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.

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.

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.

Knockdown of NCAPD3 inhibits the tumorigenesis of non-small cell lung cancer by regulation of the PI3K/Akt pathway.

BACKGROUND: Accumulating evidence indicates that aberrant non-SMC condensin II complex subunit D3 (NCAPD3) is associated with carcinogenesis of various cancers. Nevertheless, the biological role of NCAPD3 in the pathogenesis of non-small cell lung cancer (NSCLC) remains unclear. METHODS: Immunohistochemistry and Western blot were performed to assess NCAPD3 expression in NSCLC tissues and cell lines. The ability of cell proliferation, invasion, and migration was evaluated by CCK-8 assays, EdU assays, Transwell assays, and scratch wound healing assays. Flow cytometry was performed to verify the cell cycle and apoptosis. RNA-sequence and rescue experiment were performed to reveal the underlying mechanisms. RESULTS: The results showed that the expression of NCAPD3 was significantly elevated in NSCLC tissues. High NCAPD3 expression in NSCLC patients was substantially associated with a worse prognosis. Functionally, knockdown of NCAPD3 resulted in cell apoptosis and cell cycle arrest in NSCLC cells as well as a significant inhibition of proliferation, invasion, and migration. Furthermore, RNA-sequencing analysis suggested that NCAPD3 contributes to NSCLC carcinogenesis by regulating PI3K/Akt/FOXO4 pathway. Insulin-like growth factors-1 (IGF-1), an activator of PI3K/Akt signaling pathway, could reverse NCAPD3 silence-mediated proliferation inhibition and apoptosis in NSCLC cells. CONCLUSION: NCAPD3 suppresses apoptosis and promotes cell proliferation via the PI3K/Akt/FOXO4 signaling pathway, suggesting a potential use for NCAPD3 inhibitors as NSCLC therapeutics.

Forkhead O Transcription Factor 4 Restricts HBV Covalently Closed Circular DNA Transcription and HBV Replication through Genetic Downregulation of Hepatocyte Nuclear Factor 4 Alpha and Epigenetic Suppression of Covalently Closed Circular DNA via Interacting with Promyelocytic Leukemia Protein.

Nuclear located hepatitis B virus (HBV) covalently closed circular DNA (cccDNA) remains the key obstacle to cure chronic hepatitis B (CHB). In our previous investigation, it was found that FoxO4 could inhibit HBV core promoter activity through downregulating the expression of HNF4α. However, the exact mechanisms whereby FoxO4 inhibits HBV replication, especially its effect on cccDNA, remain unclear. Here, our data further revealed that FoxO4 could effectively inhibit cccDNA mediated transcription and HBV replication without affecting cccDNA level. Mechanistic study showed that FoxO4 could cause epigenetic suppression of cccDNA. Although FoxO4-mediated downregulation of HNF4α contributed to inhibiting HBV core promoter activity, it had little effect on cccDNA epigenetic regulation. Further, it was found that FoxO4 could colocalize within promyelocytic leukemia protein (PML) nuclear bodies and interact with PML. Of note, PML was revealed to be critical for FoxO4-mediated inhibition of cccDNA epigenetic modification and of the following cccDNA transcription and HBV replication. Furthermore, FoxO4 was found to be downregulated in HBV-infected hepatocytes and human liver tissues, and it was negatively correlated with cccDNA transcriptional activity in CHB patients. Together, these findings highlight the role of FoxO4 in suppressing cccDNA transcription and HBV replication via genetic downregulation of HNF4α and epigenetic suppression of cccDNA through interacting with PML. Targeting FoxO4 may present as a new therapeutic strategy against chronic HBV infection. IMPORTANCE HBV cccDNA is a determining factor for viral persistence and the main obstacle for a cure of chronic hepatitis B. Strategies that target cccDNA directly are therefore of great importance in controlling persistent HBV infection. In present investigation, we found that FoxO4 could efficiently suppress cccDNA transcription and HBV replication without affecting the level of cccDNA itself. Further, our data revealed that FoxO4 might inhibit cccDNA function via a two-part mechanism: one is to epigenetically suppress cccDNA transcription via interacting with PML, and the other is to inhibit HBV core promoter activity via the genetic downregulation of HNF4α. Of note, HBV might dampen the expression of FoxO4 for its own persistent infection. We propose that manipulation of FoxO4 may present as a potential therapeutic strategy against chronic HBV infection.

