Biomarqueurs pronostiques dans le cancer de la prostate : mieux prédire pour mieux traiter

Le cancer de la prostate (CP) est le cancer le plus fréquemment diagnostiqué en Amérique du Nord et est au troisième rang en termes de létalité chez les hommes. Suite aux traitements de première ligne, 20 à 30% des patients diagnostiqués avec un cancer localisé auront une récidive biochimique de la maladie. La déplétion androgénique mène fréquemment au développement du stade de résistance à la castration (RC). Ce dernier est associé avec une augmentation de la morbidité (métastases osseuses) et de la mortalité avec une survie moyenne inférieure à deux ans. L’évolution du CP est très hétérogène dans la population et il n’existe actuellement aucun biomarqueur pronostique permettant d’identifier les patients à risque de récurrence biochimique, de métastases osseuses et de développement d’une résistance à la castration. De nombreuses études ont démontré que les cytokines inflammatoires IL-6 et IL-8 jouent un rôle dans la pathogénèse du CP, notamment dans le développement de la résistance à la castration. Par ailleurs, les niveaux sériques élevés de ces cytokines ont été associés à un mauvais pronostic. Précédemment, notre laboratoire a démontré in vitro que la protéine IKKε entraîne une augmentation de la sécrétion de ces cytokines dans les cellules du CP et qu’elle est exprimée davantage dans les tissus de cancers plus avancés. Le premier objectif du présent mémoire fut d’évaluer dans des tissus humains la corrélation d’IKKε, IL-6 et IL-8 avec des paramètres cliniques. Nos résultats soulignent le potentiel d’IKKε comme biomarqueur tissulaire pronostique de récurrence biochimique et de métastases osseuses. Nous n’avons trouvé aucune association entre IL-6/IL-8 et les paramètres cliniques inclus dans l’étude. Le second objectif de ce projet fut d’évaluer la coexpression de ces trois molécules dans l’épithélium du CP. Nos résultats confirment les observations in vitro en mettant en évidence une forte association entre l’expression d’IKKε, IL-6 et IL-8. Le troisième objectif fut d’évaluer la relation entre les niveaux sériques et tissulaires d’IL-6 et d’IL-8. Aucune relation significative n’a été établie, suggérant que les cytokines sériques ne sont pas uniquement d’origine prostatique. En conclusion, mon projet de maîtrise aura permis de préciser le potentiel d’IKKε comme biomarqueur tissulaire pronostique et de valider pour la première fois dans des tissus humains sa co-expression avec les cytokines IL-6 et IL-8, dont le rôle dans la pathogénèse de la maladie est bien établi. Une étude plus exhaustive des voies de signalisation d’IKKε reste d’intérêt pour élucider notamment les mécanismes par lesquels IKKε stimule la production de cytokines et par quels moyens cette protéine pourrait être impliquée dans le développement d’un état résistant à la castration.

The alpha(2)delta auxiliary subunit reduces affinity of omega-conotoxins for recombinant N-type (Ca(v)2.2) calcium channels

The omega-conotoxins from fish-hunting cone snails are potent inhibitors of voltage-gated calcium channels. The omega-conotoxins MVIIA and CVID are selective N-type calcium channel inhibitors with potential in the treatment of chronic pain. The beta and alpha(2)delta-1 auxiliary subunits influence the expression and characteristics of the alpha(1B) subunit of N-type channels and are differentially regulated in disease states, including pain. In this study, we examined the influence of these auxiliary subunits on the ability of the omega-conotoxins GVIA, MVIIA, CVID and analogues to inhibit peripheral and central forms of the rat N-type channels. Although the beta3 subunit had little influence on the on- and off-rates of omega-conotoxins, coexpression of alpha(2)delta with alpha(1B) significantly reduced on- rates and equilibrium inhibition at both the central and peripheral isoforms of the N-type channels. The alpha(2)delta also enhanced the selectivity of MVIIA, but not CVID, for the central isoform. Similar but less pronounced trends were also observed for N-type channels expressed in human embryonic kidney cells. The influence of alpha(2)delta was not affected by oocyte deglycosylation. The extent of recovery from the omega-conotoxin block was least for GVIA, intermediate for MVIIA, and almost complete for CVID. Application of a hyperpolarizing holding potential ( - 120 mV) did not significantly enhance the extent of CVID recovery. Interestingly, [R10K] MVIIA and [O10K] GVIA had greater recovery from the block, whereas [K10R] CVID had reduced recovery from the block, indicating that position 10 had an important influence on the extent of omega-conotoxin reversibility. Recovery from CVID block was reduced in the presence of alpha(2)delta in human embryonic kidney cells and in oocytes expressing alpha(1B-b). These results may have implications for the antinociceptive properties of omega-conotoxins, given that the alpha(2)delta subunit is up-regulated in certain pain states.

