UCAGenomiX related publications

Du to our strong expertise in "omics" experiments and in microRNAs topics we decided to separate into 3 categories the related publications into which the Functional genomics Platform of Nice-Sophia-Antipolis is involved :
  1. Expression studies (DNA microarrays and high-throughput sequencing experiments)
  2. MicroRNA studies
  3. Miscellaneous

Mari Bernard

 mari@ipmc.cnrs.fr
 04 93 95 77 90
 660 route des lucioles 06560 Valbonne - Sophia-Antipolis

67 publications found

1. Comparative Transcriptome Profiling of Virulent and Attenuated Ehrlichia ruminantium Strains Highlighted Strong Regulation of map1- and Metabolism Related Genes.
Front Cell Infect Microbiol. 2018 May 15;8:153. doi: 10.3389/fcimb.2018.00153. eCollection 2018.
Pruneau L, Lebrigand K, Mari B, Lefrançois T, Meyer DF, Vachiery N
CIRAD, UMR ASTRE, Guadeloupe, France. ASTRE, CIRAD, INRA, University of Montpellier, Montpellier, France. Université des Antilles, Guadeloupe, France. Centre National de la Recherche Scientifique, IPMC, Université Côte d'Azur, Valbonne, France. CIRAD, UMR ASTRE, Montpellier, France.

The obligate intracellular pathogenic bacterium, Ehrlichia ruminantium, is the causal agent of heartwater, a fatal disease in ruminants transmitted by Amblyomma ticks. So far, three strains have been attenuated by successive passages in mammalian cells. The attenuated strains have improved capacity for growth in vitro, whereas they induced limited clinical signs in vivo and conferred strong protection against homologous challenge. However, the mechanisms of pathogenesis and attenuation remain unknown. In order to improve knowledge of E. ruminantium pathogenesis, we performed a comparative transcriptomic analysis of two distant strains of E. ruminantium, Gardel and Senegal, and their corresponding attenuated strains. Overall, our results showed an upregulation of gene expression encoding for the metabolism pathway in the attenuated strains compared to the virulent strains, which can probably be associated with higher in vitro replicative activity and a better fitness to the host cells. We also observed a significant differential expression of membrane protein-encoding genes between the virulent and attenuated strains. A major downregulation of map1-related genes was observed for the two attenuated strains, whereas upregulation of genes encoding for hypothetical membrane proteins was observed for the four strains. Moreover, CDS_05140, which encodes for a putative porin, displays the highest gene expression in both attenuated strains. For the attenuated strains, the significant downregulation of map1-related gene expression and upregulation of genes encoding other membrane proteins could be important in the implementation of efficient immune responses after vaccination with attenuated vaccines. Moreover, this study revealed an upregulation of gene expression for 8 genes encoding components of Type IV secretion system and 3 potential effectors, mainly in the virulent Gardel strain. Our transcriptomic study, supported by previous proteomic studies, provides and also confirms new information regarding the characterization of genes involved in E. ruminantium virulence and attenuation mechanisms.
Pubmed link : 29868509

2. Tacrolimus-induced nephrotoxicity in mice is associated with microRNA deregulation
Arch Toxicol. 2018 Jan 23. doi: 10.1007/s00204-018-2158-3
Vandenbussche C, Van der Hauwaert C, Dewaeles E, Franczak J, Hennino MF, Gnemmi V, Savary G, Tavernier Q, Nottet N, Paquet A, Perrais M, Blum D, Mari B, Pottier N, Glowacki F, Cauffiez C
EA 4483-IMPECS-IMPact of Environmental ChemicalS on Human Health, Faculté de Médecine/Pôle Recherche, Univ. Lille, 1, place de Verdun, 59045, Lille Cedex, France. Centre Hospitalier de Valenciennes-Service de Néphrologie, Médecine Interne et Vasculaire, 59300, Valenciennes, France. Département de la Recherche en Santé, CHU Lille, 59000, Lille, France. UMR-S 1172-JPArc-Centre de Recherche Jean-Pierre AUBERT Neurosciences et Cancer, Univ. Lille, 59000, Lille, France. UMR-S 1172, Inserm, 59000, Lille, France. Service d'Anatomopathologie, CHU Lille, 59000, Lille, France. CNRS, IPMC, FHU-OncoAge, Université Côte d'Azur, 06560, Valbonne, France. Service de Toxicologie et Génopathies, CHU Lille, 59000, Lille, France. Service de Néphrologie, CHU Lille, 59000, Lille, France. EA 4483-IMPECS-IMPact of Environmental ChemicalS on Human Health, Faculté de Médecine/Pôle Recherche, Univ. Lille, 1, place de Verdun, 59045, Lille Cedex, France. christelle.cauffiez@univ-lille2.fr.

Although Tacrolimus is an immunosuppressive drug widely used in renal transplantation, its chronic use paradoxically induces nephrotoxic effects, in particular renal fibrosis, which is responsible for chronic allograft dysfunction and represents a major prognostic factor of allograft survival. As molecular pathways and mechanisms involved in Tacrolimus-induced fibrogenic response are poorly elucidated, we assessed whether miRNAs are involved in the nephrotoxic effects mediated by Tacrolimus. Treatment of CD-1 mice with Tacrolimus (1 mg/kg/d for 28 days) resulted in kidney injury and was associated with alteration of a gene expression signature associated with cellular stress, fibrosis and inflammation. Tacrolimus also affected renal miRNA expression, including miRNAs previously involved in fibrotic and inflammatory processes as "fibromirs" such as miR-21-5p, miR-199a-5p and miR-214-3p. In agreement with in vivo data, Renal Proximal Tubular Epithelial cells exposed to Tacrolimus (25 and 50 µM) showed upregulation of miR-21-5p and the concomitant induction of epithelial phenotypic changes, inflammation and oxidative stress. In conclusion, this study suggests for the first time that miRNAs, especially fibromiRs, participate to Tacrolimus-induced nephrotoxic effects. Therefore, targeting miRNAs may be a new therapeutic option to counteract Tacrolimus deleterious effects on kidney.
Pubmed link : 29362864

3. A new long noncoding RNA (LncRNA) is induced in cutaneous squamous cell carcinoma and downregulates several anticancer and cell-differentiation genes in mouse.
J Biol Chem. 2017 Jun 8. pii: jbc.M117.776260. doi: 10.1074/jbc.M117.776260. [Epub ahead of print]
Ponzio G, Rezzonico R, Bourget I, Allan R, Nottet N, Popa A, Magnone V, Rios G, Mari B, Barbry P
Université Côte d’Azur, CNRS, IPMC, France. Université Côte d'Azur, CNRS, INSERM, IRCAN, France.

Keratinocyte-derived cutaneous squamous cell carcinoma (cSCC) is the most common metastatic skin cancer. Although some of the early events involved in this pathology have been identified, the subsequent steps leading to tumor development are poorly defined. We demonstrate here that the development of mouse tumors induced by the concomitant application of a carcinogen and a tumor promoter (7,12 dimethylbenz[a]anthracene [DMBA] and 12-O-tetradecanoylphorbol-13-acetate [TPA], respectively) is associated with the upregulation of a previously uncharacterized long noncoding RNA (lncRNA), termed AK144841. We found that AK144841 expression was absent from normal skin and was specifically stimulated in tumors and highly tumorigenic cells. We also found that AK144841 exists in two variants, one consisting of a large 2-kb transcript composed of four exons and one of a 1.8-kb transcript lacking the second exon. Gain- and loss-of-function studies indicated that AK144841 mainly inhibited gene expression, specifically downregulating the expression of genes of the late-cornified-envelope-1 (Lce1) family involved in epidermal terminal differentiation and of anticancer genes such as Cgref1, Brsk1, Basp1, Dusp5, Btg2, Anpep, Dhrs9, Stfa2, Tpm1, SerpinB2, Cpa4, Crct1, Cryab, Il24, Csf2, and Rgs16. Interestingly, the lack of the second exon significantly decreased AK144841's inhibitory effect on gene expression. We also noted that high AK144841 expression correlated with a low expression of the aforementioned genes and with the tumorigenic potential of cell lines. These findings suggest that AK144841 could contribute to the dedifferentiation program of tumor-forming keratinocytes and to molecular cascades leading to tumor development.
Pubmed link : 28596382

4. Rapid decay of engulfed extracellular miRNA by XRN1 exonuclease promotes transient epithelial-mesenchymal transition
Nucleic Acids Res. 2016 Dec 19. pii: gkw1284
Zangari J, Ilie M, Rouaud F, Signetti L, Ohanna M, Didier R, Roméo B, Goldoni D, Nottet N, Staedel C, Gal J, Mari B, Mograbi B, Hofman P, Brest P
Université Côte d'Azur, CNRS, INSERM, IRCAN, FHU-OncoAge, 06107 Nice France. Université Côte d'Azur, CHU-Nice, Hospital-related Biobank (BB-0033-00025), FHU-OncoAge, 06000 Nice, France. Université Côte d'Azur, INSERM, C3M, 06200 Nice, France. Université Côte d'Azur, CNRS, INSERM, IPMC, FHU-OncoAge, 06560 Valbonne, France. Université de Bordeaux, INSERM, ARNA, 33076 Bordeaux, France. Antoine Lacassagne Cancer Center, Epidemiology and Biostatistics Unit, 06189 Nice, France.

Extracellular vesicles (EVs) have been shown to play an important role in intercellular communication as carriers of DNA, RNA and proteins. While the intercellular transfer of miRNA through EVs has been extensively studied, the stability of extracellular miRNA (ex-miRNA) once engulfed by a recipient cell remains to be determined. Here, we identify the ex-miRNA-directed phenotype to be transient due to the rapid decay of ex-miRNA. We demonstrate that the ex-miR-223-3p transferred from polymorphonuclear leukocytes to cancer cells were functional, as demonstrated by the decreased expression of its target FOXO1 and the occurrence of epithelial-mesenchymal transition reprogramming. We showed that the engulfed ex-miRNA, unlike endogenous miRNA, was unstable, enabling dynamic regulation and a return to a non-invasive phenotype within 8 h. This transient phenotype could be modulated by targeting XRN1/PACMAN exonuclease. Indeed, its silencing was associated with slower decay of ex-miR-223-3p and subsequently prolonged the invasive properties. In conclusion, we showed that the 'steady step' level of engulfed miRNA and its subsequent activity was dependent on the presence of a donor cell in the surroundings to constantly fuel the recipient cell with ex-miRNAs and of XRN1 exonuclease, which is involved in the decay of these imported miRNA.
Pubmed link : 27994032

5. Membrane-bound ICAM-1 contributes to the onset of proinvasive tumor stroma by controlling acto-myosin contractility in carcinoma-associated fibroblasts
Oncotarget. 2016 Nov 25. doi: 10.18632/oncotarget.13610.
Bonan S, Albrengues J, Grasset E, Kuzet SE, Nottet N, Bourget I, Bertero T, Mari B, Meneguzzi G, Gaggioli C
INSERM U1081, CNRS UMR7284, Institute for Research on Cancer and Aging, Nice (IRCAN), University of Nice Sophia Antipolis, Medical School, F-06107, Nice, France. Institut de Pharmacologie Moléculaire et Cellulaire (IPMC), CNRS UMR7275, Sophia-Antipolis, France.

Acto-myosin contractility in carcinoma-associated fibroblasts leads to assembly of the tumor extracellular matrix. The pro-inflammatory cytokine LIF governs fibroblast activation in cancer by regulating the myosin light chain 2 activity. So far, however, how LIF mediates cytoskeleton contractility remains unknown. Using phenotypic screening assays based on knock-down of LIF-dependent genes in fibroblasts, we identified the glycoprotein ICAM-1 as a crucial regulator of stroma fibroblast proinvasive matrix remodeling. We demonstrate that the membrane-bound ICAM-1 isoform is necessary and sufficient to promote inflammation-dependent extracellular matrix contraction, which favors cancer cell invasion. Indeed, ICAM-1 mediates generation of acto-myosin contractility downstream of the Src kinases in stromal fibroblasts. Moreover, acto-myosin contractility regulates ICAM-1 expression by establishing a positive feedback signaling. Thus, targeting stromal ICAM-1 might constitute a possible therapeutic mean to counteract tumor cell invasion and dissemination.
Pubmed link : 27901489

6. MicroRNA-375/SEC23A as biomarkers of the in vitro efficacy of vandetanib.
Oncotarget. 2016 May 24;7(21):30461-78. doi: 10.18632/oncotarget.8458.
Lassalle S, Zangari J, Popa A, Ilie M, Hofman V, Long E, Patey M, Tissier F, Belléannée G, Trouette H, Catargi B, Peyrottes I, Sadoul JL, Bordone O, Bonnetaud C, Butori C, Bozec A, Guevara N, Santini J, Hénaoui IS, Lemaire G, Blanck O, Vielh P, Barbry P, Mari B, Brest P, Hofman P
1Centre Hospitalier Universitaire de Nice, Laboratory of Clinical and Experimental Pathology, Nice, France. 2Institute of Research on Cancer and Ageing of Nice (IRCAN), INSERM U1081/CNRS UMR7284, Nice, France. 3University of Nice Sophia-Antipolis, Nice, France. 4Centre Hospitalier Universitaire de Nice, Hospital Integrated Biobank (BB 0033-00025), Nice, France. 5Fédération Hospitalo-Universitaire "OncoAge", University of Nice Sophia Antipolis, Nice, France. 6Institut de Pharmacologie Moléculaire et Cellulaire IPMC, CNRS UMR7275, Sophia-Antipolis, France. 7Hôpital Universitaire de Reims - Hôpital Robert Debré, Department of Pathology, Institut Jean Godinot, Reims, France. 8Assistance Publique - Hôpitaux de Paris (AP-HP), Groupe Hospitalier Pitié-Salpêtrière, Laboratory of Pathology, Paris, France. 9Centre Hospitalier Universitaire de Bordeaux, Hôpital Universitaire de Pessac-Haut Lévêque, Laboratory of Pathology, Pessac, France. 10Centre Hospitalier Universitaire de Bordeaux, Department of Endocrinology, Pessac, France. 11Centre Antoine Lacassagne, Laboratory of Pathology, Nice, France. 12Centre Hospitalier Universitaire de Nice, Hôpital de l'Archet, Department of Endocrinology, Nice, France. 13Centre Antoine Lacassagne, Head and Neck Institute, Surgery and Otorhinolaryngology Department, Nice, France. 14Bayer CropScience SA, Research Center, Sophia Antipolis, Valbonne, France. 15Institut Gustave Roussy, Translational Research Laboratory, Department of Pathology, Villejuif, France.

In this study, we performed microRNA (miRNA) expression profiling on a large series of sporadic and hereditary forms of medullary thyroid carcinomas (MTC). More than 60 miRNAs were significantly deregulated in tumor vs adjacent non-tumor tissues, partially overlapping with results of previous studies. We focused our attention on the strongest up-regulated miRNA in MTC samples, miR-375, the deregulation of which has been previously observed in a variety of human malignancies including MTC. We identified miR-375 targets by combining gene expression signatures from human MTC (TT) and normal follicular (Nthy-ori 3-1) cell lines transfected with an antagomiR-375 inhibitor or a miR-375 mimic, respectively, and from an in silico analysis of thyroid cell lines of Cancer Cell Line Encyclopedia datasets. This approach identified SEC23A as a bona fide miR-375 target, which we validated by immunoblotting and immunohistochemistry of non-tumor and pathological thyroid tissue. Furthermore, we observed that miR-375 overexpression was associated with decreased cell proliferation and synergistically increased sensitivity to vandetanib, the clinically relevant treatment of metastatic MTC. We found that miR-375 increased PARP cleavage and decreased AKT phosphorylation, affecting both cell proliferation and viability. We confirmed these results through SEC23A direct silencing in combination with vandetanib, highlighting the importance of SEC23A in the miR-375-associated increased sensitivity to vandetanib.Since the combination of increased expression of miR-375 and decreased expression of SEC23A point to sensitivity to vandetanib, we question if the expression levels of miR-375 and SEC23A should be evaluated as an indicator of eligibility for treatment of MTC patients with vandetanib.
Pubmed link : 27036030

7. SENS-IS, a 3D reconstituted epidermis based model for quantifying chemical sensitization potency: Reproducibility and predictivity results from an inter-laboratory study.
Toxicol In Vitro. 2016 Jan 18;32:248-260. doi: 10.1016/j.tiv.2016.01.007.
Cottrez F, Boitel E, Ourlin JC, Peiffer JL, Fabre I, Henaoui IS, Mari B, Vallauri A, Paquet A, Barbry P, Auriault C, Aeby P, Groux H
1ImmunoSearch, Grasse, France. 2Agence nationale de sécurité du médicament, Vendargues, France. 3CNRS, Institute of Molecular and Cellular Pharmacology, Sophia Antipolis, France; University of Nice Sophia Antipolis, Nice, France. 4Independant Consultant, Marly, Switzerland. 5ImmunoSearch, Grasse, France. Electronic address: hgroux@immunosearch.fr.