FOXO4 mediates resistance to oxidative stress in lens epithelial cells by modulating the TRIM25/Nrf2 signaling.

Oxidative stress damage to the lens is a key factor in most cataracts. Forkhead box O 4 (FOXO4), a member of the forkhead box O family, plays a pivotal role in oxidative stress. FOXO4 is upregulated in lens of age-related cataract patients, but its role in cataract has not been elucidated. Herein, we investigated the role and mechanism of FOXO4 during oxidative stress damage in lens epithelial cells. H2O2 treatment enhanced FOXO4 expression in HLEpiC cells. Short hairpin RNAs mediated FOXO4 silence aggravated H2O2-induced cell apoptosis. In addition, upon H2O2 exposure, silencing of FOXO4 reduced SOD and CAT activities, as well as increased intracellular MDA and ROS levels. FOXO4 silencing also inhibited Nrf2 nuclear translocation, followed by reducing the expressions of Nrf2-governed antioxidant genes HO-1 and NOQ-1. Exogenous overexpression of FOXO4 was also involved in this study and exhibited opposite effects of FOXO4-silencing. Mechanistically, FOXO4 directly bound the promoter of TRIM25 and regulated its transcription, thereby activating the Nrf2 signaling. Taken together, in the condition of oxidative stress, the expression of FOXO4 showed a compensatory upregulation and it exhibited an anti-oxidative effect by modulating the transcription of TRIM25, thus activating the Nrf2 signaling. The FOXO4/TRIM25/Nrf2 axis may be associated with the pathological mechanisms of cataract.

FoxO4 mediates macrophage M2 polarization by promoting LXA4R expression in an ovalbumin-induced allergic asthma model in mice.

BACKGROUND: Asthma imposes a heavy burden due to its high prevalence. Forkhead box O4 (FoxO4) proteins participate in the modulation of cell progression. However, the role and mechanism of FoxO4 in asthma remains uncharted. METHODS: An allergic asthma model was constructed by the induction of ovalbumin and interleukin (IL)-4 in mice and monocyte/macrophage-like Raw264.7 cells, respectively. The role and mechanism of FoxO4 in asthma was determined by pathological staining, immunofluorescence assay, measurement of inflammatory cells in the blood, reverse transcription quantitative polymerase chain reaction (RT-qPCR), Western blot analysis, and flow cytometry. RESULTS: Ovalbumin treatment triggered an obvious inflammatory cell infiltration with a prominent increase in F4/80+ cell numbers. The relative messenger RNA (mRNA) and protein expressions of FoxO4 were increased in both ovalbumin-induced mice and interleukin-4 (IL-4)-induced Raw264.7 cells. Inhibition of FoxO4 via AS1842856 reduced inflammatory cell infiltration, the number of Periodic Acid Schiff+ (PAS+) goblet cells, the numbers of inflammatory cells in the blood, and the airway resistance in ovalbumin-induced mice. Besides, interference of FoxO4 decreased the number of F4/80+CD206+ cells, and the relative protein expressions of CD163 and Arg1 in vivo and in vitro. Mechanically, suppression of FoxO4 diminished the relative mRNA and protein expressions of LXA4R in both ovalbumin-induced mice and IL-4-induced Raw264.7 cells. Overexpression of LXA4R reversed the outcomes caused by repression of FoxO4, including airway resistance, the number of F4/80+ cells, the proportion of CD206+ cells in ovalbumin-induced mice, and the proportion of F4/80+CD206+ cells in IL-4-induced Raw264.7 cells. CONCLUSION: FoxO4/LXA4R axis mediated macrophage M2 polarization in allergic asthma.

