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1.
Nat Commun ; 12(1): 4502, 2021 07 23.
Artigo em Inglês | MEDLINE | ID: mdl-34301937

RESUMO

Cells in many tissues, such as bone, muscle, and placenta, fuse into syncytia to acquire new functions and transcriptional programs. While it is known that fused cells are specialized, it is unclear whether cell-fusion itself contributes to programmatic-changes that generate the new cellular state. Here, we address this by employing a fusogen-mediated, cell-fusion system to create syncytia from undifferentiated cells. RNA-Seq analysis reveals VSV-G-induced cell fusion precedes transcriptional changes. To gain mechanistic insights, we measure the plasma membrane surface area after cell-fusion and observe it diminishes through increases in endocytosis. Consequently, glucose transporters internalize, and cytoplasmic glucose and ATP transiently decrease. This reduced energetic state activates AMPK, which inhibits YAP1, causing transcriptional-reprogramming and cell-cycle arrest. Impairing either endocytosis or AMPK activity prevents YAP1 inhibition and cell-cycle arrest after fusion. Together, these data demonstrate plasma membrane diminishment upon cell-fusion causes transient nutrient stress that may promote transcriptional-reprogramming independent from extrinsic cues.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Membrana Celular/metabolismo , Núcleo Celular/metabolismo , Glicoproteínas de Membrana/metabolismo , Fatores de Transcrição/metabolismo , Transcrição Genética/genética , Proteínas do Envelope Viral/metabolismo , Proteínas Quinases Ativadas por AMP/genética , Proteínas Quinases Ativadas por AMP/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Transporte Biológico , Fusão Celular , Linhagem Celular , Linhagem Celular Tumoral , Células Cultivadas , Células Gigantes/metabolismo , Células HEK293 , Humanos , Glicoproteínas de Membrana/genética , Camundongos , RNA-Seq/métodos , Transdução de Sinais/genética , Fatores de Transcrição/genética , Proteínas do Envelope Viral/genética
2.
Gastroenterology ; 161(4): 1179-1193, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34197832

RESUMO

BACKGROUND & AIMS: Colorectal cancer (CRC) shows variable response to immune checkpoint blockade, which can only partially be explained by high tumor mutational burden (TMB). We conducted an integrated study of the cancer tissue and associated tumor microenvironment (TME) from patients treated with pembrolizumab (KEYNOTE 177 clinical trial) or nivolumab to dissect the cellular and molecular determinants of response to anti- programmed cell death 1 (PD1) immunotherapy. METHODS: We selected multiple regions per tumor showing variable T-cell infiltration for a total of 738 regions from 29 patients, divided into discovery and validation cohorts. We performed multiregional whole-exome and RNA sequencing of the tumor cells and integrated these with T-cell receptor sequencing, high-dimensional imaging mass cytometry, detection of programmed death-ligand 1 (PDL1) interaction in situ, multiplexed immunofluorescence, and computational spatial analysis of the TME. RESULTS: In hypermutated CRCs, response to anti-PD1 immunotherapy was not associated with TMB but with high clonality of immunogenic mutations, clonally expanded T cells, low activation of Wnt signaling, deregulation of the interferon gamma pathway, and active immune escape mechanisms. Responsive hypermutated CRCs were also rich in cytotoxic and proliferating PD1+CD8 T cells interacting with PDL1+ antigen-presenting macrophages. CONCLUSIONS: Our study clarified the limits of TMB as a predictor of response of CRC to anti-PD1 immunotherapy. It identified a population of antigen-presenting macrophages interacting with CD8 T cells that consistently segregate with response. We therefore concluded that anti-PD1 agents release the PD1-PDL1 interaction between CD8 T cells and macrophages to promote cytotoxic antitumor activity.

