ABSTRACT
The lethality, chemoresistance and metastatic characteristics of cancers are associated with phenotypically plastic cancer stem cells (CSCs). How the non-cell autonomous signalling pathways and cell-autonomous transcriptional machinery orchestrate the stem cell-like characteristics of CSCs is still poorly understood. Here we use a quantitative proteomic approach for identifying secreted proteins of CSCs in pancreatic cancer. We uncover that the cell-autonomous E2F1/4-pRb/RBL2 axis balances non-cell-autonomous signalling in healthy ductal cells but becomes deregulated upon KRAS mutation. E2F1 and E2F4 induce whereas pRb/RBL2 reduce WNT ligand expression (e.g. WNT7A, WNT7B, WNT10A, WNT4) thereby regulating self-renewal, chemoresistance and invasiveness of CSCs in both PDAC and breast cancer, and fibroblast proliferation. Screening for epigenetic enzymes identifies GCN5 as a regulator of CSCs that deposits H3K9ac onto WNT promoters and enhancers. Collectively, paracrine signalling pathways are controlled by the E2F-GCN5-RB axis in diverse cancers and this could be a therapeutic target for eliminating CSCs.
Subject(s)
E2F1 Transcription Factor , E2F4 Transcription Factor , Neoplastic Stem Cells , Pancreatic Neoplasms , Paracrine Communication , Humans , Neoplastic Stem Cells/metabolism , Neoplastic Stem Cells/pathology , E2F1 Transcription Factor/metabolism , E2F1 Transcription Factor/genetics , Cell Line, Tumor , Pancreatic Neoplasms/metabolism , Pancreatic Neoplasms/pathology , Pancreatic Neoplasms/genetics , E2F4 Transcription Factor/metabolism , E2F4 Transcription Factor/genetics , Animals , Carcinoma, Pancreatic Ductal/pathology , Carcinoma, Pancreatic Ductal/genetics , Carcinoma, Pancreatic Ductal/metabolism , Wnt Proteins/metabolism , Wnt Proteins/genetics , Retinoblastoma Protein/metabolism , Retinoblastoma Protein/genetics , Breast Neoplasms/metabolism , Breast Neoplasms/genetics , Breast Neoplasms/pathology , p300-CBP Transcription Factors/metabolism , p300-CBP Transcription Factors/genetics , Gene Expression Regulation, Neoplastic , Proto-Oncogene Proteins p21(ras)/genetics , Proto-Oncogene Proteins p21(ras)/metabolism , Female , Cell Proliferation , Mice , Signal Transduction , Drug Resistance, Neoplasm/geneticsABSTRACT
Pancreatic cancer is one of the most lethal malignancies worldwide. Acquiring infinite proliferation ability is a key hallmark and basis of tumorigenesis. NOP14 is an identified ribosome biogenesis protein that plays potential roles in cell proliferation. However, the function and molecular mechanism of NOP14 remain ambiguous in most human cancers. In this study, we first investigated the subcellular localization and expression of NOP14 by multiple quantitative assays in pancreatic cancer. We confirmed that NOP14 was mainly localized in nucleolus in human pancreatic cancer cells. Then we studied the regulatory effects of this nucleolus protein on tumor cell proliferation in vitro. NOP14 was demonstrated to play a dominant pro-proliferation role in pancreatic cancer. Furthermore, we identified miR17-5p as a downstream target of NOP14. Transfection of miR17-5p mimics or inhibitors rescued the down- or upregulated effect of NOP14 on cell proliferation by regulating expression of P130. In addition, NOP14 induced expression of transcription factor E2F4 independent of miR17-5p/P130 signaling, which simultaneously activated a set of targeted genes, such as CCNE1, PIM1, AKT1 etc., to promote tumor proliferation. These findings might provide novel insights for better understanding the diverse function of NOP14 in human malignancies to develop new strategies for targeted therapy.
Subject(s)
MicroRNAs , Pancreatic Neoplasms , Humans , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , Cell Line, Tumor , Pancreatic Neoplasms/pathology , MicroRNAs/genetics , Cell Proliferation/genetics , Gene Expression Regulation, Neoplastic , E2F4 Transcription Factor/genetics , E2F4 Transcription Factor/metabolism , Pancreatic NeoplasmsABSTRACT
Tumor suppressor p53 and its related proteins, p63 and p73, can be synthesized as multiple isoforms lacking part of the N- or C-terminal regions. Specifically, high expression of the ΔNp73α isoform is notoriously associated with various human malignancies characterized by poor prognosis. This isoform is also accumulated by oncogenic viruses, such as Epstein-Barr virus (EBV), as well as genus beta human papillomaviruses (HPV) that appear to be involved in carcinogenesis. To gain additional insight into ΔNp73α mechanisms, we have performed proteomics analyses using human keratinocytes transformed by the E6 and E7 proteins of the beta-HPV type 38 virus as an experimental model (38HK). We find that ΔNp73α associates with the E2F4/p130 repressor complex through a direct interaction with E2F4. This interaction is favored by the N-terminal truncation of p73 characteristic of ΔNp73 isoforms. Moreover, it is independent of the C-terminal splicing status, suggesting that it could represent a general feature of ΔNp73 isoforms (α, ß, γ, δ, ε, ζ, θ, η, and η1). We show that the ΔNp73α-E2F4/p130 complex inhibits the expression of specific genes, including genes encoding for negative regulators of proliferation, both in 38HK and in HPV-negative cancer-derived cell lines. Such genes are not inhibited by E2F4/p130 in primary keratinocytes lacking ΔNp73α, indicating that the interaction with ΔNp73α rewires the E2F4 transcriptional program. In conclusion, we have identified and characterized a novel transcriptional regulatory complex with potential implications in oncogenesis. IMPORTANCE The TP53 gene is mutated in about 50% of human cancers. In contrast, the TP63 and TP73 genes are rarely mutated but rather expressed as ΔNp63 and ΔNp73 isoforms in a wide range of malignancies, where they act as p53 antagonists. Accumulation of ΔNp63 and ΔNp73, which is associated with chemoresistance, can result from infection by oncogenic viruses such as EBV or HPV. Our study focuses on the highly carcinogenic ΔNp73α isoform and uses a viral model of cellular transformation. We unveil a physical interaction between ΔNp73α and the E2F4/p130 complex involved in cell cycle control, which rewires the E2F4/p130 transcriptional program. Our work shows that ΔNp73 isoforms can establish interactions with proteins that do not bind to the TAp73α tumor suppressor. This situation is analogous to the gain-of-function interactions of p53 mutants supporting cellular proliferation.
