Foxo in the Immune System

SL Peng

Clinical Research and Exploratory Development, Roche Palo Alto, Palo Alto, CA, USA

In addition to their key roles in cellular survival, death, GTPase (Ral) -Jun kinase and other mitogen-activated proliferation and metabolism, the Foxo subfamily of protein kinase pathways (De Ruiter et al., 2001; Essers forkhead (Fox) transcription factors play critical roles et al., 2004; Asada et al., 2007). In addition, other post- in the homeostasis of immune-relevant cells, including translational modifications may modulate Foxo activity: T cells, B cells, neutrophils and other non-lymphoid acetylation by cAMP response element-binding protein lineages that modulate inflammation in disease states such -binding protein (CBP)/p300 at the lysine residue of the as inflammatory arthritis and systemic lupus erythemato- forkhead domain, for instance, reduces transcriptional sus. This review summarizes such current and expanding activity but also sensitizes Foxo for phosphorylation by knowledge of the Foxo family members in immunity, and PKB, in contrast to deacetylation by silent information their potential as therapeutic targets in inflammatory regulator 2 (Brunet et al., 2004; Daitoku et al., 2004; disease. Motta et al., 2004; van der Horst et al., 2004; Matsuzaki Oncogene (2008) 27, 2337–2344; doi:10.1038/onc.2008.26 et al., 2005; Nakae et al., 2006). Also, ubiquitination, as regulated by ubiquitin ligases like Skp2 and deubiqui- Keywords: transcription factors; autoimmunity; T cells; tinases like USP7, further modulates Foxo proteins by B cells; arthritis; lupus promoting proteosomal degradation and/or inhibition of transcriptional activity (Matsuzaki et al., 2003; Plas and Thompson, 2003; Huang et al., 2005; van der Horst et al., 2006). The regulation of Foxo proteins therefore reflects a complex network of biochemical interactions, Biology of the Foxo transcription factor family with heavy post-translational regulation during cellular signaling. Structurally and functionally, Foxo proteins—which Beyond simply traditional transactivation by target include Foxo1 (Fkhr), Foxo3a (Fkhrl1), Foxo4 (Afx) gene binding and direct interactions with basic tran- and Foxo6 and are all mammalian homologues of the scriptional machinery (for example, So and Cleary, Caenorhabditis elegans longevity and dauer arrest gene 2003), Foxo proteins regulate target gene transcription DAF-16—remain some of the most well-understood by a diverse array of mechanisms. For instance, Foxo forkhead (Fox) family members, as illustrated by the proteins may cooperate with one of many cofactors, other reviews in this journal (reviewed in Peng, 2007). including CBP/p300, to promote histone acetylation The post-translational regulation of their activities have (reviewed in refs. van der Heide and Smidt, 2005; been exquisitely elucidated; most detailed is the kinase- Wijchers et al., 2006). Other known coactivators, mediated pathway of functional inactivation in response corepressors and/or interacting proteins include Smad, to growth factors, which involves 14-3-3-mediated STAT, PPAR, Runx, p53 and other Fox family nuclear export after Foxo phosphorylation by phos- members as well as nuclear androgen, glucocorticoid, phoinositide 3-kinase (PI3K)-protein kinase B (PKB, thyroid and retinoic acid receptors (reviewed in refs. Akt) or other cellular kinases like IkB kinase (IKK) b Coffer and Burgering, 2004; Wijchers et al., 2006; (Hu et al., 2004), serum/glucocorticoid-regulated kinase Yamamura et al., 2006; You et al., 2006b). Indeed, (Brunet et al., 2001; You et al., 2004), casein kinase 1 surprisingly, many target gene effects by Foxo have been (Rena et al., 2002, 2004), dual-specificity tyrosine-(Y)- shown to be independent of the DNA-binding domain, phosphorylation regulated kinase 1A (Woods et al., suggesting that many Foxo functions reflect largely 2001), cyclin-dependent kinase 2 (Huang et al., 2006) protein–protein interactions (Ramaswamy et al., 2002). and mammalian Ste20-like kinase (Lehtinen et al., Foxo target genes are involved in cell cycle, prolifera- 2006). tion, survival and apoptosis, and include the cell cycle Kip1 Cip1 Some phosphorylation events, however, can regulate inhibitors p27 , p21 , retinoblastoma family mem- Foxo activity independent of nuclear shuttling—for ber p130, cyclins B1 and G2 and the checkpoint protein example, phosphorylation mediated by Ras-related growth arrest and DNA damage-inducible protein (GADD) 45a; the pro-apoptotic factors Bim and Fas ligand (CD95L) (reviewed in refs. Lam et al., 2006; Correspondence: Dr SL Peng, Clinical Research and Exploratory Development, Roche Palo Alto, LLC, 3431 Hillview Avenue, M/S Wijchers et al., 2006); as well as differentiation A2-259, Palo Alto, CA 94304, USA. factors such as Id1 (Birkenkamp et al., 2007). E-mail: [email protected] Foxo proteins also protect cells from oxidative stress Immunology of Foxo SL Peng 2338 via the induction of target genes like superoxide Overall, Foxo proteins mediate cytokine withdrawal dismutase (SOD) 2 (Mn-SOD) and catalase (Kops and/or mitogen absence-induced apoptosis by inducing et al., 2002; Nemoto and Finkel, 2002). Through the known pro-apoptotic and/or cell cycle cessation target control of such critical target genes in multiple genes, like Bim and p27Kip1. Nonetheless, growing biological systems, Foxo proteins play critical roles in evidence indicates that Foxo proteins likely utilize organismal