Cold-induced FOXO1 nuclear transport aids cold survival and tissue storage.

Cold-induced injuries severely limit opportunities and outcomes of hypothermic therapies and organ preservation, calling for better understanding of cold adaptation. Here, by surveying cold-altered chromatin accessibility and integrated CUT&Tag/RNA-seq analyses in human stem cells, we reveal forkhead box O1 (FOXO1) as a key transcription factor for autonomous cold adaptation. Accordingly, we find a nonconventional, temperature-sensitive FOXO1 transport mechanism involving the nuclear pore complex protein RANBP2, SUMO-modification of transporter proteins Importin-7 and Exportin-1, and a SUMO-interacting motif on FOXO1. Our conclusions are supported by cold survival experiments with human cell models and zebrafish larvae. Promoting FOXO1 nuclear entry by the Exportin-1 inhibitor KPT-330 enhances cold tolerance in pre-diabetic obese mice, and greatly prolongs the shelf-life of human and mouse pancreatic tissues and islets. Transplantation of mouse islets cold-stored for 14 days reestablishes normoglycemia in diabetic mice. Our findings uncover a regulatory network and potential therapeutic targets to boost spontaneous cold adaptation.

Mutant FOXO1 controls an oncogenic network via enhancer accessibility.

Transcriptional dysregulation is a hallmark of diffuse large B cell lymphoma (DLBCL), as transcriptional regulators are frequently mutated. However, our mechanistic understanding of how normal transcriptional programs are co-opted in DLBCL has been hindered by a lack of methodologies that provide the temporal resolution required to separate direct and indirect effects on transcriptional control. We applied a chemical-genetic approach to engineer the inducible degradation of the transcription factor FOXO1, which is recurrently mutated (mFOXO1) in DLBCL. The combination of rapid degradation of mFOXO1, nascent transcript detection, and assessment of chromatin accessibility allowed us to identify the direct targets of mFOXO1. mFOXO1 was required to maintain accessibility at specific enhancers associated with multiple oncogenes, and mFOXO1 degradation impaired RNA polymerase pause-release at some targets. Wild-type FOXO1 appeared to weakly regulate many of the same targets as mFOXO1 and was able to complement the degradation of mFOXO1 in the context of AKT inhibition.

Temporal coordination of the transcription factor response to H2O2 stress.

Oxidative stress from excess H2O2 activates transcription factors that restore redox balance and repair oxidative damage. Although many transcription factors are activated by H2O2, it is unclear whether they are activated at the same H2O2 concentration, or time. Dose-dependent activation is likely as oxidative stress is not a singular state and exhibits dose-dependent outcomes including cell-cycle arrest and cell death. Here, we show that transcription factor activation is both dose-dependent and coordinated over time. Low levels of H2O2 activate p53, NRF2 and JUN. Yet under high H2O2, these transcription factors are repressed, and FOXO1, NF-κB, and NFAT1 are activated. Time-lapse imaging revealed that the order in which these two groups of transcription factors are activated depends on whether H2O2 is administered acutely by bolus addition, or continuously through the glucose oxidase enzyme. Finally, we provide evidence that 2-Cys peroxiredoxins control which group of transcription factors are activated.

Cellular Cutaneous Epithelioid Hemangioma Harboring the Rare GATA6::FOXO1 Gene Fusion.

ABSTRACT: Epithelioid hemangioma (EH) is a benign vascular tumor displaying diverse histomorphologies. Among these, one EH subtype comprises cellular sheets of atypical epithelioid cells, posing potential challenges in distinguishing it from malignant vascular lesions. In this case report, we present a cutaneous cellular EH that carries the rare GATA6::FOXO1 gene fusion, a recent discovery. Our aim is to provide an updated insight into the evolving knowledge of EHs while delving into the histologic and molecular characteristics of the primary differential diagnoses.