Subject(s)
Epstein-Barr Virus Infections , Papillomavirus Infections , Humans , Cell Transformation, Neoplastic , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , E2F4 Transcription Factor/genetics , E2F4 Transcription Factor/metabolism , Gene Expression , Herpesvirus 4, Human/genetics , Human Papillomavirus Viruses , Keratinocytes , Protein Isoforms/genetics , Protein Isoforms/metabolism , Tumor Suppressor Protein p53/genetics , Tumor Suppressor Protein p53/metabolism , Crk-Associated Substrate Protein/metabolism , Neoplasms/metabolismABSTRACT
Background: We aimed to evaluate the prognostic value of E2F4 expression in oral squamous cell carcinoma (OSCC) and clarify its influence on immune cell infiltration and biological functions. Methods: The Cancer Genome Atlas (TCGA) database, the STRING database, and related online tools as well as single-sample gene set enrichment analysis (ssGSEA) were used for the analyses in our study. Results: The E2F4 expression was elevated in OSCC tumor tissue compared with paracancerous tissues. The high expression of E2F4 was closely related to the poorer overall survival, disease-free survival, and progression-free interval of OSCC. In addition, pathway enrichment analyses revealed that the top 49 genes most closely related to E2F4 were strongly associated with the cell cycle. E2F4-related proteins were closely related to the following KEGG pathways: cell cycle, cellular senescence, PI3K-Akt signaling pathway, Wnt signaling pathway, and notch signaling pathway. It was also demonstrated that the E2F4 expression was negatively correlated with immune purity and strongly related to immune cell infiltration in OSCC. Conclusions: E2F4 can be used as a novel biomarker for the diagnosis and prognosis of OSCC.
Subject(s)
Carcinoma, Squamous Cell , Head and Neck Neoplasms , Mouth Neoplasms , Squamous Cell Carcinoma of Head and Neck , Humans , Carcinoma, Squamous Cell/diagnosis , Carcinoma, Squamous Cell/genetics , Carcinoma, Squamous Cell/metabolism , Carcinoma, Squamous Cell/pathology , E2F4 Transcription Factor/biosynthesis , E2F4 Transcription Factor/genetics , Head and Neck Neoplasms/diagnosis , Head and Neck Neoplasms/genetics , Head and Neck Neoplasms/metabolism , Head and Neck Neoplasms/pathology , Mouth Neoplasms/diagnosis , Mouth Neoplasms/genetics , Mouth Neoplasms/metabolism , Mouth Neoplasms/pathology , Phosphatidylinositol 3-Kinases , Prognosis , Squamous Cell Carcinoma of Head and Neck/diagnosis , Squamous Cell Carcinoma of Head and Neck/genetics , Squamous Cell Carcinoma of Head and Neck/metabolism , Squamous Cell Carcinoma of Head and Neck/pathology , Wnt Signaling PathwayABSTRACT
To investigate the relationship between the transcription factor, E2F4, and head and neck squamous cell carcinoma (HNSCC), and to preliminarily explore the signaling pathways and immunological role of E2F4. The mRNA expression of E2F4 in HNSCC was evaluated by searching Gene Expression Omnibus (GEO) and The Cancer Genome Atlas (TCGA) datasets. E2F4 protein expression was analyzed by immunohistochemistry using the CMU1h-ENT database. The association between E2F4 expression and tumor infiltration of immune cells was analyzed. Intracellular signaling by E2F4 was explored using KEGG and GO analysis. The correlation of E2F4 expression with clinical characteristics and its prognostic role were validated and analyzed in TCGA database. From the analysis of GEO and TCGA data, E2F4 expression was found to be up-regulated in HNSCC tumor tissues, and its level was associated with T, Grade, and M staging. Kaplan-Meier curve and Cox analyses indicated that the high expression of E2F4 was related to a poor prognosis. Thus, E2F4 was considered a potential prognostic factor for HNSCC. Immunohistochemical staining showed that E2F4 was mainly localized in the cell nucleus; it was highly expressed in HNSCC tissues, with a significant difference noted from that in pericancerous mucosa tissues. A correlation was observed between the differential expression of E2F4 and the immune infiltration of HNSCC. As revealed by KEGG and GO analysis, differential enrichment was found in the cell cycle, spliceosome, meiosis, microbial polysaccharide synthesis, and WNT signaling pathway, as well as in cyclic adenosine monophosphate, ERBB2, VEGF, GCNP and MYC pathways. E2F4 plays an important role in tumor progression and may be a critical biological prognostic factor for HNSCC. In addition, it functions in the nucleus as a transcription factor, regulates immune cells, and could be a promising molecular target for the diagnosis and treatment of HNSCC.
