Investigation of the Role of Novel Hormone Regulated Genes in Mammary Gland Development and Carcinogenesis

Heidi Nicole Hilton

A thesis submitted for the degree of Doctor of Philosophy in the Faculty of Medicine at the University of New South Wales

Acknowledgements

Having reached this point where I am almost finished and about to disembark the PhD rollercoaster, I am all too aware of the people that have helped me get here. First and foremost I must thank my supervisor, Chris Ormandy. Thank you for giving me enough freedom to learn and try out new things on my own, yet providing good and solid support whenever it was required. Your acceptance of me having a life outside of my PhD has made this experience far closer to enjoyable than traumatic! What I have learned, and the experiences I have gained both inside and outside of the lab, probably could not have been matched had I not worked in your group over the past 6 years, so thank you.

Roger Daly – thanks for stepping up as my co-supervisor mid-way through the event. You were definitely the one for the job! Thanks for all your help and support, and especially with getting that Goblin paper over the line in the end.

Prue Stanford – thank you for teaching me so much when I first started working at the Garvan and onwards, for being a fantastic part-time co- supervisor, and for giving me such good support and encouragement when it was sorely needed! On top of that you have also been a great friend to me. Thank you!

Sam Oakes – what can I say Sammy? It’s been extreme! Thank you so much for all your advice and encouragement with my work, not to mention all the fun times we have had in the lab, at the Green Park, and of course at conferences! I don’t think living in different cities (or countries) will ever make us lose touch!

I am indebted to Assoc Prof Liz Musgrove for helpful discussion and use of reagents, to Chehani Alles for doing such a great job with the microarray analyses in a short amount of time, to Maria Kalyuga for generating the Elf5

pHUSH and ProEx constructs, and Warren Kaplan for his great bioinformatic work on Goblin. Thank you!

Many thanks must go to all the past and present Development Group members – Matt, Jess, Fi, Cara, Heather, Wendy, Renee and Anita, and particularly Katrina, for being my ABI fairy and close friend.

And of course thank you to a number of other past and present Garvanites – Gillian for all her help in TC plus more, Tilman, Darren, Nick and Ashleigh for making work so fun, and especially Liz C, the genius with a gold heart, who has given me so much help and advice and answered my endless questions over the years!

To my family who are always there for me no matter what. Mum and Dad, thank you so much for your endless support in everything I do. I know you would be equally proud no matter what I had done. Thank you.

Ryan – perhaps my personal highlight of this PhD was that I met you during it. Thank you so much for keeping me relaxed and giving me perspective during this time, as well as countless other things. I look forward to whatever lies ahead for us.

Financial support

University Postgraduate Award (UNSW); August 2004 – January 2008

Manuscripts arising from this thesis

Oakes SR, Hilton HN, Ormandy CJ. The alveolar switch: coordinating the proliferative cues and cell fate decisions that drive the formation of lobuloalveoli from ductal epithelium. Breast Cancer Res. 8(2):207 (2006) (see Appendix I)

Hilton HN, Stanford PM, Harris J, Oakes SR, Kaplan W, Daly RJ, Ormandy CJ. KIBRA interacts with discoidin domain receptor 1 to modulate collagen-induced signalling. Biochim Biophys Acta. 1783(3);383-93 (2008) (see Appendix II)

Oakes SR, Naylor MJ, Asselin-Labat ML, Blazek KD, Gardiner-Garden M, Hilton HN, Kazlauskas M, Pritchard MA, Chodosh LA, Pfeffer PL, Lindeman GJ, Visvader JE, Ormandy CJ. The Ets transcription factor Elf5 specifies mammary alveolar cell fate. Genes Dev. 22(5):581-6 (2008) (see Appendix III)

Abbreviations -/- Knockout +/+ Wildtype 2D Two-dimensional 3D Three-dimensional AR Androgen receptor AREG Amphiregulin BLAST Basic local alignment search tool oC Degrees Celcius Ccnd1 Cyclin D1 ChIP Chromatin immunoprecipitation cDNA Complementary DNA DDR1 Discoidin domain receptor 1 DNA Deoxyribonucleic acid Dox Doxycycline dpc Days post coitus dpp Days post partum DSP Desmoplakin ECM Extracellular matrix E Estrogen EcoR Ecotrophic receptor EGF Epidermal growth factor EGFR Epidermal growth factor receptor Elf3 E74-like factor 3 Elf5 E74-like factor 5 ER Estrogen receptor ER Estrogen receptor ErbB2 v-erb-b2 erythroblastic leukemia viral oncogene homolog 2 EST Expressed sequence tag Ets E twenty-six

FACS Fluorescence activated cell sorting FAK Focal adhesion kinase FASN Fatty acid synthase FBS Foetal bovine serum FGF Fibroblast growth factor Gal Galanin GAPDH Glyceraldehyde-3-phosphate dehydrogenase GATA-3 GATA binding protein 3 GFP Green fluorescent protein GH Growth hormone GHR Growth hormone receptor h Hours HEK-293 Human embryonic kidney-293 HELU Hyperplastic enlarged lobular unit HEPES 4-(2-hydroxyethyl)-1- piperazineethanesulfonic acid HMEC Human mammary epithelial cell HRT Hormone replacement therapy IF Immunofluorescence IGF-1 Insulin-like growth factor 1 IHC Immunohistochemistry IgG Immunoglobulin G IKK Kinase IkB IP Immunoprecipitation IRES Internal ribosome entry site Jak2 Janus kinase 2 kDa Kilodalton MAPK Mitogen activated protein kinase MEC Mammary epithelial cell

min Minutes MMTV Mouse mammary tumour virus MMP Matrix metalloproteinase NaCl Sodium chloride NF1 Nuclear factor 1 NF-B Nuclear Factor-kappa B NLB Normal lysis buffer Nrg Neuregulin PBS Phosphate buffered saline PCR Polymerase chain reaction Pg Progesterone Pgr Progesterone receptor PgrA Progesterone receptor A PgrB Progesterone receptor B PI3K Phosphatidylinositol 3-kinase PKC Protein kinase C PKC Protein kinase C zeta PL Placental lactogen PRE Progesterone receptor element PTH1R Parathyroid hormone-related protein receptor PTHrP Parathyroid hormone-related protein Prl Prolactin Prlr Prolactin receptor PVDF Polyvinylidene Fluoride RACE Rapid amplification of complementary DNA ends RankL Receptor activator of NF-kB-ligand RNA Ribonucleic acid rpm Revolutions per minute

SDS Sodium dodecyl sulfate Shc Src homology 2 domain containing transforming protein SH2 Src homology 2 siRNA Small interfering RNA Socs Suppressor of cytokine signalling Sos Son of sevenless Srebp1 Sterol regulatory element-binding protein 1 Stat5 Signal transducer and activator of transcription 5 TDLU Terminal ductal lobular unit TEB Terminal end bud TGF Transforming growth factor, alpha TGF1 Transforming growth factor, beta 1 Tyr Tyrosine WAP Whey acidic protein WB Western blot Wnt Wingless-related MMTV integration site Wt Wildtype

Abstract

Mammary gland development is controlled by hormones such as progesterone and prolactin, which activate a genomic regulatory network. Identification of the components and regulatory links that comprise this network will provide the basis for defining the network's dynamic response during normal development and its perturbation during breast carcinogenesis. This thesis investigates two molecules in detail, Elf5 and KIBRA, which were identified as potential prolactin targets in a transcript profiling screen for key members in this genetic program of mammary morphogenesis.

We examined the effect of expression of Elf5, a transcription factor critical in alveolar differentiation, in a 3D culture model of non-transformed mammary epithelial MCF-10A cells. We discovered that Elf5 expression was selectively repressed over time in these cells when cultured on a basement membrane, and that Elf5 overexpression disrupted the architecture of acini resulting in luminal filling. This occurred due to an increase in the expression of epidermal growth factor receptor (EGFR) with repressed the induction of the pro- apoptotic molecule, Bim. We also observed that Elf5 is up-regulated with progesterone treatment, and that suppression of Elf5 expression in T47D breast cancer cells inhibits proliferation. Data obtained from the suppression of Elf5 expression in the presence of progesterone suggested that the role played by Elf5 in the Pg signalling pathway in T47D cells is relatively minor, and that rather than being a major downstream factor, the induction of Elf5 expression is utilised more to influence and potentiate other signalling pathways, such as the Prl pathway.

We characterised expression of KIBRA in the mammary gland and breast cancer cell lines, and observed that KIBRA was also up-regulated with progesterone treatment. Using a bioinformatic approach, we identified the tyrosine kinase receptor DDR1 as a binding partner of KIBRA. We have demonstrated that the WW domains of KIBRA bind to a PPxY motif in DDR1,

and that these molecules dissociate upon treatment with the DDR1 ligand, collagen. Finally, overexpression and knockdown studies demonstrate that KIBRA promotes the collagen-stimulated activation of the MAPK cascade. Thus KIBRA may play a role in how the reproductive state influences the mammary epithelial cell to respond to changing cell-context information, such as experienced during the tissue remodelling events of mammary gland development. Overall, the data presented in this thesis contributes to our growing knowledge of the genetic program responsible for mammary development and carcinogenesis.

