UMR3348 – Intégrité du génome, ARN et cancer

Publications de l’unité

Année de publication : 2016

Hélène Malka-Mahieu, Isabelle Girault, Margot Rubington, Melissa Leriche, Caroline Welsch, Nyam Kamsu-Kom, Qian Zhao, Laurent Desaubry, Stéphan Vagner, Caroline Robert (2016 Sep 16)

Synergistic effects of eIF4A and MEK inhibitors on proliferation of NRAS-mutant melanoma cell lines.

Cell cycle (Georgetown, Tex.) : 1-5 : DOI : 10.1080/15384101.2016.1208862 En savoir plus
Résumé

Activating mutations of the NRAS (neuroblastoma rat sarcoma viral oncogene) protein kinase, present in many cancers, induce a constitutive activation of both the RAS-RAF-MEK-ERK mitogen-activated protein kinase (MAPK) signal transduction pathway and the PI(3)K-AKT-mTOR, pathway. This in turn regulates the formation of the eIF4F eukaryotic translation initiation complex, comprising the eIF4E cap-binding protein, the eIF4G scaffolding protein and the eIF4A RNA helicase, which binds to the 7-methylguanylate cap (m(7)G) at the 5′ end of messenger RNAs. Small molecules targeting MEK (MEKi: MEK inhibitors) have demonstrated activity in NRAS-mutant cell lines and tumors, but resistance sets in most cases within months of treatment. Using proximity ligation assays, that allows visualization of the binding of eIF4E to the scaffold protein eIF4G, generating the active eIF4F complex, we have found that resistance to MEKi is associated with the persistent formation of the eIF4F complex in MEKi-treated NRAS-mutant cell lines. Furthermore, inhibiting the eIF4A component of the eIF4F complex, with a small molecule of the flavagline/rocaglate family, synergizes with inhibiting MEK to kill NRAS-mutant cancer cell lines.

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Catharina von Nicolai, Åsa Ehlén, Charlotte Martin, Xiaodong Zhang, Aura Carreira (2016 Sep 15)

A second DNA binding site in human BRCA2 promotes homologous recombination.

Nature communications : 12813 : DOI : 10.1038/ncomms12813 En savoir plus
Résumé

BRCA2 tumour-suppressor protein is well known for its role in DNA repair by homologous recombination (HR); assisting the loading of RAD51 recombinase at DNA double-strand breaks. This function is executed by the C-terminal DNA binding domain (CTD) which binds single-stranded (ss)DNA, and the BRC repeats, which bind RAD51 and modulate its assembly onto ssDNA. Paradoxically, analysis of cells resistant to DNA damaging agents missing the CTD restore HR proficiency, suggesting another domain may take over its function. Here, we identify a region in the N terminus of BRCA2 that exhibits DNA binding activity (NTD) and provide evidence for NTD promoting RAD51-mediated HR. A missense variant detected in breast cancer patients located in the NTD impairs HR stimulation on dsDNA/ssDNA junction containing substrates. These findings shed light on the function of the N terminus of BRCA2 and have implications for the evaluation of breast cancer variants.

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Amélie Trinquand, Nuno R Dos Santos, Christine Tran Quang, Francesca Rocchetti, Benedetta Zaniboni, Mohamed Belhocine, Cindy Da Costa de Jesus, Ludovic Lhermitte, Melania Tesio, Michael Dussiot, François-Loïc Cosset, Els Verhoeyen, Françoise Pflumio, Norbert Ifrah, Hervé Dombret, Salvatore Spicuglia, Lucienne Chatenoud, David-Alexandre Gross, Olivier Hermine, Elizabeth Macintyre, Jacques Ghysdael, Vahid Asnafi (2016 Sep 6)

Triggering the TCR Developmental Checkpoint Activates a Therapeutically Targetable Tumor Suppressive Pathway in T-cell Leukemia.