homeostasis. multiple, still unidentified target genes as well: for instance, microarray studies involving an inducible Foxo3a construct in p53-deficient murine embryonic fibroblast cells have identified the pro-apoptotic Bcl-2 Immunology of the Foxo transcription factors homolog Puma as a Foxo3a target gene: IL-2 withdrawal in T cells resulted in the upregulation of Puma Foxo proteins in T cells and Bim, associated with dephosphorylation of Foxo3a, Although most immunological roles for the Foxo’s have increased occupancy of Foxo3a on the Puma and Bim been inferred from the aforementioned non-immunolo- promoters, and cellular apoptosis. Knockdown of gical systems and/or Foxo-related pathways, such as Foxo3a by shRNA prevented Puma induction in studies of PI3K and PKB pathway in rheumatoid serum-starved murine embryonic fibroblasts bearing a arthritis and T cells (Fabre et al., 2006; Reedquist transcriptionally inactive form of p53 (L25Q, W26S, et al., 2006), several recent studies have begun to A135 V) (You et al., 2006a). At the same time, though, elucidate the roles of the individual Foxo’s in immunity even additional Foxo-dependent pathways appear to (summarized in Table 1 and Figure 1). In T cells, Foxo modulate overall cellular effects; the recently described proteins seem largely regulated by T cell receptor (TCR) Foxo3a target glucocorticoid-induced leucine zipper and/or CD28 ligation-induced signals, and/or IL-2, transiently protects CTLL-2 T cells from IL-2 with- explained largely by PI3K/PKB activation: in IL-2- drawal-induced apoptosis (Asselin-Labat et al., 2004). dependent mouse CTLL-2 T cells, IL-2 withdrawal Thus, multiple additional Foxo target genes likely results in the dephosphorylation of Foxo3a and Foxo4, mediate a number of feedback loops, which regulate associated with the induction of the Foxo target genes cellular homeostasis. Bim and p27Kip1—a process mimicked by a constitutively Functionally, though, the importance of the Foxo active Foxo1 (T24A/S256A/S319A; ‘3A mutant’) (Stahl proteins in T cells has only been definitively demon- et al., 2002). In the human leukemia T cell line Jurkat, strated and reported so far for Foxo3a, where Foxo3a overexpression of Foxo1 suppresses proliferation (Me- deficiency in mice leads to spontaneous, autoreactive dema et al., 2000), while overexpression of Foxo3a helper T cell activation, Th1 and Th2 cytokine produc- induces apoptosis (Brunet et al., 1999), while in primary tion and a mild lymphoproliferative, autoimmune human T cells, TCR ligation recruits p85a PI3K to the syndrome that includes inflammatory lesions in multiple immune synapse, and the subsequent tyrosine kinase end organs. Interestingly, these findings appear inde- activity correlates with nuclear exclusion and phosphor- pendent of an apoptosis phenotype, because Foxo3a- ylation of Foxo1 (Stahl et al., 2002; Fabre et al., 2005). deficient T cells seemed to perform normally in assays of Nucleofection of the 3A mutant Foxo1 impairs human activation-induced cell death, among others. Instead, T cell growth and/or proliferation, but not early Foxo3a-deficient T cells possessed unrestrained NF-kB activation as judged by CD69 expression (Fabre et al., activation, correlated with diminished levels of inhibitor 2005). In addition, CTLA4 ligation can conversely of NF-kB proteins. Foxo3a therefore likely acts as a T protect activated T cells from apoptosis, including cell quiescence, tolerance and/or suppressive factor by activation-induced cell death, correlating with Foxo3a upregulating the inhibitory components of the NF-kB phosphorylation and diminished CD95L expression pathway (Lin et al., 2004). (Pandiyan et al., 2004). Interestingly, CD95/CD95L- Such observations imply disease relevance in systemic induced expression of Bim via Foxo3a may indirectly autoimmunity: in the 16/6 idiotype anti-DNA idiotype reflect the induction of H2O2 by CD95 signaling, experimentally-induced model of systemic lupus erythe- indicating an intersection of the apoptotic and oxidative matosus in BALB/c mice, treatment with a peptide stress functionalities (Bosque et al., 2007). based on the complimentarity determining region 1 of TCR-mediated regulation of Foxo proteins is now the idiotype ameliorates several clinical parameters of known to involve at least the Vav family of guanine disease, including autoantibody titers and kidney exchange factors for Rho GTPases: Vav1- and Vav1/ disease, associated with diminished IFN-g expression 3-deficient T cells exhibit impaired phosphorylation and, among other findings, increased expression in T and 14-3-3 association of Foxo1, as well as impaired cells of Foxo3a. This appears to be due to the induction p27Kip1 downregulation, in response to TCR ligation. of the immunoregulatory cytokine TGF-b, since T cells In contrast, Vav1-deficient T cells demonstrate normal from hCDR1-treated animals expressed increased IL-2-induced Foxo1 phosphorylation and p27Kip1 TGF-b, and antibody-mediated neutralization of downregulation—suggesting that Vav1 promotes a TGF-b abrogated the hCDR1-induced upregulation of TCR-specific pathway of T cell cycle progression and Foxo3a, at least in vitro (Sela et al., 2006). proliferation by enabling the inhibition of Foxo1, Other T cell subpopulations, however, likely also presumably via the PI3K/PKB pathway (Charvet involve regulation of Foxo3a for their homeostasis. et al., 2006). Human central memory T cells, for instance, possess

