C-FOS inhibition promotes pancreatic cancer cell ferroptosis by transcriptionally regulating the expression of SLC7A11.

Cellular proto-oncogene C-Fos forms the AP-1 transcription factor by dimerizing with proto-oncogene c-Jun; this factor upregulates the transcription of genes associated with different malignancies. However, its functions in pancreatic adenocarcinoma (PAAD) remain poorly understood. In this study, the c-Fos was increased in PAAD cells and tissues through bioinformatic analysis, RT-PCR, and WB. In two PAAD cell lines, PANC-1 and BxPC-3, we performed c-Fos knockdown studies using short hairpin RNA (shRNA). Functional analysis indicated that c-Fos depletion in PAAD cells inhibits cell proliferation and promotes ferroptosis. Chromatin Immunoprecipitation (ChIP) and Dual-luciferase experiments showed that c-Fos coupled to the promoter region of SLC7A11 stimulated its transcription, providing mechanistic insight into the process. Moreover, SLC7A11 blocked the decline of proliferation and ferroptosis by c-Fos knockdown in PAAD cells. Furthermore, a xenograft nude mouse model was established to study the impact of c-Fos on tumorigenesis in vivo. Depletion of c-Fos could suppress PC tumor growth and the expressions of SLC7A11, ki-67, and 4HNE, but overexpression of SLC7A11 reversed this process. In summary, our investigation has shown that c-Fos acts as a transcriptional regulator of SLC7A11, which may enhance tumour growth in pancreatic cancer by inhibiting ferroptosis. These results indicate that c-Fos might be a promising target for treating ferroptosis in PAAD.

CIB2 mediates acquired gefitinib resistance by inducing ZEB1 expression and epithelial-mesenchymal transition.

EGFR-TKIs have been used as frontline treatment in patients with advanced non-small cell lung cancer (NSCLC) suffering from the EGFR mutation. Gefitinib, the first-generation EGFR-TKI, has greatly improved survival rates in lung cancer patients, whereas acquired gefitinib resistance is still a critical issue that needs to be overcome. In our research, high expression levels of CIB2 were found in gefitinib-resistant lung cancer cells. CIB2 knockout rendered gefitinib-resistant cells more sensitive to gefitinib, and overexpression of CIB2 in parental cells was sufficient to induce more resistance to gefitinib. Inhibition of CIB2 in gefitinib-resistant lung cancer cells significantly induced cell apoptosis. To clarify the major molecular mechanism by which CIB2 increases gefitinib resistance, we demonstrated that raised CIB2 in lung cancer cells promoted epithelial-to-mesenchymal transition (EMT) through upregulation of ZEB1. Moreover, FOSL1 transcriptionally regulated CIB2 expression. Finally, CIB2 rendered tumors resistant to gefitinib treatment in vivo. Our results explored a new mechanism: upregulated CIB2 promoted EMT through ZEB1 to regulate gefitinib resistance, which could be a candidate therapeutic target for overcoming acquired resistance to EGFR-TKIs in NSCLC patients.

Saikosaponin A Recovers Impaired Filaggrin Levels in Inflamed Skin by Downregulating the Expression of FRA1 and c-Jun.

Filaggrin (FLG) is an essential structural protein expressed in differentiated keratinocytes. Insufficient FLG expression contributes to the pathogenesis of chronic inflammatory skin diseases. Saikosaponin A (SSA), a bioactive oleanane-type triterpenoid, exerts anti-inflammatory activity. However, the effects of topically applied SSA on FLG expression in inflamed skin remain unclear. This study aimed to evaluate the biological activity of SSA in restoring reduced FLG expression. The effect of SSA on FLG expression in HaCaT cells was assessed through various biological methods, including reverse transcription PCR, quantitative real-time PCR, immunoblotting, and immunofluorescence staining. TNFα and IFNγ decreased FLG mRNA, cytoplasmic FLG protein levels, and FLG gene promoter-reporter activity compared to the control groups. However, the presence of SSA restored these effects. A series of FLG promoter-reporter constructs were generated to investigate the underlying mechanism of the effect of SSA on FLG expression. Mutation of the AP1-binding site (mtAP1) in the -343/+25 FLG promoter-reporter abrogated the decrease in reporter activities caused by TNFα + IFNγ, suggesting the importance of the AP1-binding site in reducing FLG expression. The SSA treatment restored FLG expression by inhibiting the expression and nuclear localization of FRA1 and c-Jun, components of AP1, triggered by TNFα + IFNγ stimulation. The ERK1/2 mitogen-activated protein kinase signaling pathway upregulates FRA1 and c-Jun expression, thereby reducing FLG levels. The SSA treatment inhibited ERK1/2 activation caused by TNFα + IFNγ stimulation and reduced the levels of FRA1 and c-Jun proteins in the nucleus, leading to a decrease in the binding of FRA1, c-Jun, p-STAT1, and HDAC1 to the AP1-binding site in the FLG promoter. The effect of SSA was evaluated in an animal study using a BALB/c mouse model, which induces human atopic-dermatitis-like skin lesions via the topical application of dinitrochlorobenzene. Topically applied SSA significantly reduced skin thickening, immune cell infiltration, and the expression of FRA1, c-Jun, and p-ERK1/2 compared to the vehicle-treated group. These results suggest that SSA can effectively recover impaired FLG levels in inflamed skin by preventing the formation of the repressor complex consisting of FRA1, c-Jun, HDAC1, and STAT1.

