miR-486-5p predicted adverse outcomes of SCAP and regulated K. pneumonia infection via FOXO1.

PURPOSE: Severe community-acquired pneumonia (SCAP) is a common respiratory system disease with rapid development and high mortality. Exploring effective biomarkers for early detection and development prediction of SCAP is of urgent need. The function of miR-486-5p in SCAP diagnosis and prognosis was evaluated to identify a promising biomarker for SCAP. PATIENTS AND METHODS: The serum miR-486-5p in 83 patients with SCAP, 52 healthy individuals, and 68 patients with mild CAP (MCAP) patients were analyzed by PCR. ROC analysis estimated miR-486-5p in screening SCAP, and the Kaplan-Meier and Cox regression analyses evaluated the predictive value of miR-486-5p. The risk factors for MCAP patients developing SCAP were assessed by logistic analysis. The alveolar epithelial cell was treated with Klebsiella pneumonia to mimic the occurrence of SCAP. The targeting mechanism underlying miR-486-5p was evaluated by luciferase reporter assay. RESULTS: Upregulated serum miR-486-5p screened SCAP from healthy individuals and MCAP patients with high sensitivity and specificity. Increasing serum miR-486-5p predicted the poor outcomes of SCAP and served as a risk factor for MCAP developing into SCAP. K. pneumonia induced suppressed proliferation, significant inflammation and oxidative stress in alveolar epithelial cells, and silencing miR-486-5p attenuated it. miR-486-5p negatively regulated FOXO1, and the knockdown of FOXO1 reversed the effect of miR-486-5p in K. pneumonia-treated alveolar epithelial cells. CONCLUSION: miR-486-5p acted as a biomarker for the screening and monitoring of SCAP and predicting the malignancy of MCAP. Silencing miR-486-5p alleviated inflammation and oxidative stress induced by K. pneumonia via negatively modulating FOXO1.

CAPN2 promotes apalutamide resistance in metastatic hormone-sensitive prostate cancer by activating protective autophagy.

Apalutamide, a novel endocrine therapy agent, has been shown to significantly improve the prognosis of patients with metastatic hormone-sensitive prostate cancer (mHSPC). However, resistance to apalutamide has also been reported, and the underlying mechanism for this response has yet to be clearly elucidated. First, this study established apalutamide-resistant prostate cancer (PCa) cells, and confirmed that apalutamide activated the release of calcium ions (Ca2+) and endoplasmic reticulum stress (ERS) to enhance autophagy. Second, RNA sequencing, western blotting, and immunohistochemistry revealed significantly decreased Calpain 2 (CAPN2) expression in the apalutamide-resistant PCa cells and tissues. Furthermore, immunofluorescence and transmission electron microscopy (TEM) showed that CAPN2 promoted apalutamide resistance by activating protective autophagy. CAPN2 promoted autophagy by reducing Forkhead Box O1 (FOXO1) degradation while increasing nuclear translocation via nucleoplasmic protein isolation and immunofluorescence. In addition, FOXO1 promoted protective autophagy through the transcriptional regulation of autophagy-related gene 5 (ATG5). Furthermore, a dual-fluorescence assay confirmed that transcription factor 3 (ATF3) stimulation promoted CAPN2-mediated autophagy activation via transcriptional regulation. In summary, CAPN2 activated protective autophagy by inhibiting FOXO1 degradation and promoting its nuclear translocation via transcriptional ATG5 regulation. ATF3 activation and transcriptional CAPN2 regulation jointly promoted this bioeffect. Thus, our findings have not only revealed the mechanism underlying apalutamide resistance, but also provided a promising new target for the treatment of metastatic PCa.

The Long Non-Coding RNA Gene AC027288.3 Plays a Role in Human Endometrial Stromal Fibroblast Decidualization.