Deletion of the foxO4 Gene Increases Hypoxia Tolerance in Zebrafish.

Oxygen homeostasis is an important organizing principle for understanding development, physiology, disease, and evolution. Under various physiological and pathological states, organisms experience oxygen deficiency or hypoxia. FoxO4 has been recognized as an important transcriptional regulator involved in a variety of cellular functions, including proliferation, apoptosis, differentiation, and stress resistance, but its role in hypoxia adaptation mechanisms in animals is not so clear. To explore the role of foxO4 in the hypoxia response, we detected the expression of foxO4 and the regulatory relationship between Hif1α and foxO4 under hypoxic conditions. It was found that the expression of foxO4 was up-regulated in ZF4 cells and zebrafish tissues after hypoxia treatment, and Hif1α could directly target the HRE of the foxO4 promoter to regulate foxO4 transcription, indicating that foxO4 was involved in the hypoxia response by the Hif1α-mediated pathway. Furthermore, we obtained foxO4 knockout zebrafish and found that the disruption of foxO4 increased the tolerance to hypoxia. Further research found that the oxygen consumption and locomotor activity of foxO4-/- zebrafish were lower than those of WT zebrafish, as was true for NADH content, NADH/NAD+ rate, and expression of mitochondrial respiratory chain complex-related genes. This suggests that disruption of foxO4 reduced the oxygen demand threshold of the organism, which explained why the foxO4-/- zebrafish were more tolerant to hypoxia than WT zebrafish. These results will provide a theoretical basis for further study of the role of foxO4 in the hypoxia response.

Whole-Transcriptome Sequencing of Antler Tissue Reveals That circRNA2829 Regulates Chondrocyte Proliferation and Differentiation via the miR-4286-R+1/FOXO4 Axis.

The antler is the unique mammalian organ found to be able to regenerate completely and periodically after loss, and the continuous proliferation and differentiation of mesenchymal cells and chondrocytes together complete the regeneration of the antler. Circular non-coding RNAs (circRNAs) are considered to be important non-coding RNAs that regulate body development and growth. However, there are no reports on circRNAs regulating the antler regeneration process. In this study, full-transcriptome high-throughput sequencing was performed on sika deer antler interstitial and cartilage tissues, and the sequencing results were verified and analyzed. The competing endogenous RNA (ceRNA) network related to antler growth and regeneration was further constructed, and the differentially expressed circRNA2829 was screened out from the network to study its effect on chondrocyte proliferation and differentiation. The results indicated that circRNA2829 promoted cell proliferation and increased the level of intracellular ALP. The analysis of RT-qPCR and Western blot demonstrated that the mRNA and protein expression levels of genes involved in differentiation rose. These data revealed that circRNAs play a crucial regulatory role in deer antler regeneration and development. CircRNA2829 might regulate the antler regeneration process through miR-4286-R+1/FOXO4.

[Effect and mechanism of Dahuang Zhechong Pills in improving liver aging in rats by regulating ROS-mediated PI3K/Akt/FoxO4 signaling pathway].

