Regulation Of Cyclin-dependent Kinases Research Articles

Cyclin D3 predicts disease-free survival in breast cancer.

BackgroundCyclin D3, which induces progression through the G1 phase of the cell cycle, is a regulator of Cyclin-dependent kinases 4 and 6. Previous studies revealed that abnormal expression of Cyclin D3 was found in many different cancers. However, the role of Cyclin D3 in breast cancer (BC) remains unknown. The aim of this study is to examine the expression pattern of Cyclin D3 in BC and to evaluate its biological role and clinical significance in prognosis prediction. The mechanism involved is also evaluated.MethodsImmunohistochemical staining was used to detect the expression of Cyclin D3. qRT-PCR was used to detect the mRNA level of Cyclin D3 in BC tissues and BC cell lines. Transwell assay was used to examine the role of Cyclin D3 in the migration and invasion of BC cells. Mass Spectrometry was used to search for the interacting protein with Cyclin D3. Co-Immunoprecipitation assay and GST-Pull Down assay were used to validate the interaction of Cyclin D3 and its interaction protein.ResultsThrough detecting Cyclin D3 expression in 243 breast cancer patients’ tissue array, we found Cyclin D3 expression was correlated with ER status (p = 0.000), PR status (p = 0.001), HER2 status (p = 0.002) and tumor differentiation (p = 0.045). The Kaplan–Meier survival curves indicated that the disease free survival (DFS) was significantly poor in high Cyclin D3 expression BC patients (p = 0.004). Furthermore, expression of Cyclin D3 was significantly associated with BC prognosis and was shown to be an independent prognostic marker in breast cancer (p = 0.028). By IHC staining and qPCR detection, Cyclin D3 expression was found to be down-regulated both in BC tissues and in BC cell lines compared with the corresponding normal controls. Further investigation showed Cyclin D3 was involved in the metastasis of BC cells and physically interacted with actin in vivo and in vitro.ConclusionOur studies revealed that Cyclin D3 was upregulated in breast cancer and represented a novel predictor of BC prognosis.Electronic supplementary materialThe online version of this article (doi:10.1186/s12935-015-0245-6) contains supplementary material, which is available to authorized users.

Upregulation of sex-determining region Y-box 9 (SOX9) promotes cell proliferation and tumorigenicity in esophageal squamous cell carcinoma.

Sex-determining region Y-box 9 (SOX9), a vital transcription factor, play important roles in numerous biological and pathological processes. However, the clinical significance and biological role of SOX9 expression has not been characterized in human esophageal squamous cell cancer (ESCC). Herein, we found that SOX9 was markedly upregulated, at both mRNA and protein level, in ESCC cell lines and ESCC tissues and that SOX9 expression was significantly correlated with tumor clinical stage, T classification, N classification, M classification, pathological differentiation, and shorter overall survival. The proliferation and tumorigenicity of ESCC cells were dramatically induced by SOX9 overexpression but were inhibited by SOX9 knockdown both in vitro and in vivo. Moreover, we demonstrated that upregulation of SOX9 increased the expression of phosphorylated Akt, the cyclin-dependent kinase (CDK) regulator cyclin D1, phosphorylated forkhead box O (FOXO)1, and phosphorylated FOXO3, but SOX9 downregulation decreased their expression, whereas the levels of the CDK inhibitors p21Cip1 and p27Kip1 were attenuated in SOX9-transduced cells. Taken together, our results suggest that SOX9 plays an important role in promoting the proliferation and tumorigenesis of ESCC and may represent a novel prognostic marker for the disease.

GOLPH3 is a potential therapeutic target and a prognostic indicator of poor survival in bladder cancer treated by cystectomy.

Golgi phosphoprotein 3 (GOLPH3) has been reported to be involved in the development of several human cancers. However, its clinical significance and biological role in bladder cancer remains unclear. In this study, we sought to analyze the GOLPH3 expression in bladder cancer samples and cells, and explore its clinical significance and biological role. We found that GOLPH3 was significantly increased in bladder cancer tissues and cells. Overexpression of GOLPH3 had significant correlation with poorer survival for bladder cancer patients treated by cystectomy. Knockdown of GOLPH3 inhibited the proliferation, migration and invasion of cancer cells, and tumor growth in a xenograft mouse model. GOLPH3 silencing inhibited AKT/m-TOR signaling, increased the cyclin-dependent kinase (CDK) inhibitor p27 and decreased the CDK regulator cyclin D1 and matrix metallopeptidase 9 (MMP9). Thus, GOLPH3 is likely to play important roles in bladder cancer progression via modulating AKT/mTOR signaling, and it is a novel prognostic biomarker and promising therapeutic target for bladder cancer.

