 1999, 2001; Kops the evolutionary expansion and diversification of et al., 2002b; Jacobs et al., 2003; Greer and Brunet, protein families. Since the founding member of the 2005) and the deregulation of PKB and these kinases is forkhead boxtranscription factor family, FoxA, was frequently observed in proliferative disorders and can cloned and characterized in the fruitfly Drosophila contribute significantly to tumorigenesis (Hennessy melanogaster (Weigel et al., 1989), at least 17 subclasses et al., 2005; Samuels and Ericson, 2006). The intimate of Foxtranscription factors have been identified in link between FoxO signalling and regulation of cell cycle humans. The FoxO family is symptomatic of the control is highlighted by the fact that in these instances increase in complexity required for the development of the re-introduction of FoxO expression, for example in higher organisms. Whereas only one FoxO analogue PTEN-deficient tumours, can induce cell cycle arrest (Daf-16) has been identified in , followed by cell death (Nakamura et al., 2000). four FoxO isoforms have so far been identified (FoxO1, This review will aim to describe both the mechanisms FoxO3a, FoxO4 and FoxO6) in mammalian cells (Greer by which FoxO regulate cell cycle progression and Brunet, 2005). Recent evidence would suggest that and effect checkpoint control, and how the role of these FoxO isoforms display both redundancy and these pathways in other physiological processes, such as compensation in vivo, while retaining isoform-specific senescence, cell differentiation and apoptosis, which roles in certain cell lineages, despite sharing common may play a role in cancer development and progression. consensus DNA-binding motifs and other functional Moreover, we will discuss the role of the cell cycle when domains. considering FoxO as a potential therapeutic target.

Correspondence: Professor EW-F Lam, Department of Oncology, FoxO transcription factors and cell cycle control Cancer Research UK Labs, MRC Cyclotron Building, Imperial College London, Hammersmith Hospital Campus, Du Cane Road, London W12 0NN, UK. Cell proliferation involves entry into the cell cycle from E-mail: [email protected] a resting or quiescent state () and successful FoxO and cell cycle control KK Ho et al 2301 progression through G1 (gap phase 1), S (DNA the first components of the cell cycle machinery to be synthesis), G2 (gap phase 2) and M (mitosis) of the cell induced in response to mitogenic stimulation, thus cycle. The phase transitions of the cell cycle are under providing a link between proliferative signals and the stringent control by a complexset of cell cycle cell cycle. These cyclin D-CDK4/6 holoenzymes are regulatory proteins, and progression through each phase believed to be crucial for driving cells through the of the cell cycle is controlled primarily by the (Matsushime et al., 1992; Bates et al., cooperative activity of specific cyclin-dependent kinases 1994; Meyerson and Harlow, 1994), a location at late (CDKs), and their regulatory subunits, the cyclins G1 phase after which cells often become independent of (Sherr, 1996; Sherr and Roberts, 1999). In fact, one of growth factors and refractory to inhibitory signals. the first functions discovered for mammalian FoxO Overexpression of the constitutively active nuclear form proteins was their ability to regulate the G1/S phase of FoxO1 or FoxO3a has been shown to repress CDK4 transition (Medema et al., 2000; Dijkers et al., 2000b) activity and induce cell cycle arrest at G1 phase (Figure 1). (Ramaswamy et al., 2002; Schmidt et al., 2002). Conversely, ectopic expression of cyclin D1 can partially overcome the FoxO-induced cell cycle arrest, implicat- FoxO and the G1/S phase transition ing the downregulation of D-type cyclins as a mechan- The progression of the cell cycle from G1 to S phase is ism of FoxO-induced cell cycle inhibition (Ramaswamy dependent primarily upon the activation of et al., 2002). At present, there is no evidence supporting transcription factors through the sequential hyperpho- FoxO proteins suppress D-type cyclin expression sphorylation of the retinoblastoma (pRB) family of through direct binding to their promoters; how- proteins (that is, pRB, p107 and p130) mediated by the ever, FoxO proteins can indirectly downregulate D-type cyclin-D/E-CDK complexes. During early G1 phase, the cyclins by upregulating the transcription repressor Bcl-6, expression levels of D-type cyclins are upregulated by a known transcriptional repressor of cyclin D2 gene mitogenic signals, leading to increased levels of active (Fernandez de Mattos et al., 2004). It is also worth D-type cyclin-CDK4/6 complexes (Sherr, 1995, 1996; mentioning that the PI3K–PKB signalling pathway has Sherr and Roberts, 1999). In fact, the D-type cyclins are been directly implicated in both cyclin D1 gene transcription and protein stabilization (Diehl et al., 1998; Muise-Helmericks et al., 1998; Gille and Down- ward, 1999). Thus, an elevated level of PI3K–PKB activity, coupled with the concomitant FoxO inactiva- tion will induce cyclin D1 at multiple levels, resulting in aberrantly high levels of cyclin D1-CDK4/6 activities and premature S phase entry. Nevertheless, current evidence would suggest that regulation of cyclin D expression is not the sole mechanism through which FoxO regulates G1/S phase transition and that regula- tion of G1/S phase transition by FoxO also relies heavily upon the cyclin-dependent kinase inhibitors (CKIs). Cyclin-dependent kinase inhibitors function through inhibiting the activity as well as the assembly of cyclin- CDK complexes. There are two distinct classes of