During the secretory phase of the menstrual cycle, endometrial fibroblast cells begin to change into large epithelial-like cells called decidual cells in a process called decidualization. This differentiation continues more broadly in the endometrium and forms the decidual tissue during early pregnancy. The cells undergoing decidualization as well as the resulting decidual cells, support successful implantation and placentation during early pregnancy. This study was carried out to identify new potentially important long non-coding RNA (lncRNA) genes that may play a role in human endometrial stromal fibroblast cells (hESF) undergoing decidualization in vitro, and several were found. The expression of nine was further characterized. One of these, AC027288.3, showed a dramatic increase in the expression of hESF cells undergoing decidualization. When AC027288.3 expression was targeted, the ability of the cells to undergo decidualization as determined by the expression of decidualization marker protein-coding genes was significantly altered. The most affected markers of decidualization whose expression was significantly reduced were FOXO1, FZD4, and INHBA. Therefore, AC027288.3 may be a major upstream regulator of the WNT-FOXO1 pathway and activin-SMAD3 pathways previously shown as critical for hESF decidualization. Finally, we explored possible regulators of AC027288.3 expression during human ESF decidualization. Expression was regulated by cAMP and progesterone. Our results suggest that AC027288.3 plays a role in hESF decidualization and identifies several other lncRNA genes that may also play a role.

MicroRNA-721 regulates gluconeogenesis via KDM2A-mediated epigenetic modulation in diet-induced insulin resistance in C57BL/6J mice.

BACKGROUND: Aberrant gluconeogenesis is considered among primary drivers of hyperglycemia under insulin resistant conditions, with multiple studies pointing towards epigenetic dysregulation. Here we examine the role of miR-721 and effect of epigenetic modulator laccaic acid on the regulation of gluconeogenesis under high fat diet induced insulin resistance. RESULTS: Reanalysis of miRNA profiling data of high-fat diet-induced insulin-resistant mice model, GEO dataset (GSE94799) revealed a significant upregulation of miR-721, which was further validated in invivo insulin resistance in mice and invitro insulin resistance in Hepa 1-6 cells. Interestingly, miR-721 mimic increased glucose production in Hepa 1-6 cells via activation of FOXO1 regulated gluconeogenic program. Concomitantly, inhibition of miR-721 reduced glucose production in palmitate induced insulin resistant Hepa 1-6 cells by blunting the FOXO1 induced gluconeogenesis. Intriguingly, at epigenetic level, enrichment of the transcriptional activation mark H3K36me2 got decreased around the FOXO1 promoter. Additionally, identifying targets of miR-721 using miRDB.org showed H3K36me2 demethylase KDM2A as a potential target. Notably, miR-721 inhibitor enhanced KDM2A expression which correlated with H3K36me2 enrichment around FOXO1 promoter and the downstream activation of the gluconeogenic pathway. Furthermore, inhibition of miR-721 in high-fat diet-induced insulin-resistant mice resulted in restoration of KDM2A levels, concomitantly reducing FOXO1, PCK1, and G6PC expression, attenuating gluconeogenesis, hyperglycemia, and improving glucose tolerance. Interestingly, the epigenetic modulator laccaic acid also reduced the hepatic miR-721 expression and improved KDM2A expression, supporting our earlier report that laccaic acid attenuates insulin resistance by reducing gluconeogenesis. CONCLUSION: Our study unveils the role of miR-721 in regulating gluconeogenesis through KDM2A and FOXO1 under insulin resistance, pointing towards significant clinical and therapeutic implications for metabolic disorders. Moreover, the promising impact of laccaic acid highlights its potential as a valuable intervention in managing insulin resistance-associated metabolic diseases.

An extensive analysis of the prognostic and immune role of FOXO1 in various types of cancer.

Forkhead Box O1 (FOXO1) has been reported to play important roles in many tumors. However, FOXO1 has not been studied in pan-cancer. The purpose of this study was to reveal the roles of FOXO1 in pan-cancer (33 cancers in this study). Through multiple public platforms, a pan-cancer analysis of FOXO1 was conducted to obtained FOXO1 expression profiles in various tumors to explore the relationship between FOXO1 expression and prognosis of these tumors and to disclose the potential mechanism of FOXO1 in these tumors. FOXO1 was associated with the prognosis of multiple tumors, especially LGG (low grade glioma), OV (ovarian carcinoma), and KIRC (kidney renal clear cell carcinoma). FOXO1 might play the role of an oncogenic gene in LGG and OV, while playing the role of a cancer suppressor gene in KIRC. FOXO1 expression had a significant correlation with the infiltration of some immune cells in LGG, OV, and KIRC. By combining FOXO1 expression and immune cell infiltration, we found that FOXO1 might influence the overall survival of LGG through the infiltration of myeloid dendritic cells or CD4+ T cells. Functional enrichment analysis and gene set enrichment analysis showed that FOXO1 might play roles in tumors through immunoregulatory interactions between a lymphoid and a non-lymphoid cell, TGF-beta signaling pathway, and transcriptional misregulation in cancer. FOXO1 was associated with the prognosis of multiple tumors, especially LGG, OV, and KIRC. In these tumors, FOXO1 might play its role via the regulation of the immune microenvironment.

