FOXO4-DRI made headlines in 2017 when Dutch researchers showed it could make old and prematurely aged mice look and function younger again - thicker fur, better kidney function, more stamina - by clearing out senescent cells, the cells that stop dividing as we age but refuse to die and instead pump out inflammatory chemicals that damage nearby tissue. Since then, other labs have tested it in mice for low testosterone, declining sperm quality, and thick scar tissue, and structural biologists have mapped out exactly how it locks onto its molecular target. But it has never been given to a human being in a published study, and one high-profile study found that clearing out these old cells with FOXO4-DRI can actually backfire and make certain diseases worse. It remains a research compound, not a medicine.
How strong is the evidence?
Every study specifically on FOXO4-DRI is a mouse experiment, a cell-culture/tissue experiment, or a molecular structural analysis - there are no human trials of any kind. The main finding (that killing senescent cells with this peptide improves how old mice look and function) has been reproduced by more than one lab in more than one mouse model - natural aging, premature-aging genetic mice, low-testosterone aging, and lab-grown scar tissue - which is a reassuring sign of consistency. But a 2023 study in a major cardiology journal found that the same strategy can worsen blood vessel disease in certain lung conditions, showing the approach isn't uniformly safe even in animals. Because there is zero human safety or dosing data, this sits firmly in the preclinical (animal/lab-only) category.
Uses
What people use it for
Senolytic research - clearing aged cells to restore tissue function
Animal / labThe core idea researchers are testing: selectively kill senescent ("zombie") cells that build up with age, so surrounding tissue can work better. In old and prematurely aging mice, this restored fitness, fur growth, and kidney function.
Blunting damage from chemotherapy
Animal / labChemo drugs like doxorubicin create a wave of senescent cells as a side effect. In mice, FOXO4-DRI reduced this chemo-related damage by clearing those cells out.
Reversing age-related low testosterone in animal models
Animal / labResearchers used it to clear out senescent testosterone-producing cells (Leydig cells) in old mice, aiming at a mouse version of age-related low testosterone.
Improving fertility markers in aged animals
Animal / labIn aged mice, clearing senescent Leydig cells with FOXO4-DRI was linked to better sperm quality, an early-stage idea for age-related fertility decline.
Shrinking thick, overgrown scar tissue (keloids)
Animal / labIn lab-grown keloid scar tissue and scar cells, FOXO4-DRI triggered death of the senescent scar-forming cells that keep keloids aggressive and prone to coming back.
Potential benefits
What it may help with
Restored overall condition in old and prematurely aged mice
Animal / labIn the foundational 2017 study, mice with a fast-aging genetic condition, and separately naturally old mice, showed better overall fitness, thicker fur, and improved kidney function after treatment.
Studies:28340339Reduced chemotherapy-related tissue damage in mice
Animal / labFOXO4-DRI neutralized some of the toxic side effects of the chemo drug doxorubicin in treated mice by clearing the senescent cells that drug creates.
Studies:28340339Improved testosterone-related cell function in aged mice
Animal / labIn naturally aged mice, the peptide improved the microenvironment of the testes and eased age-related declines in testosterone production, by killing off senescent hormone-producing cells.
Studies:31959736Better sperm quality in aged mice
Animal / labA follow-up study found FOXO4-DRI improved sperm quality and signs of active sperm production in old mice, again by clearing senescent Leydig cells.
Studies:39025385Killed off aggressive scar-tissue cells in lab samples
Animal / labIn keloid (overgrown scar) tissue and cells grown in the lab, FOXO4-DRI pushed senescent scar-forming cells into cell death, a proposed way to make keloids less likely to keep growing or return.
Studies:39994346
What to watch for
Side effects & risks
- Serious
Can worsen blood vessel disease in the lungs (in mice)
A 2023 study found that clearing senescent cells with FOXO4-DRI (or a different senolytic drug) in certain mouse models actually made pulmonary hypertension - high blood pressure in the lung's blood vessels - worse, by also killing off senescent cells that were protecting the blood vessel lining. This is a real, documented downside, not just a theoretical one.
- Moderate
Removing "aged" cells can remove ones that were doing useful work
Senescent cells aren't purely harmful - some help with wound healing, tissue repair, and protecting blood vessels. Wiping them out with a senolytic can strip away that benefit along with the harm. This is a general concern raised by senolytic researchers rather than a side effect measured in a specific FOXO4-DRI human study.
- Serious
Completely unknown safety profile in humans
No published human study has given FOXO4-DRI to a person, so there's no data on allergic reactions, injection-site effects, organ toxicity, or interactions with other health conditions at the doses people might be tempted to use.
Dosing
Dosing — what studies used
There is no established human dose for FOXO4-DRI - it has never been used in a published human study. The animal research we reviewed describes giving it to mice and calls it "well tolerated," but the abstracts available to us don't spell out a specific milligram dose, injection schedule, or treatment length. Because of that, we can't responsibly print a dosing protocol. Anyone using FOXO4-DRI outside of a laboratory is working from an unverified regimen, not a research-backed one - this should be treated as a serious gap, not a minor detail.
Do not treat any dose you see online (forums, vendor sites, social media) as backed by these studies - the actual published animal research does not report exact dosing details in the material we reviewed, and there is no human dosing data at all.
These figures describe what researchers used in studies. They are not a recommendation or a prescription.
Mechanism
How it works
FOXO4-DRI is a short, lab-built peptide (a small chain of amino acids, the building blocks of proteins) designed to copy part of a natural protein called FOXO4. Inside old, worn-out cells that refuse to die (called senescent cells), FOXO4 grabs onto another protein called p53 and holds it inside the cell's control center, the nucleus. Normally p53 is the protein that tells a damaged cell to self-destruct, but trapped there it can't do its job, so the worn-out cell survives indefinitely and keeps releasing inflammatory chemicals into nearby tissue. FOXO4-DRI acts like a decoy: it pushes its way into that FOXO4-p53 partnership and breaks it apart. Once freed, p53 leaves the nucleus and triggers the cell's own self-destruct program, but only in cells that were already senescent - healthy young cells are left alone. That selective cell-killing action is why it's called a "senolytic" (senescence + lytic, meaning cell-destroying).
Who should avoid it
- Anyone with active cancer or a history of cancer - FOXO4-DRI works by interfering with p53, one of the body's main tumor-suppressor proteins, and disrupting that system carries a theoretical cancer-related risk that hasn't been ruled out.
- Anyone with lung, heart, or blood vessel disease - the one specific safety study on record found this class of treatment can worsen blood vessel problems in the lungs in certain animal disease models.
- Pregnant or breastfeeding people - no safety data exists at all.
- Anyone expecting a medically supervised, dosed treatment - none exists; this is a research chemical, not an approved or clinically dosed product.
Interactions to know
- Not established in humans - no published human interaction data exists.
- Animal studies combined it with the chemotherapy drug doxorubicin to test whether it reduces chemo-related tissue damage, but this is not the same as knowing how it interacts with medications in people.
The papers that matter most
Key studies
The foundational study: designed the FOXO4-DRI peptide, showed it kills senescent cells and restored fitness, fur density, and kidney function in fast-aging and naturally old mice, and blunted chemo drug damage.
Targeted Apoptosis of Senescent Cells Restores Tissue Homeostasis in Response to Chemotoxicity and Aging
An important caution: clearing senescent cells with FOXO4-DRI (or a comparable senolytic drug) worsened blood vessel disease in the lungs in specific mouse models, showing the strategy isn't universally safe.
Eliminating Senescent Cells Can Promote Pulmonary Hypertension Development and Progression
In aged mice, the peptide cleared senescent testosterone-producing cells and eased age-related declines in testosterone production.
FOXO4-DRI alleviates age-related testosterone secretion insufficiency by targeting senescent Leydig cells in aged mice
Follow-up study showing improved sperm quality and testicular function in old mice after clearing senescent Leydig cells.
FOXO4-DRI improves spermatogenesis in aged mice through reducing senescence-associated secretory phenotype secretion from Leydig cells
In lab-grown keloid (overgrown scar) tissue, FOXO4-DRI killed off senescent scar-forming cells, suggesting a possible approach to stubborn scarring.
FOXO4-DRI induces keloid senescent fibroblast apoptosis by promoting nuclear exclusion of upregulated p53-serine 15 phosphorylation
Detailed molecular mapping of exactly how FOXO4-DRI binds and disrupts the FOXO4-p53 interaction, confirming and refining the mechanism first proposed in 2017.
