E2F1 Expression Research Articles

IGF-I protects porcine granulosa cells from hypoxia-induced apoptosis by promoting homologous recombination repair through the PI3K/AKT/E2F8/RAD51 pathway.

Severe hypoxia induced by vascular compromise (ovarian torsion, surgery), obliteration of vessels (aging, chemotherapy, particularly platinum drugs) can cause massive follicle atresia. On the other hand, hypoxia increases the occurrence of DNA double-strand breaks (DSBs) and triggers cellular damage repair mechanisms; however, if the damage is not promptly repaired, it can also induce the apoptosis program. Insulin-like growth factor-I (IGF-I) is a polypeptide hormone that plays essential roles in stimulating mammalian follicular development. Here, we report a novel role for IGF-I in protecting hypoxic GCs from apoptosis by promoting DNA repair through the homologous recombination (HR) process. Indeed, the hypoxic environment within follicles significantly inhibited the efficiency of HR-directed DNA repair. The presence of IGF-I-induced HR pathway to alleviate hypoxia-induced DNA damage and apoptosis primarily through upregulating the expression of the RAD51 recombinase. Importantly, we identified a new transcriptional regulator of RAD51, namely E2F8, which mediates the protective effects of IGF-I on hypoxic GCs by facilitating the transcriptional activation of RAD51. Furthermore, we demonstrated that the PI3K/AKT pathway is crucial for IGF-I-induced E2F8 expression, resulting in increased RAD51 expression and enhanced HR activity, which mitigates hypoxia-induced DNA damage and thereby protects against GCs apoptosis. Together, these findings define a novel mechanism of IGF-I-mediated GCs protection by activating the HR repair through the PI3K/AKT/E2F8/RAD51 pathway under hypoxia.

Identification of arnicolide C as a novel chemosensitizer to suppress mTOR/E2F1/FANCD2 axis in non-small cell lung cancer.

The mammalian target of rapamycin (mTOR) pathway plays critical roles in intrinsic chemoresistance by regulating Fanconi anaemia complementation group D2 (FANCD2) expression. However, the mechanisms by which mTOR regulates FANCD2 expression and related inhibitors are not clearly elucidated. Extracts of Centipeda minima (C.minima) showed promising chemosensitizing effects by inhibiting FANCD2 activity. Here, we have aimed to identify the bioactive chemosensitizer in C.minima extracts and elucidate its underlying mechanism. The chemosensitizing effects of arnicolide C (ArC), a bioactive compound in C.minima, on non-small cell lung cancer (NSCLC) were investigated using immunoblotting, immunofluorescence, flow cytometry, the comet assay, small interfering RNA (siRNA) transfection and animal models. The online SynergyFinder software was used to determine the synergistic effects of ArC and chemotherapeutic drugs on NSCLC cells. ArC had synergistic cytotoxic effects with DNA cross-linking drugs such as cisplatin and mitomycin C in NSCLC cells. ArC treatment markedly decreased FANCD2 expression in NSCLC cells, thus attenuating cisplatin-induced FANCD2 nuclear foci formation, leading to DNA damage and apoptosis. ArC inhibited the mTOR pathway and attenuated mTOR-mediated expression of E2F1, a critical transcription factor of FANCD2. Co-administration of ArC and cisplatin exerted synergistic anticancer effects in the A549 xenograft mouse model by suppressing mTOR/FANCD2 signalling in tumour tissues. ArC suppressed DNA cross-linking drug-induced DNA damage response by inhibiting the mTOR/E2F1/FANCD2 signalling axis, serving as a chemosensitizing agent. This provides insight into the anticancer mechanisms of ArC and offers a potential combinatorial anticancer therapeutic strategy.

Predictive role of E2F6 in cancer prognosis and responses of immunotherapy

BackgroundE2F6 is a member of the E2F transcription factor family. Numerous studies have demonstrated that E2F6 is critical to cancer development and progression, but its role in cancer immunotherapy remains unclear. MethodsGenotype-Tissue Expression (GTEx) and The Cancer Genome Atlas (TCGA) databases were used to obtain RNA-seq data for cancer and normal tissues, and we utilized the cBioPortal to analyze E2F6 genomic alterations in pan-cancer. The protein localization of E2F6 was obtained using the Human Protein Atlas (HPA), and the upregulation of E2F6 expression in clinical glioblastoma multiforme (GBM) tissues was detected by Western blot analysis. The ComPPI website was used to analyze the protein interaction information of E2F6. To evaluate the role of E2F6 in pan-cancer prognosis, we used univariate Cox regression and Kaplan-Meier methods, and gene set enrichment analysis (GSEA) was utilized to identify markers associated with E2F6 expression in tumors. TIMER 2.0 was used to study E2F6-related immune cell infiltration in tumor tissues, and the correlation of E2F6 with immunotherapy biomarkers was investigated using Spearman correlation analysis. The role of E2F6 in the cell cycle was analyzed by flow cytometry, and the Cell Counting Kit-8 (CCK-8) and colony formation assays were utilized to determine the proliferative ability of cells. ResultsIn most tumor types, E2F6 was highly expressed and was a good predictor of prognosis. E2F6 was significantly related to markers of immune activation, tumor immune cell infiltration, and immune regulators. Furthermore, E2F6 knockdown significantly attenuated the proliferative ability of glioma cells. Finally, E2F6 effectively predicted anti-programmed cell death 1 (PD1) treatment response. ConclusionE2F6 is an effective biomarker that predicts the prognosis of cancer patients treated with anti-immune checkpoint therapy.