Table of Contents

Abbreviations.……………………………………………………………………………..7

Abstract…………………………………………………………………………………….11

CHAPTER 1: INTRODUCTION ...... 17

Background ...... 18

Overview of Mammary Gland Cellular Morphology...... 19

Key Stages of Post-natal Mammary Gland Morphogenesis ...... 21 Ductal branching morphogenesis ...... 21 Alveolar morphogenesis...... 22 Involution...... 24

Hormonal Regulation of Mammary Development ...... 25 Regulation of ductal morphogenesis ...... 26 Regulation of alveolar morphogenesis ...... 29 Regulation of lactation and involution ...... 31

Mammary development and stem cells ...... 33

Signalling Pathways Involved in Alveolar Morphogenesis ...... 34 Molecular modulators of prolactin and progesterone induced alveolar morphogenesis ...... 34 The MAPK pathway ...... 36 Other factors involved in alveolar morphogenesis ...... 42

The Ets Transcription Factor Family and Elf5 ...... 46

Prolactin and Progesterone in Mammary Carcinogenesis ...... 50

Project Aims...... 52

CHAPTER 2: MATERIALS AND METHODS ...... 55

DNA Methods...... 56 Expression vector construction ...... 56 Site-directed mutagenesis ...... 57

Cell Culture Methods ...... 58

Cell lines ...... 58 HEK-293 cells ...... 58 MCF-10A cells...... 58 T47D and BT474 cells...... 58 Other cell lines ...... 59 Transient transfections ...... 59 Production of retrovirus ...... 59 Infection of MCF-10A cells ...... 59 Three-dimensional (3D) culture...... 60 siRNA duplexes and transfection...... 60 Ligand stimulation of cells...... 61 Progesterone receptor transactivation assay...... 61 Cell proliferation assays...... 62 Growth curve analysis ...... 62 MTT assays ...... 62 Flow Cytometry ...... 62

RNA Methods...... 63 Extraction, quantitation and analysis ...... 63

Protein Methods ...... 64 Antibody preparation...... 64 Cell lysate preparation and protein extraction...... 64 From monolayer cultures ...... 64 From 3D cultures ...... 65 From mammary glands ...... 65 Immunoprecipitation ...... 66 Western blot analysis ...... 66 Immunohistochemistry ...... 68

Immunofluorescence and microscopy ...... 68 Fixation and staining protocol of 2D cultures ...... 68 Fixation and staining protocol of 3D cultures ...... 69 Microscopy ...... 69

Bioinformatic Methods ...... 70 Statistical analysis ...... 70 Microarray analysis ...... 70 Functional Annotation Analysis ...... 71

CHAPTER 3: EFFECT OF ELF5 EXPRESSION ON MCF-10A CELLS IN 3D CULTURE...... 72

Introduction ...... 73

Results ...... 77 Generation of constitutive Elf5 overexpressing stable cell lines...... 77

Investigation of the effect of Elf5 expression on the proliferation of MCF-10A cells in two-dimensional culture...... 79 Investigation of the effect of Elf5 expression on acinar morphogenesis in three-dimensional culture ...... 81 Mechanism of Elf5 action on lumen formation in 3D culture ...... 89 Generation of inducible Elf5 overexpressing stable cell lines ...... 93 Effect of inducible Elf5 overexpression on levels of EGFR and Bim expression...... 96 Role of the ERK MAPK pathway in repression of Bim expression by Elf5 in acinar morphogenesis ...... 98

Discussion ...... 99

CHAPTER 4: INVESTIGATION OF THE ROLE OF ELF5 IN THE HUMAN BREAST CANCER CELL LINE, T47D...... 102

Introduction ...... 103

Results ...... 105 Hormonal regulation of Elf5 ...... 105 Regulation of Elf5 expression by sex-steroid hormones in the mouse mammary gland ...... 105 Regulation of human Elf5 expression by progestins in breast cancer cells ...... 106 Regulation of Elf5 expression by co-treatment of T47D breast cancer cells with estrogen and progestin...... 109 Regulation of Elf5 expression by growth hormone in T47D cells ...... 110 Effects of down-regulation of Elf5 expression in T47D breast cancer cells by siRNA...... 111 Changes in cellular morphology in T47D cells with suppressed Elf5 expression...... 112 Effect of suppression of Elf5 expression on cell cycle progression in T47D cells...... 112 Role of Elf5 in progestin-induced differentiation in T47Ds ...... 117 Identification of potential downstream targets of Elf5 in T47Ds ...... 119

Discussion ...... 137

CHAPTER 5: EXPRESSION PROFILE AND HORMONAL REGULATION OF KIBRA ...... 140

Introduction ...... 141

Results ...... 143 Generation of reagents ...... 143 Cloning of KIBRA ...... 143 Validation of KIBRA antibody ...... 144

Downregulation of KIBRA expression by RNA interference ...... 145 KIBRA expression in mammary gland and breast ...... 146 Expression of KIBRA protein in human breast cancer cell lines ...... 146 Localisation of KIBRA as visualised by immunofluorescence ...... 146 Localisation of KIBRA as visualised by immunohistochemistry...... 150 Effect of hormonal stimulation on KIBRA gene expression ...... 152 KIBRA is not a co-activator of the progesterone receptor...... 155 Effect of KIBRA overexpression levels on cell growth...... 156

Discussion ...... 161

CHAPTER 6: IDENTIFICATION AND CHARACTERISATION OF KIBRA INTERACTION WITH DDR1 ...... 163

Introduction ...... 164

Results ...... 166 Use of bioinformatic approach to identify KIBRA interacting partners ...... 166 KIBRA interacts with DDR1 in a collagen-regulated manner ...... 168 The WW domains of KIBRA bind the PPxY motif in DDR1 ...... 171 KIBRA interacts with DDR1 and PKC in a trimeric complex ...... 172 Modulation of KIBRA expression has downstream effects on the collagen- stimulated activation of ERK MAPK cascade ...... 173

Discussion ...... 177

CHAPTER 7: DISCUSSION ...... 181

Overview ...... 182

A Role for Elf5 in Proliferation and Survival of Mammary Epithelial Cells ...... 182

A Potential Role for Elf5 in Breast Cancer?...... 189

KIBRA and Modulation of ECM Signalling ...... 192

Summary and Conclusions...... 196

REFERENCES ...... 197

APPENDICES……………………………………………………………………233

Chapter 1: Introduction

Background

According to recent statistical reports, incidence rates of breast cancer in New South Wales, Australia have remained constant over the past 10 years, and mortality rates have fallen (Tracey et al. 2007). Breast cancer represents 30% of female cancer diagnoses, and remains the most common malignancy and cause of cancer-related death in Australian women today. Encouragingly, improvements in detection and treatment have seen survival rates continue to increase (Figure 1.1), indicating that in many cases cancer has been transformed from a critical disease into a chronic one.

Figure 1.1 Breast cancer survival rates. Age standardised relative survival (%) of female breast cancer patients at one, five, ten and twenty years post-diganosis in England and Wales (1971-2003) (Cancer Research UK www.cancerresearchuk.org 2008; Coleman et al. 1999; Coleman et al. 2004)).

Although survival rates are improving, incidence rates and development of resistance to treatment are increasing, demonstrating the need for new therapies that are truly curative. The rational design of new therapies requires an understanding of the etiology of breast cancer. Cancer can be viewed as

aberrant development, or neo-organogenesis, in which normal developmental mechanisms are perturbed by oncogenic insult. A key approach to understanding breast cancer is to define the normal developmental mechanisms that operate in the mammary gland and to determine how these mechanisms are corrupted during carcinogenesis. Development of the mammary gland occurs in defined stages, involving cellular processes such as proliferation, differentiation and apoptosis, as a result of hormonal cues that regulate reproductive events. Mammary carcinogenesis involves the mutation or dysregulation of genes required for normal mammary gland development. This may result in uncontrolled cell proliferation, loss of differentiation, sustained cell division, loss of apoptosis and a capacity to migrate and invade to other tissues and organs, the hallmark features of a cancerous cell. Therefore a detailed understanding of the complex network of the signalling systems that drive normal development of the mammary gland will provide the foundation to understand how these processes are perturbed during tumorigenesis.

Overview of Mammary Gland Cellular Morphology

The mammary gland is unique in that it only reaches complete development post-natally, following pregnancy and lactation. It is comprised of two major tissue compartments – the epithelium, which includes the ducts and milk- secreting alveolar cells, and the stroma, which consists of adipocytes, fibroblasts, blood vessels, inflammatory cells and the extracellular matrix (ECM) (Richert et al. 2000). Growing ducts end in bulbous terminal end bud (TEB) structures, which extend to completely fill the fat pad, and eventually give rise to basal myoepithelial cells and luminal alveolar cells. The alveolar cells form a sphere-like single layer of epithelial cells that envelopes a circular lumen, termed alveolus, which is connected to the ductal network via a single small duct. Upon functional differentiation of these luminal alveolar cells during pregnancy, they synthesise and secrete milk into the lumen of the alveolus where it exits the body via the nipple. The architecture of this extensive

system of TEBs and ducts embedded in the stroma is depicted in Figure 1.2. Terminal differentiation of the luminal cells into milk-secreting lobuloalveolar structures occurs in distinct stages tightly regulated by hormonal cues, as described in detail in the following sections.

