Forkhead Box Protein A1 Research Articles

Redirecting the pioneering function of FOXA1 with covalent small molecules

Pioneer transcription factors (TFs) bind to and open closed chromatin, facilitating engagement by other regulatory factors involved in gene activation or repression. Chemical probes are lacking for pioneer TFs, which has hindered their mechanistic investigation in cells. Here, we report the chemical proteomic discovery of electrophilic compounds that stereoselectively and site-specifically bind the pioneer TF forkhead box protein A1 (FOXA1) at a cysteine (C258) within the forkhead DNA-binding domain. We show that these covalent ligands react with FOXA1 in a DNA-dependent manner and rapidly remodel its pioneer activity in prostate cancer cells reflected in redistribution of FOXA1 binding across the genome and directionally correlated changes in chromatin accessibility. Motif analysis supports a mechanism where the ligands relax the canonical DNA-binding preference of FOXA1 by strengthening interactions with suboptimal sequences in predicted proximity to C258. Our findings reveal a striking plasticity underpinning the pioneering function of FOXA1 that can be controlled by small molecules.

Impaired SUMOylation of FoxA1 promotes nonalcoholic fatty liver disease through down-regulation of Sirt6

Abnormal SUMOylation is implicated in non-alcoholic fatty liver disease (NAFLD) progression. Forkhead box protein A1 (FoxA1) has been shown to protect liver from steatosis, which was down-regulated in NAFLD. This study elucidated the role of FoxA1 deSUMOylation in NAFLD. NAFLD models were established in high-fat diet (HFD)-induced mice and palmitate acid (PAL)-treated hepatocytes. Hepatic steatosis was evaluated by biochemical and histological methods. Lipid droplet formation was determined by BODIPY and Oil red O staining. Target molecule levels were analyzed by RT-qPCR, Western blotting, and immunohistochemistry staining. SUMOylation of FoxA1 was determined by Ni-NTA pull-down assay and SUMOylation assay Ultra Kit. Protein interaction and ubiquitination were detected by Co-IP. Gene transcription was assessed by ChIP and dual luciferase reporter assays. Liver FoxA1 knockout mice developed severe liver steatosis, which could be ameliorated by sirtuin 6 (Sirt6) overexpression. Nutritional stresses reduced Sumo2/3-mediated FoxA1 SUMOylation at lysine residue K6, which promoted lipid droplet formation by repressing fatty acid β-oxidation. Moreover, Sirt6 was a target gene of FoxA1, and Sirt6 transcription activity was restrained by deSUMOylation of FoxA1 at site K6. Furthermore, nutritional stresses-induced deSUMOylation of FoxA1 promoted the ubiquitination and degradation of FoxA1 with assistance of murine double minute 2 (Mdm2). Finally, activating FoxA1 SUMOylation delayed the progression of NAFLD in mice. DeSUMOylation of FoxA1 at K6 promotes FoxA1 degradation and then inhibits Sirt6 transcription, thereby suppressing fatty acid β-oxidation and facilitating NAFLD development. Our findings suggest that FoxA1 SUMOylation activation might be a promising therapeutic strategy for NAFLD.

MiR-6855-5p Enhances Radioresistance and Promotes Migration of Pancreatic Cancer by Inducing Epithelial-Mesenchymal Transition via Suppressing FOXA1: Potential of Plasma Exosomal miR-6855-5p as an Indicator of Radiosensitivity in Patients with Pancreatic Cancer.

Whether radiation should be added to neoadjuvant treatment remains controversial, and liquid biopsy has not been reported to predict radioresistance in pancreatic cancer (PC). We aimed to identify microRNAs (miRNAs) governing radioresistance in PC by utilizing peripheral plasma exosome samples and to verify their usefulness as biomarkers. miRNA microarray analysis was conducted using pretreatment peripheral plasma exosomes from 10 patients with PC receiving neoadjuvant chemoradiotherapy (NACRT) in the discovery cohort. Patients were categorized into two groups (good and poor responders) based on treatment responses, and candidate miRNAs exhibiting differential expression between the two groups were identified. The radiosensitivity of PC cells was examined after miR-6855-5p overexpression. Next-generation sequencing (NGS) and TargetScan were used to explore the mechanisms of radioresistance. We investigated the correlation between miR-6855-5p expression levels in the pretreatment peripheral plasma exosomes of 28 patients in the validation cohort and the response to NACRT. miR-6855-5p expression was higher in poor responders than in good responders. miR-6855-5p induces radioresistance in PC cells. NGS showed that epithelial-mesenchymal transition (EMT) was involved in miR-6855-5p-related radioresistance. Forkhead box protein A1 (FOXA1) was identified as a direct target of miR-6855-5p using NGS and TargetScan. Clinical examination of samples from the validation cohort revealed a tendency for patients with higher expression of miR-6855-5p in peripheral plasma exosomes to exhibit increased radioresistance (r=-0.5964). miR-6855-5p regulates the radioresistance of PC by inducing EMT via suppressing FOXA1, and miR-6855-5p in peripheral plasma exosomes may be a biomarker for radioresistance of PC.

