The Contribution of the Zebrafish Model to the Understanding of Polycomb Repression in Vertebrates.

Polycomb group (PcG) proteins are highly conserved proteins assembled into two major types of complexes, PRC1 and PRC2, involved in the epigenetic silencing of a wide range of gene expression programs regulating cell fate and tissue development. The crucial role of PRC1 and PRC2 in the fundamental cellular processes and their involvement in human pathologies such as cancer attracted intense attention over the last few decades. Here, we review recent advancements regarding PRC1 and PRC2 function using the zebrafish model. We point out that the unique characteristics of the zebrafish model provide an exceptional opportunity to increase our knowledge of the role of the PRC1 and PRC2 complexes in tissue development, in the maintenance of organ integrity and in pathology.

Direct interaction of TrkA/CD44v3 is essential for NGF-promoted aggressiveness of breast cancer cells.

BACKGROUND: CD44 is a multifunctional membrane glycoprotein. Through its heparan sulfate chain, CD44 presents growth factors to their receptors. We have shown that CD44 and Tropomyosin kinase A (TrkA) form a complex following nerve growth factor (NGF) induction. Our study aimed to understand how CD44 and TrkA interact and the consequences of inhibiting this interaction regarding the pro-tumoral effect of NGF in breast cancer. METHODS: After determining which CD44 isoforms (variants) are involved in forming the TrkA/CD44 complex using proximity ligation assays, we investigated the molecular determinants of this interaction. By molecular modeling, we isolated the amino acids involved and confirmed their involvement using mutations. A CD44v3 mimetic peptide was then synthesized to block the TrkA/CD44v3 interaction. The effects of this peptide on the growth, migration and invasion of xenografted triple-negative breast cancer cells were assessed. Finally, we investigated the correlations between the expression of the TrkA/CD44v3 complex in tumors and histo-pronostic parameters. RESULTS: We demonstrated that isoform v3 (CD44v3), but not v6, binds to TrkA in response to NGF stimulation. The final 10 amino acids of exon v3 and the TrkA H112 residue are necessary for the association of CD44v3 with TrkA. Functionally, the CD44v3 mimetic peptide impairs not only NGF-induced RhoA activation, clonogenicity, and migration/invasion of breast cancer cells in vitro but also tumor growth and metastasis in a xenograft mouse model. We also detected TrkA/CD44v3 only in cancerous cells, not in normal adjacent tissues. CONCLUSION: Collectively, our results suggest that blocking the CD44v3/TrkA interaction can be a new therapeutic option for triple-negative breast cancers.

H3.3K27M Mutation Controls Cell Growth and Resistance to Therapies in Pediatric Glioma Cell Lines.

High-grade gliomas represent the most lethal class of pediatric tumors, and their resistance to both radio- and chemotherapy is associated with a poor prognosis. Recurrent mutations affecting histone genes drive the tumorigenesis of some pediatric high-grade gliomas, and H3K27M mutations are notably characteristic of a subtype of gliomas called DMG (Diffuse Midline Gliomas). This dominant negative mutation impairs H3K27 trimethylation, leading to profound epigenetic modifications of genes expression. Even though this mutation was described as a driver event in tumorigenesis, its role in tumor cell resistance to treatments has not been deciphered so far. To tackle this issue, we expressed the H3.3K27M mutated histone in three initially H3K27-unmutated pediatric glioma cell lines, Res259, SF188, and KNS42. First, we validated these new H3.3K27M-expressing models at the molecular level and showed that K27M expression is associated with pleiotropic effects on the transcriptomic signature, largely dependent on cell context. We observed that the mutation triggered an increase in cell growth in Res259 and SF188 cells, associated with higher clonogenic capacities. Interestingly, we evidenced that the mutation confers an increased resistance to ionizing radiations in Res259 and KNS42 cells. Moreover, we showed that H3.3K27M mutation impacts the sensitivity of Res259 cells to specific drugs among a library of 80 anticancerous compounds. Altogether, these data highlight that, beyond its tumorigenic role, H3.3K27M mutation is strongly involved in pediatric glioma cells' resistance to therapies, likely through transcriptomic reprogramming.