Figure 1.2 Terminal end bud and duct morphology. TEBs are composed of an outer layer of myoepithelial cells surrounding a mass of luminal epithelial cells. This TEB is encased by a sheet of basement membrane, which is in turn surrounded by the fatty stroma, consisting of adipocytes, fibroblasts, immune cells and ECM components. The cap cells (green) at the tip of the TEB are putative progenitor stem cells which can give rise to myoepithelial (blue) or luminal (red) cells.

Key Stages of Post-natal Mammary Gland Morphogenesis

Ductal branching morphogenesis

In the mouse, mammary gland morphogenesis commences before birth when the mammary anlage develops during the embryonic and foetal periods (Hovey et al. 2002). Following birth, the anlage of mammary epithelium consists of a small, branched ductal tree connected to the nipple. These ducts then undergo isometric growth until the onset of puberty when secretion of ovarian hormones induces allometric growth. This is when the TEB structures appear at the end of the epithelial ducts, and begin the process of ductal elongation and bifurcation in order to reach the periphery of the fat pad (Richert et al. 2000). As depicted in Figure 1.2, the TEB is a unique and specialised structure with rapidly dividing cells arranged into a single outer layer of undifferentiated cap cells, and multiple inner layers of luminal epithelial cells (Hovey et al. 2002). The highly proliferative luminal cells also undergo an extensive amount of concurrent apoptosis, which is important for the formation of hollow ducts enabling milk delivery during lactation (Humphreys et al. 1996; Humphreys 1999). The mammary gland branches by two processes: bifurcation of the TEBs directly into adipose tissue, and sprouting of side branches from mature ducts through basement membrane (Wiseman and Werb 2002). Each side branch terminates in a small alveolar bud. As branching proceeds the mammary fat pad is eventually filled with alveolar buds attached to the sides of ducts. Despite this, ducts never come into very close contact with each other, suggesting the diffusion of local inhibitory signals from adjacent ducts (Hovey et al. 2002). Once the elongated ducts have reached the edge of the fat pad, growth ceases, the cap cell layer vanishes, and the TEBs disappear (Brisken 2002).

Due to the limited availability of human breast tissue, the vast majority of our knowledge of mammary development has evolved from mouse models. The

morphology of the human breast differs substantially from the mouse mammary gland. Whereas humans have the one pair of nipples and glands, most mouse strains have five pairs. The human mammary gland has a more complex segmented structure starting with the nipple that contains the outlet for 5 to 10 lactiferous ducts (Cardiff and Wellings 1999). In contrast, the mouse mammary gland generally terminates in a single lactiferous duct that forms 5 to 10 secondary ducts that grow in a more linear pattern than in humans (Cardiff and Wellings 1999). The human counterpart of the mouse side branch and alveolar bud is the terminal ductal lobular unit (TDLU), and is a small unit resembling a cluster of grapes at the end of a stem, encased in a loose intra-lobular connective tissue surrounded by denser stroma (Cardiff and Wellings 1999; Brisken 2002). However despite these morphological differences, there are also many similarities between the human breast and the mouse mammary gland. In the human breast, ductal elongation and side branching also occurs during puberty, and these ducts are also lined by a bilayer of myoepithelial and luminal epithelial cells (Monaghan et al. 1990). Both the alveolar buds and TDLUs have similar compositions of epithelial cells, which are hormone responsive throughout the murine estrous or human menstrual cycle, respectively, and are the source of milk production (Cardiff and Wellings 1999). Due to these parallels, the mouse is thus an ideal model for studying mammary morphogenesis and cancer.

Alveolar morphogenesis

Alveolar morphogenesis or the development of the mouse mammary epithelium during pregnancy results in the formation of alveolar structures primed for milk secretion during lactation. This involves rapid and global proliferation of the epithelial cells within the ductal branches and developing alveoli, which increases both epithelial cell number and surface area, actions essential for sufficient milk production during lactation. This initial proliferative phase is instigated by an increase in the level of serum prolactin (Prl) and progesterone (Pg) (Brisken 2002). These hormones activate the ‘alveolar

switch’, a genetic program that coordinates changes in epithelial cell proliferation, migration, differentiation and deletion within the many tissue types of the mammary gland.

Following this massive proliferation phase, cell differentiation becomes dominant from mid-pregnancy as the gland begins to move into the secretory initiation phase (Richert et al. 2000). The developing alveoli cleave and polarise to form a spherical layer of epithelial cells surrounding a hollow lumen, connected to the ductal network via a single small duct. Each individual alveolus is surrounded by a basket-like architecture of contractile myoepithelial cells. These contractile cells are responsible for the movement of milk out of the alveoli and through the ducts during lactation (Richert et al. 2000). The myoepithelium of the alveoli is discontinuous so that the luminal cells can directly contact the underlying basement membrane, which forms part of the ECM. Some cells of the ductal network also contact the basement membrane. This contact is required in order for complete lobuloalveolar differentiation to occur (Streuli et al. 1995; Fata et al. 2004), seen morphologically by the appearance of lipid droplets (Neville et al. 2002) and by the initiation of gene expression in a defined order (Rudolph et al. 2003). Nearing parturition, alveolar tight junctions close, and milk and colostrum proteins move into the alveolar lumen. This occurs in preparation for active milk secretion post partum, which marks the onset of the secretory activation phase (Nguyen et al. 2001).

The epithelial expansion is paralleled by equally dramatic changes in other tissue compartments. Adipocytes lose their lipid content and remain as long projections scattered throughout the alveolar epithelium (Neville et al. 1998). A huge expansion of the vasculature also occurs within the stroma, to provide the large quantities of energy, sugars, amino acids and solutes required for milk production (Djonov et al. 2001). Developmental events are also elicited elsewhere in the animal, for example the gut and liver enlarge dramatically to cope with the energy needs of gestation and lactation. The brain is

programmed for correct maternal behaviour by Prl (Lucas et al. 1998). Thus the alveolar switch is part of a larger mechanism controlling all aspects of adaptation to pregnancy and lactation.

Involution

Following lactation, further tissue remodelling occurs during involution when the milk-secreting epithelial cells undergo cell death once they are no longer required. It is a two-step process whereby the first phase is marked by cells undergoing apoptosis and shedding into the lumen of the alveoli, followed by the second phase, which is marked by remodelling of the surrounding stroma and replacement of the secretory epithelium by adipocytes (Watson 2006). During the second phase of involution, further apoptosis is induced by degradation of the basement membrane and ECM by matrix metalloproteases (MMPs) (Pullan et al. 1996; Green and Lund 2005). Following this, the involuted mammary gland returns to a predominantly quiescent state that resembles the pre-pregnant state, only to redevelop with the next pregnancy. A schematic diagram representing the different developmental stages of the mammary gland is depicted in Figure 1.3 over the page.

Figure 1.3 Post-natal organogenesis of the mammary gland. The pink ovals depict the mammary fat pad (stroma) and the solid black circle represents the nipple from which the ducts originate. The growing ducts end in terminal end buds (TEBs) during puberty. In the mature virgin, the entire fat pad is filled with an extensive ductal system with side branches. The onset of pregnancy induces increased cell proliferation and the formation of alveolar buds which grow and differentiate into milk-secreting alveoli at the end of pregnancy. During lactation, the luminal cells of fully mature alveoli synthesise and secrete milk components into the lumen. After weaning the mammary gland undergoes apoptosis (involution), returning the gland to a pre-pregnant state.

Hormonal Regulation of Mammary Development

The initial and very restricted development of the mammary gland in the embryo is independent of hormonal regulation, demonstrated by a lack of any obvious embryonic mammary phenotype in either of the knockout mouse models of the estrogen receptor (ER), prolactin receptor (Prlr), growth hormone receptor (GHR) or progesterone receptor (Pgr) (Hennighausen and Robinson 2001; Hovey et al. 2002; Hens and Wysolmerski 2005). The anlage

is, however, sensitive to maternal hormonal stimulation as lactation is observed in some newborns, both male and female. These secretions are termed “witches’ milk” following English medieval folklore where they were said to be stolen by witches to feed their familiar spirits, or as an extension of the ability of witches to milk inanimate objects. The interesting paradox raised by this observation is that these hormones do not elicit morphological development (as they will during puberty) but can initiate lactation from the anlage.

Embryonic mammary development requires the expression of parathyroid hormone-related protein (PTHrP) in the embryonic mammary bud, and of its receptor (PTH1R) in the mammary mesenchyme, which are essential in order for the mesenchyme to acquire a specialised mammary fate (Wysolmerski et al. 1998; Foley et al. 2001b). In the absence of PTHrP signalling, mammary epithelial cells (MECs) revert to an epidermal fate and no mammary ducts or nipple are formed (Foley et al. 2001b). PTHrP is also critical in maintaining sexual dimorphism, as it induces androgen receptor (AR) expression within the mammary mesenchyme (Dunbar and Wysolmerski 1999). Thus in males, the expression of androgens from the fetal testes bind to the AR in the mammary mesenchyme, resulting in the destruction of the nipple and mammary bud (Dunbar and Wysolmerski 1999). In addition to PTHrP, signalling pathways downstream of wingless-type MMTV integration site family (Wnt) and fibroblast growth factor 10 (FGF10) are also necessary for mammary gland development in the mouse embryo (Mailleux et al. 2002; Chu et al. 2004). In contrast to embryonic development, post-natal mammary development is tightly controlled by hormonal signals elicited by the ovaries and pituitary gland, which will now be discussed in detail.