Upregulation of the AMPK-FOXO1-PDK4 pathway is a primary mechanism of pyruvate dehydrogenase activity reduction in tafazzin-deficient cells.

Barth syndrome (BTHS) is a rare disorder caused by mutations in the TAFAZZIN gene. Previous studies from both patients and model systems have established metabolic dysregulation as a core component of BTHS pathology. In particular, features such as lactic acidosis, pyruvate dehydrogenase (PDH) deficiency, and aberrant fatty acid and glucose oxidation have been identified. However, the lack of a mechanistic understanding of what causes these conditions in the context of BTHS remains a significant knowledge gap, and this has hindered the development of effective therapeutic strategies for treating the associated metabolic problems. In the current study, we utilized tafazzin-knockout C2C12 mouse myoblasts (TAZ-KO) and cardiac and skeletal muscle tissue from tafazzin-knockout mice to identify an upstream mechanism underlying impaired PDH activity in BTHS. This mechanism centers around robust upregulation of pyruvate dehydrogenase kinase 4 (PDK4), resulting from hyperactivation of AMP-activated protein kinase (AMPK) and subsequent transcriptional upregulation by forkhead box protein O1 (FOXO1). Upregulation of PDK4 in tafazzin-deficient cells causes direct phospho-inhibition of PDH activity accompanied by increased glucose uptake and elevated intracellular glucose concentration. Collectively, our findings provide a novel mechanistic framework whereby impaired tafazzin function ultimately results in robust PDK4 upregulation, leading to impaired PDH activity and likely linked to dysregulated metabolic substrate utilization. This mechanism may underlie previously reported findings of BTHS-associated metabolic dysregulation.

NEK2 promotes the development of ovarian endometriosis and impairs decidualization by phosphorylating FOXO1.

Ovarian endometriosis is a common gynecological disease, and one of its most significant symptoms is infertility. In patients with endometriosis, defects in endometrial decidualization lead to impaired endometrial receptivity and embryo implantation, thus affecting early pregnancy and women's desire to have children. However, the mechanisms underlying the development of endometriosis and its associated defective decidualization are unclear. We find that NEK2 expression is increased in the ectopic and eutopic endometrium of patients with endometriosis. Meanwhile, NEK2 interacts with FOXO1 and phosphorylates FOXO1 at Ser184, inhibiting the stability of the FOXO1 protein. Importantly, NEK2-mediated phosphorylation of FOXO1 at Ser184 promotes cell proliferation, migration, invasion and impairs decidualization. Furthermore, INH1, an inhibitor of NEK2, inhibits the growth of ectopic lesions in mouse models of endometriosis and promotes endometrial decidualization in mouse models of artificially induced decidualization. Taken together, these findings indicate that NEK2 regulates the development of endometriosis and associated disorders of decidualization through the phosphorylation of FOXO1, providing a new therapeutic target for its treatment.

Comprehensive data mining reveals RTK/RAS signaling pathway as a promoter of prostate cancer lineage plasticity through transcription factors and CNV.