CKIs, the Cip/Kip family (that is, p21Cip1, p27Kip1 and p57Kip2) and the INK4 family (that is, p15INK4b, p16INK4a, p18INK4c and p191NK4d). While the Cip/Kip family members form inhibitory heterotrimeric complexes with cyclin-CDK, the INK4 members complexonly with CDK4 and CDK6, and in doing so, excluding their cyclin D regulatory subunits (Sherr and Roberts, 1999, 2004; Sherr, 2001). FoxO proteins have been shown to directly upregulate the Cip/Kip members p21Cip1 and p27Kip1 transcription (Medema et al., 2000; Seoane et al., 2004; Gomis et al., 2006; Katayama et al., 2007), and thus may inhibit CDK-cyclin A, E and D activity. Hence, the rescue of FoxO-induced cell cycle arrest by the over- Figure 1 Cell cycle regulation through FoxO transcription factors. FoxO factors stabilize G1 and G2/M checkpoints by the expression of cyclin D may result from a titration effect activation (dotted lines) of a number of cell cycle regulatory upon the CKIs upregulated by FoxO, rather than proteins, including p130, members of the Cip/Kip and INK4 CKI rescuing the transcriptional downregulation of cyclin families, cyclin G and Gadd45a. In addition, FoxO factors can also D. Most recently, the two INK4 CDK4/6-specific facilitate cell cycle arrest through the inhibition (solid lines) of the INK4b INK4d D-type cyclins and FoxM, a subgroup of Fox inhibitors p15 and p19 have also been shown which is known to positively drive G2/M phase transition. Detailed to be FoxO targets in inducing G1 cell cycle arrest mechanisms are described in the main text. (Katayama et al., 2007). Consistent with this finding,

Oncogene FoxO and cell cycle control KK Ho et al 2302 inhibition of PKB signalling by PI3K inhibitors, a et al., 2001). If this were true, then FoxO expression PDK1 inhibitor or expression of a dominant-negative would appear to arrest cells at G1, while at the same PKB increased expression of p15INK4b and p19INK4d but time promoting cell cycle progression during G2 and M not p16INK4a and p18INK4c. Similarly, in p15INK4b/ or phases. However, subsequent microarray evidence has p19INK4d/ mouse embryo fibroblasts, treatment with the revealed that the overexpression of FoxO1 decreases the PI3K inhibitor LY294002 does not result in an increase expression of (for example, CDK2, cyclin B1, in cell population at G1 phase. cyclin B2, CDC2 and NEK2) essential for G2 and M The functional consequence of FoxO-mediated dis- transition, although the mode of action is yet to be fully ruption of cyclin-CDK complexformation is to decrease determined (Takano et al., 2007). Cyclin G2, an the phosphorylation of the pRB family proteins, and unconventional cyclin that facilitates G2/M arrest by hence E2F transcriptional activity. However, FoxO may inhibiting the cyclin B/CDC2(CDK1) complex, has also also directly target the pRB family members p107 and been identified as the direct target of FoxO proteins p130 (also called pRB2) (Bandara et al., 1994; Giacinti (Martinez-Gac et al., 2004). Moreover, in response to and Giordano, 2006; Macaluso et al., 2006); mammalian DNA damage, FoxO3a has also been shown to activate FoxO3a and FoxO4 have been shown to directly the expression of the growth arrest and DNA damage activate the transcription of the p130 gene, which can response gene GADD45a to mediate G2/M cell cycle induce cells to undergo cell cycle arrest and enter a arrest and trigger DNA repair (Tran et al., 2002). quiescent state (Kops et al., 2002b). Interestingly, some Recently, one of the pro-survival Bcl-2 family members, cell cycle regulators, including p107, pRB, -3, Bcl-XL, which is an important downstream target of cyclin D1, cyclin E1/2, cyclin A1/2, CDK2 and CDC2 FoxO (Tang et al., 2002), has been shown to colocalize are themselves transcriptional targets of E2F- and pRB- and bind to CDC2 during G2/M phase progression and related proteins (Dalton, 1992; Furukawa et al., 1994; its overexpression stabilizes a G2/M arrest senescence Shan et al., 1994; Ohtani et al., 1995; Schulze et al., programme in surviving cells after DNA damage 1995; Sears et al., 1997; Araki et al., 2003; Cobrinik, (Schmitt et al., 2007). Thus, these data would suggest 2005). During cell cycle entry, the cyclin D-CDK4/6 that FoxO can directly regulate a series of cell cycle kinase has been shown to trigger pRB protein partial proteins, and depending on stimuli, control both G1/S phosphorylation, resulting in the derepression of a small and G2/M phase progression. However, while it is number of E2F genes, including cyclin E. Expression of evident that FoxO activation can directly induce cyclin E then activates CDK2 to further phosphorylate multiple changes in cell cycle regulatory factors, the pRB proteins, leading to the expression of the full evidence is also emerging of a complextranscriptional array of E2F-regulated cell cycle regulatory genes. network in which the regulation of cell cycle progression Therefore, FoxO-mediated D-type cyclin repression, by FoxO requires the direct binding to other transcrip- CKI and p130 activation will ultimately lead to the tional factors and/or cofactors, in a process that may be downregulation of a whole host of E2F-regulated cell disrupted during tumorigenesis and contribute to cycle regulators essential for cell cycle entry. It is disease development. therefore evident that FoxO activation can directly induce multiple changes in factors associated with G1/S FoxO transcriptional networks and cell cycle phase transition, through downregulating activating The transforming growth factor b (TGFb) superfamily factors and upregulating repressors, cumulating in the of cytokines regulates a wide variety of cellular