Comparative Transcriptome Analyses in Mantle Cell Lymphoma (MCL): Evidence of E2F1 As a Major Target in Aggressive Blastoid MCL Model

Background Mantle cell lymphoma (MCL) is a highly aggressive B-cell lymphoma characterized by the translocation t(11;14)(q13;q32) which triggers the dysregulation of the cyclin D1 by placing its coding gene CC1ND1 under the control of an immunoglobulin promoter. One of the major consequences of this molecular event is the disturbance of the cyclin D/Rb pathway leading to Rb inactivation and activation the elongation factor E2F1 mainly implicated in cell cycle progression. The mainstay of MCL therapies remains the chemo-immunotherapy followed by autologous transplantation but recently several novel therapies have been developed such as BTK inhibitors, Venetoclax, CDK4/6 inhibitors and more recently CAR-T therapies. However, patients with aggressive forms of MCL exhibit low response rates, requiring the identification of novel therapeutic targets. Materials and Methods We have previously generated an MCL cell line (UPN-1) from a patient with a blastoid variant of MCL presenting unique morphological and cytogenetics characteristics (M'kacher et al, Oncogene 2003). This cell line has been shown to harbor a TP53 mutation and a highly increased sensitivity to radiation despite the absence of functional p53. The molecular basis of this phenomenon has not been explored. In this work, we further characterized UPN-1 cell line using the Clarium S human microarray which has been compared to the gene expression profile of other existing MCL cell lines (GRANTA-519, JEKO-1, JVM-2, MAVEK-1, MINO, REC-1, SP-49, SP-53, Z-138). After mathematical correction, a supervised analysis was performed between triplicate samples of UPN-1 and a group constituted with all other MCL cell lines. We have then set up in vitro assays to determine the effect of a direct E2F1 inhibitor on the proliferative potential of UPN-1. Results The comparative transcriptome analyses revealed a list of 47 genes specifically upregulated in UPN-1. Unsupervised classification allowed to discriminate UPN-1 samples from other MCL cell lines based on the expression of these 47 genes. These data suggested a specific expression profile of UPN-1 as compared to other cell lines. Among the upregulated genes, E2F1 elongation factor was found to be highly upregulated in UPN-1 as compared to other MCL cell lines along with positive enrichment of E2F targets which are mainly implicated in cell cycle progression (Figure 1). Interestingly, UPN-1 has a low level of BCL-2 as compared to other MCL lines that could explain its radio sensitivity. However, other signaling pathways involved in the inhibition of apoptosis were found to be present in UPN-1 by geneset enrichment analysis. The expression of CDKN1A (p21), major cell cycle regulator and known as a marker of aggressive MCL, was found to be low in UPN-1. A repression of immune response pathway such as NFKB with low expression of NFKBIA was identified. Finally, the expression of PIK3CD which encodes for a therapeutic target in MCL (such as PI3KDelta inhibitor Idelalisib) was also significantly repressed in UPN-1 as compared to nine other MCL cell lines. To evaluate the effect of the direct inhibition of E2F1, we treated UPN-1 cells with a targeted E2F1 inhibitor 5‘-Deoxy-5‘-(methylthio)adenosine (MTA) which is a protein methylation inhibitor. MTT assays revealed a 50% reduction of proliferation at + 4 hours in comparison to the vehicle-treated conditions (P<0.0001).Annexin assays showed an increase in early apoptosis (36.6 %) at + 8 hours with subsequent rise in late apoptotic cells (64.5 %) observed after 24 hours of treatment. Interestingly, qRT-PCR analyses of MTA-treated cells exhibited a decrease in the expression of downstream factors associated with E2F1, such as RAD51, BRCA1, CDK2, CCNB2, RANBP1. Conclusions We report here for the first time the comparative transcriptome analysis of the unique aggressive blastoid MCL line UPN-1 as compared to other MCL lines described. This cell line exhibited a major upregulation of E2F1 expression and a down regulation of the cell cycle inhibitor CDKN1A(p21). The potential effects of the direct inhibition of E2F1 using the targeted inhibitor MTA demonstrated a reduction in cell proliferation and induction of apoptosis, suggesting the possibility of associating clinically acceptable E2F1 inhibition strategies to other current therapies.