Regulation of ductal morphogenesis

Ductal elongation and TEB growth is dependent upon estrogen (E) and growth hormone (GH) (Daniel et al. 1987; Silberstein et al. 1994; Kleinberg 1997).

This development is triggered by an increase in ovarian production of E, acting through the estrogen receptor alpha (ER). It also requires secretion of GH by the pituitary gland, which acts through the GHR in the stroma to induce production of insulin-like growth factor 1 (IGF-1), which, in turn, stimulates mammary development in a paracrine manner (Ruan et al. 1992; Kleinberg et al. 2000). Indeed, ER-/-, GHR-/- and IGF-1-/- mouse models display impaired ductal elongation and reduced numbers of TEBs (Ruan and Kleinberg 1999; Curtis Hewitt et al. 2000; Gallego et al. 2001). IGF-1-/- mice fail to develop TEBs and ducts when treated with E and GH, and in addition, long-term treatment of IGF-1-/- mice with IGF-1 and E increases TEB formation and ductal elongation, demonstrating the synergy between these hormones and growth factors that is required during ductal morphogenesis (Ruan and Kleinberg 1999).

Tertiary side branches form in response to stimulation by Pg, further filling out the mature ductal tree (Atwood et al. 2000). Pg mediates its effects on the mammary gland by binding to the Pgr, of which there are two isoforms, PgrA and PgrB, which are expressed from a single gene. Loss of both Pgr isoforms in mice blocks tertiary side branching in the mammary gland following puberty (Lydon et al. 1995), and it was subsequently shown that loss of PgrB was necessary and sufficient to mediate these effects (Mulac-Jericevic et al. 2003). Not every cell in the mammary epithelium expresses Pgr, but those that do are segregated from, but located adjacent to proliferating cells (Silberstein et al. 1996; Seagroves et al. 2000). Thus, in the normal mammary gland, Pgr- positive cells do not appear to proliferate, but instead may be responsible for the expression and/or secretion of locally acting growth factors that stimulate proliferation of adjacent Pgr-negative cells (Brisken et al. 1998; Seagroves et al. 2000). Signalling by Wnt family proteins are important in mediating the paracrine effects of Pg that result in epithelial cell proliferation and side- branching of the adult mammary gland. This was demonstrated by Brisken et al. in 2000, when Pg was shown to directly induce Wnt4 expression, and that

Wnt4 was also required for side-branching (Brisken et al. 2000). Pg may also indirectly influence normal primary and secondary branching by synergising with IGF-1 action (Ruan et al. 2005).

Influences of the mammary stromal environment are crucial during ductal development (reviewed in Silberstein 2001). Indeed, the extent of ductal side branching differs between stroma of different mouse strains. This has been demonstrated by mammary stromal-epithelial recombination, whereby epithelium from a strain such as C57BL/6 which shows no side branching, results in a highly side branched morphology when recombined with the stroma of the highly side branched 129 strain (Naylor and Ormandy 2002). The important influence of the stroma is further demonstrated by epidermal growth factor receptor (EGFR) expression being required in the stroma, and not the epithelium, in order for normal mammary ductal growth and branching morphogenesis to occur (Wiesen et al. 1999). Although the stromal background is strongly stimulatory, neighbouring ductal branches avoid one another. This maintenance of proper ductal spacing is due to local inhibition of both ductal elongation and lateral branching elicited by transforming growth factor -1 (TGF1) (Silberstein et al. 1990; Pierce et al. 1993).

A number of knockout and transgenic mouse models have demonstrated a role for other molecules, both epithelial and stromal, in mediating ductal side- branching of the mammary epithelium. These include amphiregulin (AREG) (Luetteke et al. 1999), the disintegrin and metalloproteinase, ADAM17 (Sternlicht et al. 2005), C/EBP (Grimm and Rosen 2003), activin/inhibin (Robinson and Hennighausen 1997), DDR1 (Vogel et al. 2001), and the matrix metalloproteases, MMP2 and MMP3 (Wiseman et al. 2003). Once the complex network of ducts has been formed and the TEBs have reached the periphery of the mammary fat pad, the TEBs of the mature adult virgin regress and differentiate into quiescent alveolar buds, ready for a second wave of hormonal stimulation upon pregnancy.

Regulation of alveolar morphogenesis

The formation of the milk secreting structures during pregnancy is dependent on a synergy between Prl and Pg signalling (Neville et al. 2002). These hormones trigger an initial wave of cell proliferation during early pregnancy (Traurig 1967). The Pgr knockout mouse demonstrated that Pg is required for alveolar morphogenesis, and epithelial recombination experiments demonstrated that Pgr in the mammary epithelium, not the stroma, was essential for epithelial cell proliferation (Brisken et al. 1998). Mammary gland chimeras made from wildtype and Pgr-/- MECs, demonstrated that Pgr-/- epithelial cells proliferate in response to Pg (Brisken 2002). As not all mammary epithelial cells express Pgr, and are unable to respond to Pg directly, they must therefore respond to a paracrine factor from Pgr+/+ cells. Indeed, in the epithelium, proliferating cells segregate with Pgr positive cells (Conneely et al. 2003). This is also true for ER positive cells (Clarke et al. 1997). Further, steroid receptor positive cells are in close proximity to proliferating cells, indicating that proliferation is mediated, at least in part, by a paracrine mechanism. This heterogeneous receptor patterning observed in the luminal epithelium is required for complete lobuloalveolar development (Grimm et al. 2002).

As described in the previous section, Pgr is essential in ductal branching morphogenesis, and this role continues in the extensive branching that continues throughout pregnancy. Wnt4 continues to act in a paracrine fashion to stimulate epithelial ductal side branching during early pregnancy (Brisken et al. 2000). Although Wnt4-/- mammary epithelial transplants displayed impaired side branching in these experiments, normal lobuloalveolar proliferation was observed during the later half of pregnancy, indicating that other factors mediating proliferation in late pregnancy are likely to be involved.

In addition to Wnt4, Receptor activator of NF-B-ligand (RankL) is also a target of the Pgr signalling pathway and may be another paracrine factor responsible

for cellular proliferation in steroid receptor negative cells. The RankL target, Nuclear Factor-kappa B (NF-B), is required for Cyclin D1 (Ccnd1) activation via the kinase IkB (IKK) in neighbouring proliferating cells (Cao et al. 2001). Germ-line deletion of both RankL and its receptor (Rank) in mice resulted in failed alveolar morphogenesis due to reduced proliferation and increased apoptosis of alveolar epithelial cells (Fata et al. 2000). These effects were mediated by PI3K/Akt, demonstrating that this pathway is also essential for the formation of lobuloalveolar structures (Fata et al. 2000). The RankL/NF- B/Ccnd1 pathway is now known to be crucial for the formation of alveolar structures during pregnancy (Cao and Karin 2003), and NF-B is essential for Pg driven proliferation within alveoli (Conneely et al. 2003). RankL also co- localises with Pgrs in response to pregnancy levels of E and Pg, indicating this is an important part of the response. In primary MEC cultures, Pg acts in synergy with E to increase Ccnd1 transcription resulting in increased proliferation (Said et al. 1997). Together these data indicate that Pg may drive the proliferation of neighbouring cells via RankL/NF-B resulting in Ccnd1 transcription. In addition to the impaired ductal proliferation displayed in the PgrB-/- mouse model, alveoli also failed to develop during pregnancy, possibly an effect mediated via activation of RankL (Mulac-Jericevic et al. 2003).

In mice, pituitary Prl stimulation of ovarian Pg assists in maintaining the required levels of Pg during early pregnancy (Binart et al. 2000). In addition, up-regulation of Pgr expression by Prl, and Prlr expression by Pg, suggests that these hormones may interact in a synergistic manner to control alveolar development. Prlr knockout mice (Prlr-/-) have demonstrated the importance of this receptor during mammary development (Ormandy et al. 1997a). Similarly to Pgr, experiments with Prlr-/- mice have shown that the presence of Prlr in the epithelial cells, not the stroma, is essential for normal lobuloalveolar differentiation (Brisken et al. 1999). Prlr-/- mammary transplants fail to develop lobuloalveoli and produce milk proteins during pregnancy, illustrating that Prlr is essential in the mammary epithelium during alveolar morphogenesis.