Prostate cancer lineage plasticity is a key driver in the transition to neuroendocrine prostate cancer (NEPC), and the RTK/RAS signaling pathway is a well-established cancer pathway. Nevertheless, the comprehensive link between the RTK/RAS signaling pathway and lineage plasticity has received limited investigation. In particular, the intricate regulatory network governing the interplay between RTK/RAS and lineage plasticity remains largely unexplored. The multi-omics data were clustered with the coefficient of argument and neighbor joining algorithm. Subsequently, the clustered results were analyzed utilizing the GSEA, gene sets related to stemness, multi-lineage state datasets, and canonical cancer pathway gene sets. Finally, a comprehensive exploration of the data based on the ssGSEA, WGCNA, GSEA, VIPER, prostate cancer scRNA-seq data, and the GPSAdb database was conducted. Among the six modules in the clustering results, there are 300 overlapping genes, including 3 previously unreported prostate cancer genes that were validated to be upregulated in prostate cancer through RT-qPCR. Function Module 6 shows a positive correlation with prostate cancer cell stemness, multi-lineage states, and the RTK/RAS signaling pathway. Additionally, the 19 leading-edge genes of the RTK/RAS signaling pathway promote prostate cancer lineage plasticity through a complex network of transcriptional regulation and copy number variations. In the transcriptional regulation network, TP63 and FOXO1 act as suppressors of prostate cancer lineage plasticity, whereas RORC exerts a promoting effect. This study provides a comprehensive perspective on the role of the RTK/RAS pathway in prostate cancer lineage plasticity and offers new clues for the treatment of NEPC.

mir-182-5p regulates all three phases of inflammation, proliferation, and remodeling during cutaneous wound healing.

Wound healing is a highly programmed process, in which any abnormalities result in scar formation. MicroRNAs are potent regulators affecting wound repair and scarification. However, the function of microRNAs in wound healing is not fully understood. Here, we analyzed the expression and function of microRNAs in patients with cutaneous wounds. Cutaneous wound biopsies from patients with either hypertrophic scarring or normal wound repair were collected during inflammation, proliferation, and remodeling phases. Fourteen candidate microRNAs were selected for expression analysis by qRT-PCR. The expression of genes involved in inflammation, angiogenesis, proliferation, and migration were measured using qRT-PCR. Cell cycle and scratch assays were used to explore the proliferation and migration rates. Flow cytometry analysis was employed to examine TGF-ß, αSMA and collagen-I expression. Target gene suggestion was performed using Enrichr tool. The results showed that miR-16-5p, miR-152-3p, miR-125b-5p, miR-34c-5p, and miR-182-5p were revealed to be differentially expressed between scarring and non-scarring wounds. Based on the expression patterns obtained, miR-182-5p was selected for functional studies. miR-182-5p induced RELA expression synergistically upon IL-6 induction in keratinocytes and promoted angiogenesis. miR-182-5p prevented keratinocyte migration, while overexpressed TGF-ß3 following induction of inflammation. Moreover, miR-182-5p enhanced fibroblast proliferation, migration, differentiation, and collagen-1 expression. FoxO1 and FoxO3 were found to potentially serve as putative gene targets of miR-182-5p. In conclusion, miR-182-5p is differentially expressed between scarring and non-scarring wounds and affect the behavior of cells involved in cutaneous wound healing. Deregulated expression of miR-182-5p adversely affects the proper transition of wound healing phases, resulting in scar formation.

Ghrelin alleviates intestinal ischemia-reperfusion injury by activating the GHSR-1α/Sirt1/FOXO1 pathway.

Ischemia-reperfusion (IR) injury is primarily characterized by the restoration of blood flow perfusion and oxygen supply to ischemic tissue and organs, but it paradoxically leads to tissue injury aggravation. IR injury is a challenging pathophysiological process that is difficult to avoid clinically and frequently occurs during organ transplantation, surgery, shock resuscitation, and other processes. The major causes of IR injury include increased levels of free radicals, calcium overload, oxidative stress, and excessive inflammatory response. Ghrelin is a newly discovered brain-intestinal peptide with anti-inflammatory and antiapoptotic effects that improve blood supply. The role and mechanism of ghrelin in intestinal ischemia-reperfusion (IIR) injury remain unclear. We hypothesized that ghrelin could attenuate IIR-induced oxidative stress and apoptosis. To investigate this, we established IIR by using a non-invasive arterial clip to clamp the root of the superior mesenteric artery (SMA) in mice. Ghrelin was injected intraperitoneally at a dose of 50 µg/kg 20 min before IIR surgery, and [D-Lys3]-GHRP-6 was injected intraperitoneally at a dose of 12 nmol/kg 20 min before ghrelin injection. We mimicked the IIR process with hypoxia-reoxygenation (HR) in Caco-2 cells, which are similar to intestinal epithelial cells in structure and biochemistry. Our results showed that ghrelin inhibited IIR/HR-induced oxidative stress and apoptosis by activating GHSR-1α. Moreover, it was found that ghrelin activated the GHSR-1α/Sirt1/FOXO1 signaling pathway. We further inhibited Sirt1 and found that Sirt1 was critical for ghrelin-mediated mitigation of IIR/HR injury. Overall, our data suggest that pretreatment with ghrelin reduces oxidative stress and apoptosis to attenuate IIR/HR injury by binding with GHSR-1α to further activate Sirt1.