The disordered p53 transactivation domain is the target of FOXO4 and the senolytic compound FOXO4-DRI
Bottom line
FOXO4-DRI is one of the most famous experimental "senolytic" peptides in aging science, and its animal results are genuinely striking - but it has never been tested in a human clinical trial, has no established dose, and one solid study found it can make certain diseases worse rather than better. This belongs in a research lab, not in a personal injection routine.
Research papers
Studies we have on file for FOXO4-DRI. Tap a title to open it on PubMed. Labels like “animal” or “human trial” are rough guides.
40 papers
Eliminating Senescent Cells Can Promote Pulmonary Hypertension Development and Progression.
Senescent cells (SCs) are involved in proliferative disorders, but their role in pulmonary hypertension remains undefined. We investigated SCs in patients with pulmonary arterial hypertension and the role of SCs in animal pulmonary hypertension models. We investigated senescence (p16, p21) and DNA damage (γ-H2AX, 53BP1) markers in patients with pulmonary arterial hypertension and murine models. We monitored p16 activation by luminescence imaging in p16-luciferase (p16LUC/+) knock-in mice. SC clearance was obtained by a suicide gene (p16 promoter-driven killer gene construct in p16-ATTAC mice), senolytic drugs (ABT263 and cell-permeable FOXO4-p53 interfering peptide [FOXO4-DRI]), and p16 inactivation in p16LUC/LUC mice. We investigated pulmonary hypertension in mice exposed to normoxia, chronic hypoxia, or hypoxia+Sugen, mice overexpressing the serotonin transporter (SM22-5-HTT+), and rats given monocrotaline. Patients with pulmonary arterial hypertension compared with controls exhibited high lung p16, p21, and γ-H2AX protein levels, with abundant vascular cells costained for p16, γ-H2AX, and 53BP1. Hypoxia increased thoracic bioluminescence in p16LUC/+ mice. In wild-type mice, hypoxia increased lung levels of senescence and DNA-damage markers, senescence-associated secretory phenotype components, and p16 staining of pulmonary endothelial cells (P-ECs, 30% of lung SCs in normoxia), and pulmonary artery smooth muscle cells. SC elimination by suicide gene or ABT263 increased the right ventricular systolic pressure and hypertrophy index, increased vessel remodeling (higher dividing proliferating cell nuclear antigen-stained vascular cell counts during both normoxia and hypoxia), and markedly decreased lung P-ECs. Pulmonary hemodynamic alterations and lung P-EC loss occurred in older p16LUC/LUC mice, wild-type mice exposed to Sugen or hypoxia+Sugen, and SM22-5-HTT+ mice given either ABT263 or FOXO4-DRI, compared with relevant controls. The severity of monocrotaline-induced pulmonary hypertension in rats was decreased slightly by ABT263 for 1 week but was aggravated at 3 weeks, with loss of P-ECs. Elimination of senescent P-ECs by senolytic interventions may worsen pulmonary hemodynamics. These results invite consideration of the potential impact on pulmonary vessels of strategies aimed at controlling cell senescence in various contexts.
Targeted Apoptosis of Senescent Cells Restores Tissue Homeostasis in Response to Chemotoxicity and Aging.
The accumulation of irreparable cellular damage restricts healthspan after acute stress or natural aging. Senescent cells are thought to impair tissue function, and their genetic clearance can delay features of aging. Identifying how senescent cells avoid apoptosis allows for the prospective design of anti-senescence compounds to address whether homeostasis can also be restored. Here, we identify FOXO4 as a pivot in senescent cell viability. We designed a FOXO4 peptide that perturbs the FOXO4 interaction with p53. In senescent cells, this selectively causes p53 nuclear exclusion and cell-intrinsic apoptosis. Under conditions where it was well tolerated in vivo, this FOXO4 peptide neutralized doxorubicin-induced chemotoxicity. Moreover, it restored fitness, fur density, and renal function in both fast aging XpdTTD/TTD and naturally aged mice. Thus, therapeutic targeting of senescent cells is feasible under conditions where loss of health has already occurred, and in doing so tissue homeostasis can effectively be restored.
FOXO transcription factors as therapeutic targets in human diseases.
Forkhead box (FOX)O proteins are transcription factors (TFs) with four members in mammals designated FOXO1, FOXO3, FOXO4, and FOXO6. FOXO TFs play a pivotal role in the cellular adaptation to diverse stress conditions. FOXO proteins act as context-dependent tumor suppressors and their dysregulation has been implicated in several age-related diseases. FOXO3 has been established as a major gene for human longevity. Accordingly, FOXO proteins have emerged as potential targets for the therapeutic development of drugs and geroprotectors. In this review, we provide an overview of the most recent advances in our understanding of FOXO regulation and function in various pathological conditions. We discuss strategies targeting FOXOs directly or by the modulation of upstream regulators, shedding light on the most promising intervention points. We also reveal the most relevant clinical indications and discuss the potential, trends, and challenges of modulating FOXO activity for therapeutic purposes.
Damaged brain accelerates bone healing by releasing small extracellular vesicles that target osteoprogenitors.
Clinical evidence has established that concomitant traumatic brain injury (TBI) accelerates bone healing, but the underlying mechanism is unclear. This study shows that after TBI, injured neurons, mainly those in the hippocampus, release osteogenic microRNA (miRNA)-enriched small extracellular vesicles (sEVs), which targeted osteoprogenitors in bone to stimulate bone formation. We show that miR-328a-3p and miR-150-5p, enriched in the sEVs after TBI, promote osteogenesis by directly targeting the 3'UTR of FOXO4 or CBL, respectively, and hydrogel carrying miR-328a-3p-containing sEVs efficiently repaires bone defects in rats. Importantly, increased fibronectin expression on sEVs surface contributes to targeting of osteoprogenitors in bone by TBI sEVs, thereby implying that modification of the sEVs surface fibronectin could be used in bone-targeted drug delivery. Together, our work unveils a role of central regulation in bone formation and a clear link between injured neurons and osteogenitors, both in animals and clinical settings.
FOXO transcription factors as mediators of stress adaptation.
The forkhead box protein O (FOXO, consisting of FOXO1, FOXO3, FOXO4 and FOXO6) transcription factors are the mammalian orthologues of Caenorhabditis elegans DAF-16, which gained notoriety for its capability to double lifespan in the absence of daf-2 (the gene encoding the worm insulin receptor homologue). Since then, research has provided many mechanistic details on FOXO regulation and FOXO activity. Furthermore, conditional knockout experiments have provided a wealth of data as to how FOXOs control development and homeostasis at the organ and organism levels. The lifespan-extending capabilities of DAF-16/FOXO are highly correlated with their ability to induce stress response pathways. Exogenous and endogenous stress, such as cellular redox stress, are considered the main drivers of the functional decline that characterizes ageing. Functional decline often manifests as disease, and decrease in FOXO activity indeed negatively impacts on major age-related diseases such as cancer and diabetes. In this context, the main function of FOXOs is considered to preserve cellular and organismal homeostasis, through regulation of stress response pathways. Paradoxically, the same FOXO-mediated responses can also aid the survival of dysfunctional cells once these eventually emerge. This general property to control stress responses may underlie the complex and less-evident roles of FOXOs in human lifespan as opposed to model organisms such as C. elegans.
The disordered p53 transactivation domain is the target of FOXO4 and the senolytic compound FOXO4-DRI.
A central process contributing to the phenotype of aging is cellular senescence. We recently identified the FOXO4 - p53 axis as pivotal in maintaining the viability of senescent cells, and that senescent cells can be targeted selectively with the senolytic peptide FOXO4-DRI. Here, we solve the solution NMR structural models of the p53 transactivation domain in complex with the FOXO4 forkhead domain and in complex with FOXO4-DRI. Strikingly, we find that the disordered FOXO4-DRI binds to the disordered p53TAD2 and forms a transiently folded complex. In this complex, both, the FOXO4-derived region and the cationic cell permeability peptide contribute to the interaction. Furthermore, we show that p53 phosphorylation enhances the affinity for both FOXO4 and FOXO4-DRI. Summarizing we provide a detailed characterization of the interaction of p53 with FOXO4 and FOXO4-DRI which is the basis for development of p53 inhibitors to treat diseases linked to cellular senescence such as cancers.
