DOCSLIB.ORG

Whole-Genome Rnai Screen Highlights Components of The

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Whole-Genome Rnai Screen Highlights Components of The Whole-genome RNAi screen highlights components of PNAS PLUS the endoplasmic reticulum/Golgi as a source of resistance to immunotoxin-mediated cytotoxicity Matteo Pasettoa,1,2, Antonella Antignania,1, Pinar Ormanoglub, Eugen Buehlerb, Rajarshi Guhab, Ira Pastana,3, Scott E. Martinb, and David J. FitzGeralda,3 aLaboratory of Molecular Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892-4264; and bDivision of Preclinical Innovation, National Center for Advancing Translational Sciences, National Institutes of Health, Rockville, MD 20850 Contributed by Ira Pastan, February 2, 2015 (sent for review October 6, 2014; reviewed by Arthur Frankel and Wayne I. Lencer) Immunotoxins (antibody–toxin fusion proteins) target surface EF2 no longer functions at the elongation step of protein trans- antigens on cancer cells and kill these cells via toxin-mediated in- lation. Death results from a combination of events: the loss of hibition of protein synthesis. To identify genes controlling this short-lived survival proteins (e.g., Mcl1), triggering apoptosis, process, an RNAi whole-genome screen (∼22,000 genes at three and stress responses that cannot be executed because protein siRNAs per gene) was conducted via monitoring the cytotoxicity of translation is shut down (21). Regarding the constituents of the the mesothelin-directed immunotoxin SS1P. SS1P, a Pseudomonas pathway responsible for transporting the immunotoxin from the exotoxin-based immunotoxin, was chosen because it is now in surface to the cytosol, there are only a handful of experimentally clinical trials and has produced objective tumor regressions in established components. These proteins include the surface patients. High and low concentrations of SS1P were chosen to target itself, the protease furin, and KDELR2 (discussed below). allow for the identification of both mitigators and sensitizers. As In addition, within the cytosol, the diphthamide pathway re- expected, silencing known essential genes in the immunotoxin sponsible for the multistep posttranslational modification of EF2, pathway, such as mesothelin, furin, KDEL receptor 2, or members converting histidine 715 to diphthamide, is necessary (22, 23). of the diphthamide pathway, protected cells. Of greater interest Beyond these components, the total number of constituents is was the observation that many RNAi targets increased immuno- unknown, although it is under study (24). Silencing of genes MEDICAL SCIENCES toxin sensitivity, indicating that these gene products normally con- associated with the immunotoxin pathway would likely change tribute to inefficiencies in the killing pathway. Of the top sen- cell sensitivity. Furthermore, should these gene products be ame- sitizers, many genes encode proteins that locate to either the nable to regulation via small molecular drugs, alterations of killing endoplasmic reticulum (ER) or Golgi and are annotated as part of activity might be possible via chemical intervention (25, 26). the secretory system. Genes related to the ER-associated degrada- Mesothelin is expressed on mesotheliomas, as well as on other tion system were not among high-ranking mitigator or sensitizer epithelial cancers (27–32). The immunotoxin SS1P was designed candidates. However, the p97 inhibitor eeyarestatin 1 enhanced immunotoxin killing. Our results highlight potential targets for to kill cells displaying this surface protein and is currently under chemical intervention that could increase immunotoxin killing of clinical evaluation. Of note, recent results from a phase 1 trial cancer cells and enhance our understanding of toxin trafficking. combining SS1P with pemetrexed and cisplatin reported a 60% RNAi | toxin | immunotoxin | screen | genome Significance ntibody-based cancer therapeutics are designed to kill target To increase understanding of how antibody–toxin fusion pro- Acells, ideally causing little damage to normal cells. These teins (immunotoxins) kill cells, we used RNAi, striving to reduce molecules extend from intact IgG molecules through antibody– the expression level of all human genes. Some RNAi treatments drug conjugates to antibody–toxin fusions (1–4). When antibodies resulted in resistance to immunotoxins and some caused in- are tasked with delivering radionuclides or T cells, surface binding creased sensitivity. We focused on target genes that caused is usually