UMR3348 – Intégrité du génome, ARN et cancer

Publications de l’unité

Année de publication : 2016

Tony Sourisseau, Carole Helissey, Céline Lefebvre, Florence Ponsonnailles, Hélène Malka-Mahieu, Ken A Olaussen, Fabrice André, Stephan Vagner, Jean-Charles Soria (2016 Jan 29)

Translational regulation of the mRNA encoding the ubiquitin peptidase USP1 involved in the DNA damage response as a determinant of Cisplatin resistance.

Cell cycle (Georgetown, Tex.) : 295-302 : DOI : 10.1080/15384101.2015.1120918 En savoir plus
Résumé

Cisplatin (cis-diaminedichloroplatin (II), CDDP) is part of the standard therapy for a number of solid tumors including Non-Small-Cell Lung Cancer (NSCLC). The initial response observed is in most cases only transient and tumors quickly become refractory to the drug. Tumor cell resistance to CDDP relies on multiple mechanisms, some of which still remain unknown. In search for such mechanisms, we examined the impact of CDDP on mRNA translation in a sensitive and in a matched resistant NSCLC cell line. We identified a set of genes whose mRNAs are differentially translated in CDDP resistant vs. sensitive cells. The translation of the mRNA encoding the Ubiquitin-Specific Peptidase 1 (USP1), a Ubiquitin peptidase with important function in multiple DNA repair pathways, is inhibited by CDDP exposure in the sensitive cells, but not in the resistant cells. This lack of down-regulation of USP1 expression at the translational level plays a primary role in CDDP resistance since inhibition of USP1 expression or activity by siRNA or the small molecule inhibitor ML323, respectively is sufficient to re-sensitize resistant cells to CDDP. We involved the USP1 mRNA translation as a major mechanism of CDDP resistance in NSCLC cells and suggest that USP1 could be evaluated as a candidate predictive marker and as a therapeutic target to overcome CDDP resistance. More generally, our results indicate that analysis of gene expression at the level of mRNA translation is a useful approach to identify new determinants of CDDP resistance.

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M Blosa, C Bursch, S Weigel, M Holzer, C Jäger, C Janke, R T Matthews, T Arendt, M Morawski (2016 Jan 1)

Reorganization of Synaptic Connections and Perineuronal Nets in the Deep Cerebellar Nuclei of Purkinje Cell Degeneration Mutant Mice.

Neural plasticity : 2828536 : DOI : 10.1155/2016/2828536 En savoir plus
Résumé

The perineuronal net (PN) is a subtype of extracellular matrix appearing as a net-like structure around distinct neurons throughout the whole CNS. PNs surround the soma, proximal dendrites, and the axonal initial segment embedding synaptic terminals on the neuronal surface. Different functions of the PNs are suggested which include support of synaptic stabilization, inhibition of axonal sprouting, and control of neuronal plasticity. A number of studies provide evidence that removing PNs or PN-components results in renewed neurite growth and synaptogenesis. In a mouse model for Purkinje cell degeneration, we examined the effect of deafferentation on synaptic remodeling and modulation of PNs in the deep cerebellar nuclei. We found reduced GABAergic, enhanced glutamatergic innervations at PN-associated neurons, and altered expression of the PN-components brevican and hapln4. These data refer to a direct interaction between ECM and synapses. The altered brevican expression induced by activated astrocytes could be required for an adequate regeneration by promoting neurite growth and synaptogenesis.

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Année de publication : 2015

Mónica T Fernandes, Marinella N Ghezzo, André B Silveira, Ravi K Kalathur, Vanda Póvoa, Ana R Ribeiro, Sílvia R Brandalise, Emmanuel Dejardin, Nuno L Alves, Jacques Ghysdael, João T Barata, José Andres Yunes, Nuno R dos Santos (2015 Dec 1)

Lymphotoxin-β receptor in microenvironmental cells promotes the development of T-cell acute lymphoblastic leukaemia with cortical/mature immunophenotype.