MicroRNA-365-targeted nuclear factor I/B transcriptionally represses cyclin-dependent kinase 6 and 4 to inhibit the progression of cutaneous squamous cell carcinoma

Cyclin-dependent kinases are either post-transcriptionally regulated by interacting with cyclins and cyclin-dependent kinase inhibitors or are transcriptionally regulated by transcription factors, but the latter mechanism has not been extensively investigated. Dysregulated transcription factors resulting from aberrantly expressed microRNAs play critical roles in tumor development and progression. Our previous work identified miR-365 as an oncogenic microRNA that promotes the development of cutaneous squamous cell carcinoma via repression of cyclin-dependent kinase 6, while miR-365 also targets nuclear factor I/B. However, the underlying mechanism(s) of the interaction between nuclear factor I/B and cyclin-dependent kinase 6 are unclear. In this work, we demonstrate that miR-365-regulated nuclear factor I/B transcriptionally inhibits cyclin-dependent kinases 6 and 4 by binding to their promoter regions. In vivo and in vitro experiments demonstrate that the loss of nuclear factor I/B after miR-365 expression or treatment with small interfering RNAs results in the upregulation of cyclin-dependent kinases 6 and 4. This upregulation, in turn, enhances the phosphorylation of retinoblastoma protein and tumor progression. Characterizing this transcriptional repression of cyclin-dependent kinases 6 and 4 by nuclear factor I/B contributes to the understanding of the transcriptional regulation of cyclin-dependent kinases by transcription factors and also facilitates the development of new therapeutic regimens to improve the clinical treatment of cutaneous squamous cell carcinoma.

Determining the Functions of HIV-1 Tat and a Second Magnesium Ion in the CDK9/Cyclin T1 Complex: A Molecular Dynamics Simulation Study.

The current paradigm of cyclin-dependent kinase (CDK) regulation based on the well-established CDK2 has been recently expanded. The determination of CDK9 crystal structures suggests the requirement of an additional regulatory protein, such as human immunodeficiency virus type 1 (HIV-1) Tat, to exert its physiological functions. In most kinases, the exact number and roles of the cofactor metal ions remain unappreciated, and the repertoire has thus gained increasing attention recently. Here, molecular dynamics (MD) simulations were implemented on CDK9 to explore the functional roles of HIV-1 Tat and the second Mg2+ ion at site 1 (Mg1 2+). The simulations unveiled that binding of HIV-1 Tat to CDK9 not only stabilized hydrogen bonds (H-bonds) between ATP and hinge residues Asp104 and Cys106, as well as between ATP and invariant Lys48, but also facilitated the salt bridge network pertaining to the phosphorylated Thr186 at the activation loop. By contrast, these H-bonds cannot be formed in CDK9 owing to the absence of HIV-1 Tat. MD simulations further revealed that the Mg1 2+ ion, coupled with the Mg2 2+ ion, anchored to the triphosphate moiety of ATP in its catalytic competent conformation. This observation indicates the requirement of the Mg1 2+ ion for CDK9 to realize its function. Overall, the introduction of HIV-1 Tat and Mg1 2+ ion resulted in the active site architectural characteristics of phosphorylated CDK9. These data highlighted the functional roles of HIV-1 Tat and Mg1 2+ ion in the regulation of CDK9 activity, which contributes an important complementary understanding of CDK molecular underpinnings.

Novel arylazopyrazole inhibitors of cyclin-dependent kinases

Here, we describe new 4-arylazo-3,5-diamino-1H-pyrazole derivatives developed from CAN508, one of the first inhibitors to show preference for transcriptional regulator cyclin-dependent kinase 9. By substituting nitrogen in the pyrazole ring and employing a heteroatom in the 4-aryl ring, we obtained more potent derivatives differing in their CDK-selectivity profiles. The antiproliferative and anti-CDK kinase activities of the novel arylazopyrazoles were examined. The cellular effect of compound IVc was studied on MCF-7 cells synchronized by various methods and compared with other selective CDK inhibitors. The results demonstrated that IVc shows a preference for CDK4 and CDK1. In contrast to cytostatic effects induced by IVc in MCF-7 and K562 cells, we observed apoptotic activities in the RPMI-8226 cell line, which were confirmed by detecting active caspases by different biochemical assays.