3.
Curr Protoc ; 1(3): e71, 2021 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-33657274

RESUMO

Intracellular signaling processes are frequently based on direct interactions between proteins and organelles. A fundamental strategy to elucidate the physiological significance of such interactions is to utilize optical dimerization tools. These tools are based on the use of small proteins or domains that interact with each other upon light illumination. Optical dimerizers are particularly suitable for reproducing and interrogating a given protein-protein interaction and for investigating a protein's intracellular role in a spatially and temporally precise manner. Described in this article are genetic engineering strategies for the generation of modular light-activatable protein dimerization units and instructions for the preparation of optogenetic applications in mammalian cells. Detailed protocols are provided for the use of light-tunable switches to regulate protein recruitment to intracellular compartments, induce intracellular organellar membrane tethering, and reconstitute protein function using enhanced Magnets (eMags), a recently engineered optical dimerization system. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Genetic engineering strategy for the generation of modular light-activated protein dimerization units Support Protocol 1: Molecular cloning Basic Protocol 2: Cell culture and transfection Support Protocol 2: Production of dark containers for optogenetic samples Basic Protocol 3: Confocal microscopy and light-dependent activation of the dimerization system Alternate Protocol 1: Protein recruitment to intracellular compartments Alternate Protocol 2: Induction of organelles' membrane tethering Alternate Protocol 3: Optogenetic reconstitution of protein function Basic Protocol 4: Image analysis Support Protocol 3: Analysis of apparent on- and off-kinetics Support Protocol 4: Analysis of changes in organelle overlap over time.


Assuntos
Optogenética , Organelas , Animais , Organelas/metabolismo , Multimerização Proteica , Transporte Proteico , Transdução de Sinais
4.
Elife ; 92020 11 11.
Artigo em Inglês | MEDLINE | ID: mdl-33174843

RESUMO

Light-inducible dimerization protein modules enable precise temporal and spatial control of biological processes in non-invasive fashion. Among them, Magnets are small modules engineered from the Neurospora crassa photoreceptor Vivid by orthogonalizing the homodimerization interface into complementary heterodimers. Both Magnets components, which are well-tolerated as protein fusion partners, are photoreceptors requiring simultaneous photoactivation to interact, enabling high spatiotemporal confinement of dimerization with a single excitation wavelength. However, Magnets require concatemerization for efficient responses and cell preincubation at 28°C to be functional. Here we overcome these limitations by engineering an optimized Magnets pair requiring neither concatemerization nor low temperature preincubation. We validated these 'enhanced' Magnets (eMags) by using them to rapidly and reversibly recruit proteins to subcellular organelles, to induce organelle contacts, and to reconstitute OSBP-VAP ER-Golgi tethering implicated in phosphatidylinositol-4-phosphate transport and metabolism. eMags represent a very effective tool to optogenetically manipulate physiological processes over whole cells or in small subcellular volumes.


Assuntos
Transporte Biológico , Proteínas Fúngicas/efeitos da radiação , Luz , Optogenética , Organelas/metabolismo , Engenharia de Proteínas , Animais , Células COS , Chlorocebus aethiops , Dimerização , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Células HeLa , Humanos , Cinética , Metabolismo dos Lipídeos , Camundongos Endogâmicos C57BL , Organelas/genética , Fosfatos de Fosfatidilinositol/metabolismo , Multimerização Proteica , Estabilidade Proteica , Transporte Proteico
5.
Nat Methods ; 17(2): 225-231, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31907447

RESUMO

Combining the molecular specificity of fluorescent probes with three-dimensional imaging at nanoscale resolution is critical for investigating the spatial organization and interactions of cellular organelles and protein complexes. We present a 4Pi single-molecule switching super-resolution microscope that enables ratiometric multicolor imaging of mammalian cells at 5-10-nm localization precision in three dimensions using 'salvaged fluorescence'. Imaging two or three fluorophores simultaneously, we show fluorescence images that resolve the highly convoluted Golgi apparatus and the close contacts between the endoplasmic reticulum and the plasma membrane, structures that have traditionally been the imaging realm of electron microscopy. The salvaged fluorescence approach is equally applicable in most single-objective microscopes.


