E2F Proteins Research Articles

Cancer Cell enters reversible quiescence through Intracellular Acidification to resist Paclitaxel Cytotoxicity.

Cancer cells can enter quiescent or dormant state to resist anticancer agents while maintaining the potential of reactivation. However, the molecular mechanism underlying quiescence entry and reactivation remains largely unknown. In this paper, cancer cells eventually entered a reversible quiescent state to resist long-term paclitaxel (PTX) stress. The quiescent cells were characterized with Na+/H+ exchanger 1 (NHE1) downregulation and showed acidic intracellular pH (pHi). Accordingly, decreasing pHi by NHE1 inhibitor could induce cell enter quiescence. Further, acidic pHi could activate the ubiquitin-proteasome system and inhibiting proteasome activity by MG132 prevented cells entering quiescence. In addition, we show that after partial release, the key G1-S transcription factor E2F1 protein level was not recovered, while MCM7 protein returned to normal level in the reactivated cells. More importantly, MCM7 knockdown inhibited G1/S genes transcription and inhibited the reactivated proliferation. Taken together, this study demonstrates a regulatory function of intracellular acidification and subsequent protein ubiquitination on quiescence entry, and reveals a supportive effect of MCM7 on the quiescence-reactivated proliferation.

A Novel Expression Profile of Cell Cycle and DNA Repair Proteins in Nonfunctioning Pituitary Adenomas.

The molecular mechanisms underlying the formation of nonfunctioning pituitary adenomas (NFAs) are largely unknown. In this study, we aimed to understand the relationship between NFAs and functional pituitary adenomas and the possible role of proteins involved in cell cycle, senescence, and DNA damage control mechanisms in the etiology of NFA. We analyzed pATM-S1981, pRb-S608, Rb, pE2F1-S364, p16, E2F1, p73, cyclin D1, and CHEK2 protein expression (in a group of 20 patients with acromegaly, 18 patients with Cushing's disease (CD), and 29 NFA patients) by immunohistochemistry and their relevant mRNA expression by qRT-PCR (in a group of 7 patients with acromegaly, 7 patients with CD, and 7 NFA patients). The clinical and histopathological results on the patients were statistically evaluated. pE2F1-S364 protein expression in the CD group was significantly lower than that in the NFA and acromegaly groups (p = 0.025, p = 0.034, respectively). However, the expression of the p16 protein was lower than in the NFA group than in the CD and acromegaly groups (p = 0.030, p = 0.033, respectively), and E2F1 protein expression was significantly higher in the NFA group than in the CD group (p = 0.025). p73 protein expression in patients with acromegaly was significantly higher (p = 0.031) than that in the CD group. CHEK2 mRNA expression in the CD group was significantly higher than that in the acromegaly group (p = 0.012). The selective and tumor-specific associations between E2F1, pE2F1-S364, CHEK2, and p73 mRNA and protein levels indicate their involvement in pituitary adenoma formation in NFA, CD, and acromegaly patients.

Reciprocal regulation of miR-1205 and E2F1 modulates progression of laryngeal squamous cell carcinoma

The burgeoning functions of many microRNAs (miRs) have been well study in cancer. However, the level and function of miR-1205 in laryngeal squamous cell cancer remains unknown. In the current research, we validated that miR-1205 was notably downregulated in human laryngeal squamous cell carcinoma (LSCC) samples in comparison with tissues adjacent to LSCC, and correlated with T stage, lymph node metastasis, and clinical stage. Using Kaplan–Meier analysis indicates that high expression of miR-1205 has a favorable prognosis for patients with LSCC. Functional assays show that enforced miR-1205 expression attenuates the migration, growth, and invasion of LSCC cells. And E2F1 is verified to be a target of miR-1205, while E2F1 binds to miR-1205 promoter and transcriptionally inhibits miR-1205 expression. Overexpression of E2F1 reverses the inhibitory impacts of miR-1205 on LSCC cells in part. Importantly, E2F1 is abnormally increased in LSCC tissues, and its protein levels were inversely relevant to miR-1205 expression. High E2F1 protein level is in connection with clinical stage, T stage, lymph node metastasis, and poor prognosis. Consequently, reciprocal regulation of miR-1205 and E2F1 plays a crucial role in the progression of LSCC, suggesting a new miR-1205/E2F1-based clinical application for patients of LSCC.