Cyclin D1 in human neuroblastic tumors recapitulates its developmental expression: An immunohistochemical study

The protein cyclin D1 (CD1), which belongs to a family of proteins functioning as regulators of CDKs (cyclin-dependent kinases) throughout the cell cycle, has been immunohistochemically detected in a wide variety of human malignant tumors. The aim of the present study was to investigate immunohistochemically the expression and distribution of CD1 in the developing human peripheral sympathetic nervous system (PSNS) and in childhood peripheral neuroblastic tumors (neuroblastomas, ganglioneuroblastomas, and ganglioneuromas). The above mentioned fetal and neoplastic tissues represent an in vivo model in which undifferentiated neuroblastic cells undergo ganglion cell differentiation. During development, a strong nuclear expression of CD1 was restricted to neuroblasts, disappearing progressively from the maturing ganglion cells with increasing gestational age. In neoplastic tissues, CD1 immunoreactivity was restricted to neuroblastic cell component of all neuroblastomas and ganglioneuroblastomas, whereas it was absent or only focally detectable in maturing/mature ganglion cell component of differentiating neuroblastomas, ganglioneuroblastomas, and ganglioneuromas. We conclude that CD1 is a reliable marker, which can be used routinely to stain neuroblastic cells in both developing and neoplastic tissues. Furthermore, our results indicate that CD1 expression in childhood peripheral neuroblastic tumors recapitulates the changes during normal development of PSNS, as previously reported for Bcl-2 oncoprotein, c-ErbB2, insulin-like growth factor 2, β-2-microglobulin, and cathepsin D. This is consistent with the current view that childhood peripheral neuroblastic tumors exhibit gene expression profiles mirroring those occurring during PSNS development.

1α,25 dihydroxi-vitamin D3 modulates CDK4 and CDK6 expression and localization

We recently reported that the vitamin D receptor (VDR) and p38 MAPK participate in pro-differentiation events triggered by 1α,25(OH)2-vitamin D3 [1,25D] in skeletal muscle cells. Specifically, our studies demonstrated that 1,25D promotes G0/G1 arrest of cells inducing cyclin D3 and cyclin dependent kinases inhibitors (CKIs) p21Waf1/Cip1 and p27Kip1 expression in a VDR and p38 MAPK dependent manner. In this work we present data indicating that cyclin-dependent kinases (CDKs) 4 and 6 also play a role in the mechanism by which 1,25D stimulates myogenesis. To investigate VDR involvement in hormone regulation of CDKs 4 and 6, we significantly reduced its expression by the use of a shRNA against mouse VDR, generating the skeletal muscle cell line C2C12-VDR. Investigation of changes in cellular cycle regulating proteins by immunoblotting showed that the VDR is involved in the 1,25D –induced CDKs 4 and 6 protein levels at 6 h of hormone treatment. CDK4 levels remains high during S phase peak and G0/G1 arrest while CDK6 expression decreases at 12 h and increases again al 24 h. The up-regulation of CDKs 4 and 6 by 1,25D (6 h) was abolished in C2C12 cells pre-treated with the ERK1/2 inhibitor, UO126. Moreover, CDKs 4 and 6 expression induced by the hormone nor was detected when α and β isoforms of p38 MAPK were inhibited by compound SB203580. Confocal images show that there is not co-localization between VDR and CDKs at 6 h of hormone treatment, however CDK4 and VDR co-localizates in nucleus after 12 h of 1,25D exposure. Of relevance, at this time 1,25D promotes CDK6 localization in a peri-nuclear ring. Our data demonstrate that the VDR, ERK1/2 and p38 MAPK are involved in the control of CDKs 4 and 6 by 1,25D in skeletal muscle cells sustaining the operation of a VDR and MAPKs –dependent mechanism in hormone modulation of myogenesis.

