MicroRNA-99a-5p suppresses breast cancer progression and cell-cycle pathway through downregulating CDC25A

In this study, we aimed to explore the association between miR-99a-5p and CDC25A in breast cancer and the regulatory mechanisms of miR-99a-5p on breast cancer. The expressions of messenger RNA and microRNAs in breast cancer tissues and adjacent tissues were analyzed by the Cancer Genome Atlas microarray analysis. Quantitative real-time polymerase chain reaction was conducted to find out the expression levels of miR-99a-5p and CDC25A. The expression levels of proteins (CDC25A, ki67, cyclin D1, p21, BAX, BCL-2, BCL-XL, MMP2, and MMP9) were determined by Western blot analysis. The relationship between miR-99a-5p and CDC25A was predicted and verified by bioinformatics analysis and dual luciferase assay. After transfection, cell proliferation, invasion, and apoptosis of breast cancer tissues were, respectively, observed by cell counting kit-8 assay, transwell assay, and flow cytometry (FCM). Furthermore, the relationship among miR-99a-5p, CDC25A, and cell-cycle progression was determined by FCM assay. The nude mouse transplantation tumor experiment was performed to verify the influence of miR-99a-5p on breast cancer cell in vivo. The expression of miR-99a-5p in breast cancer tissues and cells was significantly downregulated, whereas CDC25A expression was upregulated. MiR-99a-5p targeted CDC25A and suppressed its expression in breast cancer cells. Overexpression of miR-99a-5p and decreased expression of CDC25A could suppress breast cancer cell proliferation and invasion and facilitate apoptosis. Cell-cycle progression was significantly activated by downregulated miR-99a-5p and upregulated CDC25A. Moreover, miR-99a-5p overexpression repressed the expressions of CDC25A, marker ki67, and Cyclin D1 proteins, whereas it upregulated the expression of p21 protein. MicroRNA-99a-5p suppresses breast cancer progression and cell-cycle pathway through downregulating CDC25A.     

CDC25B associates with a centrin 2-containing complex and is involved in maintaining centrosome integrity

Background information. CDC25 (cell division cycle 25) phosphatases function as activators of CDK (cyclin‐dependent kinase)–cyclin complexes to regulate progression through the CDC. We have recently identified a pool of CDC25B at the centrosome of interphase cells that plays a role in regulating centrosome numbers. Results. In the present study, we demonstrate that CDC25B forms a close association with Ctn (centrin) proteins at the centrosome. This interaction involves both N‐ and C‐terminal regions of CDC25B and requires CDC25B binding to its CDK—cyclin substrates. However, the interaction is not dependent on the enzyme activity of CDC25B. Although CDC25B appears to bind indirectly to Ctn2, this association is pertinent to CDC25B localization at the centrosome. We further demonstrate that CDC25B plays a role in maintaining the overall integrity of the centrosome, by regulating the centrosome levels of multiple centrosome proteins, including that of Ctn2. Conclusions. Our results therefore suggest that CDC25B associates with a Ctn2‐containing multiprotein complex in the cytoplasm, which targets it to the centrosome, where it plays a role in maintaining the centrosome levels of Ctn2 and a number of other centrosome components.     

Prognostic implication of CDC25A and cyclin E expression on primary breast cancer patients

Defect in cell cycle control is a hallmark character of cancer. We have investigated the association of Ki67 labeling index, cyclin E and CDC25A expressions with clinical follow-up data in breast carcinomas. Flow cytometry was used to detect gene amplification of cyclins in 44 tumor tissue with invasive breast carcinomas. Multivariate Cox proportional hazard ratio test was used to show the correlations. Cyclin E or CDC25A were upregulated in 34% of the tumors. Among the whole total material, expression of cyclin E and of CDC25A were found upregulated in 31.9% and 39.4% of cells, respectively. Both CDC25A and cyclin E protein expression levels were correlated with Ki67 expression level (p < 0.001). In addition, the expression of CDC25A was associated significantly with poor survival (P = 0.028), whereas no correlation was found with cyclin E. These findings suggest a possible prognostic value for CDC25A as a cell cycle marker and may imply in characteristic of high risk breast cancer patients.     