Other hormones and growth factors can influence alveolar morphogenesis. The neuronal peptide galanin (Gal) regulates Prl secretion from the pituitary lactotrophs (Wynick et al. 1993). In addition, the mammary epithelium is responsive to Gal, as it augments alveolar morphogenesis in mammary explants in the presence of Prl (Naylor et al. 2003). Gal-/- mice show increased levels of the inhibitory phosphorylated form of Prl (Naylor et al. 2005), and are unable to nurse pups due to failed secretory activation (Wynick et al. 1998). Therefore Gal has dual actions: firstly an indirect role by modulating pituitary Prl and phosphorylated Prl release; and secondly a direct cell autonomous role in the formation of lobuloalveoli during pregnancy. GH may act in combination with Prl to mediate alveolar proliferation. GH treatment restores alveolar morphogenesis but inhibits lactation in Prlr+/- mammary glands (Allan et al. 2002). Placental lactogen (PL) is released from the placenta during pregnancy and can fully compensate for Prl, allowing alveolar morphogenesis in Prl-/- mice (Horseman 1999). Some of the key hormones and their mediators which control ductal and alveolar morphogenesis are depicted in Figure 1.4. The downstream targets and molecular modulators of Prl and Pg signalling will be discussed in more detail later in this chapter.

Regulation of lactation and involution

Just prior to lactation, Pg suppresses active milk secretion. Withdrawal of Pg in the presence of high levels of Prl induces the onset of secretory activation, characterised by closure of tight junctions and the movement of milk and lipids into the alveolar lumina for transport down the ducts and out through the nipple (Neville et al. 2002). Milk ejection maintains lactation, together with Prl stimulation of the luminal epithelial cells to continue milk secretion, and oxytocin stimulation of the contractile myo-epithelial cells to continue milk ejection (Neville et al. 2002). After termination of the suckling stimulus and withdrawal of Prl stimulation, the mammary gland undergoes rapid apoptosis, and together with remodelling of the ECM, the ductal framework is left remaining in readiness for a subsequent pregnancy.

Figure 1.4 Hormonal regulation of mammary morphogenesis. A Representative mammary wholemount images (Carmine alum stain) of (i) virgin, (ii) 12 days post coitus (dpc), (iii) 18 dpc and (iv) 1 day post partum. B Schematic diagrams of corresponding mammary gland development stages. C Key hormones and their mediators which regulate mammary morphogenesis. Solid arrows indicate direct interactions; broken arrows indicate indirect interactions. Wholemount images were taken from Oakes et al. 2006.

Mammary development and stem cells

During pregnancy, ductal branches grow and proliferate to yield differentiated and functional alveoli, which following weaning are then removed by apoptotic cell death during the involution phase, only to re-develop with the next pregnancy. The cyclical nature of this tissue remodelling is striking. This observation first led researchers to hypothesise that mammary tissue must contain persistent self-renewing mammary stem cells (reviewed in Smalley and Ashworth 2003). The ability of small epithelial transplants to recapitulate a complete and fully functional epithelial mammary gland reinforced this view (DeOme et al. 1959). The presence of a single mammary stem cell was indicated by limiting dilution experiments and the existence of committed progenitor cells was demonstrated by transplants that showed limited developmental capacity (Smith and Boulanger 2003). This cell was recently isolated and elegantly demonstrated to be capable of producing a renewable and complete mammary epithelium (Shackleton et al. 2006). Thus it is hypothesised, based on a paradigm developed in the hematopoietic system, that a primary mammary epithelial stem cell gives rise to a hierarchy of epithelial progenitor cell lineages to ultimately produce the different cells found in the mammary epithelium (Smalley and Clarke 2005; Stingl et al. 2005). The flux of cells through these lineages is likely to be controlled by, and in turn control, the patterns of gene expression that comprise the alveolar switch. Integrating our knowledge of gene expression patterns with the emerging knowledge regarding stem cell lineages and their interactions offers us an unprecedented opportunity to understand this phase of mammary development.

Signalling Pathways Involved in Alveolar Morphogenesis

Molecular modulators of prolactin and progesterone induced alveolar morphogenesis

Members of the prolactin- and progesterone-signalling pathways are essential for normal alveolar morphogenesis (Hennighausen and Robinson 2005). Prlr dimerisation occurs after Prl binding and leads to the phosphorylation of the associated Janus kinase (Jak2) (Han et al. 1997; Pezet et al. 1997), which in turn phosphorylates specific residues on the Prlr (Lebrun et al. 1995). Stat5 is then recruited to the receptor and is phosphorylated by Jak2 (DaSilva et al. 1996). Phosphorylated Stat5 is then translocated to the nucleus where it can activate transcription of multiple genes (Wakao et al. 1995) involved in a variety of processes during alveolar morphogenesis, including establishment of epithelial polarity and cell-cell interactions, stromal epithelial interactions and milk protein expression during lactation. Both isoforms of Stat5, Stat5a and Stat5b, result in lobuloalveolar defects when knocked out in mice, and this phenotype is more severe in combined Stat5a/Stat5b knockout animals (Liu et al. 1995; Liu et al. 1997; Teglund et al. 1998).

Pg is also a critical regulator of Stat5 in the mammary gland. Stat5 expression increases with E and Pg treatment in ovariectomized mice, and co-localises extensively with PgR positive cells, suggesting it may be a target of PgR (Santos et al. 2008). Stat5 is up-regulated and translocated to the nucleus with Pg treatment in human breast cancer cells (Lange et al. 1998; Richer et al. 1998), and an interaction has been reported between PgR and Stat5, either directly, or via binding on the beta-casein promoter (Richer et al. 1998; Buser et al. 2007). This interaction may be pivotal in the mechanism of how Pg antagonises the lactogenic effect of Prl observed in the mammary gland during pregnancy.

One class of genes activated by the Prl-signalling pathway are the suppressor of cytokine signalling (Socs) members, which act to shut down the Prl- signalling pathway. In a negative feedback loop, Prl induces expression of Socs1 and Socs2, which attenuates Stat5 activation by direct binding of Jak2 and preventing its activation of Stat5 (Hanada et al. 2001; Harris et al. 2006). Socs1 knockout mice show precocious development during pregnancy, and heterozygous Socs1+/- mice can restore the lobuloalveolar defects present in Prlr+/- mice (Lindeman et al. 2001). Socs2-/- mice show no developmental defects and complete loss of Socs2 is required to rescue lactation in Prlr+/- females (Harris et al. 2006). Socs2 shows the most robust transcriptional response to modulation of prolactin (Harris et al. 2006), with little change in Socs1 detectable.

Transcript profiling of prolactin receptor knockout mammary glands identified a panel of genes that require Prlr-mediated signalling for increased expression during early pregnancy (Ormandy et al. 2003; Harris et al. 2004). Laminin and two members of the collagen family were identified. These molecules are cell adhesion components of the ECM and play an important part in the epithelial- stromal signalling required for full lobuloalveolar differentiation and gene expression (Streuli et al. 1995; Rudolph et al. 2003). Alveolar morphogenesis induced by Prl involves the establishment of polarity and cell-cell communication. The maintenance of cellular polarity is regulated by the closure of tight junctions, and the expression of tight junction proteins Claudin- 3 and Claudin-7 was reduced in Prlr-/- mammary transplants. The gap junction protein Connexin-26 was also identified and is involved in the exchange of small ions and metabolites. Recently, Connexin-26 was shown to be important in full lobuloalveolar development and in the prevention of alveolar cellular apoptosis (Bry et al. 2004).

As discussed earlier, Wnt4 and RankL are targets of the Pgr signalling pathway. Wnt4 was also down regulated in Prlr-/- transplants, indicating that it is potentially a target of Prlr signalling also. The downstream target of Wnt, -

catenin, has specific actions in both the luminal and myoepithelial compartments of the epithelium, and as a component of cell-cell junctions, appears to have a role in signalling to luminal epithelial cells (Imbert et al. 2001; Teulière et al. 2005). Indeed, activation of -catenin within the basal epithelial cells results in premature differentiation of the luminal epithelium during pregnancy and persistent proliferation resulting in tumors. These tumors consisted predominantly of undifferentiated basal cells, which were amplified in response to -catenin activation, thereby implicating this molecule in cell fate decisions in the mammary gland (Teulière et al. 2005).

RankL was also identified as a potential Prl-regulated gene (Ormandy et al. 2003; Srivastava et al. 2003). Ccnd1 null mutants exhibit significantly delayed alveolar cell proliferation and impaired lactation, which was shown to be epithelial cell autonomous (Fantl et al. 1999). Interestingly, Prl can induce Ccnd1 expression via induction of Igf2, independent of RankL induction (Brisken et al. 2002). The similarities between Prl and Pg mediated effects on both RankL and Wnt signalling is further evidence of the co-operation of these pathways for alveolar cell proliferation during early pregnancy.

Gene expression profiling of Prl-/- mice has also identified unique targets of mammary development. Expression of tryptophan hydroxylase, the rate- limiting enzyme in serotonin biosynthesis, is increased by prolactin during pregnancy and lactation. Accumulation of serotonin due to milk engorgement experienced during weaning or experimentally via teat sealing, inhibits milk gene expression and can induce involution, providing a mechanism that is put in place by prolactin to stop lactation at weaning (Matsuda et al. 2004).