Unraveling the role of Cdk1 in postnatal neurogenesis

Event Abstract Back to Event Unraveling the role of Cdk1 in postnatal neurogenesis Quentin Marlier1*, Sebastien Verteneuil1*, Renaud Vandenbosch1, Nicolas Caron2, Philipp Kaldis3, Laurent Nguyen1 and Brigitte Malgrange1 1 GIGA Neurosciences-University of Liege, Belgium 2 Affichem, France 3 Institute of Molecular and Cell Biology, Singapore Age-related neurological disorders, including stroke and neurodegenerative diseases are becoming a major health care problem in many countries as average life expectancy is increasing. One appealing therapeutic strategy to treat neurological disorders would consist of recruiting endogenous neural precursor cells (NPCs) to replace the lost neurons. NPCs are present in two specific areas of the postnatal brain, the dentate gyrus (DG) of the hippocampus and the subventricular zone (SVZ) of the lateral ventricles. These NPCs give rise to neurons throughout life, a phenomenon termed postnatal neurogenesis. A prerequisite for the development of new therapeutic strategies involving postnatal NPCs is a better understanding of their molecular regulation. In this context, both extrinsic factors and intrinsic factors have been identified. For instance, the family of cell cycle regulators cyclin-dependent kinases (Cdks) are key regulators of postnatal neurogenesis since genetic invalidation of interphase Cdk2 or Cdk6 leads to a decrease of NPCs proliferation. Here, we investigated the role of Cdk1, an essential Cdk for M-phase in several tissues, in postnatal neurogenesis. Towards that purpose, we crossed mice bearing a conditional Cdk1 allele (Cdk1lox) with mice expressing a tamoxifen-inducible form of the Cre-recombinase under the control of the Sox2 promoter (Sox2CreER), allowing us to specifically delete Cdk1 in Sox2+ NPCs in the postnatal brain. Following Cdk1 loss, our preliminary results show a decrease in the percentage of proliferating Sox2+ cells as well as a decrease in the number of phospho-histone H3+ (ph-h3) in the DG, but there is no difference in the number of cells positive for the apoptotic marker activated-caspase 3. Interestingly, we also observed a long-term depletion of the pool of Sox2+ in the absence of Cdk1. Altogether, these data suggest a crucial role for Cdk1 in postnatal neurogenesis, but further investigations are required to identify its precise requirement in this process. Keywords: adult neurogenesis, Cell Cycle, Cdk1, Neural Stem Cells, proliferation Conference: 11th National Congress of the Belgian Society for Neuroscience, Mons, Belgium, 22 May - 22 May, 2015. Presentation Type: Poster presentation Topic: Neuroscience Citation: Marlier Q, Verteneuil S, Vandenbosch R, Caron N, Kaldis P, Nguyen L and Malgrange B (2015). Unraveling the role of Cdk1 in postnatal neurogenesis. Front. Neurosci. Conference Abstract: 11th National Congress of the Belgian Society for Neuroscience. doi: 10.3389/conf.fnins.2015.89.00069 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 30 Apr 2015; Published Online: 05 May 2015. * Correspondence: Mr. Quentin Marlier, GIGA Neurosciences-University of Liege, Liège, 4000, Belgium, quentin.marlier@ulg.ac.be Mr. Sebastien Verteneuil, GIGA Neurosciences-University of Liege, Liège, 4000, Belgium, sebastien.verteneuil@hotmail.fr Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Quentin Marlier Sebastien Verteneuil Renaud Vandenbosch Nicolas Caron Philipp Kaldis Laurent Nguyen Brigitte Malgrange Google Quentin Marlier Sebastien Verteneuil Renaud Vandenbosch Nicolas Caron Philipp Kaldis Laurent Nguyen Brigitte Malgrange Google Scholar Quentin Marlier Sebastien Verteneuil Renaud Vandenbosch Nicolas Caron Philipp Kaldis Laurent Nguyen Brigitte Malgrange PubMed Quentin Marlier Sebastien Verteneuil Renaud Vandenbosch Nicolas Caron Philipp Kaldis Laurent Nguyen Brigitte Malgrange Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Proteomic Analysis of the Human Cyclin-dependent Kinase Family Reveals a Novel CDK5 Complex Involved in Cell Growth and Migration