Oncogene Immunology of Foxo SL Peng 2339 Table 1 Roles of Foxo proteins in immune cells Foxo Cell type Proposed function Evidence Reference

Foxo1 CTLL-2 T cells Bim and p27Kip1 induction Ectopic expression of (Stahl et al., 2002) CA Jurkat T cells Suppression of proliferation Ectopic expression (Medema et al., 2000) Primary mouse T Regulation of p27Kip1 in response to Correlation in Vav1/3 (Charvet et al., 2006) TCR ligation knockout Primary human T Supression of proliferation but not early Ectopic expression of (Fabre et al., 2005) activation CA Primary human T Induction of Bim in response to CD95/ Correlation (Bosque et al., 2007) CD95L via H2O2 Primary human Treg Modulation of proliferative capacity Correlation (Crellin et al., 2007) Primary mouse B cells Cell-cycle arrest via cyclin G2 and Rb Ectopic expression (Chen et al., 2006; Yusuf p130 et al., 2004) Primary human PMNs Modulation of neutrophil survival in Correlation (Crossley, 2003) response to fMLP via Mcl-1

Foxo3a CTLL-2 T cells IL-2 withdrawal-induced Bim and Correlation (Stahl et al., 2002) p27Kip1 CTLL-2 T cells IL-2 withdrawal-induced GILZ Correlation (Asselin-Labat et al., 2004) MC/9, FDC-P1, TF-1 Cytokine withdrawal-induced CD95L, Correlation (Behzad et al., 2007) cells Bim, p27 Jurkat T cells Apoptosis Ectopic expression (Brunet et al., 1999) Primary mouse T IL-2 withdrawal-induced Puma, Bim Correlation (You et al., 2006a) Primary mouse T Tolerance or quiescence maintenance via Knockout (Lin et al., 2004) IkB Primary mouse T Modulation of apoptosis susceptibility Correlation during (Pandiyan et al., 2004) via CD95L CTLA4 ligation Primary human TCM Modulation of apoptosis susceptibility Correlation (Riou et al., 2007) and survival Primary human Treg Modulation of proliferative capacity Correlation (Crellin et al., 2007) Ba/F3 pre-B cells Apoptosis Ectopic expression (Dijkers et al., 2002) v-Abl transformed pre-B Cell cycle via p27Kip1 Correlation during con- (Jacobsen et al., 2006) ditional v-Abl inactivation Primary mouse B cells Cell cycle arrest via cyclin G2 and Rb Ectopic expression (Chen et al., 2006; Yusuf p130 et al., 2004) Primary mouse DCs Tolerance and IFN-g sensitivity via siRNA studies during (Fallarino et al., 2004) SOD2 regulation CTLA4-Ig treatment Primary mouse PMNs Inﬂammation-tolerant survival via Arthritis and peritonitis (Jonsson et al., 2005) CD95L regulation models in knockout mice Primary human PMNs Modulation of neutrophil survival in Correlation (Crossley, 2003) response to fMLP

Foxo4 CTLL-2 T cells IL-2 withdrawal-induced Bim and Correlation (Stahl et al., 2002) p27Kip1 Primary human PMNs Modulation of neutrophil survival in Correlation (Crossley, 2003) response to fMLP

Abbreviations: CA, constitutively-active mutant; DC, dendritic cell; fMLP, formyl-Met-Leu-Phe; GILZ, glucocorticoid-induced leucine zipper; PMN, polymorphonuclear neutrophil; Rb, retinoblastoma protein; SOD, superoxide dismutase. ‘Correlation’ indicates an indirect demonstration of the Foxo’s involvement, e.g., a correlation between Foxo phosphorylation and p27Kip1 expression in Vav-deﬁcient T cells (Charvet et al., 2006). Adapted from (Peng, 2007).

increased resistance to spontaneous and CD95-induced central memory T cell survival is driven by Foxo3a apoptosis than effector memory T cells, increased levels inactivation, which presumably results from the con- of phosphorylated Foxo3a and diminished quantities of vergence of TCR and cytokine signaling (Riou et al., several Foxo3a target genes as judged by microarray 2007). analysis, such as the proapoptotic target Bim. Pharma- A converse role for Foxo3a may participate in human cological inhibition of the Foxo3a kinases IKK and regulatory T cells, which exhibit impaired PKB phos- PKB in vitro rendered human central memory T cells phorylation in association with reduced levels of more susceptible to apoptosis, mimicking effector phosphorylated Foxo1 and Foxo3a. Interestingly, en- memory T cells. These ﬁndings suggest that human forced expression of an activated (PH domain-deﬁcient)

Oncogene Immunology of Foxo SL Peng 2340

Figure 1 A working model for Foxo protein functions in lymphocytes. Mitogenic signals such as interleukin-2 or T cell receptor (TCR) ligation promote the nuclear exclusion of Foxo proteins by leading to their phosphorylation via kinases such as protein kinase B (PKB, Akt), IkB kinase (IKK), serum/glucocorticoid-regulated kinase (SGK), casein kinase (CK) 1, dual-speciﬁcity tyrosine-(Y)- phosphorylation-regulated kinase (DYRK) 1A and mammalian Ste20-like (MST). Additional regulatory mechanisms include acetylation by cAMP response element-binding protein (CREB)-binding protein (CBP)/p300 versus deacetylation by silent information regulator (Sir) 2, as well as ubiquitination, as regulated by ubiquitin ligases like Skp2 and deubiquitinases like USP7, leading eventually to proteosomal degradation. In the absence of such inhibitory processes, Foxo proteins shuttle into the nucleus, where they transactivate target genes, which mediate multiple cellular processes, including cell cycle arrest, apoptosis, quiescence and immune tolerance. Note that many aspects of this model have been derived from studies in non-immunological systems, and have been extrapolated to lymphocytes here. Adapted from (Peng, 2007).