KDM3B inhibitors disrupt the oncogenic activity of PAX3-FOXO1 in fusion-positive rhabdomyosarcoma.

Fusion-positive rhabdomyosarcoma (FP-RMS) is an aggressive pediatric sarcoma driven primarily by the PAX3-FOXO1 fusion oncogene, for which therapies targeting PAX3-FOXO1 are lacking. Here, we screen 62,643 compounds using an engineered cell line that monitors PAX3-FOXO1 transcriptional activity identifying a hitherto uncharacterized compound, P3FI-63. RNA-seq, ATAC-seq, and docking analyses implicate histone lysine demethylases (KDMs) as its targets. Enzymatic assays confirm the inhibition of multiple KDMs with the highest selectivity for KDM3B. Structural similarity search of P3FI-63 identifies P3FI-90 with improved solubility and potency. Biophysical binding of P3FI-90 to KDM3B is demonstrated using NMR and SPR. P3FI-90 suppresses the growth of FP-RMS in vitro and in vivo through downregulating PAX3-FOXO1 activity, and combined knockdown of KDM3B and KDM1A phenocopies P3FI-90 effects. Thus, we report KDM inhibitors P3FI-63 and P3FI-90 with the highest specificity for KDM3B. Their potent suppression of PAX3-FOXO1 activity indicates a possible therapeutic approach for FP-RMS and other transcriptionally addicted cancers.

Type 1 interferons and Foxo1 down-regulation play a key role in age-related T-cell exhaustion in mice.

Foxo family transcription factors are critically involved in multiple processes, such as metabolism, quiescence, cell survival and cell differentiation. Although continuous, high activity of Foxo transcription factors extends the life span of some species, the involvement of Foxo proteins in mammalian aging remains to be determined. Here, we show that Foxo1 is down-regulated with age in mouse T cells. This down-regulation of Foxo1 in T cells may contribute to the disruption of naive T-cell homeostasis with age, leading to an increase in the number of memory T cells. Foxo1 down-regulation is also associated with the up-regulation of co-inhibitory receptors by memory T cells and exhaustion in aged mice. Using adoptive transfer experiments, we show that the age-dependent down-regulation of Foxo1 in T cells is mediated by T-cell-extrinsic cues, including type 1 interferons. Taken together, our data suggest that type 1 interferon-induced Foxo1 down-regulation is likely to contribute significantly to T-cell dysfunction in aged mice.

IGF-1 Regulates Skeletal Muscle Degradation and Remolding in Ventilator-Induced Diaphragmatic Dysfunction by Mediating FOXO1 Expression.