inhibition of E2F transcriptional activity. functions, including cell cycle progression, and dereg- ulation of this pathway is associated with tumorigenesis. FoxO and G2/M phase transition For example, TGFb has been reported to inhibit the During G1/S phase transition, cells pass through the proliferation of primary malignant epithelial cells, by restriction point and become committed to cell cycle inducing cell cycle arrest through activation of the Smad progression, and often independent of growth factors group of transcription factors, which are frequently and refractory to inhibitory signal. However, cell cycle mutated in tumours (Miyaki and Kuroki, 2003). arrest at the G2/M checkpoint is a critical component of Evidence would suggest that a key component of TGFb stress response, allowing DNA damage repair (Kauf- signalling is the formation of a Smad-FoxO3a complex, mann, 1995; Molinari, 2000; Stark and Taylor, 2006). which is required to induce the expression of the p21Cip1 Moreover, deficiencies in the G2/M phase checkpoint gene (Seoane et al., 2004). In addition, the p15INK4b gene control are often associated with genomic instability and has also been demonstrated to be target of the FoxO- tumorigenesis (Lobrich and Jeggo, 2007). FoxO trans- Smad complex, in a mechanism dependent upon the cription factors have also been reported to have a role in CCAAT/enhancer-binding protein b (C/EBPb) tran- the late phases of the mammalian cell cycle. Activation scription factor (Gomis et al., 2006). The regulation of of FoxO3a during S phase transition will induce a G2/M the FoxO-Smad complex by C/EBPb may be disrupted cell cycle arrest, although this effect may not be during tumorigenesis; the cytostatic effects of TGFb are mutually exclusive with the regulation of G1/S phase lost in half of the metastatic breast cancer patient cells as transition by FoxO. In contrast, a prior report has a result of the expression of high levels of the C/EBPb demonstrated that FoxO proteins activate the expres- inhibitory isoform LIP. In addition to regulating G1/S sion of genes, such as cyclin B and polo-like kinase, phase transition, FoxO-Smad complexes have also been important for G2 and M phase progression (Alvarez implicated in regulating the G2/M checkpoint by acting

Oncogene FoxO and cell cycle control KK Ho et al 2303 as co-regulators of the stress response genes in tissue, evidence suggests that FoxM1 and FoxO3a keratinocytes, including GADD45a and GADD45b, may cooperate to regulate ERa gene transcription which have a role in G2/M cell cycle control. Thus, (Madureira et al., 2006). FoxO represents a point of convergence of the PI3K– PKB pathway with the TGFb signalling cascade to regulate cell cycle progression. Interestingly, the induc- FoxO, cell cycle, senescence and lifespan tion of p21Cip1 expression by the FoxO-Smad complexes is antagonized by another forkhead subfamily member FoxO- FoxG1 during the development of the telencephalic neuroepithelium and in glioblastoma brain tumour cells, Cellular or replicative senescence is believed to be an suggesting that the Foxfamilies may interact to important mechanism of tumour suppression, and FoxO determine cell fate (Seoane et al., 2004). Moreover, the has a crucial role in regulating this process by tissue-specific expression pattern of FoxG1, primarily controlling the expression of a number of cell cycle being found in brain tissue, points to a scenario whereby regulators. PI3K inhibition using chemical inhibitors, the cell lineage influences not only the outcome but also such as wortmannin or LY294002, can induce a cell the regulation of FoxO signalling. cycle arrest similar to cellular senescence in primary et al In fact, it is becoming apparent that FoxO may mouse embryo fibroblasts (Collado ., 2000), Kip1 expression. Moreover, interact with other Foxproteins to influence cell cycle. through FoxO-induced p27 overexpression of FoxO or p27Kip1 in primary mouse For example, a recent transcriptional profiling study embryo fibroblasts can recapitulate this phenotype, identified FoxM1 to be repressed by FoxO3a (Delpuech promoting premature cell cycle arrest, changes in cell et al., 2007) and it was shown that the FoxM1 gene morphology, increases in senescence-associated b-galac- expression can be negatively regulated by a FoxO3a tosidase activity and decreased ability to re-enter cell target gene Mxi1 (Delpuech et al., 2007). FoxM1 has a critical function in G2 and M transition by regulating cycle in response to mitogenic stimulation. FoxO also targets p130, but does so primarily in the absence of the expression of various G2/M cell cycle regulatory p27Kip1, through a compensatory mechanism by which genes, including polo-like kinase, cyclin B, cyclin A, Kip1 CDC25B and CDC2 (Wang et al., 2002a, b; Yoshida p130 can functionally substitute for the loss of p27 (Collado et al., 2000). The ability of FoxO to induce et al., 2007), and loss of FoxM is associated with G2/M G0/G1 arrest is lessened in p27Kip1 and p130 double- checkpoint arrest and loss of mitotic spindle integrity deficient fibroblasts (Kops et al., 2002b), suggesting that (Laoukili et al., 2005; Wonsey and Follettie, 2005). both p27Kip1 and p130 are important for mediating Overexpression of FoxM1 through chromosomal FoxO-dependent, cellular senescence-associated G0/G1 amplification or the activation of transcription, for arrest. Further evidence for a role of FoxO in cellular example, through Gli-1 is associated with the develop- in vivo ment and progression of many cancer types, including senescence is supported by a recent study by Courtois-Cox et al. (2006), demonstrating