Role and molecular mechanisms of HuangQiSiJunZi decoction for treating triple-negative breast cancer as explored via network pharmacology and bioinformatics analyses.

OBJECTIVE: In this study, we evaluated the molecular mechanisms of HuangQiSiJunZi Decoction (HQSJZD) for treating triple-negative breast cancer (TNBC) using network pharmacology and bioinformatics analyses. METHODS: Effective chemical components together with action targets of HQSJZD were selected based on the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP). Meanwhile, differentially expressed genes (DEGs) were extracted from TNBC sample data in The Cancer Genome Atlas (TCGA) database. Additionally, we built a protein-protein interaction (PPI) network and acquired hub genes. Gene Expression Omnibus(GEO) datasets were utilized to verify the accuracy of hub gene expression. Additionally, enrichment analyses were conducted on key genes. Furthermore, TNBC severity-related high-risk factors were screened through univariate together with multivariate Cox regressions; next, the logistic regression prediction model was built. Moreover, differential levels of 22 immune cell types in TNBC tissues compared with normal tissues were analyzed. The hub gene levels within pan-cancer and the human body were subsequently visualized and analyzed. Finally, quantitative PCR (RT-qPCR) was used to validate the correlation of the hub genes in TNBC cells. RESULTS: The study predicted 256 targets of active ingredients and 1791 DEGs in TNBC, and obtained 16 hub genes against TNBC. The prognostic signature based on FOS, MMP9, and PGR was independent in predicting survival. A total of seven types of immune cells, such as CD4 + memory T cells, showed a significant difference in infiltration (p < 0.05), and immune cells were related to the hub genes. The HPA database was adopted for hub gene analyses, and as determined, FOS was highly expressed in most human organs. The results of RT-qPCR validation for the FOS hub gene were consistent with those of bioinformatic analyses. CONCLUSION: HQSJZD might regulate the interleukin-17 and aging pathways via FOS genes to increase immune cell infiltration in TNBC tissues, and thus, may treat TNBC and improve the prognosis. The FOS genes are likely to be a new marker for TNBC.

Expression of c-jun/c-fos mRNA Indicates Persistent Myocardial Stretch During Asphyxia-Induced Cardiac Arrest.

Right ventricular dysfunction is a key clinical issue for the viability of donation-after-circulatory-death (DCD) heart transplantation. DCD hearts with volume overload have the potential to exhibit aggravated right ventricular dysfunction following heart transplantation. The c-jun/c-fos mRNAs are genes that immediately respond to myocardial cell stretch. We assessed myocardial cell stretch during asphyxia-induced cardiac arrest by measuring c-jun/c-fos mRNA expression levels. The trachea was dissected and ligated to initiate asphyxiation in anesthetized Wistar rats under paralyzed ventilation. The hearts were harvested at 4 time points: 0, 15, 30, and 45 minutes after the termination of ventilation. Free walls of the right and left ventricles and the interventricular septum were sectioned. Total RNA was extracted from these tissues, and cDNA was synthesized using reverse transcription. The c-jun/c-fos mRNA expression levels were quantified using the droplet digital polymerase chain reaction method. In the left ventricle, c-jun/c-fos expression levels rapidly increased at 15 minutes, but the expression levels returned to the baseline level at 30 minutes after tracheal ligation. In contrast, in the right ventricle, c-jun/c-fos expression levels gradually increased and peaked 30 minutes after tracheal ligation. Myocardial cell stretching in the right ventricle is prolonged after asphyxia-induced cardiac arrest compared to that in the left ventricle, which may lead to right ventricular dysfunction after DCD heart transplantation.

Rhein Alleviates Doxorubicin-Induced Myocardial Injury by Inhibiting the p38 MAPK/HSP90/c-Jun/c-Fos Pathway-Mediated Apoptosis.