Assuntos
Imagem Óptica , Frações Subcelulares/metabolismo , Animais , Humanos , Organelas/metabolismo
6.
Biochim Biophys Acta Gene Regul Mech ; 1863(6): 194445, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-31654804

RESUMO

Interactions between cancer cells and non-cancer cells composing the tumour microenvironment play a primary role in determining cancer progression and shaping the response to therapy. The qualitative and quantitative characterisation of the different cell populations in the tumour microenvironment is therefore crucial to understand its role in cancer. In recent years, many experimental and computational approaches have been developed to identify the cell populations composing heterogeneous tissue samples, such as cancer. In this review, we describe the state-of-the-art approaches for the quantification of non-cancer cells from bulk and single-cell cancer transcriptomic data, with a focus on immune cells. We illustrate the main features of these approaches and highlight their applications for the analysis of the tumour microenvironment in solid cancers. We also discuss techniques that are complementary and alternative to RNA sequencing, particularly focusing on approaches that can provide spatial information on the distribution of the cells within the tumour in addition to their qualitative and quantitative measurements. This article is part of a Special Issue entitled: Transcriptional Profiles and Regulatory Gene Networks edited by Dr. Federico Manuel Giorgi and Dr. Shaun Mahony.


Assuntos
Neoplasias/genética , RNA-Seq/métodos , Análise de Célula Única/métodos , Microambiente Tumoral/genética , Biomarcadores/metabolismo , Humanos , Neoplasias/imunologia
7.
Nat Commun ; 10(1): 3101, 2019 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-31308377

RESUMO

The identification of cancer-promoting genetic alterations is challenging particularly in highly unstable and heterogeneous cancers, such as esophageal adenocarcinoma (EAC). Here we describe a machine learning algorithm to identify cancer genes in individual patients considering all types of damaging alterations simultaneously. Analysing 261 EACs from the OCCAMS Consortium, we discover helper genes that, alongside well-known drivers, promote cancer. We confirm the robustness of our approach in 107 additional EACs. Unlike recurrent alterations of known drivers, these cancer helper genes are rare or patient-specific. However, they converge towards perturbations of well-known cancer processes. Recurrence of the same process perturbations, rather than individual genes, divides EACs into six clusters differing in their molecular and clinical features. Experimentally mimicking the alterations of predicted helper genes in cancer and pre-cancer cells validates their contribution to disease progression, while reverting their alterations reveals EAC acquired dependencies that can be exploited in therapy.


Assuntos
Adenocarcinoma/genética , Antineoplásicos/uso terapêutico , Biomarcadores Tumorais/genética , Neoplasias Esofágicas/genética , Perfilação da Expressão Gênica/métodos , Medicina de Precisão/métodos , Antineoplásicos/farmacologia , Biomarcadores Tumorais/antagonistas & inibidores , Biologia Computacional/métodos , Conjuntos de Dados como Assunto , Progressão da Doença , Dosagem de Genes , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Instabilidade Genômica , Humanos , Aprendizado de Máquina , Modelos Genéticos , Família Multigênica/efeitos dos fármacos , Taxa de Mutação , Polimorfismo de Nucleotídeo Único
8.
Nat Commun ; 10(1): 2350, 2019 05 28.
Artigo em Inglês | MEDLINE | ID: mdl-31138815

RESUMO

Endothelial cell migration, proliferation and survival are triggered by VEGF-A activation of VEGFR2. However, how these cell behaviors are regulated individually is still unknown. Here we identify Endophilin-A2 (ENDOA2), a BAR-domain protein that orchestrates CLATHRIN-independent internalization, as a critical mediator of endothelial cell migration and sprouting angiogenesis. We show that EndoA2 knockout mice exhibit postnatal angiogenesis defects and impaired front-rear polarization of sprouting tip cells. ENDOA2 deficiency reduces VEGFR2 internalization and inhibits downstream activation of the signaling effector PAK but not ERK, thereby affecting front-rear polarity and migration but not proliferation or survival. Mechanistically, VEGFR2 is directed towards ENDOA2-mediated endocytosis by the SLIT2-ROBO pathway via SLIT-ROBO-GAP1 bridging of ENDOA2 and ROBO1. Blocking ENDOA2-mediated endothelial cell migration attenuates pathological angiogenesis in oxygen-induced retinopathy models. This work identifies a specific endocytic pathway controlling a subset of VEGFR2 mediated responses that could be targeted to prevent excessive sprouting angiogenesis in pathological conditions.