Deconstructing the Clonal Advantage and Clonal Stability of 5q- Candidate Genes in Del(5q) MDS on a Single Cell Level

Hematopoietic Stem/Progenitor cells (HSPCs) with 5q haploinsufficiency in del(5q) myelodysplastic syndrome (MDS) acquire a clonal advantage in the bone marrow and out-compete normal hematopoiesis. A critical, yet unsolved question remains: how does genetic haploinsufficiency in del(5q) cells contribute to the clonal advantage of HSPCs? We investigated the role of haploinsufficiency for three candidate genes in the common deleted region on chromosome 5 (Csnk1a1, Egr1 and Apc) in direct competition with each other and wild-type (wt) cells on a single cell level by employing a novel lentiviral genetic barcoding strategy. We introduced genotype and cell-specific barcodes into HSPCs from murine models haploinsufficient for Csnk1a1, Egr1 or Apc. Barcoded HSPCs were sort-purified, genotypes mixed and subsequently competitively transplanted into lethally irradiated mice and re-transplanted after 16 weeks in a secondary transplant. The barcoded progeny was reliably recovered from peripheral blood and relative contribution of the barcoded clones to differentiated blood lineages was followed over 32 weeks. Despite heterogeneity in clonal evolution among the mice, all haploinsufficient clones had the potential to outcompete wt clones (3 of 5 mice in the primary transplant, 3 out of 4 mice in the secondary transplant). Csnk1a1 haploinsufficient clones showed the largest clonal abundance and clonal persistence. Expansion of oligoclonal Csnk1a1 haploinsufficient HSPCs was further enhanced in the secondary transplant in all mice. Egr1 haploinsufficient clones showed potential for prominent oligoclonal expansion in one mouse, but decreased in abundance in all secondary transplants. Apc haploinsufficient clones showed persistence but not expansion in 3 out of 5 mice. These results were validated by conventional competitive transplants, which demonstrated that Csnk1a1 and Egr1 haploinsufficient cells achieved the highest advantage over wt hematopoiesis in the primary transplant and more enhanced in the secondary transplant. Since Csnk1a1 regulates β-catenin protein stability, we hypothesized that the clonal expansion of Csnk1a1 haploinsufficient HSPCs is dependent on β-catenin levels. We performed a second genetic barcoding competitive transplant, comparing Csnk1a1-/+ HSPC directly to double haploinsufficient Csnk1a1-/+/Ctnnb1-/+ (β-catenin encoding) HSPCs. We included additional double haploinsufficient mutants Csnk1a1-/+/Apc-/+ and Csnk1a1-/+/Egr1-/+. Results showed pronounced expansion of Csnk1a1-/+ clones, while Csnk1a1-/+/Ctnnb1-/+ clones were outcompeted over time, suggesting that the advantage of Csnk1a1-/+ clones is β-catenin dependent. Csnk1a1-/+/Egr1-/+ and Csnk1a1-/+/Apc-/+ clones were less advantageous than Csnk1a1-/+ clones. To further investigate the mechanism of clonal fitness in Csnk1a1-/+ haploinsufficient HSPCs, we performed droplet based single cell RNA sequencing of Csnk1a1-/+ and wt Lin-Sca1+cKit+ (LSK) HSPCs. Csnk1a1 -/+ LSK were characterized by a higher fraction of cells expressing cell cycle genes compared to wt cells. In line, transcriptional alterations in the most primitive HSCs suggest that the clonal advantage is conveyed by canonical Wnt signaling activating downstream targets such E2F proteins. Csnk1a1-/+ haploinsufficient multipotent progenitors and myeloid/lymphoid primed progenitors expressed marked upregulation of metabolic pathways, mitochondrial respiration, cell cycle and differentiation, ubiquitination/proteasome system and deregulation of ribosome biogenesis. In conclusion, we demonstrate using a novel genetic barcoding approach in a competitive transplant setting that Csnk1a1-/+ haploinsufficient HSPCs have the potential for oligoclonal expansion and clonal persistence. Wnt/β-catenin signaling plays a central role in the clonal expansion. Interestingly, in Csnk1a1 haploinsufficiency the HSC state is preserved and the increased proliferation and metabolic activation are hallmark features of differentiating progenitor cells at MPP stage, increasing with cell cycle activation, thus ensuring clonal stability and preventing HSC exhaustion over time. Disclosures Brümmendorf: University Hospital of the RWTH Aachen: Employment; Janssen: Consultancy; Pfizer: Consultancy, Research Funding; Merck: Consultancy; Novartis: Consultancy, Research Funding; Ariad: Consultancy. Ebert:Celgene: Research Funding; Deerfield: Research Funding; Broad Institute: Other: Contributor to a patent filing on this technology that is held by the Broad Institute..