British journal of haematology : 736-51 : DOI : 10.1111/bjh.13760 En savoir plus
Résumé

Lymphotoxin-mediated activation of the lymphotoxin-β receptor (LTβR; LTBR) has been implicated in cancer, but its role in T-cell acute lymphoblastic leukaemia (T-ALL) has remained elusive. Here we show that the genes encoding lymphotoxin (LT)-α and LTβ (LTA, LTB) are expressed in T-ALL patient samples, mostly of the TAL/LMO molecular subtype, and in the TEL-JAK2 transgenic mouse model of cortical/mature T-ALL (Lta, Ltb). In these mice, expression of Lta and Ltb is elevated in early stage T-ALL. Surface LTα1 β2 protein is expressed in primary mouse T-ALL cells, but only in the absence of microenvironmental LTβR interaction. Indeed, surface LT expression is suppressed in leukaemic cells contacting Ltbr-expressing but not Ltbr-deficient stromal cells, both in vitro and in vivo, thus indicating that dynamic surface LT expression in leukaemic cells depends on interaction with its receptor. Supporting the notion that LT signalling plays a role in T-ALL, inactivation of Ltbr results in a significant delay in TEL-JAK2-induced leukaemia onset. Moreover, young asymptomatic TEL-JAK2;Ltbr(-/-) mice present markedly less leukaemic thymocytes than age-matched TEL-JAK2;Ltbr(+/+) mice and interference with LTβR function at this early stage delayed T-ALL development. We conclude that LT expression by T-ALL cells activates LTβR signalling in thymic stromal cells, thus promoting leukaemogenesis.

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Juan S Martinez, Céline Baldeyron, Aura Carreira (2015 Nov 13)

Molding BRCA2 function through its interacting partners.

Cell cycle (Georgetown, Tex.) : 3389-95 : DOI : 10.1080/15384101.2015.1093702 En savoir plus
Résumé

The role of the tumor suppressor BRCA2 has been shaped over 2 decades thanks to the discovery of its protein and nucleic acid partners, biochemical and structural studies of the protein, and the functional evaluation of germline variants identified in breast cancer patients. Yet, the pathogenic and functional effect of many germline mutations in BRCA2 remains undetermined, and the heterogeneity of BRCA2-associated tumors challenges the identification of causative variants that drive tumorigenesis. In this review, we propose an overview of the established and emerging interacting partners and functional pathways attributed to BRCA2, and we speculate on how variants altering these functions may contribute to cancer susceptibility.

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Diana Passaro, Christine Tran Quang, Jacques Ghysdael (2015 Sep 12)

Calcineurin/CXCR4 in T-ALL.

Oncoscience : 781-2 : DOI : 10.18632/oncoscience.238 En savoir plus
Résumé

The calcineurin/NFAT signaling pathway is implicated in a wide variety of biological processes, acting as a bridge pathway between calcium signals and gene expression. Although its role as an effector of immune responses figured prominently in early studies, it forms just one part of a larger picture. Indeed calcineurin has been shown to participate in the development and function of e.g. the immune, cardiovascular, nervous and musculoskeletal systems, and dysregulation of calcineurin/NFAT signaling contributes to pathologies affecting these tissues, in particular cancer [1].