Protection of CDC25 phosphatases against oxidative stress in breast cancer cells: Evaluation of the implication of the thioredoxin system

Abstract

Reactive oxygen species regulate protein functionality. Cell cycle CDC25 phosphatases are targets of such oxidative regulation in vitro. We sought to evaluate if a thioredoxin (trx)-dependent redox regulation of CDC25 exists in cancer cells. For that purpose, we used MCF7 and MDA-MB 231 breast cancer cells, which express trx1 differentially, together with two trx/thioredoxin reductase (trxR) inhibitors, Auranofin and Acrolein. Auranofin could induce a full trxR inhibition associated with ROS production in both cell lines. Acrolein could provoke similar effects only in MDA-MB 231 cells with a low trx1 expression. Simultaneous trx1 oxidation and trxR inactivation occurred only in the presence of Acrolein and resulted in a G2-M cell cycle arrest, without full CDC25 inhibition in MDA-MB 231 cells. Our data suggest that the maintenance of CDC25 activity does not fully rely on the trx system in breast cancer cells, even in the presence of a major oxidative stress.     

A novel CDC25A/DYRK2 regulatory switch modulates cell cycle and survival

The cell division cycle 25A (CDC25A) phosphatase is a key regulator of cell cycle progression that acts on the phosphorylation status of Cyclin–Cyclin-dependent kinase complexes, with an emergent role in the DNA damage response and cell survival control. The regulation of CDC25A activity and its protein level is essential to control the cell cycle and maintain genomic integrity. Here we describe a novel ubiquitin/proteasome-mediated pathway negatively regulating CDC25A stability, dependent on its phosphorylation by the serine/threonine kinase DYRK2. DYRK2 phosphorylates CDC25A on at least 7 residues, resulting in its degradation independent of the known CDC25A E3 ubiquitin ligases. CDC25A in turn is able to control the phosphorylation of DYRK2 at several residues outside from its activation loop, thus affecting DYRK2 localization and activity. An inverse correlation between DYRK2 and CDC25A protein amounts was observed during cell cycle progression and in response to DNA damage, with CDC25A accumulation responding to the manipulation of DYRK2 levels or activity in either physiological scenario. Functional data show that the pro-survival activity of CDC25A and the pro-apoptotic activity of DYRK2 could be partly explained by the mutual regulation between both proteins. Moreover, DYRK2 modulation of CDC25A expression and/or activity contributes to the DYRK2 role in cell cycle regulation. Altogether, we provide evidence suggesting that DYRK2 and CDC25A mutually control their activity and stability by a feedback regulatory loop, with a relevant effect on the genotoxic stress pathway, apoptosis, and cell cycle regulation.Subject terms: Proteins, Cell biology, Proteomics     

CDC25B is associated with the risk of hepatocellular carcinoma,but not related to persistent infection of hepatitis B virus in a Chinese population

The cell division cycle 25 (CDC25) gene members, including CDC25A, CDC25B and CDC25C, are reported to be associated with several human cancers. Here, we aim to investigate the association of functional polymorphisms of CDC25 gene family with the risk of hepatocellular carcinoma (HCC) and persistent infection of Hepatitis B virus (HBV) in a Chinese HBV-related population. First, we used bioinformatics tools to systematically screen functional polymorphisms within CDC25 gene family. Second, we evaluated the effects of candidate polymorphisms by recruiting 790 HCC cases, 709 persistent HBV carriers (PHC), and 741 subjects with HBV natural clearance (SHNC). MassARRAY platform was used for genotyping. At last, we conducted functional prediction and assay to further explore the pathogenic mechanism of the identified polymorphism. Our results demonstrated that CDC25B rs2295348 played a protective role in HCC risk in a HBV-related Chinese population (adjusted odds ratio [OR]?=?0.77, 95% confidence interval [CI] 0.65–0.93, P?=?0.006). It showed a more significantly reduced HCC risk in the SHNC population (adjusted OR?=?0.73, 95% CI 0.59–0.89, P?=?0.002). However, we did not observe the association between CDC25B rs2295348 and the risk of persistent HBV infection. Further functional prediction and assay demonstrated that the mutant A allele of CDC25B rs2295348 might significantly decrease gene expression to modify the HCC risk. Our results suggest that CDC25B rs2295348 may confer a protective effect on HCC risk in a HBV-related Chinese population, but do not influence the susceptibility to persistent HBV infection.

     

Overexpression of the <Emphasis Type="Italic">Arabidopsis</Emphasis> anaphase promoting complex subunit <Emphasis Type="Italic">CDC27a</Emphasis> increases growth rate and organ size

The Anaphase Promoting Complex (APC) controls CDK activity by targeting the ubiquitin-dependent proteolysis of S-phase and mitosis-promoting cyclins. Here, we report that the ectopic expression of the Arabidopsis CDC27a, an APC subunit, accelerates plant growth and results in plants with increased biomass production. CDC27a overexpression was associated to apical meristem restructuration, protoplasts with higher ³H-thimidine incorporation and altered cell-cycle marker expression. Total protein extracts immunoprecipitated with a CDC27a antibody showed ubiquitin ligase activity, indicating that the Arabidopsis CDC27a gets incorporated into APC complexes. These results indicate a role of AtCDC27a in regulation of plant growth and raise the possibility that the activity of the APC and the rates of plant cell division could be regulated by the concentration of the CDC27a subunit. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. Cristian Antonio Rojas and Nubia Barbosa Eloy contributed equally to this work.     