Subject(s)
E2F4 Transcription Factor , Head and Neck Neoplasms , Biomarkers, Tumor/genetics , Biomarkers, Tumor/immunology , E2F4 Transcription Factor/genetics , E2F4 Transcription Factor/immunology , Head and Neck Neoplasms/diagnosis , Head and Neck Neoplasms/genetics , Head and Neck Neoplasms/immunology , Humans , Prognosis , Squamous Cell Carcinoma of Head and Neck/diagnosis , Squamous Cell Carcinoma of Head and Neck/genetics , Squamous Cell Carcinoma of Head and Neck/immunologyABSTRACT
BACKGROUND: Recent studies have shown that curcumin can reduce the symptoms of hydrocephalus. However, the underlying mechanisms remain unclear. Our previous studies demonstrated that E2F transcription factor 4 (E2F4) protein plays an important role in hydrocephalus; hence, we hypothesized that E2F4 may involve in curcumin mediated anti-hydrocephalus benefits. METHODS: E2F4 expression and functions in different human tissues and cell lines were determined and analyzed using the all RNA-seq and ChIP-seq sample and signature search database and ChIP-atlas database. Hydrocephalus mouse model was established through stereotactic injection of shE2F4 into frontal cortex. Mice were treated with curcumin, and then hydrocephalus severity, the expression of E2F4, and downstream targets were analyzed. RESULTS: E2F4 was highly expressed in the nervous system, which was downregulated in the bran of hydrocephalus patients. Knockdown E2F4 in mice could mimic the phenotype of human hydrocephalus. Upon curcumin administration, E2F4 expression level was increased, and the hydrocephalus severity score was significantly decreased in mouse model. Mechanistically, curcumin attenuated hydrocephalus through activating E2F4 signaling pathway. CONCLUSION: Curcumin suppresses hydrocephalus progression via activation of E2F4, which could be a target for hydrocephalus treatment.
Subject(s)
Curcumin , Animals , Cell Line , Curcumin/pharmacology , E2F4 Transcription Factor/genetics , E2F4 Transcription Factor/metabolism , Humans , MiceABSTRACT
Alzheimer's disease (AD) has a complex etiology, which requires a multifactorial approach for an efficient treatment. We have focused on E2 factor 4 (E2F4), a transcription factor that regulates cell quiescence and tissue homeostasis, controls gene networks affected in AD, and is upregulated in the brains of Alzheimer's patients and of APPswe/PS1dE9 and 5xFAD transgenic mice. E2F4 contains an evolutionarily conserved Thr-motif that, when phosphorylated, modulates its activity, thus constituting a potential target for intervention. In this study, we generated a knock-in mouse strain with neuronal expression of a mouse E2F4 variant lacking this Thr-motif (E2F4DN), which was mated with 5xFAD mice. Here, we show that neuronal expression of E2F4DN in 5xFAD mice potentiates a transcriptional program consistent with the attenuation of the immune response and brain homeostasis. This correlates with reduced microgliosis and astrogliosis, modulation of amyloid-ß peptide proteostasis, and blocking of neuronal tetraploidization. Moreover, E2F4DN prevents cognitive impairment and body weight loss, a known somatic alteration associated with AD. We also show that our finding is significant for AD, since E2F4 is expressed in cortical neurons from Alzheimer patients in association with Thr-specific phosphorylation, as evidenced by an anti-E2F4/anti-phosphoThr proximity ligation assay. We propose E2F4DN-based gene therapy as a promising multifactorial approach against AD.