Vimentin Promotes the Aggressiveness of Triple Negative Breast Cancer Cells Surviving Chemotherapeutic Treatment.

Tremendous data have been accumulated in the effort to understand chemoresistance of triple negative breast cancer (TNBC). However, modifications in cancer cells surviving combined and sequential treatment still remain poorly described. In order to mimic clinical neoadjuvant treatment, we first treated MDA-MB-231 and SUM159-PT TNBC cell lines with epirubicin and cyclophosphamide for 2 days, and then with paclitaxel for another 2 days. After 4 days of recovery, persistent cells surviving the treatment were characterized at both cellular and molecular level. Persistent cells exhibited increased growth and were more invasive in vitro and in zebrafish model. Persistent cells were enriched for vimentinhigh sub-population, vimentin knockdown using siRNA approach decreased the invasive and sphere forming capacities as well as Akt phosphorylation in persistent cells, indicating that vimentin is involved in chemotherapeutic treatment-induced enhancement of TNBC aggressiveness. Interestingly, ectopic vimentin overexpression in native cells increased cell invasion and sphere formation as well as Akt phosphorylation. Furthermore, vimentin overexpression alone rendered the native cells resistant to the drugs, while vimentin knockdown rendered them more sensitive to the drugs. Together, our data suggest that vimentin could be considered as a new targetable player in the ever-elusive status of drug resistance and recurrence of TNBC.

The effect of Activin pathway modulation on the expression of both pluripotency and differentiation markers during early zebrafish development compared with other vertebrates.

Activin-like factors control many developmental processes, including pluripotency maintenance and differentiation. Although Activin-like factors' action in mesendoderm induction has been demonstrated in zebrafish, their involvement in preserving the stemness remains unknown. To investigate the role of maternal Activin-like factors, their effects were promoted or blocked using synthetic human Activin A or SB-431542 treatments respectively until the maternal to zygotic transition. To study the role of zygotic Activin-like factors, SB-431542 treatment was also applied after the maternal to zygotic transition. The effect of the pharmacological modulations of the Activin/Smad pathway was then studied on the mRNA expressions of the ndr1, ndr2, tbxta (no tail/ntl) as the differentiation index, mych, nanog, and oct4 (pou5f3) as the pluripotency markers of the zebrafish embryonic cells as well as sox17 as a definitive endoderm marker. Expression of the target genes was measured at the 16-cell, 256-cell, 1K-cell, oblong, dome, and shield stages using the real-time quantitative polymerase chain reaction (RT-qPCR). Activation of the maternal Activin signaling pathway led to an increase in zygotic expression of the tbxta, particularly marked at the oblong stage. In other words, promotion of the maternal Activin/Smad pathway induced differentiation by advancing the major peaks of ndr1 and nanog, thereby eliciting tbxta expression. Whereas suppression of the maternal or zygotic Activin/Smad pathway sustained the pluripotency by preventing the major peaks of ndr1 and nanog as well as tbxta encoding.

Genetic Engineering of Zebrafish in Cancer Research.

Zebrafish (Danio rerio) is an excellent model to study a wide diversity of human cancers. In this review, we provide an overview of the genetic and reverse genetic toolbox allowing the generation of zebrafish lines that develop tumors. The large spectrum of genetic tools enables the engineering of zebrafish lines harboring precise genetic alterations found in human patients, the generation of zebrafish carrying somatic or germline inheritable mutations or zebrafish showing conditional expression of the oncogenic mutations. Comparative transcriptomics demonstrate that many of the zebrafish tumors share molecular signatures similar to those found in human cancers. Thus, zebrafish cancer models provide a unique in vivo platform to investigate cancer initiation and progression at the molecular and cellular levels, to identify novel genes involved in tumorigenesis as well as to contemplate new therapeutic strategies.

Enhancement of Breast Cancer Cell Aggressiveness by lncRNA H19 and its Mir-675 Derivative: Insight into Shared and Different Actions.