PKB isoform, which presumably would increase demonstrated by in vitro studies involving ectopic phosphorylated Foxo species, partially reversed the expression (Chen et al., 2006). Here, B cell regulation suppressive capacity of regulatory T cells in vitro, of Foxo activity does not appear to be limited to post- associated with restoration of their ability to produce translational phosphorylation: BCR crosslinking also several cytokines such as IFN-g, but not IL-2, without suppresses Foxo1 mRNA expression in a PI3K- and affecting expression levels of Foxp3, CTLA4, CD25, or Btk-dependent manner, whereas the induction of granzymes A/B (Crellin et al., 2007). Foxo proteins phosphorylated Foxo1 is Btk-independent, at least at therefore play diverse roles in multiple T cell subsets Ser256 and at least in mouse B cells. Foxo3a and Foxo4 (Table 1). mRNAs are also suppressed by BCR ligation (Hinman et al., 2007). Finally, in v-Abl-transformed pre-B cells from bcl-2 transgenic SCID mice, temperature-sensitive Foxo proteins in B cells inactivation of v-Abl results in cell cycle arrest and an Analogous pathways likely regulate Foxo proteins in B increased nuclear abundance of Foxo3a and p27. cells, although emerging data clearly indicate speciﬁc Reactivation reinitiates cell cycle entry, associated with differences (Table 1). Overexpression of Foxo3a in the diminished Foxo3a and p27. Interestingly, this reactiva- mouse pre-B cell line Ba/F3 can also induce apoptosis tion is associated with attenuated NF-kB activity, (Dijkers et al., 2002) and in mouse primary B cells, BCR suggesting that the NF-kB-inhibiting function of Foxo3a cross-linking induces nuclear exportation of Foxo1 in a may not play a critical physiological role, at least in this manner that can be inhibited by the PI3K inhibitor system (Jacobsen et al., 2006). Thus, B cells may utilize LY294002 (Yusuf et al., 2004). Retroviral transduction both translational and post-translational mechanisms to of A3 mutant versions of Foxo1 or Foxo3a into modulate Foxo activity, which, as in T cells, also activated primary mouse B cells resulted in a modest regulates cell survival and proliferation, but perhaps via cell cycle arrest and annexin V positivity, suggesting that less of an effect on the NF-kB pathway. optimal B cell proliferation requires inactivation of the Foxo proteins. This cell cycle arrest appears to reﬂect cooperation of Foxo proteins with the transcription Foxo proteins in non-lymphoid lineages factor dEF1 to induce the expression of the cell-cycle Nonetheless, many Foxo pathways appear to be arrest genes cyclin G2 and retinoblastoma p130, as analogous across multiple immune-relevant cell lineages.