Activation of the calcineurin/NFAT pathway was first observed in human lymphoma, as well as in mouse models of T cell acute lymphoblastic leukemia (T-ALL) and xenotransplanted human T-ALL [2]. In T-ALL, calcineurin activation is independent of preTCR/TCR signaling (the major calcineurin activator in normal T cell progenitors), but strongly depends upon micro-environmental signals. The fundamental, intrinsic role of calcineurin in T-ALL was demonstrated in several mouse models, in which conditional calcineurin genetic deletion was restricted to leukemic cells. Calcineurin was found essential to the physical and functional interactions that leukemic cells establish with supportive stromal cells, with its deletion resulting in impaired leukemia propagation, reduced cell survival, proliferation, migration and homing [3]. The therapeutic relevance of these findings was highlighted by preclinical studies showing strong anti-leukemic effects of calcineurin inhibitors (namely cyclosporin A or tacrolimus [FK506]) and long-term leukemia remission in a mouse T-ALL model when vincristine treatment was combined with calcineurin genetic inactivation [2, 3]. However, available calcineurin inhibitors appear suboptimal as potential therapeutic agents since they are associated with a number of toxic side effects [4], show clear off-target effects in T-ALL cells [3] and are expected to interfere with the anti-tumor immune response.

A recently developed, alternative option is to identify and target molecular pathways acting downstream of calcineurin and critical to T-ALL maintenance [5]. Our global transcriptomic analysis identified a large number of calcineurin-dependent genes in T-ALL, involved in an array of biological function, including the de-repression of known tumor suppressive pathways (e.g. CDKN1A) [3]. Although of high biological interest, these deregulations are not easily accessible for targeted therapy. In contrast, genes/proteins implicated in the adhesion/migration to the bone marrow microenvironment are promising candidates (i) for a thorough understanding of the factors that contribute to microenvironment-mediated support of leukemia progression and (ii) for the design of niche-targeted therapies. Along these lines, we linked calcineurin-dependent regulation of the adhesive/migratory properties of T-ALL cells to a boost of CXCR4 surface expression and the subsequent ability of the leukemic cells to respond to CXCL12 [5]. Upregulation of CXCR4 cell surface expression was also demonstrated in diagnostic T-ALL cases and primary xenograft in NSG mice [5][6]. The mechanism by which calcineurin affects CXCR4 trafficking is partially explained by the Cn-dependent up-regulation of cortactin [5], an actin-binding protein implicated in the regulation of endosomal trafficking [7]. Because actin polymerization is required for CXCR4 and other chemokine receptors trafficking to recycling vesicles, inhibition of cortactin expression in calcineurin-deficient T-ALL cells likely results in impaired actin dynamics in this endosomal compartment. Further investigation of the intrinsic function of CXCR4 in murine and human T-ALL revealed an important role of CXCL12/CXCR4 signaling in both survival/proliferation and homing/migration of leukemic cells to the supportive bone marrow niche [5][6]. Intravital multiphoton imaging and genetic studies revealed a strong interaction between T-ALL cells and CXCL12-expressing niche(s), and an essential supportive function of CXCL12 produced by vascular endothelial cells [6]. Local CXCL12 production, in addition to induction of CXCR4-dependent signaling cues will result in activation of T-ALL cells specific integrins, further stabilizing adhesion to integrin ligands expressing niche(s) and induction of additional pro-survival signals. In this scenario, the nature of the niche cells expressing the integrin ligands requires further characterization.

Strikingly, CXCR4 is also critical to leukemia initiating cell activity (LIC) in murine T-ALL and human xenografts [5][6], highlighting an unexpected, fundamental function of microenvironmental signals for T-ALL maintenance and progression. Many inhibitors of CXCL12 or CXCR4 have been developed and are tested in clinical studies in other pathological contexts, in particular other hematological malignancies [8]. However, only in T-ALL anti-CXCR4 monotherapy shows strong efficacy, suggesting a strong dependence of these tumor cells on Editorial CXCR4 signaling [6]. As relapse in T-ALL remains a challenging issue, these new data call for clinical trials to incorporate CXCR4 antagonists either as single agents following induction therapy, or as part of the first induction therapy regimen or later, during the consolidation phase.

In conclusion, the calcineurin/NFAT pathway acts as a fundamental bridge between microenvironmental-derived signals and T-ALL cells, mediating a complex crosstalk that is so far only partially dissected, but that already lead to the identification of novel targets of therapeutic relevance to T-ALL treatment.