Doxorubicin (Dox) has been limited in clinical application due to its cardiac toxicity that varies with the dose. This study aimed to explore how Rhein modulates Dox-induced myocardial toxicity. The general condition and echocardiographic changes of mice were observed to evaluate cardiac function and structure, with myocardial cell injury and apoptosis checked by TUNEL and HE staining. The ELISA assessed markers of myocardial damage and inflammation. The TCMSP and SwissTargetPrediction databases were used to retrieve Rhein's targets while GeneCards was used to find genes related to Dox-induced myocardial injury. Intersection genes were analyzed by Protein-Protein Interaction Networks. The core network genes underwent GO and KEGG enrichment analysis using R software. Western blot was used to detect protein expression. Compared to the Dox group, there was no remarkable difference in heart mass /body mass ratio in the Rhein+Dox group. However, heart mass/tibia length increased. Mice in the Rhein+Dox group had significantly increased LVEF, LVPWs, and LVFS compared to those in the Dox group. Myocardial cell damage, inflammation, and apoptosis significantly reduced in the Rhein+Dox group compared to the model group. Eleven core network genes were selected. Further, Rhein+Dox group showed significantly downregulated expression of p38/p-p38, HSP90AA1, c-Jun/p-c-Jun, c-Fos/p-c-Fos, Bax, and cleaved-caspase-3/caspase-3 while Bcl-2 expression significantly upregulated compared to the Dox group. The study suggests that Rhein mediates cardioprotection against Dox-induced myocardial injury, at least partly, by influencing multiple core genes in the MAPK signaling pathway to inhibit myocardial cell apoptosis.

FOSL1 promotes stem cell­like characteristics and anoikis resistance to facilitate tumorigenesis and metastasis in osteosarcoma by targeting SOX2.

Metastasis is the leading cause of cancer­related death in osteosarcoma (OS). OS stem cells (OSCs) and anoikis resistance are considered to be essential for tumor metastasis formation. However, the underlying mechanisms involved in the maintenance of a stem­cell phenotype and anoikis resistance in OS are mostly unknown. Fos­like antigen 1 (FOSL1) is important in maintaining a stem­like phenotype in various cancers; however, its role in OSCs and anoikis resistance remains unclear. In the present study, the dynamic expression patterns of FOSL1 were investigated during the acquisition of cancer stem­like properties using RNA sequencing, PCR, western blotting and immunofluorescence. Flow cytometry, tumor­sphere formation, clone formation assays, anoikis assays, western blotting and in vivo xenograft and metastasis models were used to further investigate the responses of the stem­cell phenotype and anoikis resistance to FOSL1 overexpression or silencing in OS cell lines. The underlying molecular mechanisms were evaluated, focusing on whether SOX2 is crucially involved in FOSL1­mediated stemness and anoikis in OS. FOSL1 expression was observed to be upregulated in OSCs and promoted tumor­sphere formation, clone formation and tumorigenesis in OS cells. FOSL1 expression correlated positively with the expression of stemness­related factors (SOX2, NANOG, CD117 and Stro1). Moreover, FOSL1 facilitated OS cell anoikis resistance and promoted metastases by regulating the expression of apoptosis related proteins BCL2 and BAX. Mechanistically, FOSL1 upregulated SOX2 expression by interacting with the SOX2 promoter and activating its transcription. The results also showed that SOX2 is critical for FOSL1­mediated stem­like properties and anoikis resistance. The current findings indicated that FOSL1 is an important regulator that promotes a stem cell­like phenotype and anoikis resistance to facilitate tumorigenesis and metastasis in OS by regulating the transcription of SOX2. Thus, FOSL1 might represent an attractive target for therapeutic interventions in OS.

FOS+ Macrophages Promote Chronic Rejection of Cardiac Transplantation.

OBJECTIVES: Chronic rejection remains the leading cause of progressive decline in graft function. Accumulating evidence indicates that macrophages participate in chronic rejection dependent on CD40-CD40L. The FOS family members are critical in inflammatory and immune responses. However, the mechanisms underlying the role of FOS family members in chronic rejection remain unclear. In this study, we aimed to elucidate the role and underlying mechanisms of FOS-positive macrophages regulated by CD40 that mediate chronic allograft rejection. MATERIALS AND METHODS: We downloaded publicly accessible chronic rejection kidney transplant single-cell sequencing datasets from the gene expression omnibus database. Differentially expressed genes between the CD40hi and CD40low macrophage chronic rejection groups were analyzed. We established a chronic rejection mouse model by using CTLA-4-Ig. We treated bone marrow-derived macrophages with an anti-CD40 antibody. We assessed expression of the FOS family by flow cytometry, real-time quantitative polymerase chain reaction, Western blotting, and immunofluorescence. We identified altered signaling pathways by using RNA sequencing analysis. We detected DNA specifically bound to transcription factors by using ChIP-sequencing, with detection of the degree of graft fibrosis and survival. RESULTS: FOS was highly expressed on CD40hi macrophages in patients with chronic transplantrejection. Mechanistically, we showed that CD40 activated NF-κB2 translocation into the nucleus to upregulate c-Fos and FosB expression, thus promoting chronic rejection of cardiac transplant.We showed thatNF-κB2 regulated c-Fos and FosB expression by binding to the c-fos and fosb promoter regions. Inhibition of c-Fos/activator protein-1 decreased graft fibrosis and prolonged graft survival. CONCLUSIONS: CD40 may activate transcription factor NF-κB2 translocation into the nucleus of macrophages to upregulate c-Fos and FosB expression, thus promoting chronic rejection of cardiac transplant. Inhibition of c-Fos/activator protein-1 decreased grafts fibrosis and prolonged graft survival.