The SENS-IS test protocol for the in vitro detection of sensitizers is based on a reconstructed human skin model (Episkin) as the test system and on the analysis of the expression of a large panel of genes. Its excellent performance was initially demonstrated with a limited set of test chemicals. Further studies (described here) were organized to confirm these preliminary results and to obtain a detailed statistical analysis of the predictive capacity of the assay. A ring-study was thus organized and performed within three laboratories, using a test set of 19 blind coded chemicals. Data analysis indicated that the assay is robust, easily transferable and offers high predictivity and excellent within- and between-laboratories reproducibility. To further evaluate the predictivity of the test protocol according to Cooper statistics a comprehensive test set of 150 chemicals was then analyzed. Again, data analysis confirmed the excellent capacity of the SENS-IS assay for predicting both hazard and potency characteristics, confirming that this assay should be considered as a serious alternative to the available in vivo sensitization tests.
Pubmed link : 26795242

8. Knockout of Vdac1 activates hypoxia-inducible factor through reactive oxygen species generation and induces tumor growth by promoting metabolic reprogramming and inflammation.
Cancer Metab. 2015 Aug 26;3:8. doi: 10.1186/s40170-015-0133-5. eCollection 2015.
Brahimi-Horn MC, Giuliano S, Saland E, Lacas-Gervais S, Sheiko T, Pelletier J, Bourget I, Bost F, Féral C, Boulter E, Tauc M, Ivan M, Garmy-Susini B, Popa A, Mari B, Sarry JE, Craigen WJ, Pouysségur J, Mazure NM
1Institute for Research on Cancer and Aging of Nice, CNRS-UMR 7284-Inserm U1081, University of Nice Sophia-Antipolis, Centre Antoine Lacassagne, 33 Ave de Valombrose, 06189 Nice, France. 2Centre de Recherche en Cancérologie de Toulouse, INSERM-UPSIII U1037, Oncopole, Toulouse, 31037 Cedex 1 France. 3Centre Commun de Microscopie Appliquée, University of Nice Sophia-Antipolis, 28 Ave Valombrose, 06103 Nice, France. 4Department of Molecular and Human Genetics, Baylor College of Medicine, One Baylor Plaza, MS BCM225, Houston, TX 77030 USA. 5Institute for Research on Cancer and Aging of Nice, CNRS-UMR 7284-Inserm U1081, University of Nice Sophia-Antipolis, 28 Ave de Valombrose, 06107 cedex 02 Nice, France. 6INSERM U1065, Centre Méditerranéen de Médecine Moléculaire (C3M), Team Cellular and Molecular Physiopathology of Obesity and Diabetes, and University of Nice Sophia-Antipolis, Nice, France. 7Faculté de Médecine, LP2M - CNRS UMR-7370, Université de Nice Sophia Antipolis, 28 Avenue de Valombrose, Nice, 06107 cedex 2 France. 8Department of Microbiology and Immunology, Indiana University School of Medicine, Indianapolis, IN 46202 USA. 9Institute of Metabolic and Cardiovascular Diseases, INSERM U1048, Rangueil Hospital, 1 Avenue Professeur Jean Poulhes, BP 84225, 31432 Cedex 4 Toulouse, France. 10Institut de Pharmacologie Moléculaire et Cellulaire (IPMC), Centre National de la Recherche Scientifique, CNRS UMR 7275, Sophia Antipolis, & University of Nice Sophia-Antipolis, Nice, France. 11Institute for Research on Cancer and Aging of Nice, CNRS-UMR 7284-Inserm U1081, University of Nice Sophia-Antipolis, Centre Antoine Lacassagne, 33 Ave de Valombrose, 06189 Nice, France ; Centre Scientifique de Monaco (CSM), Monte Carlo, Sophia Antipolis, Monaco.

Mitochondria are more than just the powerhouse of cells; they dictate if a cell dies or survives. Mitochondria are dynamic organelles that constantly undergo fusion and fission in response to environmental conditions. We showed previously that mitochondria of cells in a low oxygen environment (hypoxia) hyperfuse to form enlarged or highly interconnected networks with enhanced metabolic efficacy and resistance to apoptosis. Modifications to the appearance and metabolic capacity of mitochondria have been reported in cancer. However, the precise mechanisms regulating mitochondrial dynamics and metabolism in cancer are unknown. Since hypoxia plays a role in the generation of these abnormal mitochondria, we questioned if it modulates mitochondrial function. The mitochondrial outer-membrane voltage-dependent anion channel 1 (VDAC1) is at center stage in regulating metabolism and apoptosis. We demonstrated previously that VDAC1 was post-translationally C-terminal cleaved not only in various hypoxic cancer cells but also in tumor tissues of patients with lung adenocarcinomas. Cells with enlarged mitochondria and cleaved VDAC1 were also more resistant to chemotherapy-stimulated cell death than normoxic cancer cells. RESULTS: Transcriptome analysis of mouse embryonic fibroblasts (MEF) knocked out for Vdac1 highlighted alterations in not only cancer and inflammatory pathways but also in the activation of the hypoxia-inducible factor-1 (HIF-1) signaling pathway in normoxia. HIF-1α was stable in normoxia due to accumulation of reactive oxygen species (ROS), which decreased respiration and glycolysis and maintained basal apoptosis. However, in hypoxia, activation of extracellular signal-regulated kinase (ERK) in combination with maintenance of respiration and increased glycolysis counterbalanced the deleterious effects of enhanced ROS, thereby allowing Vdac1 (-/-) MEF to proliferate better than wild-type MEF in hypoxia. Allografts of RAS-transformed Vdac1 (-/-) MEF exhibited stabilization of both HIF-1α and HIF-2α, blood vessel destabilization, and a strong inflammatory response. Moreover, expression of Cdkn2a, a HIF-1-target and tumor suppressor gene, was markedly decreased. Consequently, RAS-transformed Vdac1 (-/-) MEF tumors grew faster than wild-type MEF tumors. CONCLUSIONS: Metabolic reprogramming in cancer cells may be regulated by VDAC1 through vascular destabilization and inflammation. These findings provide new perspectives into the understanding of VDAC1 in the function of mitochondria not only in cancer but also in inflammatory diseases.
Pubmed link : 26322231

9. Tetraspanin CD63 acts as a pro-metastatic factor via β-catenin stabilization.
Int J Cancer. 2015 May 15;136(10):2304-15. doi: 10.1002/ijc.29296. Epub 2014 Nov 11.
Seubert B, Cui H, Simonavicius N, Honert K, Schäfer S, Reuning U, Heikenwalder M, Mari B, Krüger A
Institute for Experimental Oncology and Therapy Research and Institute of Molecular Immunology, Klinikum rechts der Isar der Technische Universität München, München, Germany.

The tetraspanin CD63 is implicated in pro-metastatic signaling pathways but, so far, it is unclear, how CD63 levels affect the tumor cell phenotype. Here, we investigated the effect of CD63 modulation in different metastatic tumor cell lines. In vitro, knock down of CD63 induced a more epithelial-like phenotype concomitant with increased E-cadherin expression, downregulation of its repressors Slug and Zeb1, and decreased N-cadherin. In addition, β-catenin protein was markedly reduced, negatively affecting expression of the target genes MMP-2 and PAI-1. β-catenin inhibitors mimicked the epithelial phenotype induced by CD63 knock down. Inhibition of β-catenin upstream regulators PI3K/AKT or GSK3β could rescue the mesenchymal phenotype underlining the importance of the β-catenin pathway in CD63-regulated cell plasticity. CD63 knock down-induced phenotypical changes correlated with a decrease of experimental metastasis whereas CD63 overexpression enhanced the tumor cell-intrinsic metastatic potential. Taken together, our data show that CD63 is a crucial player in the regulation of the tumor cell-intrinsic metastatic potential by affecting cell plasticity.
Pubmed link : 25354204

10. Forkhead Box F1 represses cell growth and inhibits COL1 and ARPC2 expression in lung fibroblasts in vitro.
Am J Physiol Lung Cell Mol Physiol. 2014 Dec 1;307(11):L838-47. doi: 10.1152/ajplung.00012.2014. Epub 2014 Sep 26.
Melboucy-Belkhir S, Pradère P, Tadbiri S, Habib S, Bacrot A, Brayer S, Mari B, Besnard V, Mailleux A, Guenther A, Castier Y, Mal H, Crestani B, Plantier L
1INSERM UMR1152, Labex Inflamex, Paris, France; 2INSERM UMR1152, Labex Inflamex, Paris, France; Assistance-Publique-Hôpitaux de Paris, Hôpital Bichat-Claude Bernard, DHU FIRE, Service de Pneumologie A, Paris, France; Université Paris Diderot, PRES Sorbonne Paris Cité, Paris, France; 3Institut de Pharmacologie Moléculaire et Cellulaire, CNRS UMR7275, Valbonne, France; 4University of Giessen Lung Centre, Department of Internal Medicine, Giessen, Germany; Lung Clinic Waldhof-Elgershausen, Greifenstein, Germany; 5Université Paris Diderot, PRES Sorbonne Paris Cité, Paris, France; Assistance-Publique-Hôpitaux de Paris, Hôpital Bichat-Claude Bernard, Service de Chirurgie Thoracique et Transplantation Pulmonaire, Paris, France; 6Université Paris Diderot, PRES Sorbonne Paris Cité, Paris, France; Assistance-Publique-Hôpitaux de Paris, Hôpital Bichat-Claude Bernard, Service de Pneumologie B et Transplantation Pulmonaire, Paris, France; and. 7INSERM UMR1152, Labex Inflamex, Paris, France; Université Paris Diderot, PRES Sorbonne Paris Cité, Paris, France; Assistance-Publique-Hôpitaux de Paris, Hôpital Bichat-Claude Bernard, Service de Physiologie-Explorations Fonctionnelles, Paris, France. laurent.plantier@inserm.fr.

Aberrant expression of master phenotype regulators or alterations in their downstream pathways in lung fibroblasts may play a central role in idiopathic pulmonary fibrosis (IPF). Interrogating IPF fibroblast transcriptome datasets, we identified Forkhead Box F1 (FOXF1), a DNA-binding protein required for lung development, as a candidate actor in IPF. Thus we determined FOXF1 expression levels in fibroblasts cultured from normal or IPF lungs in vitro, and explored FOXF1 functions in these cells using transient and stable loss-of-function and gain-of-function models. FOXF1 mRNA and protein were expressed at higher levels in IPF fibroblasts compared with normal fibroblasts (mRNA: +44%, protein: +77%). Immunohistochemistry showed FOXF1 expression in nuclei of bronchial smooth muscle cells, endothelial cells, and lung fibroblasts including fibroblastic foci of IPF lungs. In normal lung fibroblasts, FOXF1 repressed cell growth and expression of collagen-1 (COL1) and actin-related protein 2/3 complex, subunit 2 (ARPC2). ARPC2 knockdown inhibited cell growth and COL1 expression, consistent with FOXF1 acting in part through ARPC2 repression. In IPF fibroblasts, COL1 and ARPC2 repression by FOXF1 was blunted, and FOXF1 did not repress growth. FOXF1 expression was induced by the antifibrotic mediator prostaglandin E2 and repressed by the profibrotic cytokine transforming growth factor-β1 in both normal and IPF lung fibroblasts. Ex vivo, FOXF1 knockdown conferred CCL-210 lung fibroblasts the ability to implant in uninjured mouse lungs. In conclusion, FOXF1 functions and regulation were consistent with participation in antifibrotic pathways. Alterations of pathways downstream of FOXF1 may participate to fibrogenesis in IPF fibroblasts.
Pubmed link : 25260753

11. Tissue inhibitor of metalloproteinases-1 induces a pro-tumourigenic increase of miR-210 in lung adenocarcinoma cells and their exosomes.
Oncogene. 2014 Sep 29. doi: 10.1038/onc.2014.300.
Cui H, Seubert B, Stahl E, Dietz H, Reuning U, Moreno-Leon L, Ilie M, Hofman P, Nagase H, Mari B, Krüger A
1Institut für Experimentelle Onkologie und Therapieforschung, Klinikum Rechts der Isar der Technischen Universität München, Ismaninger Straße 22, Munich, Germany. 2Physik Department, Walter Schottky Institute, Technische Universität München, Am Coulombwall 4a, Munich, Germany. 3Klinische Forschergruppe der Frauenklinik, Klinikum Rechts der Isar der Technischen Universität München, Ismaninger Straße 22, Munich, Germany. 41] University of Nice Sophia-Antipolis, Nice, France [2] Institut de Pharmacologie Moléculaire et Cellulaire, Centre National de la Recherche Scientifique, UMR 6097 CNRS/UNSA, Sophia Antipolis, France. 51] University of Nice Sophia-Antipolis, Nice, France [2] Laboratory of Clinical and Experimental Pathology and Hospital Integrated Biobank, Centre Hospitalier Universitaire de Nice, Nice, France. 6Kennedy Institute of Rheumatology, Nuffield Department of Orthopaedics, Rheumatology and Muscolosketal Sciences, University of Oxford, London, UK.

Tissue inhibitor of metalloproteinases-1 (TIMP-1) recently emerged as a pro-metastatic factor highly associated with poor prognosis in a number of cancers. This correlation seemed paradox as TIMP-1 is best described as an inhibitor of pro-tumourigenic matrix metalloproteinases. Only recently, TIMP-1 has been revealed as a signalling molecule that can regulate cancer progression independent of its inhibitory properties. In the present study, we demonstrate that an increase of both exogenous and endogenous TIMP-1 led to the upregulation of miR-210 in a CD63/PI3K/AKT/HIF-1-dependent pathway in lung adenocarcinoma cells. TIMP-1 induced P110/P85 PI3K-signalling and AKT phosphorylation. It also led to increase of HIF-1α protein levels positively correlating with HIF-1-regulated mRNA expression and upregulation of the microRNA miR-210. Downstream targets of miR-210, namely FGFRL1, E2F3, VMP-1, RAD52 and SDHD, were decreased in the presence of TIMP-1. Upon the overexpression of TIMP-1 in tumour cells, miR-210 was accumulated in exosomes in vitro and in vivo. These exosomes promoted tube formation activity in human umbilical vein endothelial cell (HUVECs), which was reflected in increased angiogenesis in A549L-derived tumour xenografts. Activation and elevation of PI3K, AKT, HIF-1A and miR-210 in tumours additionally confirmed our in vitro data. This new pro-tumourigenic signalling function of TIMP-1 may explain why elevated TIMP-1 levels in lung cancer patients are highly correlated with poor prognosis.Oncogene advance online publication, 29 September 2014; doi:10.1038/onc.2014.300.
Pubmed link : 25263437

12. MicroRNA target identification: lessons from hypoxamiRs.
Antioxid Redox Signal. 2014 Sep 10;21(8):1249-68. doi: 10.1089/ars.2013.5648. Epub 2014 Feb 3.
Bertero T, Robbe-Sermesant K, Le Brigand K, Ponzio G, Pottier N, Rezzonico R, Mazure NM, Barbry P, Mari B
Institut de Pharmacologie Moléculaire et Cellulaire (IPMC) , Centre National de la Recherche Scientifique, CNRS UMR 7275, Sophia Antipolis, France .

SIGNIFICANCE: MicroRNAs (miRNAs) are small noncoding RNAs that have emerged as key regulators of many physiological and pathological processes, including those relevant to hypoxia such as cancer, neurological dysfunctions, myocardial infarction, and lung diseases. RECENT ADVANCES: During the last 5 years, miRNAs have been shown to play a role in the regulation of the cellular response to hypoxia. The identification of several bona fide targets of these hypoxamiRs has underlined their pleiotropic functions and the complexity of the molecular rules directing miRNA::target transcript pairing. CRITICAL ISSUES: This review outlines the main in silico and experimental approaches used to identify the targetome of hypoxamiRs and presents new recent relevant methodologies for future studies. FUTURE DIRECTIONS: Since hypoxia plays key roles in many pathophysiological conditions, the precise characterization of regulatory hypoxamiRs networks will be instrumental both at a fundamental level and for their future potential therapeutic applications.
Pubmed link : 24111877

13. Blocking lipid synthesis overcomes tumor regrowth and metastasis after antiangiogenic therapy withdrawal.
Cell Metab. 2014 Aug 5;20(2):280-94. doi: 10.1016/j.cmet.2014.05.022. Epub 2014 Jul 10.
Sounni NE, Cimino J, Blacher S, Primac I, Truong A, Mazzucchelli G, Paye A, Calligaris D, Debois D, De Tullio P, Mari B, De Pauw E, Noel A
1Laboratory of Tumor and Developmental Biology, GIGA-CANCER, University of Liege, 4000 Liege, Belgium. Electronic address: nesounni@ulg.ac.be. 2Laboratory of Tumor and Developmental Biology, GIGA-CANCER, University of Liege, 4000 Liege, Belgium; Mass Spectrometry Laboratory, GIGA-R, Department of Chemistry, University of Liege, 4000 Liege, Belgium. 3Laboratory of Tumor and Developmental Biology, GIGA-CANCER, University of Liege, 4000 Liege, Belgium. 4Mass Spectrometry Laboratory, GIGA-R, Department of Chemistry, University of Liege, 4000 Liege, Belgium. 5Laboratory of Drug Research Center, University of Liege, 4000 Liege, Belgium. 6UMR-7275 CNRS, University of Nice Sophia-Antipolis, Institute of Molecular and Cellular Pharmacology, 06560 Valbonne, France.