Breast cancer is a major public health problem and the leading world cause of women death by cancer. Both the recurrence and mortality of breast cancer are mainly caused by the formation of metastasis. The long non-coding RNA H19, the precursor of miR-675, is involved in breast cancer development. The aim of this work was to determine the implication but, also, the relative contribution of H19 and miR-675 to the enhancement of breast cancer metastatic potential. We showed that both H19 and miR-675 increase the invasive capacities of breast cancer cells in xenografted transgenic zebrafish models. In vitro, H19 and miR-675 enhance the cell migration and invasion, as well as colony formation. H19 seems to induce the epithelial-to-mesenchymal transition (EMT), with a decreased expression of epithelial markers and an increased expression of mesenchymal markers. Interestingly, miR-675 simultaneously increases the expression of both epithelial and mesenchymal markers, suggesting the induction of a hybrid phenotype or mesenchymal-to-epithelial transition (MET). Finally, we demonstrated for the first time that miR-675, like its precursor H19, increases the stemness properties of breast cancer cells. Altogether, our data suggest that H19 and miR-675 could enhance the aggressiveness of breast cancer cells through both common and different mechanisms.

The Polycomb Orthologues in Teleost Fishes and Their Expression in the Zebrafish Model.

The Polycomb Repressive Complex 1 (PRC1) is a chromatin-associated protein complex involved in transcriptional repression of hundreds of genes controlling development and differentiation processes, but also involved in cancer and stem cell biology. Within the canonical PRC1, members of Pc/CBX protein family are responsible for the targeting of the complex to specific gene loci. In mammals, the Pc/CBX protein family is composed of five members generating, through mutual exclusion, different PRC1 complexes with potentially distinct cellular functions. Here, we performed a global analysis of the cbx gene family in 68 teleost species and traced the distribution of the cbx genes through teleost evolution in six fish super-orders. We showed that after the teleost-specific whole genome duplication, cbx4, cbx7 and cbx8 are retained as pairs of ohnologues. In contrast, cbx2 and cbx6 are present as pairs of ohnologues in the genome of several teleost clades but as singletons in others. Furthermore, since zebrafish is a widely used vertebrate model for studying development, we report on the expression of the cbx family members during zebrafish development and in adult tissues. We showed that all cbx genes are ubiquitously expressed with some variations during early development.

[Cancer cell transplantation in zebrafish: From translational research to personalized medicine]. / La transplantation de cellules tumorales chez le poisson zèbre : de la recherche translationnelle à la médecine personnalisée.

Primarily used in genetic studies of development, the zebrafish (Danio rerio) has rapidly emerged as a promising animal model of human cancer. Cancer cell transplantation in zebrafish constitutes a key platform for clinical research since it allows to study cellular and molecular events involved in various aspects of tumorigenesis and to evaluate the efficacy of therapeutic molecules in vivo. Applied to patient-derived cells, the xenotransplantation approach in zebrafish allows to define the most appropriate therapeutic strategies for specific alterations found in patients in the context of personalized medicine. This review discusses the zebrafish transplantation model for the study of cancer development and drug discovery.

Protein quality control in the nucleolus safeguards recovery of epigenetic regulators after heat shock.

Maintenance of epigenetic modifiers is of utmost importance to preserve the epigenome and consequently appropriate cellular functioning. Here, we analyzed Polycomb group protein (PcG) complex integrity in response to heat shock (HS). Upon HS, various Polycomb Repressive Complex (PRC)1 and PRC2 subunits, including CBX proteins, but also other chromatin regulators, are found to accumulate in the nucleolus. In parallel, binding of PRC1/2 to target genes is strongly reduced, coinciding with a dramatic loss of H2AK119ub and H3K27me3 marks. Nucleolar-accumulated CBX proteins are immobile, but remarkably both CBX protein accumulation and loss of PRC1/2 epigenetic marks are reversible. This post-heat shock recovery of pan-nuclear CBX protein localization and reinstallation of epigenetic marks is HSP70 dependent. Our findings demonstrate that the nucleolus is an essential protein quality control center, which is indispensable for recovery of epigenetic regulators and maintenance of the epigenome after heat shock.

[The zebrafish model in oncology]. / Le modèle poisson zèbre dans la lutte contre le cancer.