Assuntos
Aciltransferases/genética , Células Endoteliais/metabolismo , Neovascularização Fisiológica/genética , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Animais , Movimento Celular/genética , Polaridade Celular/genética , Proliferação de Células/genética , Sobrevivência Celular/genética , Endocitose/genética , Células Endoteliais/citologia , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Sistema de Sinalização das MAP Quinases , Camundongos , Camundongos Knockout , Proteínas do Tecido Nervoso/metabolismo , Receptores Imunológicos/metabolismo , Vasos Retinianos/citologia , Vasos Retinianos/crescimento & desenvolvimento , Quinases Ativadas por p21/metabolismo
9.
J Cell Biol ; 217(10): 3577-3592, 2018 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-30087126

RESUMO

INPP5K (SKIP) is an inositol 5-phosphatase that localizes in part to the endoplasmic reticulum (ER). We show that recruitment of INPP5K to the ER is mediated by ARL6IP1, which shares features of ER-shaping proteins. Like ARL6IP1, INPP5K is preferentially localized in ER tubules and enriched, relative to other ER resident proteins (Sec61ß, VAPB, and Sac1), in newly formed tubules that grow along microtubule tracks. Depletion of either INPP5K or ARL6IP1 results in the increase of ER sheets. In a convergent but independent study, a screen for mutations affecting the distribution of the ER network in dendrites of the PVD neurons of Caenorhabditis elegans led to the isolation of mutants in CIL-1, which encodes the INPP5K worm orthologue. The mutant phenotype was rescued by expression of wild type, but not of catalytically inactive CIL-1. Our results reveal an unexpected role of an ER localized polyphosphoinositide phosphatase in the fine control of ER network organization.


Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/enzimologia , Dendritos/enzimologia , Retículo Endoplasmático/enzimologia , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatases/metabolismo , Animais , Células COS , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Chlorocebus aethiops , Dendritos/genética , Retículo Endoplasmático/genética , Deleção de Genes , Células HeLa , Humanos , Camundongos , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatases/genética
10.
Proc Natl Acad Sci U S A ; 115(10): E2238-E2245, 2018 03 06.
Artigo em Inglês | MEDLINE | ID: mdl-29463750

RESUMO

Methods to acutely manipulate protein interactions at the subcellular level are powerful tools in cell biology. Several blue-light-dependent optical dimerization tools have been developed. In these systems one protein component of the dimer (the bait) is directed to a specific subcellular location, while the other component (the prey) is fused to the protein of interest. Upon illumination, binding of the prey to the bait results in its subcellular redistribution. Here, we compared and quantified the extent of light-dependent dimer occurrence in small, subcellular volumes controlled by three such tools: Cry2/CIB1, iLID, and Magnets. We show that both the location of the photoreceptor protein(s) in the dimer pair and its (their) switch-off kinetics determine the subcellular volume where dimer formation occurs and the amount of protein recruited in the illuminated volume. Efficient spatial confinement of dimer to the area of illumination is achieved when the photosensitive component of the dimerization pair is tethered to the membrane of intracellular compartments and when on and off kinetics are extremely fast, as achieved with iLID or Magnets. Magnets and the iLID variants with the fastest switch-off kinetics induce and maintain protein dimerization in the smallest volume, although this comes at the expense of the total amount of dimer. These findings highlight the distinct features of different optical dimerization systems and will be useful guides in the choice of tools for specific applications.