The aryl hydrocarbon receptor and FOS mediate cytotoxicity induced by Acinetobacter baumannii.

Acinetobacter baumannii is a pathogenic and multidrug-resistant Gram-negative bacterium that causes severe nosocomial infections. To better understand the mechanism of pathogenesis, we compare the proteomes of uninfected and infected human cells, revealing that transcription factor FOS is the host protein most strongly induced by A. baumannii infection. Pharmacological inhibition of FOS reduces the cytotoxicity of A. baumannii in cell-based models, and similar results are also observed in a mouse infection model. A. baumannii outer membrane vesicles (OMVs) are shown to activate the aryl hydrocarbon receptor (AHR) of host cells by inducing the host enzyme tryptophan-2,3-dioxygenase (TDO), producing the ligand kynurenine, which binds AHR. Following ligand binding, AHR is a direct transcriptional activator of the FOS gene. We propose that A. baumannii infection impacts the host tryptophan metabolism and promotes AHR- and FOS-mediated cytotoxicity of infected cells.

c-Myc-XRCC2-FOS axis promotes the proliferation and the resistance to Doxorubicin of NSCLC.

Lung cancer represents one of the most prevalent malignant neoplasms, commanding an alarming incidence and mortality rate globally. Non-small cell lung cancer (NSCLC), constituting approximately 80 %-90 % of all lung cancer cases, is the predominant pathological manifestation of this disease, with a disconcerting 5-year survival rate scarcely reaching 10 %. Extensive prior investigations have elucidated that the aberrant expression of X-ray repair cross-complementing gene 2 (XRCC2), a critical meiotic gene intricately involved in the DNA damage repair process, is intimately associated with tumorigenesis. Nevertheless, the precise roles and underlying mechanistic pathways of XRCC2 in NSCLC remain largely elusive. In the present study, we discerned an overexpression of XRCC2 within NSCLC patient tissues, particularly in high-grade samples, when juxtaposed with normal tissues. Targeted knockdown of XRCC2 notably impeded the proliferation of NSCLC both in vitro and in vivo. Comprehensive RNA sequencing and flow rescue assays unveiled that XRCC2 augments the proliferation of NSCLC cells through the down-regulation of FOS expression. Moreover, the c-Myc gene was definitively identified as an XRCC2 transcriptional factor by means of chromatin immunoprecipitation (ChIP) and luciferase reporter assays, whereby pharmacological attenuation of c-Myc expression, in conjunction with Doxorubicin, synergistically curtailed NSCLC cell growth both in vitro and in vivo. Collectively, our findings proffer critical insights into the novel c-Myc-XRCC2-FOS axis in promoting both proliferation and resistance to Doxorubicin in NSCLC cells, thereby extending a promising avenue for potential new diagnostic strategies and therapeutic interventions in NSCLC.

Comparative transcriptome reveals EphA2 and c-Fos as key factors driving enhanced replication in high-passage porcine deltacoronavirus strain.

Porcine deltacoronavirus (PDCoV), a cross-species transmissible enterovirus, frequently induces severe diarrhea and vomiting symptoms in piglets, which not only pose a significant menace to the global pig industry but also a potential public safety risk. In a previous study, we isolated a vaccine candidate, PDCoV CZ2020-P100, by passaging a parental PDCoV strain in vitro, exhibiting attenuated virulence and enhanced replication. However, the factors underlying these differences between primary and passaged strains remain unknown. In this study, we present the transcriptional landscapes of porcine kidney epithelial cells (LLC-PK1) cells infected with PDCoV CZ2020-P1 strain and P100 strain using the RNA-sequencing. We identified 105 differentially expressed genes (DEGs) in P1-infected cells and 295 DEGs in P100-infected cells. Enrichment analyses indicated that many DEGs showed enrichment in immune and inflammatory responses, with a more and higher upregulation of DEGs enriched in the P100-infected group. Notably, the DEGs were concentrated in the MAPK pathway within the P100-infected group, with significant upregulation in EphA2 and c-Fos. Knockdown of EphA2 and c-Fos reduced PDCoV infection and significantly impaired P100 replication compared to P1, suggesting a novel mechanism in which EphA2 and c-Fos are highly involved in passaged virus replication. Our findings illuminate the resemblances and distinctions in the gene expression patterns of host cells infected with P1 and P100, confirming that EphA2 and c-Fos play key roles in high-passage PDCoV replication. These results enhance our understanding of the changes in virulence and replication capacity during the process of passaging.