Cancer discovery : 972-85 : DOI : 10.1158/2159-8290.CD-15-0675 En savoir plus
Résumé

Cancer onset and progression involves the accumulation of multiple oncogenic hits, which are thought to dominate or bypass the physiologic regulatory mechanisms in tissue development and homeostasis. We demonstrate in T-cell acute lymphoblastic leukemia (T-ALL) that, irrespective of the complex oncogenic abnormalities underlying tumor progression, experimentally induced, persistent T-cell receptor (TCR) signaling has antileukemic properties and enforces a molecular program resembling thymic negative selection, a major developmental event in normal T-cell development. Using mouse models of T-ALL, we show that induction of TCR signaling by high-affinity self-peptide/MHC or treatment with monoclonal antibodies to the CD3ε chain (anti-CD3) causes massive leukemic cell death. Importantly, anti-CD3 treatment hampered leukemogenesis in mice transplanted with either mouse- or patient-derived T-ALLs. These data provide a strong rationale for targeted therapy based on anti-CD3 treatment of patients with TCR-expressing T-ALL and demonstrate that endogenous developmental checkpoint pathways are amenable to therapeutic intervention in cancer cells.

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Simon Gemble, Géraldine Buhagiar-Labarchède, Rosine Onclercq-Delic, Denis Biard, Sarah Lambert, Mounira Amor-Guéret (2016 Aug 15)

A balanced pyrimidine pool is required for optimal Chk1 activation to prevent ultrafine anaphase bridge formation.

Journal of cell science : 3167-77 : DOI : 10.1242/jcs.187781 En savoir plus
Résumé

Cytidine deaminase (CDA) deficiency induces an excess of cellular dCTP, which reduces basal PARP-1 activity, thereby compromising complete DNA replication, leading to ultrafine anaphase bridge (UFB) formation. CDA dysfunction has pathological implications, notably in cancer and in Bloom syndrome. It remains unknown how reduced levels of PARP-1 activity and pyrimidine pool imbalance lead to the accumulation of unreplicated DNA during mitosis. We report that a decrease in PARP-1 activity in CDA-deficient cells impairs DNA-damage-induced Chk1 activation, and, thus, the downstream checkpoints. Chemical inhibition of the ATR-Chk1 pathway leads to UFB accumulation, and we found that this pathway was compromised in CDA-deficient cells. Our data demonstrate that ATR-Chk1 acts downstream from PARP-1, preventing the accumulation of unreplicated DNA in mitosis, and, thus, UFB formation. Finally, delaying entry into mitosis is sufficient to prevent UFB formation in both CDA-deficient and CDA-proficient cells, suggesting that both physiological and pathological UFBs are derived from unreplicated DNA. Our findings demonstrate an unsuspected requirement for a balanced nucleotide pool for optimal Chk1 activation both in unchallenged cells and in response to genotoxic stress.

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Hélène Colman, Catherine Le Berre-Scoul, Céline Hernandez, Sandra Pierredon, Audrey Bihouée, Rémi Houlgatte, Stephan Vagner, Arielle R Rosenberg, Cyrille Féray (2016 May 27)

Correction for Colman et al., Genome-Wide Analysis of Host mRNA Translation during Hepatitis C Virus Infection.

Journal of virology : 5846 : DOI : 10.1128/JVI.00507-16 En savoir plus
Résumé

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Diana Passaro, Christine Tran Quang, Jacques Ghysdael (2016 May 1)

Microenvironmental cues for T-cell acute lymphoblastic leukemia development.

