Towards definition of an ECM parts list: An advance on GO categories

Those of us interested in the extracellular matrix (ECM) are faced with significant challenges of definition. ECM proteins are large, complex and assembled into crosslinked insoluble matrices. This has meant that defining the biochemical composition of ECMs has been difficult. Nonetheless, protein chemistry and molecular biology have defined many familiar ECM proteins — collagens, proteoglycans, laminins, thrombospondins, tenascins, fibronectins, etc. With the completion of many genomes it should now be possible to develop complete “parts lists” for the ECM. Such lists are needed for analyzing data from “omic” approaches such as expression arrays, latest-generation sequencing and proteomics. These approaches generate long lists and it is typically necessary to extract from those lists the genes/proteins of interest. Anyone who attempts to do this using the commonly used gene ontology (GO) categories soon discovers that they are largely useless for defining ECM proteins. Many ECM proteins are unannotated and those which are, are sorted, with little evidence of logic or consistency, into diverse categories such “extracellular matrix,” “basement membrane,” “cell surface” and many others. The human and mouse orthologs are often found in different categories and attempts to use GO categories to extract a complete list of ECM genes or proteins from a data set are unsatisfactory at best

Extracellular matrix signatures of human primary metastatic colon cancers and their metastases to liver

Background: Colorectal cancer is the third most frequently diagnosed cancer and the third cause of cancer deaths in the United States. Despite the fact that tumor cell-intrinsic mechanisms controlling colorectal carcinogenesis have been identified, novel prognostic and diagnostic tools as well as novel therapeutic strategies are still needed to monitor and target colon cancer progression. We and others have previously shown, using mouse models, that the extracellular matrix (ECM), a major component of the tumor microenvironment, is an important contributor to tumor progression. In order to identify candidate biomarkers, we sought to define ECM signatures of metastatic colorectal cancers and their metastases to the liver. Methods: We have used enrichment of extracellular matrix (ECM) from human patient samples and proteomics to define the ECM composition of primary colon carcinomas and their metastases to liver in comparison with normal colon and liver samples. Results: We show that robust signatures of ECM proteins characteristic of each tissue, normal and malignant, can be defined using relatively small samples from small numbers of patients. Comparisons with gene expression data from larger cohorts of patients confirm the association of subsets of the proteins identified by proteomic analysis with tumor progression and metastasis. Conclusions: The ECM protein signatures of metastatic primary colon carcinomas and metastases to liver defined in this study, offer promise for development of diagnostic and prognostic signatures of metastatic potential of colon tumors. The ECM proteins defined here represent candidate serological or tissue biomarkers and potential targets for imaging of occult metastases and residual or recurrent tumors and conceivably for therapies. Furthermore, the methods described here can be applied to other tumor types and can be used to investigate other questions such as the role of ECM in resistance to therapy

Ezrin phosphorylation on tyrosine 477 regulates invasion and metastasis of breast cancer cells

Background The membrane cytoskeletal crosslinker, ezrin, a member of the ERM family of proteins, is frequently over-expressed in human breast cancers, and is required for motility and invasion of epithelial cells. Our group previously showed that ezrin acts co-operatively with the non-receptor tyrosine kinase, Src, in deregulation of cell-cell contacts and scattering of epithelial cells. In particular, ezrin phosphorylation on Y477 by Src is specific to ezrin within the ERM family, and is required for HGF-induced scattering of epithelial cells. We therefore sought to examine the role of Y477 phosphorylation in ezrin on tumor progression. Methods Using a highly metastatic mouse mammary carcinoma cell line (AC2M2), we tested the effect of over-expressing a non-phosphorylatable form of ezrin (Y477F) on invasive colony growth in 3-dimensional Matrigel cultures, and on local invasion and metastasis in an orthotopic engraftment model. Results AC2M2 cells over-expressing Y477F ezrin exhibited delayed migration in vitro, and cohesive round colonies in 3-dimensional Matrigel cultures, compared to control cells that formed invasive colonies with branching chains of cells and numerous actin-rich protrusions. Moreover, over-expression of Y477F ezrin inhibits local tumor invasion in vivo. Whereas orthotopically injected wild type AC2M2 tumor cells were found to infiltrate into the abdominal wall and visceral organs within three weeks, tumors expressing Y477F ezrin remained circumscribed, with little invasion into the surrounding stroma and abdominal wall. Additionally, Y477F ezrin reduces the number of lung metastatic lesions. Conclusions Our study implicates a role of Y477 ezrin, which is phosphorylated by Src, in regulating local invasion and metastasis of breast carcinoma cells, and provides a clinically relevant model for assessing the Src/ezrin pathway as a potential prognostic/predictive marker or treatment target for invasive human breast cancer.Canadian Breast Cancer Research Alliance (BEE, 017374)Canadian Institutes of Health Research (BEE, 102644)Physicians Society Inc.Association pour le développement de la recherche sur le cancer (France

Global proteomic analysis of extracellular matrix in mouse and human brain highlights relevance to cerebrovascular disease