sufficient. However, when delivering toxic payloads, cells to be more immunotoxin-sensitive. Results highlight antibody internalization to specific intracellular pathways plays genes that naturally protect cells from the action of immuno- a critical role in determining cellular susceptibility (5–8). toxins. Of interest, many of these genes encode proteins that Immunotoxin design includes an antibody fragment, either Fv reside in the endoplasmic reticulum and Golgi, and are part of or Fab, fused with a protein toxin (4, 9–11). The antibody frag- the cell’s secretion system. Should these gene products be ment binds a surface antigen leading to internalization followed amenable to regulation via small molecular drugs, enhance- by cleavage via the cellular protease, furin, which begins sepa- ment of killing might be possible via chemical intervention. rating the toxin from the antibody Fv (12, 13). Separation is completed by a reduction step that generates a C-terminal toxin Author contributions: A.A., I.P., S.E.M., and D.J.F. designed research; M.P., A.A., and P.O. performed research; P.O., E.B., R.G., S.E.M., and D.J.F. analyzed data; and A.A., I.P., S.E.M., fragment of 35 kDa (14). This fragment contains a KDEL-like and D.J.F. wrote the paper. sequence at the C terminus, which is necessary for cell killing Reviewers: A.F., UT Southwestern; and W.I.L., Harvard Medical School. (15). The need for a KDEL-like sequence implicates the endo- The authors declare no conflict of interest. plasmic reticulum (ER) as an essential organelle in the toxin Data deposition: The sequence reported in this paper has been deposited in the PubChem pathway and suggests that the toxin travels to the ER via a ret- database (assay ID 1117281). rograde pathway. KDEL-receptor 2 (KDELR2) has been shown 1M.P. and A.A. contributed equally to this work. to interact with Pseudomonas exotoxin (PE)-derived immuno- 2Present address: Department of Pathology and Diagnosis, Ospedale GB Rossi, 37134 toxins (16). From the ER, the C-terminal toxin fragment trans- Verona, Italy. locates to the cell cytosol, where it ADP-ribosylates elongation 3To whom correspondence may be addressed. Email: [email protected] or fitzgerd@ factor 2 (EF2). Only EF2 that is modified posttranslationally by helix.nih.gov. a multistep diphthamide pathway is susceptible to toxin-mediated This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. ADP ribosylation (17–20). Cells succumb because ADP-ribosylated 1073/pnas.1501958112/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1501958112 PNAS Early Edition | 1of8 Downloaded by guest on October 1, 2021 response rate in patients with pleural mesothelioma and a 77% Table 2. Top 24 ranked genes from the genome-wide screen for response rate at the maximum tolerated dose (33). Mesothelin is sensitizers expressed on KB cells at about 10,000 copies per cell, rendering RSA rank Symbol Gene_ID RSA rank Symbol Gene_ID these cells moderately sensitive to the SS1P immunotoxin with an IC50 of 10–20 ng/mL. 1 RPL10A 4736 13 CD48 962 RNAi results in the loss of mRNA from target genes (34). To 2 SLC33A1 9197 14 PKDREJ 10343 identify genes involved in immunotoxin-mediated killing, we 3 HDDC3 374659 15 hCG_31916 653702 undertook a whole-genome screen where three siRNAs per gene 4 RPL38 6169 16 RPL24 6152 were added before the addition of a high or low concentration of 5 BICC1 80114 17 EIF3I 8668 immunotoxin. Genes were scored as “mitigators” or “sensitizers” 6 COPB1 1315 18 RBM13 84549 based on their protection from or enhancement of immunotoxin 7 ZNF37A 7587 19 CPEB4 80315 action. To validate selected targets, additional siRNAs were 8 FUCA1 2517 20 STX19 415117 tested via dose–response analysis of immunotoxin action. Here, 9 RPLP1 6176 21 CHIT1 1118 we confirm the roles of essential genes in the immunotoxin 10 DYNLT1 6993 22 HTR4 3360 pathway and identify previously unknown members of the toxin 11 ARL1 400 23 LOC388532 388532 pathway, notably those members that usually protect cells from 12 COPE 11316 24 ARF4 378 immunotoxin action but, when reduced in expression, lead to the sensitization of cells. most significant mitigator genes determined by Haystack (Dataset Results S3). Also among the top candidates were genes that could rea- Validation of Initial RNAi Screen. To identify genes involved in sonably be expected to participate in the immunotoxin pathway. immunotoxin intoxication, a whole-genome RNAi screen was These genes were components of the endocytic pathway, in- performed on KB cells that express surface mesothelin and are cluding the clathrin coat protein AP2M1 and dynamin 2. Addi- moderately susceptible to the immunotoxin SS1P. Cells were tional members of the clathrin coat were also ranked highly transfected with three siRNAs corresponding to each of ∼22,000 (RSA: P < 0.001), including AP2A1 and AP2S1 (Dataset S1). human genes and then treated with either a “high” concentration Proapoptotic genes were also among the top candidates, in- of immunotoxin, where the silencing of essential genes would cluding a top RSA-ranked candidate, BAK1, and Haystack “