Oncogene Immunology of Foxo SL Peng 2341 For instance, in the hematopoietic cell lines MC/9, Interestingly, a Foxo3a haplotype has recently been FDC-P1 and TF-1, cytokine withdrawal results in reported to associate with erosive activity in adult increased CD95L, Bim and p27 expression, correlating rheumatoid arthritis (Hughes et al., 2006), where with Foxo3a phosphorylation (Behzad et al., 2007). cell lineage-specific inactivation of Foxo family Also, animals bearing combined deficiencies in Foxo1, members has also been observed in synovial fibroblast- Foxo3a and Foxo4 (Foxo-deficient) develop deficiencies like synoviocytes, T cells and macrophages (Ludikhuize in hematopoietic stem cells due to an impaired resistance et al., 2007). Such findings suggest that Foxo3a to oxidative stress (Tothova et al., 2007), indicating that may be a key target for therapeutic modulation Foxo proteins play key roles in the regulation of stem in rheumatoid and/or perhaps other inflammatory cell differentiation and development. arthritides. In non-obese diabetic mice, the tolerogenic potential of IFN-g is lost in CD8 þ dendritic cells, apparently due to peroxynitrite-mediated nitration of STAT1 which impairs the IFN-g signaling pathway. Treatment of Potential considerations for Foxo proteins in non-obese diabetic dendritic cells with CTLA4-Ig inflammatory diseases in vitro confers tolerogenic properties due to increased Foxo3a activity, as judged by siRNA and luciferase It is tempting to speculate upon the utility of Foxo reporter assays—which results in increased superoxide proteins as targets of therapeutic intervention in dismutase 2 (Mn-superoxide dismutase) expression, inflammatory diseases, since they appear to play such and an improvement in IFN-g-dependent tryptophan key roles in cells relevant to inflammatory disease catabolism. This pathway likely involves CTLA4-Ig- pathogenesis. Blockade of Foxo, or at least of Foxo3a, induced activation of phosphatase and tensin homolog activity in inflammatory arthritis, for instance, may be and inactivation of PKB, through which the increased of particular interest, but on the other hand, amplifica- superoxide dismutase 2 activity diminishes peroxy- tion of Foxo activity may be desired in diseases such as nitrite formation which in turn relaxes the STAT1 systemic lupus erythematosus or type 1 diabetes. For impairment (Fallarino et al., 2004). Modulation of optimal disease positioning, continued studies would be Foxo pathways in dendritic cells therefore may required to explore both the breadth of diseases in which possess therapeutic value in a variety of inflammatory Foxo family members participate, as well as the relative diseases. context-dependent roles of the various individual Foxo In poylmorphonuclear neutrophils, Foxo proteins family members. appear to play critical roles in the regulation of cellular At the same time, chemical tractability of the Foxo survival. In human poylmorphonuclear neutrophils, proteins for the generation of agonist or antagonist exposure to the bacterially derived chemoattractant molecules is and will likely remain a developmental formyl-Met-Leu-Phe results in phosphorylation of issue, since non-ligand transcription factors in general Foxo1, Foxo3a and Foxo4, apparently via PI3K and remain highly challenging targets for small molecule mitogen-activated protein kinase -dependent pathways intervention due to their large biologically-relevant as judged by pharmacological inhibitors in vitro interfaces (reviewed in Aud and Peng, 2006). Recent (Crossley, 2003). Interestingly, formyl-Met-Leu-Phe success with other transcription factor families, such as also induces the Bcl-2-related survival factor Mcl-1, AP-1, however, offer hope for the development of small which is capable of forming a complex with Foxo1 and molecular modulators of Foxo activity (Tsuchida et al., may therefore coordinate neutrophil survival via mod- 2006). Theoretically, other strategies successfully used to ulation of Foxo activity. However, it is interesting target other transcription factors, such as decoy to note that PI3Kg-deficient neutrophils have only oligonucleotides or short interfering RNA molecules, modestly diminished levels of phospho-Foxo1, implying may be successful here, but these interventions would be redundancy among the PI3K isoforms in activating limited by the current pharmacokinetic limitations of PKB to inactivate Foxo proteins and/or perhaps the such molecules in vivo, and also would be restricted to presence of non-PI3K pathways to Foxo inactivation in Foxo antagonism. On the other hand, intervention upon neutrophils (Yang et al., 2003). other, more chemically tractable targets within the Foxo Nonetheless, at least Foxo3a clearly plays critical pathway may hold promise, as suggested by studies with roles in neutrophil survival, as demonstrated inhibitors of CK1 or PI3K (Rena et al., 2004; Yau et al., by Foxo3a-deficient mice which are resistant to 2005), and library screens which have yielded phosphor- both thioglycollate-induced peritonitis and the passive ylation-dependent inhibitors of Foxo nuclear export K/B N serum transfer model of arthritis, two (Kau et al., 2003; Georgiades and Clardy, 2005; neutrophilic models of inflammation (Jonsson Schroeder et al., 2005). Challenges of substrate specifi- et al., 2005). Foxo3a-deficient neutrophils appear city will likely pervade, since multiple Fox proteins, both to enter target organs, but undergo apoptosis due Foxo and non-Foxo, likely share multiple up- and to an inability to suppress the upregulation of CD95L. down-stream pathway regulators, yet the diverse func- Thus, Foxo3a is required to prevent inflammation- tions of Fox family members in the immune system induced apoptosis of neutrophils via CD95L, suggests that high target specificity will be required for surprisingly demonstrating an anti-apoptotic function clinical success (reviewed in refs. Coffer and Burgering, for Foxo3a via its ability to suppress CD95L expression. 2004; Jonsson and Peng, 2005).

Oncogene Immunology of Foxo SL Peng 2342 Conclusions Still, as is the case for Foxo3a and neutrophils, extrapolation from transformed cell line data can be Emerging data indicate that Foxo proteins play key misleading, since Foxo proteins can promote apoptosis roles in the regulation of inflammation, but significant via CD95/CD95L in some cellular systems, but in vivo knowledge gaps remain; whereas Foxo3a modulates Foxo3a in fact protects against apoptosis via CD95/ helper T cell quiescence and inflammatory neutrophil CD95L in primary cells—thus Foxo proteins may likely survival, the specific roles of the other Foxo proteins, either enhance or repress the transcription of target and the roles of Foxo3a in other cell lineages, remains genes, perhaps through the recruitment of gene-specific still incompletely understood. Importantly, many cur- coactivators or corepressors (Jonsson et al., 2005). rent models of Foxo functions in the immune system Furthermore, the relative importance of additional path- have been extrapolated from more fundamental biolo- ways beyond mitogen-induced PI3K pathways will require gical systems, such as cancer or metabolism and it additional study: for instance, the ability of the C5b-9 remains unclear how frequently parallels may be drawn complement complex to regulate Foxo activity in correla- (Figure 1). Nonetheless it is intriguing to consider the tion with proliferation, at least in some cell types (Fosbrink possibility that Foxo proteins, by virtue of their roles in et al., 2006), suggests that tissue- and/or context-specific diverse biological processes, mediate molecularly the pathways and functions exist for the Foxo proteins, intersection and impact of non-immune biological requiring significant future studies for complete elucida- systems and the immune system—for example, given tion. Continued evaluation on the Foxo proteins in the importance of Foxo in metabolism of glucose and immunity, correlated with their involvement in other other endocrine systems, it is tempting to envision Foxo biological systems, will therefore hopefully elucidate as a key point of intersection for endocrine—immune disease-relevant, tissue-specific pathways, bearing in- interactions (Zhuang et al., 2006). sight into disease pathogenesis and/or therapies.