Assuntos
Proteínas de Arabidopsis/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Criptocromos/metabolismo , Citoplasma/efeitos da radiação , Fotorreceptores Microbianos/química , Ligação Proteica/efeitos da radiação , Animais , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/química , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Criptocromos/química , Criptocromos/genética , Citoplasma/química , Citoplasma/genética , Citoplasma/metabolismo , Células HeLa , Humanos , Cinética , Camundongos , Mitocôndrias/química , Mitocôndrias/genética , Mitocôndrias/metabolismo , Mitocôndrias/efeitos da radiação , Neurospora crassa/química , Neurospora crassa/metabolismo , Neurospora crassa/efeitos da radiação , Fotorreceptores Microbianos/genética , Fotorreceptores Microbianos/metabolismo , Multimerização Proteica/efeitos da radiação
11.
Sci Rep ; 7(1): 8585, 2017 08 17.
Artigo em Inglês | MEDLINE | ID: mdl-28819307

RESUMO

MicroRNAs (miRNAs) are important regulators of diverse physiological and pathophysiological processes. MiRNA families and clusters are two key features in miRNA biology. Here we explore the use of CRISPR/Cas9 as a powerful tool to delineate the function and regulation of miRNA families and clusters. We focused on four miRNA clusters composed of miRNA members of the same family, homo-clusters or different families, hetero-clusters. Our results highlight different regulatory mechanisms in miRNA cluster expression. In the case of the miR-497~195 cluster, editing of miR-195 led to a significant decrease in the expression of the other miRNA in the cluster, miR-497a. Although no gene editing was detected in the miR-497a genomic locus, computational simulation revealed alteration in the three dimensional structure of the pri-miR-497~195 that may affect its processing. In cluster miR-143~145 our results imply a feed-forward regulation, although structural changes cannot be ruled out. Furthermore, in the miR-17~92 and miR-106~25 clusters no interdependency in miRNA expression was observed. Our findings suggest that CRISPR/Cas9 is a powerful gene editing tool that can uncover novel mechanisms of clustered miRNA regulation and function.


Assuntos
Sistemas CRISPR-Cas , Edição de Genes , MicroRNAs/metabolismo , Animais , Células Cultivadas , Simulação por Computador , Perfilação da Expressão Gênica , Células HEK293 , Humanos , Camundongos , MicroRNAs/genética , Família Multigênica , Músculo Liso Vascular/citologia
12.
J Vis Exp ; (124)2017 06 26.
Artigo em Inglês | MEDLINE | ID: mdl-28671644

RESUMO

The protocols described here are designed to allow researchers to study cell communication without altering the integrity of the environment in which the cells are located. Specifically, they have been developed to analyze the electrical activity of excitable cells, such as spinal neurons. In such a scenario, it is crucial to preserve the integrity of the spinal cell, but it is also important to preserve the anatomy and physiological shape of the systems involved. Indeed, the comprehension of the manner in which the nervous system-and other complex systems-works must be based on a systemic approach. For this reason, the live zebrafish embryo was chosen as a model system, and the spinal neuron membrane voltage changes were evaluated without interfering with the physiological conditions of the embryos. Here, an approach combining the employment of zebrafish embryos with a FRET-based biosensor is described. Zebrafish embryos are characterized by a very simplified nervous system and are particularly suited for imaging applications thanks to their transparency, allowing for the employment of fluorescence-based voltage indicators at the plasma membrane during zebrafish development. The synergy between these two components makes it possible to analyze the electrical activity of the cells in intact living organisms, without perturbing the physiological state. Finally, this non-invasive approach can co-exist with other analyses (e.g., spontaneous movement recordings, as shown here).


Assuntos
Potenciais da Membrana/fisiologia , Neurônios Motores/fisiologia , Peixe-Zebra/embriologia , Animais , Comunicação Celular , Embrião não Mamífero
13.
Oncotarget ; 8(23): 37619-37632, 2017 Jun 06.
Artigo em Inglês | MEDLINE | ID: mdl-28430577

RESUMO

Cohesin is a multi-protein complex that tethers sister chromatids during mitosis and mediates DNA repair, genome compartmentalisation and regulation of gene expression. Cohesin subunits frequently acquire cancer loss-of-function alterations and act as tumour suppressors in several tumour types. This has led to increased interest in cohesin as potential target in anti-cancer therapy. Here we show that the loss-of-function of STAG2, a core component of cohesin and an emerging tumour suppressor, leads to synthetic dependency of mutated cancer cells on its paralog STAG1. STAG1 and STAG2 share high sequence identity, encode mutually exclusive cohesin subunits and retain partially overlapping functions. We inhibited STAG1 and STAG2 in several cancer cell lines where the two genes have variable mutation and copy number status. In all cases, we observed that the simultaneous blocking of STAG1 and STAG2 significantly reduces cell proliferation. We further confirmed the synthetic lethal interaction developing a vector-free CRISPR system to induce STAG1/STAG2 double gene knockout. We provide strong evidence that STAG1 is a promising therapeutic target in cancers with inactivating alterations of STAG2.


