A Cell Motility Screen Reveals Role for MARCKS-Related Protein in Adherens Junction Formation and Tumorigenesis
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{"title"=>"A cell motility screen reveals role for MARCKS-related protein in adherens junction formation and tumorigenesis", "type"=>"journal", "authors"=>[{"first_name"=>"Alexander E.", "last_name"=>"Finlayson", "scopus_author_id"=>"55253184000"}, {"first_name"=>"Kevin W.", "last_name"=>"Freeman", "scopus_author_id"=>"7202892014"}], "year"=>2009, "source"=>"PLoS ONE", "identifiers"=>{"issn"=>"19326203", "isbn"=>"1932-6203", "doi"=>"10.1371/journal.pone.0007833", "pui"=>"355724744", "sgr"=>"70849127012", "pmid"=>"19924305", "scopus"=>"2-s2.0-70849127012"}, "id"=>"094d9998-44ff-39cc-a796-0e175deefd32", "abstract"=>"Invasion through the extracellular matrix (ECM) is important for wound healing, immunological responses and metastasis. We established an invasion-based cell motility screen using Boyden chambers overlaid with Matrigel to select for pro-invasive genes. By this method we identified antisense to MARCKS related protein (MRP), whose family member MARCKS is a target of miR-21, a microRNA involved in tumor growth, invasion and metastasis in multiple human cancers. We confirmed that targeted knockdown of MRP, in both EpRas mammary epithelial cells and PC3 prostate cancer cells, promoted in vitro cell migration that was blocked by trifluoperazine. Additionally, we observed increased immunofluoresence of E-cadherin, beta-catenin and APC at sites of cell-cell contact in EpRas cells with MRP knockdown suggesting formation of adherens junctions. By wound healing assay we observed that reduced MRP supported collective cell migration, a type of cell movement where adherens junctions are maintained. However, destabilized adherens junctions, like those seen in EpRas cells, are frequently important for oncogenic signaling. Consequently, knockdown of MRP in EpRas caused loss of tumorigenesis in vivo, and reduced Wnt3a induced TCF reporter signaling in vitro. Together our data suggest that reducing MRP expression promotes formation of adherens junctions in EpRas cells, allowing collective cell migration, but interferes with oncogenic beta-catenin signaling and tumorigenesis.", "link"=>"http://www.mendeley.com/research/cell-motility-screen-reveals-role-marcksrelated-protein-adherens-junction-formation-tumorigenesis", "reader_count"=>44, "reader_count_by_academic_status"=>{"Professor > Associate Professor"=>5, "Researcher"=>18, "Student > Doctoral Student"=>3, "Student > Ph. D. Student"=>12, "Student > Postgraduate"=>2, "Other"=>1, "Student > Bachelor"=>2, "Professor"=>1}, "reader_count_by_user_role"=>{"Professor > Associate Professor"=>5, "Researcher"=>18, "Student > Doctoral Student"=>3, "Student > Ph. D. Student"=>12, "Student > Postgraduate"=>2, "Other"=>1, "Student > Bachelor"=>2, "Professor"=>1}, "reader_count_by_subject_area"=>{"Engineering"=>1, "Biochemistry, Genetics and Molecular Biology"=>11, "Agricultural and Biological Sciences"=>23, "Medicine and Dentistry"=>5, "Design"=>1, "Chemistry"=>1, "Social Sciences"=>2}, "reader_count_by_subdiscipline"=>{"Design"=>{"Design"=>1}, "Engineering"=>{"Engineering"=>1}, "Medicine and Dentistry"=>{"Medicine and Dentistry"=>5}, "Chemistry"=>{"Chemistry"=>1}, "Social Sciences"=>{"Social Sciences"=>2}, "Agricultural and Biological Sciences"=>{"Agricultural and Biological Sciences"=>23}, "Biochemistry, Genetics and Molecular Biology"=>{"Biochemistry, Genetics and Molecular Biology"=>11}}, "reader_count_by_country"=>{"Canada"=>1, "Uruguay"=>1, "United Kingdom"=>1, "Germany"=>1, "Spain"=>1}, "group_count"=>7}