The long noncoding RNA lncNB1 promotes tumorigenesis by interacting with ribosomal protein RPL35

The majority of patients with neuroblastoma due to MYCN oncogene amplification and consequent N-Myc oncoprotein over-expression die of the disease. Here our analyses of RNA sequencing data identify the long noncoding RNA lncNB1 as one of the transcripts most over-expressed in MYCN-amplified, compared with MYCN-non-amplified, human neuroblastoma cells and also the most over-expressed in neuroblastoma compared with all other cancers. lncNB1 binds to the ribosomal protein RPL35 to enhance E2F1 protein synthesis, leading to DEPDC1B gene transcription. The GTPase-activating protein DEPDC1B induces ERK protein phosphorylation and N-Myc protein stabilization. Importantly, lncNB1 knockdown abolishes neuroblastoma cell clonogenic capacity in vitro and leads to neuroblastoma tumor regression in mice, while high levels of lncNB1 and RPL35 in human neuroblastoma tissues predict poor patient prognosis. This study therefore identifies lncNB1 and its binding protein RPL35 as key factors for promoting E2F1 protein synthesis, N-Myc protein stability and N-Myc-driven oncogenesis, and as therapeutic targets.

E2F1 acetylation directs p300/CBP-mediated histone acetylation at DNA double-strand breaks to facilitate repair

E2F1 and retinoblastoma (RB) tumor-suppressor protein not only regulate the periodic expression of genes important for cell proliferation, but also localize to DNA double-strand breaks (DSBs) to promote repair. E2F1 is acetylated in response to DNA damage but the role this plays in DNA repair is unknown. Here we demonstrate that E2F1 acetylation creates a binding motif for the bromodomains of the p300/KAT3B and CBP/KAT3A acetyltransferases and that this interaction is required for the recruitment of p300 and CBP to DSBs and the induction of histone acetylation at sites of damage. A knock-in mutation that blocks E2F1 acetylation abolishes the recruitment of p300 and CBP to DSBs and also the accumulation of other chromatin modifying activities and repair factors, including Tip60, BRG1 and NBS1, and renders mice hypersensitive to ionizing radiation (IR). These findings reveal an important role for E2F1 acetylation in orchestrating the remodeling of chromatin structure at DSBs to facilitate repair.

Circulating miR-200a is a novel molecular biomarker for early-stage renal cell carcinoma