The MAPK pathway

In addition to activating the Jak2/Stat5 signalling cascade, binding of Prl to the Prlr also stimulates the MAPK cascade. Dimerisation of ligand-bound Prlr activates Jak2, which generates docking sites for Src homology domain 2

(SH2)-containing molecules, like SHC, and stimulates the Grb2/SOS-Ras-Raf- MAPK cascade (Erwin et al. 1995). Upon Prl treatment of MECs, Raf is rapidly activated, quickly followed by activation and phosphorylation of MEK and MAPK, mediating the Prl-induced proliferation that is observed in MECs (Das and Vonderhaar 1996). Although Jak2 is crucial for activation of Stat5 signalling, it is dispensable for the Prl-induced activation of the MAPK cascade, which instead may be mediated by the tyrosine kinase, c-Src, and focal adhesion kinase (FAK) (Acosta et al. 2003; Sakamoto et al. 2007).

Progesterone and synthetic progestins also rapidly activate the Src/Ras/MAPK cascade in MECs (Skildum et al. 2005). Progestins can activate c-Src either by direct binding by Pgr (Boonyaratanakornkit et al. 2001), or via an interaction between Pgr and ER (Ballaré et al. 2003). This cross-talk with ER is essential for Pg-induced proliferation of breast cancer cells (Migliaccio et al. 1998; Vicent et al. 2008). Pg activation of the PI3K/Akt pathway has been shown to be required for Pg-induced cell proliferation in the mammary gland (Ballaré et al. 2006). Prl can also activate the PI3K/Akt pathway, which has been demonstrated to promote proliferation (Fresno Vara et al. 2001; Chakravarti et al. 2005), and survival in the mammary gland (Bailey et al. 2004).

In addition to cross-talk with other steroid hormone signals, Pg also can enhance growth factor signalling, as demonstrated by the selective potentiation of EGF-stimulated MAPK activation by pre-treatment of human breast cancer cells with Pg (Lange et al. 1998). The ultimate targets of activation of this signalling pathway are not well-defined, but likely to include transcription factors and co-factors involved in cell cycle control and proliferative responses (Ballaré et al. 2006). It has been demonstrated that Prl and Pg synergistically stimulate the proliferation of mouse MECs in vivo (Hovey et al. 2001), and although the mechanisms underlying their co-operative effect is not known, it is likely that cross-talk between the Jak2-Stat5 pathway and mitogenic stimulation of the MAPK cascade is involved. A summary of the key regulators in these signalling cascades is depicted in Figure 1.5.

Figure 1.5 Prolactin and progesterone signalling pathways. Binding of Prl to Prlr induces receptor dimerisation and activation of Jak2. Stat5 is then recruited and phosphorylated, and then dimerises and translocates to the nucleus to activate the transcription of genes involved in alveolar morphogenesis, such as milk protein genes. Jak2 activation can also mediate phosphorylation of Shc, followed by association of Grb2 and SOS resulting in the binding and activation of Ras and Raf and subsequently, the MAPK signalling pathway. Attenuation of Prlr signalling is mediated by members of the Socs gene family through inhibition of Jak2 tyrosine kinase activity. Alternatively, Prl binding of Prlr can directly induce activation of c-Src, which mediates the activation of the MAPK cascade, required for cell proliferation. Progesterone regulates several intracellular effectors by up-regulating and inducing nuclear localisation of Stat5, increasing the phosphorylation of Jak2 and Shc, and potentiating EGF-stimulated MAPK activity. Prl and Pg can also promote mammary proliferation and survival, through activation of the PI3K/Akt pathway.

Transcription factors involved in alveolar morphogenesis Prl and Pg and other factors induce the transcription of genes via activation of target transcription factors. These include Stat5 and the steroid hormone receptors as discussed previously, which bind to DNA and result in the transcription of genes involved in many aspects of alveolar morphogenesis. Further, some of these target genes are transcription factors also, which act to induce the expression of genes or groups of genes involved in lobuloalveolar development. An example includes the transcription factor Srebf1, which was identified from transcript profiling experiments on 3 mouse models of failed secretory activation (Naylor et al. 2005). Srebf1 controls the expression of a number of key lipid metabolism genes (Horton et al. 2002), that showed reduced expression concomitantly with decreased Srebf1 expression (Naylor et al. 2005). Some transcription factors which appear to be involved in alveolar morphogenesis and regulation of cellular proliferation include the Homeobox genes, Helix-loop-helix genes, Stats, Tcf/Lef family, NF-B, C/EBP family, Nuclear factor family and the Ets transcription factors (Coletta et al. 2004).

Pg and Prl are hypothesised to influence the expression of -catenin, via induction of the Wnt pathway as discussed earlier. -catenin regulates the activity of the Tcf/Lef family of transcription factors which appear to mediate - catenin signalling and therefore may play a role during alveolar morphogenesis (Hatsell et al. 2003). Inhibition of -catenin results in alveolar apoptosis and greatly reduced milk production capacity. Mice lacking Lef-1 demonstrate a failure to form the alveolar bud at embryonic day 13. The expression of Lef-1 was co-expressed with -catenin, and shows a similar expression pattern in response to PTHrP (Foley et al. 2001a). Thus Lef-1 may act to mediate the actions of -catenin, although its effects during alveolar morphogenesis are still unclear.

The nuclear factor (NF1) family of transcriptions factors also play a role in functional differentiation as they regulate the transcription of milk protein

genes such as those encoding whey acidic protein (WAP), -lactalbumin and - lactoglobulin (Murtagh et al. 2003). The NF1-C2 isoform member of this family induces the expression of the milk genes caboxyl ester lipase and WAP. Prl regulates the protein expression of NF1-C2 in NMuMG cells, and its expression is reduced in the nucleus of Prlr-/- luminal cells at mid-pregnancy, indicating that NF1-C2 may be regulated by Prl signalling during pregnancy and involved in expression of milk genes in preparation for lactation (Johansson et al. 2005).

The helix-loop-helix transcription factors Id1 and Id2 have varying expression in the mammary gland. Id1 expression is increased during early pregnancy, remains low during lactation and rises again at involution. Unlike Id1, Id2 remains high during lactation, indicating that these isoforms have specific functional roles during alveolar morphogenesis (Desprez et al. 2003). Id1 is specifically expressed by the expanding epithelium during the alveolar proliferative phase and is inversely correlated with the expression of -Casein, therefore it appears to be an important factor during early alveolar proliferation. Id1 also regulates Clusterin, a gene involved in the regulation of cell-cell interactions. Additionally, lobuloalveolar development is severely impaired in Id2 knockout mice. Reduced proliferation and increased apoptosis has been observed in mammary epithelium lacking Id2, resulting in the failure to form alveolar structures and consequently failure of lactation (Mori et al. 2000). Id2 also promotes differentiation in MEC cultures, indicating Id2 is essential for the differentiation of the mammary epithelium (Desprez et al. 2003)

The transcription factor NF-kB discussed earlier in this review is essential for Pg induced alveolar cell proliferation resulting in Cyclin D1 transcription (Cao and Karin 2003; Conneely et al. 2003). NF-B can also induce the transcription many genes involved in the regulation of apoptosis. NF-B levels are induced during pregnancy, decline during lactation and are re-induced during lactation

implying a role in mammary gland remodelling. It is also hypothesised that NF- B is an essential “checkpoint” of apoptosis, whose actions are dependent on association with specific transcriptional regulators. Thus NF-B is an important transcription factor controlling both proliferation and apoptosis in the epithelium during pregnancy (Clarkson and Watson 1999).

The C/EBP family of proteins appear to be important regulators of alveolar morphogenesis. (For review see Grimm and Rosen 2003). C/EBP and C/EBP isoforms are increased during pregnancy and decline during lactation, indicating that they play a critical role in alveolar morphogenesis, and early milk gene expression. Transplantation experiments have revealed that C/ebp is required in epithelial cells for normal lobuloalveolar development during pregnancy, and C/EBP knockout mice display phenotypes similar to Pgr, Prlr, Stat5a/b, Ccnd1, Id2 and RankL knockouts (Grimm and Rosen 2003). Interestingly Pgr expression was dramatically increased in the mammary glands of C/EBP null mice, and in addition, the expression of Pgr was unusually uniform within the epithelium (Seagroves et al. 2000). These effects were associated with a 10-fold decrease in the rate of proliferation. There was however no change in the expression of C/EBP in the mammary glands of Pgr knockout mice, indicating that C/EBP is upstream of Pgr, and possibly controls the spatial distribution of epithelial cells, which influence proliferation in alveolar progenitors (Seagroves et al. 2000). C/EBP null epithelium significantly increased TGF and Smad2 signalling, and this pathway is known to inhibit cellular proliferation (Barcellos-Hoff and Ewan 2000). Cell cycle progression in C/EBP null MECs was blocked at the G1/S transition, preventing these cells from proliferating in response to early pregnancy levels progesterone and estrogen (Grimm et al. 2005). Therefore, C/EBP is essential for controlling cell fate decisions within the mammary gland, including attenuating Pgr expression resulting in mammary epithelial cell differentiation during pregnancy.

