UNIVERSITY OF CALGARY Identification of Prolactin-Regulated Factors Contributing to Breast Cancer-Mediated Osteoclastogenesis

UNIVERSITY OF CALGARY
Identification of Prolactin-Regulated Factors Contributing to
Breast Cancer-Mediated Osteoclastogenesis
by
Amanda Nicole Forsyth
A THESIS
SUBMITTED TO THE FACULTY OF GRADUATE STUDIES
IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE
DEGREE OF MASTER OF SCIENCE
GRADUATE PROGRAM IN BIOLOGICAL SCIENCES
CALGARY, ALBERTA
SEPTEMBER, 2015
© Amanda Nicole Forsyth 2015

ii
Abstract
High serum levels of prolactin increase risk of breast cancer as well as increase
metastasis in breast cancer patients. Bone is a preferential site of breast cancer metastasis
and breast tumors are known to secrete soluble factors that enhance bone breakdown by
stimulating differentiation and maturation of osteoclasts. This project conducted gene and
cytokine arrays to elucidate the mechanism by which prolactin contributes to breast
cancer-mediated osteoclastogenesis. Here we demonstrate that sonic hedgehog is a
prolactin-regulated cytokine in breast cancer cells and is part of the mechanism that
induces osteoclast differentiation. Furthermore, sonic hedgehog is expressed more
frequently in breast invasive carcinoma tissue than normal tissue, supporting its role in
tumor progression and metastasis. This mechanism can lead to the development of novel
therapies to help alleviate osteolysis experienced by breast cancer patients.

iii
Acknowledgements
I extend my deepest appreciation to my supervisor, Dr. Carrie Shemanko, for her
support, extreme patience and infectious enthusiasm towards academic sciences and
cancer research. My experience in her laboratory has provided me the inspiration and
means to pursue a career as a clinician-scientist. To that end, I also thank the Cumming
School of Medicine Leaders in Medicine Program for allowing me to simultaneously
pursue medical school and this graduate degree. I would like to thank my committee
members, Drs. Andre Buret and Frank Jirik, for their encouragement and their ability to
show me aspects of this project that I had not yet thought of. Thank you to the
supervisors and students of the Cobb, Hansen, Habibi and Muench labs for teaching me
different scientific techniques, and for the use of your equipment to conduct them. I thank
Dr. Kyla Flanagan for imparting her knowledge of statistics to me.
Thank you to the members of the Shemanko lab, past and present, who have
provided guidance and made coming into school every day fun. To the graduate students
Anna Urbanska and Ashley Sutherland, thank you for showing me the ropes of the lab
and laying the groundwork for my project. I thank Odul Karayazi-Atici for endlessly
fielding my questions and, more importantly, for being my friend. Thank you to Lin Su
for always helping when needed and teaching me so many techniques. Ying Ying Cong,
thanks for being such a cheerful, friendly presence in the lab. I am indebted to the
undergraduate students who contributed to this project: Heather Gibling, Vanessa Lam,
Jessica Howlett, Sara Mirzai, and Laurel Grant. I thank Heather Gibling in particular for
her humour, friendship, and for putting on a copy-editor’s cap and ridding my thesis of
poor grammar.
A very special thank you is extended to my family and friends. Mom and Dad, thank
you for every step you have taken to ensure that the only thing limiting my success is
myself. Grandma and Grandpa, thank you for checking in on me to make sure I am
keeping up with “thesising”. Thank you to my talented sister Heidi Forsyth, who took
my chicken scratch diagrams and turned them into the beautiful illustrations present in
this thesis. I thank Winston for providing a furry shoulder to bury my head in after a
rough day. Finally, I thank Ryan Greig for loving me at my best and worst. I could not
have chosen a better, more supportive partner.