BACKGROUND: Mechanical ventilation (MV) sustains life in critically ill patients by providing adequate alveolar ventilation. However, prolonged MV could induce inspiratory muscle atrophy known as ventilator-induced diaphragmatic dysfunction (VIDD). Insulin-like growth factor (IGF)-1 has been proven to play crucial roles in regulating skeletal muscle size and function. Meanwhile, the forkhead box protein O1 (FOXO1) has been linked to muscle atrophy. This study aimed to explore the effect of IGF-1 on muscle degradation and remodeling in VIDD and delved into the association of the underlying mechanism involving FOXO1. METHODS: VIDD models were established by treating rats with MV. Adeno-associated virus (AAV) was used for transfection to construct IGF-1 and/or FOXO1 overexpressed rats. There were four groups in this study: normal rats (NC), normal rats with MV treatment (MV), IGF-1-overexpressed rats with MV treatment (MV+IGF-1), and rats overexpressing both IGF-1 and FOXO1 with MV treatment (MV+IGF-1+FOXO1). Protein levels were measured by western blot or enzyme-linked immunosorbent assay (ELISA), and mRNA levels were detected by real-time reverse transcriptase-polymerase chain reaction (RT-qPCR). IGF-1 and FOXO1 expression were validated by detecting mRNA and protein levels. Diaphragmatic muscle contractility and morphometry were tested using stimulating electrodes in conjunction with hematoxylin and eosin (H&E) staining. Interleukin (IL)-6 and carbonylated protein were used for evaluating muscle atrophy and oxidation, respectively. Protein degradation was determined by troponin-I level and tyrosine release. Apoptosis was assessed using the terminal deoxynucleotidyl transferase-mediated uridine 5'-triphosphate (UTP) nick-end labeling (TUNEL) assay, alongside markers like Bax, B-cell lymphoma 2 (BCL-2), and Cleaved Caspase-3. Atrogin-1, muscle RING finger 1 (MURF1), neuronally expressed developmentally downregulated 4 (NEDD4), muscle ubiquitin ligase of SCF complex in atrophy-1 (MUSA1), and ubiquitinated protein was used to determine proteolysis. Additionally, protein synthesis was measured by assessing the rates of mixed muscle protein (MMP) and myosin heavy chain (MHC). RESULTS: MV treatment caused IGF-1 downregulation (p < 0.01) and FOXO1 upregulation (p < 0.01). The IGF-1 upregulation downregulated FOXO1 in the MV+IGF-1 group (p < 0.001) while IGF-1 and FOXO1 were both upregulated in the MV+IGF-1+FOXO1 group (p < 0.001). The treatment of MV decreased muscle contractility and cross-sectional areas of diaphragm muscle fibers (p < 0.01). Additionally, IL-6, troponin-1, tyrosine release, carbonylated protein, TUNEL positive nuclei, Bax, Cleaved Caspase-3, Atrogin-1, MURF1, neuronally expressed developmentally downregulated 4 (NEDD4), MUSA1, and ubiquitinated protein levels increased significantly in MV group (p < 0.001) while levels of BCL-2, fractional synthetic rate of MMP and MHC, and type I and type II MHC protein mRNA expression decreased in MV group (p < 0.001). All of these alterations were reversed in the MV+IGF-1 group (p < 0.01), while the IGF-1-induced reversion was disrupted in the MV+IGF-1+FOXO1 group (p < 0.01). CONCLUSIONS: IGF-1 may protect diaphragmatic muscles from VIDD-induced structural damage and function loss by downregulating FOXO1. This action suppresses muscle breakdown and facilitates muscle remodeling in diaphragmatic muscles affected by VIDD.

The Roles and Regulatory Mechanisms of Tight Junction Protein Cingulin and Transcription Factor Forkhead Box Protein O1 in Human Lung Adenocarcinoma A549 Cells and Normal Lung Epithelial Cells.

Tight junction (TJ) protein cingulin (CGN) and transcription factor forkhead box protein O1 (FOXO1) contribute to the development of various cancers. Histone deacetylase (HDAC) inhibitors have a potential therapeutic role for some cancers. HDAC inhibitors affect the expression of both CGN and FOXO1. However, the roles and regulatory mechanisms of CGN and FOXO1 are unknown in non-small cell lung cancer (NSCLC) and normal human lung epithelial (HLE) cells. In the present study, to investigate the effects of CGN and FOXO1 on the malignancy of NSCLC, we used A549 cells as human lung adenocarcinoma and primary human lung epithelial (HLE) cells as normal lung tissues and performed the knockdown of CGN and FOXO1 by siRNAs. Furthermore, to investigate the detailed mechanisms in the antitumor effects of HDAC inhibitors for NSCLC via CGN and FOXO1, A549 cells and HLE cells were treated with the HDAC inhibitors trichostatin A (TSA) and Quisinostat (JNJ-2648158). In A549 cells, the knockdown of CGN increased bicellular TJ protein claudin-2 (CLDN-2) via mitogen-activated protein kinase/adenosine monophosphate-activated protein kinase (MAPK/AMPK) pathways and induced cell migration, while the knockdown of FOXO1 increased claudin-4 (CLDN-4), decreased CGN, and induced cell proliferation. The knockdown of CGN and FOXO1 induced cell metabolism in A549 cells. TSA and Quisinostat increased CGN and tricellular TJ protein angulin-1/lipolysis-stimulated lipoprotein receptor (LSR) in A549. In normal HLE cells, the knockdown of CGN and FOXO1 increased CLDN-4, while HDAC inhibitors increased CGN and CLDN-4. In conclusion, the knockdown of CGN via FOXO1 contributes to the malignancy of NSCLC. Both HDAC inhibitors, TSA and Quisinostat, may have potential for use in therapy for lung adenocarcinoma via changes in the expression of CGN and FOXO1.