Rab11 in recycling endosomes regulates the sorting and basolateral transport of E-cadherin

E-cadherin plays an essential role in cell polarity and cell-cell adhesion; however, the pathway for delivery of E-cadherin to the basolateral membrane of epithelial cells has not been fully characterized. We first traced the post-Golgi, exocytic transport of GFP-tagged E-cadherin (Ecad-GFP) in unpolarized cells. In live cells, Ecad-GFP was found to exit the Golgi complex in pleiomorphic tubulovesicular carriers, which, instead of moving directly to the cell surface, most frequently fused with an intermediate compartment, subsequently identified as a Rab11-positive recycling endosome. In MDCK cells, basolateral targeting of E-cadherin relies on a dileucine motif. Both E-cadherin and a targeting mutant, Delta S1-E-cadherin, colocalized with Rab11 and fused with the recycling endosome before diverging to basolateral or apical membranes, respectively. In polarized and unpolarized cells, coexpression of Rab11 mutants disrupted the cell surface delivery of E-cadherin and caused its mistargeting to the apical membrane, whereas apical Delta S1-E-cadherin was unaffected. We thus demonstrate a novel pathway for Rab11 dependent, dileucine-mediated, mu 1B-independent sorting and basolateral trafficking, exemplified by E-cadherin. The recycling endosome is identified as an intermediate compartment for the post-Golgi trafficking and exocytosis of E-cadherin, with a potentially important role in establishing and maintaining cadherin-based adhesion.

Regulated Cleavages at the West Nile Virus NS4A-2K-NS4B Junctions Play a Major Role in Rearranging Cytoplasmic Membranes and Golgi Trafficking of the NS4A Protein

A common feature associated with the replication of most RNA viruses is the formation of a unique membrane environment encapsulating the viral replication complex. For their part, flaviviruses are no exception, whereupon infection causes a dramatic rearrangement and induction of unique membrane structures within the cytoplasm of infected cells. These virus-induced membranes, termed paracrystalline arrays, convoluted membranes, and vesicle packets, all appear to have specific functions during replication and are derived from different organelles within the host cell. The aim of this study was to identify which protein(s) specified by the Australian strain of West Nile virus, Kunjin virus (KUNV), are responsible for the dramatic membrane alterations observed during infection. Thus, we have shown using immunolabeling of ultrathin cryosections of transfected cells that expression of the KUNV polyprotein intermediates NS4A-4B and NS213-34A, as well as that of individual NS4A proteins with and without the C-terminal transmembrane domain 2K, resulted in different degrees of rearrangement of cytoplasmic membranes. The formation of the membrane structures characteristic for virus infection required coexpression of an NS4A-NS4B cassette with the viral protease NS2B-3pro which was shown to be essential for the release of the individual NS4A and NS4B proteins. Individual expression of NS4A protein retaining the C-terminal transmembrane domain 2K resulted in the induction of membrane rearrangements most resembling virus-induced structures, while removal of the 2K domain led to a less profound membrane rearrangement but resulted in the redistribution of the NS4A protein to the Golgi apparatus. The results show that cleavage of the KUNV polyprotein NS4A-4B by the viral protease is the key initiation event in the induction of membrane rearrangement and that the NS4A protein intermediate containing the uncleaved C-terminal transmembrane domain plays an essential role in these membrane rearrangements.

Gene network and proteomic analyses of cardiac responses to pathological and physiological stress

Background—The molecular mechanisms underlying similarities and differences between physiological and pathological left ventricular hypertrophy (LVH) are of intense interest. Most previous work involved targeted analysis of individual signaling pathways or screening of transcriptomic profiles. We developed a network biology approach using genomic and proteomic data to study the molecular patterns that distinguish pathological and physiological LVH. Methods and Results—A network-based analysis using graph theory methods was undertaken on 127 genome-wide expression arrays of in vivo murine LVH. This revealed phenotype-specific pathological and physiological gene coexpression networks. Despite >1650 common genes in the 2 networks, network structure is significantly different. This is largely because of rewiring of genes that are differentially coexpressed in the 2 networks; this novel concept of differential wiring was further validated experimentally. Functional analysis of the rewired network revealed several distinct cellular pathways and gene sets. Deeper exploration was undertaken by targeted proteomic analysis of mitochondrial, myofilament, and extracellular subproteomes in pathological LVH. A notable finding was that mRNA–protein correlation was greater at the cellular pathway level than for individual loci. Conclusions—This first combined gene network and proteomic analysis of LVH reveals novel insights into the integrated pathomechanisms that distinguish pathological versus physiological phenotypes. In particular, we identify differential gene wiring as a major distinguishing feature of these phenotypes. This approach provides a platform for the investigation of potentially novel pathways in LVH and offers a freely accessible protocol (http://sites.google.com/site/cardionetworks) for similar analyses in other cardiovascular diseases.

beta-Arrestin1 modulates lymphoid enhancer factor transcriptional activity through interaction with phosphorylated dishevelled proteins.