NEIL3 promotes cell proliferation of ccRCC via the cyclin D1-Rb-E2F1 feedback loop regulation

Nei endonuclease VIII-like 3 (NEIL3), a novel tumor-related gene, is differentially expressed and involved in pathophysiological processes in multiple tumors. However, the potential biological functions and molecular mechanisms of NEIL3 in human clear cell renal cell carcinoma (ccRCC) have not been identified. In this research, we demonstrated that NEIL3, transcriptionally activated by E2F1, served as an oncogene to facilitate cell proliferation and cell cycle progression and contribute to tumorigenesis via the cyclin D1-Rb-E2F1 feedback loop in ccRCC. First, we found that NEIL3 expression was upregulated in ccRCC tissues and cell lines compared with matched adjacent nontumor tissues and renal tubular epithelial cells and was also positively correlated with adverse clinicopathological characteristics, such as advanced cancer stages and higher tumor grades, and acted as an independent prognostic marker in ccRCC. Mechanistically, we demonstrated that NEIL3 promoted cell proliferation, DNA replication and cell cycle progression in vitro and tumor growth in vivo. Furthermore, we found that NEIL3 overexpression activated the cyclin D1-Rb-E2F1 pathway, and the E2F1 upregulation transcriptionally activated NEIL3 expression, thus forming a feedback loop. In addition, there was a positive correlation between NEIL3 and E2F1 expression in clinical specimens of ccRCC. Taken together, our results suggest that NEIL3 serves as a proto-oncogene in ccRCC and presents as a novel candidate for ccRCC diagnosis and treatment.

Transcription Factor E2F7 Hampers the Killing Effect of NK Cells against Colorectal Cancer Cells via Activating RAD18 Transcription.

As a pivotal defensive line against multitudinous malignant tumors, natural killer (NK) cells exist in the tumor microenvironment (TME). RAD18 E3 Ubiquitin Protein Ligase (RAD18) has been reported to foster the malignant progression of multiple cancers, but its effect on NK function has not been mined. Here, the study was designed to mine the mechanism by which RAD18 regulates the killing effect of NK cells on colorectal cancer (CRC) cells. Expression of E2F Transcription Factor 7 (E2F7) and RAD18 in CRC tissues, their correlation, binding sites, and RAD18 enrichment pathway were analyzed by bioinformatics. Expression of E2F7 and RAD18 in cells was assayed by qRT-PCR and western blot. Dual-luciferase assay and chromatin immunoprecipitation (ChIP) assay verified the regulatory relationship between E2F7 and RAD18. CCK-8 assay was utilized to assay cell viability, colony formation assay to detect cell proliferation, lactate dehydrogenase (LDH) test to assay NK cell cytotoxicity, ELISA to assay levels of granulocyte-macrophage colony-stimulating factor (GM-CSF), tumor necrosis factor-α (TNF-α) and interferon-γ (IFN-γ), and immunofluorescence to detect expression of toxic molecules perforin and granzyme B. High expression of RAD18 and E2F7 was found in CRC tissues and cells. Silencing RAD18 could hamper the proliferation of CRC cells, foster viability and cytotoxicity of NK cells, and increase the secretion of GM-CSF, TNF-α, IFN-γ as well as the expression of perforin and granzyme B. Additionally, ChIP and dual-luciferase reporter assay ascertained the binding relationship between RAD18 promoter region and E2F7. E2F7 could activate the transcription of RAD18, and silencing RAD18 reversed the inhibitory effect of E2F7 overexpression on NK cell killing. This work clarified the inhibitory effect of the E2F7/RAD18 axis on NK cell killing in CRC, and proffered a new direction for immunotherapy of CRC in targeted immune microenvironment.

ARID3A and ARID3B exert direct regulatory control over the long non-coding RNAs (lncRNAs) MALAT1 and NORAD within the context of non-small cell lung cancer (NSCLC)

Lung cancer, known for its high mortality rates and poor prognosis, remains one of the most prevalent cancer types. Early detection and effective treatment methods are crucial for improving survival rates. Non-small cell lung cancer (NSCLC) accounts for approximately 85 % of all lung cancer cases. Long non-coding RNAs (lncRNAs), which play vital roles in various biological processes, have been implicated in the development of cancer and can impact key therapeutic targets in different cancer types. In NSCLC, the dysregulation of specific lncRNAs, such as MALAT1 and NORAD, has been associated with neoplastic initiation, progression, metastasis, tumor angiogenesis, chemoresistance, and genomic instability. Both MALAT1 and NORAD directly regulate the expression of the transcription factor E2F1, thereby influencing cell cycle progression. Additionally, MALAT1 has been reported to affect the expression of p53 target genes, leading to cell cycle progression through the repression of p53 promoter activity. NORAD, on the other hand, is indirectly regulated by p53. The AT-rich interaction domain (ARID) family of DNA-binding proteins, particularly ARID3A and ARID3B, are involved in various biological processes such as cell proliferation, differentiation, and development. They also play significant roles in E2F-dependent transcription and are transcriptional targets of p53. The intricate balance between promoting cellular proliferation through the pRB-E2F pathway and inducing growth arrest through the p53 pathway underscores the crucial regulatory role of ARID3A, ARID3B, and their interaction with lncRNAs MALAT1 and NORAD. In this study, we aimed to investigate the potential interactive and functional connections among ARID3A, ARID3B, MALAT1, and NORAD in NSCLC, considering their involvement in the pRB-E2F and p53 pathways. Our findings strongly suggest that ARID3A and ARID3B play a regulatory role in controlling MALAT1 and NORAD in NSCLC. Specifically, our study demonstrates that the activities of MALAT1 and NORAD were markedly increased upon the overexpression of ARID3A and ARID3B. Therefore, we can conclude that ARID3A and ARID3B likely contribute significantly to the oncogenic functions of MALAT1 and NORAD in NSCLC. Consequently, targeting ARID3A and ARID3B could hold promise as a therapeutic approach in NSCLC, given their direct control over the expression of MALAT1 and NORAD.

