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Breast Cancer HUGL and the Role of Polarity in Breast Cancer Item Type text; Electronic Dissertation Authors Russ, Atlantis Dawn Publisher The University of Arizona. Rights Copyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author. Download date 25/09/2021 10:34:17 Link to Item http://hdl.handle.net/10150/293491 HUGL AND THE ROLE OF POLARITY IN BREAST CANCER By Atlantis Dawn Russ ________________________________________________________________________ A Dissertation Submitted to the Faculty of the GRADUATE INTERDISCIPLINARY PROGRAM IN GENETICS In Partial Fulfillment of the Requirements For the Degree of DOCTOR OF PHILOSOPHY In the Graduate College THE UNIVERSITY OF ARIZONA 2013 2 THE UNIVERSITY OF ARIZONA GRADUATE COLLEGE As members of the Dissertation Committee, we certify that we have read the dissertation prepared by Atlantis Dawn Russ entitled HUGL AND THE ROLE OF POLARITY IN BREAST CANCER and recommend that it be accepted as fulfilling the dissertation requirement for the Degree of Doctor of Philosophy Dr. Joyce Schroeder ____________________________________ Date: 03/22/2013 Dr. Daniela Zarnescu ___________________________________ Date: 03/22/2013 Dr. Tom Doetschman ___________________________________ Date: 03/22/2013 Dr. Kimberly McDermott ________________________________ Date: 03/22/2013 Final approval and acceptance of this dissertation is contingent upon the candidate’s submission of the final copies of the dissertation to the Graduate College. I hereby certify that I have read this dissertation prepared under my direction and recommend that it be accepted as fulfilling the dissertation requirement. ________________________________________________ Date: _____________ Dissertation Director: Dr. Joyce Schroeder ________________________________________________ Date: _____________ Dissertation Director: Dr. Daniela Zarnescu 3 STATEMENT BY AUTHOR This dissertation has been submitted in partial fulfillment of requirements for an advanced degree at The University of Arizona and is deposited in the University Library to be made available to borrowers under rules of the library. Brief quotations from this dissertation are allowable without special permission, provided that accurate acknowledgement of source is made. Requests for permission for extended quotation from or reproduction of this manuscript in whole or in part may be granted by the head of the major department of the Dean of the Graduate College when in his or her judgment the proposed use of the material is in the interests of scholarship. In all other instances, however, permission must be obtained from the author. SIGNED: Atlantis Dawn Russ 4 TABLE OF CONTENTS LIST OF FIGURES ............................................................................................................ 7 LIST OF TABLES .............................................................................................................. 9 ABSTRACT ...................................................................................................................... 10 I. INTRODUCTION ........................................................................................................ 12 Lethal Giant Larvae ....................................................................................................... 13 Discovery of Lgl.................................................................................................... 13 Major polarity protein complexes ......................................................................... 15 Lgl orthologs in fly, mouse, and human ............................................................... 18 Drosophila (lgl) ................................................................................................. 18 Mouse (Mgl1 and Mgl2) ................................................................................... 18 Human (HUGL1 and HUGL2) .......................................................................... 19 Phenotypes of Lgl loss indicate varied roles ................................................................. 20 Role in apicobasal polarity .................................................................................... 20 Role in front rear polarity and migration .............................................................. 22 Role in planar cell polarity .................................................................................... 23 Role in asymmetric cell division ........................................................................... 24 Role in Hippo pathway .......................................................................................... 26 Role in miRNA pathway ...................................................................................... 29 Lgl and cancer ............................................................................................................... 32 Polarity proteins and mammalian cancer .............................................................. 32 Loss of HUGL1 and HUGL2 in human cancers ................................................... 33 HUGL1 and HUGL2 in breast cancer ........................................................................... 35 Structure of the mammary gland ........................................................................... 35 Hallmarks of cancer .............................................................................................. 36 Cancer of the breast ............................................................................................... 37 Epithelial to Mesenchymal Transition .................................................................. 38 Stem cell hypothesis .............................................................................................. 40 3-D cell culture to model polarity ......................................................................... 43 Statement of the Problem .............................................................................................. 45 II. MATERIALS AND METHODS ................................................................................ 47 Human tissue immunofluorescence .............................................................................. 48 Cell culture .................................................................................................................... 48 Plasmid transfections..................................................................................................... 49 Viral shRNA transductions ........................................................................................... 49 MTT Assays .................................................................................................................. 50 5 TABLE OF CONTENTS – CONTINUED Acinar culture and slide preparation ............................................................................. 50 Light, fluorescent, and confocal microscopy ................................................................ 51 Western blots ................................................................................................................. 52 Antibodies ..................................................................................................................... 53 Image quantification ...................................................................................................... 54 Drosophila genetics ....................................................................................................... 54 Adult wing and eye sample preparations ...................................................................... 55 Brain dissection and imaging ........................................................................................ 56 RNA extraction, microarrays, and RT-PCR validation................................................. 56 Real-Time PCR Array ................................................................................................... 58 Microarray analysis, miRNA target matching and GO-term analysis .......................... 59 Animal husbandry ......................................................................................................... 60 Mouse genotyping ......................................................................................................... 60 Whole mounts ............................................................................................................... 61 III. HUGL1 AND HUGL2 IN MAMMARY EPITHELIAL CELLS: POLARITY, PROLIFERATION, AND DIFFERENTIATION. ........................................................... 62 Abstract ......................................................................................................................... 63 Introduction ................................................................................................................... 65 Results ........................................................................................................................... 69 HUGL1 AND HUGL2 have restricted expression in breast epithelium and loss induces a mesenchymal phenotype ....................................................................... 69 HUGL1 AND HUGL2 control acinar formation in Matrigel ............................... 74 HUGL1 AND HUGL2 inhibit proliferation in normal and transformed cells ..... 79 HUGL1
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