BackgroundCirculating microRNAs (miRNAs) have been recognized as novel molecular markers for renal cell carcinoma (RCC) diagnosis. However, the clinical impact of circulating miRNA has rarely been evaluated for the early detection of RCC. Moreover, miRNAs also play important roles in regulating RCC tumorigenesis. The aim of this study is to assess the serum signatures of the epithelial–mesenchymal transition (EMT)-related miRNA-200 family in RCC patients and evaluate their diagnostic usefulness for early-stage RCC, and further explore the functions of specifically altered miRNAs that regulate pathological genes during the RCC tumorigenesis.MethodsThe serum levels of three miR-200 miRNAs (miR-200a, miR-200b and miR-200c) were firstly measured in 26 RCC patients and 26 noncancer controls of the training phase using quantitative real-time polymerase chain reaction (RT-qPCR). Markedly dysregulated miRNAs in RCC patients were subsequently verified by RT-qPCR in an independent validation phase that consist of 73 patients and 73 controls. Differentially expressed miRNAs were further examined in RCC urine samples and corresponding controls. Additionally, the mechanisms underlying the altered miRNAs involved in RCC tumorigenesis were also elucidated.ResultsOf the three examined miRNAs, only miR-200a showed consistently decreased levels in serum of the RCC patients, especially in those with stage I disease, as compared with controls (P < 0.001). MiR-200a is also frequently downregulated in RCC urine samples. Mechanistic investigations revealed that E2F3, an oncogene with strong proliferative and cell cycle regulation potential, is a direct target gene of miR-200a in RCC. E2F3 protein levels in RCC tissues and cell lines were increased and inversely associated with miR-200a levels. Upregulation of miR-200a decreased the expression of E2F3, with consequent repressed RCC cell proliferation and induced cell cycle arrest at G0/G1 phase in vitro. In contrast, inhibition of miR-200a in RCC cells resulted in increased expression of E2F3 and enhanced cell proliferation, and promoted G1/S transition.ConclusionsSerum miR-200a has the potential to be served as a novel noninvasive auxiliary diagnostic biomarker for the early detection of RCC, and the identified novel molecular interaction between miR-200a and E2F3 may provide insights into novel therapies for RCC.

Phenotypic transition of tumor cells between epithelial- and mesenchymal-like state during adaptation to acidosis

ABSTRACT Acidic microenvironment is an important feature of solid tumors that contributes to malignant transformation. Low extracellular pH could promote epithelial-mesenchymal transition (EMT) thereby endowing tumor cells with higher invasive capability. However, the relation between EMT and tumor cell proliferation under long-term acidic condition is still not fully understood. Here, we show that tumor cells have undergone a phenotypic transition from EMT to mesenchymal-epithelial transition (MET) during adaptation to acidosis, and is closely related with cell proliferative state. Under early stage of acidic stress, tumor cells entered a non-cycling quiescent state with mesenchymal phenotype and expressed high level of stemness genes. Whereas, after long-term acid culture (2 months), acid-adapted cells resumed proliferating but lost mesenchymal phenotype. Further, our results show that the acid-adapted cells have distinct proliferative mechanism from non-acid cells, as the G1-S transcriptional factor E2F1 protein was not recovered in the adapted cells. Meanwhile, mini-chromosome maintenance 7 (MCM7) is shown to regulate the EMT to MET phenotypic transition, and is required for proliferation of the adapted cells under acidic condition. MCM7 Knockdown promoted mesenchymal phenotype and inhibited proliferation of the acid-adapted cells. While, MCM7 overexpression inhibited acid-induced EMT and supported tumor cell proliferation under acidic condition. Thus, our study provides evidence that tumor cells display phenotypic plasticity that allows them to survive acid stress.

Potential Effect of SOX2 on the Cell Cycle of Wharton's Jelly Stem Cells (WJSCs).

The connective tissue of the umbilical cord contains stem cells called Wharton's jelly cells. These cells express core transcription factors (NANOG, OCT4, and SOX2). The protein product of the SOX2 gene controls the cell cycle by interacting with cyclin D (directly and indirectly) and cycle inhibitors—p21 and p27, as well as two E2f3 protein isoforms. The aim of the study was to analyze the effect of SOX2 on the cell cycle of stem cells of Wharton's jelly. The material for the study was the stem cells of Wharton's jelly isolated from 20 umbilical cords collected during childbirth. The stem cells collected were subjected to cytometric analysis, cell culture, and RNA isolation. cDNA was the starting material for the analysis of gene expression: SOX2, CCND1, CDK4, and CDKN1B. The studies indicate a high proliferative potential of the Wharton's jelly stem cells and the inhibitory effect of SOX2 on the expression of the CCND1 and CDK4 gene.

The broken cycle: E2F dysfunction incancer.