The expression of the Ets transcription factor sub family Pea3, are elevated at the onset of pregnancy but decline during mid pregnancy to low levels at lactation and involution suggesting a role in early pregnancy induced ductal outgrowth. There are 3 members of the Pea3 subfamily, which are expressed by both the myoepithelium and the luminal cells, however their expression varies during pregnancy suggesting multiple signalling roles during alveolar morphogenesis. The expression of all members of the family remains in the myoepithelium during pregnancy, however the expression of the ER81 member declines in the luminal epithelium 7 days after impregnation. Increased numbers of dividing cells was observed in the terminal end buds of Pea3 knockout mice, and mammary gland transplants of Pea3 knockout epithelium displayed reduced mammary branching during pregnancy suggesting a role for Pea3 in progenitor cell differentiation (Kurpios et al. 2003).

Other factors involved in alveolar morphogenesis

The receptor tyrosine kinase, ErbB (epidermal growth factor) family and their ligands, are important mediators of all aspects of mammary development. There are four receptors: EGFR/ErbB/Her1, ErbB2/Her2/neu, ErbB3/Her3 and ErbB4/Her4, which are activated by a variety of ligands inducing activation via dimerisation and cross phosphorylation. ErbB ligands share a 50 amino acid domain, which is homologous to EGF. Mice expressing a truncated dominant negative allele of ErbB2 did not exhibit a phenotype until late pregnancy where alveoli failed to expand and distend, indicating that ErbB2 is critical for secretory activation. Conditional deletion of ErbB4 within the mammary gland at pregnancy demonstrated a critical role for this receptor during alveolar morphogenesis (Long et al. 2003). Alveolar expansion was reduced from 13.5 days post coitus in mammary epithelium lacking ErbB4 resulting in incomplete alveolar development and failure to nurse pups due to reduced milk gene expression. Alveolar proliferation was attenuated and Stat5 phosphorylation was abolished. The ErbB4 ligand Neuregulin/Heregulin-1 (Nrg) promotes lobuloalveolar development and the expression of milk genes when used in

mammary gland explants (Yang et al. 1995), indicating a role for this ligand in lobuloalveolar development. In addition, mice that lack the alpha form of Nrg show a similar phenotype to ErbB4 knockout with reduced alveolar proliferation and differentiation, demonstrated by reduced -casein expression in reduced alveoli expansion (Li et al. 2002).

Other ErbB ligands also appear to have overlapping functions for mammary gland development. Amphiregulin (AREG) null animals have reduced alveolar development, however the phenotype was much more severe in a triple mutant including knockouts of TGF and EGF (all ligands of the ErbB family), indicating overlapping and compensatory roles for these ligands during alveolar morphogenesis (Luetteke et al. 1999). Triple mutants developed poorly organised and differentiated alveoli, had reduced milk protein expression and often pups born to these mice did not survive. AREG loss was also associated with reduced Stat5 phosphorylation. Our transcript profiling experiments demonstrated that AREG was down-regulated in Prlr-/- epithelium (Ormandy et al. 2003), indicating that AREG may be modulated by Prlr signalling. These data together indicate important roles for the ErbB receptors and ligands during alveolar morphogenesis. The overlapping phenotypes observed in Prlr, Pgr and ErbB knockout mice suggest there may be some cross talk between these receptors, which is yet to be fully understood.

The cell surface receptors, 1-integrins, which are present on luminal epithelial cells, are essential mediators of ECM signalling via its ligands collagen and laminin (Zutter et al. 1998). Mammary epithelium in mice lacking 1-integrin in the luminal cells, displayed reduced proliferation and alveolar disorganisation (Li et al. 2005). The focal adhesion kinase, which is important in protein complexes that connect the ECM to the actin cytoskeleton was also reduced in these mice. Conditional deletion of 1 integrin during early pregnancy and late pregnancy demonstrates that this molecule was important for both the formation of lobuloalveolar structures and for functional

differentiation (Zutter et al. 1998). In these mammary glands, luminal epithelium becomes dissociated from the basement membrane, and cellular polarity is compromised as luminal epithelial cells protrude into the alveolar luminal space. In addition, Prl stimulated milk protein expression via phosphorylation of Stat5 was largely absent in primary mammary epithelial cells lacking 1-integrin, indicating that it is essential for Prl-induced activation of Stat5 (Naylor and Streuli 2006).

The cytokine transforming growth factor-1 (TGF1) is an important regulator of mammary cell proliferation during pregnancy (Barcellos-Hoff and Ewan 2000). TGF1 is restricted to the luminal epithelial cells and can control cell proliferation via phosphorylation of Smad following Tgf- receptor activation (Massagué and Chen 2000). TGF1 heterozygote mice display accelerated lobuloalveolar development due to increased proliferation, indicating that the expression of TGF1 restricts alveolar cell proliferation. Epithelial cell proliferation was increased more than 15-fold in TGF1 null ovariectomised animals treated with E and Pg compared to wildtype mice (Ewan et al. 2002). In animals treated with E and Pg, TGF1 expression was restricted to the steroid receptor positive epithelial cells, indicating that TGF1 may play an important role in restricting epithelial cell proliferation in these cells (Ewan et al. 2005). Some of the major proteins and pathways involved in the molecular control of alveolar morphogenesis are depicted in Figure 1.6.

Figure 1.6 Molecular control of alveolar morphogenesis. Signalling from the progesterone receptor (Pgr) and prolactin receptor (Prlr) is essential for alveolar morphogenesis in pregnancy. Increases in serum progesterone (Pg) and prolactin (Prl) result in luminal proliferation during early pregnancy, which continues throughout gestation. Wnt4 and RankL are transcribed in response to Pgr signalling in co-operation with Prl signalling, and appear to stimulate proliferation of neighbouring cells via paracrine mechanisms. RankL binds to its receptor Rank in a neighbouring cell and activates the RankL/NF-B pathway, resulting in cyclin-D1 (Ccnd1) transcription and proliferation. Wnt4 binds and activates its target -catenin, which has specific roles for both luminal and myo-epithelium for cell fate decisions involving proliferation and differentiation. Prl binds to Prlr and activates Jak2/Stat5 cascade, resulting in transcription of various genes, including milk proteins essential in lactation and proteins that regulate their own pathway (Socs proteins). Laminin in the extracellular matrix binds to 1-integrin when contact between the basement membrane and the luminal epithelium is established, and is essential for the maintenance of alveolar cell polarity and differentiation. ErbB receptors and their ligands complement Prlr signalling as activation of ErbB4 results in Stat5 phosphorylation and translocation to the nucleus. (GJ = gap junction; TJ = tight junction; L = lipid droplet).

The Ets Transcription Factor Family and Elf5

The E26 transformation-specific (Ets) family is one of the largest families of transcription factors, and is unique to metazoans. Family members are identified by a highly conserved DNA binding domain (the Ets domain), which is a winged helix-turn-helix structure, and binds to sites containing a central GGA motif (Sharrocks et al. 1997). Many Ets transcription factors contain a pointed domain, which serves as a protein-protein interaction motif, most contain a transcription activation domain, and some possess a repressor domain (Sharrocks 2001). The Ets domain has been highly conserved in both sequence and function between species, and it is the regions outside of this domain that vary more widely between family members, and which are likely to contribute to functional differences between Ets proteins. For example, most Ets proteins transactivate target promoters, while some are transrepressors. Overall, the Ets family is divided into 11 subfamilies based on their structure, which are listed in Table 1.1 on the following page.

Ets functional activity is modulated at multiple levels. It is dependent on specific protein interactions, and signalling pathways, such as the MAP kinases (ERK, p38 and JNK), converging on the Ets transcription factors to regulate their DNA-binding activity, subcellular localisation, transactivation potential and specification of downstream target genes (Seth and Watson 2005). The end result is that with over 400 genes identified as potential targets of the 27 different human Ets genes (Seth and Watson 2005), this family of transcription factors plays a critical role in the regulation of normal biological processes. In particular, these include differentiation, proliferation, migration, hormone responses and tumorigenesis of multiple tissues (reviewed in (Oikawa and Yamada 2003; Hsu et al. 2004; Gutierrez-Hartmann et al. 2007)).

Table 1.1 The Ets family of transcription factors. The subfamilies, their respective members and a schematic of the arrangement of their functional domains are depicted. (AD – transcriptional activation domain; ETS – DNA binding domain; RD – transcriptional repressor domain). Adapted from (Gutierrez-Hartmann et al. 2007).

Subfamily Members Structure ETS Ets-1, Ets-2, Pointed

ERG Erg, Fli1, Fev

ELG Gabp, Elg

ELF Elf1, Nerf/Elf2, Mef/Elf4

ESE ESE-1/ESX/Elf3, ESE-2/Elf5, ESE-3/EHF ERF ERF/PE2, ETV-3/PE1

TEL Tel/ETV-6, Tel-2/ETV-7

PEA3 PEA3/E1AF/ETV-4, ERM/ETV- 5, ER81/ETV-1, ER71/Etv-2 SPI PU.1/SP1, SPIB, SPIC

TCF Elk-1, SAP1/Elk-4, NET/SAP2/Elk-3, LIN PDEF PDEF/SPDEF/PSE

Our lab became interested in Ets transcription factors after identifying Elf5 (e74-like factor 5 or ESE-2) as showing reduced expression in response to a loss of Prlr, in addition to up-regulation in a cell based model of positive prolactin action (Harris et al. 2006). Elf5 is an epithelial specific member of the Elf subfamily of Ets transcription factors, and is closely related to the epithelial specific Elf3 (ESE-1) and Ehf (ESE-3) (see Table 1.1; (Zhou et al. 1998; Oettgen et al. 1999). The predicted protein products of mouse Elf5 and human ESE-2 are 95% identical and are expressed as 2 isoforms produced by alternative start sites. Such high conservation of sequence implies similar conservation of function (Zhou et al. 1998).