Although cell culture and mouse models will remain a cornerstone of cancer research, the unique capabilities of the zebrafish outline the potential of this model for shedding light on cancer biology in vivo. Zebrafish develops cancers spontaneously, after chemical mutagenesis or through genetic manipulations. Furthermore, zebrafish cancers are similar to human tumors at the histological and molecular levels allowing the study of tumor initiation, progression and heterogeneity. Xenotransplantation of human cancer cells in embryos or adult zebrafish presents the advantage of following cancer cell behavior in vivo. Finally, zebrafish embryos are used in molecule screens and contribute to the identification of novel anti-cancer therapeutic strategies. Here, we review different involvements of the zebrafish model in cancer research.

The role of long non-coding RNAs in genome formatting and expression.

Long non-coding RNAs (lncRNAs) are transcripts without protein-coding potential but having a pivotal role in numerous biological functions. Long non-coding RNAs act as regulators at different levels of gene expression including chromatin organization, transcriptional regulation, and post-transcriptional control. Misregulation of lncRNAs expression has been found to be associated to cancer and other human disorders. Here, we review the different types of lncRNAs, their mechanisms of action on genome formatting and expression and emphasized on the multifaceted action of the H19 lncRNA.

Diverse involvement of EZH2 in cancer epigenetics.

EZH2 is the catalytic subunit of Polycomb Repressor Complex 2 (PRC2) which catalyzes methylation of histone H3 at lysine 27 (H3K27me) and mediates gene silencing of target genes via local chromatin reorganization. Numerous evidences show that EZH2 plays a critical role in cancer initiation, progression and metastasis, as well as in cancer stem cell biology. Indeed, EZH2 dysregulation alters gene expression programs in various cancer types. The molecular mechanisms responsible for EZH2 alteration appear to be diverse and depending on the type of cancer. Furthermore, accumulating evidences indicate that EZH2 could also act as a PRC2-independent transcriptional activator in cancer. In this review, we address the current understanding of the oncogenic role of EZH2, including the mechanisms of EZH2 dysregulation in cancer and progresses in therapeutic approaches targeting EZH2.

PRC1 components exhibit different binding kinetics in Polycomb bodies.

BACKGROUND INFORMATION: Polycomb group (PcG) proteins keep the memory of cell identity by maintaining the repression of numerous target genes. They accumulate into nuclear foci called Polycomb bodies, which function in Drosophila cells as silencing compartments where PcG target genes convene. PcG proteins also exert their activities elsewhere in the nucleoplasm. In mammalian cells, the dynamic organisation and function of Polycomb bodies remain to be determined. RESULTS: Fluorescently tagged PcG proteins CBXs and their partners BMI1 and RING1 form foci of different sizes and intensities in human U2OS cells. Fluorescence recovery after photobleaching (FRAP) analysis reveals that PcG dynamics outside foci is governed by diffusion as complexes and transient binding. In sharp contrast, recovery curves inside foci are substantially slower and exhibit large variability. The slow binding component amplitudes correlate with the intensities and sizes of these foci, suggesting that foci contained varying numbers of binding sites. CBX4-green fluorescent protein (GFP) foci were more stable than CBX8-GFP foci; yet the presence of CBX4 or CBX8-GFP in the same focus had a minor impact on BMI1 and RING1 recovery kinetics. CONCLUSION: We propose that FRAP recovery curves provide information about PcG binding to their target genes outside foci and about PcG spreading onto chromatin inside foci.

Interaction proteomics analysis of polycomb proteins defines distinct PRC1 complexes in mammalian cells.