Recent studies have shown that the occurrence and development of common liver diseases, including non-alcoholic fatty liver disease, cirrhosis, and liver cancer, are related to liver aging(LA). Therefore, to explore the effect and mechanism of Dahuang Zhechong Pills(DHZCP), a traditional classic prescription in improving LA with multiple targets, the present study randomly divided 24 rats into a normal group, a model group, a DHZCP group, and a vitamin E(VE) group, with six rats in each group. The LA model was induced by continuous intraperitoneal injection of D-galactose(D-gal) in rats. For the LA model rats, the general situation was evaluated by aging phenotype and body weight(BW). LA was assessed by the pathological characteristics of hepatocyte senescence, hepatic function indexes, the staining characteristics of phosphorylated histone family 2A variant(γ-H2AX), and the expression levels of cell cycle arrest proteins(P21, P53, P16) and senescence-associated secretory phenotype(SASP) in the liver. The activation of the reactive oxygen species(ROS)-mediated phosphatidylinositol-3 kinase(PI3K)/protein kinase B(Akt)/forkhead box protein O4(FoxO4) signaling pathway was estimated by hepatic ROS expression feature and the protein expression levels of the key signaling molecules in the PI3K/Akt/FoxO4 signaling pathway. The results showed that after the treatment with DHZCP or VE for 12 weeks, for the DHZCP and VE groups, the characterized aging phenotype, BW, pathological characteristics of hepatocyte senescence, hepatic function indexes, relative expression of ROS in the liver, protein expression levels of key signaling molecules including p-PI3K, p-Akt, and FoxO4 in the liver, staining characteristics of γ-H2AX, and the protein expression levels of P16, P21, P53, interleukin-6(IL-6), and tumor necrosis factor-α(TNF-α) in the liver were improved, and the effects of DHZCP and VE were similar. Based on the D-gal-induced LA model in rats, this study demonstrates that DHZCP can ameliorate LA with multiple targets in vivo, and its effects and mechanism are related to regulating the activation of the ROS-mediated PI3K/Akt/FoxO4 signaling pathway in the liver. These findings are expected to provide new pharmacological evidence for the treatment of DHZCP in aging-related liver diseases.

FOXO4 peptide targets myofibroblast ameliorates bleomycin-induced pulmonary fibrosis in mice through ECM-receptor interaction pathway.

Pulmonary fibrosis (PF) is a progressive interstitial lung disease with limited treatment options. The incidence and prevalence of PF is increasing with age, cell senescence has been proposed as a pathogenic driver, the clearance of senescent cells could improve lung function in PF. FOXO4-D-Retro-Inverso (FOXO4-DRI), a synthesis peptide, has been reported to selectively kill senescent cells in aged mice. However, it remains unknown if FOXO4-DRI could clear senescent cells in PF and reverse this disease. In this study, we explored the effect of FOXO4-DRI on bleomycin (BLM)-induced PF mouse model. We found that similar as the approved medication Pirfenidone, FOXO4-DRI decreased senescent cells, downregulated the expression of senescence-associated secretory phenotype (SASP) and attenuated BLM-induced morphological changes and collagen deposition. Furthermore, FOXO4-DRI could increase the percentage of type 2 alveolar epithelial cells (AEC2) and fibroblasts, and decrease the myofibroblasts in bleomycin (BLM)-induced PF mouse model. Compared with mouse and human lung fibroblast cell lines, FOXO4-DRI is inclined to kill TGF-ß-induced myofibroblast in vitro. The inhibited effect of FOXO4-DRI on myofibroblast lead to a downregulation of extracellular matrix (ECM) receptor interaction pathway in BLM-induced PF. Above all, FOXO4-DRI ameliorates BLM-induced PF in mouse and may be served as a viable therapeutic option for PF.

FBXO7 triggers caspase 8-mediated proteolysis of the transcription factor FOXO4 and exacerbates neuronal cytotoxicity.

Parkinson's disease (PD) is characterized by the progressive loss of midbrain dopamine neurons in the substantia nigra. Mutations in the F-box only protein 7 gene (Fbxo7) have been reported to cause an autosomal recessive form of early-onset familial PD. FBXO7 is a part of the SKP1-Cullin1-F-box (SCF) E3 ubiquitin ligase complex, which mediates ubiquitination of numerous substrates. FBXO7 also regulates mitophagy, cell growth, and proteasome activity. A member of the FOXO family, the transcription factor FOXO4, is also known to modulate several cellular responses, including cell cycle progression and apoptosis; however, the relationship between FBXO7 and FOXO4 has not been investigated. In this study, we determined that FBXO7 binds to FOXO4 and negatively regulates intracellular FOXO4 levels. Interestingly, we also found that FBXO7-mediated degradation of FOXO4 did not occur through either of two major proteolysis systems, the ubiquitin-proteasome system or the lysosome-autophagy pathway, although it was blocked by a caspase 8-specific inhibitor and caspase 8-knockdown. Moreover, intracellular FOXO4 levels were greatly reduced in dopaminergic MN9D cells following treatment with neurotoxic 6-hydroxydopamine (6-OHDA), which was produced upon FBXO7-mediated and caspase 8-mediated proteolysis. Taken together, these results suggest that FOXO4 is negatively regulated in FBXO7-linked PD through caspase 8 activation, suppressing the cytoprotective effect of FOXO4 during 6-OHDA-induced neuronal cell death.