that onco- cancers of the breast, liver, prostate, brain and lung (Teh gene-induced senescence also involves the repression of et al., 2002; Kalinichenko et al., 2004; Kalin et al., 2006; Kim et al., 2006; Liu et al., 2006; Madureira et al., 2006). the PI3K–PKB signalling pathway and induction of FoxO. In the report, the authors provided experimental Other mechanisms of interaction may exist between evidence that oncogene-induced senescence occurs FoxM1 and FoxO3a. For instance, the inhibition of in vivo using a ras oncogene hyperactivation system FoxO may also be associated with the activation of triggered by NF1 functional deletion and showed that FoxM1; DYRK1 inhibits FoxO (Woods et al., 2001), senescent population of cells can be detected in vivo in while also activating Gli-1, and thereby promoting benign and not advanced tumours, supporting a genuine FoxM1 expression. In addition, FoxO3a-induced role of cellular senescence in tumour suppression, expression of CKIs may inhibit CDK-cyclin complexes, especially during early carcinogenesis and hyperplasia. which are required for the activation and nuclear This oncogene-induced cellular senescence has also been localization of FoxM1 (Wang et al., 2002a, b, 2005; Luscher-Firzlaff et al., 2006; Wierstra and Alves, 2006). shown to be mediated through the repression of the PI3K–PKB signalling pathway. Moreover, the expres- However, while induction of FoxO may repress FoxM1 sion of a constitutively activated FoxO1 mutant, which expression, the significance of this repression for the FoxO-induced cell cycle arrest and/or apoptosis remains cannot be inactivated by PKB, rapidly triggered cellular senescence associated with the hypophosphorylation to be determined. Moreover, current evidence would and thus activation of the suggest that the regulation of CKIs and pro-apoptotic pRB, a hallmark for G0/G1 cell cycle arrest. These genes by FoxO are primarily responsible for these cell- discoveries highlight the pivotal role of FoxO-mediated fate outcomes. However, the interaction between FoxM cell cycle arrest in cellular senescence and tumour and FoxO remains an intriguing prospect, and the suppression. equilibrium between proliferation and cell cycle arrest may be maintained in part by cooperation and interac- tion of these factors. For example, in addition to FoxO- FoxO-stem cell renewal binding oestrogen in breast cancer cells (Schuur In addition to being a potent tumour suppressor et al., 2001; Zhao et al., 2001), which is critical in mechanism, cellular senescence also functions to control regulating the proliferation and differentiation of breast the proliferative competence of stem cell and progenitor

Oncogene FoxO and cell cycle control KK Ho et al 2304 pools. The cell cycle has an essential function in opment is probably due to the fact that FoxO regulates controlling self-renewal during stem cell development, genes controlling early cell cycle phases when the and this is particularly evident in the stem cells of the decisions to proliferate and differentiate are made, haematopoietic system, a tissue with high volumes of reflecting the particular importance of the G1 check- cell turnover. An important feature of haematopoietic point in regulating the high volumes of cell turnover stem cells (HSCs) is their ability to maintain a resting, or observed in the haematopoietic system. quiescent state (G0), only entering the cell cycle after long intervals, and thereby preventing proliferative exhaustion (Myatt and Lam, 2007a). Recent evidence FoxO-organismal senescence and longevity suggests that FoxO family members play a critical role FoxO proteins also have a central role in regulating in maintenance of the HSC pool; simultaneous deletion organismal (senescence), but the outcomes of of three FoxO isoforms in the HSCs of mice led to a FoxO activation appear to be different from those for significant rise in the numbers of the mature peripheral cellular senescence. In multicellular organisms, FoxO blood cells, and an expansion of haematopoietic cells of proteins function to induce cell cycle exit, and thereby myeloid, lymphoid and erythroid origins (Tothova et al., counteracting senescence and promoting longevity. For 2007). Conversely, the bone marrow of FoxO-null mice example, in C. elegans, dauer is the long-lived species possessed a smaller HSC pool, which is required for compared with larva, and its formation is induced by sustaining the haematopoietic system. This decrease in food deprivation or stress. Dauer formation requires the HSCs is associated with a reduced ability to repopulate activation of the FoxO homologue Daf-16 to induce recipient bone marrow after serial transplantations and G0/G1 cell cycle arrest through the induction of the a reciprocal increase in the number of more differen- nematode Cip/Kip inhibitor, Cki-1 (Boxem and van den tiated haematopoietic populations. This was paralleled Heuvel, 2001). Expression of Cki-1 can induce prema- by an increased exit from quiescence and a higher rate of turely cell cycle arrest in G1, whereas knocking down of cell proliferation. These observations are consistent with Cki-1 activity by RNA interference promotes S phase previous in vitro studies showing that FoxO activation entry (Boxem and van den Heuvel, 2001). It is also can culminate in a G0/G1 cell cycle arrest mediated by worth mentioning that this role of FoxO is also transcriptional regulation of p21Cip1, p27Kip1, cyclin D conserved in the fruitfly Drosophila (Kramer et al., and p130. Taken together, these results indicate that the 2003). When nutrients are limited, the insulin–insulin- ability of FoxO to