Cyclin-dependent kinases (CDKs) are the catalytic subunits of a family of mammalian heterodimeric serine/threonine kinases that play critical roles in the control of cell-cycle progression, transcription, and neuronal functions. However, the functions, substrates, and regulation of many CDKs are poorly understood. To systematically investigate these features of CDKs, we conducted a proteomic analysis of the CDK family and identified their associated protein complexes in two different cell lines using a modified SAINT (Significance Analysis of INTeractome) method. The mass spectrometry data were deposited to ProteomeXchange with identifier PXD000593 and DOI 10.6019/PXD000593. We identified 753 high-confidence candidate interaction proteins (HCIPs) in HEK293T cells and 352 HCIPs in MCF10A cells. We subsequently focused on a neuron-specific CDK, CDK5, and uncovered two novel CDK5-binding partners, KIAA0528 and fibroblast growth factor (acidic) intracellular binding protein (FIBP), in non-neuronal cells. We showed that these three proteins form a stable complex, with KIAA0528 and FIBP being required for the assembly and stability of the complex. Furthermore, CDK5-, KIAA0528-, or FIBP-depleted breast cancer cells displayed impaired proliferation and decreased migration, suggesting that this complex is required for cell growth and migration in non-neural cells. Our study uncovers new aspects of CDK functions, which provide direction for further investigation of these critical protein kinases.

C/EBPα is an essential collaborator in Hoxa9/Meis1-mediated leukemogenesis.

Homeobox A9 (HOXA9) is a homeodomain-containing transcription factor that plays a key role in hematopoietic stem cell expansion and is commonly deregulated in human acute leukemias. A variety of upstream genetic alterations in acute myeloid leukemia (AML) lead to overexpression of HOXA9, almost always in association with overexpression of its cofactor meis homeobox 1 (MEIS1) . A wide range of data suggests that HOXA9 and MEIS1 play a synergistic causative role in AML, although the molecular mechanisms leading to transformation by HOXA9 and MEIS1 remain elusive. In this study, we identify CCAAT/enhancer binding protein alpha (C/EBPα) as a critical collaborator required for Hoxa9/Meis1-mediated leukemogenesis. We show that C/EBPα is required for the proliferation of Hoxa9/Meis1-transformed cells in culture and that loss of C/EBPα greatly improves survival in both primary and secondary murine models of Hoxa9/Meis1-induced leukemia. Over 50% of Hoxa9 genome-wide binding sites are cobound by C/EBPα, which coregulates a number of downstream target genes involved in the regulation of cell proliferation and differentiation. Finally, we show that Hoxa9 represses the locus of the cyclin-dependent kinase inhibitors Cdkn2a/b in concert with C/EBPα to overcome a block in G1 cell cycle progression. Together, our results suggest a previously unidentified role for C/EBPα in maintaining the proliferation required for Hoxa9/Meis1-mediated leukemogenesis.

A role for Drosophila Cyclin J in oogenesis revealed by genetic interactions with the piRNA pathway

Cyclin J (CycJ) is a poorly characterized member of the Cyclin superfamily of cyclin-dependent kinase regulators, many of which regulate the cell cycle or transcription. Although CycJ is conserved in metazoans its cellular function has not been identified and no mutant defects have been described. In Drosophila, CycJ transcript is present primarily in ovaries and very early embryos, suggesting a role in one or both of these tissues. The CycJ gene (CycJ) lies immediately downstream of armitage (armi), a gene involved in the Piwi-associated RNA (piRNA) pathways that are required for silencing transposons in the germline and adjacent somatic cells. Mutations in armi result in oogenesis defects but a role for CycJ in oogenesis has not been defined. Here we assessed oogenesis in CycJ mutants in the presence or absence of mutations in armi or other piRNA pathway genes. CycJ null ovaries appeared normal, indicating that CycJ is not essential for oogenesis under normal conditions. In contrast, armi null ovaries produced only two egg chambers per ovariole and the eggs had severe axis specification defects, as observed previously for armi and other piRNA pathway mutants. Surprisingly, the CycJ armi double mutant failed to produce any mature eggs. The double null ovaries generally had only one egg chamber per ovariole and the egg chambers frequently contained an overabundance of differentiated germline cells. Production of these compound egg chambers could be suppressed with CycJ transgenes but not with mutations in the checkpoint gene mnk, which suppress oogenesis defects in armi mutants. The CycJ null showed similar genetic interactions with the germline and somatic piRNA pathway gene piwi, and to a lesser extent with aubergine (aub), a member of the germline-specific piRNA pathway. The strong genetic interactions between CycJ and piRNA pathway genes reveal a role for CycJ in early oogenesis. Our results suggest that CycJ is required to regulate egg chamber production or maturation when piRNA pathways are compromised.