Transcriptional activation of SIRT5 by FOXA1 reprograms glycolysis to facilitate the malignant progression of diffuse large B-cell lymphoma

Diffuse large B-cell lymphoma (DLBCL) is the most common diagnosed subtype of lymphoma with high invasiveness and heterogeneity. Glycolysis is involved in regulating DLBCL progression. We aimed to explore the role of forkhead box protein A1 (FOXA1) in DLBCL and the mechanisms related to sirtuine5 (SIRT5) and glycolysis. FOXA1 expression in DLBCL cells was analyzed. Then, the proliferation and apoptosis of DLBCL cells were detected using Cell Counting Kit-8 (CCK-8), 5-ethynyl-2′-deoxyuridine (EDU) staining and flow cytometry analysis following FOXA1 or SIRT5 knockdown. The glycolysis was assessed by measuring extracellular acidification rate (ECAR), glucose consumption and lactate secretion. Immunoblotting was employed to examine the expression of apoptosis- and glycolysis-related proteins. Additionally, luciferase reporter assay and chromatin immunoprecipitation (ChIP) assay were conducted to test the combination of FOXA1 to SIRT5 promotor region. Subsequently, SIRT5 expression was upregulated to conduct rescue assays. Finally, the effects of FOXA1 downregulation on the growth and glycolysis in OCI-ly7 tumor-bearing mice were examined. As a result, FOXA1 was upregulated in DLBCL cells and FOXA1 or SIRT5 knockdown inhibited the proliferation, accelerated the apoptosis and suppressed glycolysis reprograming in DLBCL cells. Importantly, FOXA1 could transcriptionally activate SIRT5 expression in DLBCL cells. Besides, SIRT5 overexpression counteracted the effects of FOXA1 deficiency on the proliferation, apoptosis and glycolysis reprogramming in DLBCL cells. Furthermore, FOXA1 knockdown inhibited the tumor growth, suppressed the glycolysis reprogramming and downregulated SIRT5 expression in vivo. In summary, FOXA1 could transcriptionally activate SIRT5 to reprogram glycolysis, thereby facilitating the malignant progression of DLBCL.

ACAT2 negatively modulated by FOXA2 suppresses ferroptosis to expedite the aggressive phenotypes of endometrial cancer cells.

Endometrial cancer (EC) remains a prevalent gynecological disease with a continuously rising incidence and fatality rate. Acyl coenzyme A: cholesterol acyltransferase 2 (ACAT2) has been commonly perceived as a tumor promoter in multiple human malignancies. This study was conducted to specify the role and mechanism of ACAT2 in EC, which has not been covered. The expression and prognostic significance of ACAT2 in EC samples were respectively analyzed by the ENCORI and Kaplan-Meier plotter databases. RT-qPCR and western blot examined ACAT2 and forkhead box protein A2 (FOXA2) expression in EC cells. The CCK-8 method, colony formation, and EdU staining assays detected cell proliferation. The cell cycle was detected by flow cytometry analysis. Wound healing and Transwell assays, respectively, estimated cell migration and invasion. The thiobarbituric acid reactive species (TBARS) method and BODIPY 581/591 C11 probe detected lipid peroxidation levels. FerroOrange staining estimated intracellular iron level. Western blot examined the expression of epithelial-mesenchymal transition (EMT) and ferroptosis-associated proteins. The human TFDB database predicted the binding of FOXA2 with the ACAT2 promoter, which was substantiated by ChIP and luciferase reporter assays. As a result, ACAT2 expression was increased in EC tissues and cells and associated with poor survival outcomes in EC patients. ACAT2 deletion might hinder EC cell proliferation, migration, invasion, and EMT while stimulating cell cycle arrest. Moreover, ACAT2 silencing promoted the ferroptosis of EC cells. Also, FOXA2 inactivated the transcription of ACAT2 through binding with the ACAT2 promoter. FOXA2 interference could promote the proliferation, migration, invasion, EMT, cell cycle, and ferroptosis of ACAT2-silenced EC cells, which was partially reversed by the ferroptosis activator erastin. Conclusively, ACAT2 transcriptionally inactivated by FOXA2 might contribute to the malignant progression of EC via the inhibition of ferroptosis.

