Trends in Cell Biology
ReviewUnravelling the tumor-suppressive functions of FOXO proteins
Introduction
The forkhead box O (FOXO) class of transcription factors consists of four members: FOXO1, FOXO3a, FOXO4 and FOXO6. FOXO proteins represent a subfamily of the larger group of forkhead transcription factors that are all characterized by a highly conserved DNA-binding domain – the forkhead domain – which is sometimes referred to as a winged-helix domain 1, 2. Although the forkhead family is defined by this highly conserved DNA-binding domain, the remainder of the sequences of the various family members show less homology, which could explain their differential regulation and cellular function. FOXO proteins can function as monomeric transcription factors, and a consensus DNA-binding sequence has been identified [3]. However, as with most, if not all, transcription factors, binding of FOXO proteins to coactivators (also known as repressors) and combined binding of FOXO proteins with other gene regulators {e.g. SMAD proteins [4], the histone acetyltransferase p300, the cAMP response element-binding (CREB)-binding protein (CBP) [5] and the cellular tumor antigen p53 [6]; reviewed in Ref. [7]} ultimately determines the outcome of gene regulation by the FOXO proteins. Gene regulation by FOXO proteins can specify several cell fates. For example, FOXO activation can result in upregulation of genes that regulate cell-cycle progression (e.g. those encoding cyclin-dependent kinase inhibitors p21Cip1 [4] and p27Kip1 [8], and the retinoblastoma-like protein p130Rb2 [9]) and thereby can lead to G1-phase cell-cycle arrest. Furthermore, gene products involved in DNA-damage repair (e.g. the growth arrest and DNA-damage-inducible protein GADD45 [10]) or in preventing the generation of excessive levels of reactive oxygen species (ROS) {e.g. manganese superoxide dismutase (MnSOD) and the peroxidase catalase [11]} can be induced by FOXO proteins. Presumably, this prevents or delays cells arrested in the G1 phase from entering cell-death programs. Alternatively, FOXO proteins can regulate the onset of apoptosis by enhancing transcription of genes encoding the apoptotic regulators tumor necrosis factor ligand 6, also known as Fas ligand (FasL) [12], B-cell lymphoma 6 (BCL6) [13] and the Bcl-2-like protein 11 (BIM) [14]. Thus, FOXO proteins can determine cell survival and cell death under specific conditions. FOXO proteins have also been implicated in the control of differentiation of (hematopoietic) stem cells [15] and timing of the maturation of oocytes [16].
FOXO function is regulated predominantly by two modes of cellular signaling. First, insulin signaling regulates FOXO activity through the phosphoinositide 3-kinase (PI3K)–protein kinase B (PKB; or AKT) signaling pathway. PKB directly phosphorylates FOXO proteins on three conserved serine/threonine residues and this results in binding of the adaptor protein 14-3-3 to FOXO proteins [17]. Binding of 14-3-3 coincides with nuclear exit of FOXO proteins and, consequently, with loss of FOXO-mediated transcription. Second, cellular-stress signaling, especially oxygen stress, regulates FOXO activity through stress kinases such as c-Jun N-terminal kinase (JNK, also known as mitogen-activated protein kinase 8) [18] and other modifying enzymes such as acetyl transferases, CBP and p300 [5], the NAD-dependent deacetylase SIRT1 (silent mating type information regulation 2 homolog 1) 19, 20, 21 and (de)ubiquitylating enzymes Skp2 (S-phase kinase-associated protein 2) [22], MDM2 (murine double minute 2) [23] and USP7 (ubiquitin-specific peptidase 7; also known as herpes-associated ubiquitin-specific protease, HAUSP) [24]. Insulin signaling inactivates FOXO proteins, whereas stress signaling initially activates the FOXO proteins, most probably to mediate stress resistance and to prevent premature cell death. However, when cells are unable to manage a level of stress that is too high or enduring, FOXO proteins can initiate cell death by inducing the transcription of pro-apoptotic genes and, eventually, a shutdown of FOXO-mediated protection through the inactivation of FOXO at high levels of (oxidative) stress also occurs. Because of their ability to regulate the cell cycle, stress repair and cell death, it has long been suggested that FOXO proteins are tumor suppressors and, indeed, FOXO function is inhibited in most human cancers owing to hyperactive PKB and the latter is due to mutations in RAS, PTEN or PI3K. As we explain, recent evidence in animal models has shown that FOXO proteins are indeed true tumor suppressors.