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Carsten Janke, Michel O Steinmetz (2015 Aug 13)

Optochemistry to control the microtubule cytoskeleton.

The EMBO journal : 2114-6 : DOI : 10.15252/embj.201592415 En savoir plus
Résumé

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Sara Chiker, Vincent Pennaneach, Damarys Loew, Florent Dingli, Denis Biard, Fabrice P Cordelières, Simon Gemble, Sophie Vacher, Ivan Bieche, Janet Hall, Marie Fernet (2015 Aug 3)

Cdk5 promotes DNA replication stress checkpoint activation through RPA-32 phosphorylation, and impacts on metastasis free survival in breast cancer patients.

Cell cycle (Georgetown, Tex.) : 3066-78 : DOI : 10.1080/15384101.2015.1078020 En savoir plus
Résumé

Cyclin dependent kinase 5 (Cdk5) is a determinant of PARP inhibitor and ionizing radiation (IR) sensitivity. Here we show that Cdk5-depleted (Cdk5-shRNA) HeLa cells show higher sensitivity to S-phase irradiation, chronic hydroxyurea exposure, and 5-fluorouracil and 6-thioguanine treatment, with hydroxyurea and IR sensitivity also seen in Cdk5-depleted U2OS cells. As Cdk5 is not directly implicated in DNA strand break repair we investigated in detail its proposed role in the intra-S checkpoint activation. While Cdk5-shRNA HeLa cells showed altered basal S-phase dynamics with slower replication velocity and fewer active origins per DNA megabase, checkpoint activation was impaired after a hydroxyurea block. Cdk5 depletion was associated with reduced priming phosphorylations of RPA32 serines 29 and 33 and SMC1-Serine 966 phosphorylation, lower levels of RPA serine 4 and 8 phosphorylation and DNA damage measured using the alkaline Comet assay, gamma-H2AX signal intensity, RPA and Rad51 foci, and sister chromatid exchanges resulting in impaired intra-S checkpoint activation and subsequently higher numbers of chromatin bridges. In vitro kinase assays coupled with mass spectrometry demonstrated that Cdk5 can carry out the RPA32 priming phosphorylations on serines 23, 29, and 33 necessary for this checkpoint activation. In addition we found an association between lower Cdk5 levels and longer metastasis free survival in breast cancer patients and survival in Cdk5-depleted breast tumor cells after treatment with IR and a PARP inhibitor. Taken together, these results show that Cdk5 is necessary for basal replication and replication stress checkpoint activation and highlight clinical opportunities to enhance tumor cell killing.

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Magali Grange, Marilyn Giordano, Amandine Mas, Romain Roncagalli, Guylène Firaguay, Jacques A Nunes, Jacques Ghysdael, Anne-Marie Schmitt-Verhulst, Nathalie Auphan-Anezin (2015 Aug 1)

Control of CD8 T cell proliferation and terminal differentiation by active STAT5 and CDKN2A/CDKN2B.

Immunology : 543-57 : DOI : 10.1111/imm.12471 En savoir plus
Résumé

CD8 T cells used in adoptive immunotherapy may be manipulated to optimize their effector functions, tissue-migratory properties and long-term replicative potential. We reported that antigen-stimulated CD8 T cells transduced to express an active form of the transcription factor signal transducer and activator of transcription 5 (STAT5CA) maintained these properties upon adoptive transfer. We now report on the requirements of STAT5CA-expressing CD8 T cells for cell survival and proliferation in vivo. We show that STAT5CA expression allows for greater expansion of T cells in vivo, while preserving dependency on T-cell-receptor-mediated tonic stimulation for their in vivo maintenance and return to a quiescent stage. STAT5CA expression promotes the formation of a large pool of effector memory T cells that respond upon re-exposure to antigen and present an increased sensitivity to γc receptor cytokine engagement for STAT5 phosphorylation. In addition, STAT5CA expression prolongs the survival of what would otherwise be short-lived terminally differentiated KLRG1-positive effector cells with up-regulated expression of the senescence-associated p16(INK) (4A) transcripts. However, development of a KLRG1-positive CD8 T cell population was independent of either p16(INK) (4A) or p19(ARF) expression (as shown using T cells from CDKN2A(-/-) mice) but was associated with expression of transcripts encoding p15(INK) (4B) , another protein involved in senescence induction. We conclude that T-cell-receptor- and cytokine-dependent regulation of effector T cell homeostasis, as well as mechanisms leading to senescent features of a population of CD8 T cells are maintained in STAT5CA-expressing CD8 T cells, even for cells that are genetically deficient in expression of the tumour suppressors p16(INK) (4A) and p19(ARF) .