Circ_0084443 Inhibits Wound Healing Via Repressing Keratinocyte Migration Through Targeting the miR-17-3p/FOXO4 Axis.

Keratinocyte migration is a crucial process during skin wound healing, and circular RNAs are associated with keratinocyte migration. The purpose of our study was to clarify the role of circ_0084443 in wound healing. The levels of circ_0084443, microRNA (miR)-17-3p, and forkhead box protein O4 (FOXO4) were examined by quantitative reverse transcription-PCR. Cell migration was detected via wound scratch assay or transwell assay. The protein expression was measured using western blot. The binding analysis between miR-17-3p and circ_0084443 or FOXO4 was determined by dual-luciferase reporter assay and RNA Immunoprecipitation assay. TGF-ß1 decreased the levels of circ_0084443 and FOXO4 while increased the miR-17-3p expression in keratinocytes by a concentration-dependent manner. Circ_0084443 acted as a miR-17-3p sponge and circ_0084443 overexpression alleviated TGF-ß1-induced migration of keratinocytes by sponging miR-17-3p. FOXO4 was a target for miR-17-3p. The downregulation of miR-17-3p suppressed cell migration in TGF-ß1-induced cells by increasing the FOXO4 level. Circ_0084443 positively regulated the FOXO4 expression by sponging miR-17-3p. Circ_0084443 suppressed the TGFß signaling pathway by affecting the miR-17-3p/FOXO4 axis. These results exhibited that circ_0084443 suppressed the TGF-ß1-induced keratinocyte migration by regulating the miR-17-3p/FOXO4 axis, suggesting the application potential of circ_0084443 in wound-healing-related diseases.

Targeting Multiple Homeostasis-Maintaining Systems by Ionophore Nigericin Is a Novel Approach for Senolysis.

Within the present study we proposed a novel approach for senolysis based on the simultaneous disturbance of the several homeostasis-maintaining systems in senescent cells including intracellular ionic balance, energy production and intracellular utilization of damaged products. Of note, we could not induce senolysis by applying ouabain, amiloride, valinomycin or NH4Cl-compounds that modify each of these systems solely. However, we found that ionophore nigericin can disturb plasma membrane potential, intracellular pH, mitochondrial membrane potential and autophagy at once. By affecting all of the tested homeostasis-maintaining systems, nigericin induced senolytic action towards stromal and epithelial senescent cells of different origins. Moreover, the senolytic effect of nigericin was independent of the senescence-inducing stimuli. We uncovered that K+ efflux caused by nigericin initiated pyroptosis in senescent cells. According to our data, the higher sensitivity of senescent cells compared to the control ones towards nigericin-induced death was partially mediated by the lower intracellular K+ content in senescent cells and by their predisposition towards pyroptosis. Finally, we proposed an interval dosing strategy to minimize the negative effects of nigericin on the control cells and to achieve maximal senolytic effect. Hence, our data suggest ionophore nigericin as a new senotherapeutic compound for testing against age-related diseases.

Hyalinizing epithelioid tumors with OGT-FOXO fusions. A case report of a non-acral soft tissue mass harboring a novel FOXO4 gene rearrangement.