Structural plasticity of the FOXO-DBD:p53-TAD interaction.
The transcription factors FOXO4 and p53 regulate aging, and their deregulation has been linked to several diseases, including cancer. Under stress conditions, cellular senescence is promoted by p53 sequestration and senescence-associated protein p21 transcriptional upregulation induced by interactions between the FOXO4 Forkhead DNA-binding domain and the p53 transactivation domain. However, the molecular details of these interactions remain unclear. Here, we report that these interactions between p53 and FOXO4 domains are highly heterogeneous. The p53 transactivation domain primarily interacts with the region formed by the N-terminal helical bundle of the FOXO4 Forkhead domain but retains a substantial degree of flexibility in the complex. In addition, NMR data-driven molecular simulations suggest that p53 interacts with FOXO4 through multiple binding modes. Overall, our findings not only provide the structural insights into interactions between FOXO4 and p53 but also highlight their potential as targets for developing senolytic compounds.
Human FOX gene family (Review).
Human Forkhead-box (FOX) gene family consists of at least 43 members, including FOXA1, FOXA2, FOXA3, FOXB1, FOXC1, FOXC2, FOXD1, FOXD2, FOXD3, FOXD4, FOXD5 (FOXD4L1), FOXD6 (FOXD4L3), FOXE1, FOXE2, FOXE3, FOXF1, FOXF2, FOXG1 (FOXG1B), FOXH1, FOXI1, FOXJ1, FOXJ2, FOXJ3, FOXK1, FOXK2, FOXL1, FOXL2, FOXM1, FOXN1, FOXN2 (HTLF), FOXN3 (CHES1), FOXN4, FOXN5 (FOXR1), FOXN6 (FOXR2), FOXO1 (FOXO1A), FOXO2 (FOXO6), FOXO3 (FOXO3A), FOXO4 (MLLT7), FOXP1, FOXP2, FOXP3, FOXP4, and FOXQ1. FOXE3-FOXD2 (1p33), FOXQ1-FOXF2-FOXC1 (6p25.3), and FOXF1-FOXC2-FOXL1 (16q24.1) loci are FOX gene clusters within the human genome. Members of FOX subfamilies A-G, I-L and Q were grouped into class 1 FOX proteins, while members of FOX subfamilies H and M-P were grouped into class 2 FOX proteins. C-terminal basic region within the FOX domain was the common feature of class 1 FOX proteins. FOXH1 and FOXO1 mRNAs are expressed in human embryonic stem (ES) cells. FOXC1, FOXC2, FOXE1, FOXE3, FOXL2, FOXN1, FOXP2 and FOXP3 genes are mutated in human congenital disorders. FOXA1 gene is amplified and over-expressed in esophageal and lung cancer. FOXM1 gene is up-regulated in pancreatic cancer and basal cell carcinoma due to the transcriptional regulation by Sonic Hedgehog (SHH) pathway. FOXO1 gene is fused to PAX3 or PAX7 genes in rhabdomyosarcoma. FOXO3 and FOXO4 genes are fused to MLL gene in hematological malignancies. Deregulation of FOX family genes leads to congenital disorders, diabetes mellitus, or carcinogenesis. Expression profiles, genetic alterations and epigenetic changes of FOX family genes as well as binding proteins and target genes of FOX family transcription factors should be comprehensively investigated to develop novel therapeutics and preventives for human diseases.
FOXOs: masters of the equilibrium.
Forkhead box O (FOXO) transcription factors (TFs) are a subclass of the larger family of forkhead TFs. Mammalians express four members FOXO1, FOXO3, FOXO4, and FOXO6. The interest in FOXO function stems mostly from their observed role in determining lifespan, where in model organisms, increased FOXO activity results in extended lifespan. FOXOs act as downstream of several signaling pathway and are extensively regulated through post-translational modifications. The transcriptional program activated by FOXOs in various cell types, organisms, and under various conditions has been described and has shed some light on what the critical transcriptional targets are in mediating FOXO function. At the cellular level, these studies have revealed a role for FOXOs in cell metabolism, cellular redox, cell proliferation, DNA repair, autophagy, and many more. The general picture that emerges hereof is that FOXOs act to preserve equilibrium, and they are important for cellular homeostasis. Here, we will first briefly summarize the general knowledge of FOXO regulation and possible functions. We will use genomic stability to illustrate how FOXOs ensure homeostasis. Genomic stability is critical for maintaining genetic integrity, and therefore preventing disease. However, genomic mutations need to occur during lifetime to enable evolution, yet their accumulation is believed to be causative to aging. Therefore, the role of FOXO in genomic stability may underlie its role in lifespan and aging. Finally, we will come up with questions on some of the unknowns in FOXO function, the answer(s) to which we believe will further our understanding of FOXO function and ultimately may help to understand lifespan and its consequences.
Introduction to FOXO Biology.
Forkhead box O (FOXO) proteins are a family of transcription factors with four members in mammals, namely FOXO1, FOXO3a, FOXO4, and FOXO6. FOXO factors, originally identified as downstream regulators of the insulin pathway, are known to bind to the promoters of a broad variety of target genes and control several processes of key importance for cellular homeostasis including cellular energy production, oxidative stress resistance, and cell viability and proliferation. Accordingly, deregulation of FOXO proteins has been shown to play an essential role in metabolic disorders, human longevity, and the suppression of tumors. As the activity of these transcription factors is controlled by posttranslational modifications, inactivation of FOXOs occurs mostly due to the overactivation of their upstream modifying enzymes providing a wealth of possibilities for restoring FOXO activity pharmaceutically.
Hyperandrogen-induced imbalance of FOXO4-AR regulatory loop contributes to ovulatory disorders in polycystic ovary syndrome.
Polycystic ovary syndrome (PCOS) is the leading cause of anovulatory infertility, and its underlying mechanisms remain largely unknown. Our study aimed to investigate the role of FOXO4 in PCOS and its possible regulatory mechanisms. Decreased FOXO4 and CDKN1A expressions and increased androgen receptor (AR) and CCND1 expressions were observed in granulosa cells (GCs) from patients with PCOS. Luteinizing hormone (LH) surge induced upregulation of FOXO4 and CDKN1A and downregulation of AR and CCND1 in vitro and in vivo. FOXO4 inhibited cell proliferation and cell cycle progression and partially mediated the induction of CCND1 and CDKN1A expressions by LH surge. Knockdown of FOXO4 in rat ovaries led to a PCOS-like model, and hyperandrogenism was responsible for reduced FOXO4 expression in ovaries of PCOS in vitro and in vivo. AR-mediated androgen action is known to play a key role in the development of PCOS. Notably, AR repressed FOXO4 expression by binding to its promoter, whereas FOXO4 inhibited AR protein levels through protease degradation, thus establishing a regulatory loop between AR and FOXO4 that was disrupted by hyperandrogenism. This study demonstrates that hyperandrogenism inhibits LH surge formation and disrupts the regulatory balance between FOXO4 and AR, which may contribute to the continued exacerbation of PCOS.
FOXO Transcription Factors: A Brief Overview.
Forkhead box O (FOXO) transcription factors constitute a mammalian family of proteins, comprising FOXO1, FOXO3, FOXO4, and FOXO6. Originally recognized as downstream regulators within the insulin pathway, FOXO factors exhibit the ability to bind to diverse target gene promoters, thereby governing crucial facets of cellular homeostasis. These encompass cellular energy generation, resilience against oxidative stress, and the modulation of cell viability and proliferation. The dysregulation of FOXO proteins has been established as pivotal in metabolic disorders, human longevity, and the inhibition of tumorigenesis. Notably subject to posttranslational modifications for regulation, FOXO inactivation predominantly arises from excessive activation of their upstream modifying enzymes, presenting a plethora of potential avenues for pharmaceutical reinstatement of FOXO activity.
Pseudokinase STK40 promotes TH1 and TH17 cell differentiation by targeting FOXO transcription factors.