Tea Polysaccharide Ameliorates High-Fat Diet-Induced Renal Tubular Ectopic Lipid Deposition via Regulating the Dynamic Balance of Lipogenesis and Lipolysis.

Renal tubular ectopic lipid deposition (ELD) plays a significant role in the development of chronic kidney disease, posing a great threat to human health. The present work aimed to explore the intervention effect and potential molecular mechanism of a purified tea polysaccharide (TPS3A) on renal tubular ELD. The results demonstrated that TPS3A effectively improved kidney function and slowed the progression of tubulointerstitial fibrosis in high-fat-diet (HFD)-exposed ApoE-/- mice. Additionally, TPS3A notably suppressed lipogenesis and enhanced lipolysis, as shown by the downregulation of lipogenesis markers (SREBP-1 and FAS) and the upregulation of lipolysis markers (HSL and ATGL), thereby reducing renal tubular ELD in HFD-fed ApoE-/- mice and palmitic-acid-stimulated HK-2 cells. The AMPK-SIRT1-FoxO1 axis is a core signal pathway in regulating lipid deposition. Consistently, TPS3A significantly increased the levels of phosphorylated-AMPK, SIRT1, and deacetylation of Ac-FoxO1. However, these effects of TPS3A on lipogenesis and lipolysis were abolished by AMPK siRNA, SIRT1 siRNA, and FoxO1 inhibitor, resulting in exacerbated lipid deposition. Taken together, TPS3A shows promise in ameliorating renal tubular ELD by inhibiting lipogenesis and promoting lipolysis through the AMPK-SIRT1-FoxO1 signaling pathway.

The mechanosensory channel PIEZO1 functions upstream of angiopoietin/TIE/FOXO1 signaling in lymphatic development.

Lymphedema is a debilitating disease with no effective cure and affects an estimated 250 million individuals worldwide. Prior studies have identified mutations in piezo-type mechanosensitive ion channel component 1 (PIEZO1), angiopoietin 2 (ANGPT2), and tyrosine kinase with Ig-like and EGF-like domains 1 (TIE1) in patients with primary lymphedema. Here, we identified crosstalk between these molecules and showed that activation of the mechanosensory channel PIEZO1 in lymphatic endothelial cells (LECs) caused rapid exocytosis of the TIE ligand ANGPT2, ectodomain shedding of TIE1 by disintegrin and metalloproteinase domain-containing protein 17 (ADAM17), and increased TIE/PI3K/AKT signaling, followed by nuclear export of the transcription factor FOXO1. These data establish a functional network between lymphedema-associated genes and provide what we believe to be the first molecular mechanism bridging channel function with vascular signaling and intracellular events culminating in transcriptional regulation of genes expressed in LECs. Our study provides insights into the regulation of lymphatic function and molecular pathways involved in human disease.

Lycopene Promotes Osteogenesis and Reduces Adipogenesis through Regulating FoxO1/PPARγ Signaling in Ovariectomized Rats and Bone Marrow Mesenchymal Stem Cells.

Recent interest in preventing the development of osteoporosis has focused on the regulation of redox homeostasis. However, the action of lycopene (LYC), a strong natural antioxidant compound, on osteoporotic bone loss remains largely unknown. Here, we show that oral administration of LYC to OVX rats for 12 weeks reduced body weight gain, improved lipid metabolism, and preserved bone quality. In addition, LYC treatment inhibited ROS overgeneration in serum and bone marrow in OVX rats, and in BMSCs upon H2O2 stimulation, leading to inhibiting adipogenesis and promoting osteogenesis during bone remodeling. At the molecular level, LYC improved bone quality via an increase in the expressions of FoxO1 and Runx2 and a decrease in the expressions of PPARγ and C/EBPα in OVX rats and BMSCs. Collectively, these findings suggest that LYC attenuates osteoporotic bone loss through promoting osteogenesis and inhibiting adipogenesis via regulation of the FoxO1/PPARγ pathway driven by oxidative stress, presenting a novel strategy for osteoporosis management.