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Simon Gemble, Akshay Ahuja, Géraldine Buhagiar-Labarchède, Rosine Onclercq-Delic, Julien Dairou, Denis S F Biard, Sarah Lambert, Massimo Lopes, Mounira Amor-Guéret (2015 Jul 16)

Pyrimidine Pool Disequilibrium Induced by a Cytidine Deaminase Deficiency Inhibits PARP-1 Activity, Leading to the Under Replication of DNA.

PLoS genetics : e1005384 : DOI : 10.1371/journal.pgen.1005384 En savoir plus
Résumé

Genome stability is jeopardized by imbalances of the dNTP pool; such imbalances affect the rate of fork progression. For example, cytidine deaminase (CDA) deficiency leads to an excess of dCTP, slowing the replication fork. We describe here a novel mechanism by which pyrimidine pool disequilibrium compromises the completion of replication and chromosome segregation: the intracellular accumulation of dCTP inhibits PARP-1 activity. CDA deficiency results in incomplete DNA replication when cells enter mitosis, leading to the formation of ultrafine anaphase bridges between sister-chromatids at « difficult-to-replicate » sites such as centromeres and fragile sites. Using molecular combing, electron microscopy and a sensitive assay involving cell imaging to quantify steady-state PAR levels, we found that DNA replication was unsuccessful due to the partial inhibition of basal PARP-1 activity, rather than slower fork speed. The stimulation of PARP-1 activity in CDA-deficient cells restores replication and, thus, chromosome segregation. Moreover, increasing intracellular dCTP levels generates under-replication-induced sister-chromatid bridges as efficiently as PARP-1 knockdown. These results have direct implications for Bloom syndrome (BS), a rare genetic disease combining susceptibility to cancer and genomic instability. BS results from mutation of the BLM gene, encoding BLM, a RecQ 3′-5′ DNA helicase, a deficiency of which leads to CDA downregulation. BS cells thus have a CDA defect, resulting in a high frequency of ultrafine anaphase bridges due entirely to dCTP-dependent PARP-1 inhibition and independent of BLM status. Our study describes previously unknown pathological consequences of the distortion of dNTP pools and reveals an unexpected role for PARP-1 in preventing DNA under-replication and chromosome segregation defects.

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Diana Passaro, Marta Irigoyen, Claire Catherinet, Stéphanie Gachet, Cindy Da Costa De Jesus, Charlène Lasgi, Christine Tran Quang, Jacques Ghysdael (2015 Jun 8)

CXCR4 Is Required for Leukemia-Initiating Cell Activity in T Cell Acute Lymphoblastic Leukemia.

Cancer cell : 769-79 : DOI : 10.1016/j.ccell.2015.05.003 En savoir plus
Résumé

Impaired cell migration has been demonstrated in T cell acute lymphoblastic leukemia (T-ALL) cells upon calcineurin inactivation, among other phenotypic traits including increased apoptosis, inhibition of cell proliferation, and ultimately inhibition of leukemia-initiating cell (LIC) activity. Herein we demonstrate that the chemokine receptor CXCR4 is essential to the LIC activity of T-ALL leukemic cells both in NOTCH-induced mouse T-ALL and human T-ALL xenograft models. We further demonstrate that calcineurin regulates CXCR4 cell-surface expression in a cortactin-dependent manner, a mechanism essential to the migratory properties of T-ALL cells. Because 20%-25% of pediatric and over 50% of adult patients with T-ALL do not achieve complete remission and relapse, our results call for clinical trials incorporating CXCR4 antagonists in T-ALL treatment.