Phenotypic and proteomic characterization of the human erythroid progenitor continuum reveal dynamic changes in cell cycle and in metabolic pathways.

Human erythropoiesis is a complex process leading to the production of 2.5 million red blood cells per second. Following commitment of hematopoietic stem cells to the erythroid lineage, this process can be divided into three distinct stages: erythroid progenitor differentiation, terminal erythropoiesis, and reticulocyte maturation. We recently resolved the heterogeneity of erythroid progenitors into four different subpopulations termed EP1-EP4. Here, we characterized the growth factor(s) responsiveness of these four progenitor populations in terms of proliferation and differentiation. Using mass spectrometry-based proteomics on sorted erythroid progenitors, we quantified the absolute expression of ~5500 proteins from EP1 to EP4. Further functional analyses highlighted dynamic changes in cell cycle in these populations with an acceleration of the cell cycle during erythroid progenitor differentiation. The finding that E2F4 expression was increased from EP1 to EP4 is consistent with the noted changes in cell cycle. Finally, our proteomic data suggest that the protein machinery necessary for both oxidative phosphorylation and glycolysis is present in these progenitor cells. Together, our data provide comprehensive insights into growth factor-dependence of erythroid progenitor proliferation and the proteome of four distinct populations of human erythroid progenitors which will be a useful framework for the study of erythroid disorders.

Mesenchymal stem cell-regulated miRNA-mRNA landscape in acute-on-chronic liver failure

BackgroundAcute-on-chronic liver failure (ACLF) is a major challenge in the field of hepatology. While mesenchymal stem cell (MSC) therapy can improve the prognosis of patients with ACLF, the molecular mechanisms through which MSCs attenuate ACLF remain poorly understood. We performed global miRNA and mRNA expression profiling via next-generation sequencing of liver tissues from MSC-treated ACLF mice to identify important signaling pathways and major factors implicated in ACLF alleviation by MSCs. MethodsCarbon tetrachloride-induced ACLF mice were treated with saline or mouse bone marrow-derived MSCs. Mouse livers were subjected to miRNA and mRNA sequencing. Related signal transduction pathways were obtained through Gene Set Enrichment Analysis. Functional enrichment, protein-protein interaction, and immune infiltration analyses were performed for the differentially expressed miRNA target genes (DETs). Hub miRNA and mRNA associated with liver injury were analyzed using LASSO regression. The expression levels of hub genes were subjected to Pearson's correlation analysis and verified using RT-qPCR. The biological functions of hub genes were verified in vitro. ResultsThe tricarboxylic acid cycle and peroxisome proliferator-activated receptor pathways were activated in the MSC-treated groups. The proportions of liver-infiltrating NK resting cells, M2 macrophages, follicular helper T cells, and other immune cells were altered after MSC treatment. The expression levels of six miRNAs and 10 transcripts correlated with the degree of liver injury. miR-27a-5p was downregulated in the mouse liver after MSC treatment, while its target gene E2f2 was upregulated. miR-27a-5p inhibited E2F2 expression, suppressed G1/S phase transition and proliferation of hepatocytes, in addition to promoting their apoptosis. ConclusionsThis is the first comprehensive analysis of miRNA and mRNA expression in the liver tissue of ACLF mice after MSC treatment. The results revealed global changes in hepatic pathways and immune subpopulations. The miR-27a-5p/E2F2 axis emerged as a central regulator of the MSC-induced attenuation of ACLF. The current findings improve our understanding of the molecular mechanisms through which MSCs alleviate ACLF.

E2F1-mediated Up-regulation of NCAPG Promotes Hepatocellular Carcinoma Development by Inhibiting Pyroptosis.