One aspect of the function of the beta-arrestins is to serve as scaffold or adapter molecules coupling G-protein coupled receptors (GPCRs) to signal transduction pathways distinct from traditional second messenger pathways. Here we report the identification of Dishevelled 1 and Dishevelled 2 (Dvl1 and Dvl2) as beta-arrestin1 (betaarr1) interacting proteins. Dvl proteins participate as key intermediates in signal transmission from the seven membrane-spanning Frizzled receptors leading to inhibition of glycogen synthase kinase-3beta (GSK-3beta), stabilization of beta-catenin, and activation of the lymphoid enhancer factor (LEF) transcription factor. We find that phosphorylation of Dvl strongly enhances its interaction with betaarr1, suggesting that regulation of Dvl phosphorylation and subsequent interaction with betaarr1 may play a key role in the activation of the LEF transcription pathway. Because coexpression of the Dvl kinases, CK1epsilon and PAR-1, with Dvl synergistically activates LEF reporter gene activity, we reasoned that coexpression of betaarr1 with Dvl might also affect LEF-dependent gene activation. Interestingly, whereas betaarr1 or Dvl alone leads to low-level stimulation of LEF (2- to 5-fold), coexpression of betaarr1 with either Dvl1 or Dvl2 leads to a synergistic activation of LEF (up to 16-fold). Additional experiments with LiCl as an inhibitor of GSK-3beta kinase activity indicate that the step affected by betaarr1 is upstream of GSK-3beta and most likely at the level of Dvl. These results identify betaarr1 as a regulator of Dvl-dependent LEF transcription and suggest that betaarr1 might serve as an adapter molecule that can couple Frizzled receptors and perhaps other GPCRs to these important transcription pathways.

Data for the co-expression and purification of human recombinant CaMKK2 in complex with calmodulin in Escherichia coli

Calcium/calmodulin-dependent kinase kinase 2 (CaMKK2) has been implicated in a range of conditions and pathologies from prostate to hepatic cancer. Here, we describe the expression in Escherichia coli and the purification protocol for the following constructs: full-length CaMKK2 in complex with CaM, CaMKK2 'apo', CaMKK2 (165-501) in complex with CaM, and the CaMKK2 F267G mutant. The protocols described have been optimized for maximum yield and purity with minimal purification steps required and the proteins subsequently used to develop a fluorescence-based assay for drug binding to the kinase, "Using the fluorescent properties of STO-609 as a tool to assist structure-function analyses of recombinant CaMKK2"

Mutations in SNX14 cause a distinctive autosomal-recessive cerebellar ataxia and intellectual disability syndrome

Intellectual disability and cerebellar atrophy occur together in a large number of genetic conditions and are frequently associated with microcephaly and/or epilepsy. Here we report the identification of causal mutations in Sorting Nexin 14 (SNX14) found in seven affected individuals from three unrelated consanguineous families who presented with recessively inherited moderate-severe intellectual disability, cerebellar ataxia, early-onset cerebellar atrophy, sensorineural hearing loss, and the distinctive association of progressively coarsening facial features, relative macrocephaly, and the absence of seizures. We used homozygosity mapping and whole-exome sequencing to identify a homozygous nonsense mutation and an in-frame multiexon deletion in two families. A homozygous splice site mutation was identified by Sanger sequencing of SNX14 in a third family, selected purely by phenotypic similarity. This discovery confirms that these characteristic features represent a distinct and recognizable syndrome. SNX14 encodes a cellular protein containing Phox (PX) and regulator of G protein signaling (RGS) domains. Weighted gene coexpression network analysis predicts that SNX14 is highly coexpressed with genes involved in cellular protein metabolism and vesicle-mediated transport. All three mutations either directly affected the PX domain or diminished SNX14 levels, implicating a loss of normal cellular function. This manifested as increased cytoplasmic vacuolation as observed in cultured fibroblasts. Our findings indicate an essential role for SNX14 in neural development and function, particularly in development and maturation of the cerebellum.

Variation in myogenic differentiation 1 mRNA abundance is associated with beef tenderness in Nelore cattle.

The myogenic differentiation 1 gene (MYOD1) has a key role in skeletal muscle differentiation and composition through its regulation of the expression of several muscle-specific genes. We first used a general linear mixed model approach to evaluate the association of MYOD1 expression levels on individual beef tenderness phenotypes. MYOD1 mRNA levels measured by quantitative polymerase chain reactions in 136 Nelore steers were significantly associated (P ? 0.01) with Warner?Bratzler shear force, measured on the longissimus dorsi muscle after 7 and 14 days of beef aging. Transcript abundance for the muscle regulatory gene MYOD1 was lower in animals with more tender beef. We also performed a coexpression network analysis using whole transcriptome sequence data generated from 30 samples of longissimus muscle tissue to identify genes that are potentially regulated by MYOD1. The effect of MYOD1 gene expression on beef tenderness may emerge from its function as an activator of muscle-specific gene transcription such as for the serum response factor (C-fos serum response element-binding transcription factor) gene (SRF), which determines muscle tissue development, composition, growth and maturation.