Assuntos
Antígenos Nucleares/genética , Proliferação de Células/genética , Proteínas Nucleares/genética , Proteínas Supressoras de Tumor/genética , Antígenos Nucleares/metabolismo , Sistemas CRISPR-Cas , Proteínas de Ciclo Celular , Linhagem Celular Tumoral , Edição de Genes/métodos , Regulação Neoplásica da Expressão Gênica , Humanos , Mutação com Perda de Função , Células MCF-7 , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patologia , Proteínas Nucleares/metabolismo , Ligação Proteica , Interferência de RNA , Proteínas Supressoras de Tumor/metabolismo
14.
Nat Commun ; 8: 14536, 2017 03 06.
Artigo em Inglês | MEDLINE | ID: mdl-28262662

RESUMO

Shrm4, a protein expressed only in polarized tissues, is encoded by the KIAA1202 gene, whose mutations have been linked to epilepsy and intellectual disability. However, a physiological role for Shrm4 in the brain is yet to be established. Here, we report that Shrm4 is localized to synapses where it regulates dendritic spine morphology and interacts with the C terminus of GABAB receptors (GABABRs) to control their cell surface expression and intracellular trafficking via a dynein-dependent mechanism. Knockdown of Shrm4 in rat severely impairs GABABR activity causing increased anxiety-like behaviour and susceptibility to seizures. Moreover, Shrm4 influences hippocampal excitability by modulating tonic inhibition in dentate gyrus granule cells, in a process involving crosstalk between GABABRs and extrasynaptic δ-subunit-containing GABAARs. Our data highlights a role for Shrm4 in synaptogenesis and in maintaining GABABR-mediated inhibition, perturbation of which may be responsible for the involvement of Shrm4 in cognitive disorders and epilepsy.


Assuntos
Hipocampo/metabolismo , Proteínas dos Microfilamentos/genética , Proteínas do Tecido Nervoso/genética , Neurônios/metabolismo , Receptores de GABA-A/genética , Receptores de GABA-B/genética , Transmissão Sináptica/genética , Animais , Giro Denteado/metabolismo , Giro Denteado/patologia , Giro Denteado/ultraestrutura , Embrião de Mamíferos , Epilepsia/genética , Epilepsia/metabolismo , Epilepsia/patologia , Regulação da Expressão Gênica , Células HEK293 , Hipocampo/patologia , Hipocampo/ultraestrutura , Humanos , Injeções Intraventriculares , Deficiência Intelectual/genética , Deficiência Intelectual/metabolismo , Deficiência Intelectual/patologia , Proteínas dos Microfilamentos/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Inibição Neural , Neurogênese/genética , Neurônios/patologia , Neurônios/ultraestrutura , Cultura Primária de Células , Ratos , Ratos Wistar , Receptor Cross-Talk , Receptores de GABA-A/metabolismo , Receptores de GABA-B/metabolismo , Sinapses/metabolismo , Sinapses/patologia , Sinapses/ultraestrutura
15.
Mol Cancer Res ; 15(3): 304-316, 2017 03.
Artigo em Inglês | MEDLINE | ID: mdl-28031408