Subject(s)
Alzheimer Disease , Cognitive Dysfunction , E2F4 Transcription Factor , Alzheimer Disease/drug therapy , Alzheimer Disease/genetics , Alzheimer Disease/metabolism , Alzheimer Disease/therapy , Amyloid beta-Peptides/metabolism , Amyloid beta-Protein Precursor/metabolism , Animals , Cognitive Dysfunction/drug therapy , Cognitive Dysfunction/genetics , Cognitive Dysfunction/metabolism , Disease Models, Animal , E2F4 Transcription Factor/genetics , E2F4 Transcription Factor/metabolism , Mice , Mice, TransgenicABSTRACT
BACKGROUND: Cervical cancer is the most common gynecological cancer worldwide and is associated with high morbidity and mortality. Despite improvements in therapeutic strategies, the network regulation mechanism remains unclear and the treatment effect is not satisfactory. Therefore, there is a need to continue studying the mechanism of cervical cancer to explore effective gene targets and precise targeted therapy drugs. METHODS: First, three paired tissues (cancer tissues and noncancerous tissues) from patients with cervical squamous cell carcinoma were collected, grouped, and analyzed by microarray. Second, differentially expressed mRNAs (DEMs) and differentially expressed lncRNAs (DELs) (|fold change| ≥ 2 and p < 0.05) between the two groups were screened. For DEMs, functional annotation and pathway analysis were performed using DAVID. Functional prediction of DELs was then performed and their cis-regulatory and trans-regulatory networks were explored. RESULTS: Function prediction of DELs (both up-regulated and down-regulated) shows that the highest frequency Cellular Component (CC) item is cytosol, the highest frequency Molecular function (MF) item is mitotic cell cycle and the highest frequency Biological Process (BP) item is protein binding. Through cis-regulation analysis of DELs, the cis-regulatory relationship of 96 DELs was predicted. The lncRNA-trans-regulation network analysis suggested that E2F4 may be the core transcription factor in the lncRNA-TF regulatory network in cervical cancer. CONCLUSIONS: The lncRNA-TF regulatory network plays an important role in the occurrence and progression of cervical cancer, and E2F4 may be a critical transcription factor in the regulatory network.
Subject(s)
MicroRNAs , RNA, Long Noncoding , Uterine Cervical Neoplasms , E2F4 Transcription Factor/genetics , E2F4 Transcription Factor/metabolism , Female , Gene Expression Regulation, Neoplastic , Gene Regulatory Networks/genetics , Humans , MicroRNAs/genetics , RNA, Long Noncoding/genetics , RNA, Long Noncoding/metabolism , RNA, Messenger/metabolism , Transcription Factors/genetics , Uterine Cervical Neoplasms/geneticsABSTRACT
To improve cancer precision medicine, prognostic and predictive biomarkers are critically needed to aid physicians in deciding treatment strategies in a personalized fashion. Due to the heterogeneous nature of cancer, most biomarkers are expected to be valid only in a subset of patients. Furthermore, there is no current approach to determine the applicability of biomarkers. In this study, we propose a framework to improve the clinical application of biomarkers. As part of this framework, we develop a clinical outcome prediction model (CPM) and a predictability prediction model (PPM) for each biomarker and use these models to calculate a prognostic score (P-score) and a confidence score (C-score) for each patient. Each biomarker's P-score indicates its association with patient clinical outcomes, while each C-score reflects the biomarker applicability of the biomarker's CPM to a patient and therefore the confidence of the clinical prediction. We assessed the effectiveness of this framework by applying it to three biomarkers, Oncotype DX, MammaPrint, and an E2F4 signature, which have been used for predicting patient response, pathologic complete response versus residual disease to neoadjuvant chemotherapy (a classification problem), and recurrence-free survival (a Cox regression problem) in breast cancer, respectively. In both applications, our analyses indicated patients with higher C scores were more likely to be correctly predicted by the biomarkers, indicating the effectiveness of our framework. This framework provides a useful approach to develop and apply biomarkers in the context of cancer precision medicine.
Subject(s)
Biomarkers, Tumor/genetics , Breast Neoplasms/diagnosis , Breast Neoplasms/genetics , Diagnosis, Computer-Assisted/methods , E2F4 Transcription Factor/genetics , Precision Medicine/methods , Breast Neoplasms/drug therapy , Databases, Genetic , Female , Humans , Models, Theoretical , Neoadjuvant Therapy , Prognosis , ROC Curve , Treatment OutcomeABSTRACT
Senescence, a stable state of growth arrest, affects many physiological and pathophysiological processes, especially aging. Previous work has indicated that transcription factors (TFs) play a role in regulating senescence. However, a systematic study of regulatory TFs during replicative senescence (RS) using multi-omics analysis is still lacking. Here, we generated time-resolved RNA-seq, reduced representation bisulfite sequencing (RRBS) and ATAC-seq datasets during RS of mouse skin fibroblasts, which demonstrated that an enhanced inflammatory response and reduced proliferative capacity were the main characteristics of RS in both the transcriptome and epigenome. Through integrative analysis and genetic manipulations, we found that transcription factors E2F4, TEAD1 and AP-1 are key regulators of RS. Overexpression of E2f4 improved cellular proliferative capacity, attenuated SA-ß-Gal activity and changed RS-associated differentially methylated sites (DMSs). Moreover, knockdown of Tead1 attenuated SA-ß-Gal activity and partially altered the RS-associated transcriptome. In addition, knockdown of Atf3, one member of AP-1 superfamily TFs, reduced Cdkn2a (p16) expression in pre-senescent fibroblasts. Taken together, the results of this study identified transcription factors regulating the senescence program through multi-omics analysis, providing potential therapeutic targets for anti-aging.