FOXO1 reshapes neutrophils to aggravate acute brain damage and promote late depression after traumatic brain injury.

BACKGROUND: Neutrophils are traditionally viewed as first responders but have a short onset of action in response to traumatic brain injury (TBI). However, the heterogeneity, multifunctionality, and time-dependent modulation of brain damage and outcome mediated by neutrophils after TBI remain poorly understood. METHODS: Using the combined single-cell transcriptomics, metabolomics, and proteomics analysis from TBI patients and the TBI mouse model, we investigate a novel neutrophil phenotype and its associated effects on TBI outcome by neurological deficit scoring and behavioral tests. We also characterized the underlying mechanisms both in vitro and in vivo through molecular simulations, signaling detections, gene expression regulation assessments [including dual-luciferase reporter and chromatin immunoprecipitation (ChIP) assays], primary cultures or co-cultures of neutrophils and oligodendrocytes, intracellular iron, and lipid hydroperoxide concentration measurements, as well as forkhead box protein O1 (FOXO1) conditional knockout mice. RESULTS: We identified that high expression of the FOXO1 protein was induced in neutrophils after TBI both in TBI patients and the TBI mouse model. Infiltration of these FOXO1high neutrophils in the brain was detected not only in the acute phase but also in the chronic phase post-TBI, aggravating acute brain inflammatory damage and promoting late TBI-induced depression. In the acute stage, FOXO1 upregulated cytoplasmic Versican (VCAN) to interact with the apoptosis regulator B-cell lymphoma-2 (BCL-2)-associated X protein (BAX), suppressing the mitochondrial translocation of BAX, which mediated the antiapoptotic effect companied with enhancing interleukin-6 (IL-6) production of FOXO1high neutrophils. In the chronic stage, the "FOXO1-transferrin receptor (TFRC)" mechanism contributes to FOXO1high neutrophil ferroptosis, disturbing the iron homeostasis of oligodendrocytes and inducing a reduction in myelin basic protein, which contributes to the progression of late depression after TBI. CONCLUSIONS: FOXO1high neutrophils represent a novel neutrophil phenotype that emerges in response to acute and chronic TBI, which provides insight into the heterogeneity, reprogramming activity, and versatility of neutrophils in TBI.

Polystyrene nanoplastics with different functional groups and charges have different impacts on type 2 diabetes.

BACKGROUND: Increasing attention is being paid to the environmental and health impacts of nanoplastics (NPs) pollution. Exposure to nanoplastics (NPs) with different charges and functional groups may have different adverse effects after ingestion by organisms, yet the potential ramifications on mammalian blood glucose levels, and the risk of diabetes remain unexplored. RESULTS: Mice were exposed to PS-NPs/COOH/NH2 at a dose of 5 mg/kg/day for nine weeks, either alone or in a T2DM model. The findings demonstrated that exposure to PS-NPs modified by different functional groups caused a notable rise in fasting blood glucose (FBG) levels, glucose intolerance, and insulin resistance in a mouse model of T2DM. Exposure to PS-NPs-NH2 alone can also lead the above effects to a certain degree. PS-NPs exposure could induce glycogen accumulation and hepatocellular edema, as well as injury to the pancreas. Comparing the effect of different functional groups or charges on T2DM, the PS-NPs-NH2 group exhibited the most significant FBG elevation, glycogen accumulation, and insulin resistance. The phosphorylation of AKT and FoxO1 was found to be inhibited by PS-NPs exposure. Treatment with SC79, the selective AKT activator was shown to effectively rescue this process and attenuate T2DM like lesions. CONCLUSIONS: Exposure to PS-NPs with different functional groups (charges) induced T2DM-like lesions. Amino-modified PS-NPs cause more serious T2DM-like lesions than pristine PS-NPs or carboxyl functionalized PS-NPs. The underlying mechanisms involved the inhibition of P-AKT/P-FoxO1. This study highlights the potential risk of NPs pollution on T2DM, and provides a new perspective for evaluating the impact of plastics aging.