The extracellular matrix (ECM) is a key interface between the cerebrovasculature and adjacent brain tissues. Deregulation of the ECM contributes to a broad range of neurological disorders. However, despite this importance, our understanding of the ECM composition remains very limited mainly due to difficulties in its isolation. To address this, we developed an approach to extract the cerebrovascular ECM from mouse and human post-mortem normal brain tissues. We then used mass spectrometry with off-line high-pH reversed-phase fractionation to increase the protein detection. This identified more than 1000 proteins in the ECM-enriched fraction, with > 66% of the proteins being common between the species. We report 147 core ECM proteins of the human brain vascular matrisome, including collagens, laminins, fibronectin and nidogens. We next used network analysis to identify the connection between the brain ECM proteins and cerebrovascular diseases. We found that genes related to cerebrovascular diseases, such as COL4A1, COL4A2, VCAN and APOE were significantly enriched in the cerebrovascular ECM network. This provides unique mechanistic insight into cerebrovascular disease and potential drug targets. Overall, we provide a powerful resource to study the functions of brain ECM and highlight a specific role for brain vascular ECM in cerebral vascular disease

Quantitative proteomics identify Tenascin-C as a promoter of lung cancer progression and contributor to a signature prognostic of patient survival

The extracellular microenvironment is an integral component of normal and diseased tissues that is poorly understood owing to its complexity. To investigate the contribution of the microenvironment to lung fibrosis and adenocarcinoma progression, two pathologies characterized by excessive stromal expansion, we used mouse models to characterize the extracellular matrix (ECM) composition of normal lung, fibrotic lung, lung tumors, and metastases. Using quantitative proteomics, we identified and assayed the abundance of 113 ECM proteins, which revealed robust ECM protein signatures unique to fibrosis, primary tumors, or metastases. These analyses indicated significantly increased abundance of several S100 proteins, including Fibronectin and Tenascin-C (Tnc), in primary lung tumors and associated lymph node metastases compared with normal tissue. We further showed that Tnc expression is repressed by the transcription factor Nkx2-1, a well-established suppressor of metastatic progression. We found that increasing the levels of Tnc, via CRISPR-mediated transcriptional activation of the endogenous gene, enhanced the metastatic dissemination of lung adenocarcinoma cells. Interrogation of human cancer gene expression data revealed that high TNC expression correlates with worse prognosis for lung adenocarcinoma, and that a three-gene expression signature comprising TNC, S100A10, and S100A11 is a robust predictor of patient survival independent of age, sex, smoking history, and mutational load. Our findings suggest that the poorly understood ECM composition of the fibrotic and tumor microenvironment is an underexplored source of diagnostic markers and potential therapeutic targets for cancer patients

Reduced SPAG17 Expression in Systemic Sclerosis Triggers Myofibroblast Transition and Drives Fibrosis

Systemic sclerosis (SSc) is a clinically heterogeneous fibrotic disease with no effective treatment. Myofibroblasts are responsible for unresolving synchronous skin and internal organ fibrosis in SSc, but the drivers of sustained myofibroblast activation remain poorly understood. Using unbiased transcriptome analysis of skin biopsies, we identified the downregulation of SPAG17 in multiple independent cohorts of patients with SSc, and by orthogonal approaches, we observed a significant negative correlation between SPAG17 and fibrotic gene expression. Fibroblasts and endothelial cells explanted from SSc skin biopsies showed reduced chromatin accessibility at the SPAG17 locus. Remarkably, mice lacking Spag17 showed spontaneous skin fibrosis with increased dermal thickness, collagen deposition and stiffness, and altered collagen fiber alignment. Knockdown of SPAG17 in human and mouse fibroblasts and microvascular endothelial cells was accompanied by spontaneous myofibroblast transformation and markedly heightened sensitivity to profibrotic stimuli. These responses were accompanied by constitutive TGF-β pathway activation. Thus, we discovered impaired expression of SPAG17 in SSc and identified, to our knowledge, a previously unreported cell-intrinsic role for SPAG17 in the negative regulation of fibrotic responses. These findings shed fresh light on the pathogenesis of SSc and may inform the search for innovative therapies for SSc and other fibrotic conditions through SPAG17 signaling

Discutindo a educação ambiental no cotidiano escolar: desenvolvimento de projetos na escola formação inicial e continuada de professores

A presente pesquisa buscou discutir como a Educação Ambiental (EA) vem sendo trabalhada, no Ensino Fundamental e como os docentes desta escola compreendem e vem inserindo a EA no cotidiano escolar., em uma escola estadual do município de Tangará da Serra/MT, Brasil. Para tanto, realizou-se entrevistas com os professores que fazem parte de um projeto interdisciplinar de EA na escola pesquisada. Verificou-se que o projeto da escola não vem conseguindo alcançar os objetivos propostos por: desconhecimento do mesmo, pelos professores; formação deficiente dos professores, não entendimento da EA como processo de ensino-aprendizagem, falta de recursos didáticos, planejamento inadequado das atividades. A partir dessa constatação, procurou-se debater a impossibilidade de tratar do tema fora do trabalho interdisciplinar, bem como, e principalmente, a importância de um estudo mais aprofundado de EA, vinculando teoria e prática, tanto na formação docente, como em projetos escolares, a fim de fugir do tradicional vínculo “EA e ecologia, lixo e horta”.Facultad de Humanidades y Ciencias de la Educació