The cyclin-dependent kinase (CDK)-RB-E2F axis forms the core transcriptional machinery driving cell cycle progression, dictating the timing and fidelity of genome replication and ensuring genetic material is accurately passed through each cell division cycle. The ultimate effectors of this axis are members of a family of eight distinct E2F genes encoding transcriptional activators and repressors. E2F transcriptional activity is tightly regulated throughout the cell cycle via transcriptional and translational regulation, post-translational modifications, protein degradation, binding to cofactors and subcellular localization. Alterations in one or more key components of this axis (CDKs, cyclins, CDK inhibitors and the RB family of proteins) occur in virtually all cancers and result in heightened oncogenic E2F activity, leading to uncontrolled proliferation. In this Review, we discuss the activities of E2F proteins with an emphasis on the newest atypical E2F family members, the specific and redundant functions of E2F proteins, how misexpression of E2F transcriptional targets promotes cancer and both current and developing therapeutic strategies being used to target this oncogenic pathway.

Connexin 43 plays an important role in the transformation of cholangiocytes with Clonochis sinensis excretory-secretory protein and N-nitrosodimethylamine.

BackgroundClonorchis sinensis is a group I bio-carcinogen responsible for cholangiocarcinoma (CHCA) in humans. However, the mechanism by which C. sinensis promotes carcinogenesis is unclear.MethodologyUsing the human cholangiocyte line H69, we investigated cell proliferation and gap junction protein expression after stimulation with the hepatotoxin N-nitrosodimethylamine (NDMA) and/or excretory-secretory products (ESP) of C. sinensis, which induce inflammation. NDMA and ESP treatment increased proliferation by 146% and the proportion of cells in the G2/M phase by 37%. Moreover, the expression of the cell proliferation-related proteins E2F1, Ki-67, and cancer related protein cytokeratin 19 and Cox-2 increased in response to combined treatment with NDMA and ESP. The gap-junction proteins connexin (Cx) 43 and Cx26 increased. In contrast, Cx32 expression decreased in cells treated with NDMA and ESP. Silencing of Cx43 reduced cell proliferation and significantly suppressed Cx26 and Cox-2 expression.ConclusionsThese results suggest that Cx43 is an important factor in CHCA induced by C. sinensis ESP and NDMA and further investigations targeting this pathway may allow prevention of this deadly disease.

Loss of the tumor suppressor BIN1 enables ATM Ser/Thr kinase activation by the nuclear protein E2F1 and renders cancer cells resistant to cisplatin

The tumor suppressor bridging integrator 1 (BIN1) is a corepressor of the transcription factor E2F1 and inhibits cell-cycle progression. BIN1 also curbs cellular poly(ADP-ribosyl)ation (PARylation) and increases sensitivity of cancer cells to DNA-damaging therapeutic agents such as cisplatin. However, how BIN1 deficiency, a hallmark of advanced cancer cells, increases cisplatin resistance remains elusive. Here, we report that BIN1 inactivates ataxia telangiectasia-mutated (ATM) serine/threonine kinase, particularly when BIN1 binds E2F1. BIN1 + 12A (a cancer-associated BIN1 splicing variant) also inhibited cellular PARylation, but only BIN1 increased cisplatin sensitivity. BIN1 prevented E2F1 from transcriptionally activating the human ATM promoter, whereas BIN1 + 12A did not physically interact with E2F1. Conversely, BIN1 loss significantly increased E2F1-dependent formation of MRE11A/RAD50/NBS1 DNA end-binding protein complex and efficiently promoted ATM autophosphorylation. Even in the absence of dsDNA breaks (DSBs), BIN1 loss promoted ATM-dependent phosphorylation of histone H2A family member X (forming γH2AX, a DSB biomarker) and mediator of DNA damage checkpoint 1 (MDC1, a γH2AX-binding adaptor protein for DSB repair). Of note, even in the presence of transcriptionally active (i.e. proapoptotic) TP53 tumor suppressor, BIN1 loss generally increased cisplatin resistance, which was conversely alleviated by ATM inactivation or E2F1 reduction. However, E2F2 or E2F3 depletion did not recapitulate the cisplatin sensitivity elicited by E2F1 elimination. Our study unveils an E2F1-specific signaling circuit that constitutively activates ATM and provokes cisplatin resistance in BIN1-deficient cancer cells and further reveals that γH2AX emergence may not always reflect DSBs if BIN1 is absent.