Baicalin Maintains Articular Cartilage Homeostasis and Alleviates Osteoarthritis by Activating FOXO1.

Baicalin has been acknowledged for its anti-inflammatory properties. However, its potential impact on osteoarthritis (OA) has not yet been explored. Therefore, our study aimed to examine the effects of Baicalin on OA, both in laboratory and animal models. To evaluate its efficacy, human chondrocytes affected by OA were treated with interleukin-1ß and/or Baicalin. The effects were then assessed through viability tests using the cell counting kit-8 (CCK-8) method and flow cytometry. In addition, we analyzed the expressions of various factors such as FOXO1, autophagy, apoptosis, and cartilage synthesis and breakdown to corroborate the effects of Baicalin. We also assessed the severity of OA through analysis of tissue samples. Our findings demonstrate that Baicalin effectively suppresses inflammatory cytokines and MMP-13 levels caused by collagenase-induced osteoarthritis, while simultaneously preserving the levels of Aggrecan and Col2. Furthermore, Baicalin has been shown to enhance autophagy. Through the use of FOXO1 inhibitors, lentivirus-mediated knockdown, and chromatin immunoprecipitation, we verified that Baicalin exerts its protective effects by activating FOXO1, which binds to the Beclin-1 promoter, thereby promoting autophagy. In conclusion, our results show that Baicalin has potential as a therapeutic agent for treating OA (Clinical Trial Registration number: 2023-61).

FoxO1 promotes ovarian cancer by increasing transcription and METTL14-mediated m6A modification of SMC4.

The transcription factor forkhead box protein O1 (FoxO1) is closely related to the occurrence and development of ovarian cancer (OC), however its role and molecular mechanisms remain unclear. Herein, we found that FoxO1 was highly expressed in clinical samples of OC patients and was significantly correlated with poor prognosis. FoxO1 knockdown inhibited the proliferation of OC cells in vitro and in vivo. ChIP-seq combined with GEPIA2 and Kaplan-Meier database analysis showed that structural maintenance of chromosome 4 (SMC4) is a downstream target of FoxO1, and FoxO1 promotes SMC4 transcription by binding to its -1400/-1390 bp promoter. The high expression of SMC4 significantly blocked the tumor inhibition effect of FoxO1 knockdown. Furtherly, FoxO1 increased SMC4 mRNA abundance by transcriptionally activating methyltransferase-like 14 (METTL14) and increasing SMC4 m6A methylation on its coding sequence region. The Cancer Genome Atlas dataset analysis confirmed a significant positive correlation between FoxO1, SMC4, and METTL14 expression in OC. In summary, this study revealed the molecular mechanisms of FoxO1 regulating SMC4 and established a clinical link between the expression of FoxO1/METTL14/SMC4 in the occurrence of OC, thus providing a potential diagnostic target and therapeutic strategy.

FOXO1 regulates bovine skeletal muscle cells differentiation by targeting MYH3.