Subject(s)
Cellular Senescence , E2F4 Transcription Factor , TEA Domain Transcription Factors , Transcription Factor AP-1 , Aging , Animals , Cellular Senescence/genetics , E2F4 Transcription Factor/genetics , Fibroblasts/metabolism , Mice , TEA Domain Transcription Factors/genetics , TEA Domain Transcription Factors/metabolism , Transcription Factor AP-1/genetics , Transcription Factor AP-1/metabolism , TranscriptomeABSTRACT
BACKGROUND: Long non-coding RNAs (lncRNAs) are closely associated with the pathogenesis of numerous diseases including cancers. LncRNA AGAP2 Antisense RNA 1 (AGAP2-AS1) has been found to participate in the tumorigenesis of several kinds of human cancers. Nonetheless, its potential function in colorectal cancer (CRC) was still poorly investigated. METHODS: The expression level of RNAs or proteins was assessed by RT-qPCR or western blot analysis. Functional experiments were performed to analyze the role of AGAP2-AS1 in CRC in vitro and in vivo. Mechanism investigations were fulfilled to determine the potential mechanism of the molecules. RESULTS: AGAP2-AS1 expression was significantly elevated in CRC cells and could be transcriptionally activated by E2F Transcription Factor 4 (E2F4). Down-regulated AGAP2-AS1 could weaken CRC cell growth, migration, invasion, and epithelial-mesenchymal transition (EMT). MicroRNA-182-5p (miR-182-5p) was the target downstream molecule of AGAP2-AS1. Furthermore, Cofilin 1 (CFL1) was proved as the target of miR-182-5p. Mechanically, AGAP2-AS1 could boost the CFL1 expression via competitively binding to miR-182-5p in CRC. Importantly, CFL1 restoration could counteract the in vitro and in vivo suppression of depleted AGAP2-AS1 on CRC progression. CONCLUSION: E2F4-stimulated AGAP2-AS1 aggravated CRC development through regulating miR-182-5p/CFL1 axis, implying that AGAP2-AS1 might become a potent new target for future therapies for CRC.
Subject(s)
Colorectal Neoplasms , GTP-Binding Proteins/genetics , GTPase-Activating Proteins/genetics , MicroRNAs , RNA, Long Noncoding , Cell Line, Tumor , Cell Movement , Cell Proliferation , Cofilin 1/genetics , Cofilin 1/metabolism , Colorectal Neoplasms/genetics , Colorectal Neoplasms/pathology , E2F4 Transcription Factor/genetics , E2F4 Transcription Factor/metabolism , Gene Expression Regulation, Neoplastic , Humans , MicroRNAs/genetics , MicroRNAs/metabolism , RNA, Long Noncoding/genetics , RNA, Long Noncoding/metabolism , Up-RegulationABSTRACT
CADASIL is a small vessel disease caused by mutations in NOTCH3 that lead to an odd number of cysteines in the EGF-like repeat domain, causing protein misfolding and aggregation. The main symptoms are migraine, psychiatric disturbances, recurrent strokes and dementia, being executive function characteristically impaired. The molecular pathways altered by this receptor aggregation need to be studied further. A genome-wide transcriptome study (four cases paired with three healthy siblings) was carried out, in addition to a qRT-PCR for validation purposes (ten new cases and eight new controls). To study the expression profile by cell type of the significant mRNAs found, we performed an in situ hybridization (ISH) (nine cases and eight controls) and a research in the Single-nuclei Brain RNA-seq expression browser (SNBREB). Pathway analysis enrichment was carried out with Gene Ontology and Reactome. Neuropsychological tests were performed in five of the qRT-PCR cases. The two most significant differentially expressed mRNAs (BANP, p-value = 7.23 × 10-4 and PDCD6IP, p-value = 8.36 × 10-4) were selected for the validation study by qRT-PCR. Additionally, we selected two more mRNAs (CAMK2G, p-value = 4.52 × 10-3 and E2F4, p-value = 4.77 × 10-3) due to their association with ischemic neuronal death. E2F4 showed differential expression in the genome-wide transcriptome study and in the qRT-PCR (p = 1.23 × 10-3), and it was upregulated in CADASIL cases. Furthermore, higher E2F4 expression was associated with worse executive function (p = 2.04 × 10-2) and attention and information processing speed (IPS) (p = 8.73 × 10-2). In situ hibridization showed E2F4 expression in endothelial and vascular smooth vessel cells. In silico studies indicated that E2F4 is also expressed in brain endothelial cells. Among the most significant pathways analyzed, there was an enrichment of vascular development, cell adhesion and vesicular machinery terms and autophagy process. E2F4 is more highly expressed in the skin biopsy of CADASIL patients compared to controls, and its expression is present in endothelial cells and VSMCs. Further studies are needed to understand whether E2F4 could be useful as a biomarker, to monitor the disease or be used as a therapeutic target.
Subject(s)
CADASIL/pathology , Cognitive Dysfunction/pathology , E2F4 Transcription Factor/genetics , Genome, Human , Mutation , Skin/pathology , Transcriptome , Adult , Biopsy , CADASIL/genetics , CADASIL/metabolism , Case-Control Studies , Cognitive Dysfunction/genetics , Cognitive Dysfunction/metabolism , Female , Genome-Wide Association Study , Humans , Male , Middle Aged , Skin/metabolismABSTRACT
Oxygen therapy is a common treatment in neonatal intensive care units, but long-term continuous hyperoxia ventilation may induce acute lung injury (ALI). Gasdermin D (GSDMD)-mediated pyroptosis participates in various diseases including ALI, but the role of GSDMD in hyperoxia-induced ALI is yet understood. Here, we showed a significant increase in GSDMD after exposure to high oxygen. To elucidate the molecular mechanisms involved in GSDMD regulation, we identified the core promoter of GSDMD, -98 ~ -12 bp relative to the transcriptional start site (TSS). The results of mutational analysis, overexpression or siRNA interference, EMSA and ChIP demonstrated that E2F4 and TFAP2A positively regulate the transcriptional activity of the GSDMD by binding to its promoter. However, only TFAP2A showed a regulatory effect on the expression of GSDMD. Moreover, TFAP2A was increased in the lung tissues of rats exposed to hyperoxia and showed a strong linear correlation with GSDMD. Our results indicated that TFAP2A positively regulates the GSDMD expression via binding to the promoter region of GSDMD.