Enhanced insect and fungal resistance of maize callus transgenically expressing a maize E2F regulatory gene

A maize gene coding for an E2F regulatory protein located in a quantitative trait locus (QTL) for Fusarium ear rot resistance was cloned and transgenically introduced into maize callus. Several of the transformants were more resistant to feeding by corn earworms and fall armyworms, and retarded growth of Fusarium proliferatum compared to GUS control transformants. More effective transformants contained higher levels of E2F product than GUS controls as indicated by antibody detection. Increased and decreased levels of regulated proteins were noted in E2F overexpressing compared to GUS expressing control transformants. These differentially produced proteins were previously reported to be interactors with the E2F protein, suggesting E2F is affecting the production of these proteins. Other proteins coded for by genes reported to interact with the relevant E2F protein and associated phenotypic effects that could be promoting resistance include those that would increase cell resistance to water stress, increase the presence of reactive oxygen species, and increase the density of indigestible components. Although the full complex of responsible proteins regulated by the E2F examined that promote resistance to insects and fungi remains to be determined, overproduction of the E2F in transgenic callus enhances resistance to some maize insect and fungal pests.

Lactate Activates the E2F Pathway to Promote Cell Motility by Up-Regulating Microtubule Modulating Genes.

Excess lactate production due to enhanced aerobic glycolysis is characteristic of malignant cancers, which is also intimately associated with poor cancer prognoses. Although tumor-associated lactate contributes to all major steps in carcinogenesis, its action mechanism remains obscure. To understand the molecular mechanism of the lactate-induced tumor metastatic process, we identified an array of lactate-responsive genes via transcriptome analysis of a metformin-induced hyper-glycolytic liver cancer model. Gene set enrichment analysis suggested E2F-RB pathway as the dominant regulator of the lactate-induced gene expression. We experimentally verified that lactate indeed activates E2F-mediated transcription by promoting E2F1 protein accumulation through a posttranscriptional mechanism. Literature-based analysis of target pathways potentially modulated by 136 top-ranked genes indicated that genes functioning in cell-cell or cell-matrix communications dominate the lactate-induced gene expression. Especially, those regulating microtubule functions, including a group of kinesin family members, were significantly up-regulated in lactate- and E2F1-dependent manners. Depletion of E2F1 or kinesins (KIF2C, KIF18B, KIF20A) led to deformation of microtubule structures, impairing cell motility as much as the deficit in lactate production. These results indicate that E2F pathway activation by tumor-associated lactate and subsequent transcriptional activation of microtubule functions play crucial roles in tumor metastasis, providing mechanistic clues to cell motility-directed anti-cancer strategies.

Regulation of Cell Cycle Regulatory Proteins by MicroRNAs in Uterine Leiomyoma.

The objective of this study was to determine whether miR-93, miR-29c, and miR-200c, which we previously reported to be downregulated in leiomyomas, target cell cycle regulatory proteins that influence cell proliferation. Based on TargetScan algorithm 3 cell cycle regulatory proteins namely, E2F transcription factor 1 (E2F1), Cyclin D1 (CCND1) and CDK2 which were predicted to be targets of these miRNAs were further analyzed. In 30 hysterectomy specimens, we found the expression of E2F1 and CCND1 messenger RNA (mRNA) was increased in leiomyoma as compared to matched myometrium, with no significant changes in CDK2 mRNA levels. There was a significant increase in the abundance of all 3 proteins in leiomyoma in comparison with matched myometrium. Using luciferase reporter assay, we demonstrated E2F1 and CCND1 are targets of miR-93 and CDK2 is a target of miR-29c and miR-200c. We confirmed these findings through transfection studies in which transfection of primary leiomyoma cells with miR-93 resulted in a significant decrease in the expression of E2F1 and CCND1 mRNA and protein levels, whereas knockdown of miR-93 had the opposite effect. Similarly, overexpression of miR-29c and miR-200c in leiomyoma cells inhibited the expression of CDK2 protein and mRNA, whereas knockdown of this microRNAs (miRNA) had the opposite effect. Transfection of miR-29c, miR-200c, and miR-93 in primary leiomyoma cells resulted in a time-dependent inhibition of cell proliferation and cell motility. These results collectively indicate that the 3 miRNAs known to be downregulated in fibroid tumors are critical in regulation of cell proliferation because of their effects on 3 key cell cycle regulatory proteins, which are overexpressed in uterine leiomyomas.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

