Investigation of the role of prolactin in mammary gland development and carcinogenesis. Samantha Richelle Oakes A thesis submitted for the Degree of Doctor of Philosophy in the Faculty of Medicine at the University of New South Wales. December 2006 ORIGINALITY STATEMENT ‘I hereby declare that this submission is my own work and to the best of my knowledge it contains no materials previously published or written by another person, or substantial proportions of material which have been accepted for the award of any other degree or diploma at UNSW or any other educational institution, except where due acknowledgement is made in the thesis. Any contribution made to the research by others, with whom I have worked at UNSW or elsewhere, is explicitly acknowledged in the thesis. I also declare that the intellectual content of this thesis is the product of my own work, except to the extent that assistance from others in the project's design and conception or in style, presentation and linguistic expression is acknowledged.’ Signed …………………………………………….............. Date …………………………………………….............. ii “I wish to record my unbounded admiration for the work of the experimenter in his struggle to wrest interpretable facts from an unyielding Nature who knows so well how to meet our theories with a decisive No-or with an inaudible Yes.” Hermann Weyl (1885-1955) Weyl H. (1931) Gruppentherie und Quantenmechanik p2 English translation: Robertson, HP. (1931) The Theory of Groups and Quantum Mechanics, p xx I couldn’t agree more! iii Acknowledgements As I sit here and reflect on the past few years, I cannot help but think about all of the people who have helped me along the way. Firstly, I must thank my supervisor, Chris Ormandy. Thank you for taking the chance on a mad motorcyclist, for knowing that it was not experience, but the ability to acquire knowledge that was important as a scientist. Over the past six years, with your encouragement and support, I have grown confident in my abilities as a researcher, and most important of all I have come out of it loving science more than when I began. I feel proud to have been part of your laboratory, and I could not have asked for a more compatible supervisor. I thank you for the wonderful experiences I have had over this journey. Since beginning work at the Garvan Institute, I have been humbled by working with so many incredible and fascinating people. To Roger Daly, my co-supervisor, thank you for many stimulating conversations, and your continuous support. To Jess Harris and Fi Robertson, thank you for being so welcoming when I first started and for allowing me to share your windowsill. Jess, you inspired me with your intelligence and lateral thinking, and Fi, your friendship has meant a great deal to me. To my colleague Matt Naylor, I cannot begin to thank you for all the advice and training you have given me over the course of my thesis. There may be plenty of competition between us, but I respect you in so many ways, and you push me to be a better scientist. Heres to plenty more ‘friendly’ competitions at high altitude! I also thank you for our collaboration on the secretory activation project. I thank Margaret Gardiner-Garden for her collaboration involving the statistical analysis of the work in this thesis, and to Michael Kazlauskas for his wonderful work on the Elf5 transgenic. To Prue Stanford, I treasure your advice and friendship, thank you for always being an ear to cry to. I also thank you for your collaboration with the Elf5 retroviral experiment and the immunohistochemistry. To my dear friends Heidi Hilton and Katrina Blazek, I am so privileged to have worked with you. Thank you for many wonderful scientific and non-scientific discussions, and thank you for being my stunning bridesmaids. I thank Liz Caldon for her many words of wisdom and her valuable friendship, it’s an honour working in the same lab as you. To Rhian, thank you for all of your hard work and assistance in my final year of my PhD. To Maria, Simon, Karl, Daniel, Andrea and Chehani, thank you all for being part of this journey. I also thank Sarah iv Eggleton for her amazing expertise involving immunohistochemistry. To Jay, Alex, Darren, Tillman, Nick, Gillian, Eileen, Marcello, Luke, Amanda, Ruth and everyone else in the cancer program, thank you for your help and friendship. I also would like to thank Jeffrey Green for providing the SV40T mouse, and Lewis Chodosh for the tetracycline inducible MTB mouse. Finally I have to thank my family. To Mum and Dad, I will always be thankful for your gifts to me of love, support and education. I am so proud to be your daughter and I am blessed to have you both. For this, I dedicate this thesis to Mum and Dad, as your hard work and sacrifices have contributed to my