Load more

Recommended publications
  • Kids First Pediatric Research Program (Kids First) Poster Session at ASHG Accelerating Pediatric Genomics Research Through Collaboration October 15Th, 2019
    The Gabriella Miller Kids First Pediatric Research Program (Kids First) Poster Session at ASHG Accelerating Pediatric Genomics Research through Collaboration October 15th, 2019 Background The Gabriella Miller Kids First Pediatric Research Program (Kids First) is a trans- NIH Common Fund program initiated in response to the 2014 Gabriella Miller Kids First Research Act. The program’s vision is to alleviate suffering from childhood cancer and structural birth defects by fostering collaborative research to uncover the etiology of these diseases and support data sharing within the pediatric research community. This is implemented through developing the Gabriella Miller Kids First Data Resource (Kids First Data Resource) and populating this resource with whole genome sequence datasets and associated clinical and phenotypic information. Both childhood cancers and structural birth defects are critical and costly conditions associated with substantial morbidity and mortality. Elucidating the underlying genetic etiology of these diseases has the potential to profoundly improve preventative measures, diagnostics, and therapeutic interventions. Purpose During this evening poster session, attendees will gain a broad understanding of the utility of the genomic data generated by Kids First, learn about the progress of Kids First X01 cohort projects, and observe demonstrations of the tools and functionalities of the recently launched Kids First Data Resource Portal. The session is an opportunity for the scientific community and public to engage with Kids First investigators, collaborators, and a growing community of researchers, patient foundations, and families. Several other NIH and external data efforts will present posters and be available to discuss collaboration opportunities as we work together to accelerate pediatric research.
    [Show full text]
  • The Genomic Landscape of Pancreatic and Periampullary Adenocarcinoma Vandana Sandhu1,2, David C
    Published OnlineFirst August 3, 2016; DOI: 10.1158/0008-5472.CAN-16-0658 Cancer Molecular and Cellular Pathobiology Research The Genomic Landscape of Pancreatic and Periampullary Adenocarcinoma Vandana Sandhu1,2, David C. Wedge3,4, Inger Marie Bowitz Lothe1,5, Knut Jørgen Labori6, Stefan C. Dentro3,4,Trond Buanes6,7, Martina L. Skrede1, Astrid M. Dalsgaard1, Else Munthe8, Ola Myklebost8, Ole Christian Lingjærde9, Anne-Lise Børresen-Dale1,7, Tone Ikdahl10,11, Peter Van Loo12,13, Silje Nord1, and Elin H. Kure1,2 Abstract Despite advances in diagnostics, less than 5% of patients with and Wnt signaling. By integrating genomics and transcrip- periampullary tumors experience an overall survival of five years tomics data from the same patients, we identified CCNE1 and or more. Periampullary tumors are neoplasms that arise in the ERBB2 as candidate driver genes. Morphologic subtypes of vicinity of the ampulla of Vater, an enlargement of liver and periampullary adenocarcinomas (i.e., pancreatobiliary or intes- pancreas ducts where they join and enter the small intestine. In tinal) harbor many common genomic aberrations. However, this study, we analyzed copy number aberrations using Affymetrix gain of 13q and 3q, and deletions of 5q were found specificto SNP 6.0 arrays in 60 periampullary adenocarcinomas from Oslo the intestinal subtype. Our study also implicated the use of the University Hospital to identify genome-wide copy number aber- PAM50 classifier in identifying a subgroup of patients with a rations, putative driver genes, deregulated pathways, and poten- high proliferation rate, which had impaired survival. Further- tial prognostic markers. Results were validated in a separate cohort more, gain of 18p11 (18p11.21-23, 18p11.31-32) and 19q13 derived from The Cancer Genome Atlas Consortium (n ¼ 127).
