It is well known that transcription factors regulate the expression of target genes by identifying and binding to specific DNA sequences, after which they participate in the formation of a complex signaling network to maintain cell homeostasis. Dysregulation of transcription factors leads to a variety of pathological changes in cells, results in the occurrence of various diseases, and determines the various behaviors of malignant tumors[17,18]. Among various transcription factors, FOX transcription factors are widely distributed in organisms from yeasts to humans. They are characterized by a forkhead domain (FHD) and a highly conserved DNA binding domain (DBD) that is composed of 100 amino acid residues folded into a helix-turn-helix motif with two characteristic large loops and three α helices.
Among the different types of FOX transcription factors, the four FOXO isoforms, FOXO1, FOXO3, FOXO4, and FOXO6, in mammals belong to the O subfamily of the FOX family of transcription factors. FOXOs have four common domains, including a FHD, a nuclear export sequence (NES) domain, a nuclear localization signal (NLS), and a C-terminal transactivation domain (TAD), although FOXO6 lacks the NES domain (Figure 1). All FOXOs can recognize and bind to two sequences: the Daf-16 family member-binding element (DEB), 5′-GTAAA(T/C)AA-3′, and the insulin-responsive sequence (IRE), 5′-(C/A)(A/C)AAA(C/T)AA-3′[21,22].
Regulatory mechanism of FOXOs
FOXOs function as central transcription factors that regulate many cellular processes through transcriptional activity. Unsurprisingly, FOXOs are also regulated by multiple signaling pathways involving synthesis, phosphorylation, acetylation, and ubiquitination, which mainly determine subcellular localization, transcriptional activity, and protein stability[11,22,27]. As transcription factors, FOXOs usually exist in the nuclei of quiescent or growth factor (GF)-deficient cells. When GFs are absent, FOXOs shuttle into and accumulate in the nucleus to promote cell cycle arrest, stress resistance, and apoptosis, by upregulating the transcription of a series of target genes. However, in the presence of cell GFs, FOXOs relocate to the cytoplasm for degradation by the ubiquitin-proteasome pathway.
Phosphorylation via the classical PI3K-AKT pathway: Except for FOXO6, the regulation of FOXO-dependent transcription primarily depends on shuttling between the nucleus and cytoplasm. More specifically, negative regulation by the PI3K-AKT pathway is dependent on activation by GF receptor tyrosine kinases (RTKs). Under normal physiological conditions, RTKs are activated by autophosphorylation after binding GFs or insulin, which is followed by recruitment and activation of PI3K. Then, activated PI3K catalyzes phosphatidylinositol-4,5-bisphosphate (PIP2) to phosphatidylinositol-3,4,5-trisphosphate (PIP3), which serves as the docking site for AKT and PDK1. PIP3 facilitates the translocation of PDK1 and AKT to the cell membrane, where AKT is activated by phosphorylation on threonine 308 by PDK1. Activated AKT phosphorylates FOXOs at three sites to promote the binding of nuclear 14-3-3 protein to FOXO, which results in masking of the FOXO NLS; this causes the export of FOXO from the nucleus and prevents nuclear entry, thus preventing FOXO from binding to corresponding sites on DNA and inhibiting its transcriptional activity. When the GF-PI3K-AKT pathway is constitutively activated, such as in cancer cells, the nuclear localization of FOXOs is negatively regulated, which results in the transfer of FOXOs to the cytoplasm and loss of their activity. However, in the absence of GF signals, PIP3 will be dephosphorylated by PTEN (phosphatase and tensin homolog), thereby reducing PKB/AKT activity and concomitantly resulting in the loss of FOXO phosphorylation and nuclear accumulation.
According to a previously defined mechanism, FOXOs enter the nucleus, bind to a variety of transcription cofactors, and regulate the transcription of target genes related to the cell cycle, apoptosis, the antioxidant state, metabolism, and angiogenesis. For FOXO6, phosphorylation of two residues (threonine 26 and serine 184) by AKT results in inactivation. Unlike other FOXOs, the PI3K-AKT pathway cannot affect the subcellular localization of FOXO6 due to the lack of carboxy-terminal AKT-dependent phosphorylation sites in FOXO6[11,25,29].
AKT-independent phosphorylation: Inhibition of FOXOs by the PI3K-AKT pathway is believed to enhance tumor development, while stress-activated kinases, such as c-Jun N-terminal kinase (JNK), mammalian sterile 20like kinase 1 (MST1), and protein kinase RNA-like endoplasmic reticulum kinase (PERK), play a tumor inhibitory role by promoting FOXO function in an AKT-independent manner.
Essers et al illustrated that in contrast to insulin-mediated regulation, under oxidative stress, FOXO4 is phosphorylated by JNK on threonine 447 and threonine 451 in a GTPase-dependent manner, which leads to the nuclear translocation of p-FOXO4. Specifically, the regulatory effect of JNK on FOXO activity involves phosphorylation of 14-3-3 on serine 184 to block 14-3-3 proteins from binding to FOXOs[11,31].