The molecular mechanisms responsible for the failure of antiangiogenic therapies and how tumors adapt to these therapies are unclear. Here, we applied transcriptomic, proteomic, and metabolomic approaches to preclinical models and provide evidence for tumor adaptation to vascular endothelial growth factor blockade through a metabolic shift toward carbohydrate and lipid metabolism in tumors. During sunitinib or sorafenib treatment, tumor growth was inhibited and tumors were hypoxic and glycolytic. In sharp contrast, treatment withdrawal led to tumor regrowth, angiogenesis restoration, moderate lactate production, and enhanced lipid synthesis. This metabolic shift was associated with a drastic increase in metastatic dissemination. Interestingly, pharmacological lipogenesis inhibition with orlistat or fatty acid synthase downregulation with shRNA inhibited tumor regrowth and metastases after sunitinib treatment withdrawal. Our data shed light on metabolic alterations that result in cancer adaptation to antiangiogenic treatments and identify key molecules involved in lipid metabolism as putative therapeutic targets.
Pubmed link : 25017943

14. The small heat shock protein B8 (HSPB8) confers resistance to bortezomib by promoting autophagic removal of misfolded proteins in multiple myeloma cells.
Oncotarget. 2014 Aug 15;5(15):6252-66.
Hamouda MA, Belhacene N, Puissant A, Colosetti P, Robert G, Jacquel A, Mari B, Auberger P, Luciano F
1INSERM U1065, C3M, Team 2, Nice, France; Université de Nice Sophia-Antipolis; Equipe labellisée par la Ligue Nationale Contre le Cancer. 2Dana-Farber Cancer Institute and Boston Children's Hospital, Harvard Medical School, Boston, USA. 3UMR7275 CNRS-UNS, Institut de Pharmacologie Moléculaire et Cellulaire, Valbonne, France. 4INSERM U1065, C3M, Team 2, Nice, France; Université de Nice Sophia-Antipolis; Equipe labellisée par la Ligue Nationale Contre le Cancer; These authors contributed equally to this work.

Velcade is one of the inescapable drug to treat patient suffering from multiple myeloma (MM) and resistance to this drug represents a major drawback for patients. However, the mechanisms underlying velcade resistance remain incompletely understood. We derived several U266 MM cell clones that resist to velcade. U266-resistant cells were resistant to velcade-induced cell death but exhibited a similar sensitivity to various proapoptotic stimuli. Careful analysis of proteosomal subunits and proteasome enzymatic activities showed that neither the composition nor the activity of the proteasome was affected in velcade-resistant cells. Elimination of velcade-induced poly-ubiquitinated proteins and protein aggregates was drastically stimulated in the resistant cells and correlated with increased cell survival. Inhibition of the lysosomal activity in velcade-resistant cells resulted in an increase of cell aggregates and decrease survival, indicating that aggregates are eliminated through lysosomal degradation. In addition, pangenomic profiling of velcade-sensitive and resistant cells showed that the small heat shock protein HSPB8 was overexpressed in resistant cells. Finally, gain and loss of function experiment demonstrated that HSPB8 is a key factor for velcade resistance. In conclusion, HSPB8 plays an important role for the elimination of aggregates in velcade-resistant cells that contributes to their enhanced survival.
Pubmed link : 25051369

15. Phenotypic and genotypic characterization of azacitidine-sensitive and resistant SKM1 myeloid cell lines.
Oncotarget. 2014 Jun 30;5(12):4384-91.
Cluzeau T, Dubois A, Jacquel A, Luciano F, Renneville A, Preudhomme C, Karsenti JM, Mounier N, Rohrlich P, Raynaud S, Mari B, Robert G, Auberger P
1INSERM U1065, Mediterranean Center for Molecular Medicine (C3M), Nice, France; University of Nice, Nice, France. 2INSERM U1065, Mediterranean Center for Molecular Medicine (C3M), Nice, France; University of Nice, Nice, France; Department of Clinical hematology and Transplantation, CHU of Nice, Nice, France.

In the present study, we provide a comparative phenotypic and genotypic analysis of azacitidine-sensitive and resistant SKM-1 cell lines. Morphologically, SKM1-R exhibited increase in cell size that accounts for by enhanced ploidy in a majority of cells as shown by cell cycle and karyotype analysis. No specific Single Nucleotide Polymorphism (SNP) alteration was found in SKM1-R cells compared to their SKM1-S counterpart. Comparative pangenomic profiling revealed the up-regulation of a panel of genes involved in cellular movement, cell death and survival and down-regulation of genes required for cell to cell signaling and free radical scavenging in SKM1-R cells. We also searched for mutations frequently associated with myelodysplastic syndromes (MDS) and found that both cell lines harbored mutations in TET2, ASLX1 and TP53. Collectively, our data show that despite their different morphological and phenotypic features, SKM1-S and SKM1-R cells exhibited similar genotypic characteristics. Finally, pangenomic profiling identifies new potential pathways to be targeted to circumvent AZA-resistance. In conclusion, SKM1-R cells represent a valuable tool for the validation of new therapeutic intervention in MDS.
Pubmed link : 24962689

16. miR-193b/365a cluster controls progression of epidermal squamous cell carcinoma.
Carcinogenesis. 2014 May;35(5):1110-20. doi: 10.1093/carcin/bgt490. Epub 2013 Dec 28.
Gastaldi C, Bertero T, Xu N, Bourget-Ponzio I, Lebrigand K, Fourre S, Popa A, Cardot-Leccia N, Meneguzzi G, Sonkoly E, Pivarcsi A, Mari B, Barbry P, Ponzio G, Rezzonico R
UMR 7275, Centre National de la Recherche Scientifique, Institut de Pharmacologie Moléculaire et Cellulaire, 660 route des Lucioles, F-06560 Valbonne, France.

Incidence of cutaneous squamous cell carcinomas (cSCCs) constantly increases in the Caucasian population. Developing preferentially on precancerous lesions such as actinic keratoses due to chronic sunlight exposure, cSCCs result from the malignant transformation of keratinocytes. Although a resection of the primary tumor is usually curative, a subset of aggressive cSCCs shows a high risk of recurrence and metastases. The characterization of the molecular dysfunctions involved in cSCC development should help to identify new relevant targets against these aggressive cSCCs. In that context, we have used small RNA sequencing to identify 100 microRNAs (miRNAs) whose expression was altered during chemically induced mouse skin tumorigenesis. The decreased expression of the miR-193b/365a cluster during tumor progression suggests a tumor suppressor role. Ectopic expression of these miRNAs in tumor cells indeed inhibited their proliferation, clonogenic potential and migration, which were stimulated in normal keratinocytes when these miRNAs were blocked with antisense oligonucleotides. A combination of in silico predictions and transcriptome analyses identified several target genes of interest. We validated KRAS and MAX as direct targets of miR-193b and miR-365a. Repression of these targets using siRNAs mimicked the effects of miR-193b and miR-365a, suggesting that these genes might mediate, at least in part, the tumor-suppressive action of these miRNAs.
Pubmed link : 24374827

17. ⁹⁹mTcO₄--, auger-mediated thyroid stunning: dosimetric requirements and associated molecular events.
PLoS One. 2014 Mar 24;9(3):e92729. doi: 10.1371/journal.pone.0092729. eCollection 2014.
Cambien B, Franken PR, Lamit A, Mauxion T, Richard-Fiardo P, Guglielmi J, Crescence L, Mari B, Pourcher T, Darcourt J, Bardiès M, Vassaux G
Laboratoire TIRO, UMRE 4320, iBEB, DSV, CEA, Nice, France; Université de Nice-Sophia Antipolis, Nice, France; Centre Antoine Lacassagne, Department of nuclear medicine, Nice, France. 2UMR 1037 INSERM/UPS, Centre de Recherche en Cancérologie, Toulouse, France. 3Université de Nice-Sophia Antipolis, Nice, France; Institut de Pharmacologie Moléculaire et Cellulaire-IPMC, CNRS UMR 7275, Sophia Antipolis, France.

Low-energy Auger and conversion electrons deposit their energy in a very small volume (a few nm3) around the site of emission. From a radiotoxicological point of view the effects of low-energy electrons on normal tissues are largely unknown, understudied, and generally assumed to be negligible. In this context, the discovery that the low-energy electron emitter, 99mTc, can induce stunning on primary thyrocytes in vitro, at low absorbed doses, is intriguing. Extrapolated in vivo, this observation suggests that a radioisotope as commonly used in nuclear medicine as 99mTc may significantly influence thyroid physiology. The aims of this study were to determine whether 99mTc pertechnetate (99mTcO4-) is capable of inducing thyroid stunning in vivo, to evaluate the absorbed dose of 99mTcO4- required to induce this stunning, and to analyze the biological events associated/concomitant with this effect. Our results show that 99mTcO4--mediated thyroid stunning can be observed in vivo in mouse thyroid. The threshold of the absorbed dose in the thyroid required to obtain a significant stunning effect is in the range of 20 Gy. This effect is associated with a reduced level of functional Na/I symporter (NIS) protein, with no significant cell death. It is reversible within a few days. At the cellular and molecular levels, a decrease in NIS mRNA, the generation of double-strand DNA breaks, and the activation of the p53 pathway are observed. Low-energy electrons emitted by 99mTc can, therefore, induce thyroid stunning in vivo in mice, if it is exposed to an absorbed dose of at least 20 Gy, a level unlikely to be encountered in clinical practice. Nevertheless this report presents an unexpected effect of low-energy electrons on a normal tissue in vivo, and provides a unique experimental setup to understand the fine molecular mechanisms involved in their biological effects.
Pubmed link : 24663284

18. FibromiRs: translating molecular discoveries into new anti-fibrotic drugs.
Trends Pharmacol Sci. 2014 Mar;35(3):119-26. doi: 10.1016/j.tips.2014.01.003. Epub 2014 Feb 19.
Pottier N, Cauffiez C, Perrais M, Barbry P, Mari B
1EA4483, Department of Biochemistry and Molecular Biology, Lille 2 University School of Medicine, Lille, France. Electronic address: nico_pottier@yahoo.fr. 2EA4483, Department of Biochemistry and Molecular Biology, Lille 2 University School of Medicine, Lille, France. 3INSERM U837, Jean-Pierre Aubert Research Center, Lille, France. 4CNRS, Institute of Molecular and Cellular Pharmacology, Sophia Antipolis, France; University of Nice Sophia Antipolis, Nice, France.

Fibrosis, or tissue scarring, is defined as excessive and persistent accumulation of extracellular matrix components in response to chronic tissue injury. Fibrosis is a pathological feature characterizing nearly all forms of chronic organ failure. Fibroproliferative disorders of liver, kidney, heart, and lung are frequently associated with considerable morbidity and mortality worldwide. Limited therapeutic options are available; none is yet effective in stopping the ultimate progression of the disease. This has prompted investigations for new molecular targets. Recent studies have shown aberrant expression of miRNAs (fibromiRs) during the development of fibrosis. The challenge now is to understand how these aberrantly expressed miRNAs collaborate to drive fibrogenesis. Progress in understanding how fibromiRs contribute to tissue fibrosis is necessary to translate molecular discoveries into new therapeutics for fibroproliferative diseases.
Pubmed link : 24560301

19. miR-199a-5p in idiopathic pulmonary fibrosis
Med Sci (Paris). 2013 May;29(5):461-3. doi: 10.1051/medsci/2013295006. Epub 2013 May 28.
Henaoui IS, Cauffiez C, Aubert S, Buscot M, Dewaeles E, Copin MC, Marquette CH, Barbry P, Perrais M, Pottier N, Mari B
UMR-7275 CNRS, université de Nice Sophia-Antipolis, institut de pharmacologie moléculaire et cellulaire, 660, route des Lucioles Sophia-Antipolis, 06560 Valbonne, Nice, France.


Pubmed link : 23732092

20. A novel role for the RNA-binding protein FXR1P in myoblasts cell-cycle progression by modulating p21/Cdkn1a/Cip1/Waf1 mRNA stability.
PLoS Genet. 2013 Mar;9(3):e1003367. doi: 10.1371/journal.pgen.1003367. Epub 2013 Mar 21.
Davidovic L, Durand N, Khalfallah O, Tabet R, Barbry P, Mari B, Sacconi S, Moine H, Bardoni B
Institut de Pharmacologie Moléculaire et Cellulaire, CNRS UMR 7275, Valbonne, France.

The Fragile X-Related 1 gene (FXR1) is a paralog of the Fragile X Mental Retardation 1 gene (FMR1), whose absence causes the Fragile X syndrome, the most common form of inherited intellectual disability. FXR1P plays an important role in normal muscle development, and its absence causes muscular abnormalities in mice, frog, and zebrafish. Seven alternatively spliced FXR1 transcripts have been identified and two of them are skeletal muscle-specific. A reduction of these isoforms is found in myoblasts from Facio-Scapulo Humeral Dystrophy (FSHD) patients. FXR1P is an RNA-binding protein involved in translational control; however, so far, no mRNA target of FXR1P has been linked to the drastic muscular phenotypes caused by its absence. In this study, gene expression profiling of C2C12 myoblasts reveals that transcripts involved in cell cycle and muscular development pathways are modulated by Fxr1-depletion. We observed an increase of p21--a regulator of cell-cycle progression--in Fxr1-knocked-down mouse C2C12 and FSHD human myoblasts. Rescue of this molecular phenotype is possible by re-expressing human FXR1P in Fxr1-depleted C2C12 cells. FXR1P muscle-specific isoforms bind p21 mRNA via direct interaction with a conserved G-quadruplex located in its 3' untranslated region. The FXR1P/G-quadruplex complex reduces the half-life of p21 mRNA. In the absence of FXR1P, the upregulation of p21 mRNA determines the elevated level of its protein product that affects cell-cycle progression inducing a premature cell-cycle exit and generating a pool of cells blocked at G0. Our study describes a novel role of FXR1P that has crucial implications for the understanding of its role during myogenesis and muscle development, since we show here that in its absence a reduced number of myoblasts will be available for muscle formation/regeneration, shedding new light into the pathophysiology of FSHD.
Pubmed link : 23555284

21. MiR-210 promotes a hypoxic phenotype and increases radioresistance in human lung cancer cell lines.
Cell Death Dis. 2013 Mar 14;4:e544. doi: 10.1038/cddis.2013.71.
Grosso S, Doyen J, Parks SK, Bertero T, Paye A, Cardinaud B, Gounon P, Lacas-Gervais S, Noel A, Pouysségur J, Barbry P, Mazure NM, Mari B
Institut de Pharmacologie Moléculaire et Cellulaire (IPMC), Centre National de la Recherche Scientifique, CNRS UMR 7275, Sophia Antipolis, France.

The resistance of hypoxic cells to radiotherapy and chemotherapy is a major problem in the treatment of cancer. Recently, an additional mode of hypoxia-inducible factor (HIF)-dependent transcriptional regulation, involving modulation of a specific set of micro RNAs (miRNAs), including miR-210, has emerged. We have recently shown that HIF-1 induction of miR-210 also stabilizes HIF-1 through a positive regulatory loop. Therefore, we hypothesized that by stabilizing HIF-1 in normoxia, miR-210 may protect cancer cells from radiation. We developed a non-small cell lung carcinoma (NSCLC)-derived cell line (A549) stably expressing miR-210 (pmiR-210) or a control miRNA (pmiR-Ctl). The miR-210-expressing cells showed a significant stabilization of HIF-1 associated with mitochondrial defects and a glycolytic phenotype. Cells were subjected to radiation levels ranging from 0 to 10 Gy in normoxia and hypoxia. Cells expressing miR-210 in normoxia had the same level of radioresistance as control cells in hypoxia. Under hypoxia, pmiR-210 cells showed a low mortality rate owing to a decrease in apoptosis, with an ability to grow even at 10 Gy. This miR-210 phenotype was reproduced in another NSCLC cell line (H1975) and in HeLa cells. We have established that radioresistance was independent of p53 and cell cycle status. In addition, we have shown that genomic double-strand breaks (DSBs) foci disappear faster in pmiR-210 than in pmiR-Ctl cells, suggesting that miR-210 expression promotes a more efficient DSB repair. Finally, HIF-1 invalidation in pmiR-210 cells removed the radioresistant phenotype, showing that this mechanism is dependent on HIF-1. In conclusion, miR-210 appears to be a component of the radioresistance of hypoxic cancer cells. Given the high stability of most miRNAs, this advantage could be used by tumor cells in conditions where reoxygenation has occurred and suggests that strategies targeting miR-210 could enhance tumor radiosensitization.
Pubmed link : 23492775

22. CDC25A targeting by miR-483-3p decreases CCND-CDK4/6 assembly and contributes to cell cycle arrest.
Cell Death Differ. 2013 Jun;20(6):800-11. doi: 10.1038/cdd.2013.5. Epub 2013 Feb 22.
Bertero T, Gastaldi C, Bourget-Ponzio I, Mari B, Meneguzzi G, Barbry P, Ponzio G, Rezzonico R
1] CNRS UMR 7275, IPMC, Physiological Genomics of the Eukaryotes, Valbonne, France [2] Université de Nice Sophia-Antipolis, Nice, France.