iv
Table of Contents
Abstract ............................................................................................................................... ii!
Acknowledgements ............................................................................................................ iii!
Table of Contents ............................................................................................................... iv!
List of Figures and Illustrations ....................................................................................... viii!
List of Symbols, Abbreviations and Nomenclature ............................................................ x!
Epigraph ............................................................................................................................ xii!
CHAPTER 1: INTRODUCTION ....................................................................................... 1!
1.1 Breast Cancer ............................................................................................................ 1!
1.1.1 Breast Cancer Statistics in Canada ................................................................... 1!
1.1.2 Breast Cancer Classification ............................................................................. 2!
1.2 Prolactin .................................................................................................................... 4!
1.2.1 Prolactin Signaling and Function ..................................................................... 4!
1.2.2 The Role of Prolactin in Mammary Gland Development ................................ 8!
1.2.3 The Role of Prolactin in Breast Cancer ............................................................ 8!
1.3 Bone ........................................................................................................................ 11!
1.3.1 Bone Structure and Function .......................................................................... 11!
1.3.2 Bone Remodeling ........................................................................................... 12!
1.3.3 Osteoclastogenesis and Bone Resorption ....................................................... 14!
1.3.4 Common Factors in Both the Mammary Gland and Bone ............................. 18!
1.4 Breast Cancer Metastasis to Bone .......................................................................... 18!
1.4.1 The Process of Metastasis .............................................................................. 18!
1.4.2 Mechanisms of Breast to Bone Metastasis ..................................................... 19!
1.4.3 The ‘Vicious Cycle’ of Breast Cancer Bone Metastases ................................ 20!
1.5 Hedgehog Signaling ................................................................................................ 24!
1.5.1 Hedgehog Signaling ....................................................................................... 24!
1.5.2 The Role of Hedgehog Signaling in Vertebrate Development ....................... 25!
1.5.3 The Role of Hedgehog Signaling in Cancer ................................................... 27!
CHAPTER 2: HYPOTHESIS ........................................................................................... 29!
2.1 Project Aims ........................................................................................................... 29!
CHAPTER 3: MATERIALS AND METHODS .............................................................. 31!
3.1 Cell Lines ................................................................................................................ 31!
3.1.1 Culture and Maintenance of Breast Cancer Cell Lines .................................. 31!
3.1.2 Culture and Maintenance of Osteoclast Precursors ........................................ 32!
3.2 Prolactin-Induced Breast Cancer-Mediated Osteoclastogenesis ............................ 32!
3.2.1 Preparation of Breast Cancer Cell Condition Medium ................................... 32!
3.2.1.1 Preparation of Serum Free or Reduced Serum Conditioned Medium .. 33!
3.2.1.2 Preparation of Conditioned Media Using a Prolactin Dose Response . 33!
3.2.2 Conditioned Media-Induced Differentiation of RAW264.7 Pre-Osteoclasts . 33!
3.2.3 Hedgehog Signaling Inhibition in Osteoclast Differentiation ........................ 33!
3.3 Methods of Quantifying Osteoclastogenesis .......................................................... 34!
3.3.1 Tartrate Resistant Acid Phosphatase (TRAP) and Nuclear Staining .............. 34!
3.3.2 para-Nitrophenyl Phosphate (pNPP) TRAP Activity Assay .......................... 34!

v
3.3.3 Fluorescent Staining ....................................................................................... 35!
3.3.3.1 Plasma Membrane Staining .................................................................. 35!
3.3.3.2 Actin Ring Staining .............................................................................. 36!
3.4 Gene Array .............................................................................................................. 36!
3.5 Cytokine Array ....................................................................................................... 36!
3.6 Quantitative Polymerase Chain Reaction ............................................................... 37!
3.6.1 RNA Preparation and Extraction .................................................................... 37!
3.6.2 Assessment of RNA Quantity and Quality ..................................................... 37!
3.6.3 Complimentary DNA Synthesis ..................................................................... 38!
3.6.4 Quantitative Polymerase Chain Reaction ....................................................... 38!
3.6.5 Endpoint PCR and Visualization of PCR Products ........................................ 39!
3.7 Enzyme-Linked Immunosorbent Assays (ELISA) ................................................. 40!
3.8 mRNA expression analysis of PRLR and SHH in human breast tissue ................. 40!
3.9 Statistical Analysis .................................................................................................. 41!
CHAPTER 4: RESULTS: OPTIMIZATION OF OSTEOCLAST
DIFFERENTIATION AND QUANTIFICATION .................................................. 42!
4.1 Optimization of Osteoclast Differentiation ............................................................. 42!
4.1.1 PRL Enhances Breast Cancer-Mediated Osteoclastogenesis ......................... 42!
4.1.2 Treatment of SKBR3 with hPRL for 48 hr is Optimal for Induction of
Osteoclastogenesis .......................................................................................... 42!
4.1.3 Prolactin Does Not Increase Breast Cancer Cell Proliferation During
Conditioned Media Preparation ...................................................................... 45!
4.2 Methods of Quantifying Osteoclastogenesis .......................................................... 47!
4.2.1 TRAP Staining ................................................................................................ 47!
4.2.2 para-Nitrophenyl Phosphate (pNPP) TRAP Activity Assay .......................... 47!
4.2.3 Morphological Identification using Fluorescent Microscopy ........................ 52!
CHAPTER 5: RESULTS: IDENTIFICATION OF PRL-REGULATED, BREAST
CANCER CELL SECRETED FACTORS .............................................................. 55!
5.1 Gene Array .............................................................................................................. 55!
5.1.1 Gene Array Sample Preparation and Testing ................................................. 55!
5.1.2 Regulation of Gene Transcription by PRL ..................................................... 55!
5.1.3 Gene Array Validation ................................................................................... 59!
5.1.3.1 Establishing TBP as a Reference Gene ................................................. 59!
5.1.3.2 Gene Array Validation .......................................................................... 59!
5.1.4 Gene Array Candidate Selection and Assessment .......................................... 61!
5.1.4.1 Candidate Selection .............................................................................. 61!
5.1.4.2 FLRT1 and FSTL4 are Not PRL-Regulated Genes .............................. 65!
5.2 Cytokine Array ....................................................................................................... 71!
5.2.1 Preparation of Serum Free SKBR3 and MCF7 Conditioned Media
Samples ........................................................................................................... 71!
5.2.2 PRL-Regulated, Breast Cancer Cell Secreted Candidate Selection ............... 71!
5.2.3 Luminex Cytokine Array Validation .............................................................. 80!
5.3 Detection of SHH, IHH and LIGHT in Conditioned Media Samples by ELISA ... 80!
5.4 SHH Signaling Inhibition in Osteoclast Differentiation ......................................... 87!
5.5 Expression of the PRLR and SHH in Human Breast Tumours .............................. 87!