induce cell arrest in HSC is linked to like growth factor (IGF) signalling pathway is repressed an ability to preserve self-renewal potential, preventing and the activated dFoxO promotes G1 cell cycle premature ageing and excess differentiation of HSCs. It arrest but lengthens lifespan (Giannakou et al., 2004). is generally believed that CKIs, such as p21Cip1 and Consistently, expression of dFoxO during early larval p27Kip1, set the stoichiometric thresholds for cell cycle development causes inhibition of larval growth entry and progression through G1 in response to (Giannakou et al., 2004). Thus, in terms of longevity mitogenic stimuli, such as cytokines and interactions of a eukaryotic multicellular organism, FoxO causes cell with their microenvironments. Experimental evidence cycle arrest, but at the same time increases the lifespan. from p21Cip1- and p27Kip1-deficient mice suggests that Recent data identifying Daf-16-regulated genes in these CKIs have specific roles in the regulation of C. elegans support this notion; 29 Daf-16/FoxO- quiescence of HSCs and progenitors, respectively. For regulated genes were identified that act in the insulin– instance, p21Cip1-deficient mice exhibit an increase in IGF-1 pathway to influence tumour growth, and are HSC cell cycle entry and exhaustion upon transplanta- likely to act in a cumulative manner to influence tumour tion, suggesting a role for p21Cip1 in HSC quiescence growth (Pinkston-Gosse and Kenyon, 2007). Interest- (Cheng et al., 2000b). In contrast, p27Kip1 knockout mice ingly, a large proportion of genes that affect tumour display increased progenitor, but not HSC, populations growth also affect the normal lifespan of C. elegans. (Cheng et al., 2000a). Indeed, evidence suggests that Perhaps the most intriguing question arises from the p21Cip1 and p27Kip1 are critical regulators of the increased parallels between and FoxO (van der Horst and HSC-specific entry into the cell cycle observed in the Burgering, 2007). In the above study, inhibiting the FoxO isoform-deficient mice. Although p130 deletion upregulation of nuclear pore genes by Daf-16 blocked alone has little effect on HSC and more mature p53-dependent, but not independent, cell death progenitor development, in p27Kip1 and p130 double- (Pinkston-Gosse and Kenyon, 2007). In contrast to the deficient mice there is an increased in cellularity of current understanding of the role of FoxO in C. elegans, the more mature lymphoid, erythroid and myeloid activation of p53 reduces lifespan in mouse models, but haematopoietic cells (Soeiro et al., 2006). Similar decreases cancer incidence, supporting a system of haematopoietic-related defects are detected for other antagonistic pleiotropy (van der Horst and Burgering, FoxO-regulated cell cycle regulatory genes, including 2007). Interestingly, both FoxO3a and p53 are regulated cyclin Ds and Ink4s (Myatt and Lam, 2007a). For by a similar profile of post-translational modifications example, mouse embryos that lack all three D-type and influence cell cycle through the regulation of cyclins, or both CDK4 and CDK6, die of haematopoie- GADD45 and p21Cip1. Thus, p53 and FoxO may tic abnormalities after day E14.5 (Myatt and Lam, influence tumour suppression and longevity, and a 2007a). In summary, the finding that FoxO deletion balance in activity may be controlled in part by a specifically affects HSC renewal and progenitor devel- negative feedback loop between the two proteins.

Oncogene FoxO and cell cycle control KK Ho et al 2305 Indeed, p53 may induce the expression of serum and are defective in ovarian follicular development, char- glucocorticoid-regulated kinase, resulting in the phos- acterized by age-dependent infertility, and female phorylation and nuclear exclusion of FoxO3a (Maiyar mice lacking cyclin D2, one of the critical downstream et al., 1996), and FoxO3a may inhibit p53-mediated targets of FoxO3a, are also sterile because of the repression of SIRT1 expression, which binds to, and inability of ovarian granulosa cells to proliferate deacetylates p53. (Campisi, 2005; van der Horst and normally in response to follicle-stimulating hormone Burgering, 2007). Thus, the regulation of the cell cycle (Sicinski et al., 1996). In primary rat ovarian granulosa by FoxO3a may play an important role in longevity and cells, cyclin D2 expression has been shown to be tumour suppression, in an intriguing and complex specifically induced by follicle-stimulating hormone via manner, which demands further investigation. FoxO at transcriptional level (Park et al., 2005). Taken together, these results indicate FoxO3a has a unique role in ovarian development, through modulating the FoxO, cell cycle arrest and cell-fate decisions expression of cyclin D2, and thus controlling cell cycle progression in the granulosa cells. FoxO-cell cycle and differentiation It is also becoming apparent that disruption of cell As well as playing an active role in the G1 checkpoint of differentiation through FoxO3a deregulation has a the cell cycle, CKIs are also important regulators in critical role in the development of certain cancers. mediating cell cycle arrest that precedes differentiation Expression of the oncogenic fusion protein Bcr-Abl, and apoptosis. Cumulative data from mouse and which is associated with the development of chronic nematode studies point to a critical role of FoxO3a in myeloid leukaemia, downregulates FoxO activity differentiation, primarily through modulation of the cell through Bcr-Abl-induced PI3K–PKB hyperactivation. cycle machinery, and a key feature of both is the Besides uncontrolled proliferation, another defining regulation of