Two-polarized roles of transcription factor FOSB in lung cancer progression and prognosis: dependent on p53 status.

BACKGROUND: Activator protein-1 (AP-1) represents a transcription factor family that has garnered growing attention for its extensive involvement in tumor biology. However, the roles of the AP-1 family in the evolution of lung cancer remain poorly characterized. FBJ Murine Osteosarcoma Viral Oncogene Homolog B (FOSB), a classic AP-1 family member, was previously reported to play bewilderingly two-polarized roles in non-small cell lung cancer (NSCLC) as an enigmatic double-edged sword, for which the reasons and significance warrant further elucidation. METHODS AND RESULTS: Based on the bioinformatics analysis of a large NSCLC cohort from the TCGA database, our current work found the well-known tumor suppressor gene TP53 served as a key code to decipher the two sides of FOSB - its expression indicated a positive prognosis in NSCLC patients harboring wild-type TP53 while a negative one in those harboring mutant TP53. By constructing a panel of syngeneically derived NSCLC cells expressing p53 in different statuses, the radically opposite prognostic effects of FOSB expression in NSCLC population were validated, with the TP53-R248Q mutation site emerging as particularly meaningful. Transcriptome sequencing showed that FOSB overexpression elicited diversifying transcriptomic landscapes across NSCLC cells with varying genetic backgrounds of TP53 and, combined with the validation by RT-qPCR, PREX1 (TP53-Null), IGFBP5 (TP53-WT), AKR1C3, and ALDH3A1 (TP53-R248Q) were respectively identified as p53-dependent transcriptional targets of FOSB. Subsequently, the heterogenous impacts of FOSB on the tumor biology in NSCLC cells via the above selective transcriptional targets were confirmed in vitro and in vivo. Mechanistic investigations revealed that wild-type or mutant p53 might guide FOSB to recognize and bind to distinct promoter sequences via protein-protein interactions to transcriptionally activate specific target genes, thereby creating disparate influences on the progression and prognosis in NSCLC. CONCLUSIONS: FOSB expression holds promise as a novel prognostic biomarker for NSCLC in combination with a given genetic background of TP53, and the unique interactions between FOSB and p53 may serve as underlying intervention targets for NSCLC.

Icariin alleviates the injury of Sertoli cell junction function by upregulating PKR pathway via ERα/c-fos signaling in aged mice.

ETHNOPHARMACOLOGICAL RELEVENACE: Sertoli cells are vital to maintain spermatogenesis and their function decline during aging. Epimedium has the effects of tonifying kidney-yang, strengthening bones and muscles, and expelling wind and dampness, and is commonly used in the treatment of kidney-yang deficiency, impotence and spermatorrhea. Icariin is the main active ingredients from Epimedium exhibiting delaying aging effects and improving male reproductive dysfunction. Whereas, it remains poorly understood how icariin alleviates age-associated decline in testicular function by protecting against the damage of junction function of Sertoli cells. AIM OF THE STUDY: This study aimed to evaluate the improvement effect of icariin on Sertoli cell junction function damage and explore the underlying mechanisms. MATERIALS AND METHODS: Male C57BL/6 mice and mouse Sertoli cell line TM4 cells were utilized to assess the improvement effect of icariin on aging-associated Sertoli cell junction function injury. H&E staining, transmission electron microscopy, qPCR, Western blot, molecular docking, siRNA transfection, and immunofluorescence were performed in this study. RESULTS: Dietary administration of icariin remarkly attenuated age-associated deterioration in spermatogenic function as evidenced by elevated testicular weight and index, sperm concentration and sperm viability. In addition, icariin protected Sertoli cell junction function from age-associated damage as proven by increased Sertoli cell numbers, improved tight junction ultrastructure, and upregulated junction-related proteins (ZO-1, Occludin and ß-Catenin). Moreover, icariin significantly upregulated ERα/c-fos signaling and PKR pathway in testicular Sertoli cells. Similarly, in vitro studies revealed that deletion of ERα, c-fos or PKR abolished the improvement effects of icariin on Sertoli cell junction function damage. CONCLUSIONS: Icariin effectively mitigates age-associated decline in testicular function by diminished Sertoli cell junction function damage through upregulating PKR pathway via ERα/c-fos signaling. Therefore, attenuating Sertoli cell junction function injury by the upregulation of PKR pathway via ERα/c-fos signaling probably indicates an effective target for the prevention and treatment of testicular spermatogenic function with aging.