As a subunit of the condensin complex, NCAPG has an important role in maintaining chromosome condensation, but its biological function and regulatory mechanism in hepatocellular carcinoma (HCC) remains undefined. The prognostic ability of NCAPG in HCC patients was examined by univariate and multivariate Cox regression analysis. ROC curves were plotted to compare the predictive ability of NCAPG and AFP. Double luciferase reporter system, and ChIP were used to investigate transcriptional potential of E2F1 to NCAPG. Pyroptosis was observed by scanning electron microscopy. Protein expression of NCAPG, E2F1, and major proteins constituting NLRP3 inflammasome was determined by western blotting and ELISA. An in vivo tumor formation assay was conducted to verify the in vitro results. Up-regulated NCAPG was identified in HCC tissues compared with adjacent tissue and high NCAPG was positively correlated with poor prognosis. Serum NCAPG mRNA level was a prognostic factor in HCC patients and also a diagnostic factor with higher predictive ability compared with AFP [AUROC 0.766 (95% CI: 0.650-0.881) vs. 0.649 (95% CI 0.506-0.793)]. HBx transfection resulted in concomitant up-regulation of E2F1 and NCAPG. E2F1 significantly increased the activity of luciferase reporter fused with NCAPG reporter, and the interaction of E2F1 and NCAPG gene was confirmed by ChIP. Silencing of E2F1 resulted in significant down-regulation of NCAPG. Knockdown of NCAPG promote pyroptosis mediated by NLRP3 inflammasome activation in multiple HCC cell lines and also suppressed tumorigenesis in vitro. We identified a novel role of NCAPG in the regulation of NLRP3 inflammasome-mediated pyroptosis, which was regulated by its upstream transactivator, E2F1. The role of E2F1-NCAPG-NLRP3 regulation of pyroptosis network may be a potential target in HCC treatment.

Attenuation of binuclear hepatocytes in the paracancerous liver tissue is associated with short-term recurrence of hepatocellular carcinoma post-radical surgery.

Short-term recurrence of hepatocellular carcinoma (HCC) after radical resection leads to dismal outcomes. To screen high-recurrence risk patients to provide adjuvant treatment is necessary. Herein, based on our previous research, we further focused on the changes in the abundance of binuclear hepatocytes (ABH) in the paracancerous liver tissue to discuss the relationship between the attenuation of binuclear hepatocytes and postoperative short-term recurrence, by combining with the assessment of the value of a reported independent early recurrence risk factor in HCC, protein induced by vitamin K absence or antagonist-II (PIVKA-II). A cohort of 142 paracancerous liver tissues from HCC patients who received radical resection was collected. Binuclear hepatocytes were reduced in the paracancerous liver tissues, compared with the liver tissues from normal donors. ABH was negatively correlated with clinical features such as tumor size, TNM stages, tumor microsatellite formation, venous invasion, and Alpha-fetoprotein (AFP) level, as well as the expression of E2F7 and Anillin, which are two critical regulators concerning the hepatocyte polyploidization. According to the short-term recurrence information, ABH value was laminated, and univariate and multivariate logistic regression was performed to analyze the relationship between paracancerous ABH and short-term tumor relapse. Simultaneously, the predictive effectiveness of the ABH value was compared with the preoperative PIVKA-II value. As observed, the paracancerous ABH value below 1.5% was found to be an independent risk factor for recurrence. In conclusion, the paracancerous ABH is a credible indicator of short-term recurrence of HCC patients after radical resection, and regular assessment of ABH might help to prevent short-term HCC recurrence.

Cyclosporin A inhibits prostate cancer growth through suppression of E2F8 transcription factor in a MELK‑dependent manner.

The treatment of advanced prostate cancer remains a formidable challenge due to the limited availability of effective treatment options. Therefore, it is imperative to identify promising druggable targets that provide substantial clinical benefits and to develop effective treatment strategies to overcome therapeutic resistance. Cyclosporin A (CsA) showed an anticancer effect on prostate cancer in cultured cell and xenograft models. E2F8 was identified as a master transcription factor that regulated a clinically significant CsA specific gene signature. The expression of E2F8 increased during prostate cancer progression and high levels of E2F8 expression are associated with a poor prognosis in patients with prostate cancer. MELK was identified as a crucial upstream regulator of E2F8 expression through the transcriptional regulatory network and Bayesian network analyses. Knockdown of E2F8 or MELK inhibited cell growth and colony formation in prostate cancer cells. High expression levels of E2F8 and androgen receptor (AR) are associated with a worse prognosis in patients with prostate cancer compared with low levels of both genes. The inhibition of E2F8 improved the response to AR blockade therapy. These results suggested that CsA has potential as an effective anticancer treatment for prostate cancer, while also revealing the oncogenic role of E2F8 and its association with clinical outcomes in prostate cancer. These results provided valuable insight into the development of therapeutic and diagnostic approaches for prostate cancer.

The Integration of Genome-Wide DNA Methylation and Transcriptomics Identifies the Potential Genes That Regulate the Development of Skeletal Muscles in Ducks.

DNA methylation is a pivotal epigenetic regulatory mechanism in the development of skeletal muscles. Nonetheless, the regulators responsible for DNA methylation in the development of embryonic duck skeletal muscles remain unknown. In the present study, whole genome bisulfite sequencing (WGBS) and transcriptome sequencing were conducted on the skeletal muscles of embryonic day 21 (E21) and day 28 (E28) ducks. The DNA methylation pattern was found to fall mainly within the cytosine-guanine (CG) context, with high methylation levels in the intron, exon, and promoter regions. Overall, 7902 differentially methylated regions (DMRs) were identified, which corresponded to 3174 differentially methylated genes (DMGs). By using integrative analysis of both WGBS with transcriptomics, we identified 1072 genes that are DMGs that are negatively associated with differentially expressed genes (DEGs). The gene ontology (GO) analysis revealed significant enrichment in phosphorylation, kinase activity, phosphotransferase activity, alcohol-based receptors, and binding to cytoskeletal proteins. The Kyoto Encyclopedia of Genes and Genomes (KEGGs) analysis showed significant enrichment in MAPK signaling, Wnt signaling, apelin signaling, insulin signaling, and FoxO signaling. The screening of enriched genes showed that hyper-methylation inhibited the expression of Idh3a, Got1, Bcl2, Mylk2, Klf2, Erbin, and Klhl38, and hypo-methylation stimulated the expression of Col22a1, Dnmt3b, Fn1, E2f1, Rprm, and Wfikkn1. Further predictions showed that the CpG islands in the promoters of Klhl38, Klf2, Erbin, Mylk2, and Got1 may play a crucial role in regulating the development of skeletal muscles. This study provides new insights into the epigenetic regulation of the development of duck skeletal muscles.