Inappropriate CD4+ T helper (TH) cell differentiation leads to progression of inflammatory and autoimmune diseases, yet the regulatory mechanisms governing stability and activity of transcription factors controlling TH cell differentiation remain elusive. Here, we describe how pseudokinase serine threonine kinase 40 (STK40) facilitates TH1/TH17 differentiation under pathological conditions. STK40 in T cells is dispensable for immune homeostasis in resting mice. However, mice with T cell-specific deletion of STK40 exhibit attenuated symptoms of experimental autoimmune encephalomyelitis and colitis, accompanied by diminished TH1 and TH17 cell differentiation. Mechanistically, STK40 facilitates K48-linked polyubiquitination and proteasomal degradation of FOXO1/4 through promoting their interaction with E3 ligase COP1. Inhibition of FOXO4 or FOXO1, respectively, restores differentiation potential of STK40-deficient TH1/TH17 cells. Together, our data suggest a crucial role of STK40 in TH1 and TH17 cell differentiation, thereby enabling better understanding of the molecular regulatory network of CD4+ T cell differentiation and providing effective targets for the treatment of autoimmune diseases.
FOXO4-DRI induces keloid senescent fibroblast apoptosis by promoting nuclear exclusion of upregulated p53-serine 15 phosphorylation.
Keloids are pathological scars exhibiting tumour-like aggressiveness and high recurrence rate. Here we find increased proportion of pro-inflammatory and mesenchymal fibroblast subpopulations and senescent fibroblasts, and enhanced expression of senescence-associated secretory phenotype genes using single-cell RNA sequencing analysis, as well as elevated p16 protein and more β-galactosidase-positive cells in keloids. The up-regulated p53-serine15 phosphorylation (p53-pS15) in keloids is identified by phosphospecific protein microarray and western blotting. We further demonstrate that a senolytic FOXO4-D-retro-inverso-isoform peptide (FOXO4-DRI) promotes apoptosis and decreases G0/G1 phase cells in pro-senescence models of keloid organ cultures and fibroblasts, accompanied with p53-pS15 nuclear exclusion. Our study indicates that upregulation of p53-pS15 and p16 maintains a persistent senescent microenvironment to promote cell cycle arrest and apoptosis resistance in keloid fibroblasts. FOXO4-DRI shows potential as a treatment targeting the senescence and apoptosis resistance, and holds promise as an approach to prevent the aggressiveness and relapse of keloids.
Intermolecular disulfide-dependent redox signalling.
Until recently, ROS (reactive oxygen species) were often seen as merely damaging agents. However, small, but significant, amounts of hydrogen peroxide (H2O2) are also being produced upon, for instance, NADPH-oxidase activation in response to growth factor signalling and as a by-product of mitochondrial respiration. H2O2 perturbs the local cellular redox state and this results in specific and reversible cysteine oxidation in target proteins, thereby translating the redox state into a signal that ultimately leads to an appropriate cellular response. This phenomenon of signalling through cysteine oxidation is known as redox signalling and has recently been shown to be involved in a wide range of physiological processes. Cysteine residue oxidation can lead to a range of post-translational modifications, one of which is the formation of intermolecular disulfides. In the present mini-review we will give a number of examples of proteins regulated by intermolecular disulfides and discuss a recently developed method to screen for these interactions. The consequences of the regulation of the FOXO4 (forkhead box O4) transcription factor by formation of intermolecular disulfides with both TNPO1 (transportin 1) and p300/CBP [CREB (cAMP-response-element-binding protein)-binding protein] are discussed in more detail.
FOXO4-SP6 axis controls surface epithelium commitment by mediating epigenomic remodeling.
Proper development of surface epithelium (SE) is a requisite for the normal development and function of ectodermal appendages; however, the molecular mechanisms underlying SE commitment remain largely unexplored. Here, we developed a KRT8 reporter system and utilized it to identify FOXO4 and SP6 as novel, essential regulators governing SE commitment. We found that the FOXO4-SP6 axis governs SE fate and its abrogation markedly impedes SE fate determination. Mechanistically, FOXO4 regulates SE initiation by shaping the SE chromatin accessibility landscape and regulating the deposition of H3K4me3. SP6, as a novel effector of FOXO4, activates SE-specific genes through modulating the H3K27ac deposition across their super-enhancers. Our work highlights the regulatory function of the FOXO4-SP6 axis in SE development, contributing to an improved understanding of SE fate decisions and providing a research foundation for the therapeutic application of ectodermal dysplasia.
FoxO3a and disease progression.
The Forkhead box O (FoxO) family has recently been highlighted as an important transcriptional regulator of crucial proteins associated with the many diverse functions of cells. So far, FoxO1, FoxO3a, FoxO4 and FoxO6 proteins have been identified in humans. Although each FoxO family member has its own role, unlike the other FoxO families, FoxO3a has been extensively studied because of its rather unique and pivotal regulation of cell proliferation, apoptosis, metabolism, stress management and longevity. FoxO3a alteration is closely linked to the progression of several types of cancers, fibrosis and other types of diseases. In this review, we will examine the function of FoxO3a in disease progression and also explore FoxO3a's regulatory mechanisms. We will also discuss FoxO3a as a potential target for the treatment of several types of disease.
FoxO integration of insulin signaling with glucose and lipid metabolism.
The forkhead box O family consists of FoxO1, FoxO3, FoxO4 and FoxO6 proteins in mammals. Expressed ubiquitously in the body, the four FoxO isoforms share in common the amino DNA-binding domain, known as 'forkhead box' domain. They mediate the inhibitory action of insulin or insulin-like growth factor on key functions involved in cell metabolism, growth, differentiation, oxidative stress, senescence, autophagy and aging. Genetic mutations in FoxO genes or abnormal expression of FoxO proteins are associated with metabolic disease, cancer or altered lifespan in humans and animals. Of the FoxO family, FoxO6 is the least characterized member and is shown to play pivotal roles in the liver, skeletal muscle and brain. Altered FoxO6 expression is associated with the pathogenesis of insulin resistance, dietary obesity and type 2 diabetes and risk of neurodegeneration disease. FoxO6 is evolutionally divergent from other FoxO isoforms. FoxO6 mediates insulin action on target genes in a mechanism that is fundamentally different from other FoxO members. Here, we focus our review on the role of FoxO6, in contrast with other FoxO isoforms, in health and disease. We review the distinctive mechanism by which FoxO6 integrates insulin signaling to hepatic glucose and lipid metabolism. We highlight the importance of FoxO6 dysregulation in the dual pathogenesis of fasting hyperglycemia and hyperlipidemia in diabetes. We review the role of FoxO6 in memory consolidation and its contribution to neurodegeneration disease and aging. We discuss the potential therapeutic option of pharmacological FoxO6 inhibition for improving glucose and lipid metabolism in diabetes.
Peptide Inhibitors Targeting FOXO4-p53 Interactions and Inducing Senescent Cancer Cell-specific Apoptosis.
Cellular senescence, marked by irreversible cell cycle arrest and senescence-associated secretory phenotype, contributes to aging and cancer recurrence. While chemotherapy can induce senescence in cancer cells, these therapy-induced senescent cells often resist apoptosis and promote tumor recurrence. The nuclear interaction between FOXO4 and p53 is crucial for senescent cell survival. Using NMR spectroscopy, we identified that hydrophobic interactions in the p53 transactivation domain play a key role in FOXO4 forkhead domain binding. Based on this structural information, we designed an optimized peptide inhibitor with reduced negative charges and incorporated a cationic cell-penetrating peptide for enhanced cellular delivery (CPP-CAND). CPP-CAND exhibited high selectivity for senescent cells, effectively disrupting nuclear FOXO4-p53 foci and inducing caspase-dependent apoptosis. Notably, it showed cytotoxicity against senescent cancer cells induced by different chemotherapeutic agents including doxorubicin and cisplatin. With its enhanced selectivity, l-amino acid composition, and shorter length, CPP-CAND represents a promising therapeutic candidate for targeting therapy-induced senescent cancer cells.
The role of FOXO4/NFAT2 signaling pathway in dysfunction of human coronary endothelial cells and inflammatory infiltration of vasculitis in Kawasaki disease.