Abstract PO2-06-08: Suppression of GATA2/3-FOXA1-HER3 Axis by Histone Deacetylase (HDAC) Inhibitors shows Antitumor Activity in Basal-like Breast Cancer

Abstract Introduction: Basal-like breast cancer (BLBC) represents an aggressive subtype of triple-negative breast cancer (TNBC) that exhibits a high risk of recurrence and resistance to treatment. Histone deacetylase (HDAC) inhibitors (HDACis), including panobinostat and romidepsin, have obtained approval for the treatment of hematopoietic malignancies and demonstrated effectiveness in TNBC cells. We recently found that panobinostat and romidepsin potently induced TNBC cell growth inhibition and apoptosis via downregulation of HER3 through suppression of forkhead box protein A1 (FOXA1), a pioneering transcription factor. Nonetheless, the underlying mechanism through which HDACis suppress FOXA1, thereby inhibiting HER3 expression and its downstream signaling in FOXA1/HER3 co-expressing TNBC remains elusive. Methods: Colony formation, MTS, and LIVE/DEAD cell staining assays were used to detect cell viability. Apoptosis was detected by flow cytometry assays. QRT-PCR, western blots, and immunohistochemistry were performed to determine the expression and activation of genes and/or proteins. Co-immunoprecipitation was performed to assess the interaction between proteins. Lentivirus vectors containing cDNA or shRNAs were used to overexpress or knockdown gene expression. Chromatin immunoprecipitation-quantitative PCR and dual luciferase reporter assays were performed to elucidate the regulatory role of gene transcription. Results: Elevated expression of FOXA1 was observed in BLBC specimens and cell lines tested. FOXA1 was co-expressed with HER3 and transcriptionally activated the HER3 expression in BLBC cells. HER3 formed heterodimers with epidermal growth factor receptor (EGFR), activating downstream signaling pathways in BLBC cells. Treatment with panobinostat and romidepsin resulted in growth inhibition and apoptosis in BLBC cells by downregulating FOXA1 expression and inhibiting HER3 signaling. Notably, the combination of HDACis with an EGFR inhibitor (gefitinib) or an anti-HER3 antibody synergistically enhanced the anti-survival effects on BLBC cells. Additionally, gene expression profiling datasets revealed a significant positive correlation between FOXA1 expression and the transcription factors GATA-Binding Protein 2/3 (GATA2/3). Patients with high GATA2/3-FOXA1 expression exhibited worse Relapse-Free Survival (RFS) compared to those with low expressions in BLBC via Kaplan-Meier analysis. Further investigations showed that GATA2/3 directly activated FOXA1 transcription by binding to its promoter. Moreover, ectopic expression of GATA2/3 not only restored FOXA1 expression downregulated by HDACis but also attenuated the anti-survival effects of HDACis on BLBC cells. Conclusion: HDACis exhibit potent inhibitory effects on BLBC cells via downregulation of GATA2/3-mediated repression of FOXA1 gene transcription, which in turn suppresses HER3 expression and signaling. Our findings indicate that epigenetic targeting of the GATA2/3-FOXA1-HER3 axis may be an effective therapeutic strategy for the eradication of BLBC tumors. Keywords: FOXA1, GATA2/3, HER3, HDAC inhibitors, Basal-like breast cancer Citation Format: Bolin Liu, Congcong Tan, Hui Lyu. Suppression of GATA2/3-FOXA1-HER3 Axis by Histone Deacetylase (HDAC) Inhibitors shows Antitumor Activity in Basal-like Breast Cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO2-06-08.

The relationship between cardiac oxidative stress, inflammatory cytokine response, cardiac pump function, and prognosis post-myocardial infarction

This study delves into the potential connections between cardiac oxidative stress, inflammatory cytokine response, cardiac pump function, and prognosis in individuals following myocardial infarction. A total of 276 patients were categorized into two groups: the control group (n = 130) and the observation group (n = 146), based on the drug intervention strategies. The control group received standard drug treatment, while the observation group received early drug intervention targeting antioxidant and anti-inflammatory treatment in addition to standard treatment. Serum levels of inflammatory factors, including tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and interleukin-9 (IL-6), were assessed using enzyme-linked immuno sorbent assay (ELISA) kits. The Forkhead Box Protein A2 (FOX2) reagent was used to determine the overall oxidation level. Left Ventricular End-Diastolic Diameter (LVEDD), Left Ventricular Ejection Fraction (LVEF), and End-Systolic Diameter (ESD) were measured using Doppler ultrasound. The observation group exhibited significantly reduced serum levels of TNF-α, IL-1β, and IL-6 compared to the control group (P < 0.05). Moreover, the observation group exerted lower total oxidation levels, OSI, EDD, and ESD compared to the control group (P < 0.05), while the LVEF and TAS levels in the observation group were higher than those in the control group (P < 0.05). Remarkably, the observation group experienced a significant reduction in the incidences of reinfarction, heart failure, arrhythmia, and abnormal valve function compared to the control group (P < 0.05). Decreased cardiac pump function and a more unfavorable prognosis were associated with elevated levels of cardiac oxidative stress and inflammatory factors (P < 0.05). Timely intervention with appropriate medications have a crucial effect in decreasing inflammatory marker levels, mitigating oxidative pressure, and enhancing cardiac pumping capacity and overall prognosis.