Abstract Disruption of contact inhibition and serum afflux that occur after a tissue injury activate cell cycle, which then stops when confluence is reached again. Although the events involved in cell cycle entry have been widely documented, those managing cell cycle exit have remained so far ill defined. We have identified that the final stage of wound closure is preceded in keratinocytes by a strong accumulation of miR-483-3p, which acts as a mandatory signal triggering cell cycle arrest when confluence is reached. Blocking miR-483-3p accumulation strongly delays cell cycle exit, maintains cells into a proliferative state and retards their differentiation program. Using two models of cell cycle synchronization (i.e. mechanical injury and serum addition), we show that an ectopic upregulation of miR-483-3p blocks cell cycle progression in early G1 phase. This arrest results from a direct targeting of the CDC25A phosphatase by miR-483-3p, which can be impeded using an anti-miRNA against miR-483-3p or a protector that blocks the complex formation between miR-483-3p and the 3'-untranslated region (UTR) of CDC25A transcript. We show that the miRNA-induced silencing of CDC25A increases the tyrosine phosphorylation status of CDK4/6 cyclin-dependent kinases which, in turn, abolishes CDK4/6 capacity to associate with D-type cyclins. This prevents CDK4/6 kinases' activation, impairs downstream events such as cyclin E stimulation and sequesters cells in early G1. We propose this new regulatory process of cyclin-CDK association as a general mechanism coupling miRNA-mediated CDC25A invalidation to CDK post-transcriptional modifications and cell cycle control.
Pubmed link : 23429262

23. miR-199a-5p Is upregulated during fibrogenic response to tissue injury and mediates TGFbeta-induced lung fibroblast activation by targeting caveolin-1.
PLoS Genet. 2013 Feb;9(2):e1003291. doi: 10.1371/journal.pgen.1003291. Epub 2013 Feb 14.
Lino Cardenas CL, Henaoui IS, Courcot E, Roderburg C, Cauffiez C, Aubert S, Copin MC, Wallaert B, Glowacki F, Dewaeles E, Milosevic J, Maurizio J, Tedrow J, Marcet B, Lo-Guidice JM, Kaminski N, Barbry P, Luedde T, Perrais M, Mari B, Pottier N
EA4483, Faculté de Médecine de Lille, Pole Recherche, Lille, France.

As miRNAs are associated with normal cellular processes, deregulation of miRNAs is thought to play a causative role in many complex diseases. Nevertheless, the precise contribution of miRNAs in fibrotic lung diseases, especially the idiopathic form (IPF), remains poorly understood. Given the poor response rate of IPF patients to current therapy, new insights into the pathogenic mechanisms controlling lung fibroblasts activation, the key cell type driving the fibrogenic process, are essential to develop new therapeutic strategies for this devastating disease. To identify miRNAs with potential roles in lung fibrogenesis, we performed a genome-wide assessment of miRNA expression in lungs from two different mouse strains known for their distinct susceptibility to develop lung fibrosis after bleomycin exposure. This led to the identification of miR-199a-5p as the best miRNA candidate associated with bleomycin response. Importantly, miR-199a-5p pulmonary expression was also significantly increased in IPF patients (94 IPF versus 83 controls). In particular, levels of miR-199a-5p were selectively increased in myofibroblasts from injured mouse lungs and fibroblastic foci, a histologic feature associated with IPF. Therefore, miR-199a-5p profibrotic effects were further investigated in cultured lung fibroblasts: miR-199a-5p expression was induced upon TGFβ exposure, and ectopic expression of miR-199a-5p was sufficient to promote the pathogenic activation of pulmonary fibroblasts including proliferation, migration, invasion, and differentiation into myofibroblasts. In addition, we demonstrated that miR-199a-5p is a key effector of TGFβ signaling in lung fibroblasts by regulating CAV1, a critical mediator of pulmonary fibrosis. Remarkably, aberrant expression of miR-199a-5p was also found in unilateral ureteral obstruction mouse model of kidney fibrosis, as well as in both bile duct ligation and CCl4-induced mouse models of liver fibrosis, suggesting that dysregulation of miR-199a-5p represents a general mechanism contributing to the fibrotic process. MiR-199a-5p thus behaves as a major regulator of tissue fibrosis with therapeutic potency to treat fibroproliferative diseases.
Pubmed link : 23459460

24. The 3' UTR of FMR1 mRNA is a target of miR-101, miR-129-5p and miR-221: implications for the molecular pathology of FXTAS at the synapse.
Hum Mol Genet. 2013 May 15;22(10):1971-82. doi: 10.1093/hmg/ddt044. Epub 2013 Feb 5.
Zongaro S, Hukema R, D'Antoni S, Davidovic L, Barbry P, Catania MV, Willemsen R, Mari B, Bardoni B
CNRS UMR 7275, Institute of Molecular and Cellular Pharmacology, Valbonne Sophia-Antipolis, France.

While FMR1 is silenced in Fragile X syndrome (FXS) patients carrying the full mutation, its expression is elevated (2-8 fold) in premutated individuals. These people may develop the Fragile X-associated Tremor/Ataxia syndrome (FXTAS), a late onset neurodegenerative disorder characterized by ataxia and parkinsonism. In addition, people carrying the premutation can be affected by a set of neurological and behavioral disorders during young age. Problems of memory have been detected in these patients as well as in the mouse models for FXTAS. To date little is known concerning the metabolism of FMR1 mRNA, notwithstanding the importance of the finely tuned regulation of the expression of this gene. In the present study, we identified three microRNAs that specifically target the 3' UTR of FMR1 and can modulate its expression throughout the brain particularly at the synapse where their expression is very high. The expression level of miR-221 is reduced in synaptosomal preparations of young FXTAS mice suggesting a general deregulation of transcripts located at the synapse of these mice. By transcriptome analysis, we show here a robust deregulation of the expression levels of genes involved in learning, memory and autistic behavior, Parkinson disease and neurodegeneration. These findings suggest the presence of a synaptopathy in these animals. Interestingly, many of those deregulated mRNAs are target of the same microRNAs that modulate the expression of FMR1 at the synapse.
Pubmed link : 23390134

25. Dkk3 is a component of the genetic circuitry regulating aldosterone biosynthesis in the adrenal cortex.
Hum Mol Genet. 2012 Nov 15;21(22):4922-9. doi: 10.1093/hmg/dds333. Epub 2012 Aug 23.
El Wakil A, Bandulik S, Guy N, Bendahhou S, Zennaro MC, Niehrs C, Mari B, Warth R, Barhanin J, Lalli E
Institut de Pharmacologie Moléculaire et Cellulaire CNRS, Valbonne, France.

Primary aldosteronism (PA, autonomous aldosterone production from the adrenal cortex) causes the most common form of secondary arterial hypertension (HT), which is also the most common curable form of HT. Recent studies have highlighted an important role of mutations in genes encoding potassium channels in the pathogenesis of PA, both in human disease and in animal models. Here, we have exploited the unique features of the hyperaldosteronemic phenotype of Kcnk3 null mice, which is dependent on sexual hormones, to identify genes whose expression is modulated in the adrenal gland according to the dynamic hyperaldosteronemic phenotype of those animals. Genetic inactivation of one of the genes identified by our strategy, dickkopf-3 (Dkk3), whose expression is increased by calcium influx into adrenocortical cells, in the Kcnk3 null background results in the extension of the low-renin, potassium-rich diet insensitive hyperaldosteronemic phenotype to the male sex. Compound Kcnk3/Dkk3 animals display an increased expression of Cyp11b2, the rate-limiting enzyme for aldosterone biosynthesis in the adrenal zona glomerulosa (ZG). Our data show that Dkk3 can act as a modifier gene in a mouse model for altered potassium channel function and suggest its potential involvement in human PA syndromes.
Pubmed link : 22918120

26. Hypoxia-inducible miR-210 regulates the susceptibility of tumor cells to lysis by cytotoxic T cells.
Cancer Res. 2012 Sep 15;72(18):4629-41. Epub 2012 Sep 7.
Noman MZ, Buart S, Romero P, Ketari S, Janji B, Mari B, Mami-Chouaib F, Chouaib S
Cancer Res. 2012 Sep 15;72(18):4629-41. Epub 2012 Sep 7.

Hypoxia in the tumor microenvironment plays a central role in the evolution of immune escape mechanisms by tumor cells. In this study, we report the definition of miR-210 as a miRNA regulated by hypoxia in lung cancer and melanoma, documenting its involvement in blunting the susceptibility of tumor cells to lysis by antigen-specific cytotoxic T lymphocytes (CTL). miR-210 was induced in hypoxic zones of human tumor tissues. Its attenuation in hypoxic cells significantly restored susceptibility to autologous CTL-mediated lysis, independent of tumor cell recognition and CTL reactivity. A comprehensive approach using transcriptome analysis, argonaute protein immunoprecipitation, and luciferase reporter assay revealed that the genes PTPN1, HOXA1, and TP53I11 were miR-210 target genes regulated in hypoxic cells. In support of their primary importance in mediating the immunosuppressive effects of miR-210, coordinate silencing of PTPN1, HOXA1, and TP53I11 dramatically decreased tumor cell susceptibility to CTL-mediated lysis. Our findings show how miR-210 induction links hypoxia to immune escape from CTL-mediated lysis, by providing a mechanistic understanding of how this miRNA mediates immunosuppression in oxygen-deprived regions of tumors where cancer stem-like cells and metastatic cellular behaviors are known to evolve.
Pubmed link : 22962263

27. "Seed-Milarity" confers to hsa-miR-210 and hsa-miR-147b similar functional activity.
PLoS One. 2012;7(9):e44919. doi: 10.1371/journal.pone.0044919. Epub 2012 Sep 13.
Bertero T, Grosso S, Robbe-Sermesant K, Lebrigand K, Henaoui IS, Puissegur MP, Fourre S, Zaragosi LE, Mazure NM, Ponzio G, Cardinaud B, Barbry P, Rezzonico R, Mari B
Institut de Pharmacologie Moléculaire et Cellulaire-IPMC, Centre National de la Recherche Scientifique, CNRS UMR 7275, Sophia Antipolis, France.

Specificity of interaction between a microRNA (miRNA) and its targets crucially depends on the seed region located in its 5'-end. It is often implicitly considered that two miRNAs sharing the same biological activity should display similarity beyond the strict six nucleotide region that forms the seed, in order to form specific complexes with the same mRNA targets. We have found that expression of hsa-miR-147b and hsa-miR-210, though triggered by different stimuli (i.e. lipopolysaccharides and hypoxia, respectively), induce very similar cellular effects in term of proliferation, migration and apoptosis. Hsa-miR-147b only shares a "minimal" 6-nucleotides seed sequence with hsa-miR-210, but is identical with hsa-miR-147a over 20 nucleotides, except for one base located in the seed region. Phenotypic changes induced after heterologous expression of miR-147a strikingly differ from those induced by miR-147b or miR-210. In particular, miR-147a behaves as a potent inhibitor of cell proliferation and migration. These data fit well with the gene expression profiles observed for miR-147b and miR-210, which are very similar, and the gene expression profile of miR-147a, which is distinct from the two others. Bioinformatics analysis of all human miRNA sequences indicates multiple cases of miRNAs from distinct families exhibiting the same kind of similarity that would need to be further characterized in terms of putative functional redundancy. Besides, it implies that functional impact of some miRNAs can be masked by robust expression of miRNAs belonging to distinct families.
Pubmed link : 23028679

28. On the Pro-Metastatic Stress Response to Cancer Therapies: Evidence for a Positive Co-Operation between TIMP-1, HIF-1α, and miR-210.
Front Pharmacol. 2012;3:134. doi: 10.3389/fphar.2012.00134. Epub 2012 Jul 12.
Cui H, Grosso S, Schelter F, Mari B, Kruger A
Klinikum Rechts der Isar der Technischen Universität München, Institut für Experimentelle Onkologie und Therapieforschung München, Germany.

In contrast to expectations in the past that tumor starvation or unselective inhibition of proteolytic activity would cure cancer, there is accumulating evidence that microenvironmental stress, such as hypoxia or broad-spectrum inhibition of metalloproteinases can promote metastasis. In fact, malignant tumor cells, due to their genetic and epigenetic instability, are predisposed to react to stress by adaptation and, if the stress persists, by escape and formation of metastasis. Recent recognition of the concepts of dynamic evolution as well as population and systems biology is extremely helpful to understand the disappointments of clinical trials with new drugs and may lead to paradigm-shifts in therapy strategies. This must be complemented by an increased understanding of molecular mechanism involved in stress response. Here, we review new roles of Hypoxia-inducible factor-1 (HIF-1), one transcription factor regulating stress response-related gene expression: HIF-1 is crucial for invasion and metastasis, independent from its pro-survival function. In addition, HIF-1 mediates pro-metastatic microenvironmental changes of the proteolytic balance as triggered by high systemic levels of tissue inhibitor of metalloproteinases-1 (TIMP-1), typical for many aggressive cancers, and regulates the metabolic switch to glycolysis, notably via activation of the microRNA miR-210. There is preliminary evidence that TIMP-1 also induces miR-210. Such positive-regulatory co-operation of HIF-1α, miR-210, and TIMP-1, all described to correlate with bad prognosis of cancer patients, opens new perspectives of gaining insight into molecular mechanisms of metastasis-inducing evasion of tumor cells from stress.
Pubmed link : 22807917

29. Distinct epithelial gene expression phenotypes in childhood respiratory allergy.
Eur Respir J. 2012 May;39(5):1197-205. Epub 2011 Oct 17.
Giovannini-Chami L, Marcet B, Moreilhon C, Chevalier B, Illie MI, Lebrigand K, Robbe-Sermesant K, Bourrier T, Michiels JF, Mari B, Crénesse D, Hofman P, de Blic J, Castillo L, Albertini M, Barbry P
CNRS and University of Nice Sophia Antipolis, Institut de Pharmacologie Moléculaire et Cellulaire, UMR 7275, Sophia Antipolis, 06560 Sophia Antipolis, France.

Epithelial cell contribution to the natural history of childhood allergic respiratory disease remains poorly understood. Our aims were to identify epithelial pathways that are dysregulated in different phenotypes of respiratory allergy. We established gene expression signatures of nasal brushings from children with dust mite-allergic rhinitis, associated or not associated with controlled or uncontrolled asthma. Supervised learning and unsupervised clustering were used to predict the different subgroups of patients and define altered signalling pathways. These profiles were compared with those of primary cultures of human nasal epithelial cells stimulated with either interleukin (IL)-4, IL-13, interferon (IFN)-α, IFN-β or IFN-γ, or during in vitro differentiation. A supervised method discriminated children with allergic rhinitis from healthy controls (prediction accuracy 91%), based on 61 transcripts, including 21 T-helper cell (Th) type 2-responsive genes. This method was then applied to predict children with controlled or uncontrolled asthma (prediction accuracy 75%), based on 41 transcripts: nine of them, which were down-regulated in uncontrolled asthma, are directly linked to IFN. This group also included GSDML, which is genetically associated with asthma. Our data revealed a Th2-driven epithelial phenotype common to all children with dust mite allergic rhinitis. It highlights the influence of epithelially expressed molecules on the control of asthma, in association with atopy and impaired viral response.
Pubmed link : 22005912

30. Expression of a truncated active form of VDAC1 in lung cancer associates with hypoxic cell survival and correlates with progression to chemotherapy resistance.
Cancer Res. 2012 Apr 15;72(8):2140-50. doi: 10.1158/0008-5472.CAN-11-3940. Epub 2012 Mar 2.
Brahimi-Horn MC, Ben-Hail D, Ilie M, Gounon P, Rouleau M, Hofman V, Doyen J, Mari B, Shoshan-Barmatz V, Hofman P, Pouysségur J, Mazure NM
Institute of Developmental Biology and Cancer Research, University of Nice, CNRS-UMR 6543, Centre Antoine Lacassagne, Nice, France.