The Ca+/NFAT (Nuclear factor of activated T cells) signaling pathway activation is implicated in the pathogenesis of Kawasaki disease (KD); however, we lack detailed information regarding the regulatory network involved in the human coronary endothelial cell dysfunction and cardiovascular lesion development. Herein, we aimed to use mouse and endothelial cell models of KD vasculitis in vivo and in vitro to characterize the regulatory network of NFAT pathway in KD. Among the NFAT gene family, NFAT2 showed the strongest transcriptional activity in peripheral blood mononuclear cells (PBMCs) from patients with KD. Then, NFAT2 overexpression and knockdown experiments in Human coronary artery endothelial cells (HCAECs) indicated that NFAT2 overexpression disrupted endothelial cell homeostasis by regulation of adherens junctions, whereas its knockdown protected HCAECs from such dysfunction. Combined analysis using RNA-sequencing and transcription factor (TF) binding site analysis in the NFAT2 promoter region predicted regulation by Forkhead box O4 (FOXO4). Western blotting, chromatin immunoprecipitation, and luciferase assays validated that FOXO4 binds to the promoter and transcriptionally represses NFAT2. Moreover, Foxo4 knockout increased the extent of inflamed vascular tissues in a mouse model of KD vasculitis. Functional experiments showed that inhibition NFAT2 relieved Foxo4 knockout exaggerated vasculitis in vivo. Our findings revealed the FOXO4/NFAT2 axis as a vital pathway in the progression of KD that is associated with endothelial cell homeostasis and cardiovascular inflammation development.
The FoxO4/DKK3 axis represses IFN-γ expression by Th1 cells and limits antimicrobial immunity.
Forkhead box O transcriptional factors, especially FoxO1 and FoxO3a, play critical roles in physiologic and pathologic immune responses. However, the function of FoxO4, another main member of the FoxO family, in lymphoid cells is still poorly understood. Here, we showed that loss of FoxO4 in T cells augmented IFN-γ production of Th1 cells in vitro. Correspondingly, conditional deletion of FoxO4 in CD4+ T cells enhanced T cell-specific responses to Listeria monocytogenes infection in vivo. Genome-wide occupancy and transcriptomic analyses identified Dkk3 (encoding the Dickkopf-3 protein) as a direct transcriptional target of FoxO4. Consistent with the FoxO4-DKK3 relationship, recombinant DKK3 protein restored normal levels of IFN-γ production in FoxO4-deficient Th1 cells through the downregulation of lymphoid enhancer-binding factor 1 (Lef1) expression. Together, our data suggest a potential FoxO4/DKK3 axis in Th1 cell differentiation, providing what we believe to be an important insight and supplement for FoxO family proteins in T lymphocyte biology and revealing a promising target for the treatment of immune-related diseases.
Multiomic analysis implicates FOXO4 in genetic regulation of chick lens fiber cell differentiation.
A classic model for identification of novel differentiation mechanisms and pathways is the eye lens that consists of a monolayer of quiescent epithelial cells that are the progenitors of a core of mature fully differentiated fiber cells. The differentiation of lens epithelial cells into fiber cells follows a coordinated program involving cell cycle exit, expression of key structural proteins and the hallmark elimination of organelles to achieve transparency. Although multiple mechanisms and pathways have been identified to play key roles in lens differentiation, the entirety of mechanisms governing lens differentiation remain to be discovered. A previous study established that specific chromatin accessibility changes were directly associated with the expression of essential lens fiber cell genes, suggesting that the activity of transcription factors needed for expression of these genes could be regulated through binding access to the identified chromatin regions. Sequence analysis of the identified chromatin accessible regions revealed enhanced representation of the binding sequence for the transcription factor FOXO4 suggesting a direct role for FOXO4 in expression of these genes. FOXO4 is known to regulate a variety of cellular processes including cellular response to metabolic and oxidative stress, cell cycle withdrawal, and homeostasis, suggesting a previously unidentified role for FOXO4 in the regulation of lens cell differentiation. To further evaluate the role of FOXO4 we employed a multiomics approach to analyze the relationship between genome-wide FOXO4 binding, the differentiation-specific expression of key genes, and chromatin accessibility. To better identify active promoters and enhancers we also examined histone modification through analysis of H3K27ac. Specific methods included CUT&RUN (FOXO4 binding and H3K27ac modification), RNA-seq (differentiation state specific gene expression), and ATAC-seq (chromatin accessibility). CUT&RUN identified 20,966 FOXO4 binding sites and 33,921 H3K27ac marked regions across the lens fiber cell genome. RNA-seq identified 956 genes with significantly greater expression levels in fiber cells compared to epithelial cells (log2FC > 0.7, q < 0.05) and 2548 genes with significantly lower expression levels (log2FC < -0.7, q < 0.05). Integrated analysis identified 1727 differentiation-state specific genes that were nearest neighbors to at least one FOXO4 binding site, including genes encoding lens gap junctions (GJA1, GJA3), lens structural proteins (BFSP1, CRYBB1, ASL1), and genes required for lens transparency (HSF4, NRCAM). Multiomics analysis comparing the identified FOXO4 binding sites in published ATAC-seq data revealed that chromatin accessibility was associated with FOXO4-dependent gene expression during lens differentiation. The results provide evidence for an important requirement for FOXO4 in the regulated expression of key genes required for lens differentiation and link epigenetic regulation of chromatin accessibility and H3K27ac histone modification with the function of FOXO4 in controlling lens gene expression during lens fiber cell differentiation.
Hypoxia-induced FOXO4/LDHA axis modulates gastric cancer cell glycolysis and progression.
We previously identified forkhead box (FOX) O4 mRNA as a predictor in gastric cancer (GC). However, the underlying mechanism has yet to be elucidated. We aimed to illustrate the mechanism by which FOXO4 regulated glycolysis under hypoxia in GC. FOXO4 protein expression was investigated by immunohistochemical staining of 252 GC and their normal adjacent tissues. We restored or silenced FOXO4 expression in GC cell lines to explore the underlying mechanisms. FOXO4 was downregulated in GC. Loss of FOXO4 expression was validated in univariate and multivariate survival analysis as an independent prognostic predictor for overall survival (P < 0.05) and disease-free survival (P<0.05). Restored FOXO4 expression significantly impaired the glycolysis rate in GC cells, while silencing FOXO4 expression enhanced glycolysis rate. FOXO4 expression was inversely associated with maximum standardized uptake value in mice models and patient samples. Mechanistically, FOXO4 bound to the glycolytic enzyme lactate dehydrogenase (LDH)A promoter and inactivated its activity in a dose-dependent manner (P < 0.05). Finally, we determined that FOXO4 was a transcriptional target of hypoxia-inducible factor (HIF) -1α, which is central in response to hypoxia. Our data suggested that FOXO4 plays a key role in the regulation of glycolysis in GC, and disrupting the HIF-1α-FOXO4-LDHA axis might be a promising therapeutic strategy for GC.
The multifaceted impact of physical exercise on FoxO signaling pathways.
This review explores the multifaceted impact of physical exercise on FoxO signaling pathways, which play a central role in cellular homeostasis, stress response, metabolism, and longevity. Exercise influences FoxO proteins-particularly FoxO1, FoxO3, FoxO4, and FoxO6-through diverse mechanisms, including phosphorylation, acetylation, and ubiquitination, determining their localization, transcriptional activity, and stability. Regular exercise modulates FoxO signaling by activating pathways like PI3K/AKT, AMPK, SIRT1, and IGF-1, promoting cellular resilience against oxidative stress, apoptosis, and metabolic dysfunction. The review highlights how exercise-induced modulation of FoxO pathways contributes to improved insulin sensitivity, muscle hypertrophy, cardiovascular health, neuroprotection, and reduced risks of chronic diseases, including metabolic syndrome, neurodegeneration, cardiovascular disease, and cancer. Additionally, it addresses the role of exercise in preventing muscle atrophy under various conditions, such as pharmacological interventions, aging, disease, and dietary factors. By enhancing FoxO signaling, exercise promotes anabolic processes, mitochondrial function, autophagy, and antioxidant defenses. Understanding the intricate relationship between exercise and FoxO pathways offers insights into developing therapeutic strategies to mitigate disease progression.
Dynamic FoxO transcription factors.
Forkhead box O (FoxO) transcription factors FoxO1, FoxO3a, FoxO4 and FoxO6, the mammalian orthologs of Caenorhabditis elegans DAF-16, are emerging as an important family of proteins that modulate the expression of genes involved in apoptosis, the cell cycle, DNA damage repair, oxidative stress, cell differentiation, glucose metabolism and other cellular functions. FoxO proteins are regulated by multiple mechanisms. They undergo inhibitory phosphorylation by protein kinases such as Akt, SGK, IKK and CDK2 in response to external and internal stimuli. By contrast, they are activated by upstream regulators such as JNK and MST1 under stress conditions. Their activities are counterbalanced by the acetylases CBP and p300 and the deacetylase SIRT1. Also, whereas polyubiquitylation of FoxO1 and FoxO3a leads to their degradation by the proteasome, monoubiquitylation of FoxO4 facilitates its nuclear localization and augments its transcriptional activity. Thus, the potent functions of FoxO proteins are tightly controlled by complex signaling pathways under physiological conditions; dysregulation of these proteins may ultimately lead to disease such as cancer.