A high concentrate diet inhibits forkhead box protein A2 expression, and induces oxidative stress, mitochondrial dysfunction and mitochondrial unfolded protein response in the liver of dairy cows

High-concentrate diet induce subacute ruminal acidosis (SARA) and cause liver damage in ruminants. It has been reported that forkhead box protein A2 (FOXA2) can enhance mitochondrial membrane potential but its function in mitochondrial dysfunction induced by high concentrate diets is still unknown. Therefore, the aim of this study was to elucidate the effect of high-concentrate (HC) diet on hepatic FOXA2 expression, mitochondrial unfolded protein response (UPRmt), mitochondrial dysfunction and oxidative stress. A total of 12 healthy mid-lactation Holstein cows were selected and randomized into 2 groups: the low concentrate (LC) diet group (concentrate:forage = 4:6) and HC diet group (concentrate:forage = 6:4). The trial lasted 21 d. The rumen fluid, blood and liver tissue were collected at the end of the experiment. The results showed that the rumen fluid pH level was reduced in the HC group and the pH was lower than 5.6 for more than 4 h/d, indicating that feeding HC diets successfully induced SARA in dairy cows. Both FOXA2 mRNA and protein abundance were significantly reduced in the liver of the HC group compared with the LC group. The activity of antioxidant enzymes (CAT, G6PDH, T-SOD, Cu/Zn SOD, Mn SOD) and mtDNA copy number in the liver tissue of the HC group decreased, while the level of H2O2 significantly increased, this increase was accompanied by a decrease in oxidative phosphorylation (OXPHOS). The balance of mitochondrial division and fusion was disrupted in the HC group, as evidenced by the decreased mRNA level of OPA1, MFN1, and MFN2 and increased mRNA level of Drp1, Fis1, and MFF. At the same time, HC diet downregulated the expression level of SIRT1, SIRT3, PGC-1α, TFAM, and Nrf 1 to inhibit mitochondrial biogenesis. The HC group induced UPRmt in liver tissue by upregulating the mRNA and protein levels of CLPP, LONP1, CHOP, Hsp10, and Hsp60. In addition, HC diet could increase the protein abundance of Bax, CytoC, Caspase 3 and Cleaved-Caspase 3, while decrease the protein abundance of Bcl-2 and the Bcl-2/Bax ratio. Overall, our study suggests that the decreased expression of FOXA2 may be related to UPRmt, mitochondrial dysfunction, oxidative stress, and apoptosis in the liver of dairy cows fed a high concentrate diet.

Staufen1 Represses the FOXA1-Regulated Transcriptome by Destabilizing FOXA1 mRNA in Colorectal Cancer Cells.

Transcription factors play key roles in development and disease by controlling gene expression. Forkhead box A1 (FOXA1), is a pioneer transcription factor essential for mouse development and functions as an oncogene in prostate and breast cancer. In colorectal cancer (CRC), FOXA1 is significantly downregulated and high FOXA1 expression is associated with better prognosis, suggesting potential tumor suppressive functions. We therefore investigated the regulation of FOXA1 expression in CRC, focusing on well-differentiated CRC cells, where FOXA1 is robustly expressed. Genome-wide RNA stability assays identified FOXA1 as an unstable mRNA in CRC cells. We validated FOXA1 mRNA instability in multiple CRC cell lines and in patient-derived CRC organoids, and found that the FOXA1 3'UTR confers instability to the FOXA1 transcript. RNA pulldowns and mass spectrometry identified Staufen1 (STAU1) as a potential regulator of FOXA1 mRNA. Indeed, STAU1 knockdown resulted in increased FOXA1 mRNA and protein expression due to increased FOXA1 mRNA stability. Consistent with these data, RNA-seq following STAU1 knockdown in CRC cells revealed that FOXA1 targets were upregulated upon STAU1 knockdown. Collectively, this study uncovers a molecular mechanism by which FOXA1 is regulated in CRC cells and provides insights into our understanding of the complex mechanisms of gene regulation in cancer.

Effects of angiotensin receptor blockers and angiotensin-converting enzyme inhibitors on respiratory Mucin-5ac, Mucin-C5b and Forkhead box protein-A2 of a hamster tracheal mucosa.