Protection of CDC25 phosphatases against oxidative stress in breast cancer cells: evaluation of the implication of the thioredoxin system

Reactive oxygen species regulate protein functionality. Cell cycle CDC25 phosphatases are targets of such oxidative regulation in vitro. We sought to evaluate if a thioredoxin (trx)-dependent redox regulation of CDC25 exists in cancer cells. For that purpose, we used MCF7 and MDA-MB 231 breast cancer cells, which express trx1 differentially, together with two trx/thioredoxin reductase (trxR) inhibitors, Auranofin and Acrolein. Auranofin could induce a full trxR inhibition associated with ROS production in both cell lines. Acrolein could provoke similar effects only in MDA-MB 231 cells with a low trx1 expression. Simultaneous trx1 oxidation and trxR inactivation occurred only in the presence of Acrolein and resulted in a G2-M cell cycle arrest, without full CDC25 inhibition in MDA-MB 231 cells. Our data suggest that the maintenance of CDC25 activity does not fully rely on the trx system in breast cancer cells, even in the presence of a major oxidative stress.     

Domains of the Saccharomyces cerevisiae CDC25 gene controlling mitosis and meiosis

Summary The cell division cycle gene CDC25 was replaced by various disrupted and deleted mutant copies. Mutants disrupted at a central position of the gene, or lacking 532 residues within the amono-terminal half of the gene product grow normally in glucose, but not in acetate media, and they fail to sporulate as homozygous diploids. Disruptions or deletions within the carboxy-terminal half are lethal, except for the deletion of the 38 carboxy-terminal residues, which are required for sporulation but not for growth in glucose or acetate media. It is concluded that distinct domains of the CDC25 gene product are involved in the control of mitosis and/or meiosis.     

2-(Thienothiazolylimino)-1,3-thiazolidin-4-ones inhibit cell division cycle 25 A phosphatase

Cell division cycle dual phosphatases (CDC25) are essential enzymes that regulate cell progression in cell cycle. Three isoforms exist as CDC25A, B and C. Over-expression of each CDC25 enzyme is found in cancers of diverse origins. Thiazolidinone derivatives have been reported to display anti-proliferative activities, bactericidal activities and to reduce inflammation process. New 2-(thienothiazolylimino)-1,3-thiazolidin-4-ones were synthesized and evaluated as inhibitors of CDC25 phosphatase. Among the molecules tested, compound 6 inhibited CDC25A with an IC₅₀ estimated at 6.2 ± 1.0 μM. The binding of thiazolidinone derivative 6 onto CDC25A protein was reversible. In cellulo, compound 6 treatment led to MCF7 and MDA-MB-231 cell growth arrest. To our knowledge, it is the first time that such 4-thiazolidinone derivatives are characterized as CDC25 potential inhibitor.     

A murine CDC25/ras-GRF-related protein implicated in ras regulation

A partial cDNA encoding a novel putative p2, ras guanine nucleotide release-inducing factor (GRF), GRF2, was amplified from murine embryonic stem cells. The presumptive catalytic region of GRF2 is related to the yeast Ras GRF encoded by CDC25. GRF2 is 80% identical to murine CDC25Mm/ras-GRF, but is more similar to yeast CDC25 than to other ras GRFs related to the Drosophila son of sevenless gene product. A 9-kb GRF2 messenger RNA was highly expressed in brain, but GRF2-specific antibodies recognized apparent GRF2 proteins in various mouse tissues in addition to brain. Thus GRF2 represents a novel widely-expressed protein that is highly related to CDC25Mm/ras-GRF, at least in its catalytic domain. Both GRF2 and CDC25Mm/ras-GRF are expressed in murine embryonic stem cells, suggesting that different Ras activators may regulate ras-dependent proliferation and differentiation in early mouse development. © 1993Wiley-Liss, Inc.     