Polycomb group (PcG) proteins maintain transcriptional repression of hundreds of genes involved in development, signaling or cancer using chromatin-based epigenetic mechanisms. Biochemical studies in Drosophila have revealed that PcG proteins associate in at least two classes of protein complexes known as Polycomb repressive complexes 1 and 2 (PRC1 and PRC2). Drosophila core PRC1 is composed of four subunits, Polycomb (Pc), Sex combs extra (Sce), Polyhomeotic (Ph), and Posterior sex combs (Psc). Each of these proteins has multiple orthologs in vertebrates classified respectively as the CBX, RING1/RNF2, PHC, and BMI1/PCGF families. Mammalian genomes encode five CBX family members (CBX2, CBX4, CBX6, CBX7, and CBX8) that are believed to have distinct biological functions. Here, we applied a tandem affinity purification (TAP) approach coupled with tandem mass spectrometry (MS/MS) methodologies in order to identify interacting partners of CBX family proteins under the same experimental conditions. Our analysis identified with high confidence about 20 proteins co-eluted with CBX2 and CBX7 tagged proteins, about 40 with CBX4, and around 60 with CBX6 and CBX8. We provide evidences that the CBX family proteins are mutually exclusive and define distinct PRC1-like protein complexes. CBX proteins also interact with different efficiencies with the other PRC1 components. Among the novel CBX interacting partners, protein kinase 2 associates with all CBX-PRC1 protein complexes, whereas 14-3-3 proteins specifically bind to CBX4. 14-3-3 protein binding to CBX4 appears to modulate the interaction between CBX4 and the BMI1/PCGF components of PRC1, but has no effect on CBX4-RING1/RNF2 interaction. Finally, we suggest that differences in CBX protein interactions would account, at least in part, for distinct subnuclear localization of the CBX family members.

The zebrafish genes encoding the Polycomb repressive complex (PRC) 1.

Polycomb repression controls regulation of hundreds of genes involved in development, signalling or cancer and is mediated by essentially two classes of chromatin-associated protein complexes, the Polycomb repressive complexes 1 and 2 (PRC1 and PRC2). PRC2 trimethylates histone H3 at Lysine 27 and this H3K27me3 epigenetic mark serves as a docking site for the PRC1 protein complex. Drosophila core PRC1 is composed of four subunits, Polycomb (Pc), Posterior sex combs (Psc), Polyhomeotic (Ph), and Sex combs extra (Sce). Each of these proteins has multiple orthologs in vertebrates. In particular, mammalian genomes encode five Pc family members (CBX2, 4, 6, 7 and 8), six Psc family members (BMI1, PCGF1, 2, 3, 5, and 6), three Ph family members (PHC1, 2 and 3) and two Sce family members (RING1 and RNF2) generating an enormous scope for potential combinatorial diversity. In order to identify the corresponding PRC1 genes in zebrafish, homology searches were undertaken and allowed the identification of a total of 19 genes. Using phylogenetic, gene organization and gene location analyses, these genes were classified. The zebrafish genes encoding the PRC1 protein complex include 8 Pc orthologs (cbx2, cbx4, cbx6a, cbx6b, cbx7a, cbx7b, cbx8a and cbx8b), 6 Psc orthologs (bmi1a, bmi1b, pcgf1, pcgf5a, pcgf5b and pcgf6), 4 Ph orthologs (phc1, phc2a, phc2b and phc3) and a single Sce ortholog (rnf2). Our results indicate that the potentially high number of distinct PRC1 protein complexes generated by the components combinatorial appeared early in the vertebrate evolution. In addition to conserved gene organization and syntenies, transcript analyses revealed that transcriptional regulation leading to various isoforms syntheses is also conserved at genes encoding the PRC1 components, highlighting a possible important biological role of these isoforms.

Functional characterization of human Polycomb-like 3 isoforms identifies them as components of distinct EZH2 protein complexes.

PcG (Polycomb group) proteins are conserved transcriptional repressors essential to regulate cell fate and to maintain epigenetic cellular memory. They work in concert through two main families of chromatin-modifying complexes, PRC1 (Polycomb repressive complex 1) and PRC2-4. In Drosophila, PRC2 contains the H3K27 histone methyltransferase E(Z) whose trimethylation activity towards PcG target genes is stimulated by PCL (Polycomb-like). In the present study, we have examined hPCL3, one of its three human paralogues. Through alternative splicing, hPCL3 encodes a long isoform, hPCL3L, containing an N-terminal TUDOR domain and two PHDs (plant homeodomains) and a smaller isoform, hPCL3S, lacking the second PHD finger (PHD2). By quantitative reverse transcription-PCR analyses, we showed that both isoforms are widely co-expressed at high levels in medulloblastoma. By co-immunoprecipitation analyses, we demonstrated that both isoforms interact with EZH2 through their common TUDOR domain. However, the hPCL3L-specific PHD2 domain, which is better conserved than PHD1 in the PCL family, is also involved in this interaction and implicated in the self-association of hPCL3L. Finally, we have demonstrated that both hPCL3 isoforms are physically associated with EZH2, but in different complexes. Our results provide the first evidence that the two hPCL3 isoforms belong to different complexes and raise important questions about their relative functions, particularly in tumorigenesis.