Effects of angiotensin receptor blockers and angiotensin-converting enzyme inhibitors on respiratory Mucin-5ac, Mucin-C5b and Forkhead box protein-A2 of a hamster tracheal mucosa.

LSD1 inhibitor hinders the demethylation of FOXA1 to inhibit prostate cancer progression

Abnormal activation of androgen receptor (AR) occurs in prostate cancer (PC) progression and metastasis. Lysine-specific demethylase 1 (LSD1), the first demethylating enzyme, regulates AR-mediated transcriptional activity. Considering the effect of Forkhead box protein A1 (FOXA1) on the expression of AR, estrogen receptor (ER) and tumor suppressor genes, this study investigated the demethylation of FOXA1 upon treatment with LSD1 inhibitors and assessed the biological behaviors of PC cells. PC cells were cultured and infected with viruses. After transient transfection, CWR22-RV1-Cas9 cells were selected by puromycin with expression of LSD1 detected by Western blot. Apart from measurement of formaldehyde production, immunoprecipitation and chromatin immunoprecipitation (ChIP) were performed, followed by ATAC-seq detection, and Western blot. The data indicated the association between LSD1-binding sites and high levels of FOXA1. LSD1 inhibitor treatment resulted in a dramatic decline in overall FOXA1 binding, significantly reducing chromosomal accessibility and also increasing lysine-methylated FOXA1 level, but it failed to affect H3K4me2 levels at LSD1-FOXA1 occupied sites. Overexpression of LSD1-WT obtained reverse outcome. Besides, LSD1 inhibition diminished binding of FOXA1 and restored lysine-methylation of FOXA1 in methylation-deficient cells with mutant K270R. Moreover, silencing of LSD1 suppressed CWR22-RV1 tumor growth, resulting in increased H3K4me2 and decreased AR-FL/V7 gene expression. K270me is demethylated by LSD1. LSD1 inhibitor disrupts FOXA1 chromatin association, blocks FOXA1 K270-demethylation and hinders AR binding, thereby suppressing PC cell growth.

Transcription Factors HNF1A, HNF4A, and FOXA2 Regulate Hepatic Cell Protein N-Glycosylation

Hepatocyte nuclear factor 1 alpha (HNF1A), hepatocyte nuclear factor 4 alpha (HNF4A), and forkhead box protein A2 (FOXA2) are key transcription factors that regulate a complex gene network in the liver, creating a regulatory transcriptional loop. The Encode and ChIP-Atlas databases identify the recognition sites of these transcription factors in many glycosyltransferase genes. Our in silico analysis of HNF1A, HNF4A, and FOXA2 binding to the ten candidate glyco-genes studied in this work confirms a significant enrichment of these transcription factors specifically in the liver. Our previous studies identified HNF1A as a master regulator of fucosylation, glycan branching, and galactosylation of plasma glycoproteins. Here, we aimed to functionally validate the role of the three transcription factors on downstream glyco-gene transcriptional expression and the possible effect on glycan phenotype. We used the state-of-the-art clustered regularly interspaced short palindromic repeats/dead Cas9 (CRISPR/dCas9) molecular tool for the downregulation of the HNF1A, HNF4A, and FOXA2 genes in HepG2 cells—a human liver cancer cell line. The results show that the downregulation of all three genes individually and in pairs affects the transcriptional activity of many glyco-genes, although downregulation of glyco-genes was not always followed by an unambiguous change in the corresponding glycan structures. The effect is better seen as an overall change in the total HepG2 N-glycome, primarily due to the extension of biantennary glycans. We propose an alternative way to evaluate the N-glycome composition via estimating the overall complexity of the glycome by quantifying the number of monomers in each glycan structure. We also propose a model showing feedback loops with the mutual activation of HNF1A–FOXA2 and HNF4A–FOXA2 affecting glyco-genes and protein glycosylation in HepG2 cells.

SENP1 knockdown-mediated CTCF SUMOylation enhanced its stability and alleviated lipopolysaccharide-evoked inflammatory injury in human lung fibroblasts via regulation of FOXA2 transcription