FoxO3a by pro-survival and nutrient characteristic of myeloid leukaemic cells is that they responsive pathways, respectively. are trapped in an undifferentiated state. Overexpression After hatching, C. elegans only commence development of FoxO3a alone induces erythroid differentiation in the presence of nutrients, and in a nutrient-deprived in leukaemic cells and this is in turn antagonized by environment the larvae remain in a developmentally ectopic expression of Id1 (inhibitor of differentiation 1), arrested state (L1 arrest). Critically, mutants lacking suggesting that FoxO3a promotes haematopoietic Daf-16, the C. elegans homologue of the mammalian differentiation by targeting Id1. Indeed, in vivo Id1 gene FoxO transcription factors, fail to arrest cell cycle and promoter-binding assay showed that FoxO3a suppresses development leading to rapid lethality in the absence of Id1 through direct binding to its promoter (Birkenkamp nutrients. The failure of Daf-16 mutants to arrest at L1 et al., 2007). Interestingly, the FoxO3a–Id1 promoter is at least partly due to their failure to activate the interaction is dependent on the consensus forkhead transcription of the nematode Kip/Cip CKI, Cki-1, in DNA recognition element. How this interaction results the larval stem cells in response to starvation (Baugh in Id1 repression is not fully characterized, but it has and Sternberg, 2006). In mammals, the PI3K–PKB been proposed that this repression is achieved through cascade is the major pro-survival nutrient responsive the recruitment of the histone deacetylase 1/mSin3a pathway, which has been demonstrated to be a critical complex(PJ Coffer and EW Lam, unpublished work). regulator of b-cell mass in the pancreas (Holz and In line with this positive role in the promotion of Chepurny, 2005), and mice lacking functional FoxO1 in haematopoietic differentiation, FoxO3a has also been the pancreas are diabetic and have a lower pancreatic shown to induce erythroid differentiation and cellular islet b-cell mass (Martinez et al., 2006). In a manner senescence through Id1 (Birkenkamp et al., 2007). reminiscent of nematode developmental defects, the Current evidence suggests that Id proteins also function disruption of FoxO signalling manifests primarily downstream of c-, and promote cell cycle progres- through cell cycle deregulation. Previous studies on sion through upregulation of cyclin D1 expression and D-type cyclin-deficient mice have shown that cyclin D2, repression of p16INK4a expression, culminating in the and to a lesser extent cyclin D1, is required for adult inactivation of the p16INK4a–pRB tumour suppressor b-cell proliferation (Kushner et al., 2005). Furthermore, pathway (Swarbrick et al., 2005). Thus, these findings overexpression of a constitutively active PKB, the direct establish FoxO proteins as key transcriptional regula- negative regulator of FoxO, in b-cells leads to cell tors of the PI3K–PKB pathway that control the cell proliferation, which is associated with increased cyclin cycle, which in turn modulates cell proliferation and D1 and D2 levels and CDK4 activity. Thus, these development. Similarly, transgenic and gene deletion findings again provide evidence to suggest that in adult studies have also shown that FoxO1 plays a crucial role mammals and nematode larvae, disruption of FoxO in liver fibrosis, a process that causes hepatic stellate leads to developmental defects associated with disrup- cells to transdifferentiate from a quiescent phenotype to tion of cell cycle through regulation of the CKIs and a proliferating collagen-producing myofibroblast-like cyclin D family members, respectively. Indeed, gene phenotype (Adachi et al., 2007). Constitutively active knockout studies in mice have also revealed that the FoxO1 has been demonstrated to inhibit hepatic stellate cyclin D gene family plays a central role in the regulation cell proliferation, via cell cycle arrest at the G1 phase, of the interplay between cell cycle and development by while dominant-negative FoxO1 promotes proliferation FoxO3a. For example, FoxO3a-deficient female mice of hepatic stellate cells, even in the presence of the PI3K

Oncogene FoxO and cell cycle control KK Ho et al 2306 inhibitor LY294002. Furthermore, FoxO1 þ / mice have survival by inducing cell cycle arrest and quiescence in been found to be more susceptible to liver fibrosis response to oxidative stress (Medema et al., 2000; Kops compared with their wild-type counterparts and may be et al., 2002a, b; Brunet et al., 2004; Essers et al., 2004). associated with the induction of p27Kip1 by FoxO1. It is At the same time, the CDK inhibitor p27Kip1, the interesting to note that FoxM1 is also a critical regulator prominent downstream target of FoxO, which blocks of hepatic cell fate, and that while FoxO3a promotes progression of cells from late G1 to S phase, has also differentiation and growth arrest, FoxM1 is required been implicated directly in the induction of apoptosis by for hepatic cell proliferation and liver regeneration FoxO (Wang et al., 1997; Wu et al., 1999; Dijkers et al., (Krupczak-Hollis et al., 2003; Wang et al., 2003). Thus, 2000b). These observations illustrate the complexity of whether FoxO1 may regulate FoxM1 expression in the interplay among cell cycle checkpoints, apoptosis hepatic cells, in a manner similar to the regulation of and senescence (Figure 2). It is most likely that the FoxM1 by FoxO3a (Delpuech et al., 2007), is an physiological response triggered by FoxO transcription interesting and as yet unexplored proposition. factors is often determined by the cellular context as well as the cofactors involved.