The growth and development of bovine skeletal muscle and beef yield is closely intertwined. Our previous research found that forkhead box O1 (FOXO1) plays an important role in the regulation of beef muscle formation, but its specific mechanism is still unknown. In this study, we aimed to clarify the regulatory mechanism of FOXO1 in proliferation and differentiation of bovine skeletal muscle cells (BSMCs). The results showed that interfering with FOXO1 can promote proliferation and the cell G1/S phase of BSMCs by up-regulating the expression of PCNA, CDK1, CDK2, CCNA2, CCNB1, CCND1 and CCNE2. Besides, interfering with FOXO1 inhibited the apoptosis of BSMCs by up-regulating the expression of anti-apoptosis gene BCL2, while simultaneously down-regulating the expression of the pro-apoptosis genes BAD and BAX. Inversely, interfering with FOXO1 can promote the differentiation of BSMCs by up-regulating the expression of myogenic differentiation marker genes MYOD, MYOG, MYF5, MYF6 and MYHC. Furthermore, RNA-seq combined with western bolt, immunofluorescence and chromatin immunoprecipitation analysis showed that FOXO1 could regulate BSMCs differentiation process by influencing PI3K-Akt, Relaxin and TGF-beta signaling pathways, and target MYH3 for transcriptional inhibition. In conclusion, this study provides a basis for studying the role and molecular mechanism of FOXO1 in BSMCs.

Genetic variants in the FOXO1 and ZNF469 genes are associated with keratoconus in Sweden: a case-control study.

BACKGROUND: Keratoconus (KC) is characterized by pathological thinning and bulging of the cornea that may lead to visual impairment. The etiology of sporadic KC remains enigmatic despite intensive research in recent decades. The purpose of this study was to examine the relationship between previously highlighted genetic variants associated with KC and sporadic KC in a Swedish cohort. METHODS: A total of 176 patients (age 16-70 years) with sporadic KC diagnosed by Scheimpflug-topography (Pentacam) were included. The control group (n = 418; age 70 years) was a subsample originating from the Gothenburg H70 Birth Cohort Studies of ageing. Extraction of DNA from blood samples was performed according to standard procedures, and genotyping was performed using competitive allele specific PCR (KASP) technology. A total of 11 single nucleotide polymorphisms (SNPs) were selected for analysis. RESULTS: Statistically significant associations (p = 0.005) were found between the SNPs rs2721051 and rs9938149 and sporadic KC. These results replicate earlier research that found associations between genetic variants in the FOXO1 and BANP-ZNF469 genes and sporadic KC in other populations. CONCLUSION: Genetic variations in the FOXO1 and BANP-ZNF469 genes may be involved in the pathogenesis of sporadic KC.

Brg1/PRMT5 nuclear complex epigenetically regulates FOXO1 in IPF mesenchymal progenitor cells.

We have discovered intrinsically fibrogenic mesenchymal progenitor cells (MPCs) in the human idiopathic pulmonary fibrosis (IPF) lung. IPF MPCs display a durably distinct transcriptome, suggesting that they have undergone epigenetic modifications. Prior studies indicate that the chromatin remodeler Brg1 associates with the arginine methyltransferase PRMT5 to epigenetically regulate transcription factors. We hypothesize that a Brg1/PRMT5 nuclear complex epigenetically regulates critical nodes in IPF MPC self-renewal signaling networks. IPF and control MPCs were isolated from primary mesenchymal cell lines established from IPF and control patients. RNA-sequencing identified increased expression of the FOXO1 transcription factor in IPF MPCs compared with controls, a result we confirmed by Q-PCR and Western blot analysis. Immunoprecipitation identified a CD44/Brg1/PRMT5 nuclear complex in IPF MPCs. Chromatin immunoprecipitation assays showed that PRMT5 and its methylation mark H3R2me2 are enriched on the FOXO1 promoter. We show that loss of Brg1 and PRMT5 function decreases FOXO1 expression and impairs IPF MPC self-renewal, and that loss of FOXO1 function decreases IPF MPC self-renewal and expression of the SOX2 and OCT4 stemness markers. Our findings indicate that the FOXO1 gene is overexpressed in IPF MPCs in a CD44/Brg1/PRMT5 nuclear complex-dependent manner. Our data suggest that Brg1 alters chromatin accessibility, enriching PRMT5 occupancy on the FOXO1 promoter, and PRMT5 methylates histone H3 arginine 2 (H3R2) on the FOXO1 promoter, increasing its expression. Our data are in accord with the concept that this coordinated interplay is responsible for promoting IPF MPC self-renewal and maintaining a critical pool of fibrogenic MPCs that drive IPF progression.NEW & NOTEWORTHY Our research offers valuable understanding regarding the epigenetic control of IPF MPC. The data we obtained strongly support the idea that the coordination between chromatin remodeling and histone methylation plays a key role in regulating transcription factors. Specifically, our findings indicate that FOXO1, an essential transcription factor, likely governs the self-renewal of IPF MPC, which is crucial for maintaining a critical pool of fibrogenic MPCs. This interplay could be an important therapeutic target.