BackgroundExcessive inflammation is the main cause of treatment failure in neonatal pneumonia (NP). CCCTC-binding factor (CTCF) represents an important node in various inflammatory diseases. In the present study, we tried to clarify the function and underlying molecular mechanism of CTCF on an in vitro cellular model of NP, which was generated by simulating the human lung fibroblast cell line WI-38 with lipopolysaccharide (LPS). MethodsThe SUMOylation level and protein interaction were verified by Co-immunoprecipitation assay. Cell viability was measured by Cell Counting Kit-8 assay. Inflammatory factors were examined by Enzyme-linked immunosorbent assay. Cell apoptosis was evaluated by TUNEL assay. The binding activity of CTCF to target promoter was tested by chromatin immunoprecipitation and luciferase reporter assay. ResultsLPS treatment restrained cell viability, promoted the production of inflammatory factors, and enhanced cell apoptosis. CTCF overexpression played anti-inflammatory and anti-apoptotic roles. Furthermore, CTCF was modified by SUMOylation with small ubiquitin-like modifier protein 1 (SUMO1). Interfering with sumo-specific protease 1 (SENP1) facilitated CTCF SUMOylation and protein stability, thus suppressing LPS-evoked inflammatory and apoptotic injuries. Moreover, CTCF could bind to the forkhead box protein A2 (FOXA2) promoter region to promote FOXA2 expression. The anti-inflammatory and anti-apoptotic roles of CTCF are associated with FOXA2 activation. In addition, SENP1 knockdown increased FOXA2 expression by enhancing the abundance and binding ability of CTCF. ConclusionsSUMOylation of CTCF by SENP1 knockdown enhanced its protein stability and binding ability and it further alleviated LPS-evoked inflammatory injury in human lung fibroblasts by positively regulating FOXA2 transcription.

FOXA1/MND1/TKT axis regulates gastric cancer progression and oxaliplatin sensitivity via PI3K/AKT signaling pathway

BackgroundDrug resistance is a main factor affecting the chemotherapy efficacy of gastric cancer (GC), in which meiosis plays an important role. Therefore, it is urgent to explore the effect of meiosis related genes on chemotherapy resistance.MethodsThe expression of meiotic nuclear divisions 1 (MND1) in GC was detected by using TCGA and clinical specimens. In vitro and in vivo assays were used to investigate the effects of MND1. The molecular mechanism was determined using luciferase reporter assay, CO-IP and mass spectrometry (MS).ResultsThrough bioinformatics, we found that MND1 was highly expressed in platinum-resistant samples. In vitro experiments showed that interference of MND1 significantly inhibited the progression of GC and increased the sensitivity to oxaliplatin. MND1 was significantly higher in 159 GC tissues in comparison with the matched adjacent normal tissues. In addition, overexpression of MND1 was associated with worse survival, advanced TNM stage, and lower pathological grade in patients with GC. Further investigation revealed that forkhead box protein A1 (FOXA1) directly binds to the promoter of MND1 to inhibit its transcription. CO-IP and MS assays showed that MND1 was coexpressed with transketolase (TKT). In addition,TKT activated the PI3K/AKT signaling axis and enhanced the glucose uptake and lactate production in GC cells.ConclusionsOur results confirm that FOXA1 inhibits the expression of MND1, which can directly bind to TKT to promote GC progression and reduce oxaliplatin sensitivity through the PI3K/AKT signaling pathway.

Characterisation of forkhead box protein A3 as a key transcription factor for hepatocyte regeneration

Liver regeneration is vital for the recovery of liver function after injury, yet the underlying mechanism remains to be elucidated. Forkhead box protein A3 (FOXA3), a member of the forkhead box family, plays important roles in endoplasmic reticulum stress sensing, and lipid and glucose homoeostasis, yet its functions in liver regeneration are unknown. Here, we explored whether Foxa3 regulates liver regeneration via acute and chronic liver injury mice models. We further characterised the molecular mechanism by chromatin immunoprecipitation sequencing and rescue experiments invivo and invitro. Then, we assessed the impact of Foxa3 pharmacological activation on progression and termination of liver regeneration. Finally, we confirmed the Foxa3-Cebpb axis in human liver samples. Foxa3 is dominantly expressed in hepatocytes and cholangiocytes and is induced upon partial hepatectomy (PH) or carbon tetrachloride (CCl4) administration. Foxa3 deficiency in mice decreased cyclin gene levels and delayed liver regeneration after PH, or acute or chronic i.p. CCl4 injection. Conversely, hepatocyte-specific Foxa3 overexpression accelerated hepatocytes proliferation and attenuated liver damage in an CCl4-induced acute model. Mechanistically, Foxa3 directly regulates Cebpb transcription, which is involved in hepatocyte division and apoptosis both invivo and invitro. Of note, Cebpb overexpression in livers of Foxa3-deficient mice rescued their defects in cell proliferation and regeneration upon CCl4 treatment. In addition, pharmacological induction of Foxa3 via cardamonin speeded up hepatocyte proliferation after PH, without interfering with liver regeneration termination. Finally, Cebpb and Ki67 levels had a positive correlation with Foxa3 expression in human chronic disease livers. These data characterise Foxa3 as a vital regulator of liver regeneration, which may represent an essential factor to maintain liver mass after liver injury by governing Cebpb transcription. Liver regeneration is vital for the recovery of liver function after chemical insults or hepatectomy, yet the underlying mechanism remains to be elucidated. Herein, via invitro and invivo models and analysis, we demonstrated that Forkhead box protein A3 (FOXA3), a Forkhead box family member, maintained normal liver regeneration progression by governing Cebpb transcription and proposed cardamonin as a lead compound to induce Foxa3 and accelerate liver repair, which signified that FOXA3 may be a potential therapeutic target for further preclinical study on treating liver injury.