FoxO-cell cycle and apoptosis Cell cycle checkpoints are activated in response to genotoxic stress induced by most chemotherapeutic Targeting FoxO as a therapeutic strategy––a role for cell drugs (Lukas et al., 2004; Bartkova et al., 2005). Thus, cycle regulation in addition to preceding differentiation, cell cycle arrest may also be induced by genotoxic stress and lead to A common feature of tumour development is the apoptosis. Indeed, most chemotherapeutic drugs are deregulation of cell cycle, leading to an uncoupling of reported to arrest or delay cell cycle progression, and if the cell cycle machinery to apoptosis and differentiation. the DNA damage or other cellular defects are extensive, The critical role of FoxO proteins in cell cycle regulation the cell will undergo either apoptosis or cellular and cell-fate determination makes these transcription senescence (Lukas et al., 2004; Bartkova et al., 2005). factors an attractive target for therapeutic intervention. Moreover, cancer cells may be considered to display an Indeed, recent evidence indicates that FoxO transcrip- enhanced or higher level of basal stress, such as tion factors play a key role in mediating the cytostatic increased levels of reactive oxygen species or stress and cytotoxic effects of various chemotherapeutic drugs. kinase activity, and it is precisely the deregulation of cell For example, paclitaxel (Taxol), an anticancer drug that cycle checkpoints that allow cancer cells to tolerate such targets the spindle checkpoint, activates FoxO3a in cellular conditions, while simultaneously promoting breast cancer cells to induce G2/M arrest and apoptosis genomic instability and tumour progression (Giles, (Sunters et al., 2003, 2006). Moreover, in paclitaxel- 2006; Wu, 2006; Fruehauf and Meyskens, 2007). One resistant breast cancer cells the G2/M cell cycle such mechanism by which cancer cells can tolerate checkpoint and apoptosis are uncoupled in the resistant cellular stress is the deactivation of FoxO3a, which cells, possibly through a loss of FoxO3a activation would be anticipated to be activated by oxidative stress and stress kinases, both of which may converge through c-Jun N-terminal kinase-mediated phosphorylation of FoxO3a (Essers et al., 2004, 2005; Vogt et al., 2005; Huang and Tindall, 2007). Thus, it is not perhaps surprising that the re-introduction of FoxO3a can induce tumour cell kill in cancer cells that have repressed Self renewal FoxO3a activity (Modur et al., 2002), and also that Cell division activation of FoxO transcription factors has a role in mediating the cytostatic and cytotoxic functions of Cell cycle chemotherapeutic drugs in some cancer types. However, Arrest the cell cycle arrest induced by FoxO can have an influence on the eventual outcome in terms of cell fate and may also promote DNA damage repair and stress resistance (Tran et al., 2002). For example, it has been Development demonstrated that abolishing the Apoptosis can augment the efficacy of some of the anticancer chemotherapeutic drugs and increase the level of cell death (Teyssier et al., 1999; Kokkinakis et al., 2006). In a similar manner, treatment of cells with radiation in Differentiation Senescence combination with caffeine, which activates CDC2 through the CDC25C phosphatase, results in ablation Figure 2 Cell fate coupled to FoxO-mediated cell cycle arrest. Alteration in the activity or expression of FoxO can promote many of G2/M checkpoint and increased apoptosis (Russell cell-fate outcomes that are frequently associated with changes in et al., 1995; Yao et al., 1996). FoxO family members the cell cycle. A number of cell fates regulated by changes in FoxO have also been shown to promote mammalian cell activity and concomitant cell cycle arrest are shown.

Oncogene FoxO and cell cycle control KK Ho et al 2307 (Sunters et al., 2003). Thus, in this instance, the the promoter of AR, while promoting binding of regulation of the G2/M checkpoint, and subsequent FoxO3a to the p27Kip1 promoter, thus changing andro- apoptosis, by FoxO3 is a critical component of gen receptor and p27Kip1 expression, leading to the paclitaxel-induced cell death. inhibition of cell proliferation and the induction of Regulation of the G2/M checkpoint by FoxO1 also apoptosis. A mechanism by which FoxO3a has been plays a critical role in the response of cancer cells to the reported to differentially affect target is DNA damage-inducing topoisomerase I inhibitor agent through the recruitment of histone deacetylase to the irinotecan, CPT-11, which has been reported to induce FoxO3a-p300/CBP complex, an event which has been G2/M cell cycle arrest, followed by senescence or hypothesized to differentially affect FoxO3a target apoptosis in colon cells (Bhonde et al., gene expression (Brunet et al., 2004; van der Heide 2006a, b). In response to CPT-11, FoxO1 promotes and Smidt, 2005). Sirtuins, the nicotinamide adenine G2/M cell cycle arrest and apoptosis independently of dinucleotide þ -dependent protein deacetylases, have p53, and FoxO1 silencing diminishes CPT-11-induced been proposed to repress apoptosis and counteract cell cycle arrest and apoptosis (Huang et al., 2006). oxidative and genotoxic stresses via FoxO (Brunet et al., According to a recent report, FoxO may facilitate the 2004; Kobayashi et al., 2005). Moreover, inhibition of cytostatic effect of CPT-11 via a mechanism involving HDACs has been reported to induce growth arrest, the pro-survival protein Bcl-XL. Besides the conven- activation of apoptotic pathways, autophagic cell death tional antiapoptotic function, Bcl-XL has recently been and senescence in transformed cells (Xu et