Subject(s)
Acute Lung Injury/genetics , E2F4 Transcription Factor/genetics , Intracellular Signaling Peptides and Proteins/genetics , Oxygen/adverse effects , Phosphate-Binding Proteins/genetics , Transcription Factor AP-2/genetics , A549 Cells , Acute Lung Injury/chemically induced , Acute Lung Injury/pathology , Animals , Animals, Newborn , Cell Hypoxia/drug effects , Gene Expression Regulation/genetics , Humans , Intensive Care Units, Neonatal , Oxygen/therapeutic use , Promoter Regions, Genetic/genetics , Pyroptosis/genetics , Rats , Transcription Initiation SiteABSTRACT
Transcriptional regulation of DNA repair is of outmost importance for the restoration of DNA integrity upon genotoxic stress. Here we report that the potent environmental carcinogen benzo[a]pyrene (B[a]P) activates a cellular DNA damage response resulting in transcriptional repression of mismatch repair (MMR) genes (MSH2, MSH6, EXO1) and of RAD51, the central homologous recombination repair (HR) component, ultimately leading to downregulation of MMR and HR. B[a]P-induced gene repression is caused by abrogated E2F1 signalling. This occurs through proteasomal degradation of E2F1 in G2-arrested cells and downregulation of E2F1 mRNA expression in G1-arrested cells. Repression of E2F1-mediated transcription and silencing of repair genes is further mediated by the p21-dependent E2F4/DREAM complex. Notably, repression of DNA repair is also observed following exposure to the active B[a]P metabolite BPDE and upon ionizing radiation and occurs in response to a p53/p21-triggered, irreversible cell cycle arrest marking the onset of cellular senescence. Overall, our results suggest that repression of MMR and HR is an early event during genotoxic-stress induced senescence. We propose that persistent downregulation of DNA repair might play a role in the maintenance of the senescence phenotype, which is associated with an accumulation of unrepairable DNA lesions.
Subject(s)
Benzo(a)pyrene/toxicity , Carcinogens/toxicity , Cellular Senescence/genetics , DNA/genetics , E2F1 Transcription Factor/genetics , E2F4 Transcription Factor/genetics , Cell Cycle Checkpoints , Cell Line, Transformed , Cell Line, Tumor , Cyclin-Dependent Kinase Inhibitor p21/genetics , Cyclin-Dependent Kinase Inhibitor p21/metabolism , DNA/metabolism , DNA Damage , DNA Mismatch Repair/drug effects , DNA Mismatch Repair/radiation effects , DNA Repair Enzymes/genetics , DNA Repair Enzymes/metabolism , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , E2F1 Transcription Factor/metabolism , E2F4 Transcription Factor/metabolism , Epithelial Cells/cytology , Epithelial Cells/drug effects , Epithelial Cells/metabolism , Epithelial Cells/radiation effects , Exodeoxyribonucleases/genetics , Exodeoxyribonucleases/metabolism , Fibroblasts/cytology , Fibroblasts/drug effects , Fibroblasts/metabolism , Fibroblasts/radiation effects , Gamma Rays , Humans , Kv Channel-Interacting Proteins/genetics , Kv Channel-Interacting Proteins/metabolism , MCF-7 Cells , MutS Homolog 2 Protein/genetics , MutS Homolog 2 Protein/metabolism , Rad51 Recombinase/genetics , Rad51 Recombinase/metabolism , Recombinational DNA Repair/drug effects , Recombinational DNA Repair/radiation effects , Repressor Proteins/genetics , Repressor Proteins/metabolism , Signal TransductionABSTRACT
Feedback regulation plays an important role in the regulation of molecular processes. Although feedback regulatory mechanisms that generate potential-specific dynamic behavior, such as oscillation and switch-like activation, have been found, their significant contribution to the signal transduction system has not been fully explored. In this study, I focused on the feedback regulation of signal molecules like transcription factor (TF)-associated target genes controlled after transcription (named TF-target feedback genes). I statistically analyzed the static network of signal transduction pathways and TF-target feedbacks to investigate their presence in upstream signal molecules of TFs in 394 different cell types, including 146 primary cells, 111 tissues, and 137 cell lines. The directed network of signal transduction utilized pathways annotated in KEGG, and the TF-target genes estimated per individual cells were used. Feedback enrichment analysis of upstream signal molecules of TF was performed to investigate whether TF-target genes are upstream of their TF and form a feedback loop in signal transduction. The study revealed the difference in the number of TF-target feedbacks between cells, while each cell had at least 11 significant TF-target feedbacks and invariably involved the E2F transcription factor 4 feedback within the cell cycle. The findings suggest the possibility of the regulation of the TF-associated signal transduction by the TF itself at the transcription level.