Multiple factors to assist human-derived induced pluripotent stem cells to efficiently differentiate into midbrain dopaminergic neurons.

Midbrain dopaminergic neurons play an important role in the etiology of neurodevelopmental and neurodegenerative diseases. They also represent a potential source of transplanted cells for therapeutic applications. In vitro differentiation of functional midbrain dopaminergic neurons provides an accessible platform to study midbrain neuronal dysfunction and can be used to examine obstacles to dopaminergic neuronal development. Emerging evidence and impressive advances in human induced pluripotent stem cells, with tuned neural induction and differentiation protocols, makes the production of induced pluripotent stem cell-derived dopaminergic neurons feasible. Using SB431542 and dorsomorphin dual inhibitor in an induced pluripotent stem cell-derived neural induction protocol, we obtained multiple subtypes of neurons, including 20% tyrosine hydroxylase-positive dopaminergic neurons. To obtain more dopaminergic neurons, we next added sonic hedgehog (SHH) and fibroblast growth factor 8 (FGF8) on day 8 of induction. This increased the proportion of dopaminergic neurons, up to 75% tyrosine hydroxylase-positive neurons, with 15% tyrosine hydroxylase and forkhead box protein A2 (FOXA2) co-expressing neurons. We further optimized the induction protocol by applying the small molecule inhibitor, CHIR99021 (CHIR).This helped facilitate the generation of midbrain dopaminergic neurons, and we obtained 31-74% midbrain dopaminergic neurons based on tyrosine hydroxylase and FOXA2 staining. Thus, we have established three induction protocols for dopaminergic neurons. Based on tyrosine hydroxylase and FOXA2 immunostaining analysis, the CHIR, SHH, and FGF8 combined protocol produces a much higher proportion of midbrain dopaminergic neurons, which could be an ideal resource for tackling midbrain-related diseases.

Lysosome-dependent FOXA1 ubiquitination contributes to luminal lineage of advanced prostate cancer.

Changes in FOXA1 (forkhead box protein A1) protein levels are well associated with prostate cancer (PCa) progression. Unfortunately, direct targeting of FOXA1 in progressive PCa remains challenging due to variations in FOXA1 protein levels, increased FOXA1 mutations at different stages of PCa, and elusive post-translational FOXA1 regulating mechanisms. Here, we show that SKP2 (S-phase kinase-associated protein 2) catalyzes K6- and K29-linked polyubiquitination of FOXA1 for lysosomal-dependent degradation. Our data indicate increased SKP2:FOXA1 protein ratios in stage IV human PCa compared to stages I-III, together with a strong inverse correlation (r = -0.9659) between SKP2 and FOXA1 levels, suggesting that SKP2-FOXA1 protein interactions play a significant role in PCa progression. Prostate tumors of Pten/Trp53 mice displayed increased Skp2-Foxa1-Pcna signaling and colocalization, whereas disruption of the Skp2-Foxa1 interplay in Pten/Trp53/Skp2 triple-null mice demonstrated decreased Pcna levels and increased expression of Foxa1 and luminal positive cells. Treatment of xenograft mice with the SKP2 inhibitor SZL P1-41 decreased tumor proliferation, SKP2:FOXA1 ratios, and colocalization. Thus, our results highlight the significance of the SKP2-FOXA1 interplay on the luminal lineage in PCa and the potential of therapeutically targeting FOXA1 through SKP2 to improve PCa control.

FOXA2 attenuates lipopolysaccharide‑induced pneumonia by inhibiting the inflammatory response, oxidative stress and apoptosis through blocking of p38/STAT3 signaling.