    [Show full text]
  • Insights Into Diphthamide, Key Diphtheria Toxin Effector
    Toxins 2013, 5, 958-968; doi:10.3390/toxins5050958 OPEN ACCESS toxins ISSN 2072-6651 www.mdpi.com/journal/toxins Brief Report Insights into Diphthamide, Key Diphtheria Toxin Effector Wael Abdel-Fattah 1,†, Viktor Scheidt 1,†, Shanow Uthman 2, Michael J. R. Stark 3 and Raffael Schaffrath 1,2,* 1 Institut für Biologie, FG Mikrobiologie, Universität Kassel, Kassel D-34132, Germany; E-Mails: [email protected] (W.A.-F.); [email protected] (V.S.) 2 Department of Genetics, University of Leicester, Leicester LE1 7RH, UK; E-Mail: [email protected] 3 Centre for Gene Regulation & Expression, University of Dundee, Dundee, DD1 5EH, Scotland; E-Mail: [email protected] † These authors contributed equally to this work. * Author to whom correspondence should be addressed; E-Mail: [email protected]; Tel.: +49-561-804-4175; Fax: +49-561-804-4337. Received: 14 March 2013; in revised form: 17 April 2013 / Accepted: 26 April 2013 / Published: 3 May 2013 Abstract: Diphtheria toxin (DT) inhibits eukaryotic translation elongation factor 2 (eEF2) by ADP-ribosylation in a fashion that requires diphthamide, a modified histidine residue on eEF2. In budding yeast, diphthamide formation involves seven genes, DPH1-DPH7. In an effort to further study diphthamide synthesis and interrelation among the Dph proteins, we found, by expression in E. coli and co-immune precipitation in yeast, that Dph1 and Dph2 interact and that they form a complex with Dph3. Protein-protein interaction mapping shows that Dph1-Dph3 complex formation can be dissected by progressive DPH1 gene truncations. This identifies N- and C-terminal domains on Dph1 that are crucial for diphthamide synthesis, DT action and cytotoxicity of sordarin, another microbial eEF2 inhibitor.
    [Show full text]
  • Building the Vertebrate Codex Using the Gene Breaking Protein Trap Library
    Genetics, Development and Cell Biology Publications Genetics, Development and Cell Biology 2020 Building the vertebrate codex using the gene breaking protein trap library Noriko Ichino Mayo Clinic Gaurav K. Varshney National Institutes of Health Ying Wang Iowa State University Hsin-kai Liao Iowa State University Maura McGrail Iowa State University, [email protected] See next page for additional authors Follow this and additional works at: https://lib.dr.iastate.edu/gdcb_las_pubs Part of the Molecular Genetics Commons, and the Research Methods in Life Sciences Commons The complete bibliographic information for this item can be found at https://lib.dr.iastate.edu/ gdcb_las_pubs/261. For information on how to cite this item, please visit http://lib.dr.iastate.edu/howtocite.html. This Article is brought to you for free and open access by the Genetics, Development and Cell Biology at Iowa State University Digital Repository. It has been accepted for inclusion in Genetics, Development and Cell Biology Publications by an authorized administrator of Iowa State University Digital Repository. For more information, please contact [email protected]. Building the vertebrate codex using the gene breaking protein trap library Abstract One key bottleneck in understanding the human genome is the relative under-characterization of 90% of protein coding regions. We report a collection of 1200 transgenic zebrafish strains made with the gene- break transposon (GBT) protein trap to simultaneously report and reversibly knockdown the tagged genes. Protein trap-associated mRFP expression shows previously undocumented expression of 35% and 90% of cloned genes at 2 and 4 days post-fertilization, respectively. Further, investigated alleles regularly show 99% gene-specific mRNA knockdown.