Section snippets
FOXO proteins in monogenic chromosomal translocations
The FOXO transcription factors were initially discovered as chromosomal translocation partners found in some forms of leukemia [MLL (myeloid/lymphoid or mixed lineage leukemia)–FOXO4 and MLL–FOXO3] and alveolar rhabdomyosarcomas (PAX3–FOXO1, PAX7–FOXO1) (for a review see Ref. [25]). Their participation in these oncogenic chromosomal translocations is not unique, however, and, in tumors, both MLL and the paired box protein PAX combine with several other translocation partners to yield
Indications of the tumor-suppressor functions of FOXO proteins from other systems
From in vitro assays, it is known that FOXO proteins, when mutated at the residue(s) required for inactivation by PKB-mediated phosphorylation, can block colony formation in RAS-transformed cells or cells deficient for the phosphatase and tensin homolog (PTEN) [8]. More recently, it was demonstrated that, in classical combined MYC and RAS oncogenic transformation, the need for RAS could be replaced with expression of a dominant-negative form of FOXO (dnFOXO). This dnFOXO comprises the FOXO
FOXO proteins are bona fide tumor suppressors
The first orthotopic in vivo evidence for FOXO as a negative regulator of uncontrolled cell-growth came from studies in the nematode Caenorhabditis elegans. The molecular mechanism(s) that control FOXO expression seem to have been conserved during evolution. The activity of DAF-16 (abnormal dauer formation protein 16), the C. elegans ortholog of FOXO, is also negatively regulated by insulin-type signaling through the PI3K–PKB pathway. In addition, DAF-16 is also regulated by stress through
DNA-damage response and oxidative stress scavenging
An important means of p53-mediated tumor suppression is the DNA-damage-repair response. Damaged DNA can lead to aneuploidy and/or chromosomal instability, which is believed to be a major contributor to tumor progression and development of drug resistance. The p53-dependent DNA-damage response is mediated through signaling by means of the Ser/Thr kinases ATM (ataxia telangiectasia mutated) and ATR (ataxia telangiectasia and Rad3 related); indeed, loss of the gene encoding ATM also cooperates
FOXO, cancer and stem-cell depletion
Besides the spontaneous occurrence of tumors in the FOXO-triple-knockout mice, the occurrence of hematopoietic stem-cell depletion has also been described in this model [15] (extensively reviewed in Ref. [64]). The authors suggest that this is partly caused by elevated levels of ROS in the FOXO-knockout stem cells because the depletion can be partially reversed by treatment with the antioxidant NAC. The observation that loss of FOXO leads to stem-cell depletion seems contradictory to the
Autophagy
In mammalian cells, FOXO proteins have been shown to regulate autophagy positively during nutrient starvation through transcriptional regulation of BNIP3 (BCL2/adenovirus E1B 19 kDa-interacting protein 3), LC3 (microtubule-associated protein 1A/1B-light chain 3), ATG12 (autophagy-related 12 homolog) and GABA(A)-receptor-associated protein-like 1 (GABARAPL1; also known as GBRL1) [65]. The regulation of this pathway has been implicated in FOXO-induced muscle wasting. By contrast, in C. elegans,
FOXO and the response to hypoxia
The observation that loss of FOXO can perturb the proliferation–apoptosis balance in homeostasis explains how cells can become tumorigenic, but, for cancer to become malignant, angiogenesis is required. When cells become deprived of oxygen owing to insufficient blood supply, for instance during rapid growth, as occurs in both embryonic and tumorigenic tissues, the hypoxia-inducible factor (HIF) complex becomes stabilized and active. The functional HIF complex consists of α (HIF1A) and β (aryl
Concluding remarks – FOXO as a therapeutic target for the treatment of cancer?
The recent demonstrations with in vivo models of loss of FOXO function show that FOXO proteins are indeed true tumor suppressors. In this review, we outline what functions of FOXO proteins might contribute to their anti-tumorigenic actions. Figure 3 summarizes these tumor-suppressive functions and highlights when during cancer development these functions might exert their effects. Some of the tumor-suppressive roles of FOXO proteins might function at different stages of tumor formation. For
Acknowledgements
Work in the Burgering laboratory is supported by the Dutch Cancer Foundation (KWF), the Netherlands Organization for Scientific Research (NWO) and the Dutch Digestive Disorders Foundation (Maag-Lever-Darmstichting).
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