Resistance to chemotherapy-induced apoptosis of tumor cells represents a major hurdle to efficient cancer therapy. Although resistance is a characteristic of tumor cells that evolve in a low oxygen environment (hypoxia), the mechanisms involved remain elusive. We observed that mitochondria of certain hypoxic cells take on an enlarged appearance with reorganized cristae. In these cells, we found that a major mitochondrial protein regulating metabolism and apoptosis, the voltage-dependent anion channel 1 (VDAC1), was linked to chemoresistance when in a truncated (VDAC1-ΔC) but active form. The formation of truncated VDAC1, which had a similar channel activity and voltage dependency as full-length, was hypoxia-inducible factor-1 (HIF-1)-dependent and could be inhibited in the presence of the tetracycline antibiotics doxycycline and minocycline, known inhibitors of metalloproteases. Its formation was also reversible upon cell reoxygenation and associated with cell survival through binding to the antiapoptotic protein hexokinase. Hypoxic cells containing VDAC1-ΔC were less sensitive to staurosporine- and etoposide-induced cell death, and silencing of VDAC1-ΔC or treatment with the tetracycline antibiotics restored sensitivity. Clinically, VDAC1-ΔC was detected in tumor tissues of patients with lung adenocarcinomas and was found more frequently in large and late-stage tumors. Together, our findings show that via induction of VDAC1-ΔC, HIF-1 confers selective protection from apoptosis that allows maintenance of ATP and cell survival in hypoxia. VDAC1-ΔC may also hold promise as a biomarker for tumor progression in chemotherapy-resistant patients.
Pubmed link : 22389449

31. B-cell regulator of immunoglobulin heavy chain transcription (Bright)/ARID3a is a direct target of the oncomir microRNA-125b in progenitor B-cells.
Leukemia. 2012 Apr 3. doi: 10.1038/leu.2012.95.
Puissegur MP, Eichner R, Quelen C, Coyaud E, Mari B, Lebrigand K, Broccardo C, Nguyen-Khac F, Bousquet M, Brousset P
1] Institut National de la Sante et de la Recherche Medicale, U563, Centre de Physiopathologie de Toulouse-Purpan, Toulouse, France [2] Universite Paul Sabatier, Toulouse, France.

B-cell acute lymphoblastic leukemia (B-ALL) is often associated with chromosomal translocations leading to the deregulation of proto-oncogenes. MicroRNAs can also be affected by chromosomal alterations and thus contribute to carcinogenesis. The microRNA miR-125b-1 is over-expressed in B-ALL cases with the t(11;14)(q24;q34) translocation, therefore we sought to determine the role of this microRNA in B-cell fate. We used murine pre-BI cells alongside murine and human leukemic B-cell lines to show that miR-125b expression enhances proliferation by targeting Bright/ARID3a, an activator of immunoglobulin heavy-chain transcription. Accordingly, this target gene was down-regulated in B-ALL patients with the t(11;14)(q24;q34) translocation. Repression of Bright/ARID3A blocked differentiation and conferred a survival advantage to Ba/F3 cells under IL3 starvation. In addition, over-expression of miR-125b protected pre-BI and leukemic B-cell lines from apoptosis through blockade of caspase activation via a mechanism that was independent of p53 and BAK1. In summary, miR-125b can act as an oncogene in B-ALL by targeting ARID3a and mediating its repression, thus leading to a blockage in differentiation, increased proliferation and inhibition of apoptosis.Leukemia accepted article preview online, 3 April 2012; doi:10.1038/leu.2012.95.
Pubmed link : 22469780

32. Spt6 levels are modulated by PAAF1 and proteasome to regulate the HIV-1 LTR.
Retrovirology. 2012 Feb 8;9:13. doi: 10.1186/1742-4690-9-13.
Nakamura M, Basavarajaiah P, Rousset E, Beraud C, Latreille D, Henaoui IS, Lassot I, Mari B, Kiernan R
Laboratoire de Régulation des Gènes, Institut de Génétique Humaine, CNRS UPR1142, Montpellier, France.

BACKGROUND: Tat-mediated activation of the HIV-1 promoter depends upon a proteasome-associated factor, PAAF1, which dissociates 26S proteasome to produce 19S RP that is essential for transcriptional elongation. The effect of PAAF1 on proteasome activity could also potentially shield certain factors from proteolysis, which may be implicated in the transcriptional co-activator activity of PAAF1 towards the LTR. RESULTS: Here, we show that Spt6 is targeted by proteasome in the absence of PAAF1. PAAF1 interacts with the N-terminus of Spt6, suggesting that PAAF1 protects Spt6 from proteolysis. Depletion of either PAAF1 or Spt6 reduced histone occupancy at the HIV-1 promoter, and induced the synthesis of aberrant transcripts. Ectopic Spt6 expression or treatment with proteasome inhibitor partially rescued the transcription defect associated with loss of PAAF1. Transcriptional profiling followed by ChIP identified a subset of cellular genes that are regulated in a similar fashion to HIV-1 by Spt6 and/or PAAF1, including many that are involved in cancer, such as BRCA1 and BARD1. CONCLUSION: These results show that intracellular levels of Spt6 are fine-tuned by PAAF1 and proteasome, which is required for HIV-1 transcription and extends to cellular genes implicated in cancer.
Pubmed link : 22316138

33. Global gene expression profiling of Ehrlichia ruminantium at different stages of development.
FEMS Immunol Med Microbiol. 2012 Feb;64(1):66-73. doi: 10.1111/j.1574-695X.2011.00901.x. Epub 2011 Dec 8.
Pruneau L, Emboulé L, Gely P, Marcelino I, Mari B, Pinarello V, Sheikboudou C, Martinez D, Daigle F, Lefrançois T, Meyer DF, Vachiery N
CIRAD, UMR CMAEE, Guadeloupe, France.

Ehrlichia ruminantium (ER), the causative agent of heartwater on ruminants, is an obligate intracellular bacterium transmitted by ticks of the genus Amblyomma. Previous studies have shown that early stages of development may be critical for Ehrlichia pathogenicity. To gain insights into the biology of intracellular ER, we determined the genome-wide transcriptional profile of ER replicating inside bovine aortic endothelial cells using DNA microarrays. At intermediate and late stages of infection (reticulate and elementary bodies, respectively), a total of 54 genes were differentially expressed. Among them, we measured by q-RTPCR the overexpression of 11 of 14 genes. A number of genes involved in metabolism, nutrient exchange, and defense mechanisms, including those involved in resistance to oxidative stress, were significantly induced in ER reticulate bodies. This is consistent with the oxidative stress condition and nutrient starvation that seem to occur in Ehrlichia-containing vacuoles. During the lysis stage of development, when ER is infectious, we showed the overexpression of a transcription factor, dksA, which is also known to induce virulence in other pathogens such as Salmonella typhimurium. Our results suggest a possible role of these genes in promoting ER development and pathogenicity.
Pubmed link : 22098128

34. MiR-129-5p is required for histone deacetylase inhibitor-induced cell death in thyroid cancer cells.
Endocr Relat Cancer. 2011 Nov 14;18(6):711-9. doi: 10.1530/ERC-10-0257. Print 2011 Dec.
Brest P, Lassalle S, Hofman V, Bordone O, Gavric Tanga V, Bonnetaud C, Moreilhon C, Rios G, Santini J, Barbry P, Svanborg C, Mograbi B, Mari B, Hofman P
Nice, France.

The molecular mechanism responsible for the antitumor activity of histone deacetylase inhibitors (HDACi) remains elusive. As HDACi have been described to alter miRNA expression, the aim of this study was to characterize HDACi-induced miRNAs and to determine their functional importance in the induction of cell death alone or in combination with other cancer drugs. Two HDACi, trichostatin A and vorinostat, induced miR-129-5p overexpression, histone acetylation and cell death in BCPAP, TPC-1, 8505C, and CAL62 cell lines and in primary cultures of papillary thyroid cancer (PTC) cells. In addition, miR-129-5p alone was sufficient to induce cell death and knockdown experiments showed that expression of this miRNA was required for HDACi-induced cell death. Moreover, miR-129-5p accentuated the anti-proliferative effects of other cancer drugs such as etoposide or human α-lactalbumin made lethal for tumor cells (HAMLET). Taken together, our data show that miR-129-5p is involved in the antitumor activity of HDACi and highlight a miRNA-driven cell death mechanism.
Pubmed link : 21946411

35. Can the microRNA signature distinguish between thyroid tumors of uncertain malignant potential and other well-differentiated tumors of the thyroid gland?
Endocr Relat Cancer. 2011 Sep 13;18(5):579-94. Print 2011 Oct.
Lassalle S, Hofman V, Ilie M, Bonnetaud C, Puisségur MP, Brest P, Loubatier C, Guevara N, Bordone O, Cardinaud B, Lebrigand K, Rios G, Santini J, Franc B, Mari B, Al Ghuzlan A, Vielh P, Barbry P, Hofman P
INSERM ERI-21/EA4319, University of Nice Sophia Antipolis, 06107 Nice, France.

The term 'thyroid tumors of uncertain malignant potential' (TT-UMP) was coined by surgical pathologists to define well-differentiated tumors (WDT) showing inconclusive morphological evidence of malignancy or benignity. We have analyzed the expression of microRNA (miRNA) in a training set of 42 WDT of different histological subtypes: seven follicular tumors of UMP (FT-UMP), six WDT-UMP, seven follicular thyroid adenomas (FTA), 11 conventional papillary thyroid carcinomas (C-PTC), five follicular variants of PTC (FV-PTC), and six follicular thyroid carcinomas (FTC), which led to the identification of about 40 deregulated miRNAs. A subset of these altered miRNAs was independently validated by qRT-PCR, which included 18 supplementary TT-UMP (eight WDT-UMP and ten FT-UMP). Supervised clustering techniques were used to predict the first 42 samples. Based on the four possible outcomes (FTA, C-PTC, FV-PTC, and FTC), about 80% of FTA and C-PTC and 50% of FV-PTC and FTC samples were correctly assigned. Analysis of the independent set of 18 WDT-UMP by quantitative RT-PCR for the selection of the six most discriminating miRNAs was unable to separate FT-UMP from WDT-UMP, suggesting that the miRNA signature is insufficient in characterizing these two clinical entities. We conclude that considering FT-UMP and WDT-UMP as distinct and specific clinical entities may improve the diagnosis of WDT of the thyroid gland. In this context, a small set of miRNAs (i.e. miR-7, miR-146a, miR-146b, miR-200b, miR-221, and miR-222) appears to be useful, though not sufficient per se, in distinguishing TT-UMP from other WDT of the thyroid gland.
Pubmed link : 21778212

36. miR-483-3p controls proliferation in wounded epithelial cells.
FASEB J. 2011 Sep;25(9):3092-105. Epub 2011 Jun 15.
Bertero T, Gastaldi C, Bourget-Ponzio I, Imbert V, Loubat A, Selva E, Busca R, Mari B, Hofman P, Barbry P, Meneguzzi G, Ponzio G, Rezzonico R
INSERM U634, IFR50, Faculté de Médecine, France.

The mechanisms that regulate keratinocyte migration and proliferation in wound healing remain largely unraveled, notably regarding possible involvements of microRNAs (miRNAs). Here we disclose up-regulation of miR-483-3p in 2 distinct models of wound healing: scratch-injured cultures of human keratinocytes and wounded skin in mice. miR-483-3p accumulation peaks at the final stage of the wound closure process, consistent with a role in the arrest of "healing" progression. Using an in vitro wound-healing model, videomicroscopy, and 5-bromo-2'-uridine incorporation, we observed that overexpression of miR-483-3p inhibits keratinocyte migration and proliferation, whereas delivery of anti-miR-483-3p oligonucleotides sustains keratinocyte proliferation beyond the closure of the wound, compared with irrelevant anti-miR treatment. Expression profiling of keratinocytes transfected with miR-483-3p identified 39 transcripts that were both predicted targets of miR-483-3p and down-regulated after miR-483-3p overexpression. Luciferase reporter assays, Western blot analyses, and silencing by specific siRNAs finally established that kinase MK2, cell proliferation marker MKI67, and transcription factor YAP1 are direct targets of miR-483-3p that control keratinocyte proliferation. miR-483-3p-mediated down-regulation of MK2, MKI67, and YAP1 thus represents a novel mechanism controlling keratinocyte growth arrest at the final steps of reepithelialization.
Pubmed link : 21676945

37. CYR61 downregulation reduces osteosarcoma cell invasion, migration, and metastasis.
J Bone Miner Res. 2011 Jul;26(7):1533-42. doi: 10.1002/jbmr.343.
Fromigue O, Hamidouche Z, Vaudin P, Lecanda F, Patino A, Barbry P, Mari B, Marie PJ
Laboratory of Osteoblast Biology and Pathology, INSERM, Paris, France. olivia.fromigue@inserm.fr

Osteosarcoma is the most common primary tumor of bone. The rapid development of metastatic lesions and resistance to chemotherapy remain major mechanisms responsible for the failure of treatments and the poor survival rate for patients. We showed previously that the HMGCoA (3-hydroxy-3-methylglutaryl-coenzyme A) reductase inhibitor statin exhibits antitumoral effects on osteosarcoma cells. Here, using microarray analysis, we identify Cyr61 as a new target of statins. Transcriptome and molecular analyses revealed that statins downregulate Cyr61 expression in human and murine osteosarcoma cells. Cyr61 silencing in osteosarcoma cell lines enhanced cell death and reduced cell migration and cell invasion compared with parental cells, whereas Cyr61 overexpression had opposite effects. Cyr61 expression was evaluated in 231 tissue cores from osteosarcoma patients. Tissue microarray analysis revealed that Cyr61 protein expression was higher in human osteosarcoma than in normal bone tissue and was further increased in metastatic tissues. Finally, tumor behavior and metastasis occurrence were analyzed by intramuscular injection of modified osteosarcoma cells into BALB/c mice. Cyr61 overexpression enhanced lung metastasis development, whereas cyr61 silencing strongly reduced lung metastases in mice. The results reveal that cyr61 expression increases with tumor grade in human osteosarcoma and demonstrate that cyr61 silencing inhibits in vitro osteosarcoma cell invasion and migration as well as in vivo lung metastases in mice. These data provide a novel molecular target for therapeutic intervention in metastatic osteosarcoma.
Pubmed link : 21312266

38. Small RNA sequencing reveals miR-642a-3p as a novel adipocyte-specific microRNA and miR-30 as a key regulator of human adipogenesis.
Genome Biol. 2011 Jul 18;12(7):R64.
Zaragosi LE, Wdziekonski B, Brigand KL, Villageois P, Mari B, Waldmann R, Dani C, Barbry P
Centre National de la Recherche Scientifique, Institut de Pharmacologie Moléculaire et Cellulaire, UMR-6097, 660 Route des Lucioles, Valbonne Sophia-Antipolis 06560, France.

BACKGROUND: In severe obesity, as well as in normal development, the growth of adipose tissue is the result of an increase in adipocyte size and numbers, which is underlain by the stimulation of adipogenic differentiation of precursor cells. A better knowledge of the pathways that regulate adipogenesis is therefore essential for an improved understanding of adipose tissue expansion. As microRNAs (miRNAs) have a critical role in many differentiation processes, our study aimed to identify the role of miRNA-mediated gene silencing in the regulation of adipogenic differentiation. RESULTS: We used deep sequencing to identify small RNAs that are differentially expressed during adipogenesis of adipose tissue-derived stem cells. This approach revealed the un-annotated miR-642a-3p as a highly adipocyte-specific miRNA. We then focused our study on the miR-30 family, which was also up-regulated during adipogenic differentiation and for which the role in adipogenesis had not yet been elucidated. Inhibition of the miR-30 family blocked adipogenesis, whilst over-expression of miR-30a and miR-30d stimulated this process. We additionally showed that both miR-30a and miR-30d target the transcription factor RUNX2, and stimulate adipogenesis via the modulation of this major regulator of osteogenesis. CONCLUSIONS: Overall, our data suggest that the miR-30 family plays a central role in adipocyte development. Moreover, as adipose tissue-derived stem cells can differentiate into either adipocytes or osteoblasts, the down-regulation of the osteogenesis regulator RUNX2 represents a plausible mechanism by which miR-30 miRNAs may contribute to adipogenic differentiation of adipose tissue-derived stem cells.
Pubmed link : 21767385

39. Impact of microRNA in normal and pathological respiratory epithelia.
Methods Mol Biol. 2011;741:171-91.
Giovannini-Chami L, Grandvaux N, Zaragosi LE, Robbe-Sermesant K, Marcet B, Cardinaud B, Coraux C, Berthiaume Y, Waldmann R, Mari B, Barbry P
CNRS, Université de Nice Sophia Antipolis, IPMC, UMR6097, Sophia Antipolis, France. chami@ipmc.cnrs.fr

Extensive sequencing efforts, combined with ad hoc bioinformatics developments, have now led to the identification of 1222 distinct miRNAs in human (derived from 1368 distinct genomic loci) and of many miRNAs in other multicellular organisms. The present chapter is aimed at describing a general experimental strategy to identify specific miRNA expression profiles and to highlight the functional networks operating between them and their mRNA targets, including several miRNAs deregulated in cystic fibrosis and during differentiation of airway epithelial cells.
Pubmed link : 21594785

40. A synonymous variant in IRGM alters a binding site for miR-196 and causes deregulation of IRGM-dependent xenophagy in Crohn's disease.
Nat Genet. 2011 Mar;43(3):242-5. Epub 2011 Jan 30.
Brest P, Lapaquette P, Souidi M, Lebrigand K, Cesaro A, Vouret-Craviari V, Mari B, Barbry P, Mosnier JF, Hébuterne X, Harel-Bellan A, Mograbi B, Darfeuille-Michaud A, Hofman P
INSERM ERI-21, EA4319, Faculty of Medicine, Nice, France.