Discovery of a novel hybrid coumarin-hydroxamate conjugate targeting the HDAC1-Sp1-FOSL2 signaling axis for breast cancer therapy.

BACKGROUND: Breast cancer is one of the most lethal cancers in women. Despite significant advances in the diagnosis and treatment of breast cancer, many patients still succumb to this disease, and thus, novel effective treatments are urgently needed. Natural product coumarin has been broadly investigated since it reveals various biological properties in the medicinal field. Accumulating evidence indicates that histone deacetylase inhibitors (HDACIs) are promising novel anti-breast cancer agents. However, most current HDACIs exhibit only moderate effects against solid tumors and are associated with severe side effects. Thus, to develop more effective HDACIs for breast cancer therapy, hydroxamate of HDACIs was linked to coumarin core, and coumarin-hydroxamate hybrids were designed and synthesized. METHODS: A substituted coumarin moiety was incorporated into the classic hydroxamate HDACIs by the pharmacophore fusion strategy. ZN444B was identified by using the HDACI screening kit and cell viability assay. Molecular docking was performed to explore the binding mode of ZN444B with HDAC1. Western blot, immunofluorescent staining, cell viability, colony formation and cell migration and flow cytometry assays were used to analyze the anti-breast cancer effects of ZN444B in vitro. Orthotopic studies in mouse models were applied for preclinical evaluation of efficacy and toxicity in vivo. Proteomic analysis, dual-luciferase reporter assay, chromatin immunoprecipitation, co-immunoprecipitation, immunofluorescent staining assays along with immunohistochemical (IHC) analysis were used to elucidate the molecular basis of the actions of ZN444B. RESULTS: We synthesized and identified a novel coumarin-hydroxamate conjugate, ZN444B which possesses promising anti-breast cancer activity both in vitro and in vivo. A molecular docking model showed that ZN444B binds to HDAC1 with high affinity. Further mechanistic studies revealed that ZN444B specifically decreases FOS-like antigen 2 (FOSL2) mRNA levels by inhibiting the deacetylase activity of HDAC1 on Sp1 at K703 and abrogates the binding ability of Sp1 to the FOSL2 promoter. Furthermore, FOSL2 expression positively correlates with breast cancer progression and metastasis. Silencing FOSL2 expression decreases the sensitivity of breast cancer cells to ZN444B treatment. In addition, ZN444B shows no systemic toxicity in mice. CONCLUSIONS: Our findings highlight the potential of FOSL2 as a new biomarker and therapeutic target for breast cancer and that targeting the HDAC1-Sp1-FOSL2 signaling axis with ZN444B may be a promising therapeutic strategy for breast cancer.

FOSL2-mediated transcription of ISG20 induces M2 polarization of macrophages and enhances tumorigenic ability of glioblastoma cells.

BACKGROUND: Interferon stimulated exonuclease gene 20 (ISG20) has been reported to be correlated with macrophage infiltration in glioblastoma (GBM) in previous bioinformatics-based studies. This study explores the exact effect of ISG20 on macrophage polarization in GBM. METHODS: ISG20 expression in GBM tissues and cells was determined by RT-qPCR and/or immunohistochemistry. GBM cells were co-cultured with M0 macrophages (PMA-stimulated THP-1 cells) in vitro, followed by flow cytometry and ELISA to analyze the M2 polarization of macrophages. Fluorescence-contained GBM cells were intracranially injected into nude mice along with M0 macrophages to generate orthotopic xenograft tumor models. Upstream regulator of ISG20 was predicted using bioinformatics. Loss- or gain-of-function assays of Fos like 2 (FOSL2) and ISG20 were performed in GBM cells. DNA methylation level of FOSL2 was analyzed by bisulfite sequencing analysis. RESULTS: ISG20 was found highly expressed in GBM tissues and cells. ISG20 silencing in GBM cells decreased CD206 and CD163 levels in the co-cultured macrophages and reduced secretion of IL-10 and TGF-ß. It also enhanced survival of nude mice bearing xenograft tumors, blocked tumor growth, and suppressed M2 polarization of macrophages in vivo. FOSL2, highly expressed in GBM, bound to the ISG20 promoter to activate its transcription. FOSL2 silencing similarly blocked M2 polarization of macrophages, which was negated by ISG20 overexpression. The high FOSL2 expression in GBM was attributed to DNA hypomethylation. CONCLUSION: This study demonstrates that FOSL2 is highly expressed in GBM due to DNA hypomethylation. It activates transcription of ISG20, thus promoting M2 polarization of macrophages and GBM development.