achievements, this degree being the greatest of them all. To my brother Steven, ‘bro’, thank you for your love and support, it means the world to me. Thank you also to Nanna, Pa, Greg, Collette and Vicky, you all have made me who I am today. Finally I thank my husband, Brad. I know it hasn’t been easy, but you have helped me through one of the hardest experiences in my life. For that, I will be eternally grateful. Sammy v Abbreviations -casein Casein alpha Acyl ATP citrate lyase Aldo3 Aldolase 3, C Angptl4 Angiopoietin-like 4 AP-1 Activator protein 1 -Casein casein beta bGH Bovine growth hormone BrdU 5-Bromo-2’-deoxyuridine Btn1a1 Butyrophilin, subfamily 1, member A1 Ccnd1 Cyclin D1 CD24a CD24a antigen Cdc6 Cell division cycle 6 homolog (S. cerevisiae) Cebp CCAAT/enhancer binding protein (C/EBP), beta Cebp CCAAT/enhancer binding protein (C/EBP), delta Cel Carboxyl ester lipase CFP Cleared fat pad Cidea Cell death-inducing DNA fragmentation factor, alpha subunit-like effector A Cldn3 Claudin 3 Copz1 Coatomer protein complex, subunit zeta 1 Cyp51 Cytochrome P450, family 51 -Casein Casein delta Dox Doxycycline dpc days post coitus dpp days post partum E Estrogen ECM Extracellular matrix EGF Epidermal growth factor EGFP Enhanced green fluorescent protein EGFR/ErbB/Her1 Epidermal growth factor receptor/v-erb-b2 erythroblastic leukemia viral oncogene homolog Ehf Ets homologous factor Elf3 (ESE-1) E74-like factor 3 Elf5 (ESE-2) E74-like factor 5 Elf5-/- E74-like factor 5 knockout Elf5+/- E74-like factor 5 heterozygote Elovl5 Elongation of very long chain fatty acids 5 Er Estrogen receptor ER Estrogen receptor alpha Erbb2/Her2 v-erb-b2 erythroblastic leukemia viral oncogene homolog 2/Hairy-related 2 ErbB3/Her3 v-erb-b2 erythroblastic leukemia viral oncogene homolog 3/Hairy-related 3 vi Erbb4/Her4 v-erb-b2 erythroblastic leukemia viral oncogene homolog 4/Hairy-related 4 Erg v-ets erythroblastosis virus E26 oncogene homolog Fak Focal adhesion kinase FCS Fetal calf serum Fdps Farnesyl diphosphate synthase Fos FBJ osteosarcoma oncogene Foxa1/HNF-3 Forkhead box A1 gene Fyn Fyn proto-oncogene G1p2/Isg15 ISG15 ubiquitin-like modifier Gal Galanin Gal-/- Galanin knockout Gal+/+ Galanin wildtype Gapdh Glyceraldehyde-3-phosphate dehydrogenase Gata3 GATA binding protein 3 GFP Green fluorescent protein GH Growth hormone GM-CSF Granulocyte-macrophage colony-stimulating factor Grb2 Growth factor bound protein 2 HandE Haematoxylin and Eosin HGMIN High-grade mammary intraepithelial neoplasia Id1 Inhibitor of DNA binding 1 Id2 Inhibitor of DNA binding 2 Igf2 Insulin-like growth factor 2 IKK (IKK) Kinase IkB Ink4a (Cdkn2a) Cyclin-dependent kinase inhibitor 2A IRES Internal ribosome entry site Jak2 Janus kinase 2 Jun Jun oncogene -casein Casein kappa Kcna1 Potassium voltage-gated ion channel, shaker-related subfamily 1 Lalba Lactalbumin Ldlr Lipoprotein receptor Lef1 Lymphoid enhancer binding factor 1 LGMIN Low-grade mammary intraepithelial neoplasia MapK/Erk Mitogen activate protein kinase Mcmds 2-7 Mini chromosome maintenance deficient 2-7 homolog (S. cerevisiae) MEC Mammary epithelial cell MMTV Mouse mammary tumor virus MTB (MMTV-rtTA-pA) Mouse mammary tumor virus-tetracycline-dependent transactivator-polyadenylation NF1/CTF Nuclear factor 1/CCAAT-binding transcription factor NF-B Nuclear Factor-kappa B NMU Nitrosomethylurea vii Nrg Neuregulin Oct1 Octamer-binding transcription factor-1 Orc6 Origin recognition complex, subunit 6-like Pcna Proliferating cell nuclear antigen PCR Polymerase chain reaction Pea3 Ets domain protein 3 Pg Progesterone Pgr/PR Progesterone receptor PgrA Progesterone receptor A PgrB Progesterone receptor B PI3K phosphatidylinositol 3-kinase PKB/Akt Protein kinase B PL Placental lactogen pMapK/pErk phosphorylated Mitogen activate protein kinase pPKB/pAkt phosphorylated Protein kinase B p-Prl phosphorylated Prolactin Prl Prolactin Prlr Prolactin receptor Prlr-/- Prolactin receptor knockout Prlr+/- Prolactin receptor heterozygote Prlr+/+ Prolactin receptor wildtype PTHrP Parathyroid hormone-related protein PTHrPr Parathyroid hormone-related protein receptor Raf v-raf-1 murine leukemia viral oncogene homolog 1 Rank Receptor activator of NF-kB RankL Receptor activator of NF-kB-ligand Ras Ras proto-oncogene Rfc3 Replicating factor C 3 Rfc4 (Rfc 1 4) Replicating factor C 4 Rfc5 (Rfc 1 5) Replicating factor C 5 Scd2 Stearoyl-CoA desaturase 2 Shc Src homology 2 domain containing transforming protein SHC src homology collagen siRNA small interfering RNA Slc34a2 Solute carrier family 34 (sodium phosphate), member 2 Slc39a8 Solute carrier family 39 (zinc transporter), member 8 Smad Mothers against decapentaplegic homolog Socs1 Suppressor of cytokine signalling 1 Socs2 Suppressor of cytokine signalling 2 Sox4 Sry-related gene a4