Pneumonia is a severe inflammatory disease of the lung. Forkhead box protein A2 (FOXA2) has been demonstrated to serve an important regulatory role in various pulmonary diseases; however, the role of FOXA2 in pneumonia remains to be elucidated. The present study aimed to explore the functional effects and regulatory mechanism of FOXA2 in pneumonia. An in vitro pneumonia model was induced using lipopolysaccharide (LPS) in WI-38 cells. The mRNA and protein expression levels of FOXA2 were determined by reverse transcription-quantitative PCR and western blotting, respectively. Cell viability was assessed using a Cell Counting Kit-8 assay. Inflammatory cytokines were evaluated using ELISA kits and oxidative stress markers were assessed using a malondialdehyde assay kit, superoxide dismutase assay kit and CATalase assay kit. Cell apoptosis was evaluated using flow cytometry and the caspase3 activity was determined. Western blotting was performed to examine the protein expression levels of endoplasmic reticulum stress (ERS)-associated factors. For a rescue assay, a p38 MAPK activator, U46619, was used to investigate the regulatory mechanism of FOXA2 involving p38/STAT3 signaling. FOXA2 was downregulated in LPS-induced WI-38 cells. FOXA2 overexpression alleviated LPS-induced inflammation, oxidative stress, apoptosis and ERS in WI-38 cells. Furthermore, the inhibitory effects of FOXA2 on inflammation, oxidative stress and apoptosis, as well as ERS in LPS-induced WI-38 cells were partly weakened by additional treatment with U46619. In conclusion, FOXA2 served a protective role against LPS-induced pneumonia by regulating p38/STAT3 signaling, providing a novel idea for the development of targeted therapeutic strategies for pneumonia.

Gambogenic acid inhibits proliferation and ferroptosis by targeting the miR-1291/FOXA2 and AMPKα/SLC7A11/GPX4 axis in colorectal cancer.

The present study aims to investigate the mechanism of the nature compound gambogenic acid (GNA) on the apoptosis and ferroptosis in colorectal cancer (CRC).The effect of GNA on the proliferation of CRC cell lines were detected by MTT and clonogenic assay. The xenograft tumor model was established, and the inhibition effect of GNA were evaluated by observing the tumor growth. The endoplasmic reticulum (ER) of HCT116 was observed by using the ER tracker. The TargrtScan database was used to predict the miRNA binding sites. The level of miRNA with GNA treatment was explored by real-time quantitative PCR. The effect of ferroptosis were evaluated by detect the expression of reactive oxygen species (ROS), intracellular ferrous iron (Fe2+ ), malondialdehyde (MDA), glutathione (GSH), subunit solute carrier family 7 member 11 (SLC7A11), glutathione peroxidase (GPX)4, transferrin, and ferritin by Western blot.GNA isolated from gamboge can inhibit the growth and proliferation of CRC cell lines in a concentration-dependent manner. GNA activated ER stress by upregulating miR-1291, and miR-1291 targeted the forkhead box protein A2 (FOXA2). GNA also induced ROS production and mediated the Fenton reaction by activating transferrin to increase Fe2+ , thus inducing ferroptosis. In addition, GNA could induce ferroptosis through the depletion of GSH and GPX4. Furthermore, GNA treatment regulated iron metabolism by activating AMPKα/SLC7A11/GPX4 signaling. In conclusion, GNA activated ER stress via miR-1291 and induced ferroptosis in CRC cells and might be a new inducer of ferroptosis, which can expand the efficacy of chemotherapy drugs.

E3 ubiquitin ligase neural precursor cell-expressed developmentally downregulated gene 4 motivates FOXA1 ubiquitination and restrains proliferation of diffuse large B-cell lymphoma cells via the Wnt/β-Catenin pathway.

Neural precursor cell-expressed developmentally downregulated gene 4 (NEDD4) is an E3 ubiquitin ligase that recognizes substrates via protein-protein interactions and takes part in tumor development. This study aims to clarify NEDD4's functions in diffuse large B-cell lymphoma (DLBCL) and its downstream mechanisms. Collection of 53 DLBCL tissues and adjacent normal lymphoid tissues, and detection of NEDD4 and Forkhead box protein A1 (FOXA1) in the tissues were conducted. The selection of DLBCL cells was for FARAGE, and test of cells' advancement was after transfection. Analysis of NEDD4 and FOXA1's link, and test of Wnt/β-catenin pathway were implemented. In vivo tumor xenograft experiments were put into effect. Detection of the pathological conditions of tumor tissues and the positive Ki67 in the family was implemented. It came out NEDD4 was reduced in DLBCL tissues and cell lines, and FOXA1 was elevated; Enhancing NEDD4 or repressing FOXA1 refrained DLBCL cells' advancement; NEDD4 could combine with FOXA1 and trigger its ubiquitination and degradation; NEDD4 inactivates the Wnt/β-catenin pathway by motivating FOXA1 ubiquitination; NEDD4 enhancement refrained DLBCL growth in vivo. In conclusion, the E3 ubiquitin ligase NEDD4 accelerates FOXA1 ubiquitination but refrains DLBCL cell proliferation via the Wnt/β-Catenin pathway.