TNFα Induces Multidrug Resistance-Associated Protein 4 Expression through p38-E2F1-Nrf2 Signaling in Obstructive Cholestasis.

PurposeTo explore the molecular mechanism of the upregulation of multidrug resistance-associated protein 4 (MRP4) in cholestasis.Materials and MethodsThe mRNA and protein levels of MRP4 in liver samples from cholestatic patients were determined by quantitative real-time PCR and Western blot. In human hepatoma HepG2 cells, electrophoretic mobility shift assay (EMSA) was used to determine the affinity of nuclear factor-E2-related factor (Nrf2) binding to MRP4 promoter. Dual-luciferase reporter assay was used to detect the binding of tumor necrosis factor α (TNFα) to the promotor of E2F1. The bile duct ligation mouse models were established using male C57BL/6 mice.ResultsThe mRNA and protein levels of MRP4 were significantly increased in cholestatic patients. TNFα treatment induced the expression of MRP4 and Nrf2 and enhanced cell nuclear extract binding activity to MRP4 promoter, as demonstrated by EMSA. Nrf2 knockdown reduced MRP4 mRNA levels in both HepG2 and Hep-3B cells. In addition, TNFα increased Rb phosphorylation and expression of MRP4 and Nrf2 and activated E2F1 and phosphorylated p38 in HepG2 and Hep-3B cells. These effects were markedly inhibited by pretreatment with E2F1 siRNA. Dual-luciferase reporter assay validated that TNFα induces the transcription of E2F1. Furthermore, the expression of MRP4, Nrf2, E2F1, and p-p38 proteins was improved with treatment of TNFα in a mouse model of cholestasis. E2F1 siRNA lentivirus or SB 203580 (p38 inhibitor) inhibited these positive effects.ConclusionOur findings indicated that TNFα induces hepatic MRP4 expression through activation of the p38-E2F1-Nrf2 signaling pathway in human obstructive cholestasis.

Long noncoding RNA LINC00511 contributes to breast cancer tumourigenesis and stemness by inducing the miR-185-3p/E2F1/Nanog axis

BackgroundEmerging evidence have illustrated the vital role of long noncoding RNAs (lncRNAs) long intergenic non-protein coding RNA 00511 (LINC00511) on the human cancer progression and tumorigenesis. However, the role of LINC00511 in breast cancer tumourigenesis is still unknown. This research puts emphasis on the function of LINC00511 on the breast cancer tumourigenesis and stemness, and investigates the in-depth mechanism.MethodsThe lncRNA and RNA expression were measured using RT-PCR. Protein levels were measured using western blotting analysis. CCK-8, colony formation assays and transwell assay were performed to evaluate the cell proliferation ability and invasion. Sphere-formation assay was also performed for the stemness. Bioinformatic analysis, chromatin immunoprecipitation (ChIP) and luciferase reporter assays were carried to confirm the molecular binding.ResultsLINC00511 was measured to be highly expressed in the breast cancer specimens and the high-expression was correlated with the poor prognosis. Functionally, the gain and loss-of-functional experiments revealed that LINC00511 promoted the proliferation, sphere-formation ability, stem factors (Oct4, Nanog, SOX2) expression and tumor growth in breast cancer cells. Mechanically, LINC00511 functioned as competing endogenous RNA (ceRNA) for miR-185-3p to positively recover E2F1 protein. Furthermore, transcription factor E2F1 bind with the promoter region of Nanog gene to promote it transcription.ConclusionIn conclusion, our data concludes that LINC00511/miR-185-3p/E2F1/Nanog axis facilitates the breast cancer stemness and tumorigenesis, providing a vital insight for them.