Subject(s)
Feedback, Physiological , Gene Expression Profiling/methods , Gene Expression Regulation , Gene Regulatory Networks/genetics , Signal Transduction/genetics , Transcription Factors/genetics , Cell Line, Tumor , Cells, Cultured , E2F1 Transcription Factor/genetics , E2F1 Transcription Factor/metabolism , E2F4 Transcription Factor/genetics , E2F4 Transcription Factor/metabolism , Humans , NF-kappa B p50 Subunit/genetics , NF-kappa B p50 Subunit/metabolism , NFATC Transcription Factors/genetics , NFATC Transcription Factors/metabolism , T-Lymphocytes/metabolism , Transcription Factors/metabolismABSTRACT
Esophageal cancer represents the eighth most frequently occurring cancer, as well as the sixth most widespread cause of cancer-related deaths. In recent years, accumulating evidence has implicated long non-coding RNAs in the progression of esophageal squamous cell carcinoma (ESCC). The aim of the present study was to investigate the potential involvement and underlying mechanisms of LINC00337 in ESCC. Expression patterns of LINC00337 and targeting protein for Xenopus kinesin-like protein 2 (TPX2) in ESCC tissues and cells were detected using RT-qPCR and immunohistochemical staining. Next, the interactions among LINC00337, E2F4, and TPX2 were identified using chromatin immunoprecipitation, dual-luciferase reporter, and RNA-binding protein immunoprecipitation assays, suggesting that LINC00337 could recruit E2F4 to enhance the transcription of TPX2. Thereafter, the regulatory roles of LINC00337 and TPX2 in ESCC were analyzed by altering the expression of LINC00337 or TPX2 in ESCC cells following treatment with cisplatin (DDP). The levels of autophagy-related proteins Beclin1 and LC3II/LC3I, viability, autophagy, apoptosis, and chemoresistance of ESCC cells to DDP were measured following transfection treatment with different plasmids. Additionally, the role of the LINC00337/E2F4/TPX2 axis was assessed in vivo by measuring tumor formation in nude mice. The results demonstrated that LINC00337 upregulated TPX2, consequently leading to elevated levels of Beclin1 and LC3II/LC3I, promoted cell viability and autophagy, while inhibiting apoptosis and chemosensitivity to DDP in ESCC. In sum, the current study evidenced that the overexpression of LINC00337 could potentially enhance ESCC cell autophagy and chemoresistance to DDP via the upregulation of TPX2 by recruiting E2F4. Thus, LINC00337 may serve as a potential candidate for the treatment of ESCC.
Subject(s)
Cell Cycle Proteins/metabolism , Cisplatin/pharmacology , Drug Resistance, Neoplasm , E2F4 Transcription Factor/metabolism , Esophageal Neoplasms/pathology , Esophageal Squamous Cell Carcinoma/pathology , Microtubule-Associated Proteins/metabolism , RNA, Long Noncoding/genetics , Aged , Animals , Antineoplastic Agents/pharmacology , Apoptosis , Biomarkers, Tumor/genetics , Biomarkers, Tumor/metabolism , Cell Cycle Proteins/genetics , Cell Proliferation , E2F4 Transcription Factor/genetics , Esophageal Neoplasms/drug therapy , Esophageal Neoplasms/genetics , Esophageal Neoplasms/metabolism , Esophageal Squamous Cell Carcinoma/drug therapy , Esophageal Squamous Cell Carcinoma/genetics , Esophageal Squamous Cell Carcinoma/metabolism , Female , Gene Expression Regulation, Neoplastic , Humans , Male , Mice , Mice, Inbred BALB C , Mice, Nude , Microtubule-Associated Proteins/genetics , Middle Aged , Prognosis , Survival Rate , Tumor Cells, Cultured , Xenograft Model Antitumor AssaysABSTRACT
Acute myeloid leukaemia (AML) is an aggressive and mostly incurable haematological malignancy with frequent relapse after an initial response to standard chemotherapy. Therefore, novel therapies are urgently required to improve AML clinical outcome. Here, we aim to study the dysregulation of a particular transcription factor, E2F4, and its role in the progression of AML. In this study, human clinical data from the Gene Expression Profiling Interactive Analysis (GEPIA) revealed that increased E2F4 expression was associated with poor prognosis in AML patients. Moreover, the experimental results showed that E2F4 was aberrantly overexpressed in human AML patients and cell lines. Depletion of E2F4 inhibited the proliferation, induced the differentiation and suppressed the growth of AML cells in a nude mouse model. By contrast, overexpression of E2F4 promoted the proliferation and inhibited the differentiation of AML cells in vitro. Additionally, E2F4 expression not only is positively correlated with EZH2 but also can bind to EZH2. RNA microarray results also showed that E2F4 can regulate MAPK signalling pathway. EZH2 can reverse the inhibitory effect of E2F4 silencing on MAPK signaling pathway. In summary, our data suggest that E2F4 may be a potential therapeutic target for AML therapy.