al., 2007). shown to stabilize G2/M cell cycle arrest through direct This raises the possibility that inhibition of sirtuin interaction with CDC2 (Schmitt et al., 2007). Analogous activity may also sensitize cancer cells to chemothera- to cyclin D2, FoxO-induced Bcl-6 activation represses peutic drugs through favouring apoptosis over stress Bcl-XL at the transcriptional level (Tang et al., 2002). resistance following cell cycle arrest induced by FoxO The activation of FoxO1 by CPT-11 is impeded by activation (Lam et al., 2006; Myatt and Lam, 2007b). In CDK2 which phosphorylates FoxO1, causing the summary, the importance of FoxO proteins in cell cycle nuclear exclusion of the transcription factor (Huang checkpoint regulation, and subsequent cell-fate determi- et al., 2006). As CDK2 is a critical component in S phase nation, supports the applicability of employing FoxO cell cycle entry, its direct role in antagonizing the regulation as a future target for therapeutic intervention cytostatic effect of CPT-11 indicates that FoxO is an in cancer. integration point between cell cycle checkpoint and apoptosis. The above examples illustrate that loss of FoxO activity may result in a reduction of sensitivity of cancer Conclusions cells to cytotoxic drugs through a loss of cell cycle regulation (Sunters et al., 2003, 2006). In a number of FoxO proteins play a central role in controlling cell fate circumstances, cancer cells may be sensitized to chemo- (Greer and Brunet, 2005; Lam et al., 2006). Multiple and therapeutic drugs by the reintroduction or reactivation divergent signalling pathways converge to influence of FoxO to restore cell cycle control. For example, FoxO activity, the net outcome of which leads to FoxO3a has been identified as a critical cellular target of changes in the expression of genes important for cell- gefitinib (Iressa), a small molecule inhibitor of epidermal fate determination, which is rarely observed without growth factor receptor, which causes growth delay in accompanying FoxO-induced changes in the cell cycle cancer cell lines and human tumour xenografts expres- sing high levels of epidermal growth factor receptor (Krol et al., 2007). In resistant breast cancer cells, Senescence reintroduction of active FoxO3a can partially restore Differentiation cell proliferative arrest and sensitivity to gefitinib. In line Apoptosis with this observation, it has also been shown that the Quiescence introduction of an active form of FoxO4 sensitizes HER2-overexpressing cells to apoptosis induced by the chemotherapeutic agent 2-methoxyestradiol, and it is Kip1 believed that the restoration of FoxO4-induced p27 FoxO is at least in part responsible for such observation (Yang et al., 2005). An emerging theme is that the consequences of Apoptosis FoxO3a activation depends on cellular environment and can lead to a switch in FoxO3a-mediated gene expression profile. The antiproliferative and pro- Senescence apoptotic compound 3,30-diindolylmethane has been Figure 3 Cell-fate decisions tied to specific cell cycle phases. The reported to regulate PKB and in induction of certain cell fate by FoxO is frequently coupled to exit from specific phases of the cell cycle. Cell fates and associated prostate cancer cells. Interestingly, treatment of prostate phases are shown. Perturbation of these pathways is often involved 0 cancer cell lines with 3,3 -diindolylmethane inhibits in hyperproliferative disorders, including cancer, as well as FoxO3a phosphorylation and binding of FoxO3a to promoting resistance to cellular stress or apoptosis.

Oncogene FoxO and cell cycle control KK Ho et al 2308 (Figure 3). Indeed, cell proliferation, survival, differ- FoxO in these processes has been highlighted by the entiation, tissue and organ development, cell renewal, observation that in both animal and cellular experi- cellular senescence, organism ageing, metastasis and mental models depletion of FoxO proteins, or their stress responses are all tied to FoxO-dependent downstream cell cycle targets, including p27Kip1,p21Cip1 changes in the cell cycle machinery. As such, while and p130 can deregulate cell-fate programmes. The FoxO may directly regulate genes important in these realization of FoxO as a critical regulator of cell-fate processes, such as the pro-apoptotic gene Bim in determination has paved a new road for future apoptosis (Dijkers et al., 2000a; Essafi et al., 2005), investigation in this field and we believe the next modulation of the cell cycle often provides a permissive challenge will be defining the mechanism by which cellular environment through which cell fate is FoxO proteins decipher upstream signals to influence specified. For example, in the absence of growth or cell-fate outcome. In many circumstances, the physio- survival stimuli, cells commonly arrest at G1 before logical responses coupled to cell cycle arrest triggered by withdrawing from the cell cycle (quiescence), and G1 FoxO transcription factors are likely to be determined arrest is similarly associated with development, cells by the cellular context, the developmental status, the undergoing apoptosis, cell differentiation or cellular intensity or duration of the signal and the cofactors senescence. In response to stress, arrest at the G1 or involved. Undoubtedly, advances in the coming G2/M checkpoint may allow for cell detoxification years should greatly aid the quest of understanding and/or repair preceding cell cycle re-entry. In fact, these exciting signalling pathways and their clinical the absolute dependence of the cell cycle functions of implications.

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