RESUMO

Understanding the mechanism of metastatic dissemination is crucial for the rational design of novel therapeutics. The secreted protein acidic and rich in cysteine (SPARC) is a matricellular glycoprotein which has been extensively associated with human breast cancer aggressiveness although the underlying mechanisms are still unclear. Here, shRNA-mediated SPARC knockdown greatly reduced primary tumor growth and completely abolished lung colonization of murine 4T1 and LM3 breast malignant cells implanted in syngeneic BALB/c mice. A comprehensive study including global transcriptomic analysis followed by biological validations confirmed that SPARC induces primary tumor growth by enhancing cell cycle and by promoting a COX-2-mediated expansion of myeloid-derived suppressor cells (MDSC). The role of SPARC in metastasis involved a COX-2-independent enhancement of cell disengagement from the primary tumor and adherence to the lungs that fostered metastasis implantation. Interestingly, SPARC-driven gene expression signatures obtained from these murine models predicted the clinical outcome of patients with HER2-enriched breast cancer subtypes. In total, the results reveal that SPARC and its downstream effectors are attractive targets for antimetastatic therapies in breast cancer.Implications: These findings shed light on the prometastatic role of SPARC, a key protein expressed by breast cancer cells and surrounding stroma, with important consequences for disease outcome. Mol Cancer Res; 15(3); 304-16. ©2016 AACR.


Assuntos
Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Neoplasias Mamárias Experimentais/metabolismo , Neoplasias Mamárias Experimentais/patologia , Osteonectina/metabolismo , Receptor ErbB-2/metabolismo , Animais , Neoplasias da Mama/enzimologia , Neoplasias da Mama/genética , Processos de Crescimento Celular/fisiologia , Linhagem Celular Tumoral , Feminino , Humanos , Células MCF-7 , Neoplasias Mamárias Experimentais/enzimologia , Neoplasias Mamárias Experimentais/genética , Camundongos , Camundongos Endogâmicos BALB C , Metástase Neoplásica , Osteonectina/genética , Prognóstico , Receptor ErbB-2/genética , Resultado do Tratamento
16.
Bioinformatics ; 33(8): 1248-1249, 2017 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-28003259

RESUMO

Summary: : Detecting significant associations between genetic variants and disease may prove particularly challenging when the variants are rare in the population and/or act together with other variants to cause the disease. We have developed a statistical framework named Mutation Enrichment Gene set Analysis of Variants (MEGA-V) that specifically detects the enrichments of genetic alterations within a process in a cohort of interest. By focusing on the mutations of several genes contributing to the same function rather than on those affecting a single gene, MEGA-V increases the power to detect statistically significant associations. Availability and Implementation: MEGA-V is available at https://github.com/ciccalab/MEGA. Contact: francesca.ciccarelli@kcl.ac.uk. Supplementary information: Supplementary data are available at Bioinformatics online.


Assuntos
Mutação , Software , Estudos de Coortes , Interpretação Estatística de Dados , Humanos
17.
Nat Commun ; 7: 12072, 2016 07 05.
Artigo em Inglês | MEDLINE | ID: mdl-27377421

RESUMO

Synchronous colorectal cancers (syCRCs) are physically separated tumours that develop simultaneously. To understand how the genetic and environmental background influences the development of multiple tumours, here we conduct a comparative analysis of 20 syCRCs from 10 patients. We show that syCRCs have independent genetic origins, acquire dissimilar somatic alterations, and have different clone composition. This inter- and intratumour heterogeneity must be considered in the selection of therapy and in the monitoring of resistance. SyCRC patients show a higher occurrence of inherited damaging mutations in immune-related genes compared to patients with solitary colorectal cancer and to healthy individuals from the 1,000 Genomes Project. Moreover, they have a different composition of immune cell populations in tumour and normal mucosa, and transcriptional differences in immune-related biological processes. This suggests an environmental field effect that promotes multiple tumours likely in the background of inflammation.