Distinct epigenomic landscapes underlie tissue-specific memory T cell differentiation.

The memory CD8+ T cell pool contains phenotypically and transcriptionally heterogeneous subsets with specialized functions and recirculation patterns. Here, we examined the epigenetic landscape of CD8+ T cells isolated from seven non-lymphoid organs across four distinct infection models, alongside their circulating T cell counterparts. Using single-cell transposase-accessible chromatin sequencing (scATAC-seq), we found that tissue-resident memory T (TRM) cells and circulating memory T (TCIRC) cells develop along distinct epigenetic trajectories. We identified organ-specific transcriptional regulators of TRM cell development, including FOSB, FOS, FOSL1, and BACH2, and defined an epigenetic signature common to TRM cells across organs. Finally, we found that although terminal TEX cells share accessible regulatory elements with TRM cells, they are defined by TEX-specific epigenetic features absent from TRM cells. Together, this comprehensive data resource shows that TRM cell development is accompanied by dynamic transcriptome alterations and chromatin accessibility changes that direct tissue-adapted and functionally distinct T cell states.

HNRNPA2B1 stabilizes NFATC3 levels to potentiate its combined actions with FOSL1 to mediate vasculogenic mimicry in GBM cells.

BACKGROUND: Vasculogenic mimicry (VM) is an enigmatic physiological feature that influences blood supply within glioblastoma (GBM) tumors for their sustained growth. Previous studies identify NFATC3, FOSL1 and HNRNPA2B1 as significant mediators of VEGFR2, a key player in vasculogenesis, and their molecular relationships may be crucial for VM in GBM. AIMS: The aim of this study was to understand how NFATC3, FOSL1 and HNRNPA2B1 collectively influence VM in GBM. METHODS: We have investigated the underlying gene regulatory mechanisms for VM in GBM cell lines U251 and U373 in vitro and in vivo. In vitro cell-based assays were performed to explore the role of NFATC3, FOSL1 and HNRNPA2B1 in GBM cell proliferation, VM and migration, in the context of RNA interference (RNAi)-mediated knockdown alongside corresponding controls. Western blotting and qRT-PCR assays were used to examine VEGFR2 expression levels. CO-IP was employed to detect protein-protein interactions, ChIP was used to detect DNA-protein complexes, and RIP was used to detect RNA-protein complexes. Histochemical staining was used to detect VM tube formation in vivo. RESULTS: Focusing on NFATC3, FOSL1 and HNRNPA2B1, we found each was significantly upregulated in GBM and positively correlated with VM-like cellular behaviors in U251 and U373 cell lines. Knockdown of NFATC3, FOSL1 or HNRNPA2B1 each resulted in decreased levels of VEGFR2, a key growth factor gene that drives VM, as well as the inhibition of proliferation, cell migration and extracorporeal VM activity. Chromatin immunoprecipitation (ChIP) studies and luciferase reporter gene assays revealed that NFATC3 binds to the promoter region of VEGFR2 to enhance VEGFR2 gene expression. Notably, FOSL1 interacts with NFATC3 as a co-factor to potentiate the DNA-binding capacity of NFATC3, resulting in enhanced VM-like cellular behaviors. Also, level of NFATC3 protein in cells was enhanced through HNRNPA2B1 binding of NFATC3 mRNA. Furthermore, RNAi-mediated silencing of NFATC3, FOSL1 and HNRNPA2B1 in GBM cells reduced their capacity for tumor formation and VM-like behaviors in vivo. CONCLUSION: Taken together, our findings identify NFATC3 as an important mediator of GBM tumor growth through its molecular and epistatic interactions with HNRNPA2B1 and FOSL1 to influence VEGFR2 expression and VM-like cellular behaviors.

Choline supplementation in early life improves and low levels of choline can impair outcomes in a mouse model of Alzheimer's disease.