References

Asada S, Daitoku H, Matsuzaki H, Saito T, Sudo T, Mukai H et al. Crellin NK, Garcia RV, Levings MK. (2007). Altered activation of (2007). Mitogen-activated protein kinases, Erk and p38, phosphor- AKT is required for the suppressive function of human ylate and regulate Foxo1. Cell Signal 19: 519–527. CD4+CD25+ T regulatory cells. Blood 109: 2014–2022. Asselin-Labat ML, David M, Biola-Vidamment A, Lecoeuche D, Crossley LJ. (2003). Neutrophil activation by fMLP regulates FOXO Zennaro MC, Bertoglio J et al. (2004). GILZ, a new target for the (forkhead) transcription factors by multiple pathways, one of which transcription factor FoxO3, protects T lymphocytes from inter- includes the binding of FOXO to the survival factor Mcl-1. J Leuk leukin-2 withdrawal-induced apoptosis. Blood 104: 215–223. Biol 74: 583–592. Aud D, Peng SL. (2006). Mechanisms of disease: transcription factors DaitokuH, Hatta M, MatsuzakiH, Aratani S, Ohshima T, Miyagishi in inﬂammatory arthritis. Nat Clin Prac Rheum 2: 434–442. M et al. (2004). Silent information regulator 2 potentiates Foxo1- Behzad H, Jamil S, Denny TA, Duronio V. (2007). Cytokine-mediated mediated transcription through its deacetylase activity. Proc Natl FOXO3a phosphorylation suppresses FasL expression in hemo- Acad Sci USA 101: 10042–10047. poietic cell lines: investigations of the role of Fas in apoptosis due to De Ruiter ND, Burgering BM, Bos JL. (2001). Regulation of the cytokine starvation. Cytokine 38: 74–83. forkhead transcription factor AFX by Ral-dependent phosphoryla- Birkenkamp KU, Essaﬁ A, van der Vos KE, da Costa M, Hui RC, tion of threonines 447 and 451. Mol Cell Biol 21: 8225–8235. Holstege F et al. (2007). FOXO3a induces differentiation of Bcr- Dijkers PF, Birkenkamp KU, Lam EW, Thomas NS, Lammers JW, Abl-transformed cells through transcriptional downregulation of Koenderman L et al. (2002). FKHR-L1 can act as a critical effector Id1. J Biol Chem 282: 2211–2220. of cell death induced by cytokine withdrawal: protein kinase B- Bosque A, Aguilo JI, Alava MA, Paz-Artal E, Naval J, Allende LM enhanced cell survival through maintenance of mitochondrial et al. (2007). The induction of Bim expression in human T-cell blasts integrity. J Cell Biol 156: 531–542. is dependent on nonapoptotic Fas/CD95 signaling. Blood 109: Essers MA, Weijzen S, de Vries-Smits AM, Saarloos I, de Ruiter ND, 1627–1635. Bos JL et al. (2004). FOXO transcription factor activation by Brunet A, Bonni A, Zigmond MJ, Lin MZ, Juo P, Hu LS et al. (1999). oxidative stress mediated by the small GTPase Ral and JNK. Akt promotes cell survival by phosphorylating and inhibiting a EMBO J 23: 4802–4812. forkhead transcription factor. Cell 96: 857–868. Fabre S, Lang V, Bismuth G. (2006). PI3-kinase and the control of T Brunet A, Park J, Tran H, Hu LS, Hemmings BA, Greenberg ME. cell growth and proliferation by Foxos. Bull Cancer 93: E36–E38. (2001). Protein kinase SGK mediates survival signals by phosphor- Fabre S, Lang V, Harriague J, Jobart A, Unterman TG, Trautmann A ylating the forkhead transcription factor FKHRL1 (FOXO3a). Mol et al. (2005). Stable activation of phosphatidylinositol 3-kinase in Cell Biol 21: 952–965. the T cell immunological synapse stimulates Akt signaling to FoxO1 Brunet A, Sweeney LB, Sturgill JF, Chua KF, Greer PL, Lin Y et al. nuclear exclusion and cell growth control. J Immunol 174: (2004). Stress-dependent regulation of FOXO transcription factors 4161–4171. by the SIRT1 deacetylase. Science 303: 2011–2015. Fallarino F, Bianchi R, Orabona C, Vacca C, Belladonna ML, Fioretti Charvet C, Canonigo AJ, Becart S, Maurer U, Miletic AV, Swat W MC et al. (2004). CTLA-4-Ig activates forkhead transcription et al. (2006). Vav1 promotes T cell cycle progression by linking factors and protects dendritic cells from oxidative stress in nonobese TCR/CD28 costimulation to FOXO1 and p27kip1 expression. J diabetic mice. J Exp Med 200: 1051–1062. Immunol 177: 5024–5031. Fosbrink M, Niculescu F, Rus V, Shin ML, Rus H. (2006). C5b-9- Chen J, Yusuf I, Andersen HM, Fruman DA. (2006). FOXO induced endothelial cell proliferation and migration are dependent transcription factors cooperate with dEF1 to activate growth on Akt inactivation of forkhead transcription factor FOXO1. J Biol suppressive genes in B lymphocytes. J Immunol 176: 2711–2721. Chem 281: 19009–19018. Coffer PJ, Burgering BM. (2004). Forkhead-box transcription factors Georgiades SN, Clardy J. (2005). Total synthesis of psammaplysenes A and their role in the immune system. Nature Rev Immunol 4: and B, naturally occurring inhibitors of FOXO1a nuclear export. 889–899. Org Lett 7: 4091–4094.