E2F8 exerts cancer-promoting effects by transcriptionally activating RRM2 and E2F8 knockdown synergizes with WEE1 inhibition in suppressing lung adenocarcinoma

Ribonucleotide reductase (RR) is a rate-limiting enzyme that facilitates DNA replication and repair by reducing nucleotide diphosphates (NDPs) to deoxyribonucleotide diphosphates (dNDPs) and is thereby crucial for cell proliferation and cancer development. The E2F family of transcription factors includes key regulators of gene expression involved in cell cycle control. In this study, E2F8 expression was significantly increased in most cancer tissues of lung adenocarcinoma (LUAD) patients and was correlated with the expression of RRM2 through database and clinical samples analysis. The protein expression of E2F8 and RRM2 were positively correlated with tumor–node–metastasis (TNM) pathological stage, and high expression of E2F8 and RRM2 predicted a low 5-year overall survival rate in LUAD patients. Overexpression and knockdown experiments showed that E2F8 was essential for LUAD cell proliferation, DNA synthesis, and cell cycle progression, which were RRM2-dependent. Reporter gene, ChIP-qPCR, and DNA pulldown–Western blot assays indicated that E2F8 activated the transcription of the RRM2 gene by directly binding with the RRM2 promoter in LUAD cells. Previous studies indicated that inhibition of WEE1 kinase can suppress the phosphorylation of CDK1/2 and promote the degradation of RRM2. We further showed here that the combination of E2F8 knockdown with MK-1775, an inhibitor of WEE1 being evaluated in clinical trials, synergistically suppressed proliferation and promoted apoptosis of LUAD cells in vitro and in vivo. Thus, this study reveals a novel role of E2F8 as a proto-oncogenic transcription activator by activating RRM2 expression in LUAD, and targeting both the transcription and degradation mechanisms of RRM2 could produce a synergistic inhibitory effect for LUAD treatment in addition to conventional inhibition of RR enzyme activity.

Silencing geranylgeranyltransferase I inhibits the migration and invasion of salivary adenoid cystic carcinoma through RhoA/ROCK1/MLC signaling and suppresses proliferation through cell cycle regulation.

Geranylgeranyltransferase type I (GGTase-I) significantly affects Rho proteins, such that the malignant progression of several cancers may be induced. Nevertheless, the effect and underlying mechanism of GGTase-I in the malignant progression of salivary adenoid cystic carcinoma (SACC) remain unclear. This study primarily aimed to investigate the role and mechanism of GGTase-I in mediating the malignant progression of SACC. The level of GGTase-I gene in cells was stably knocked down by short hairpin RNA-EGFP-lentivirus. The effects of GGTase-I silencing on the migration, invasion, and spread of cells were examined, the messenger RNA levels of GGTase-I and RhoA genes of SACC cells after GGTase-I knockdown were determined, and the protein levels of RhoA and RhoA membrane of SACC cells were analyzed. Moreover, the potential underlying mechanism of silencing GGTase-I on the above-mentioned aspects in SACC cells was assessed by examining the protein expression of ROCK1, MLC, p-MLC, E-cadherin, Vimentin, MMP2, and MMP9. Furthermore, the underlying mechanism of SACC cells proliferation was investigated through the analysis of the expression of cyclinD1, MYC, E2F1, and p21CIP1/WAF1 . Besides, the change of RhoA level in SACC tissues compared with normal paracancer tissues was demonstrated through quantitative reverse-transcription polymerase chain reaction and western blot experiments. Next, the effect after GGTase-I silencing was assessed through the subcutaneous tumorigenicity assay. As indicated by the result of this study, the silencing of GGTase-I significantly reduced the malignant progression of tumors in vivo while decreasing the migration, invasion, and proliferation of SACC cells and RhoA membrane, Vimentin, ROCK1, p-MLC, MMP2, MMP9, MYC, E2F1, and CyclinD1 expression. However, the protein expression of E-cadherin and p21CIP1/WAF1 was notably upregulated. Subsequently, no significant transform of RhoA and MLC proteins was identified. Furthermore, RhoA expression in SACC tissues was significantly higher than that in paracancerous tissues. As revealed by the results of this study, GGTase-I shows a correlation with the proliferation of SACC through the regulation of cell cycle and may take on vital significance in the migration and invasion of SACC by regulating RhoA/ROCK1/MLC signaling pathway. GGTase-I is expected to serve as a novel exploration site of SACC.