TGF-β-Induced (TGFBI) Protein in Melanoma: A Signature of High Metastatic Potential

Tumor-produced extracellular matrix (ECM) proteins can be key elements in tumor growth and metastasis. Transforming growth factor beta-inducible (TGFBI) protein is a secreted ECM component that can have dual function in cancer, acting as tumor suppressor or promoter. Although TGFBI is expressed in human melanoma cells, the exact role it might have in melanoma metastasis remains elusive. Assessing the expression and secretion of TGFBI, we show that human metastatic melanomas express and secrete significantly higher amounts of TGFBI, compared with nevus lesions and primary melanoma tumors. Intravenous injection of highly metastatic human melanoma cells expressing shRNA that targets TGFBI assigns a critical role for TGFBI in the formation of melanoma distal metastases in nude mice. In vivo assays demonstrate that TGFBI silencing does not interfere with melanoma cells' dissemination to distal sites but rather with their proliferation and outgrowth within new microenvironment. In line, TGFBI silencing increases melanoma cells motility/invasion/extravasation in vitro but interferes with their progression through the cell cycle, drastically reducing their proliferation. Furthermore, we show that TGFBI is a regulator of cyclins and cyclin-dependent kinases in melanoma. Collectively, our data describe a mechanism of melanoma metastatic outgrowth via promotion of growth/survival by the ECM protein TGFBI.

Proteomics of a fuzzy organelle: interphase chromatin.

Chromatin proteins mediate replication, regulate expression, and ensure integrity of the genome. So far, a comprehensive inventory of interphase chromatin has not been determined. This is largely due to its heterogeneous and dynamic composition, which makes conclusive biochemical purification difficult, if not impossible. As a fuzzy organelle, it defies classical organellar proteomics and cannot be described by a single and ultimate list of protein components. Instead, we propose a new approach that provides a quantitative assessment of a protein's probability to function in chromatin. We integrate chromatin composition over a range of different biochemical and biological conditions. This resulted in interphase chromatin probabilities for 7635 human proteins, including 1840 previously uncharacterized proteins. We demonstrate the power of our large-scale data-driven annotation during the analysis of cyclin-dependent kinase (CDK) regulation in chromatin. Quantitative protein ontologies may provide a general alternative to list-based investigations of organelles and complement Gene Ontology.

Convergence Of Hypoxia and TGFβ Pathways On Cell Cycle Regulation In Human Hematopoietic Stem/Progenitor Cells

Hematopoietic stem cells reside within specialized hypoxic niches in the bone marrow where they are kept in a relative quiescent state. One of the key pathways activated under low oxygen conditions is the Hypoxia Induced Factor (HIF) pathway. HIF1 and HIF2 act as oxygen sensors that are degraded by the Von Hippel Lindau (VHL) tumor suppressor protein under normoxic conditions but not when oxygen levels are low, resulting in stabilization of the HIF proteins, translocation to the nucleus and initiation of target gene transcription.Although it has been shown that HIF1 and 2 fulfill essential roles in the regulation of HSC fate, little is known about the mechanisms that are involved. Here, we set out to investigate the effects of hypoxia, HIF1 and HIF2 on gene transcription in human hematopoietic stem/progenitor cells. Cord blood (CB) CD34+ cells were cultured under low oxygen conditions (2%), or were lentivirally transduced with constitutively active HIF1(P402/564) and HIF2(P405/531) constructs under normoxic conditions and after 24 hrs transcriptome changes were analyzed by Illumina BeadArray analysis. This provided the possibility to identify common hypoxia-HIF1-HIF2 gene signatures, but also the identification of specific target genes that were exclusively regulated by HIF1, HIF2 or hypoxia. Geneset enrichment analysis (GSEA) using Gene Ontology genesets revealed that overexpression of HIF1 and -2 resulted in significant enrichment for known pathways like “hypoxia induced signaling”, but unexpectedly also for the Transforming Growth Factor beta (TGFβ) pathway. GSEA using a published dataset of TGFβ stimulated CB CD34+ cells indeed confirmed a high correlation between hypoxia target genes and TGFβ induced genes. Two of the most significantly upregulated genes in both gene sets were the cyclin dependent kinase inhibitor CDKN1C (p57kip2) and Regulator of G-protein signaling (RGS)1.q-RT-PCR analysis demonstrated enhanced expression of CDKN1C by hypoxia treatment or HIF overexpression under normoxia in combination with TGFβ stimulation. Although it was demonstrated that CD34+cells cultured under hypoxic conditions secreted high levels of latent TGFβ, no rescue of the hypoxia induced cell cycle arrest was demonstrated by knockdown of SMAD4, arguing against direct effects of hypoxia-induced secreted TGFβ on cell cycle quiescence.RGS1 is a member of the RGS family, involved in the negative regulation of G-protein coupled receptor signaling. Overexpression studies under normoxic conditions in CB CD34+cells demonstrated a decrease of SDF1-mediated migration. Furthermore, overexpression of RGS1 attenuated SDF1 and GM-CSF-induced ERK phosphorylation whereas the GM-CSF-induced STAT5 tyrosine phosphorylation was unaffected. These findings indicate that RGS1 can interfere with specific signaling pathways involved in the regulation of cell proliferation and migration.Analysis of the CDKN1C as well as the RGS1 promoters revealed binding sites for both HIF and SMAD2/3/4 in the proximal part, suggesting that both pathways can indeed converge on the regulation of these important proteins that control cell cycle progression and the response to stimulatory cytokines in human stem/progenitor cells. Disclosures:No relevant conflicts of interest to declare.