    [Show full text]
  • Variation in Protein Coding Genes Identifies Information
    bioRxiv preprint doi: https://doi.org/10.1101/679456; this version posted June 21, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. Animal complexity and information flow 1 1 2 3 4 5 Variation in protein coding genes identifies information flow as a contributor to 6 animal complexity 7 8 Jack Dean, Daniela Lopes Cardoso and Colin Sharpe* 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 Institute of Biological and Biomedical Sciences 25 School of Biological Science 26 University of Portsmouth, 27 Portsmouth, UK 28 PO16 7YH 29 30 * Author for correspondence 31 [email protected] 32 33 Orcid numbers: 34 DLC: 0000-0003-2683-1745 35 CS: 0000-0002-5022-0840 36 37 38 39 40 41 42 43 44 45 46 47 48 49 Abstract bioRxiv preprint doi: https://doi.org/10.1101/679456; this version posted June 21, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. Animal complexity and information flow 2 1 Across the metazoans there is a trend towards greater organismal complexity. How 2 complexity is generated, however, is uncertain. Since C.elegans and humans have 3 approximately the same number of genes, the explanation will depend on how genes are 4 used, rather than their absolute number.
    [Show full text]
  • Supplementary Materials
    Supplementary materials Supplementary Table S1: MGNC compound library Ingredien Molecule Caco- Mol ID MW AlogP OB (%) BBB DL FASA- HL t Name Name 2 shengdi MOL012254 campesterol 400.8 7.63 37.58 1.34 0.98 0.7 0.21 20.2 shengdi MOL000519 coniferin 314.4 3.16 31.11 0.42 -0.2 0.3 0.27 74.6 beta- shengdi MOL000359 414.8 8.08 36.91 1.32 0.99 0.8 0.23 20.2 sitosterol pachymic shengdi MOL000289 528.9 6.54 33.63 0.1 -0.6 0.8 0 9.27 acid Poricoic acid shengdi MOL000291 484.7 5.64 30.52 -0.08 -0.9 0.8 0 8.67 B Chrysanthem shengdi MOL004492 585 8.24 38.72 0.51 -1 0.6 0.3 17.5 axanthin 20- shengdi MOL011455 Hexadecano 418.6 1.91 32.7 -0.24 -0.4 0.7 0.29 104 ylingenol huanglian MOL001454 berberine 336.4 3.45 36.86 1.24 0.57 0.8 0.19 6.57 huanglian MOL013352 Obacunone 454.6 2.68 43.29 0.01 -0.4 0.8 0.31 -13 huanglian MOL002894 berberrubine 322.4 3.2 35.74 1.07 0.17 0.7 0.24 6.46 huanglian MOL002897 epiberberine 336.4 3.45 43.09 1.17 0.4 0.8 0.19 6.1 huanglian MOL002903 (R)-Canadine 339.4 3.4 55.37 1.04 0.57 0.8 0.2 6.41 huanglian MOL002904 Berlambine 351.4 2.49 36.68 0.97 0.17 0.8 0.28 7.33 Corchorosid huanglian MOL002907 404.6 1.34 105 -0.91 -1.3 0.8 0.29 6.68 e A_qt Magnogrand huanglian MOL000622 266.4 1.18 63.71 0.02 -0.2 0.2 0.3 3.17 iolide huanglian MOL000762 Palmidin A 510.5 4.52 35.36 -0.38 -1.5 0.7 0.39 33.2 huanglian MOL000785 palmatine 352.4 3.65 64.6 1.33 0.37 0.7 0.13 2.25 huanglian MOL000098 quercetin 302.3 1.5 46.43 0.05 -0.8 0.3 0.38 14.4 huanglian MOL001458 coptisine 320.3 3.25 30.67 1.21 0.32 0.9 0.26 9.33 huanglian MOL002668 Worenine
    [Show full text]
  • Novel and Highly Recurrent Chromosomal Alterations in Se´Zary Syndrome