LINC01980 induced by TGF-beta promotes hepatocellular carcinoma metastasis via miR-376b-5p/E2F5 axis

Hepatocellular carcinoma (HCC) is one of the most aggressive human malignancies worldwide. However, the molecular mechanism of HCC metastasis is largely unknown. Long non-coding RNA (lncRNA) plays a key role in gene regulation, and dysregulation of lncRNA is critical to cancer metastasis. LINC01980 has been reported in ESCC recently, but the mechanism underlying its function in HCC is still unknown. In this study, we found that LINC01980 was upregulated and associated with notably poor overall survival in HCC patients. Functionally, LINC01980 played a carcinogenic role and promoted HCC metastasis. Mechanically, LINC01980 enhanced the E2F5 expression via competitively binding miR-376b-5p, thereby inducing epithelial-mesenchymal transition and promoting HCC cells migration and invasion. In addition, LINC01980-mediated HCC cells metastasis was dependent on E2F5. What's more, TGF-β activated LINC01980 transcription through the canonical TGF-β/SMAD signaling pathway in HCC. In conclusion, LINC01980, activated by the canonical TGF-β/SMAD pathway, promoted HCC metastasis via miR-376b-5p/E2F5 axis. Therefore, LINC01980 might be a potential prognostic biomarker and therapeutic target of HCC.

Targeting the fibroblast growth factor pathway in molecular subtypes of castration-resistant prostate cancer.

Androgen receptor (AR) pathway inhibition remains the cornerstone for prostate cancer therapies. However, castration-resistant prostate cancer (CRPC) tumors can resist AR signaling inhibitors through AR amplification and AR splice variants in AR-positive CRPC (ARPC), and conversion to AR-null phenotypes, such as double-negative prostate cancer (DNPC) and small cell or neuroendocrine prostate cancer (SCNPC). We have shown previously that DNPC can bypass AR-dependence through fibroblast growth factor receptor (FGFR) signaling. However, the role of the FGFR pathway in other CRPC phenotypes has not been elucidated. RNA-Seq analysis was conducted on patient metastases, LuCaP patient-derived xenograft (PDX) models, and CRPC cell lines. Cell lines (C4-2B, VCaP, and 22Rv1) and ex vivo LuCaP PDX tumor cells were treated with enzalutamide (ENZA) and FGFR inhibitors (FGFRi) alone or in combination and sensitivity was determined using cell viability assays. In vivo efficacy of FGFRi in ARPC, DNPC, and SCNPC were evaluated using PDX models. RNA-Seq analysis of FGFR signaling in metastatic specimens, LuCaP PDX models, and CRPC cell lines revealed significant FGF pathway activation in AR-low PC (ARLPC), DNPC, and SCNPC tumors. In vitro/ex vivo analysis of erdafitinib and CH5183284 demonstrated robust and moderate growth suppression of ARPC, respectively. In vivo studies using four ARPC PDX models showed that combination ENZA and CH5183284 significantly suppressed tumor growth. Additional in vivo studies using four ARPC PDX models revealed that erdafitinib monotherapy was as effective as ENZA in suppressing tumor growth, and there was limited combination benefit. Furthermore, two of three DNPC models and two of four SCNPC models responded to CH5183284 monotherapy, suggesting FGFRi responses were model dependent. RNA-Seq and gene set enrichment analysis of end-of-study ARPC tumors treated with FGFRi displayed decreased expression of E2F and MYC target genes and suppressed G2M checkpoint genes, whereas end-of-study SCNPC tumors had heterogeneous transcriptional responses. Although FGFRi treatments suppressed tumor growth across CRPC phenotypes, our analyses did not identify a single pathway or biomarker that would identify tumor response to FGFRi. This is very likely due to the array of FGFR1-4 expression and tumor phenotypes present in CRPC. Nevertheless, our data nominate the FGFR pathway as a clinically actionable target that promotes tumor growth in diverse phenotypes of treatment-refractory metastatic CRPC.

Down-regulation of human papillomavirus E6 oncogene and antiproliferative effect of Schisandra chinensis and Pueraria lobata natural extracts on Hela cell line