KPNA2 promotes cell proliferation and tumorigenicity in epithelial ovarian carcinoma through upregulation of c-Myc and downregulation of FOXO3a

Karyopherin alpha 2 (KPNA2), a member of the karyopherin family, has a central role in nucleocytoplasmic transport and is overexpressed in many cancers. Our previous study identified KPNA2 as significantly upregulated in epithelial ovarian carcinoma (EOC), correlating with poor survival of patients. However, the precise mechanism of this effect remains unclear. The aim of the present study was to examine the role of KPNA2 in the proliferation and tumorigenicity of EOC cells, and its clinical significance in tumor progression. Real-time quantitative RT-PCR analysis revealed high expression levels of KPNA2 in 162 out of 191 (84.8%) fresh EOC tissues, which was significantly correlated with International Federation of Gynecology and Obstetrics (FIGO) stage, differentiation, histological type, recurrence, and prognosis of EOC patients. Our results showed that upregulation of KPNA2 expression significantly increased the proliferation and tumorigenicity of EOC cells (EFO-21 and SK-OV3) in vitro and in vivo, by promoting cell growth rate, foci formation, soft agar colony formation, and tumor formation in nude mice. By contrast, knockdown of KPNA2 effectively suppressed the proliferation and tumorigenicity of these EOC cells in vitro and in vivo. Our results also indicated that the molecular mechanisms of the effect of KPNA2 in EOC included promotion of G1/S cell cycle transition through upregulation of c-Myc, enhanced transcriptional activity of c-Myc, activation of Akt activity, suppression of FOXO3a activity, downregulation of cyclin-dependent kinase (CDK) inhibitor p21Cip1 and p27Kip1, and upregulation of CDK regulator cyclin D1. Our results show that KPNA2 has an important role in promoting proliferation and tumorigenicity of EOC, and may represent a novel prognostic biomarker and therapeutic target for this disease.

Dynamics of SIN Asymmetry Establishment

Timing of cell division is coordinated by the Septation Initiation Network (SIN) in fission yeast. SIN activation is initiated at the two spindle pole bodies (SPB) of the cell in metaphase, but only one of these SPBs contains an active SIN in anaphase, while SIN is inactivated in the other by the Cdc16-Byr4 GAP complex. Most of the factors that are needed for such asymmetry establishment have been already characterized, but we lack the molecular details that drive such quick asymmetric distribution of molecules at the two SPBs. Here we investigate the problem by computational modeling and, after establishing a minimal system with two antagonists that can drive reliable asymmetry establishment, we incorporate the current knowledge on the basic SIN regulators into an extended model with molecular details of the key regulators. The model can capture several peculiar earlier experimental findings and also predicts the behavior of double and triple SIN mutants. We experimentally tested one prediction, that phosphorylation of the scaffold protein Cdc11 by a SIN kinase and the core cell cycle regulatory Cyclin dependent kinase (Cdk) can compensate for mutations in the SIN inhibitor Cdc16 with different efficiencies. One aspect of the prediction failed, highlighting a potential hole in our current knowledge. Further experimental tests revealed that SIN induced Cdc11 phosphorylation might have two separate effects. We conclude that SIN asymmetry is established by the antagonistic interactions between SIN and its inhibitor Cdc16-Byr4, partially through the regulation of Cdc11 phosphorylation states.