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Marin Barisic, Ricardo Silva e Sousa, Suvranta K Tripathy, Maria M Magiera, Anatoly V Zaytsev, Ana L Pereira, Carsten Janke, Ekaterina L Grishchuk, Helder Maiato (2015 May 15)

Mitosis. Microtubule detyrosination guides chromosomes during mitosis.

Science (New York, N.Y.) : 799-803 : DOI : 10.1126/science.aaa5175 En savoir plus
Résumé

Before chromosomes segregate into daughter cells, they align at the mitotic spindle equator, a process known as chromosome congression. Centromere-associated protein E (CENP-E)/Kinesin-7 is a microtubule plus-end-directed kinetochore motor required for congression of pole-proximal chromosomes. Because the plus-ends of many astral microtubules in the spindle point to the cell cortex, it remains unknown how CENP-E guides pole-proximal chromosomes specifically toward the equator. We found that congression of pole-proximal chromosomes depended on specific posttranslational detyrosination of spindle microtubules that point to the equator. In vitro reconstitution experiments demonstrated that CENP-E-dependent transport was strongly enhanced on detyrosinated microtubules. Blocking tubulin tyrosination in cells caused ubiquitous detyrosination of spindle microtubules, and CENP-E transported chromosomes away from spindle poles in random directions. Thus, CENP-E-driven chromosome congression is guided by microtubule detyrosination.

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Saed Mohebi, Sarah A E Lambert, Antony M Carr (2015 Apr 29)

Analyzing the Response to Dysfunction Replication Forks Using the RTS1 Barrier System in Fission Yeast.

Methods in molecular biology (Clifton, N.J.) : 239-59 : DOI : 10.1007/978-1-4939-2596-4_15 En savoir plus
Résumé

The study of how eukaryotic cells overcome problems associated with dysfunctional DNA replication forks is assisted by experimental systems that allow site-specific replication fork arrest. Here we provide protocols for the use of the fission yeast RTS1 replication fork barrier. The RTS1 barrier is a directional, or polar, replication fork barrier that evolved to ensure directional replication of the fission yeast mating-type locus. We have moved the 859 bp RTS1 sequence to another locus in the genome and demonstrated that it arrests replication forks in a dysfunctional confirmation and that replication is restarted within ~20 min by the action of homologous recombination. We describe here the barrier constructs currently available, the methods by which we regulate the activity of the barrier, how to synchronize cells for analysis of replication intermediates by 2D gel electrophoresis, and the use of a replication slippage assay to measure fork fidelity.

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Nathalie Delgehyr, Alice Meunier, Marion Faucourt, Montserrat Bosch Grau, Laetitia Strehl, Carsten Janke, Nathalie Spassky (2015 Apr 4)

Ependymal cell differentiation, from monociliated to multiciliated cells.

Methods in cell biology : 19-35 : DOI : 10.1016/bs.mcb.2015.01.004 En savoir plus
Résumé

Primary and motile cilia differ in their structure, composition, and function. In the brain, primary cilia are immotile signalling organelles present on neural stem cells and neurons. Multiple motile cilia are found on the surface of ependymal cells in all brain ventricles, where they contribute to the flow of cerebrospinal fluid. During development, monociliated ependymal progenitor cells differentiate into multiciliated ependymal cells, thus providing a simple system for studying the transition between these two stages. In this chapter, we provide protocols for immunofluorescence staining of developing ependymal cells in vivo, on whole mounts of lateral ventricle walls, and in vitro, on cultured ependymal cells. We also provide a list of markers we currently use to stain both types of cilia, including proteins at the ciliary membrane and tubulin posttranslational modifications of the axoneme.