    Research Article Novel and Highly Recurrent Chromosomal Alterations in Se´zary Syndrome Maarten H. Vermeer,1 Remco van Doorn,1 Remco Dijkman,1 Xin Mao,3 Sean Whittaker,3 Pieter C. van Voorst Vader,4 Marie-Jeanne P. Gerritsen,5 Marie-Louise Geerts,6 Sylke Gellrich,7 Ola So¨derberg,8 Karl-Johan Leuchowius,8 Ulf Landegren,8 Jacoba J. Out-Luiting,1 Jeroen Knijnenburg,2 Marije IJszenga,2 Karoly Szuhai,2 Rein Willemze,1 and Cornelis P. Tensen1 Departments of 1Dermatology and 2Molecular Cell Biology, Leiden University Medical Center, Leiden, the Netherlands; 3Department of Dermatology, St Thomas’ Hospital, King’s College, London, United Kingdom; 4Department of Dermatology, University Medical Center Groningen, Groningen, the Netherlands; 5Department of Dermatology, Radboud University Nijmegen Medical Center, Nijmegen, the Netherlands; 6Department of Dermatology, Gent University Hospital, Gent, Belgium; 7Department of Dermatology, Charite, Berlin, Germany; and 8Department of Genetics and Pathology, Rudbeck Laboratory, University of Uppsala, Uppsala, Sweden Abstract Introduction This study was designed to identify highly recurrent genetic Se´zary syndrome (Sz) is an aggressive type of cutaneous T-cell alterations typical of Se´zary syndrome (Sz), an aggressive lymphoma/leukemia of skin-homing, CD4+ memory T cells and is cutaneous T-cell lymphoma/leukemia, possibly revealing characterized by erythroderma, generalized lymphadenopathy, and pathogenetic mechanisms and novel therapeutic targets. the presence of neoplastic T cells (Se´zary cells) in the skin, lymph High-resolution array-based comparative genomic hybridiza- nodes, and peripheral blood (1). Sz has a poor prognosis, with a tion was done on malignant T cells from 20 patients. disease-specific 5-year survival of f24% (1).
    [Show full text]
  • A Genome-Wide Screen in Mice to Identify Cell-Extrinsic Regulators of Pulmonary Metastatic Colonisation
    FEATURED ARTICLE MUTANT SCREEN REPORT A Genome-Wide Screen in Mice To Identify Cell-Extrinsic Regulators of Pulmonary Metastatic Colonisation Louise van der Weyden,1 Agnieszka Swiatkowska, Vivek Iyer, Anneliese O. Speak, and David J. Adams Wellcome Sanger Institute, Wellcome Genome Campus, Hinxton, Cambridge, CB10 1SA, United Kingdom ORCID IDs: 0000-0002-0645-1879 (L.v.d.W.); 0000-0003-4890-4685 (A.O.S.); 0000-0001-9490-0306 (D.J.A.) ABSTRACT Metastatic colonization, whereby a disseminated tumor cell is able to survive and proliferate at a KEYWORDS secondary site, involves both tumor cell-intrinsic and -extrinsic factors. To identify tumor cell-extrinsic metastasis (microenvironmental) factors that regulate the ability of metastatic tumor cells to effectively colonize a metastatic tissue, we performed a genome-wide screen utilizing the experimental metastasis assay on mutant mice. colonisation Mutant and wildtype (control) mice were tail vein-dosed with murine metastatic melanoma B16-F10 cells and microenvironment 10 days later the number of pulmonary metastatic colonies were counted. Of the 1,300 genes/genetic B16-F10 locations (1,344 alleles) assessed in the screen 34 genes were determined to significantly regulate pulmonary lung metastatic colonization (15 increased and 19 decreased; P , 0.005 and genotype effect ,-55 or .+55). mutant While several of these genes have known roles in immune system regulation (Bach2, Cyba, Cybb, Cybc1, Id2, mouse Igh-6, Irf1, Irf7, Ncf1, Ncf2, Ncf4 and Pik3cg) most are involved in a disparate range of biological processes, ranging from ubiquitination (Herc1) to diphthamide synthesis (Dph6) to Rho GTPase-activation (Arhgap30 and Fgd4), with no previous reports of a role in the regulation of metastasis.