Cell adhesion properties on chemically micropatterned boron-doped diamond surfaces.

The adhesion properties of living cells were investigated on a range of chemically modified boron-doped diamond (BDD) surfaces. We studied the influence of oxidized, H-, amine- (NH(2)-), methyl- (CH(3)-), trifluoromethyl- (CF(3)-) and vinyl- (CH(2)═CH-) terminated BDD surfaces on human osteosarcoma U2OS and mouse fibroblast L929 cells behavior. Cell-surface interactions were analyzed by fluorescence microscopy in terms of cell attachment, spreading and proliferation. U2OS cells poorly adhered on hydrophobic surfaces and their growth was blocked. In contrast, L929 cells were mainly influenced by the presence of perfluoroalkyl chains in regard to their morphology. The results were subsequently applied to selectively micropattern U2OS cells on dual hydrophobic/hydrophilic surfaces prepared by a UV/ozone lithographic approach. U2OS cells colonized preferentially hydrophilic (oxide-terminated) motifs, forming confluent arrays with distinguishable edges separating the alkyl regions.

Interaction proteomics: characterization of protein complexes using tandem affinity purification-mass spectrometry.

Most cellular processes are carried out by a multitude of proteins that assemble into multimeric complexes. Thus a precise understanding of the biological pathways that control cellular events relies on the identification and on the biochemical characterization of the proteins involved in such multimeric assemblies. Advances in MS have made possible the identification of multisubunit protein complexes isolated from cell lysates with high sensitivity and accuracy, whereas the TAP (tandem affinity purification) methodology efficiently isolates native protein complexes from cells for proteomics analysis. TAP is a generic method based on the sequential utilization of two affinity tags to purify protein assemblies. During the first purification step, the Protein A moiety of the TAP tag is bound to IgG beads, and protein components associated with the TAP-tagged protein are retrieved by TEV (tobacco etch virus) protease cleavage. This enzyme is a sequence-specific protease cleaving a seven-amino-acid recognition site located between the first and second tags. In the second affinity step, the protein complex is immobilized to calmodulin-coated beads via the CBP (calmodulin-binding peptide) of the TAP tag. The CBP-calmodulin interaction is calcium-dependent and calcium-chelating agents are used in the second elution step to release the final protein complex preparation used for protein identification by MS. The TAP-MS approach has proven to efficiently permit the characterization of protein complexes from bacteria, yeast and mammalian cells, as well as from multicellular organisms such as Caenorhabditis elegans, Drosophila and mice.

A physical and functional map of the human TNF-alpha/NF-kappa B signal transduction pathway.

Signal transduction pathways are modular composites of functionally interdependent sets of proteins that act in a coordinated fashion to transform environmental information into a phenotypic response. The pro-inflammatory cytokine tumour necrosis factor (TNF)-alpha triggers a signalling cascade, converging on the activation of the transcription factor NF-kappa B, which forms the basis for numerous physiological and pathological processes. Here we report the mapping of a protein interaction network around 32 known and candidate TNF-alpha/NF-kappa B pathway components by using an integrated approach comprising tandem affinity purification, liquid-chromatography tandem mass spectrometry, network analysis and directed functional perturbation studies using RNA interference. We identified 221 molecular associations and 80 previously unknown interactors, including 10 new functional modulators of the pathway. This systems approach provides significant insight into the logic of the TNF-alpha/NF-kappa B pathway and is generally applicable to other pathways relevant to human disease.