Scopus | Further Information

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Figshare

  • {"files"=>["https://ndownloader.figshare.com/files/875965"], "description"=>"<p>5×10<sup>5</sup> cells EpRas cells, clonal cell lines stably transfected with control shRNA (C1) or with shRNA directed to MRP (E13, 14) were injected subcutaneously into five congenic Balb/c mice per cohort (a) or into 10 mice per cohort by tail vein injection (b). Tumor weight was assessed at the indicated times. Representative H&E staining of lung sections from each group at 4× magnification (b).</p>", "links"=>[], "tags"=>["tumorigenesis", "targeted", "knockdown"], "article_id"=>546428, "categories"=>["Cancer", "Cell Biology", "Plant Biology"], "users"=>["Alexander E. Finlayson", "Kevin W. Freeman"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0007833.g005", "stats"=>{"downloads"=>1, "page_views"=>5, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Loss_of_tumorigenesis_with_targeted_knockdown_of_MRP_/546428", "title"=>"Loss of tumorigenesis with targeted knockdown of MRP.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2009-11-18 01:47:08"}
  • {"files"=>["https://ndownloader.figshare.com/files/875224"], "description"=>"<p>a) Schematic of ECM screen. b) Representative genomic PCR of isolated clones for inserts in pEyk3.1, Ld =  1 kb ladder (NEB). c) To segregate multiple retroviral integrants, retrovirus was repackaged from either invading clones or the control EGFP clones using pCL-Eco helper plasmid (top schematic). Naive EpH4 cells were infected and individual clonal lines were assayed for invasion. The two most invasive clones were determined to have both been infected with antisense to MRP (dark grey). d) Mouse mammary cell line EpH4 was stably transfected with expression pβ plasmid containing antisense MRP identified in the screen or control plasmid and subjected to invasion or migration assays. Data is mean of three independent experiments ± SD. * is p<0.01</p>", "links"=>[], "tags"=>["antisense", "mrp", "invasion-based", "motility"], "article_id"=>545684, "categories"=>["Cancer", "Cell Biology", "Plant Biology"], "users"=>["Alexander E. Finlayson", "Kevin W. Freeman"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0007833.g001", "stats"=>{"downloads"=>3, "page_views"=>6, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Isolation_of_antisense_to_MRP_in_Invasion_based_motility_screen_/545684", "title"=>"Isolation of antisense to MRP in Invasion-based motility screen.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2009-11-18 01:34:44"}
  • {"files"=>["https://ndownloader.figshare.com/files/875774"], "description"=>"<p>a) Bright field imaging of EpRas, C1, E13, and E14 at 10X. b) EpRas, C1, E13 or E14 cells were stained with primary antibodies specific to either E-cadherin (Becton Dickinson), APC (Calbiochem) or β-catenin (Becton Dickinson) and secondary antibodies Alexa fluor 488 anti-rabbit or anti-mouse (Invitrogen). Images at 100X c) Western blot analysis of EpRas, C1, E13, E14 whole cell lysates and immunoblotted with antibodies to Ras (Cell Signaling), E-cadherin and β-catenin (Becton Dickinson), and γ-tubulin (Sigma). NS =  non-specific. d) Indicated cells transfected with the TCF luciferase reporter construct TOPFLASH and transfection control pRL-CMV were treated with 0, 50 ng/mL or 100 ng/mL of recombinant WNT3a for 18 hours. All data is mean of three independent experiments ± SD.</p>", "links"=>[], "tags"=>["knockdown", "mrp", "leads", "adherens"], "article_id"=>546231, "categories"=>["Cancer", "Cell Biology", "Plant Biology"], "users"=>["Alexander E. Finlayson", "Kevin W. Freeman"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0007833.g004", "stats"=>{"downloads"=>1, "page_views"=>4, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Targeted_knockdown_of_MRP_leads_to_establishment_of_adherens_junctions_/546231", "title"=>"Targeted knockdown of MRP leads to establishment of adherens junctions.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2009-11-18 01:43:51"}