Bioinformatics procedure for investigating senolytic (anti-aging) agents: A digital signal processing technique.
Cell growth involves cell division. This stops after reaching a certain limit. Some cells become inactive and unable to undergo apoptosis (programmed cell death). These cells accumulate at sites of tissue damage or disease, thus accelerating aging. They are called senescent cells. Therapeutic interventions that can either eliminate senescent cells (senolytics) or suppress their harmful effects (senomorphics) have been developed. Senescence (aging) is caused by the inter- and intramolecular interactions between the domains of forkhead (FHD) and transactivation (TAD), as well as C-terminal region 3 (CR3) and DNA binding (DBD). On the other hand, anti-senescent/senolytic (anti-aging) activities are achieved by disrupting these interactions with CR3- and forkhead box protein O4 (FOXO4)-based peptides, such as ES2 and DRI, respectively. In this study, we use a computerized procedure based on digital signal processing to systematically analyze the inter-molecular interactions between senolytics and their targets. Informational spectrum method (ISM) is engaged. We obtained the sequences of the peptides from the interacting proteins of CR3 and FOXO4 and evaluated their ability to disrupt the inter-molecular interactions between FOXO4 and DRI and CR3 and BDB, which are responsible for senescence (aging). Our results show that the peptides have different degrees of senolytic (anti-aging) activity, depending on their affinity for CR3 and BDB, or FOXO4 and DRI. We found that enhanced senescence 2 (ES2) has a higher affinity for CR3 and BDB than FOXO4 and DRI, and that the interaction between CR3 and BDB is crucial for aging. Therefore, ES2 and other CR3-based peptides are more potent senolytics than FOXO4-based peptides. Our findings are consistent with previous studies and reveal new insights into the mechanisms of senescence and senolytics. ES2 is considered the best senolytic candidate, as it is 3-7 times more effective than DRI. We verified that ES2 has a weaker interaction with FOXO4 than CR3. However, the performance of DRI has been noted to depend on its intramolecular interactions and stability. Hence, intramolecular analyses using the digital signal processing-based technique has become very vital and will follow. CR3-based peptides are promising candidates for senolytic therapy. Senolytics are linear chains of amino acids that can target and eliminate senescent cells, which are cells that have stopped dividing and contribute to aging and age-related diseases. By using this proposed, novel computerized technique that is based on digital signal processing, senolytics can be easily analyzed and optimized for their effectiveness and safety. This provides a more rational approach to enhancing our longevity and well-being by offering interventions that can delay or reverse aging and insights that can advance the field of gerontology. This procedure also will compliment other approaches such as molecular stimulation, etc.
FOXO Transcriptional Factors and Long-Term Living.
Several pathologies such as neurodegeneration and cancer are associated with aging, which is affected by many genetic and environmental factors. Healthy aging conceives human longevity, possibly due to carrying the defensive genes. For instance, FOXO (forkhead box O) genes determine human longevity. FOXO transcription factors are involved in the regulation of longevity phenomenon via insulin and insulin-like growth factor signaling. Only one FOXO gene (FOXO DAF-16) exists in invertebrates, while four FOXO genes, that is, FOXO1, FOXO3, FOXO4, and FOXO6 are found in mammals. These four transcription factors are involved in the multiple cellular pathways, which regulate growth, stress resistance, metabolism, cellular differentiation, and apoptosis in mammals. However, the accurate mode of longevity by FOXO factors is unclear until now. This article describes briefly the existing knowledge that is related to the role of FOXO factors in human longevity.
EGF Relays Signals to COP1 and Facilitates FOXO4 Degradation to Promote Tumorigenesis.
Forkhead-Box Class O 4 (FOXO4) is involved in critical biological functions, but its response to EGF-PKB/Akt signal regulation is not well characterized. Here, it is reported that FOXO4 levels are downregulated in response to EGF treatment, with concurrent elevation of COP9 Signalosome subunit 6 (CSN6) and E3 ubiquitin ligase constitutive photomorphogenic 1 (COP1) levels. Mechanistic studies show that CSN6 binds and regulates FOXO4 stability through enhancing the E3 ligase activity of COP1, and that COP1 directly interacts with FOXO4 through a VP motif on FOXO4 and accelerates the ubiquitin-mediated degradation of FOXO4. Metabolomic studies demonstrate that CSN6 expression leads to serine and glycine production. It is shown that FOXO4 directly binds and suppresses the promoters of serine-glycine-one-carbon (SGOC) pathway genes, thereby diminishing SGOC metabolism. Evidence shows that CSN6 can regulate FOXO4-mediated SGOC gene expression. Thus, these data suggest a link of CSN6-FOXO4 axis and ser/gly metabolism. Further, it is shown that CSN6-COP1-FOXO4 axis is deregulated in cancer and that the protein expression levels of CSN6 and FOXO4 can serve as prognostic markers for cancers. The results illustrate a pathway regulation of FOXO4-mediated serine/glycine metabolism through the function of CSN6-COP1 axis. Insights into this pathway may be strategically designed for therapeutic intervention in cancers.
FoxO1 signaling in B cell malignancies and its therapeutic targeting.
FoxO transcription factors (FoxO1, FoxO3a, FoxO4, FoxO6) are a highly evolutionary conserved subfamily of the 'forkhead' box proteins. They have traditionally been considered tumor suppressors, but FoxO1 also exhibits oncogenic properties. The complex nature of FoxO1 is illustrated by its various roles in B cell development and differentiation, immunoglobulin gene rearrangement and cell-surface B cell receptor (BCR) structure, DNA damage control, cell cycle regulation, and germinal center reaction. FoxO1 is tightly regulated at a transcriptional (STAT3, HEB, EBF, FoxOs) and post-transcriptional level (Akt, AMPK, CDK2, GSK3, IKKs, JNK, MAPK/Erk, SGK1, miRNA). In B cell malignancies, recurrent FoxO1 activating mutations (S22/T24) and aberrant nuclear export and activity have been described, underscoring the potential of its therapeutic inhibition. Here, we review FoxO1's roles across B cell and myeloid malignancies, namely acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), follicular lymphoma (FL), diffuse large B cell lymphoma (DLBCL), mantle cell lymphoma (MCL), Burkitt lymphoma (BL), Hodgkin lymphoma (HL), and multiple myeloma (MM). We also discuss preclinical evidence for FoxO1 targeting by currently available inhibitors (AS1708727, AS1842856, cpd10).
CircROR1 binds HNRNPL to regulate FOXO4 pre-mRNA splicing, promoting cutaneous melanoma metastasis and serving as a therapeutic target via RNAi-loaded PEG-LNPs.
BACKGROUND: Circular RNAs (circRNAs) contribute to gene expression regulation by interacting with splicing factors, a process that is often disrupted in cancers such as cutaneous melanoma (CM). METHODS: A circRNA microarray analysis was performed to identify differentially expressed circRNAs. qRT‒PCR was conducted to confirm the expression of circROR1. CCK-8, colony formation, wound healing, and transwell assays were used to analyze proliferation, metastasis and apoptosis in CM cells. Xenograft models and IHC experiments were established to confirm the effects of circROR1 on tumor growth and metastasis in vivo. RNA sequencing and pull-down–MS experiments were performed to identify the mechanisms downstream of circROR1. Nuclear and cytoplasmic fractionation, along with FISH experiments, were conducted to determine the cellular localization of circROR1. To target circROR1 for CM treatment, we used a microfluidic strategy to develop FA-PEG(si-circ) nanoparticles for efficient siRNA delivery. RESULTS: In CM samples, circROR1 levels were positively correlated with HNRNPL levels and tumor metastasis but negatively correlated with FOXO4 protein levels. CircROR1 was prevalent in CM, and its upregulation increased the levels of factors involved in epithelial–mesenchymal transition, cell migration, and invasion. CircROR1 overexpression conferred resistance to PD-L1-antibody therapy in CM cells by downregulating PD-L1 expression. CircROR1 recruited HNRNPL, influencing its nuclear translocation, and further prevented intron retention in FOXO4 mRNA. In HNRNPL-overexpressing CM cells, circROR1 upregulation inhibited FOXO4α expression and promoted FOXO4ζ expression. Increased FOXO4α expression counteracted circROR1’s effects and suppressed metastatic behaviors. FA-PEG(si-circ) enhanced siRNA stability and efficiency, reducing CM cell lung colonization in vivo. CONCLUSION: This study identified circROR1 as an oncogenic circular RNA that plays a crucial role in tumor progression and metastasis. CircROR1-targeted nanotherapy is a promising option for the treatment of metastatic cancer.