Recurrent fusions between OGT and members of the Forkhead box (FOXO) family of genes have been recently described in three cases of hyalinizing epithelioid acral soft tissue tumors in young adults showing co-expression for EMA and CD34. Despite the lack of an established myoepithelial lineage by immunohistochemistry, these lesions have been labeled as myoepithelioma-like due to their epithelioid phenotype and sclerotic background. In this study, we report a novel FOXO4-OGT fusion identified by targeted RNA sequencing in an unclassified shoulder soft tissue mass in a 40-year-old male. The tumor showed nodular foci of increased cellularity in a uniformly hyalinized background. The neoplastic cells were mainly epithelioid and focally spindled, with eosinophilic cytoplasm and indented nuclei with mild atypia. The tumor lacked significant mitotic activity and necrosis. Immunohistochemically, the tumor showed variable positivity for EMA, pan-CK, CD34, ERG and FLI1, while it was negative for CD31, S100, SOX10, desmin, and MUC4. INI1 expression was retained. Due to its unusual histology and conflicting immunoprofile, TruSight RNA fusion panel sequencing was performed which revealed a fusion between FOXO4 exon 2 to OGT exon 2. This is the first example of a soft tissue lesion harboring OGT-related fusions occurring in a non-acral location and associated with FOXO4 gene. Its line of differentiation and biologic potential remain uncertain.

Primary myxoid and epithelioid mesenchymal tumor of the kidney with a novel GLI1-FOXO4 fusion.

To our knowledge, we describe the first mesenchymal tumor with a novel GLI1-FOXO4 fusion gene. This well-circumscribed kidney tumor displayed variably myxoid and epithelioid histologic features with a focally nodular growth pattern. The tumor cells showed bland, round to ovoid nuclei, with no overt high-grade features. The tumor showed focal immunopositivity for smooth muscle actin and Melan-A, which raised the possibility of a relationship with a perivascular epithelioid cell tumor. The clinical and morphologic features appear distinct from other reported neoplasms harboring GLI1 or FOXO4 gene rearrangements. The patient underwent radical nephrectomy and is without evidence of disease during a relatively short clinical follow-up period. However, the features of this tumor likely warrant long-term follow-up to monitor for the possibility of a late recurrence or metastasis. In addition to reporting this novel fusion-positive tumor, we also provide a brief review of GLI1 and FOXO4 gene functions in both normal and neoplastic contexts.

FOXO4 alleviates hippocampal neuronal damage in epileptic mice via the miR-138-5p/ROCK2 axis.

Epilepsy (EP) is one of the most universal neurological disorders. This study investigated the mechanism of forkhead box protein O4 (FOXO4) on hippocampal neuronal damage in EP mice. Initially, the EP mouse model and the in vitro HT-22 cell model were established. EP seizures and neuronal damage in mice were assessed. FOXO4, microRNA (miR)-138-5p, and rho-associated coiled-coil containing protein kinase 2 (ROCK2) levels in hippocampal tissues or HT-22 cells were examined. The cell viability and apoptosis of HT-22 cells were determined. The concentrations of oxidative stress markers and the levels of inflammatory cytokines in hippocampal tissues or HT-22 cells were detected. We found that FOXO4 was poorly expressed in EP. FOXO4 overexpression alleviated hippocampal neuronal damage in EP mice and improved HT-22 cell viability and inhibited apoptosis, and decreased oxidative stress and inflammation in hippocampal tissue and HT-22 cells. The bindings of miR-138-5p to FOXO4 and ROCK2 were analyzed, which showed that FOXO4 promoted miR-138-5p via binding to the miR-138-5p promoter region, and miR-138-5p inhibited ROCK2 expression. Joint experiments showed that miR-138-5p suppression or ROCK2 overexpression reversed the alleviation of FOXO4 overexpression on hippocampal neuronal damage. FOXO4 inhibited ROCK2 expression via promoting miR-138-5p expression, thus alleviating hippocampal neuronal damage in EP mice.

FoxO4 negatively modulates USP10 transcription to aggravate the apoptosis and oxidative stress of hypoxia/reoxygenation-induced cardiomyocytes by regulating the Hippo/YAP pathway.