MYCN is amplified during S phase, and c‑myb is involved in controlling MYCN expression and amplification in MYCN‑amplified neuroblastoma cell lines.

Neuroblastoma derived from primitive sympathetic neural precursors is a common type of solid tumor in infants. MYCN proto-oncogene bHLH transcription factor (MYCN) amplification and 1p36 deletion are important factors associated with the poor prognosis of neuroblastoma. Expression levels of MYCN and c-MYB proto-oncogene transcription factor (c-myb) decline during the differentiation of neuroblastoma cells; E2F transcription factor 1 (E2F1) activates the MYCN promoter. However, the underlying mechanism of MYCN overexpression and amplification requires further investigation. In the present study, potential c-Myb target genes, and the effect of c-myb RNA interference (RNAi) on MYCN expression and amplification were investigated in MYCN-amplified neuroblastoma cell lines. The mRNA expression levels and MYCN gene copy number in five neuroblastoma cell lines were determined by quantitative polymerase chain reaction. In addition, variations in potential target gene expression and MYCN gene copy number between pre- and post-c-myb RNAi treatment groups in MYCN-amplified Kelly, IMR32, SIMA and MHH-NB-11 cell lines, normalized to those of non-MYCN-amplified SH-SY5Y, were examined. To determine the associations between gene expression levels and chromosomal aberrations, MYCN amplification and 1p36 alterations in interphases/metaphases were analyzed using fluorescence in situ hybridization. Statistical analyses revealed correlations between 1p36 alterations and the expression of c-myb, MYB proto-oncogene like 2 (B-myb) and cyclin dependent kinase inhibitor 1A (p21). Additionally, the results of the present study also demonstrated that c-myb may be associated with E2F1 and L3MBTL1 histone methyl-lysine binding protein (L3MBTL1) expression, and that E2F1 may contribute to MYCN, B-myb, p21 and chromatin licensing and DNA replication factor 1 (hCdt1) expression, but to the repression of geminin (GMNN). On c-myb RNAi treatment, L3MBTL1 expression was silenced, while GMNN was upregulated, indicating G2/M arrest. In addition, MYCN gene copy number increased following treatment with c-myb RNAi. Notably, the present study also reported a 43.545% sequence identity between upstream of MYCN and Drosophila melanogaster amplification control element 3, suggesting that expression and/or amplification mechanisms of developmentally-regulated genes may be evolutionarily conserved. In conclusion, c-myb may be associated with regulating MYCN expression and amplification. c-myb, B-myb and p21 may also serve a role against chromosome 1p aberrations. Together, it was concluded that MYCN gene is amplified during S phase, potentially via a replication-based mechanism.

ZNF703 is Overexpressed in Papillary Thyroid Carcinoma Tissues and Mediates K1 Cell Proliferation.

Zinc finger protein 703 (ZNF703), a member of the NET family of transcription factors, has recently emerged as an important player in the development of several types of cancers, though its role in papillary thyroid cancer (PTC) has not been characterized. We investigated the expression of ZNF703, its association with the most common genetic mutation in PTC, BRAF V600E, and its potential use as a therapeutic target. Real-time PCR, immunohistochemical staining, and western blot analysis of ZNF703 expression were performed for 36 cases of PTC and corresponding normal thyroid tissues. ZNF703 mRNA and protein expression was found to be significantly higher in PTC compared to normal thyroid tissues (P < 0.05). Furthermore, expression was associated with the tumor size, lymph node metastasis, and advanced disease stage. Immunohistochemical results showed that there was no correlation between ZNF703 protein levels and BRAF V600E mutation. The human PTC cell line K1, which has a BRAF V600E mutation, was selected for further investigation. Using small interfering RNA (siRNA), ZNF703 was shown to contribute to the proliferation, apoptosis, and invasion of K1 cells. ZNF703-siRNA downregulated E2F1 and MMP9 protein expression and enhanced the expression of p27 protein (P < 0.05), but had no effects on BRAF V600E protein levels. These results suggest that ZNF703 may be of potential use as a new marker for PTC prognosis and therapy that functions independent of BRAF V600E expression.