Oncogene Immunology of Foxo SL Peng 2343 Hinman RM, Bushanam JN, Nichols WA, Satterthwaite AB. (2007). B unequal resistance of Th1 and Th2 cells against activation-induced cell receptor signaling down-regulates forkhead box transcription cell death by a mechanism requiring PI3 kinase function. J Exp Med factor class O 1 mRNA expression via phosphatidylinositol 3-kinase 199: 831–842. and Bruton’s tyrosine kinase. J Immunol 178: 740–747. Peng SL. (2007). Immune regulation by Foxo transcription factors. HuMC, Lee DF, Xia W, Golfman LS, Ou-YangF, Yang JY et al. Autoimmunity 40: 462–469. (2004). IkB kinase promotes tumorigenesis through inhibition of Plas DR, Thompson CB. (2003). Akt activation promotes degradation forkhead FOXO3a. Cell 117: 225–237. of tuberin and FOXO3a via the proteasome. J Biol Chem 278: Huang H, Regan KM, Lou Z, Chen J, Tindall DJ. (2006). CDK2- 12361–12366. dependent phosphorylation of FOXO1 as an apoptotic response to Ramaswamy S, Nakamura N, Sansal I, Bergeron L, Sellers WR. DNA damage. Science 314: 294–297. (2002). A novel mechanism of gene regulation and tumor suppres- Huang H, Regan KM, Wang F, Wang D, Smith DI, van Deursen JM sion by the transcription factor FKHR. Cancer Cell 2: 81–91. et al. (2005). Skp2 inhibits FOXO1 in tumor suppression through Reedquist KA, Ludikhuize J, Tak PP. (2006). Phosphoinositide 3- ubiquitin-mediated degradation. Proc Natl Acad Sci USA 102: kinase signalling and Foxo transcription factors in rheumatoid 1649–1654. arthritis. Biochem Soc Trans 34: 727–730. Hughes LB, Morrison D, Westfall AO, Tiwari HK, Alarcon GS, Conn Rena G, Bain J, Elliott M, Cohen P. (2004). D4476, a cell-permeant DL et al. (2006). Genetic and Non-genetic Factors Associated with inhibitor of CK1, suppresses the site-specific phosphorylation and Baseline Radiographic Erosions in African-Americans with Early nuclear exclusion of FOXO1a. EMBO Rep 5: 60–65. Rheumatoid Arthritis: Results from the CLEAR Registry (70th Rena G, Woods YL, Prescott AR, Peggie M, Unterman TG, Williams Annual Meeting of the American College of Rheumatology, MR et al. (2002). Two novel phosphorylation sites on FKHR that Washington, DC). John Wiley & Sons, Hoboken, NJ, USA. are critical for its nuclear exclusion. EMBO J 21: 2263–2271. Jacobsen EA, Ananieva O, Brown ML, Chang Y. (2006). Growth, RiouC, Yassine-Diab B, Van Grevenynghe J, Somogyi R, Greller LD, differentiation, and malignant transformation of pre-B cells Gagnon D et al. (2007). Convergence of TCR and cytokine mediated by inducible activation of v-Abl oncogene. J Immunol signaling leads to FOXO3a phosphorylation and drives the survival 176: 6831–6838. of CD4+ central memory T cells. J Exp Med 204: 79–91. Jonsson H, Peng SL. (2005). Forkhead transcription factors in Schroeder FC, KauTR, Silver PA, Clardy J. (2005). The psamma- immunology. Cell Molec Life Sci 62: 397–409. plysenes, specific inhibitors of FOXO1a nuclear export. J Nat Prod Jonsson H, Allen P, Peng SL. (2005). Inflammatory arthritis requires 68: 574–576. Foxo3a to prevent Fas ligand-induced neutrophil apoptosis. Nature Sela U, Dayan M, Hershkoviz R, Cahalon L, Lider O, Mozes E. Med 11: 666–671. (2006). The negative regulators Foxj1 and Foxo3a are upregulated KauTR, Schroeder F, Ramaswamy S, Wojciechowski CL, Zhao JJ, by a peptide that inhibits systemic lupus erythematosus-associated T Roberts TM et al. (2003). A chemical genetic screen identifies cell responses. Eur J Immunol 36: 2971–2980. inhibitors of regulated nuclear export of a forkhead transcription So CW, Cleary ML. (2003). Common mechanism for oncogenic factor in PTEN-deficient tumor cells. Cancer Cell 4: 463–476. activation of MLL by forkhead family proteins. Blood 101: 633–639. Kops GJ, Dansen TB, Polderman PE, Saarloos I, Wirtz KW, Coffer Stahl M, Dijkers PF, Kops GJ, Lens SM, Coffer PJ, Burgering BM PJ et al. (2002). Forkhead transcription factor FOXO3a protects et al. (2002). The forkhead transcription factor Foxo regulates quiescent cells from oxidative stress. Nature 419: 316–321. transcription of p27Kip1 and Bim in response to IL-2. J Immunol Lam EW, Francis RE, Petkovic M. (2006). FOXO transcription 168: 5024–5031. factors: key regulators of cell fate. Biochem Soc Trans 34: 722–726. Tothova Z, Kollipara R, Huntly BJ, Lee BH, Castrillon DH, Cullen Lehtinen MK, Yuan Z, Boag PR, Yang Y, Villen J, Becker EB et al. DE et al. (2007). Foxos are critical mediators of hematopoietic stem (2006). A conserved MST-FOXO signaling pathway mediates cell resistance to physiologic oxidative stress. Cell 128: 325–339. oxidative-stress responses and extends life span. Cell 125: 987–1001. Tsuchida K, Chaki H, Takakura T, Kotsubo H, Tanaka T, Aikawa Y Lin L, Hron JD, Peng SL. (2004). Regulation of NF-kB, Th activation, et al. (2006). Discovery of nonpeptidic small-molecule AP-1 and autoinflammation by the forkhead transcription factor Foxo3a. inhibitors: lead hopping based on a three-dimensional pharmaco- Immunity 21: 203–213. phore model. J Med Chem 49: 80–91. Ludikhuize J, de Launay D, Groot D, Smeets TJ, Vinkenoog M, van der Heide LP, Smidt MP. (2005). Regulation of Foxo activity by Sanders ME et al. (2007). Inhibition of forkhead box class O family CBP/p300-mediated acetylation. Trends Biochem Sci 30: 81–86. member transcription factors in rheumatoid synovial tissue. van der Horst A, de Vries-Smits AM, Brenkman AB, van Triest MH, Arthritis Rheum 56: 2180–2191. van den Broek N, Colland F et al. (2006). FOXO4 transcriptional Matsuzaki H, Daitoku H, Hatta M, Aoyama H, Yoshimochi K, activity is regulated by monoubiquitination and USP7/HAUSP. Fukamizu A. (2005). Acetylation of Foxo1 alters its DNA-binding Nature Cell Biol 8: 1064–1073. ability and sensitivity to phosphorylation. Proc Natl Acad Sci USA van der Horst A, Tertoolen LG, de Vries-Smits LM, Frye RA, 102: 11278–11283. Medema RH, Burgering BM. (2004). FOXO4 is acetylated upon Matsuzaki H, Daitoku H, Hatta M, Tanaka K, Fukamizu A. peroxide stress and deacetylated by the longevity protein hSir2(- (2003). Insulin-induced phosphorylation of FKHR (Foxo1) SIRT1). J Biol Chem 279: 28873–28879. targets to proteasomal degradation. Proc Natl Acad Sci USA 100: Wijchers PJ, Burbach JP, Smidt MP. (2006). In control of biology: of 11285–11290. mice, men and Foxes. Biochem J 397: 233–246. Medema RH, Kops GJ, Bos JL, Burgering BM. (2000). AFX-like Woods YL, Rena G, Morrice N, Barthel A, Becker W, Guo S et al. forkhead transcription factors mediate cell-cycle regulation by Ras (2001). The kinase DYRK1A phosphorylates the transcription and PKB through p27kip1. Nature 404: 782–787. factor FKHR at Ser329 in vitro, a novel in vivo phosphorylation site. Motta MC, Divecha N, Lemieux M, Kamel C, Chen D, Gu W et al. Biochem J 355: 597–607. (2004). Mammalian SIRT1 represses forkhead transcription factors. Yamamura Y, Lee WL, Inoue K, Ida H, Ito Y. (2006). RUNX3 Cell 116: 551–563. cooperates with Foxo3a to induce apoptosis in gastric cancer cells. Nakae J, Cao Y, DaitokuH, FukamizuA,Ogawa W, Yano Y et al. J Biol Chem 281: 5267–5276. (2006). The LXXLL motif of murine forkhead transcription factor Yang KY, Arcaroli J, Kupfner J, Pitts TM, Park JS, Strasshiem D FoxO1 mediates Sirt1-dependent transcriptional activity. J Clin et al. (2003). Involvement of phosphatidylinositol 3-kinase gamma Invest 116: 2473–2483. in neutrophil apoptosis. Cell Signal 15: 225–233. Nemoto S, Finkel T. (2002). Redox regulation of forkhead proteins YauCY, Wheeler JJ, SuttonKL, Hedley DW. (2005). Inhibition of through a p66shc-dependent signaling pathway. Science 295: 2450–2452. integrin-linked kinase by a selective small molecule inhibitor, Pandiyan P, Gartner D, Soezeri O, Radbruch A, Schulze-Osthoff K, QLT0254, inhibits the PI3K/PKB/mTOR, Stat3, and FKHR Brunner-Weinzierl MC. (2004). CD152 (CTLA-4) determines the pathways and tumor growth, and enhances gemcitabine-induced