17-β-Estradiol Protects Chondrocytes From Senescence and Ameliorates Osteoarthritis Progression via ERα-AKT-FOXO4 Pathway.
Osteoarthritis (OA) is a prevalent cause of joint pain in elderly individuals, and chondrocyte senescence plays a crucial role in its pathogenesis. FOXO4 has been identified as a crucial molecule in cellular senescence. However, little is known regarding its role in OA and the regulation of its expression. 17-β-Estradiol (E2) has been demonstrated to exert a protective effect in OA, yet the underlying mechanism remains largely unexplained. In this study, we reported a protective effect of E2 against multiple types of chondrocyte senescence, and this effect was mediated by oestrogen receptor α (ERα). Mechanically, E2 activated AKT and facilitated the nuclear export and the degradation of FOXO4, which played a crucial role in resisting senescence. Moreover, knockdown of FOXO4 in osteoarthritic chondrocytes alleviated cellular senescence. Furthermore, we demonstrated that intra-articular injection of E2 was effective in ameliorating surgery-induced OA in a rat model. Collectively, E2 contributed to the alleviation of chondrocyte senescence through the ERα-AKT-FOXO4 signalling pathway and ameliorated OA progression in the rat model. Our study offers a novel therapeutic approach for controlling chondrocyte senescence and provides insights into the role of E2 in treating OA.
Resveratrol inhibits bile acid-induced gastric intestinal metaplasia via the PI3K/AKT/p-FoxO4 signalling pathway.
Gastric intestinal metaplasia (GIM) is the essential pre-malignancy of gastric cancer. Chronic inflammation and bile acid reflux are major contributing factors. As an intestinal development transcription factor, caudal-related homeobox 2 (CDX2) is key in GIM. Resveratrol has potential chemopreventive and anti-tumour effects. The aim of the study is to probe the effect of resveratrol in bile acid-induced GIM. We demonstrated that resveratrol could reduce CDX2 expression in a time- and dose-dependent manner in gastric cell lines. A Cignal Finder 45-Pathway Reporter Array and TranSignal Protein/DNA Array Kit verified that resveratrol could increase Forkhead box O4 (FoxO4) activity and that Chenodeoxycholic acid (CDCA) could reduce FoxO4 activity. Furthermore, bioinformatics analysis showed that FoxO4 could bind to the CDX2 promoter, and these conjectures were supported by chromatin-immunoprecipitation (ChIP) assays. Resveratrol can activate FoxO4 and decrease CDX2 expression by increasing phospho-FoxO4 nucleus trans-location. Resveratrol could increase FoxO4 phosphorylation through the PI3K/AKT pathway. Ectopic FoxO4 expression can up-regulate FoxO4 phosphorylation and suppress CDCA-induced GIM marker expression. Finally, we found a reverse correlation between p-FoxO4 and CDX2 in tissue arrays. This study validates that resveratrol could reduce bile acid-induced GIM through the PI3K/AKT/p-FoxO4 signalling pathway and has a potential reversing effect on GIM, especially that caused by bile acid reflux.
AI-guided CAR designs and targeted pathway modulation to enhance multi-antigen CAR T cell durability and overcome antigen escape.
The persistence of CAR T cells and antigen escape remain major barriers to durable therapeutic success in hematologic malignancies. Our study integrates AI-guided design with targeted protein degradation to overcome these challenges. Utilizing an in-silico library of CAR constructs followed by an in vitro screening, we developed a predictive model, CARMSeD, which forecasts constructs prone to self-activation and dysfunction. Optimized bispecific CD20/CD19 CAR T cells demonstrate superior persistence and anti-tumor efficacy. To further improve durability, the platform incorporates a PROTAC-based module that selectively degrades AKT3, promoting FOXO4-driven mitochondrial fitness, central memory differentiation, and reduced mTOR signaling. We extended this strategy to develop a trispecific CAR T platform co-expressing a secretable CD3/CD22 bispecific engager, achieving potent tumor eradication even in CD19/CD20-negative malignancies demonstrates efficacy across patient-derived leukemia samples and solid tumor models. Together, our study introduces a next-generation AI-guided CAR T strategy that integrates structure-based optimization and intracellular modulation to improve persistence, broaden antigen coverage, and ensure durable therapeutic efficacy.
Ponicidin inhibited gallbladder cancer proliferation and metastasis by decreasing MAGEB2 expression through FOXO4.
Gallbladder cancer (GBC) is the most aggressively malignant tumor in the bile duct system. The prognosis for patients with GBC is extremely poor. Ponicidin is a diterpenoid compound extracted and purified from the traditional Chinese herb Rabdosia rubescens, and showed promising anti-cancer effects in a variety of tumors. However, Ponicidin has not been investigated in GBC. CCK-8, colony formation assay and EdU-488 DNA synthesis assay were performed to investigate the effect of Ponicidin on GBC cells proliferation. Cell invasion and migration assays and wound-healing assay were used to explore the effect of Ponicidin on invasion and migration ability of GBC cells. mRNA-seq was adopted to explore the underlying mechanisms. Western blot and immunohistochemical staining were conducted to detect the protein level. CHIP assay and dual-luciferase assay were used to validate binding motif. Nude mouse model of GBC was used to assess the anti-tumor effect and safety of Ponicidin. Ponicidin inhibited the proliferation and cell invasion and migration of GBC cells in vitro. Moreover, Ponicidin exerted anti-tumor effects by down-regulating the expression of MAGEB2. Mechanically, Ponicidin upregulated the FOXO4 expression and promoted it to accumulate in nucleus to inhibit the transcript of MAGEB2. Furthermore, Ponicidin suppressed tumor growth in the nude mouse model of GBC with excellent safety. Ponicidin may be a promising agent for the treatment of GBC effectively and safely.
The AKT1-FOXO4 axis reciprocally regulates hemochorial placentation.
Hemochorial placentation involves the differentiation of invasive trophoblast cells, specialized cells that possess the capacity to exit the placenta and invade into the uterus where they restructure the vasculature. Invasive trophoblast cells arise from a well-defined compartment within the placenta, referred to as the junctional zone in rat and the extravillous trophoblast cell column in human. In this study, we investigated roles for AKT1, a serine/threonine kinase, in placental development using a genome-edited/loss-of-function rat model. Disruption of AKT1 resulted in placental, fetal and postnatal growth restriction. Forkhead box O4 (Foxo4), which encodes a transcription factor and known AKT substrate, was abundantly expressed in the junctional zone and in invasive trophoblast cells of the rat placentation site. Foxo4 gene disruption using genome editing resulted in placentomegaly, including an enlarged junctional zone. AKT1 and FOXO4 regulate the expression of many of the same transcripts expressed by trophoblast cells, but in opposite directions. In summary, we have identified AKT1 and FOXO4 as part of a regulatory network that reciprocally controls critical indices of hemochorial placenta development.
FOXO4-DRI improves spermatogenesis in aged mice through reducing senescence-associated secretory phenotype secretion from Leydig cells.
Male ageing is always accompanied by decreased fertility. The forkhead O (FOXO) transcription factor FOXO4 is reported to be highly expressed in senescent cells. Upon activation, it binds p53 in the nucleus, preventing senescent cell apoptosis and maintaining senescent cells in situ. Leydig cells play key roles in assisting spermatogenesis. Leydig cell senescence leads to deterioration of the microenvironment of the testes and impairs spermatogenesis. In this study, we observed that FOXO4-DRI, a specific FOXO4- p53 binding blocker, induced apoptosis in senescent Leydig cells, reduced the secretion of certain Senescence-Associated Secretory Phenotype and improved the proliferation of cocultured GC-1 SPG cells. In naturally aged mice, FOXO4-DRI-treated aged mice exhibited increased sperm quality and improved spermatogenesis.