Lehtinen et al extended the molecular mechanism by which oxidative stress influences cell survival and homeostasis, by demonstrating the role of the protein kinase MST1 in oxidative stress-induced cell death. In the case of increased cellular oxidative stress, MST1 phosphorylates FOXO proteins at a conserved site to disrupt their interaction with 14-3-3 proteins, which results in FOXO nuclear translocation, and induces neuronal cell death. Soon after, Yuan et al also found that MST1-induced phosphorylation of FOXO1 at serine 212, which corresponds to serine 207 in FOXO3, disrupts the association between FOXO1 and 14-3-3 proteins. The above findings indicate that MST1-FOXO1 signaling is an important link to serum-deprivation-induced neuronal cell death.
Recently, PERK was found to be involved in endoplasmic reticulum (ER) stress related to the onset of type 2 diabetes. Imbalances between protein synthesis and folding lead to ER stress, which partially enhances FOXO activity through the PERK pathway. Interestingly, although three target sites serine 298, serine 301, and serine 303 on FOXO1 can be phosphorylated by PERK, PERK-mediated phosphorylation preferentially occurs on serine 298, which is not a target site for AKT. Phosphorylation by PERK enhances the transcriptional activity of FOXOs and counteracts the effect of Akt phosphorylation[34,35].
In addition, extracellular signalregulated kinase (ERK), p38, cyclin-dependent kinases (CDKs), adenosine monophosphate-activated protein kinase (AMPK), and IκB kinase (IκK) regulate FOXOs in an AKT-independent manner. For example, mitogen-activated protein kinases (MAPKs), ERK, and p38 jointly phosphorylate FOXO1, which results in p-FOXO1 serving as a coactivator for Ets-1. Additionally, ERK mediates the phosphorylation of FOXO3 at serine 294, serine 344, and serine 425, which permits the association of p-FOXO3 with the E3 ubiquitin ligase MDM2 (murine double minute 2). This in turn results in the ubiquitination and degradation of p-FOXO3 to promote cell proliferation and tumorigenesis. CDK2 binds to and phosphorylates FOXO1 at serine 249 in a glucose-dependent manner, and loss of CDK2 may mediate persistent insulin secretion defects through this pathway[38,39]. Lu et al proposed FO1–6nls, a FOXO1-derived peptide inhibitor of CDK1/2-mediated phosphorylation of FOXO1 at serine 249, as a potential therapeutic for the treatment of prostate cancers. AMPK phosphorylates FOXO1 and forms the AMPK/ FOXO1 axis, which is involved in multiple pathological processes, such as liver fibrosis, cardiac hypertrophy, and epithelial-mesenchymal transition (EMT). The phosphorylation of FOXO3 at serine 644 by IκK normally leads to ubiquitin-dependent proteasomal degradation, but causes cytoplasmic retention in acute myeloid leukemia.
Acetylation: Histone acetylation is an epigenetic modification that regulates numerous genes essential for various biological processes, including development and stress responses. It has been reported that calcium response element-binding protein (CBP)/p300 acetylates FOXOs to promote their phosphorylation by AKT and allows FOXOs to be retained in the cytoplasm. However, stress-induced FOXO1 acetylation also arrests FOXO1 ubiquitination and prevents FOXO1 degradation through the ubiquitin-proteasome pathway. Importantly, acetylation of FOXOs is a reversible process and can be eliminated by histone acetyltransferases and histone deacetylases (HDACs)[49,50]. For example, Sirt1, a class III HDAC, can deacetylate FOXOs and increase their transcription. However, this increased effect is eliminated quickly because of the facilitated degradation of deacetylated FOXOs through the ubiquitin-proteasome pathway.
Other posttranslational modifications: In addition to phosphorylation, acetylation, and polyubiquitination, the activity of FOXOs is regulated by other posttranslational modifications, including mono-ubiquitination, methylation, and glycosylation.
In contrast to degradation induced by polyubiquitination, mono-ubiquitination enhances FOXO activity. Interestingly, under oxidative stress, MDM2, which promotes the degradation of p-FOXO3, can induce mono-ubiquitination of FOXO4 to increase FOXO4 nuclear entry and transcriptional activity. Methylation of FOXO1 by protein arginine methyltransferase 1 (PRMT1) inhibits AKT-induced phosphorylation, and thus, promotes FOXO1 retention in the nucleus and increases the expression of downstream target genes. However, methylation of FOXO3 by the Set9 methyltransferase reduces the DNA-binding and transcriptional activities of FOXO3. O-glycosylation improves the transcriptional activity of FOXO1 without influencing its subcellular localization. Recently, N6-methyladenosine modifications of FOXO1 mRNA, reported by Jian et al, were demonstrated to mediate METTL14-induced endothelial inflammation and atherosclerosis. Shin et al identified a novel posttranslational modification of the FOXO family, O-GlcNAcylation of FOXO3 at serine 284, that impairs the ability of FOXO3 to induce subsequent cancer cell growth via abrogation of the p53 regulatory circuit.
Of course, other posttranslational modifications may exist and remain to be discovered. The transcriptional activities of FOXOs are involved in regulating the cell cycle, oxidative stress, apoptosis, and autophagy, as well as metabolic and immunoregulatory factors. Moreover, FOXO3 is closely related to longevity in humans[58-60]. The biological function of FOXO6 has not been well studied, and most research has indicated its participation in glucose and lipid metabolism. Unsurprisingly, FOXOs are involved in many aspects of malignant tumors.