Immunological reviews : 156-72 : DOI : 10.1111/imr.12402 En savoir plus
Résumé

Intensive chemotherapy regimens have led to a substantial improvement in the cure rate of patients suffering from T-cell acute lymphoblastic leukemia (T-ALL). Despite this progress, about 15% and 50% of pediatric and adult cases, respectively, show resistance to treatment or relapse with dismal prognosis, calling for further therapeutic investigations. T-ALL is an heterogeneous disease, which presents intrinsic alterations leading to aberrant expression of transcription factors normally involved in hematopoietic stem/progenitor cell development and mutations in genes implicated in the regulation of cell cycle progression, apoptosis, and T-cell development. Gene expression profiling allowed the classification of T-ALL into defined molecular subgroups that mostly reflects the stage of their differentiation arrest. So far this knowledge has not translated into novel, targeted therapy. Recent evidence points to the importance of extrinsic signaling cues in controlling the ability of T-ALL to home, survive, and proliferate, thus offering the perspective of new therapeutic options. This review summarizes the present understanding of the interactions between hematopoietic cells and bone marrow/thymic niches during normal hematopoiesis, describes the main signaling pathways implicated in this dialog, and finally highlights how malignant T cells rely on specific niches to maintain their ability to sustain and propagate leukemia.

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Gary Borisy, Rebecca Heald, Jonathon Howard, Carsten Janke, Andrea Musacchio, Eva Nogales (2016 Apr 22)

Microtubules: 50 years on from the discovery of tubulin.

Nature reviews. Molecular cell biology : 322-8 : DOI : 10.1038/nrm.2016.45 En savoir plus
Résumé

Next year will be the 50th anniversary of the discovery of tubulin. To celebrate this discovery, six leaders in the field of microtubule research reflect on key findings and technological breakthroughs over the past five decades, discuss implications for therapeutic applications and provide their thoughts on what questions need to be addressed in the near future.

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F Macari, Y El-Houfi, G Boldina, H Xu, S Khoury-Hanna, J Ollier, L Yazdani, G Zheng, I Bièche, N Legrand, D Paulet, S Durrieu, A Byström, S Delbecq, B Lapeyre, L Bauchet, J Pannequin, F Hollande, T Pan, M Teichmann, S Vagner, A David, A Choquet, D Joubert (2016 Apr 7)

TRM6/61 connects PKCα with translational control through tRNAi(Met) stabilization: impact on tumorigenesis.

Oncogene : 1785-96 : DOI : 10.1038/onc.2015.244 En savoir plus
Résumé

Accumulating evidence suggests that changes of the protein synthesis machinery alter translation of specific mRNAs and participate in malignant transformation. Here we show that protein kinase C α (PKCα) interacts with TRM61, the catalytic subunit of the TRM6/61 tRNA methyltransferase. The TRM6/61 complex is known to methylate the adenosine 58 of the initiator methionine tRNA (tRNAi(Met)), a nuclear post-transcriptional modification associated with the stabilization of this crucial component of the translation-initiation process. Depletion of TRM6/61 reduced proliferation and increased death of C6 glioma cells, effects that can be partially rescued by overexpression of tRNAi(Met). In contrast, elevated TRM6/61 expression regulated the translation of a subset of mRNAs encoding proteins involved in the tumorigenic process and increased the ability of C6 cells to form colonies in soft agar or spheres when grown in suspension. In TRM6/61/tRNAi(Met)-overexpressing cells, PKCα overexpression decreased tRNAi(Met) expression and both colony- and sphere-forming potentials. A concomitant increase in TRM6/TRM61 mRNA and tRNAi(Met) expression with decreased expression of PKCα mRNA was detected in highly aggressive glioblastoma multiforme as compared with Grade II/III glioblastomas, highlighting the clinical relevance of our findings. Altogether, we suggest that PKCα tightly controls TRM6/61 activity to prevent translation deregulation that would favor neoplastic development.

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Juan S Martinez, Catharina von Nicolai, Taeho Kim, Åsa Ehlén, Alexander V Mazin, Stephen C Kowalczykowski, Aura Carreira (2016 Mar 29)

BRCA2 regulates DMC1-mediated recombination through the BRC repeats.