Ginsenoside compound K induces ferroptosis via the FOXO pathway in liver cancer cells.

Liver cancer is a common malignant tumor worldwide, traditional Chinese medicine is one of the treatment measures for liver cancer because of its good anti-tumor effects and fewer toxic side effects. Ginsenoside CK (CK) is an active component of ginseng. This study explored the mechanism by which CK induced ferroptosis in liver cancer cells. We found that CK inhibited the proliferation of HepG2 and SK-Hep-1 cells, induced ferroptosis of cells. Ferrostatin-1, an ferroptosis inhibitor, was used to verify the role of CK in inducing ferroptosis of liver cancer cells. Network pharmacological analysis identified the FOXO pathway as a potential mechanism of CK, and western blot showed that CK inhibited p-FOXO1. In cells treated with the FOXO1 inhibitor AS1842856, further verify the involvement of the FOXO pathway in regulating CK-induced ferroptosis in HepG2 and SK-Hep-1 cells. A HepG2 cell-transplanted tumor model was established in nude mice, and CK inhibited the growth of transplanted tumors in nude mice, p-FOXO1 was decreased in tumor tissues, and SLC7A11 and GPX4 expressions were also down-regulated after CK treatment. These findings suggested that CK induces ferroptosis in liver cancer cells by inhibiting FOXO1 phosphorylation and activating the FOXO signaling pathway, thus playing an antitumor role.

Inhibition of the RPS6KA1/FoxO1 signaling axis by hydroxycitric acid attenuates HFD-induced obesity through MCE suppression.

BACKGROUND: Because obesity is associated with a hyperplasia-mediated increase in adipose tissue, inhibiting cell proliferation during mitotic clonal expansion (MCE) is a leading strategy for preventing obesity. Although (-)-hydroxycitric acid (HCA) is used to control obesity, the molecular mechanisms underlying its effects on MCE are poorly understood. PURPOSE: This study aimed to investigate the potential effects of HCA on MCE and underlying molecular mechanisms affecting adipogenesis and obesity improvements. METHODS: Preadipocyte cell line, 3T3-L1, were treated with HCA; oil red O, cell proliferation, cell cycle, and related alterations in signaling pathways were examined. High-fat diet (HFD)-fed mice were administered HCA for 12 weeks; body and adipose tissues weights were evaluated, and the regulation of signaling pathways in epidydimal white adipose tissue were examined in vivo. RESULTS: Here, we report that during MCE, HCA attenuates the proliferation of the preadipocyte cell line, 3T3-L1, by arresting the cell cycle at the G0/G1 phase. In addition, HCA markedly inhibits Forkhead Box O1 (FoxO1) phosphorylation, thereby inducing the expression of cyclin-dependent kinase inhibitor 1B and suppressing the levels of cyclin-dependent kinase 2, cyclin E1, proliferating cell nuclear antigen, and phosphorylated retinoblastoma. Importantly, we found that ribosomal protein S6 kinase A1 (RPS6KA1) influences HCA-mediated inactivation of FoxO1 and its nuclear exclusion. An animal model of obesity revealed that HCA reduced high-fat diet-induced obesity by suppressing adipocyte numbers as well as epididymal and mesenteric white adipose tissue mass, which is attributed to the regulation of RPS6KA1, FoxO1, CDKN1B and PCNA that had been consistently identified in vitro. CONCLUSIONS: These findings provide novel insights into the mechanism by which HCA regulates adipogenesis and highlight the RPS6KA1/FoxO1 signaling axis as a therapeutic target for obesity.

Aminophylline Improves Ventilator-Induced Diaphragmatic Dysfunction by Targeting IGF-1-FOXO1-MURF1 Pathway.

