miRNAs in control of oncogenic signaling in breast cancer cells Von der Fakultät Energie-, Verfahrens- und Biotechnik der Universität Stuttgart zur Erlangung der Würde eines Doktors der Naturwissen- schaften (Dr. rer. nat.) genehmigte Abhandlung Vorgelegt von Annabell Bischoff aus Segnitz Hauptberichter: Prof. Dr. Monilola Olayioye Mitberichter: Prof. Dr. Klaus Pfizenmaier Tag der mündlichen Prüfung: 12.11.2014 Institut für Zellbiologie und Immunologie Universität Stuttgart 2014 2 2 3 Eidesstattliche Erklärung Hiermit erkläre ich, Annabell Bischoff, dass ich die vorliegende Arbeit selbständig angefertigt habe. Es wurden nur die in der Arbeit ausdrücklich benannten Quellen und Hilfsmittel be- nutzt. Wörtlich oder sinngemäß übernommenes Gedankengut habe ich als solches kenntlich gemacht. I hereby assure that I performed this work independently without further help or other materi- als than stated. ______________________ ______________________ Datum, Ort Unterschrift 3 4 4 5 Table of content Eidesstattliche Erklärung ....................................................................................................... 3 Table of content ..................................................................................................................... 5 Abbreviations ........................................................................................................................ 9 Abstract ................................................................................................................................13 Zusammenfassung ...............................................................................................................15 1 Introduction ...................................................................................................................17 1.1 Breast Cancer ........................................................................................................17 1.2 Development of breast cancer ................................................................................17 1.3 ErbB receptors .......................................................................................................18 1.4 ErbB2-ErbB3 receptor dimer ..................................................................................20 1.5 PI3K/Akt signaling ..................................................................................................22 1.6 Ras-ERK (Extracellular Signal Regulated Kinase) and PLCγ signaling ..................25 1.7 Cell motility .............................................................................................................26 1.8 miRNAs ..................................................................................................................27 1.8.1 miRNA biogenesis ...........................................................................................27 1.8.2 miRNA targeting ..............................................................................................28 1.8.3 Nomenclature of miRNAs ................................................................................29 1.8.4 miRNA target prediction ..................................................................................30 1.8.5 miRNAs and their biological function ...............................................................31 1.8.6 miRNAs in cancer ...........................................................................................31 1.8.7 Aim of the thesis ..............................................................................................33 2 Materials and Methods ..................................................................................................35 2.1 Materials ................................................................................................................35 2.1.1 Equipment .......................................................................................................35 2.1.2 Chemicals and consumables ...........................................................................36 2.1.3 Buffers and solutions .......................................................................................37 2.1.4 Bacterial strain ................................................................................................38 2.1.5 Cell lines, cell culture medium .........................................................................38 2.1.6 Animals ...........................................................................................................39 2.1.7 Oligonucleotides ..............................................................................................39 2.1.8 Kits and enzymes ............................................................................................42 5 6 2.2 Methods .................................................................................................................42 2.2.1 Cell culture ......................................................................................................42 2.2.2 Cell transfection ..............................................................................................43 2.2.3 Screening workflow .........................................................................................43 2.2.4 In-Cell Western analysis (ICW) .......................................................................43 2.2.5 Plasmid Constructs – QuickChange Site directed PCR Mutagenesis ..............44 2.2.6 Transformation of E.coli ..................................................................................44 2.2.7 Purification of plasmid DNA (Mini-Prep) ..........................................................45 2.2.8 Preparation of plasmid DNA (Midi-Prep) ..........................................................45 2.2.9 Migration/Invasion (Transwell) Assay ..............................................................45 2.2.10 Impedance measurement ................................................................................45 2.2.11 Proliferation Assay ..........................................................................................46 2.2.12 Life cell imaging ..............................................................................................46 2.2.13 Luciferase Reporter Assay ..............................................................................46 2.2.14 Rac activity assay. ..........................................................................................46 2.2.15 Quantitative Real Time PCR ...........................................................................46 2.2.16 Cell lysis, SDS-PAGE and Western Blotting ....................................................47 2.2.17 FACS analysis .................................................................................................47 2.2.18 Immunofluorescence microscopy ....................................................................48 2.2.19 Animal experiment ..........................................................................................48 2.2.20 Target prediction analysis for miR-149 using KEGG........................................48 3 Results ..........................................................................................................................49 3.1 miRNA Screen .......................................................................................................49 3.1.1 Establishment of a screening procedure to monitor Akt activation upon HRG stimulation .....................................................................................................................49 3.1.2 miR-149 serves as a positive control for the screen targeting the ErbB3 3’UTR 51 3.1.3 Genome-wide miRNA screening for regulators of HRG-induced Akt activation 54 3.1.4 Identification of a miRNA-ErbB interaction network. ........................................58 3.1.5 Expression of miR-148b, miR-149, miR-326 and miR-520a-3p reduces ErbB3- expression and affects Erk and Akt signaling ................................................................61 3.1.6 Overexpression of miR-148b, miR-149, miR-326 and miR-520a-3p reduces the heregulin-driven proliferation. ........................................................................................63 3.2 miR-149 in breast cancer .......................................................................................65 6 7 3.2.1 Clinical data of miR-149 ..................................................................................65 3.2.3 miR-149 expression affects cell adhesion and cell spreading ..........................67 3.2.5 miR-149 expression affects Rac activity ..........................................................71 3.2.6 miR-149 affects migration and invasion also in the prostate cancer cell line PC3 73 3.2.7 miR-149 expression in vivo model ...................................................................74 4 Discussion .....................................................................................................................77 4.1 miRNA Screen .......................................................................................................77 4.1.1 Screening for miRNAs altering the ErbB/Akt pathway .....................................77 4.1.2 miRNAs alter heregulin-dependent Akt activation ............................................78 4.1.3 Protein target network of miRNAs negatively affecting ΔpAkt ..........................79 4.1.4 Protein target network of miRNAs enhancing ΔpAkt ........................................82 4.1.5 miRNAs regulate