Proceedings of the National Academy of Sciences of the United States of America : 3515-20 : DOI : 10.1073/pnas.1601691113 En savoir plus
Résumé

In somatic cells, BRCA2 is needed for RAD51-mediated homologous recombination. The meiosis-specific DNA strand exchange protein, DMC1, promotes the formation of DNA strand invasion products (joint molecules) between homologous molecules in a fashion similar to RAD51. BRCA2 interacts directly with both human RAD51 and DMC1; in the case of RAD51, this interaction results in stimulation of RAD51-promoted DNA strand exchange. However, for DMC1, little is known regarding the basis and functional consequences of its interaction with BRCA2. Here we report that human DMC1 interacts directly with each of the BRC repeats of BRCA2, albeit most tightly with repeats 1-3 and 6-8. However, BRC1-3 bind with higher affinity to RAD51 than to DMC1, whereas BRC6-8 bind with higher affinity to DMC1, providing potential spatial organization to nascent filament formation. With the exception of BRC4, each BRC repeat stimulates joint molecule formation by DMC1. The basis for this stimulation is an enhancement of DMC1-ssDNA complex formation by the stimulatory BRC repeats. Lastly, we demonstrate that full-length BRCA2 protein stimulates DMC1-mediated DNA strand exchange between RPA-ssDNA complexes and duplex DNA, thus identifying BRCA2 as a mediator of DMC1 recombination function. Collectively, our results suggest unique and specialized functions for the BRC motifs of BRCA2 in promoting homologous recombination in meiotic and mitotic cells.

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Anne Cammas, Magali Lacroix-Triki, Sandra Pierredon, Morgane Le Bras, Jason S Iacovoni, Marie-Paule Teulade-Fichou, Gilles Favre, Henri Roché, Thomas Filleron, Stefania Millevoi, Stéphan Vagner (2016 Mar 29)

hnRNP A1-mediated translational regulation of the G quadruplex-containing RON receptor tyrosine kinase mRNA linked to tumor progression.

Oncotarget : 7 : 16793-805 : DOI : 10.18632/oncotarget.7589 En savoir plus
Résumé

The expression and role of RNA binding proteins (RBPs) controlling mRNA translation during tumor progression remains largely uncharacterized. Analysis by immunohistochemistry of the expression of hnRNP A1, hnRNPH, RBM9/FOX2, SRSF1/ASF/SF2, SRSF2/SC35, SRSF3/SRp20, SRSF7/9G8 in breast tumors shows that the expression of hnRNP A1, but not the other tested RBPs, is associated with metastatic relapse. Strikingly, hnRNP A1, a nuclear splicing regulator, is also present in the cytoplasm of tumor cells of a subset of patients displaying exceedingly worse prognosis. Expression of a cytoplasmic mutant of hnRNP A1 leads to increased translation of the mRNA encoding the tyrosine kinase receptor RON/MTS1R, known for its function in tumor dissemination, and increases cell migration in vitro. hnRNP A1 directly binds to the 5′ untranslated region of the RON mRNA and activates its translation through G-quadruplex RNA secondary structures. The correlation between hnRNP A1 and RON tumoral expression suggests that these findings hold clinical relevance.

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Jeffrey J Nirschl, Maria M Magiera, Jacob E Lazarus, Carsten Janke, Erika L F Holzbaur (2016 Mar 22)

α-Tubulin Tyrosination and CLIP-170 Phosphorylation Regulate the Initiation of Dynein-Driven Transport in Neurons.

Cell reports : 2637-52 : DOI : 10.1016/j.celrep.2016.02.046 En savoir plus
Résumé

Motor-cargo recruitment to microtubules is often the rate-limiting step of intracellular transport, and defects in this recruitment can cause neurodegenerative disease. Here, we use in vitro reconstitution assays with single-molecule resolution, live-cell transport assays in primary neurons, computational image analysis, and computer simulations to investigate the factors regulating retrograde transport initiation in the distal axon. We find that phosphorylation of the cytoskeletal-organelle linker protein CLIP-170 and post-translational modifications of the microtubule track combine to precisely control the initiation of retrograde transport. Computer simulations of organelle dynamics in the distal axon indicate that while CLIP-170 primarily regulates the time to microtubule encounter, the tyrosination state of the microtubule lattice regulates the likelihood of binding. These mechanisms interact to control transport initiation in the axon in a manner sensitive to the specialized cytoskeletal architecture of the neuron.