Susceptibility to Crohn's disease, a complex inflammatory disease, is influenced by common variants at many loci. The common exonic synonymous SNP (c.313C>T) in IRGM, found in strong linkage disequilibrium with a deletion polymorphism, has been classified as non-causative because of the absence of an alteration in the IRGM protein sequence or splice sites. Here we show that a family of microRNAs (miRNAs), miR-196, is overexpressed in the inflammatory intestinal epithelia of individuals with Crohn's disease and downregulates the IRGM protective variant (c.313C) but not the risk-associated allele (c.313T). Subsequent loss of tight regulation of IRGM expression compromises control of intracellular replication of Crohn's disease-associated adherent invasive Escherichia coli by autophagy. These results suggest that the association of IRGM with Crohn's disease arises from a miRNA-based alteration in IRGM regulation that affects the efficacy of autophagy, thereby implicating a synonymous polymorphism as a likely causal variant.
Pubmed link : 21278745

41. MiRonTop: mining microRNAs targets across large scale gene expression studies
Bioinformatics. 2010 Oct 19
Le Brigand K, Robbe-Sermesant K, Mari B, Barbry P
CNRS, Institut de Pharmacologie Moleculaire et Cellulaire, UMR6097, 06560 Sophia-Antipolis, France.

SUMMARY: Current challenges in microRNA (miRNA) research are to improve the identification of in vivo mRNA targets and clarify the complex interplay existing between a specific miRNA and multiple biological networks. MiRonTop is an online java web tool that integrates DNA microarrays or high-throughput sequencing data to identify the potential implication of miRNAs on a specific biological system. It allows a rapid characterization of the most pertinent mRNA targets according to several existing miRNA target prediction approaches. It also provides useful representations of the enrichment scores according to the position of the target site along the 3'- UTR, where the contribution of the sites located in the vicinity of the stop codon and of the polyA tail can be clearly highlighted. It provides different graphs of miRNA enrichment associated with up- or down-regulated transcripts and different summary tables about selections of mRNA targets and their functional annotations by Gene Ontology. AVAILABILITY: http://www.microarray.fr:8080/miRonTop/index
Pubmed link : 20959382

42. miR-210 is overexpressed in late stages of lung cancer and mediates mitochondrial alterations associated with modulation of HIF-1 activity.
Cell Death Differ. 2010 Oct 1.
Puissegur MP, Mazure NM, Bertero T, Pradelli L, Grosso S, Robbe-Sermesant K, Maurin T, Lebrigand K, Cardinaud B, Hofman V, Fourre S, Magnone V, Ricci JE, Pouyssegur J, Gounon P, Hofman P, Barbry P, Mari B
[1] Institut de Pharmacologie Moleculaire et Cellulaire, CNRS UMR6097, Sophia Antipolis, France [2] University of Nice Sophia-Antipolis, Nice, France.

Following the identification of a set of hypoxia-regulated microRNAs (miRNAs), recent studies have highlighted the importance of miR-210 and of its transcriptional regulation by the transcription factor hypoxia-inducible factor-1 (HIF-1). We report here that miR-210 is overexpressed at late stages of non-small cell lung cancer. Expression of miR-210 in lung adenocarcinoma A549 cells caused an alteration of cell viability associated with induction of caspase-3/7 activity. miR-210 induced a loss of mitochondrial membrane potential and the apparition of an aberrant mitochondrial phenotype. The expression profiling of cells overexpressing miR-210 revealed a specific signature characterized by enrichment for transcripts related to 'cell death' and 'mitochondrial dysfunction', including several subunits of the electron transport chain (ETC) complexes I and II. The transcript coding for one of these ETC components, SDHD, subunit D of succinate dehydrogenase complex (SDH), was validated as a bona fide miR-210 target. Moreover, SDHD knockdown mimicked miR-210-mediated mitochondrial alterations. Finally, miR-210-dependent targeting of SDHD was able to activate HIF-1, in line with previous studies linking loss-of-function SDH mutations to HIF-1 activation. miR-210 can thus regulate mitochondrial function by targeting key ETC component genes with important consequences on cell metabolism, survival and modulation of HIF-1 activity. These observations help explain contradictory data regarding miR-210 expression and its putative function in solid tumors.
Pubmed link : 20885442

43. Innovative approach for transcriptomic analysis of obligate intracellular pathogen: selective capture of transcribed sequences of Ehrlichia ruminantium.
BMC Mol Biol. 2009 Dec 24;10:111.
Emboule L, Daigle F, Meyer DF, Mari B, Pinarello V, Sheikboudou C, Magnone V, Frutos R, Viari A, Barbry P, Martinez D, Lefrançois T, Vachiery N
UMR 15 CIRAD-INRA, Controle des maladies animales exotiques et emergentes, Site de Duclos, Prise d'Eau 97170, Petit Bourg, Guadeloupe. loic.emboule@cirad.fr

BACKGROUND: Whole genome transcriptomic analysis is a powerful approach to elucidate the molecular mechanisms controlling the pathogenesis of obligate intracellular bacteria. However, the major hurdle resides in the low quantity of prokaryotic mRNAs extracted from host cells. Our model Ehrlichia ruminantium (ER), the causative agent of heartwater, is transmitted by tick Amblyomma variegatum. This bacterium affects wild and domestic ruminants and is present in Sub-Saharan Africa and the Caribbean islands. Because of its strictly intracellular location, which constitutes a limitation for its extensive study, the molecular mechanisms involved in its pathogenicity are still poorly understood. RESULTS: We successfully adapted the SCOTS method (Selective Capture of Transcribed Sequences) on the model Rickettsiales ER to capture mRNAs. Southern Blots and RT-PCR revealed an enrichment of ER's cDNAs and a diminution of ribosomal contaminants after three rounds of capture. qRT-PCR and whole-genome ER microarrays hybridizations demonstrated that SCOTS method introduced only a limited bias on gene expression. Indeed, we confirmed the differential gene expression between poorly and highly expressed genes before and after SCOTS captures. The comparative gene expression obtained from ER microarrays data, on samples before and after SCOTS at 96 hpi was significantly correlated (R2 = 0.7). Moreover, SCOTS method is crucial for microarrays analysis of ER, especially for early time points post-infection. There was low detection of transcripts for untreated samples whereas 24% and 70.7% were revealed for SCOTS samples at 24 and 96 hpi respectively. CONCLUSIONS: We conclude that this SCOTS method has a key importance for the transcriptomic analysis of ER and can be potentially used for other Rickettsiales. This study constitutes the first step for further gene expression analyses that will lead to a better understanding of both ER pathogenicity and the adaptation of obligate intracellular bacteria to their environment.
Pubmed link : 20034374

44. miR-34b/miR-34c: a regulator of TCL1 expression in 11q- chronic lymphocytic leukaemia?
Leukemia. 2009 Nov;23(11):2174-7.
Cardinaud B, Moreilhon C, Marcet B, Robbe-Sermesant K, Lebrigand K, Mari B, Eclache V, Cymbalista F, Raynaud S, Barbry P
[1] CNRS, IPMC, UMR6097, Sophia Antipolis, France [2] Universite de Nice Sophia-Antipolis, IPMC, UMR6097, Sophia Antipolis, France [3] Universite Victor Segalen Bordeaux 2, EA 4135, Bordeaux, France E-mail: barbry@ipmc.cnrs.fr.


Pubmed link : 19536169

45. The caspase-cleaved form of LYN mediates a psoriasis-like inflammatory syndrome in mice.
EMBO J. 2009 Aug 19;28(16):2449-60. Epub 2009 Jul 9.
Marchetti S, Gamas P, Belhacene N, Grosso S, Pradelli LA, Colosetti P, Johansen C, Iversen L, Deckert M, Luciano F, Hofman P, Ortonne N, Khemis A, Mari B, Ortonne JP, Ricci JE, Auberger P
INSERM, U895, Centre Mediterraneen de Medecine Moleculaire (C3M), Team 2, Nice, France.

We showed previously that Lyn is a substrate for caspases, a family of cysteine proteases, involved in the regulation of apoptosis and inflammation. Here, we report that expression of the caspase-cleaved form of Lyn (LynDeltaN), in mice, mediates a chronic inflammatory syndrome resembling human psoriasis. Genetic ablation of TNF receptor 1 in a LynDeltaN background rescues a normal phenotype, indicating that LynDeltaN mice phenotype is TNF-alpha-dependent. The predominant role of T cells in the disease occurring in LynDeltaN mice was highlighted by the distinct improvement of LynDeltaN mice phenotype in a Rag1-deficient background. Using pan-genomic profiling, we also established that LynDeltaN mice show an increased expression of STAT-3 and inhibitory members of the NFkappaB pathway. Accordingly, LynDeltaN alters NFkappaB activity underlying a link between inhibition of NFkappaB and LynDeltaN mice phenotype. Finally, analysis of Lyn expression in human skin biopsies of psoriatic patients led to the detection of Lyn cleavage product whose expression correlates with the activation of caspase 1. Our data identify a new role for Lyn as a regulator of psoriasis through its cleavage by caspases.
Pubmed link : 19590497

46. Identification of keratinocyte growth factor as a target of microRNA-155 in lung fibroblasts: implication in epithelial-mesenchymal interactions.
PLoS One. 2009 Aug 24;4(8):e6718.
Pottier N, Maurin T, Chevalier B, Puissegur MP, Lebrigand K, Robbe-Sermesant K, Bertero T, Lino Cardenas CL, Courcot E, Rios G, Fourre S, Lo-Guidice JM, Marcet B, Cardinaud B, Barbry P, Mari B
CNRS, Institut de Pharmacologie Moleculaire et Cellulaire, UMR6097, Sophia Antipolis, France.

BACKGROUND: Epithelial-mesenchymal interactions are critical in regulating many aspects of vertebrate embryo development, and for the maintenance of homeostatic equilibrium in adult tissues. The interactions between epithelium and mesenchyme are believed to be mediated by paracrine signals such as cytokines and extracellular matrix components secreted from fibroblasts that affect adjacent epithelia. In this study, we sought to identify the repertoire of microRNAs (miRNAs) in normal lung human fibroblasts and their potential regulation by the cytokines TNF-alpha, IL-1beta and TGF-beta. METHODOLOGY/PRINCIPAL FINDINGS: MiR-155 was significantly induced by inflammatory cytokines TNF-alpha and IL-1beta while it was down-regulated by TGF-beta. Ectopic expression of miR-155 in human fibroblasts induced modulation of a large set of genes related to "cell to cell signalling", "cell morphology" and "cellular movement". This was consistent with an induction of caspase-3 activity and with an increase in cell migration in fibroblasts tranfected with miR-155. Using different miRNA bioinformatic target prediction tools, we found a specific enrichment for miR-155 predicted targets among the population of down-regulated transcripts. Among fibroblast-selective targets, one interesting hit was keratinocyte growth factor (KGF, FGF-7), a member of the fibroblast growth factor (FGF) family, which owns two potential binding sites for miR-155 in its 3'-UTR. Luciferase assays experimentally validated that miR-155 can efficiently target KGF 3'-UTR. Site-directed mutagenesis revealed that only one out of the 2 potential sites was truly functional. Functional in vitro assays experimentally validated that miR-155 can efficiently target KGF 3'-UTR. Furthermore, in vivo experiments using a mouse model of lung fibrosis showed that miR-155 expression level was correlated with the degree of lung fibrosis. CONCLUSIONS/SIGNIFICANCE: Our results strongly suggest a physiological function of miR-155 in lung fibroblasts. Altogether, this study implicates this miRNA in the regulation by mesenchymal cells of surrounding lung epithelium, making it a potential key player during tissue injury.
Pubmed link : 19701459

47. Gene expression profiling of imatinib and PD166326-resistant CML cell lines identifies Fyn as a gene associated with resistance to BCR-ABL inhibitors.
Mol Cancer Ther. 2009 Jul;8(7):1924-33. Epub 2009 Jun 30.
Grosso S, Puissant A, Dufies M, Colosetti P, Jacquel A, Lebrigand K, Barbry P, Deckert M, Cassuto JP, Mari B, Auberger P
INSERM U895, Cell Death, Differentiation and Cancer Team, Faculte de Medecine de Nice, Nice Cedex 2, France.

Imatinib is used to treat chronic myelogenous leukemia (CML), but resistance develops in all phases of this disease. The purpose of the present study was to identify the mode of resistance of newly derived imatinib-resistant (IM-R) and PD166326-resistant (PD-R) CML cells. IM-R and PD-R clones exhibited an increase in viability and a decrease in caspase activation in response to various doses of imatinib and PD166326, respectively, as compared with parental K562 cells. Resistance involved neither mutations in BCR-ABL nor increased BCR-ABL, MDR1 or Lyn expression, all known modes of resistance. To gain insight into the resistance mechanisms, we used pangenomic microarrays and identified 281 genes modulated in parental versus IM-R and PD-R cells. The gene signature was similar for IM-R and PD-R cells, accordingly with the cross-sensitivity observed for both inhibitors. These genes were functionally associated with pathways linked to development, cell adhesion, cell growth, and the JAK-STAT cascade. Especially relevant were the increased expression of the tyrosine kinases AXL and Fyn as well as CD44 and HMGA2. Small interfering RNA experiments and pharmacologic approaches identified FYN as a candidate for resistance to imatinib. Our findings provide a comprehensive picture of the transcriptional events associated with imatinib and PD166326 resistance and identify Fyn as a new potential target for therapeutic intervention in CML.
Pubmed link : 19567819

48. MicroRNAs and lung cancer: new oncogenes and tumor suppressors, new prognostic factors and potential therapeutic targets.
Curr Med Chem. 2009;16(9):1047-61.
Ortholan C, Puissegur MP, Ilie M, Barbry P, Mari B, Hofman P
Laboratory of Clinical and Experimental Pathology, Pasteur Hospital, Nice cedex 02, France.

MicroRNAs (miRNAs) are small non-protein-coding RNA that negatively control mRNA expression at a post-transcriptional level. They regulate various cellular functions and bioinformatic data suggest that they collectively control about 30% of human mRNAs. MiRNAs have been recently implicated in several carcinogenic processes, where they can act either as oncogenes or as tumor suppressors. This is the case in lung cancer, i.e. the leading cause of cancer deaths in Western countries, in which about 40-45 miRNAs have been found to be aberrantly expressed, thereby constituting a specific miRNA signature. Some of these miRNAs can play an important role in lung carcinogenesis. Indeed, some transcripts of the let-7 family that are significantly down-regulated in lung tumors have been identified as tumor suppressors through their ability to control several oncogenic pathways, including the RAS pathway. Identification of a growing number of other potential oncogenic or tumor suppressor miRNAs in lung cancers is in constant progress. Recent evidence supports the use of specific miRNA signatures to predict clinical outcome. This review aims to report the current knowledge about the role of miRNAs in lung cancer carcinogenesis, their potential for improving diagnosis and prognosis and their impact on future therapeutic strategies.
Pubmed link : 19275611

49. Transcriptional repression of microRNA genes by PML-RARA increases expression of key cancer proteins in acute promyelocytic leukemia.
Blood. 2009 Jan 8;113(2):412-21. Epub 2008 Oct 21.
Saumet A, Vetter G, Bouttier M, Portales-Casamar E, Wasserman WW, Maurin T, Mari B, Barbry P, Vallar L, Friederich E, Arar K, Cassinat B, Chomienne C, Lecellier CH
Institut de Genetique Humaine, CNRS UPR1142, Montpellier, France.