NPAS2 regulates proliferation of acute myeloid leukemia cells via CDC25A-mediated cell cycle progression and apoptosis

Promoted proliferation and associated suppression of apoptosis at various stages of myeloid differentiation are well-known features of acute myeloid leukemia (AML), but understanding of the molecular processes involved remains limited. As a crucial circadian agent, neuronal PAS domain protein 2 (NPAS2) is widely recognized as a promising predictor of clinical outcome in various malignancies. Nevertheless, the understanding of its influence on AML is insufficient. Using KD cells and expression assays, we carried out detailed investigation of the role of NPAS2 in AML in vivo and in vitro. Firstly, we found that NPAS2 expression was elevated in AML cells both in vivo and in vitro. NPAS2 knockdown via lentiviral infection clearly suppressed proliferation of MV4-11 and MOLM-14 cells. Additionally, NPAS2 knockdown caused G1/S cell cycle arrest (CCA), which inhibited CDC25A expression. Moreover, NPAS2 knockdown promoted cell death, as evidenced by increased caspase-3 cleavage, and change in Bcl2/Bax production. Excessive CDC25A expression eliminated G1/S CCA triggered by NPAS2 knockdown and death of NPAS2 knocked down MOLM and MV4-11 cells. The expression of CDC25A was stabilized by NPAS2, which induced cell cycle progression and participated in suppression of cell death by modulating caspase-3 cleavage, and expression of Bcl2/Bax. We therefore indicated NPAS2 to be a crucial modulator of survival as well as proliferation. Our research sheds light on the etiology of the proliferation of promyelocytes modulated via NPAS2 with regard to AML.     

Altered downstream target gene expression of the placental Vitamin D receptor in human idiopathic fetal growth restriction

Fetal growth restriction (FGR) affects up to 5% of pregnancies and is associated with significant perinatal complications. Maternal deficiency of vitamin D, a secosteroid hormone, is common in FGR-affected pregnancies. We recently demonstrated that decreased expression of the vitamin D receptor (VDR) in idiopathic FGR placentae could impair trophoblast growth. As strict regulation of cell-cycle genes in trophoblast cells is critical for optimal feto-placental growth, we hypothesised that pathologically decreased placental VDR contributes to aberrant regulation of cell-cycle genes. The study aims were to (i) identify the downstream cell-cycle regulatory genes of VDR in trophoblast cells, and (ii) determine if expression was changed in cases of FGR. Targeted cell-cycle gene cDNA arrays were used to screen for downstream targets of VDR in VDR siRNA-transfected BeWo and HTR-8/SVneo trophoblast-derived cell lines, and in third trimester placentae from FGR and gestation-matched control pregnancies (n = 25 each). The six candidate genes identified were CDKN2A, CDKN2D, HDAC4, HDAC6, TGFB2 and TGFB3. TGFB3 was prioritised for further validation, as its expression is largely unknown in FGR. Significantly reduced mRNA and protein expression of TGFB3 was verified in FGR placentae and the BeWo and HTR-8/SVneo trophoblast cell lines, using real-time PCR and immunoblotting respectively. In summary, decreased placental VDR expression alters the expression of regulatory cell-cycle genes in FGR placentae. Aberrant regulation of cell-cycle genes in the placental trophoblast cells may constitute a mechanistic pathway by which decreased placental VDR reduces feto-placental growth.     

CDC25A phosphatase controls meiosis I progression in mouse oocytes

CDK1 is a pivotal regulator of resumption of meiosis and meiotic maturation of oocytes. CDC25A/B/C are dual-specificity phosphatases and activate cyclin-dependent kinases (CDKs). Although CDC25C is not essential for either mitotic or meiotic cell cycle regulation, CDC25B is essential for CDK1 activation during resumption of meiosis. Cdc25a −/− mice are embryonic lethal and therefore a role for CDC25A in meiosis is unknown. We report that activation of CDK1 results in a maturation-associated decrease in the amount of CDC25A protein, but not Cdc25a mRNA, such that little CDC25A is present by metaphase I. In addition, expression of exogenous CDC25A overcomes cAMP-mediated maintenance of meiotic arrest. Microinjection of Gfp-Cdc25a and Gpf-Cdc25b mRNAs constructs reveals that CDC25A is exclusively localized to the nucleus prior to nuclear envelope breakdown (NEBD). In contrast, CDC25B localizes to cytoplasm in GV-intact oocytes and translocates to the nucleus shortly before NEBD. Over-expressing GFP-CDC25A, which compensates for the normal maturation-associated decrease in CDC25A, blocks meiotic maturation at MI. This MI block is characterized by defects in chromosome congression and spindle formation and a transient reduction in both CDK1 and MAPK activities. Lastly, RNAi-mediated reduction of CDC25A results in fewer oocytes resuming meiosis and reaching MII. These data demonstrate that CDC25A behaves differently during female meiosis than during mitosis, and moreover, that CDC25A has a function in resumption of meiosis, MI spindle formation and the MI-MII transition. Thus, both CDC25A and CDC25B are critical for meiotic maturation of oocytes.