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Mohammed-Tayyib Boudra, Celeste Bolin, Sara Chiker, Alexis Fouquin, Tomasz Zaremba, Laurence Vaslin, Denis Biard, Fabrice P Cordelières, Frédérique Mégnin-Chanet, Vincent Favaudon, Marie Fernet, Vincent Pennaneach, Janet Hall (2015 Apr 1)

PARP-2 depletion results in lower radiation cell survival but cell line-specific differences in poly(ADP-ribose) levels.

Cellular and molecular life sciences : CMLS : 72 : 1585-1597 : DOI : 10.1007/s00018-014-1765-2 En savoir plus
Résumé

Poly(ADP-ribose) polymerase-2 (PARP-2) activity contributes to a cells’ poly(ADP-ribosyl)ating potential and like PARP-1, has been implicated in several DNA repair pathways including base excision repair and DNA single strand break repair. Here the consequences of its stable depletion in HeLa, U20S, and AS3WT2 cells were examined. All three PARP-2 depleted models showed increased sensitivity to the cell killing effects on ionizing radiation as reported in PARP-2 depleted mouse embryonic fibroblasts providing further evidence for a role in DNA strand break repair. The PARP-2 depleted HeLa cells also showed both higher constitutive and DNA damage-induced levels of polymers of ADP-ribose (PAR) associated with unchanged PARP-1 protein levels, but higher PARP activity and a concomitant lower PARG protein levels and activity. These changes were accompanied by a reduced maximal recruitment of PARP-1, XRCC1, PCNA, and PARG to DNA damage sites. This PAR-associated phenotype could be reversed in HeLa cells on re-expression of PARP-2 and was not seen in U20S and AS3WT2 cells. These results highlight the complexity of the relationship between different members of the PARP family on PAR metabolism and suggest that cell model dependent phenotypes associated with the absence of PARP-2 exist within a common background of radiation sensitivity.

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C Tran Quang, S Leboucher, D Passaro, L Fuhrmann, M Nourieh, A Vincent-Salomon, J Ghysdael (2015 Feb 26)

The calcineurin/NFAT pathway is activated in diagnostic breast cancer cases and is essential to survival and metastasis of mammary cancer cells.

Cell death & disease : e1658 : DOI : 10.1038/cddis.2015.14 En savoir plus
Résumé

Nuclear factor of activated T cells 1 (NFAT1) expression has been associated with increased migratory/invasive properties of mammary tumor-derived cell lines in vitro. It is unknown, however, if NFAT activation actually occurs in breast cancer cases and whether the calcineurin/NFAT pathway is important to mammary tumorigenesis. Using a cohort of 321 diagnostic cases of the major subgroup of breast cancer, we found Cn/NFAT pathway activated in ER(-)PR(-)HER2(-) triple-negative breast cancer subtype, whereas its prevalence is less in other subgroups. Using a small hairpin RNA-based gene expression silencing approach in murine mammary tumor cell line (4T1), we show that not only NFAT1 but also NFAT2 and their upstream activator Cn are essential to the migratory and invasive properties of mammary tumor cells. We also demonstrate that Cn, NFAT1 and NFAT2 are essential to the tumorigenic and metastatic properties of these cells in mice, a phenotype which coincides with increased apoptosis in vivo. Finally, global gene expression analyses identified several NFAT-deregulated genes, many of them being previously associated with mammary tumorigenesis. In particular, we identified the gene encoding a disintegrin and metalloproteinase with thrombonspondin motifs 1, as being a potential direct target of NFAT1. Thus, our results show that the Cn/NFAT pathway is activated in diagnostic cases of breast cancers and is essential to the tumorigenic and metastatic potential of mammary tumor cell line. These results suggest that pharmacological inhibition of the Cn/NFAT pathway at different levels could be of therapeutical interest for breast cancer patients.

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