Subject(s)
E2F4 Transcription Factor/genetics , Enhancer of Zeste Homolog 2 Protein/genetics , Genes, Tumor Suppressor/physiology , Leukemia, Myeloid, Acute/genetics , MAP Kinase Signaling System/genetics , Signal Transduction/genetics , Animals , Cell Differentiation/genetics , Cell Line, Tumor , Cell Proliferation/genetics , Cells, Cultured , Epigenesis, Genetic/genetics , Gene Expression Profiling/methods , Humans , Male , Mice , Mice, Nude , THP-1 CellsABSTRACT
Follicles develop into preovulatory follicles during folliculogenesis and the majority of small yellow follicles become atretic and gets reabsorbed. In this study, based the RNA-seq results of duck ovary, epidermal growth factor receptor (EGFR) was selected as a candidate gene in follicular development and the role was explored. The results demonstrated that EGFR-P8 was the quail EGFR core promoter. It had an E2F4 binding site within EGFR core promoter. E2F4 overexpression significantly increased EGFR expression in quail granulosa cells (GCs). However, the effect was abolished when the GCs were treated with corynoxeine, an inhibitor of the mitogen-activated protein kinase/extracellular regulated protein kinase (MAPK/ERK) signaling pathway. Moreover, luciferase reporter assay and chromatin immunoprecipitation experiments showed that E2F4 upregulated the expression of EGFR expression, which increased E2 and P4 production. In addition, EGFR regulated GCs proliferation and affected follicular development. Taken together, our findings suggested that EGFR, which was regulated by E2F4, enhanced the expression of MAPK/ERK pathway components and follicular development. These results provided an important basis for an improved understanding of the MAPK/ERK pathway and new insight into the development of quail follicles.
Subject(s)
Cell Proliferation/genetics , ErbB Receptors/metabolism , Extracellular Signal-Regulated MAP Kinases/metabolism , Granulosa Cells/metabolism , Ovarian Follicle/metabolism , Ovary/metabolism , Quail/metabolism , Animals , Binding Sites/genetics , CHO Cells , Chromatin Immunoprecipitation , Cricetulus , E2F4 Transcription Factor/genetics , E2F4 Transcription Factor/metabolism , ErbB Receptors/genetics , Estradiol/metabolism , Extracellular Signal-Regulated MAP Kinases/antagonists & inhibitors , Female , Granulosa Cells/cytology , Indole Alkaloids/pharmacology , MAP Kinase Signaling System/genetics , Ovarian Follicle/cytology , Ovarian Follicle/drug effects , Ovarian Follicle/growth & development , Ovary/cytology , Progesterone/metabolism , Promoter Regions, Genetic , Protein Binding , Quail/genetics , RNA, Small Interfering , Signal Transduction/genetics , Up-RegulationABSTRACT
E2F transcription factors are central regulators of cell division and cell fate decisions. E2F4 often represents the predominant E2F activity in cells. E2F4 is a transcriptional repressor implicated in cell cycle arrest and whose repressive activity depends on its interaction with members of the RB family. Here we show that E2F4 is important for the proliferation and the survival of mouse embryonic stem cells. In these cells, E2F4 acts in part as a transcriptional activator that promotes the expression of cell cycle genes. This role for E2F4 is independent of the RB family. Furthermore, E2F4 functionally interacts with chromatin regulators associated with gene activation and we observed decreased histone acetylation at the promoters of cell cycle genes and E2F targets upon loss of E2F4 in RB family-mutant cells. Taken together, our findings uncover a non-canonical role for E2F4 that provide insights into the biology of rapidly dividing cells.
Subject(s)
E2F4 Transcription Factor/metabolism , Mouse Embryonic Stem Cells/metabolism , Retinoblastoma Protein/metabolism , Transcriptional Activation , Animals , Cell Cycle , Cell Division , E2F4 Transcription Factor/genetics , Gene Expression Regulation, Developmental , Mice , Mouse Embryonic Stem Cells/cytology , Multigene Family , Retinoblastoma Protein/geneticsABSTRACT
We previously reported that the cell cycle-related cyclin-dependent kinase 4-retinoblastoma (RB) transcriptional corepressor pathway is essential for stroke-induced cell death both in vitro and in vivo However, how this signaling pathway induces cell death is unclear. Previously, we found that the cyclin-dependent kinase 4 pathway activates the pro-apoptotic transcriptional co-regulator Cited2 in vitro after DNA damage. In the present study, we report that Cited2 protein expression is also dramatically increased following stroke/ischemic insult. Critically, utilizing conditional knockout mice, we show that Cited2 is required for neuronal cell death, both in culture and in mice after ischemic insult. Importantly, determining the mechanism by which Cited2 levels are regulated, we found that E2F transcription factor (E2F) family members participate in Cited2 regulation. First, E2F1 expression induced Cited2 transcription, and E2F1 deficiency reduced Cited2 expression. Moreover, determining the potential E2F-binding regions on the Cited2 gene regulatory sequence by ChIP analysis, we provide evidence that E2F1/4 proteins bind to this DNA region. A luciferase reporter assay to probe the functional outcomes of this interaction revealed that E2F1 activates and E2F4 inhibits Cited2 transcription. Moreover, we identified the functional binding motif for E2F1 in the Cited2 gene promoter by demonstrating that mutation of this site dramatically reduces E2F1-mediated Cited2 transcription. Finally, E2F1 and E2F4 regulated Cited2 expression in neurons after stroke-related insults. Taken together, these results indicate that the E2F-Cited2 regulatory pathway is critically involved in stroke injury.