Inhibition of FoxO1 alleviates polycystic ovarian syndrome by reducing inflammation and the immune response.

The aim of this study was to explore the role of forkhead box transcription Factor O1 (FoxO1) in chronic inflammation in polycystic ovary syndrome (PCOS). A PCOS rat model was constructed as an in vivo model by letrozole induction, and granulosa cells (GCs) from PCOS rats were isolated and cultured as an in vitro cellular model. FoxO1 was knocked down by shRNA and siRNA in the PCOS rat model and GCs model, respectively. H&E staining was conducted to evaluate the effect of FoxO1 inhibition on ovarian pathology and dysfunction in PCOS rats. The levels of inflammatory cytokines in the ovaries and uterus of PCOS rats and in GCs were assessed by ELISA. Flow cytometry was used to evaluate the changes in the contents of neutrophils and macrophages in the peripheral blood and spleen of PCOS rats. CCK-8 assays and Annexin V-FITC/PI staining were performed to evaluate the proliferation and apoptosis of GCs. The expression of genes and proteins related to the TLR4/NF-κB/NLRP3 pathway in GCs was determined by RT-qPCR and Western blotting. The results indicated that FoxO1 was highly expressed in PCOS rat model. Inhibition of FoxO1 significantly mitigated the pathological changes and dysfunction in the ovaries of PCOS rats while also suppressing inflammation and fibrosis in the ovaries and uterus. Moreover, knocking down FoxO1 facilitated the restoration of the normal ratio of neutrophils and macrophages in the peripheral blood and spleen of PCOS rats and promoted M2 polarization of macrophages. Additionally, inhibition of FoxO1 promoted the proliferation of GCs and inhibited the inflammatory response in GCs. Furthermore, FoxO1 knockdown inhibited the activation of the NF-κB pathway and the formation of the NLRP3 inflammasome in GCs. In conclusion, inhibition of FoxO1 can alleviate PCOS by inhibiting the TLR4/NF-κB/NLRP3 pathway to reduce inflammation and the immune response.

The FOXO1/G6PC axis promotes gastric cancer progression and mediates 5-fluorouracil resistance by targeting the PI3K/AKT/mTOR signaling pathway.

Gastric cancer (GC) is a prevalent malignancy of the digestive system. Distant metastasis and chemotherapy resistance are the crucial obstacles to prognosis in GC. Recent research has discovered that the glucose-6-phosphatase catalytic subunit (G6PC) plays an important role in tumor malignant development. However, little evidence has highlighted its role in GC. Herein, through a comprehensive analysis including profiling of tissue samples and functional validation in vivo and in vitro, we identify G6PC as a crucial factor in GC tumorigenesis. Importantly, we found that the FOXO1/G6PC axis could accelerate GC cell proliferation, metastasis, and 5-Fluorouracil (5-FU) resistance by targeting the PI3K/AKT/mTOR signaling pathway, implicating that as a prospective therapeutic approach in GC.

Withaferin A suppressed hepatocellular carcinoma progression through inducing IGF2BP3/FOXO1/JAK2/STAT3 pathway-mediated ROS production.