Micro(mi)RNAs are small noncoding RNAs that orchestrate many key aspects of cell physiology and their deregulation is often linked to distinct diseases including cancer. Here, we studied the contribution of miRNAs in a well-characterized human myeloid leukemia, acute promyelocytic leukemia (APL), targeted by retinoic acid and trioxide arsenic therapy. We identified several miRNAs transcriptionally repressed by the APL-associated PML-RAR oncogene which are released after treatment with all-trans retinoic acid. These coregulated miRNAs were found to control, in a coordinated manner, crucial pathways linked to leukemogenesis, such as HOX proteins and cell adhesion molecules whose expressions are thereby repressed by the chemotherapy. Thus, APL appears linked to transcriptional perturbation of miRNA genes, and clinical protocols able to successfully eradicate cancer cells may do so by restoring miRNA expression. The identification of abnormal miRNA biogenesis in cancer may therefore provide novel biomarkers and therapeutic targets in myeloid leukemias.
Pubmed link : 18941112

50. Isoform-specific contribution of protein kinase C to prion processing.
Mol Cell Neurosci. 2008 Nov;39(3):400-10. Epub 2008 Jul 29.
Alfa Cisse M, Louis K, Braun U, Mari B, Leitges M, Slack BE, Fisher A, Auberger P, Checler F, Vincent B
Institut de Pharmacologie Moleculaire et Cellulaire du CNRS, UMR6097, UNSA, Equipe labellisee Fondation pour la Recherche Medicale, Sophia-Antipolis, Valbonne, France.

The cellular prion protein (PrP(c)) undergoes a physiological cleavage between amino acids 111 and 112, thereby leading to the secretion of an amino-terminal fragment referred to as N1. This proteolytic event is either constitutive or regulated by protein kinase C (PKC) and is operated by the disintegrins ADAM9/ADAM10 or ADAM17 respectively. We recently showed that the stimulation of the M1/M3 muscarinic receptors potentiates this cleavage via the phosphorylation and activation of ADAM17. We have examined the contribution of various PKC isoforms in the regulated processing of PrP(c). First we show that the PDBu- and carbachol-stimulated N1 secretions are blocked by the general PKC inhibitor GF109203X. We establish that HEK293 and human-derived rhabdhomyosarcoma cells over-expressing constitutively active PKCalpha, PKCdelta or PKCepsilon, but not PKCzeta, produce increased amounts of N1 and harbor enhanced ability to hydrolyze the fluorimetric substrate of ADAM17, JMV2770. Conversely, over-expression of the corresponding dominant negative proteins abolishes PDBU-stimulated N1 secretion and restores N1 to levels comparable to constitutive production. Moreover, deletion of PKCalpha lowers N1 recovery in primary cultured fibroblasts. Importantly, mutation of threonine 735 of ADAM17 significantly lowers the PDBu-induced N1 formation while transient over-expression of constitutively active PKCalpha, PKCdelta or PKCepsilon, but not PKCzeta, induced both the phosphorylation of ADAM17 on its threonine residues and N1 secretion. As a corollary, T735A mutation concomitantly reversed PKCalpha-, PKCdelta- and PKCepsilon-induced ADAM17 phosphorylation and N1 recovery. Finally, we established that PKCepsilon-dependent N1 production is fully prevented by ADAM17 deficiency. Altogether, the present results provide strong evidence that the activation of PKCalpha, delta and epsilon, but not zeta, isoforms leads to increased N1 secretion via the phosphorylation and activation of ADAM17, a process that likely accounts for M1/M3 muscarinic receptors-mediated control of N1 production.
Pubmed link : 18722532

51. Gene expression profiling of human liver transplants identifies an early transcriptional signature associated with initial poor graft function.
Am J Transplant. 2008 Jun;8(6):1221-36.
Defamie V, Cursio R, Le Brigand K, Moreilhon C, Saint-Paul MC, Laurens M, Crenesse D, Cardinaud B, Auberger P, Gugenheim J, Barbry P, Mari B
CNRS, Institut de Pharmacologie Moleculaire et Cellulaire, UMR6097, 660, Route des Lucioles F-06560 Sophia Antipolis, France.

Liver ischemia-reperfusion injury occurring in orthotopic liver transplantation (OLT) may be responsible for early graft failure. Molecular mechanisms underlying initial poor graft function (IPGF) have been poorly documented in human. The purpose of this study was to identify the major transcriptional alterations occurring in human livers during OLT. Twenty-one RNA extracts derived from liver transplant biopsies taken after graft reperfusion were compared with 7 RNA derived from normal control livers. Three hundred seventy-one genes were significantly modulated and classified in molecular pathways relevant to liver metabolism, inflammatory response, cell proliferation and liver protection. Grafts were then subdivided into two groups based on their peak levels of serum aspartate amino transferase within 72 h after OLT (group 1, non-IPGF: 14 patients; group 2, IPGF: 7 patients). The two corresponding data sets were compared using a supervised prediction method. A new set of genes able to correctly classify 71% of the patients was defined. These genes were functionally associated with oxidative stress, inflammation and inhibition of cell proliferation. This study provides a comprehensive picture of the transcriptional events associated with human OLT and IPGF. We anticipate that such alterations provide a framework for the elucidation of the molecular mechanisms leading to IPGF.
Pubmed link : 18522548

52. Matrix metalloproteinase inhibition protects rat livers from prolonged cold ischemia-warm reperfusion injury.
Hepatology. 2008 Jan;47(1):177-85.
Defamie V, Laurens M, Patrono D, Devel L, Brault A, Saint-Paul MC, Yiotakis A, Barbry P, Gugenheim J, Crenesse D, Dive V, Huet, PM, Mari B
Institut de Pharmacologie Moleculaire et Cellulaire, CNRS, UMR6097, Universite de Nice-Sophia Antipolis, France.

Matrix metalloproteinases (MMPs) have been implicated in the hepatic injury induced after cold ischemia-warm reperfusion (CI-WR), by altering the extracellular matrix (ECM), but their precise role remains unknown. The hepatic MMP expression was evaluated after two conditions of CI (4oC for 24 and 42 hours: viable and nonviable livers) followed by different periods of WR, using isolated perfused rat livers. CI-WR induced moderate changes in hepatic MMP transcript levels not influenced by CI duration, while gelatinase activities accumulated in liver effluents. Therefore, the protective effect of a new phosphinic MMP inhibitor, RXP409, was tested after prolonged CI. RXP409 (10µM) was added to the UW solution and livers were preserved for 42 hours (4oC), then reperfused for 1 hour in Krebs solution (37oC), containing 20% erythrocytes. Liver viability parameters were recorded and the extent of cell necrosis was evaluated on liver biopsies, using trypan blue nuclear uptake. Treatment with RXP409 significantly improved liver function (transaminase release and bile secretion) and liver injury. In particular, the MMP inhibitor significantly modified the extent of cell death from large clusters of necrotic hepatocytes as found in control livers (2 to 60% of liver biopsies, mean: 26 ± 9%) to isolated necrotic hepatocytes as found in treated livers (0.2 to 12%, mean 3 ± 2%) (p<0.05). In conclusion, these data demonstrate that MMPs, by altering the ECM, play a major role in liver CI-WR injury leading to extensive hepatocyte necrosis and that their inhibition might prove to be a new strategy in improving preservation solutions.
Pubmed link : 18008367

53. A comparative analysis of perturbations caused by a gene knockout, a dominant negative allele, and a set of peptide aptamers.
Mol Cell Proteomics. 2007 Sep 4;
Abed N, Bickle M, Mari B, Schapira M, Sanjuan-Espana R, Robbe-Sermesant K, Moncorge O, Mouradian-Garcia S, Barbry P, Rudkin BB, Fauvarque MO, Michaud-Soret I, Colas P
CNRS UPS 2682, Station Biologique, Roscoff cedex 29682.

The study of protein function mostly relies on perturbing regulatory networks by acting upon protein expression levels or using transdominant negative agents. Here, we used the E.coli global transcription regulator Fur (ferric uptake regulator) as a case study to compare the perturbations exerted by a gene knockout, the expression of a dominant negative allele of a gene and the expression of peptide aptamers that bind a gene product. These three perturbations caused phenotypes that differed quantitatively and qualitatively from one another. The Fur peptide aptamers inhibited the activity of their target to various extents and reduced the virulence of a pathogenic E.coli strain in Drosophila. A genome-wide transcriptome analysis revealed that the "penetrance" of a peptide aptamer was comparable to that of a dominant-negative allele but lower than the "penetrance" of the gene knockout. Our work shows that comparative analysis of phenotypic and transcriptome responses to different types of perturbation can help decipher complex regulatory networks that control various biological processes.
Pubmed link : 17785351

54. Gene expression profiling in human gastric mucosa infected with Helicobacter pylori.
Mod Pathol. 2007 Sep;20(9):974-89. Epub 2007 Jul 20.
Hofman VJ, Moreilhon C, Brest PD, Lassalle S, Le Brigand K, Sicard D, Raymond J, Lamarque D, Hebuterne XA, Mari B, Barbry PJ, Hofman PM
1Faculty of Medicine, INSERM ERI-21, Nice Cedex, France [2] 2Laboratory of Clinical and Experimental Pathology, Pasteur Hospital, Nice, France.

Pathogenic mechanisms associated with Helicobacter pylori infection enhance susceptibility of the gastric epithelium to carcinogenic conversion. We have characterized the gene expression profiles of gastric biopsies from 69 French Caucasian patients, of which 43 (62%) were infected with H. pylori. The bacterium was detected in 27 of the 42 antral biopsies examined and in 16 of the 27 fundic biopsies. Infected biopsies were selected for the presence of chronic active gastritis, in absence of metaplasia and dysplasia of the gastric mucosa. Infected antral and fundic biopsies exhibited distinct transcriptional responses. Altered responses were linked with: (1) the extent of polymorphonuclear leukocyte infiltration, (2) bacterial density, and (3) the presence of the virulence factors vacA, babA2, and cagA. Robust modulation of transcripts associated with Toll-like receptors, signal transduction, the immune response, apoptosis, and the cell cycle was consistent with expected responses to Gram-negative bacterial infection. Altered expression of interferon-regulated genes (IFITM1, IRF4, STAT6), indicative of major histocompatibility complex (MHC) II-mediated and Th1-specific responses, as well as altered expression of GATA6, have previously been described in precancerous states. Upregulation of genes abundantly expressed in cancer tissues (UBD, CXCL13, LY96, MAPK8, MMP7, RANKL, CCL18) or in stem cells (IFITM1 and WFDC2) may reveal a molecular switch towards a premalignant state in infected tissues. Tissue microarray analysis of a large number of biopsies, which were either positive or negative for the cag-A virulence factor, when compared to each other and to noninfected controls, confirmed observed gene alterations at the protein level, for eight key transcripts. This study provides 'proof-of-principle' data for identifying molecular mechanisms driving H. pylori-associated carcinogenesis before morphological evidence of changes along the neoplastic progression pathway.Modern Pathology (2007) 20, 974-989; doi:10.1038/modpathol.3800930; published online 20 July 2007.
Pubmed link : 17643099

55. Relationships Between Early Inflammatory Response to Bleomycin and Sensitivity to Lung Fibrosis.
Am J Respir Crit Care Med. 2007 Aug 2;
Pottier N, Chupin C, Defamie V, Cardinaud B, Sutherland R, Rios G, Gauthier F, Wolters PJ, Berthiaume Y, Barbry P, Mari B
Institut de Pharmacologie Moleculaire et Cellulaire, UMR6097, CNRS and University of Nice Sophia Antipolis, Sophia Antipolis, France.

RATIONALE. Different sensitivities to pro-fibrotic compounds such as bleomycin are observed among mouse strains. OBJECTIVES. To identify genetic factors contributing to the outcome of lung injury. METHODS. Physiological comparison of C57BL/6 sensitive and Balb/C resistant mice challenged with intra tracheal bleomycin instillation revealed several early differences: global gene expression profiles were thus established from lungs derived from the two strains, in the absence of any bleomycin administration. MEASUREMENTS AND MAIN RESULTS. Expression of 25 genes differed between the two strains. Among them, two molecules, not previously associated with pulmonary fibrosis, were identified. The first one corresponds to dipeptidyl peptidase I (DPPI), a cysteine dipeptidyl peptidase (also known as cathepsin C) essential for the activation of serine proteinases produced by immune/inflammatory cells. The second corresponds to TIMP-3, an inhibitor of matrix metalloproteases and of ADAMs such as the TNFconverting enzyme. In functional studies performed in the bleomycin induced lung fibrosis model, the level of expression of these two genes was closely correlated with specific early events associated with lung fibrosis, namely activation of PMN-derived serine proteases and TNFalpha-dependent inflammatory syndrome. Surprisingly, genetic deletion of DPPI in the context of a C57BL/6 genetic background did not protect against bleomycin-mediated fibrosis, suggesting additional function(s) for this key enzyme. CONCLUSIONS. This study highlights the importance of the early inflammatory events that follow bleomycin instillation in the development of lung fibrosis, and describes for the first time the roles that DPPI and TIMP-3 may play in this process.
Pubmed link : 17673693

56. GAPDH and autophagy preserve survival after apoptotic cytochrome c release in the absence of caspase activation.
Cell. 2007 Jun 1;129(5):861-3.
Colell A, Ricci JE, Tait S, Milasta S, Maurer U, Bouchier-Hayes L, Fitzgerald P, Guio-Carrion A, Waterhouse NJ, Li CW, Mari B, Barbry P, Newmeyer DD, Beere HM, Green DR
Department of Cell Death and Proliferation, Institut d'Investigacions Biomediques de Barcelona (IIBB-CSIC), IDIBAPS, 08036 Barcelona, Spain.

In cells undergoing apoptosis, mitochondrial outer-membrane permeabilization (MOMP) is followed by caspase activation promoted by released cytochrome c. Although caspases mediate the apoptotic phenotype, caspase inhibition is generally not sufficient for survival following MOMP; instead cells undergo a "caspase-independent cell death" (CICD). Thus, MOMP may represent a point of commitment to cell death. Here, we identify glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a critical regulator of CICD. GAPDH-expressing cells preserved their clonogenic potential following MOMP, provided that caspase activation was blocked. GAPDH-mediated protection of cells from CICD involved an elevation in glycolysis and a nuclear function that correlated with and was replaced by an increase in Atg12 expression. Consistent with this, protection from CICD reflected an increase in and a dependence upon autophagy, associated with a transient decrease in mitochondrial mass. Therefore, GAPDH mediates an elevation in glycolysis and enhanced autophagy that cooperate to protect cells from CICD.
Pubmed link : 17540177

57. Transcriptional signature of epidermal keratinocytes subjected to in vitro scratch wounding, reveals selective roles for ERK1/2, P38 and PI3K signalling pathways.
J Biol Chem. 2007 May 18;282(20):15090-102.
Fitsialos G, Chassot AA, Turchi L, Dayem MA, Lebrigand K, Moreilhon C, Meneguzzi G, Busca R, Mari B, Barbry P, Ponzio G
Faculte de Medecine, INSERM U634, Nice 06107.

Covering denuded dermal surfaces after injury requires migration, proliferation and differentiation of skin keratinocytes. To clarify the major traits controlling these intermingled biological events, we surveyed the genomic modifications occurring during the course of a scratch wound closure of cultured human keratinocytes. Using a DNA microarray approach, we report the identification of 161 new markers of epidermal repair. Expression data, combined with functional analysis performed with specific inhibitors of ERK, p38[MAPK] and PI3 kinases, demonstrate that kinase pathways exert very selective functions by precisely controlling the expression of specific genes. Inhibition of the ERK pathway totally blocks the wound closure and inactivates many early transcription factors and EGF-type growth factors. P38[MAPK] inhibition only delays "healing", probably in line with the control of genes involved in the propagation of injury-initiated signalling. In contrast, PI3 kinase inhibition accelerates the scratch closure and potentiates the scratch-dependent stimulation of three genes related to epithelial cell transformation, namely HAS3, HBEGF and Ets1. Our results define in vitro human keratinocyte wound closure as a repair process resulting from a fine balance between positive signals controlled by ERK and p38[MAPK], and negative ones triggered by PI3 kinase. The perturbation of any of these pathways might lead to dysfunction in the healing process, similar to those observed in pathological wounding phenotypes, such as hypertrophic scars or keloids.
Pubmed link : 17363378

58. Effect of caspase inhibition on thymic apoptosis in hemorrhagic shock.
J Invest Surg. 2007 Mar-Apr;20(2):97-103
Bini R, Cursio R, Belhacene N, Giudicelli J, Ferrua B, Olivero G, Auberger P, Mari B, Gugenheim J, Cotogni P
Chirurgia d'Urgenza, Dipartimento di Discipline Medico-Chirurgiche, Universit a di Torino, Torino, Italy.