Ethnopharmacological relevanceCervical cancer is one of the most common malignancies in women that continues to be a public health problem worldwide. Human papillomavirus (HPV) infection is closely related as the causative agent of almost all cases of cervical cancer. Currently, there is no effective treatment for the persistence of HPV. Although vaccines have shown promising results in recent years, they are still a costly strategy for developing countries and have no therapeutic effect on existing infections, which is why the need arises to search for new strategies that can be used in treatment, suppressing oncogenic HPV and disease progression.Extracts of Schisandra Chinensis and Pueraria lobata have been used in traditional medicine, and it has been shown in recent years that some of their bioactive compounds have pharmacological, antioxidant, antitumor, apoptotic, and proliferation effects in HPV-positive cells. However, its mechanism of action has yet to be fully explored. Aim of the studyThe following study aimed to determine the chemical composition, antioxidant activity, and potential antiproliferative and viral oncogene effects of natural extracts of S. chinensis and P. lobata on HPV-18 positive cervical cancer cells. Materials and methodsThe HPV-18-positive HeLa cells were treated for 24 and 48 h with the ethanolic extracts of S chinensis and P. lobata. Subsequently, cell viability was evaluated using the resazurin method, the effect on the cell cycle of the extracts (1.0, 10, and 100 μg/mL) was measured by flow cytometry, the gene of expression of the E6/E7, P53, BCL-2, and E2F-1 were determined by RT-PCR and the protein expression of p53, Ki-67, x|and Bcl-2 by immunohistochemistry. Additionally, the chemical characterization of the two extracts was carried out using LC-MS, and the total phenolics content (TPC), Total flavonoid content (TFC), and DPPH radical scavenging capacity were determined. Data were analyzed using the Mann-Whitney and Kruskal Wallis U test with GraphPad Prism 6 software. ResultsThe natural extracts of Schisandra chinensis and Pueraria lobata induced down-regulation of E6 HPV oncogene (p<0.05) and a strong up-regulation of P53 (p<0.05), E2F-1 (p<0.05), and Bcl-2 (p<0.05) gene expression. Simultaneously, the natural extracts tend to increase the p53 protein levels and arrest the cell cycle of HeLa in the G1/S phase (p<0.05). Investigated extracts were characterized by the occurrence of bioactive lignans and isoflavones in S. chinensis and P. lobata, respectively. ConclusionThe extracts of S. chinensis and P. lobata within their chemical characterization mainly present lignan and isoflavone-type compounds, which are probably responsible for inhibiting the expression of the HPV E6 oncogene and inducing an increase in the expression of p53, Bcl -2 and E2F-1 producing cell cycle detection in S phase in HeLa cells. Therefore, these extracts are good candidates to continue studying their antiviral and antiproliferative potential in cells transformed by HPV.

MiR-210-3p enhances intermittent hypoxia-induced tumor progression via inhibition of E2F3.

Intermittent hypoxia (IH) is a hallmark of obstructive sleep apnea (OSA), which is related to tumorigenesis and progression. Although micro-ribonucleic acid-210-3p (miR-210-3p) is correlated with hypoxia-induced tumor development, its role in the relationship between IH and tumor function remains poorly understood. The present work focused on elucidating the molecular mechanism through which miR-210-3p drives tumor progression under IH. MiR-210-3p levels were quantified within tumor samples frompatients with lung adenocarcinoma who had or did not haveOSA. Correlations between miR-210-3p and polysomnographic variables were analyzed. For in vitro experiments, miR-210-3p was inhibited or overexpressed via transfection under IH conditions. Cell viability, growth, invasion and migration assays were carried out. For in vivo modeling of IH using mouse xenografts, a miR-210-3p antagomir was intratumorally injected, tumor biological behaviors were evaluated, and reverse transcription-quantitative polymerase chain reaction (RT-qPCR), immunohistochemistry and western blot were carried out for detecting miR-210-3p and E2F transcription factor 3 (E2F3) expression. For patients withlung adenocarcinoma and OSA, high miR-210-3p levels showed positive relation to polysomnographic variables, such as oxygen desaturation index, apnea-hypopnea index, and proportion of total sleep time with oxygen saturation in arterial blood < 90%. IH enhanced tumor viability, proliferation, migration, and invasion, downregulated E2F3 expression, and increased miR-210-3-p levels. miR-210-3p overexpression induced similar changes. These changes were reversed by miR-210-3p inhibition in vitro or miR-210-3p antagomir through intratumoral injection in vivo. IH-induced tumor development is driven through miR-210-3p by E2F3 suppression. MiR-210-3p represents a potential therapeutic target among patients with concomitant cancer and OSA.

ZNF500 abolishes breast cancer proliferation and sensitizes chemotherapy by stabilizing P53 via competing with MDM2.

Zinc finger protein 500 (ZNF500) has an unknown expression pattern and biological function in human tissues. Our study revealed that the ZNF500 mRNA and protein levels were higher in breast cancer tissues than those in their normal counterparts. However, ZNF500 expression was negatively correlated with advanced TNM stage (p = 0.018), positive lymph node metastasis (p = 0.014), and a poor prognosis (p < 0.001). ZNF500 overexpression abolished in vivo and in vitro breast cancer cell proliferation by activating the p53-p21-E2F4 signaling axis and directly interacting with p53 via its C2H2 domain. This may prevent ubiquitination of p53 in a manner that is competitive to MDM2, thus stabilizing p53. When ZNF500-∆C2H2 was overexpressed, the suppressed proliferation of breast cancer cells was neutralized in vitro and in vivo. In human breast cancer tissues, ZNF500 expression was positively correlated with p53 (p = 0.022) and E2F4 (p = 0.004) expression. ZNF500 expression was significantly lower in patients with Miller/Payne Grade 1-2 than in those with Miller/Payne Grade 3-5 (p = 0.012). ZNF500 suppresses breast cancer cell proliferation and sensitizes cells to chemotherapy.