  • {"files"=>["https://ndownloader.figshare.com/files/434300"], "description"=>"<div><p>Invasion through the extracellular matrix (ECM) is important for wound healing, immunological responses and metastasis. We established an invasion-based cell motility screen using Boyden chambers overlaid with Matrigel to select for pro-invasive genes. By this method we identified antisense to MARCKS related protein (MRP), whose family member MARCKS is a target of miR-21, a microRNA involved in tumor growth, invasion and metastasis in multiple human cancers. We confirmed that targeted knockdown of MRP, in both EpRas mammary epithelial cells and PC3 prostate cancer cells, promoted <em>in vitro</em> cell migration that was blocked by trifluoperazine. Additionally, we observed increased immunofluoresence of E-cadherin, β-catenin and APC at sites of cell-cell contact in EpRas cells with MRP knockdown suggesting formation of adherens junctions. By wound healing assay we observed that reduced MRP supported collective cell migration, a type of cell movement where adherens junctions are maintained. However, destabilized adherens junctions, like those seen in EpRas cells, are frequently important for oncogenic signaling. Consequently, knockdown of MRP in EpRas caused loss of tumorigenesis <em>in vivo</em>, and reduced Wnt3a induced TCF reporter signaling <em>in vitro</em>. Together our data suggest that reducing MRP expression promotes formation of adherens junctions in EpRas cells, allowing collective cell migration, but interferes with oncogenic β-catenin signaling and tumorigenesis.</p></div>", "links"=>[], "tags"=>["motility", "reveals", "marcks-related", "adherens", "junction", "tumorigenesis"], "article_id"=>145688, "categories"=>["Cancer", "Cell Biology"], "users"=>["Alexander E. Finlayson", "Kevin W. Freeman"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0007833", "stats"=>{"downloads"=>1, "page_views"=>38, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/A_Cell_Motility_Screen_Reveals_Role_for_MARCKS_Related_Protein_in_Adherens_Junction_Formation_and_Tumorigenesis/145688", "title"=>"A Cell Motility Screen Reveals Role for MARCKS-Related Protein in Adherens Junction Formation and Tumorigenesis", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2009-11-18 01:34:48"}
  • {"files"=>["https://ndownloader.figshare.com/files/875565"], "description"=>"<p>a) Parental EpRas cells, C1, E13 and E14 were assayed for transwell migration with or without chemical inhibitors (Ly294002 50 µM, Bis 1 µM, CSA 10 ug/mL, TFP 50 µM, PMA 100 ng/mL). The number of cells that migrated to the bottom of the transwell after 16 hours were counted and normalized to control, * is p<0.05 b) Indicated human cell lines were tranfected with shRNA to GFP (C) or two different shRNAs (1,2) to human MRP (Open Biosystems) and analyzed by transwell assay, * is p<0.01. c,d) MRP mRNA levels were analyzed by qt-PCR and normalized to actin after transfection with control shRNA or shRNAs to human MRP (1,2) (c) or basal MRP mRNA levels (d). e) PC3 cells were transfected with shRNA1 or 2 and analyzed as in (a), * is p<0.05. All data is mean of three independent experiments ± SD.</p>", "links"=>[], "tags"=>["mrp", "targeted", "knockdown", "blocked"], "article_id"=>546025, "categories"=>["Cancer", "Cell Biology", "Plant Biology"], "users"=>["Alexander E. Finlayson", "Kevin W. Freeman"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0007833.g003", "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Effects_of_MRP_targeted_knockdown_on_migration_is_blocked_by_trifluoperazine_/546025", "title"=>"Effects of MRP targeted knockdown on migration is blocked by trifluoperazine.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2009-11-18 01:40:25"}
  • {"files"=>["https://ndownloader.figshare.com/files/875427"], "description"=>"<p>a) Parental EpRas cells, clonal cell lines stably transfected with control shRNA (C1) or with shRNA (Open Biosystems) directed to mouse MRP (E13, E14). a) and left panel of b) Quantified MRP mRNA expression as assessed by Northern blot analysis and normalized to GAPDH (Northern shown). Right panel of b) The cell lines were assayed for transwell migration and normalized to EpRas, * is p<0.01 c) Confluent cells of C1 and E13 were assayed for migration into wound at 0, 6, and 18 hours after wounding. Note migration from left grid line as reference. Representative of three experiments.</p>", "links"=>[], "tags"=>["knockdown", "mrp", "epras"], "article_id"=>545884, "categories"=>["Cancer", "Cell Biology", "Plant Biology"], "users"=>["Alexander E. Finlayson", "Kevin W. Freeman"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0007833.g002", "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Targeted_knockdown_of_MRP_in_EpRas_promotes_migration_/545884", "title"=>"Targeted knockdown of MRP in EpRas promotes migration.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2009-11-18 01:38:04"}

PMC Usage Stats | Further Information

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