Differential regulation of cyclin-dependent kinase inhibitors in neuroblastoma cells

Gastrin-releasing peptide (GRP) and its receptor (GRP-R) are highly expressed in undifferentiated neuroblastoma, and they play critical roles in oncogenesis. We previously reported that GRP activates the PI3K/AKT signaling pathway to promote DNA synthesis and cell cycle progression in neuroblastoma cells. Conversely, GRP-R silencing induces cell cycle arrest. Here, we speculated that GRP/GRP-R signaling induces neuroblastoma cell proliferation via regulation of cyclin-dependent kinase (CDK) inhibitors. Surprisingly, we found that GRP/GRP-R differentially induced expressions of p21 and p27. Silencing GRP/GRP-R decreased p21, but it increased p27 expressions in neuroblastoma cells. Furthermore, we found that the intracellular localization of p21 and p27 in the nuclear and cytoplasmic compartments, respectively. In addition, we found that GRP/GRP-R silencing increased the expression and accumulation of PTEN in the cytoplasm of neuroblastoma cells where it co-localized with p27, thus suggesting that p27 promotes the function of PTEN as a tumor suppressor by stabilizing PTEN in the cytoplasm. GRP/GRP-R regulation of CDK inhibitors and tumor suppressor PTEN may be critical for tumoriogenesis of neuroblastoma.

Regulation of integrin αV subunit expression by sulfatide in hepatocellular carcinoma cells

Integrin is important in migration and metastasis of tumor cells. Changes of integrin expression and distribution will cause an alteration of cellular adhesion and migration behaviors. In this study, we investigated sulfatide regulation of the integrin αV subunit expression in hepatoma cells and observed that either exogenous or endogenous sulfatide elicited a robust upregulation of integrin αV subunit mRNA and protein expression in hepatoma cells. This regulatory effect occurred with a corresponding phosphorylation (T739) of the transcription factor Sp1. Based on the electrophoretic mobility shift assay, sulfatide enhanced the integrin αV promoter activity and strengthened the Sp1 complex super-shift. The results of chromatin immunoprecipitation analysis also indicated that sulfatide enhanced Sp1 binding to the integrin αV promoter in vivo. Silence of Sp1 diminished the stimulation of integrin αV expression by sulfatide. In the early stage of sulfatide stimulation, phosphorylation of Erk as well as c-Src was noted, and inhibition of Erk activation with either U0126 or PD98059 significantly suppressed Sp1 phosphorylation and integrin αV expression. We demonstrated that sulfatide regulated integrin αV expression and cell adhesion, which was associated with Erk activation.

Reversal of paclitaxel-chemoresistance by mixed Pluronic P105/L101 micelles in human ovarian cancer SKOV-3/PTX cells

A mixed Pluronic micelle for delivery of paclitaxel (PTX) with Pluronic P105 and L101 was capable of sensitizing resistant SKOV-3/PTX tumor cells. A significant inhibition of P-gp efflux system was observed in the range of monomer or micelle concentrations of the mixed Pluronics in the resistant tumor cells. Importantly, the mixed micellar PTX also showed enhanced apoptotic effect, and drastically changed the pharmacogenomic responses to free PTX. It was found that some important genes for apoptosis induction, such as BCL-2 antagonist and death domain receptor, displayed elevated levels; some cell cycle-arresting genes, such as cyclins, cyclin-dependent kinase regulator, cullin and forhead box M, showed significant down-regulated levels; and some clinical prognostic genes for resistance to chemotherapy and metabolism, such as drug efflux pump and cytochrome P450, were also down-regulated. It is concluded that inhibition of P-gp efflux system, enhanced induction of apoptosis, and significant changes of expression profiles of apoptosis, resistance and cell cycle arresting-related genes were responsible for PTX-resistance reversal by the mixed Pluronic P105/L101 micelles in the SKOV-3/PTX tumor cells.