BACKGROUND: The usage of life-saving mechanical ventilation (MV) could cause ventilator-induced diaphragmatic dysfunction (VIDD), increasing both mortality and morbidity. Aminophylline (AP) has the potential to enhance the contractility of animal skeletal muscle fibers and improve the activity of human respiratory muscles, and the insulin-like growth factor-1 (IGF-1)- forkhead box protein O1 (FOXO1)-muscle RING finger-1 (MURF1) pathway plays a crucial role in skeletal muscle dysfunction. This study aimed to investigate the impact of AP on VIDD and to elucidate the role of the IGF-1-FOXO1-MURF1 pathway as an underlying mechanism. METHODS: Rat models of VIDD were established through MV treatment. IGF-1 lentiviral (LV) interference (LV-IGF-1-shRNA; controlled by lentiviral negative control LV-NC) was employed to inhibit IGF-1 expression and thereby block the IGF-1-FOXO1-MURF1 pathway. Protein and mRNA levels of IGF-1, FOXO1, and MURF1 were assessed using western blot and real-time reverse transcriptase-polymerase chain reaction (RT-qPCR), respectively. Diaphragm contractility and morphometry were examined through measurement of compound muscle action potentials (CMAPs) and hematoxylin and eosin (H&E) staining. Oxidative stress was evaluated by levels of hydrogen peroxide (H2O2), superoxide dismutase (SOD), antioxidant glutathione (GSH), and carbonylated protein. Mitochondrial stability was assessed by measuring the mitochondrial membrane potential (MMP), and mitochondrial fission and mitophagy were examined through protein levels of dynamin-related protein 1 (DRP1), mitofusin 2 protein (MFN2), phosphatase and tensin homolog (PTEN)-induced kinase 1 (PINK1), and Parkin (western blot). Apoptosis was evaluated using the terminal deoxynucleotidyl transferase-mediated uridine 5'-triphosphate (UTP) nick-end labeling (TUNEL) assay and levels of Bax, B-cell lymphoma 2 (BCL-2), and Caspase-3. Levels of Atrogin-1, neuronally expressed developmentally downregulated 4 (NEDD4), and muscle ubiquitin ligase of SCF complex in atrophy-1 (MUSA1) mRNA, as well as ubiquitinated protein, were utilized to determine protein degradation. Furthermore, the SUnSET (surface sensing of translation) method was employed to determine rates of protein synthesis. RESULTS: MV treatment upregulated IGF-1 while downregulated FOXO1 and MURF1 (p < 0.05). AP administration reversed IGF-1, FOXO1 and MURF1 (p < 0.05), which was suppressed again by IGF-1 inhibition (p < 0.05), demonstrating the blockage of the IGF-1-FOXO1-MURF1 pathway. MV treatment caused decreased CMAP and cross-sectional areas of diaphragm muscle fibers, and increased time course of CMAP (p < 0.05). Additionally, oxidative stress, cell apoptosis, and protein degradation were increased and mitochondrial stability was decreased by MV treatment (p < 0.05). Conversely, AP administration reversed all these changes induced by MV, but this reversal was disrupted by the blockage of the IGF-1-FOXO1-MURF1 pathway. CONCLUSIONS: In this study, MV treatment induced symptoms of VIDD in rats, which were all effectively reversed by AP regulating the IGF-1-FOXO1-MURF1 pathway, demonstrating the potential of AP in ameliorating VIDD.

FOXO1 enhances CAR T cell stemness, metabolic fitness and efficacy.

Chimeric antigen receptor (CAR) T cell therapy has transformed the treatment of haematological malignancies such as acute lymphoblastic leukaemia, B cell lymphoma and multiple myeloma1-4, but the efficacy of CAR T cell therapy in solid tumours has been limited5. This is owing to a number of factors, including the immunosuppressive tumour microenvironment that gives rise to poorly persisting and metabolically dysfunctional T cells. Analysis of anti-CD19 CAR T cells used clinically has shown that positive treatment outcomes are associated with a more 'stem-like' phenotype and increased mitochondrial mass6-8. We therefore sought to identify transcription factors that could enhance CAR T cell fitness and efficacy against solid tumours. Here we show that overexpression of FOXO1 promotes a stem-like phenotype in CAR T cells derived from either healthy human donors or patients, which correlates with improved mitochondrial fitness, persistence and therapeutic efficacy in vivo. This work thus reveals an engineering approach to genetically enforce a favourable metabolic phenotype that has high translational potential to improve the efficacy of CAR T cells against solid tumours.