FoxO6 in glucose metabolism (FoxO6).
The forkhead box O (FoxO) subfamily has four members, namely FoxO1, FoxO3, FoxO4, and FoxO6. Unlike the other three members of the FoxO family, FoxO6 has garnered considerably less attention because of earlier reports that FoxO6 expression was limited to the brain. Recent data indicate that FoxO6 is produced in the liver of both rodents and humans. Hepatic FoxO6 activity, which remains at low basal levels in fed states, is markedly induced in fasted mice. FoxO6 activity becomes abnormally higher in the liver of mice with dietary obesity or type 2 diabetes (T2D). Genetically engineered mice with elevated FoxO6 activity in the liver exhibit prediabetes, culminating in the development of glucose intolerance, fasting hyperglycemia, and hyperinsulinemia. Conversely, inhibition of FoxO6 activity in the insulin-resistant liver results in a reduction in fasting hyperglycemia, contributing to the amelioration of hyperinsulinemia in T2D mice. These new data suggest that FoxO6 is an important regulator of hepatic glucose metabolism in response to insulin or physiological cues. Insulin inhibits FoxO6 activity by promoting its phosphorylation and disabling its activity in the nucleus without altering its subcellular distribution via a mechanism that is distinct from other members of the FoxO subfamily. In this article, we comprehensively review the role of FoxO6 in glucose metabolism in health and disease. We also address whether FoxO6 dysregulation is a contributing factor for the pathogenesis of fasting hyperglycemia and discuss whether FoxO6 is a potential therapeutic target for improving fasting hyperglycemia in T2D.
FOXO4-DRI alleviates age-related testosterone secretion insufficiency by targeting senescent Leydig cells in aged mice.
Male late-onset hypogonadism is an age-related disease, the core mechanism of which is dysfunction of senescent Leydig cells. Recent studies have shown that elimination of senescent cells can restore proper homeostasis to aging tissue. In the present study, we found that the fork head box O (FOXO) transcription factor FOXO4 was specially expressed in human Leydig cells and that its translocation to the nucleus in the elderly was related to decreased testosterone synthesis. Using hydrogen peroxide-induced senescent TM3 Leydig cells as an in vitro model, we observed that FOXO4 maintains the viability of senescent Leydig cells and suppresses their apoptosis. By disrupting the FOXO4-p53 interaction, FOXO4-DRI, a specific FOXO4 blocker, selectively induced p53 nuclear exclusion and apoptosis in senescent Leydig cells. In naturally aged mice, FOXO4-DRI improved the testicular microenvironment and alleviated age-related testosterone secretion insufficiency. These findings reveal the therapeutic potential of FOXO4-DRI for the treatment of male late-onset hypogonadism.
LncRNA GUARDIN suppresses cellular senescence through a LRP130-PGC1α-FOXO4-p21-dependent signaling axis.
The long noncoding RNA GUARDIN functions to protect genome stability. Inhibiting GUARDIN expression can alter cell fate decisions toward senescence or apoptosis, but the underlying molecular signals are unknown. Here, we show that GUARDIN is an essential component of a transcriptional repressor complex involving LRP130 and PGC1α. GUARDIN acts as a scaffold to stabilize LRP130/PGC1α heterodimers and their occupancy at the FOXO4 promotor. Destabilizing this complex by silencing of GUARDIN, LRP130, or PGC1α leads to increased expression of FOXO4 and upregulation of its target gene p21, thereby driving cells into senescence. We also found that GUARDIN expression was induced by rapamycin, an agent that suppresses cell senescence. FOS-like antigen 2 (FOSL2) acts as a transcriptional repressor of GUARDIN, and lower FOSL2 levels in response to rapamycin correlate with increased levels of GUARDIN. Together, these results demonstrate that GUARDIN inhibits p21-dependent senescence through a LRP130-PGC1α-FOXO4 signaling axis, and moreover, GUARDIN contributes to the anti-aging activities of rapamycin.
DNA nanoparticles targeting FOXO4 selectively eliminate cigarette smoke-induced senescent lung fibroblasts.
The pathogenesis and development of chronic obstructive pulmonary disease (COPD) are significantly related to cellular senescence. Strategies to eliminate senescent cells have been confirmed to benefit several senescence-related diseases. However, there are few reports of senolytic drugs in COPD management. In this study, we demonstrated elevated FOXO4 expression in cigarette smoke-induced senescent lung fibroblasts both in vitro and in vivo. Additionally, self-assembled DNA nanotubes loaded with single-stranded FOXO4 siRNA (siFOXO4-NT) were designed and synthesized to knockdown FOXO4 in senescent fibroblasts. We found that siFOXO4-NT can concentration- and time-dependently enter human lung fibroblasts (HFL-1 cells), thereby reducing FOXO4 levels in vitro. Most importantly, siFOXO4-NT selectively cleared senescent HFL-1 cells by reducing BCLXL expression and the BCL2/BAX ratio, which were increased in CSE-induced senescent HFL-1 cells. The findings from our work present a novel strategy for senolytic drug development for COPD therapy.
Quick links (PubMed)
- PMID 36515093 — 2023 · Eliminating Senescent Cells Can Promote Pulmonary Hypertension Developme…
- PMID 28340339 — 2017 · Targeted Apoptosis of Senescent Cells Restores Tissue Homeostasis in Res…
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- PMID 40425537 — 2025 · Structural plasticity of the FOXO-DBD:p53-TAD interaction.
- PMID 15492844 — 2004 · Human FOX gene family (Review).
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- PMID 30414140 — 2019 · Introduction to FOXO Biology.
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- PMID 25109988 — 2014 · Intermolecular disulfide-dependent redox signalling.
- PMID 40086444 — 2025 · FOXO4-SP6 axis controls surface epithelium commitment by mediating epige…
- PMID 25225602 — 2014 · FoxO3a and disease progression.
- PMID 28213398 — 2017 · FoxO integration of insulin signaling with glucose and lipid metabolism.
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- PMID 36700213 — 2022 · The role of FOXO4/NFAT2 signaling pathway in dysfunction of human corona…
- PMID 36106640 — 2022 · The FoxO4/DKK3 axis represses IFN-γ expression by Th1 cells and li…
- PMID 37722500 — 2023 · Multiomic analysis implicates FOXO4 in genetic regulation of chick lens …
- PMID 33463054 — 2021 · Hypoxia-induced FOXO4/LDHA axis modulates gastric cancer cell glycolysis…
- PMID 40861274 — 2025 · The multifaceted impact of physical exercise on FoxO signaling pathways.
- PMID 17646672 — 2007 · Dynamic FoxO transcription factors.
- PMID 38239718 — 2023 · Bioinformatics procedure for investigating senolytic (anti-aging) agents…
- PMID 28894507 — 2017 · FOXO Transcriptional Factors and Long-Term Living.
- PMID 33101846 — 2020 · EGF Relays Signals to COP1 and Facilitates FOXO4 Degradation to Promote …
- PMID 39533662 — 2025 · FoxO1 signaling in B cell malignancies and its therapeutic targeting.
- PMID 41530849 — 2026 · CircROR1 binds HNRNPL to regulate FOXO4 pre-mRNA splicing, promoting cut…
- PMID 41677002 — 2026 · 17-β-Estradiol Protects Chondrocytes From Senescence and Ameliorat…
- PMID 33103284 — 2021 · Resveratrol inhibits bile acid-induced gastric intestinal metaplasia via…
- PMID 41547950 — 2026 · AI-guided CAR designs and targeted pathway modulation to enhance multi-a…
- PMID 37002972 — 2023 · Ponicidin inhibited gallbladder cancer proliferation and metastasis by d…
- PMID 36607602 — 2023 · The AKT1-FOXO4 axis reciprocally regulates hemochorial placentation.
- PMID 39025385 — 2024 · FOXO4-DRI improves spermatogenesis in aged mice through reducing senesce…
- PMID 23324123 — 2013 · FoxO6 in glucose metabolism (FoxO6).
- PMID 31959736 — 2020 · FOXO4-DRI alleviates age-related testosterone secretion insufficiency by…
- PMID 32149459 — 2020 · LncRNA GUARDIN suppresses cellular senescence through a LRP130-PGC1;…
- PMID 37881696 — 2023 · DNA nanoparticles targeting FOXO4 selectively eliminate cigarette smoke-…