OBJECTIVE: This study aimed to investigate the underlying molecular mechanisms of Withaferin A (WA) in hepatocellular carcinoma (HCC). MATERIALS AND METHODS: The gene and protein expression were analyzed using RT-qPCR and western blot, respectively. The proliferation of HCC cells was evaluated by CCK-8 assays. The migrative ability of HCC cells was measured by transwell assays. RESULTS: We revealed that WA suppressed the proliferation and migration of HCC cells and inhibited IGF2BP3 (insulin like growth factor 2 mRNA binding protein 3) expression. IGF2BP3 abundance reversed the reactive oxygen species (ROS) accumulation and suppression of HCC cell proliferation and migration induced by WA. Besides, IGF2BP3 suppressed ROS production to promote the growth and migration of HCC cells. Furthermore, we found that IGF2BP3 exerted its tumor-promotive and ROS-suppressive effect on HCC cells by regulating the expression of FOXO1 (forkhead box O1). In addition, IGF2BP3-stimulated activation of JAK2 (Janus kinase 2)/STAT3 (signal transducer and activator of transcription 3) phosphorylation effectively decreased the transcription of FOXO1. FOXO1 abundance decreased the phosphorylation of JAK2 and STAT3 by increasing ROS level, forming a feedback loop for the inhibition of JAK2/STAT3 signaling activated by IGF2BP3. CONCLUSIONS: WA-induced ROS inhibited HCC cell growth and migration through the inhibition of IGF2BP3 to deactivate JAK2/STAT3 signaling, resulting in increased FOXO1 expression to further stimulate ROS production and inhibit JAK2/STAT3 signaling.

M6A demethylase ALKBH5 regulates FOXO1 mRNA stability and chemoresistance in triple-negative breast cancer.

Resistance to chemotherapy is the main reason for treatment failure and poor prognosis in patients with triple-negative breast cancer (TNBC). Although the association of RNA N6-methyladenosine (m6A) modifications with therapy resistance is noticed, its role in the development of therapeutic resistance in TNBC is not well documented. This study aimed to investigate the potential mechanisms underlying reactive oxygen species (ROS) regulation in doxorubicin (DOX)-resistant TNBC. Here, we found that DOX-resistant TNBC cells displayed low ROS levels because of increased expression of superoxide dismutase (SOD2), thus maintaining cancer stem cells (CSCs) characteristics and DOX resistance. FOXO1 is a master regulator that reduces cellular ROS in DOX-resistant TNBC cells, and knockdown of FOXO1 significantly increased ROS levels by inhibiting SOD2 expression. Moreover, the m6A demethylase ALKBH5 promoted m6A demethylation of FOXO1 mRNA and increased FOXO1 mRNA stability in DOX-resistant TNBC cells. The analysis of clinical samples revealed that the increased expression levels of ALKBH5, FOXO1, and SOD2 were significantly positively correlated with chemoresistance and poor prognosis in patients with TNBC. To our knowledge, this is the first study to highlight that ALKBH5-mediated FOXO1 mRNA demethylation contributes to CSCs characteristics and DOX resistance in TNBC cells. Furthermore, pharmacological targeting of FOXO1 profoundly restored the response of DOX-resistant TNBC cells, both in vitro and in vivo. In conclusion, we demonstrated a critical function of ALKBH5-mediated m6A demethylation of FOXO1 mRNA in restoring redox balance, which in turn promoting CSCs characteristics and DOX resistance in TNBC, and suggested that targeting the ALKBH5/FOXO1 axis has therapeutic potential for patients with TNBC refractory to chemotherapy.

TOX3 deficiency mitigates hyperglycemia by suppressing hepatic gluconeogenesis through FoxO1.