Rôle de l'ezrine dans le contrôle de la morphogenèse et de l'adhérence des cellules épithéliales

L ezrine est une protéine de liaison du cytosquelette d'actine à la membrane plasmique. Sa phosphorylation, en réponse à des signaux provenant des facteurs de croissance ou de la matrice extracellulaire, contrôle diverses étapes de la morphogenèse des cellules épithéliales. Ces signaux extracellulaires sont relayés par les kinases cytoplasmiques de la famille Src et nous avons montré que l ezrine est une cible de ces kinases. Au cours de ma thèse, j ai étudié deux nouveaux partenaires de l'ezrine phosphorylée par les kinases de la famille Src : la kinase Fes et les protéines de la famille Eps8. Nous avons montré que la kinase Fes interagit via son domaine SH2 avec la tyrosine 477 phosphorylée de l ezrine. Selon la confluence des cellules épithéliales, la forme activée de Fes se localise soit au niveau des adhérences focales soit au niveau des jonctions adhérentes. La localisation de Fes au niveau des jonctions adhérentes dépend de son interaction avec l ezrine. Ce recrutement est nécessaire, d une part pour l activation de la kinase Fes et d autre part pour la dissociation des cellules en réponse à une stimulation par l HGF (Hepatocyte Growth Factor). Cette étude nous a permis d impliquer l ezrine et la kinase Fes dans la régulation des jonctions cellulaires en aval de la voie de signalisation induite par l HGF. Les protéines de la famille Eps8 régulent le remodelage du cytosquelette d'actine en aval des voies de signalisation induites par les facteurs de croissance. Nos observations suggèrent un rôle pour l interaction ezrine/protéines de la famille Eps8 dans l organisation des filaments d actine du cortex cellulaire. Nos résultats indiquent que l interaction de l ezrine avec différents partenaires permet l organisation structurale et fonctionnelle de domaines spécifiques de la membrane plasmique des cellules épithéliales.Ezrin is a membrane-actin cytoskeleton linker that plays an essential role in epithelial cell morphogenesis. It participates to the transmission of signals that derive from either outside or inside the cells. In response to growth factors or to extracellular matrix signals, ezrin is phosphorylated by various kinases including Src kinases. We performed a twohybrid screen with ezrin phosphorylated by the Src family kinases to identify the downstream pathways activated through this phosphorylation. We analyzed two interacting partners of ezrin phosphorylated by Src: the Fes kinase and the proteins of the Eps8 family. We showed that the Fes kinase interacts, via its SH2 domain, with ezrin phosphorylated at tyrosine 477. In confluent epithelial cells, this interaction is necessary for the recruitment and the activation of the Fes kinase to the adherens junctions. If this interaction is impaired, the Fes kinase localizes to focal adhesions. As a consequence, the cells show delay in their spreading and they do no longer scatter in response to HGF treatment. Altogether, this study provides a novel mechanism whereby ezrin/Fes interaction at cell contacts plays an essential role in HGF-induced cell scattering and implicates Fes in the cross-talk between cell-cell and cell-matrix adhesion. The proteins that belong to the Eps8 family regulate the actin cytoskeleton remodeling in response to growth factor stimulations. Our observations suggest a possible role for the ezrin/Eps8 interaction in the organization of the cortical actin cytoskeleton. Altogether, our results indicate that the interaction of phosphorylated ezrin with its partners is required for the structural and functional organization of specific domains of the plasma membrane in epithelial cells.ORSAY-PARIS 11-BU Sciences (914712101) / SudocSudocFranceF

The cancer matrisome: from comprehensive characterization to biomarker discovery

Tumor progression and dissemination critically depend on support from the tumor microenvironment, the ensemble of cellular and acellular components surrounding and interacting with tumor cells. The extracellular matrix (ECM), the complex scaffolding of hundreds of proteins surrounding and organizing cells in tissues, is a major component of the tumor microenvironment. It orchestrates cellular processes including proliferation, migration, and invasion, that are highly dysregulated during cancer progression. Alterations in ECM abundance, integrity, and mechanical properties have been correlated with poorer prognosis for cancer patients. Yet the ECM proteome, or "matrisome," of tumors remained until recently largely unexplored. This review will present the recent developments in computational and proteomic technologies that have allowed the comprehensive characterization of the ECM of different tumor types and microenvironmental niches. These approaches have resulted in the definition of protein signatures distinguishing tumors from normal tissues, tumors of different stages, primary from secondary tumors, and tumors from other diseased states such as fibrosis. Moreover, recent studies have demonstrated that the levels of expression of certain genes encoding ECM and ECM-associated proteins is prognostic of cancer patient survival and can thus serve as biomarkers. Last, proteomic studies have permitted the identification of novel ECM proteins playing functional roles in cancer progression. Such proteins have the potential to be exploited as therapeutic targets