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Oestrogen Receptor-Alpha Regulates Non-Canonical Hedgehog-Signalling in the Mammary Gland

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Oestrogen Receptor-Alpha Regulates Non-Canonical Hedgehog-Signalling in the Mammary Gland Oestrogen Receptor-alpha Regulates Non-Canonical Hedgehog-Signalling in the Mammary Gland by Nadia Okolowsky A thesis submitted in conformity with the requirements for the degree of Master of Science Graduate Department of Laboratory Medicine & Pathobiology University of Toronto © Copyright by Nadia Okolowsky 2014 Oestrogen Receptor-alpha Regulates Non-Canonical Hedgehog-Signalling in the Mammary Gland Nadia Okolowsky Master of Science Department of Laboratory Medicine& Pathobiology University of Toronto 2014 Abstract Mesenchymal dysplasia (mes) mice harbour a truncation in the C-terminal region of the Hedgehog (Hh)-ligand receptor, Patched-1 (Ptch1) and display a block to mammary ductal elonga- tion at puberty. Our lab previously demonstrated that epithelial cell-directed expression of activat- ed c-src rescued this block to mammary development and induced estrogen receptor-alpha (ERα) expression. Using a genetic approach where a conditional allele of ERα was expressed on the mes background, we demonstrated that restricted expression of ERα also rescues mes mammary morphogenesis with similar kinetics as the MMTV-c-srcAct mice. We further demonstrated distinct cell-type specific canonical Hh signalling in primary mammary epithelial and mesenchymal cells, and identified a novel Erk1/2 activating Hh signalling system that requires the activities of c-src and ERα, but not Smoothened (smo). These data reveal a novel Hh-signalling cascade operating through c-src and ERα that is required for mammary gland morphogenesis at puberty. ii Acknolwedgements I would like to thank my supervisor, Professor Paul A. Hamel for offering me the opportunity to pursue graduate studies in his lab, as well as for all of the guidence and support he has given me over these last three years. I would also like to thank my advisory committee, Dr. Chi-Chung Hui and Dr. Rama Khokha for their insights and suggestions, as well as, Dr. Maria Rozakis-Adcock for participating in my thesis defense. I would also like to thank the members of the Hamel lab, Jennifer Lee, Andrew Fleet, my “duck- ling” Melissa Hicuburundi, and my “lab sister” Aaliya Tamachi, as well as the former lab members Dr. Hong Chang and Malcolm Harvey. Thank you for your friendship, kindness and collaboration. Most importantly, I would like to thank my family - Neil, Leanne, Mom and Dad - thank you for all your love and support. iii Table of Contents ABSTRACT.....................................................................................................................................ii ACKNOWLEDGEMENTS............................................................................................................iii TABLE OF CONTENTS................................................................................................................iv LIST OF FIGURES........................................................................................................................vi ABBREVIATION KEY................................................................................................................viii INTRODUCTION...........................................................................................................................1 1 ESTROGEN RECEPTOR-ALPHA..................................................................................1 1.1 ESR1 Gene and Regulation.....................................................................................2 1.2 Synthetic Ligands - SERMs and SERDs.................................................................3 1.3 ERα Structure and Function....................................................................................3 1.3.1 DNA Binding Domain.....................................................................................5 1.3.2 Ligand Binding Domain..................................................................................6 1.3.2.1 Recruitment of Co-Activators..........................................................6 1.3.2.2 Recruitment of Co-Repressors.........................................................8 1.3.2.3 Discrimination Between Agonists/Antagonists................................9 1.3.3 Activation Functions (AF-1 and AF-2)...............................................................10 2 ERα-MEDIATED SIGNAL TRANSDUCTION.............................................................12 2.1 Genomic Signalling...............................................................................................12 2.2 Non-Genomic Signalling.......................................................................................14 2.3 Reciprocal interactions of c-src and ERα..............................................................17 3 HEDGEHOG SIGNALLING..........................................................................................18 3.1 Canonical Hedgehog Signalling............................................................................19 3.1.1 Ligand Synthesis................................................................................19 3.1.2 Ligand Secretion................................................................................20 3.1.3 Ligand Reception...............................................................................21 3.1.4 Ligand Signal Transduction...............................................................22 3.1.5 Primary Cilium..................................................................................26 3.2 Non-Canonical Hedgehog Signalling....................................................................27 4 MAMMARY GLAND DEVELOPMENT.......................................................................30 iv 4.1 ERα Signalling in Mammary Gland Development...............................................33 4.2 Hedgehog Signalling in Mammary Gland Development.....................................34 RATIONALE.................................................................................................................................39 HYPOTHESIS & OBJECTIVES...................................................................................................39 MATERIALS AND METHODS.....................................................................................................41 Cell Culture........................................................................................................................41 Mice...................................................................................................................................41 Whole mount analysis.........................................................................................................42 Immunofluorescence...........................................................................................................42 Primary cell culture............................................................................................................43 Erk1/2 activation assays.....................................................................................................44 Preparation of Shh-conditioned media...............................................................................44 Luciferase Assays...............................................................................................................45 Western blotting..................................................................................................................45 qRTPCR.............................................................................................................................46 RESULTS.......................................................................................................................................47 Forced ERα expression rescues the mes mammary phenotype..........................................47 Forced ERα expression induces a proliferative state in the adult mammary gland...........53 Hh signalling in primary mouse mammary epithelial cells and fibroblasts.......................57 DISCUSSION................................................................................................................................61 Forced ERα expression rescues mes mammary gland development..................................61 ERα expression drives proliferation in adult mammary glands.........................................64 Epithelial-ERα stimulates stromal activation.....................................................................66 Canonical Hh signalling in primary mammary epithelial cells and fibroblasts..................67 Non-Canonical Hh signalling in primary mammary epithelial cells..................................69 Conclusions.........................................................................................................................70 REFERENCES ..............................................................................................................................72 v List of Figures FIGURE 1. THE STRUCTURE OF ERα........................................................................................4 FIGURE 2. SCHEMATIC OF PHOSPHORYLATION SITES OF ERα.......................................11 FIGURE 3. CANONICAL HH SIGNALLING PATHWAY...........................................................23 FIGURE 4. NON-CANONICAL HEDGEHOG SIGNALLING...................................................29 FIGURE 5. PUBESCENT MAMMARY GLAND DEVELOPMENT..........................................32 FIGURE 6. ERα ACTS GENETICALLY DOWNSTREAM OF PTCH1 IN MAMMARY GLAND DEVELOPMENT............................................................................................................39
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