BACKGROUND: Excessive hepatic glucose production is a hallmark that contributes to hyperglycemia in type 2 diabetes (T2D). The regulatory network governing this process remains incompletely understood. Here, we demonstrate that TOX3, a high-mobility group family member, acts as a major transcriptional driver for hepatic glucose production. METHODS: Tox3-overexpressed and knockout mice were constructed to explore its metabolic functions. Transcriptomic and chromatin-immunoprecipitation sequencing (ChIP-seq) were used to identify downstream targets of TOX3. Both FoxO1 silencing and inhibitor approaches were used to assess the contribution of FoxO1. TOX3 expression levels were examined in the livers of mice and human subjects. Finally, Tox3 was genetically manipulated in diet-induced obese mice to evaluate its therapeutic potential. RESULTS: Hepatic Tox3 overexpression activates the gluconeogenic program, resulting in hyperglycemia and insulin resistance in mice. Hepatocyte-specific Tox3 knockout suppresses gluconeogenesis and improves insulin sensitivity. Mechanistically, integrated hepatic transcriptomic and ChIP-seq analyses identify FoxO1 as a direct target of TOX3. TOX3 stimulates FoxO1 transcription by directly binding to and activating its promoter, whereas FoxO1 silencing abrogates TOX3-induced dysglycemia in mice. In human subjects, hepatic TOX3 expression shows a significant positive correlation with blood glucose levels under normoglycemic conditions, yet is repressed by high glucose during T2D. Importantly, hepatic Tox3 deficiency markedly protects against and ameliorates the hyperglycemia and glucose intolerance in diet-induced diabetic mice. CONCLUSIONS: Our findings establish TOX3 as a driver for excessive gluconeogenesis through activating hepatic FoxO1 transcription. TOX3 could serve as a promising target for preventing and treating hyperglycemia in T2D.

FOXO1 regulates expression of Neurod4 in the pituitary gland.

Pituitary gland function is regulated by the activity of various transcription factors that control cell fate decisions leading to cellular differentiation and hormone production. FOXO1 is necessary for normal somatotrope differentiation and function. Recent in vivo data implicate FOXO1 in the regulation of genes important for somatotrope differentiation including Gh1, Neurod4, and Pou1f1. In the current study, the somatotrope-like cell line GH3 was treated with a FOXO1 inhibitor, resulting in significant reduction in Neurod4 and Gh1 expression. Consistent with these findings, CRISPR/Cas9-mediated deletion of Foxo1 in GH3 cells significantly reduced expression of Gh1 and Neurod4. Chromatin immunoprecipitation sequencing identifies novel FOXO1 binding sites associated with the Neurod4, Gh1, and Pou1f1 genes. The FOXO1 binding site in the Neurod4 gene exhibits enhancer activity in somatotrope-like cells but not in gonadotrope-like cells. These data strongly suggest FOXO1 directly contributes to the transcriptional control of genes important for somatotrope differentiation.

Lipoteichoic acid restrains macrophage senescence via ß-catenin/FOXO1/REDD1 pathway in age-related osteoporosis.

Osteoporosis and its related fractures are common causes of morbidity and mortality in older adults, but its underlying molecular and cellular mechanisms remain largely unknown. In this study, we found that lipoteichoic acid (LTA) treatment could ameliorate age-related bone degeneration and attenuate intramedullary macrophage senescence. FOXO1 signaling, which was downregulated and deactivated in aging macrophages, played a key role in the process. Blocking FOXO1 signaling caused decreased REDD1 expression and increased phosphorylation level of mTOR, a major driver of aging, as well as aggravated bone loss and deteriorated macrophage senescence. Moreover, LTA elevated FOXO1 signaling through ß-catenin pathway while ß-catenin inhibition significantly suppressed FOXO1 signaling, promoted senescence-related protein expression, and accelerated bone degeneration and macrophage senescence. Our findings indicated that ß-catenin/FOXO1/REDD1 signaling plays a physiologically significant role that protecting macrophages from senescence during aging.