Acute myocardial infarction (AMI) is the main cause of death in the whole world. This study aimed to investigate whether forkhead box O4 (FoxO4) could negatively modulate ubiquitin specific peptidase 10 (USP10) transcription to aggravate the apoptosis and oxidative stress of hypoxia/reoxygenation (H/R)-induced cardiomyocytes through Hippo/YAP pathway. mRNA expression as well as protein expressions of USP10 and FoxO4 in H9C2 cells after H/R induction or transfection were respectively detected by Reverse transcription-quantitative (RT-q) PCR analysis and Western blot. The viability and apoptosis of H9C2 cells after H/R induction or transfection were respectively detected by CCK-8 and TUNEL assays. The expressions of lactate dehydrogenase (LDH), superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) and malondialdehyde (MDA) in H9C2 cells after H/R induction or transfection were analyzed using appropriate kits and intracellular reactive oxygen species (ROS) levels were detected using a ROS Assay Kit. Dual luciferase reporter assay and Chromatin Immunoprecipitation (ChIP) have adopted to confirm the combination of USP10 and FoxO4. Western blot was also used to analyze the expression of apoptosis-related proteins and Hippo/YAP pathway-related proteins. As a result, USP10 expression was decreased in H/R-induced H9C2 cells in a time-dependent manner. USP10 overexpression increased the viability and suppressed the apoptosis and oxidative stress of H/R-induced H9C2 cells. In addition, FoxO4 modulated USP10 transcription. FoxO4 expression was increased in H9C2 cells induced by H/R. FoxO4 overexpression could reverse the protective effects of USP10 overexpression on H/R-induced H9C2 cells by regulating the Hippo/YAP signaling pathway. In conclusion, FoxO4 negatively modulated USP10 transcription to aggravate the apoptosis and oxidative stress of H/R-induced H9C2 cells via blocking Hippo/YAP pathway.

SIRT7-mediated modulation of glutaminase 1 regulates TGF-ß-induced pulmonary fibrosis.

In the current work we show that the profibrotic actions of TGF-ß are mediated, at least in part, through a metabolic maladaptation in glutamine metabolism and how the inhibition of glutaminase 1 (GLS1) reverses pulmonary fibrosis. GLS1 was found to be highly expressed in fibrotic vs normal lung fibroblasts and the expression of profibrotic targets, cell migration, and soft agar colony formation stimulated by TGF-ß required GLS1 activity. Moreover, knockdown of SMAD2 or SMAD3 as well as inhibition of PI3K, mTORC2, and PDGFR abrogated the induction of GLS1 by TGF-ß. We further demonstrated that the NAD-dependent protein deacetylase, SIRT7, and the FOXO4 transcription factor acted as endogenous brakes for GLS1 expression, which are inhibited by TGF-ß. Lastly, administration of the GLS1 inhibitor CB-839 attenuated bleomycin-induced pulmonary fibrosis. Our study points to an exciting and unexplored connection between epigenetic and transcriptional processes that regulate glutamine metabolism and fibrotic development in a TGF-ß-dependent manner.

KLF2 induces the senescence of pancreatic cancer cells by cooperating with FOXO4 to upregulate p21.

Pancreatic cancer is one of the most common malignancies in the world. Senescence is frequently observed in the progression of pancreatic cancer. In a previous study, we showed that KLF2 inhibited the growth and migration of pancreatic cancer. However, the mechanisms are not fully understood. In this study, we showed that overexpression of KLF2 induced the senescence of pancreatic cancer cells and inhibited tumorigenesis, and knockdown of KLF2 inhibited senescence and p21 expression. In the molecular mechanism study, KLF2 was found to interact with FOXO4 and cooperated with FOXO4 to induce the expression of p21. Downregulation of p21 and FOXO4 impaired the induction of senescence by KLF2. Overall, this study revealed the functions and mechanisms of KLF2 in senescence and provided a novel explanation for the suppressive roles of KLF2 in pancreatic cancer.