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Lise Boussemart, Isabelle Girault, Hélène Malka-Mahieu, Christine Mateus, Emilie Routier, Margot Rubington, Nyam Kamsu-Kom, Marina Thomas, Gorana Tomasic, Sandrine Agoussi, Marie Breckler, Mélanie Laporte, Ludovic Lacroix, Alexander M Eggermont, Andrea Cavalcanti, Florent Grange, Julien Adam, Stéphan Vagner, Caroline Robert (2016 Mar 15)

Secondary Tumors Arising in Patients Undergoing BRAF Inhibitor Therapy Exhibit Increased BRAF-CRAF Heterodimerization.

Cancer research : 1476-84 : DOI : 10.1158/0008-5472.CAN-15-2900-T En savoir plus
Résumé

BRAF inhibitors (BRAFi) elicit therapeutic responses in metastatic melanoma, but alarmingly, also induce the formation of secondary benign and malignant skin tumors. Here, we report the emergence and molecular characterization of 73 skin and extracutaneous tumors in 31 patients who underwent BRAFi therapy. The majority of patients presented with classic epidermal tumors such as verrucous papillomas, keratoacanthomas, and squamous cell carcinomas (SCC). However, 15 patients exhibited new or rapidly progressing tumors distinct from these classic subtypes, such as lymph node metastasis, new melanomas, and genital and oral mucosal SCCs. Genotyping of the tumors revealed that oncogenic RAS mutations were found in 58% of the evaluable tumor samples (38/66) and 49% of the control tumors from patients not treated with BRAFi (30/62). Notably, proximity ligation assays demonstrated that BRAF-CRAF heterodimerization was increased in fixed tumor samples from BRAFi-treated patients compared with untreated patients. Our findings reveal that BRAF-CRAF complex formation is significantly associated with BRAFi treatment, and may therefore serve as a useful biomarker of BRAFi-induced cutaneous and extracutaneous tumor formation. Cancer Res; 76(6); 1476-84. ©2016 AACR.

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Samar Alsafadi, Alexandre Houy, Aude Battistella, Tatiana Popova, Michel Wassef, Emilie Henry, Franck Tirode, Angelos Constantinou, Sophie Piperno-Neumann, Sergio Roman-Roman, Martin Dutertre, Marc-Henri Stern (2016 Feb 5)

Cancer-associated SF3B1 mutations affect alternative splicing by promoting alternative branchpoint usage.

Nature communications : 10615 : DOI : 10.1038/ncomms10615 En savoir plus
Résumé

Hotspot mutations in the spliceosome gene SF3B1 are reported in ∼20% of uveal melanomas. SF3B1 is involved in 3′-splice site (3’ss) recognition during RNA splicing; however, the molecular mechanisms of its mutation have remained unclear. Here we show, using RNA-Seq analyses of uveal melanoma, that the SF3B1(R625/K666) mutation results in deregulated splicing at a subset of junctions, mostly by the use of alternative 3’ss. Modelling the differential junctions in SF3B1(WT) and SF3B1(R625/K666) cell lines demonstrates that the deregulated splice pattern strictly depends on SF3B1 status and on the 3’ss-sequence context. SF3B1(WT) knockdown or overexpression do not reproduce the SF3B1(R625/K666) splice pattern, qualifying SF3B1(R625/K666) as change-of-function mutants. Mutagenesis of predicted branchpoints reveals that the SF3B1(R625/K666)-promoted splice pattern is a direct result of alternative branchpoint usage. Altogether, this study provides a better understanding of the mechanisms underlying splicing alterations induced by mutant SF3B1 in cancer, and reveals a role for alternative branchpoints in disease.

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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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