Maternal choline supplementation (MCS) improves cognition in Alzheimer's disease (AD) models. However, the effects of MCS on neuronal hyperexcitability in AD are unknown. We investigated the effects of MCS in a well-established mouse model of AD with hyperexcitability, the Tg2576 mouse. The most common type of hyperexcitability in Tg2576 mice are generalized EEG spikes (interictal spikes [IIS]). IIS also are common in other mouse models and occur in AD patients. In mouse models, hyperexcitability is also reflected by elevated expression of the transcription factor ∆FosB in the granule cells (GCs) of the dentate gyrus (DG), which are the principal cell type. Therefore, we studied ΔFosB expression in GCs. We also studied the neuronal marker NeuN within hilar neurons of the DG because reduced NeuN protein expression is a sign of oxidative stress or other pathology. This is potentially important because hilar neurons regulate GC excitability. Tg2576 breeding pairs received a diet with a relatively low, intermediate, or high concentration of choline. After weaning, all mice received the intermediate diet. In offspring of mice fed the high choline diet, IIS frequency declined, GC ∆FosB expression was reduced, and hilar NeuN expression was restored. Using the novel object location task, spatial memory improved. In contrast, offspring exposed to the relatively low choline diet had several adverse effects, such as increased mortality. They had the weakest hilar NeuN immunoreactivity and greatest GC ΔFosB protein expression. However, their IIS frequency was low, which was surprising. The results provide new evidence that a diet high in choline in early life can improve outcomes in a mouse model of AD, and relatively low choline can have mixed effects. This is the first study showing that dietary choline can regulate hyperexcitability, hilar neurons, ΔFosB, and spatial memory in an animal model of AD.

[Restraint Stress-Induced Expression of Fos and Several Related Genes in the Hypothalamus of Hypertensive ISIAH Rats].

Stress can play a significant role in arterial hypertension and many other complications of cardiovascular diseases. Considerable attention is paid to the study of the molecular mechanisms involved in the body response to stressful influences, but there are still many blank spots in understanding the details. ISIAH rats model the stress-sensitive form of arterial hypertension. ISIAH rats are characterized by genetically determined enhanced activities of the hypothalamic-pituitary-adrenocortical and sympathetic-adrenomedullary systems, suggesting a functional state of increased stress reactivity. For the first time, the temporal expression patterns of Fos and several related genes were studied in the hypothalamus of adult male hypertensive ISIAH rats after a single exposure to restraint stress for 30, 60, or 120 min. Fos transcription was activated and peaked 1 h after the start of restraint stress. The time course of Fos activation coincided with that of blood pressure increase after stress. Activation of hypothalamic neurons also alters the transcription levels of several transcription factor genes (Jun, Nr4a3, Jdp2, and Ppargc1a), which are associated with the development of cardiovascular diseases. Because Fos induction is a marker of brain neuron activation, activation of hypothalamic neurons and an increase in blood pressure were concluded to accompany increased stress reactivity of the hypothalamic-pituitary-adrenocortical and sympathoadrenal systems in hypertensive ISIAH rats during short-term restraint.

Interaction of NF-κB and FOSL1 drives glioma stemness.

Glioblastoma multiforme (GBM) is the most common and malignant primary brain tumor; GBM's inevitable recurrence suggests that glioblastoma stem cells (GSC) allow these tumors to persist. Our previous work showed that FOSL1, transactivated by the STAT3 gene, functions as a tumorigenic gene in glioma pathogenesis and acts as a diagnostic marker and potential drug target in glioma patients. Accumulating evidence shows that STAT3 and NF-κB cooperate to promote the development and progression of various cancers. The link between STAT3 and NF-κB suggests that NF-κB can also transcriptionally regulate FOSL1 and contribute to gliomagenesis. To investigate downstream molecules of FOSL1, we analyzed the transcriptome after overexpressing FOSL1 in a PDX-L14 line characterized by deficient FOSL1 expression. We then conducted immunohistochemical staining for FOSL1 and NF-κB p65 using rabbit polyclonal anti-FOSL1 and NF-κB p65 in glioma tissue microarrays (TMA) derived from 141 glioma patients and 15 healthy individuals. Next, mutants of the human FOSL1 promoter, featuring mutations in essential binding sites for NF-κB were generated using a Q5 site-directed mutagenesis kit. Subsequently, we examined luciferase activity in glioma cells and compared it to the wild-type FOSL1 promoter. Then, we explored the mutual regulation between NF-κB signaling and FOSL1 by modulating the expression of NF-κB or FOSL1. Subsequently, we assessed the activity of FOSL1 and NF-κB. To understand the role of FOSL1 in cell growth and stemness, we conducted a CCK-8 assay and cell cycle analysis, assessing apoptosis and GSC markers, ALDH1, and CD133 under varying FOSL1 expression conditions. Transcriptome analyses of downstream molecules of FOSL1 show that NF-κB signaling pathway is regulated by FOSL1. NF-κB p65 protein expression correlates to the expression of FOSL1 in glioma patients, and both are associated with glioma grades. NF-κB is a crucial transcription factor activating the FOSL1 promoter in glioma cells. Mutual regulation between NF-κB and FOSL1 contributes to glioma tumorigenesis and stemness through promoting G1/S transition and inhibiting apoptosis. Therefore, the FOSL1 molecular pathway is functionally connected to NF-κB activation, enhances stemness, and is indicative that FOSL1 may potentially be a novel GBM drug target.