Oncogene Immunology of Foxo SL Peng 2344 apoptosis in human orthotopic primary pancreatic cancer xeno- YouH, Yamamoto K, Mak TW. (2006b). Regulation of transactiva- grafts. Cancer Res 65: 1497–1504. tion-independent proapoptotic activity of p53 by FOXO3a. Proc YouH, Jang Y, You-TenAI, Okada H, Liepa J, Wakeham A et al. Natl Acad Sci USA 103: 9051–9056. (2004). p53-dependent inhibition of FKHRL1 in response to DNA Yusuf I, Zhu X, Kharas MG, Chen J, Fruman DA. (2004). damage through protein kinase SGK1. Proc Natl Acad Sci USA Optimal B-cell proliferation requires phosphoinositide 3-kinase- 101: 14057–14062. dependent inactivation of FOXO transcription factors. Blood 104: YouH, Pellegrini M, TsuchiharaK, Yamamoto K, Hacker G, 784–787. Erlacher M et al. (2006a). FOXO3a-dependent regulation of Puma Zhuang Y, Li S, Li Y. (2006). dbNEI: a speciﬁc database for in response to cytokine/growth factor withdrawal. J Exp Med 203: neuro-endocrine-immune interactions. Neuro Endocrinol Lett 27: 1657–1663. 53–59.

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