    [Show full text]
  • Chromosome 17Q25 Genes, RHBDF2 and CYGB, in Ovarian Cancer
    INTERNATIONAL JOURNAL OF ONCOLOGY 40: 1865-1880, 2012 Chromosome 17q25 genes, RHBDF2 and CYGB, in ovarian cancer PAULINA M. WOJNAROWICZ1, DIANE M. PROVENCHER2-4, ANNE-MARIE MES-MASSON2,5 and PATRICIA N. TONIN1,6,7 1Department of Human Genetics, McGill University; 2Research Centre of the University of Montreal Hospital Centre (CRCHUM)/Montreal Cancer Institute; 3Division of Gynecologic Oncology, University of Montreal; 4Department of Obstetrics and Gynecology, University of Montreal; 5Department of Medicine, University of Montreal; 6The Research Institute of the McGill University Health Centre; 7Department of Medicine, McGill University, Montreal, Quebec, Canada Received November 15, 2011; Accepted December 29, 2011 DOI: 10.3892/ijo.2012.1371 Abstract. It has been proposed that the frequent loss of Introduction heterozygosity (LOH) of an entire chromosome 17 contig in epithelial ovarian cancers (EOC) is the consequence of It is well established that loss of chromosome 17 is a common the inactivation of multiple tumour suppressor genes on this occurrence in epithelial ovarian cancers (EOCs), as suggested chromosome. We report the characterization of a 453 Kb 17q25 by karyotype and loss of heterozygosity (LOH) studies (1,2). locus shown previously to exhibit a high frequency of LOH This observation, together with complementation studies in EOC samples. LOH analysis further defined the minimal involving the transfer of chromosome 17 which resulted in region of deletion to a 65 Kb interval flanked by D17S2239 reduced tumourigenicity of an EOC cell line (3), suggest that and D17S2244, which contains RHBDF2, CYGB and PRCD as this chromosome harbours tumour suppressor genes (TSGs). tumour suppressor gene candidates. Tissue specific expression Chromosome 17 contains a number of very well characterized excluded PRCD as a candidate.
    [Show full text]
  • Chemogenomic Approach Identified Yeast YLR143W As Diphthamide
    Chemogenomic approach identified yeast YLR143W as diphthamide synthetase Xiaoyang Sua, Zhewang Lina, Wei Chenb, Hong Jianga, Sheng Zhangb, and Hening Lina,1 aDepartment of Chemistry and Chemical Biology and bProteomic and Mass Spectrometry Facility, Cornell University, Ithaca, NY 14850 Edited by Wilfred A. van der Donk, Howard Hughes Medical Institute, University of Illinois at Urbana–Champaign, Urbana, IL, and accepted by the Editorial Board October 18, 2012 (received for review August 23, 2012) Many genes are of unknown functions in any sequenced genome. A Results combination of chemical and genetic perturbations has been used Cofitness Analysis Revealed That YLR143W Is Closely Related to to investigate gene functions. Here we present a case that such Diphthamide Biosynthesis. We reasoned that the strain lacking “chemogenomics” information can be effectively used to identify the unknown diphthamide synthetase gene should have high missing genes in a defined biological pathway. In particular, we cofitness values with strains lacking other diphthamide bio- identified the previously unknown enzyme diphthamide synthetase synthesis genes. To validate this approach, we first analyzed the for the last step of diphthamide biosynthesis. We found that yeast cofitness data of Δybr246w strain, which has high cofitness value protein YLR143W is the diphthamide synthetase catalyzing the last with Δdph2 (0.59624, ranked #3), Δdph4 (0.55343, ranked #7), amidation step using ammonium and ATP. Diphthamide synthetase and Δdph5 (0.57422, ranked #5) strains (Fig. 2A). DPH3 is is evolutionarily conserved in eukaryotes. The previously uncharac- a fairly small ORF (249 bp) and was not included in the original terized human gene ATPBD4 is the ortholog of yeast YLR143W and deletion strain collections.