In hemorrhagic shock (HS) an increased thymic apoptosis (TA) was described. The aim of this study was to evaluate the effect of administration of the caspase inhibitor N-benzyloxy-carbonil-Val-Ala-Asp-fluoromethylketone (Z-VAD-FMK) during the resuscitation phase on TA, organ dysfunctions, and tumor necrosis factor (TNF)-alpha release in HS. Forty rats were randomly assigned to four groups: no HS/resuscitation (sham); HS/resuscitation with shed blood and normal saline (control); HS/resuscitation with shed blood and phosphate-buffered solution (PBS) (vehicle); and HS/resuscitation with shed blood and Z-VAD-FMK (inhibitor). Rats were subjected to HS by blood removal to a MAP of 35-40 mmHg. After a 1-h shock period, the animals were resuscitated according to the protocol. At 1 and 3 h after resuscitation, transaminases, creatinine, urea, lipase, TNF-alpha, and TA were evaluated. Our study showed that a nonlethal HS is early able to induce organ dysfunctions and increased TA. Administration of Z-VAD-FMK did not significantly decrease organ dysfunctions, while it induced a significant TNF-alpha release. TA was significantly reduced by Z-VAD-FMK after 1 h, but not after 3 h. Our results suggest that postinjury caspase inhibition does not attenuate organ dysfunctions, and also does not permanently reduce TA induced by HS and resuscitation in rats.
Pubmed link : 17454394

59. Suppression of microRNA-silencing pathway by HIV-1 during virus replication.
Science. 2007 Mar 16;315(5818):1579-82.
Triboulet R, Mari B, Lin YL, Chable-Bessia C, Bennasser Y, Lebrigand K, Cardinaud B, Maurin T, Barbry P, Baillat V, Reynes J, Corbeau P, Jeang KT, Benkirane M
Laboratoire de Virologie Moleculaire, Institut de Genetique Humaine, Montpellier, France.

MicroRNAs (miRNAs) are single-stranded noncoding RNAs of 19 to 25 nucleotides that function as gene regulators and as a host cell defense against both RNA and DNA viruses. We provide evidence for a physiological role of the miRNA-silencing machinery in controlling HIV-1 replication. Type III RNAses Dicer and Drosha, responsible for miRNA processing, inhibited virus replication both in peripheral blood mononuclear cells from HIV-1-infected donors and in latently infected cells. In turn, HIV-1 actively suppressed the expression of the polycistronic miRNA cluster miR-17/92. This suppression was found to be required for efficient viral replication and was dependent on the histone acetyltransferase Tat cofactor PCAF. Our results highlight the involvement of the miRNA-silencing pathway in HIV-1 replication and latency.
Pubmed link : 17322031

60. Mycobacterial lipomannan induces granuloma macrophage fusion via a TLR2-dependent, ADAM9- and beta1 integrin-mediated pathway.
J Immunol. 2007 Mar 1;178(5):3161-9.
Puissegur MP, Lay G, Gilleron M, Botella L, Nigou J, Marrakchi H, Mari B, Duteyrat JL, Guerardel Y, Kremer L, Barbry P, Puzo G, Altare F
Laboratory of Molecular Physiology of Mycobacterial Granulomas, Department of Molecular Mechanisms of Mycobacterial Infections, Institut de Pharmacologie et Biologie Structurale, CNRS/UMR 5089, 205 Route de Narbonne, Toulouse, France.

Tuberculous granulomas are the sites of interaction between the host response and the tubercle bacilli within infected individuals. They mainly consist of organized aggregations of lymphocytes and macrophages (Mf). A predominant role of mycobacterial envelope glycolipids in granulomas formation has been recently emphasized, yet the signaling events interfering with granuloma cell differentiation remain elusive. To decipher this molecular machinery, we have recently developed an in vitro human model of mycobacterial granulomas. In this study, we provide evidence that the mycobacterial proinflammatory phosphatidyl-myo-inositol mannosides and lipomannans (LM), as well as the anti-inflammatory lipoarabinomannan induce granuloma formation, yet only the proinflammatory glycolipids induce the fusion of granuloma Mf into multinucleated giant cells (MGC). We also demonstrate that LM induces large MGC resembling those found in vivo within the granulomas of tuberculosis patients, and that this process is mediated by TLR2 and is dependent on the beta(1) integrin/ADAM9 cell fusion machinery. Our results demonstrate for the first time that the Mf differentiation stage specifically occurring within granulomatous structures (i.e., MGC formation) is triggered by mycobacterial envelope glycolipids, which are capable of inducing the cell fusion machinery. This provides the first characterization of the ontogeny of human granuloma MGC, thus resulting in a direct modulation by a particular mycobacterial envelope glycolipid of the differentiation process of granuloma Mf.
Pubmed link : 17312164

61. An open-access long oligonucleotide microarray resource for analysis of the human and mouse transcriptomes.
Nucleic Acids Res. 2006 Jul 19;34(12):e87
Le Brigand K, Russell R, Moreilhon C, Rouillard JM, Jost B, Amiot F, Magnone V, Bole-Feysot C, Rostagno P, Virolle V, Defamie V, Dessen P, Williams G, Lyons P, Rios G, Mari B, Gulari E, Kastner P, Gidrol X, Freeman TC, Barbry P
CNRS, Institut de Pharmacologie Moleculaire et Cellulaire, UMR6097, 660, route des Lucioles, F-06560 Sophia Antipolis, France.

Two collections of oligonucleotides have been designed for preparing pangenomic human and mouse microarrays. A total of 148,993 and 121,703 oligonucleotides were designed against human and mouse transcripts. Quality scores were created in order to select 25,342 human and 24,109 mouse oligonucleotides. They correspond to: (i) a BLAST-specificity score; (ii) the number of expressed sequence tags matching each probe; (iii) the distance to the 3' end of the target mRNA. Scores were also used to compare in silico the two microarrays with commercial microarrays. The sets described here, called RNG/MRC collections, appear at least as specific and sensitive as those from the commercial platforms. The RNG/MRC collections have now been used by an Anglo-French consortium to distribute more than 3500 microarrays to the academic community. Ad hoc identification of tissue-specific transcripts and a approximately 80% correlation with hybridizations performed on Affymetrix GeneChiptrade mark suggest that the RNG/MRC microarrays perform well. This work provides a comprehensive open resource for investigators working on human and mouse transcriptomes, as well as a generic method to generate new microarray collections in other organisms. All information related to these probes, as well as additional information about commercial microarrays have been stored in a freely-accessible database called MEDIANTE.
Pubmed link : 17384016

62. A survey of the signaling pathways involved in megakaryocytic differentiation of the human K562 leukemia cell line by molecular and c-DNA array analysis.
Oncogene. 2006 Feb 2;25(5):781-94
Jacquel A, Herrant M, Defamie V, Belhacene N, Colosetti P, Marchetti S, Legros L, Deckert M, Mari B, Cassuto JP, Hofman P, Auberger P
INSERM U526, Physiopathologie de la Survie et de la Mort Cellulaires, Equipe Labellisee par la Ligue Nationale contre le Cancer, Universite de Nice Sophia-Antipolis, IFR50, Faculte de Medecine, Avenue de Valombrose, 06107 Nice Cedex 2, France.

The K562 cell line serves as a model to study the molecular mechanisms associated with leukemia differentiation. We show here that cotreatment of K562 cells with PMA and low doses of SB202190 (SB), an inhibitor of the p38 MAPK pathway, induced a majority of cells to differentiate towards the megakaryocytic lineage. Electronic microscopy analysis showed that K562 cells treated with PMA+SB exhibited characteristic features of physiological megakaryocytic differentiation including the presence of vacuoles and demarcation membranes. Differentiation was also accompanied by a net increase in megakaryocytic markers and a reduction of erythroid markers, especially when both effectors were present. PMA effect was selectively mediated by new PKC isoforms. Differentiation of K562 cells by the combination of PMA and SB required Erk1/2 activation, a threshold of JNK activation and p38 MAPK inhibition. Interestingly, higher concentrations of SB, which drastically activated JNK, blocked megakaryocytic differentiation, and considerably increased cell death in the presence of PMA. c-DNA microarray membranes and PCR analysis allow us to identify a set of genes modulated during PMA-induced K562 cell differentiation. Several gene families identified in our screening, including ephrins receptors and some angiogenic factors, had never been reported so far to be regulated during megakaryocytic differentiation.
Pubmed link : 16186797

63. Cooperation of amphiregulin and insulin-like growth factor-1 inhibits Bax- and Bad-mediated apoptosis via a protein kinase C-dependent pathway in non-small cell lung cancer cells.
J Biol Chem. 2005 May 20;280(20):19757-67. Epub 2005 Mar 14.
Hurbin A, Coll JL, Dubrez-Daloz L, Mari B, Auberger P, Brambilla C, Favrot MC
Groupe de Recherche sur le Cancer du Poumon, INSERM U578, Institut Albert Bonniot, La Tronche, France.

Amphiregulin (AR) and insulin-like growth factor-1 (IGF1) are growth factors known to promote non-small cell lung cancer (NSCLC) survival. We have previously published that 1) AR and IGF1, secreted by H358 NSCLC cells, cooperate to protect those cells and H322 NSCLC cells from serum-starved apoptosis; 2) H358 cells resist Bax-induced apoptosis through an inhibition of Bax conformational change. We show here that the antiapoptotic activity of the AR/IGF1 combination is specifically abolished by the PKC inhibitors calphostin C and staurosporine, but not by the MAPK and phosphatidylinositol 3-kinase inhibitors PD98059 and wortmannin, suggesting the involvement of a PKC-dependent and MAPK- and phosphatidylinositol 3-kinase-independent survival pathway. The PKCdelta inhibitor rottlerin restores apoptosis induced by serum deprivation. In addition, phosphorylation of PKCdelta and PKCzeta/lambda, but not of PKCalpha/beta(II), increases in serum-starved H358 cells and in H322 cells treated with an AR/IGF1 combination and is blocked by calphostin C. The combination of AR and IGF1 increases p90(rsk) and Bad phosphorylation as well as inhibiting the conformational change of Bax by a PKC-dependent mechanism. Finally, PKCdelta, PKCzeta, or p90(rsk) small interfering RNAs block the antiapoptotic activity of AR/IGF1 combination but have no effect on partial apoptosis inhibition observed with each factor used alone. Constitutively active PKC expression inhibits serum deprivation-induced apoptosis, whereas a catalytically inactive form of p90(rsk) restores it. Thus, AR and IGF1 cooperate to prevent apoptosis by activating a specific PKC-p90(rsk)-dependent pathway, which leads to Bad and Bax inactivation. This signaling pathway is different to that used by single factor.
Pubmed link : 15767261

64. Tumor cell-mediated induction of the stromal factor stromelysin-3 requires heterotypic cell contact-dependent activation of specific protein kinase C isoforms.
J Biol Chem. 2005 Jan 14;280(2):1272-83. Epub 2004 Oct 27.
Louis K, Guerineau N, Fromigue O, Defamie V, Collazos A, Anglard P, Shipp MA, Auberger P, Joubert D, Mari B
INSERM U526, IFR50, Faculte de Medecine Pasteur, 06107 Nice, France.

Stromelysin-3 (ST3, MMP-11) has been shown to be strongly overexpressed in stromal fibroblasts of most invasive human carcinomas. However, the molecular mechanisms leading to ST3 expression in nonmalignant fibroblasts remain unknown. The aim of the present study was to analyze the signaling pathways activated in normal pulmonary fibroblasts after their interaction with non-small cell lung cancer (NSCLC) cells and leading to ST3 expression. The use of selective signaling pathway inhibitors showed that conventional and novel protein kinase Cs (PKC) were required for ST3 induction, whereas Src kinases exerted a negative control. We observed by both conventional and real time confocal microscopy that green fluorescent protein-tagged PKCalpha and PKCepsilon, but not PKCdelta, transfected in fibroblasts, accumulate selectively at the cell-cell contacts between fibroblasts and tumor cells. In agreement, RNAi-mediated depletion of PKCalpha and PKCepsilon, but not PKCdelta significantly decreased co-culture-dependent ST3 production. Finally, a tetracycline-inducible expression model allowed us to confirm the central role of these PKC isoforms and the negative regulatory function of c-Src in the control of ST3 expression. Altogether, our data emphasize signaling changes occurring in the tumor microenvironment that may define new stromal targets for therapeutic intervention.
Pubmed link : 15509588

65. Escherichia coli cytotoxic necrotizing factor 1 inhibits intestinal epithelial wound healing in vitro after mechanical injury.
Infect Immun. 2004 Oct;72(10):5733-40.
Brest P, Turchi L, Le'Negrate G, Berto F, Moreilhon C, Mari B, Ponzio G, Hofman P
Equipe INSERM 0215, Faculte de Medecine, Nice, France.

Cytotoxic necrotizing factor type 1 (CNF1) from Escherichia coli activates the small GTP-binding proteins of the Rho family (Rho, Rac, and Cdc42) by catalyzing their deamidation at a specific glutamine residue. Since RhoA, Rac, and Cdc42 play a pivotal role in cell migration during the early phase of wound repair, we investigated whether CNF1 was able to interfere with wound healing in intestinal epithelial monolayers (T84 cells). After mechanical injury, we found that CNF1 blocks epithelial wound repair within 48 h. This effect was characterized by cell elongation and filopodium formation on the leading edge, in association with permanent phosphorylation of the focal adhesion kinase (FAK) via Rho activation. Moreover, inhibition of Rho kinase with Y-27632 decreased CNF1-mediated permanent FAK phosphorylation, leading to complete restitution of wound repair within 24 h. In addition, we found that CNF1 induced upregulation of mitogen-activated protein kinases (MAPK) activation. Moreover, activation of Rac and MAPK by CNF1 increased matrix metalloproteinase 9 expression in wounded T84 monolayers. Taken together, these results provide evidence that CNF1 strongly impairs intestinal epithelial wound healing.
Pubmed link : 15385472

66. Gene expression profiling of normal human pulmonary fibroblasts following coculture with non-small-cell lung cancer cells reveals alterations related to matrix degradation, angiogenesis, cell growth and survival.
Oncogene. 2003 Nov 20;22(52):8487-97.
Fromigue O, Louis K, Dayem M, Milanini J, Pages G, Tartare-Deckert S, Ponzio G, Hofman P, Barbry P, Auberger P, Mari B
INSERM U526, IFR50, Faculte de Medecine Pasteur, Nice, France.

Increasing evidence supports a major role for the microenvironment in carcinoma formation and progression. The influence of the stroma is partly mediated by signalling between epithelial tumor cells and neighboring fibroblasts. However, the molecular mechanisms underlying these interactions are largely unknown. To mimic the initial steps of invasive carcinoma in which tumor cells come in contact with normal stromal cells, we used a coculture model of non-small-cell lung cancer tumor cells and normal pulmonary fibroblasts. Using DNA filter arrays, we first analysed the overall modification of gene expression profile after a 24 h period of coculture. Next, we focused our interest on the transcriptome of the purified fibroblastic fraction of coculture using both DNA filter arrays and a laboratory-made DNA microarray. These experiments allowed the identification of a set of modulated genes coding for growth and survival factors, angiogenic factors, proteases and protease inhibitors, transmembrane receptors, kinases and transcription regulators that can potentially affect the regulation of matrix degradation, angiogenesis, invasion, cell growth and survival. This study represents to our knowledge the first attempt to dissect early global gene transcription occurring in a tumor-stroma coculture model and should help to understand better some of the molecular mechanisms involved in heterotypic signalling between epithelial tumor cells and fibroblasts.
Pubmed link : 14627989

67. Active stromelysin-3 (MMP-11) increases MCF-7 survival in three-dimensional Matrigel culture via activation of p42/p44 MAP-kinase.
Int J Cancer. 2003 Sep 1;106(3):355-63.
Fromigue O, Louis K, Wu E, Belhacene N, Loubat A, Shipp M, Auberger P, Mari B
INSERM U526, Equipe labellisee LNC, Faculte de Medecine Pasteur, Avenue de Valombrose, 06107 Nice Cedex 2, France.

Stromelysin-3 (ST3) has the characteristic structure of matrix metalloproteinases (MMP), but its substrate specificity and pattern of expression differ markedly from that of other MMP family members. ST3 was originally isolated on the basis of its expression in primary breast cancers and has been shown to be overexpressed in virtually all primary carcinomas, suggesting that ST3 participates in the initial development of epithelial malignancies. Recent data using murine models reported that ST3 expression was able to increase tumor take by suppressing cell apoptosis. Our present goal was to set up an in vitro model in which we could study this new function. For this purpose, we analyzed survival of MCF-7 transfectants expressing either wild-type or catalytically inactive ST3 (ST3wt or ST3cat-) in three-dimensional (3-D) culture conditions by inclusion in Matrigel. In such conditions, that mimic the in vivo microenvironment, we found a marked decrease in the percentage of cell death when active ST3 was expressed (ST3wt transfectants vs. ST3cat- or vector only transfectants) as assessed by FACS and TUNEL analysis. The addition of batimastat, a broad spectrum MMP inhibitor, reversed the increased cell survival in ST3wt transfectants, confirming that ST3 enzymatic activity was required for this effect. Finally, we analyzed the expression of anti- and pro-apoptotic proteins as well as activation of cell survival pathways and we found that ST3-mediated cell survival was accompanied by activation of both p42/p44 MAPK and AKT. Our data confirm and extend the anti-apoptotic function of ST3 and provide a useful model to dissect this new role and identify new physiological substrates.
Pubmed link : 12845673