Assuntos
Neoplasias Colorretais/genética , Heterogeneidade Genética , Mutação em Linhagem Germinativa , Proteínas de Neoplasias/genética , Neoplasias Primárias Múltiplas/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Estudos de Casos e Controles , Células Clonais , Neoplasias Colorretais/imunologia , Neoplasias Colorretais/patologia , Citocinas/genética , Citocinas/imunologia , Feminino , Predisposição Genética para Doença , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Masculino , Proteínas de Neoplasias/imunologia , Neoplasias Primárias Múltiplas/imunologia , Neoplasias Primárias Múltiplas/patologia , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/imunologia , Receptores Toll-Like/genética , Receptores Toll-Like/imunologia
18.
Sci Rep ; 6: 24515, 2016 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-27079797

RESUMO

The pathogenic role of SOD1 mutations in amyotrophic lateral sclerosis (ALS) was investigated using a zebrafish disease model stably expressing the ALS-linked G93R mutation. In addition to the main pathological features of ALS shown by adult fish, we found remarkably precocious alterations in the development of motor nerve circuitry and embryo behavior, and suggest that these alterations are prompted by interneuron and motor neuron hyperexcitability triggered by anomalies in the persistent pacemaker sodium current INaP. The riluzole-induced modulation of INaP reduced spinal neuron excitability, reverted the behavioral phenotypes and improved the deficits in motor nerve circuitry development, thus shedding new light on the use of riluzole in the management of ALS. Our findings provide a valid phenotype-based tool for unbiased in vivo drug screening that can be used to develop new therapies.


Assuntos
Potenciais de Ação/efeitos dos fármacos , Potenciais de Ação/genética , Esclerose Amiotrófica Lateral/genética , Neurônios Motores/efeitos dos fármacos , Neurônios Motores/fisiologia , Fenilglioxal/análogos & derivados , Superóxido Dismutase/genética , Esclerose Amiotrófica Lateral/diagnóstico , Animais , Animais Geneticamente Modificados , Modelos Animais de Doenças , Expressão Gênica , Locomoção , Atividade Motora/efeitos dos fármacos , Músculos/patologia , Mutação , Junção Neuromuscular/metabolismo , Fenótipo , Fenilglioxal/farmacologia , Riluzol/farmacologia , Medula Espinal/patologia , Peixe-Zebra
19.
Sci Rep ; 4: 7033, 2014 Nov 13.
Artigo em Inglês | MEDLINE | ID: mdl-25391455

RESUMO

Correlative light electron microscopy (CLEM) combines the advantages of light and electron microscopy, thus making it possible to follow dynamic events in living cells at nanometre resolution. Various CLEM approaches and devices have been developed, each of which has its own advantages and technical challenges. We here describe our customized patterned glass substrates, which improve the feasibility of correlative fluorescence/confocal and scanning electron microscopy.

20.
PLoS One ; 9(10): e108826, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25295618

RESUMO

To optimise the efficiency of cell machinery, cells can use the same protein (often called a hub protein) to participate in different cell functions by simply changing its target molecules. There are large data sets describing protein-protein interactions ("interactome") but they frequently fail to consider the functional significance of the interactions themselves. We studied the interaction between two potential hub proteins, ICln and 4.1R (in the form of its two splicing variants 4.1R80 and 4.1R135), which are involved in such crucial cell functions as proliferation, RNA processing, cytoskeleton organisation and volume regulation. The sub-cellular localisation and role of native and chimeric 4.1R over-expressed proteins in human embryonic kidney (HEK) 293 cells were examined. ICln interacts with both 4.1R80 and 4.1R135 and its over-expression displaces 4.1R from the membrane regions, thus affecting 4.1R interaction with ß-actin. It was found that 4.1R80 and 4.1R135 are differently involved in regulating the swelling activated anion current (ICl,swell) upon hypotonic shock, a condition under which both isoforms are dislocated from the membrane region and thus contribute to ICl,swell current regulation. Both 4.1R isoforms are also differently involved in regulating cell morphology, and ICln counteracts their effects. The findings of this study confirm that 4.1R plays a role in cell volume regulation and cell morphology and indicate that ICln is a new negative regulator of 4.1R functions.


Assuntos
Proteínas do Citoesqueleto/metabolismo , Proteínas ELAV/metabolismo , Proteínas de Membrana/metabolismo , Isoformas de Proteínas/metabolismo , Linhagem Celular , Citoesqueleto/metabolismo , Proteína Semelhante a ELAV 2 , Células HEK293 , Humanos , Ligação Proteica
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