    [Show full text]
  • Building the Vertebrate Codex Using the Gene Breaking Protein Trap Library
    TOOLS AND RESOURCES Building the vertebrate codex using the gene breaking protein trap library Noriko Ichino1, MaKayla R Serres1, Rhianna M Urban1, Mark D Urban1, Anthony J Treichel1, Kyle J Schaefbauer1, Lauren E Greif1, Gaurav K Varshney2,3, Kimberly J Skuster1, Melissa S McNulty1, Camden L Daby1, Ying Wang4, Hsin-kai Liao4, Suzan El-Rass5, Yonghe Ding1,6, Weibin Liu1,6, Jennifer L Anderson7, Mark D Wishman1, Ankit Sabharwal1, Lisa A Schimmenti1,8,9, Sridhar Sivasubbu10, Darius Balciunas11, Matthias Hammerschmidt12, Steven Arthur Farber7, Xiao-Yan Wen5, Xiaolei Xu1,6, Maura McGrail4, Jeffrey J Essner4, Shawn M Burgess2, Karl J Clark1*, Stephen C Ekker1* 1Department of Biochemistry and Molecular Biology, Mayo Clinic, Rochester, United States; 2Translational and Functional Genomics Branch, National Human Genome Research Institute, National Institutes of Health, Bethesda, United States; 3Functional & Chemical Genomics Program, Oklahoma Medical Research Foundation, Oklahoma City, United States; 4Department of Genetics, Development and Cell Biology, Iowa State University, Ames, United States; 5Zebrafish Centre for Advanced Drug Discovery & Keenan Research Centre for Biomedical Science, Li Ka Shing Knowledge Institute, St. Michael’s Hospital, Unity Health Toronto & University of Toronto, Toronto, Canada; 6Department of Cardiovascular Medicine, Mayo Clinic, Rochester, United States; 7Department of Embryology, Carnegie Institution for Science, Baltimore, United States; 8Department of Clinical Genomics, Mayo Clinic, Rochester, United States; 9Department of Otorhinolaryngology, Mayo Clinic, Rochester, United States; 10Genomics and Molecular Medicine Unit, CSIR– 11 *For correspondence: Institute of Genomics and Integrative Biology, Delhi, India; Department of [email protected] (KJC); Biology, Temple University, Philadelphia, United States; 12Institute of Zoology, [email protected] (SCE) Developmental Biology Unit, University of Cologne, Cologne, Germany Competing interests: The authors declare that no competing interests exist.
    [Show full text]
  • A Genome-Wide Screen in Mice to Identify Cell-Extrinsic Regulators of Pulmonary Metastatic Colonisation
    bioRxiv preprint doi: https://doi.org/10.1101/2020.02.10.941401; this version posted February 10, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 1 A genome-wide screen in mice to identify cell-extrinsic regulators of pulmonary 2 metastatic colonisation 3 4 5 Louise van der Weyden1*, Agnieszka Swiatkowska1, Vivek Iyer1, Anneliese O. Speak1, David J. 6 Adams1 7 8 9 1Wellcome Sanger Institute, Wellcome Genome Campus, Hinxton, Cambridge, CB10 1SA, 10 United Kingdom 11 12 13 *Corresponding author 14 Louise van der Weyden 15 Experimental Cancer Genetics 16 Wellcome Sanger Institute 17 Wellcome Genome Campus 18 Hinxton 19 Cambridge 20 CB10 1SA 21 United Kingdom 22 Tel: +44-1223-834-244 23 Email: [email protected] 24 25 26 KEYWORDS: metastasis, metastatic colonisation, microenvironment, B16-F10, lung, mutant, 27 mouse. 28 29 30 31 1 bioRxiv preprint doi: https://doi.org/10.1101/2020.02.10.941401; this version posted February 10, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 32 ABSTRACT 33 34 Metastatic colonisation, whereby a disseminated tumour cell is able to survive and 35 proliferate at a secondary site, involves both tumour cell-intrinsic and -extrinsic factors.
    [Show full text]