Elf5 is expressed specifically in the luminal cells of mammary tissue (Harris et al. 2006), and its expression is increased dramatically during pregnancy, to levels that far exceed those seen in other tissues. Elf5 can also bind to an Ets like domain in the proximal promoter of Whey acidic protein (Wap) and induce its expression independently of lactogenic hormones, indicating that Elf5 may be an important mediator of alveolar differentiation during mid-pregnancy (Thomas et al. 2000). Elf5 knockout (Elf5-/-) mice die in utero due to a placentation defect (Donnison et al. 2005). Elf5 heterozygous (Elf5+/-) mice did not lactate due to failed alveolar development, and in some mice where alveoli had formed, differentiation into functional secretory units was severely impaired (Zhou et al. 2005). Mammary epithelial cell proliferation was reduced throughout alveolar morphogenesis and secretory activation, and mammary epithelial transplants demonstrated that this effect was cell autonomous. The level of Elf5 expression is reduced in Prlr+/- glands, however there is no similar reduction in the expression of Prlr in Elf5+/- glands, indicating that Elf5 is downstream of the Prlr (Zhou et al. 2005). MECs from Prlr-/- mammary glands fail to form lobuloalveoli during pregnancy when transplanted into the cleared fat pad of hosts with a normal endocrine milieu. Retroviral re-expression of Elf5 in Prlr-/- MECs followed by transplantation to a cleared fat pad resulted in a rescue of alveolar morphogenesis. MECs expressing high levels of Elf5 proliferated and differentiated into distended, milk filled alveoli (Harris et al.

2006). Thus re-expression of Elf5 in Prlr-/- MECs could completely compensate for the loss of the Prlr signalling cascade. Prlr-/- MECs expressing lower levels of Elf5 showed development that passed alveolar formation but failed during secretory initiation, mimicking the situation seen in Elf5+/- and Prlr+/- mice. Elf5 is a key mediator of structural and functional development of lobuloalveoli (Harris et al. 2006). Elf5 would thus appear to be a master-regulator of the alveolar switch required for alveolar morphogenesis.

So far, the mechanism of Elf5 action remains largely unknown. Elf5 is a critical regulator of milk protein synthesis, as recently demonstrated where the knockout of Elf5 in the epithelium results in almost no expression of milk proteins during pregnancy, and conversely, overexpression of Elf5 in an inducible transgenic model caused precocious development and milk secretion in virgin mice (Oakes et al. 2008 – See Appendix II), however how this occurs remains to be determined. In the murine mammary epithelial cell line, HC11, Stat5 has been shown to modulate Elf5 transcription (Oakes 2006), and Elf5 appeared to be downstream of Stat5. The Elf5 promoter has several Stat5 binding sites, or ‘GAS elements’, and it likely that Stat5 dimers translocate to the nucleus and bind to the GAS element of the Elf5 promoter to induce its transcription, although this has not yet been demonstrated explicitly (Hennighausen and Robinson 2008). The molecular mechanisms downstream of the Prlr which lead to Elf5 expression, and subsequently, those downstream of Elf5 which lead to milk protein synthesis requires further investigation. One of the specific aims of this thesis is to further understand the role and mechanism of Elf5 action in human normal mammary and breast cancer cell lines.

Prolactin and Progesterone in Mammary Carcinogenesis

Prolactin has often been implicated in the initiation and/or progression of breast cancer (Clevenger et al. 2003). For example in human studies, it has been shown that high serum Prl levels positively correlate with a 30% increased risk of breast cancer in women, and an 80% increased risk if the tumors were ER/Pgr positive (Hankinson et al. 1999; Tworoger et al. 2004; Tworoger et al. 2007). In a separate study, elevated serum Prl levels correlated with tumor size and stage, and risk of developing metastatic disease (Bhatavdekar et al. 2000). Studies carried out in rodent models have shown that raised serum prolactin is a complete carcinogen (Welsch and Nagasawa 1977). More recent approaches include the generation of mice which overexpress Prl within MECs under the control of a hormonally non-responsive promoter, neu-related lipocalin, and the demonstration that the majority of these transgenic mice developed mammary carcinomas, compared to only 5% of non-transgenic controls (Rose-Hellekant et al. 2003). Furthermore, the presence of the Prlr in the mammary epithelium is important for the early progression of pre-invasive lesions to invasive carcinoma (Oakes et al. 2007). In addition to this, in vitro studies have shown that Prl increases cell proliferation (Liby et al. 2003; Chakravarti et al. 2005), and inhibits apoptosis (Clevenger et al. 2003) in breast cancer cell lines. In contrast, local overexpression of human Prl in a differentiating murine mammary gland has been shown to induce morphological and functional defects, but not carcinoma (Manhès et al. 2006), and Prl can suppress invasion capacity of breast cancer cells (Nouhi et al. 2006). The mechanisms behind these observations are not clear, and so although Prl was at one time thought to directly induce breast cancer, the involvement of Prl in mammary carcinogenesis remains a controversial subject (Goodman and Bercovich 2008).

Produced in many tissues and acting in various mechanisms and roles, the involvement of Prl in carcinogenesis could occur by any number of ways. One

direct mechanism could involve the stimulation of cell proliferation and induction of cyclin D1 expression and MAPK activation by Prl, as previously described. This mitogenic action of Prl, combined with its role as a pro-survival factor and inhibitor of apoptosis (Clevenger et al. 2003; Perks et al. 2004), indicate possible methods by which Prl may contribute to carcinogenesis. Alternatively, perhaps Prl has more of an indirect role in carcinogenesis via modulation of the action of other hormones. For example, exogenous Prl modulates the expression of Pgr by human breast cancer cells (Ormandy et al. 1997b), while endogenous Prl can modulate ER levels (Gutzman et al. 2004). Prl may also act by transactivation or interaction with growth factor or receptor tyrosine kinase signalling pathways during carcinogenesis. Much attention has been paid to the role of EGF signalling in breast tumorigenesis (reviewed in (Gschwind et al. 2004)), and synergy between this pathway and Prl signalling has recently been demonstrated. Prl treatment has been shown to induce tyrosine phosphorylation of EGFR and ErbB2, leading to the activation of ERK/MAPK in breast cancer cell lines (Huang et al. 2006). Also, Prl potentiated the ability of TGF, a ligand of EGFR, to induce mammary neoplasia in a transgenic mouse model co-expressing both genes (Arendt et al. 2006). This observation was associated with increased ERK/MAPK phosphorylation, compared to TGF alone, suggesting that these two important pathways can synergise during the development of disease.

Progesterone and its synthetic analogs has also been implicated in the etiology and pathogenesis of human breast cancer, and too, remains a controversial subject. Depending on the time of administration and the dosage used, Pg can either promote, or provide protection, from mammary tumorigenesis. This most likely reflects whether Pg is exerting proliferative or anti-proliferative effects, dependent on the cell context (Musgrove et al. 1991; Groshong et al. 1997; Lange et al. 1999). For example, the addition of progestin to estrogen in hormone replacement therapy (HRT) can increase the risk of breast cancer, compared with E alone (Ross et al. 2000; Schairer et al. 2000; Campagnoli et

al. 2005). Also, Pg can reverse the anti-tumorigenic effects of the antiestrogen, tamoxifen, via a Pgr-mediated mechanism (Robinson and Jordan 1987), and ablation of Pgr is associated with a protective effect against breast carcinogenesis in mice (Lydon et al. 1999). Conversely, the strong protective factor against breast cancer provided from a first full-term pregnancy occurring at an early age, can be recapitulated in rodents by low dose treatments of E plus Pg (Sivaraman and Medina 2002). Neither E nor Pg will induce this response alone. In the normal adult mammary gland, Pgr is expressed by approximately 10% of epithelial cells, which are non-proliferating (Clarke et al. 1997), however in breast cancer proliferating cells can also be Pgr-positive, suggesting that signalling pathways involved in normal mammary development are reactivated during cancer progression (Lange 2008).

In summary, accumulating data indicate that Prl and Pg are involved in the etiology of at least some breast tumors, although at which point in the carcinogenic process they exert their effects is not known, and further studies must be conducted in order to confirm this. Therefore, it is of importance that we continue to investigate the target genes and signalling networks downstream of these hormones, in order to further understand the precise mechanisms which regulate normal mammary development, and potential carcinogenesis.

Project Aims

As outlined above, a combination of pituitary and ovarian hormones tightly regulate normal mammary gland development and influence breast tumorigenesis, however the mechanisms that underlie these actions remain to be elucidated. The challenge now is to dissect the various signalling networks and delineate the different mechanisms downstream of these hormones, in order to increase our overall understanding of how they regulate mammary gland function. The broad aim of this thesis is to therefore investigate the role

of novel genes downstream of the Prl- and Pg-signalling pathways in normal mammary gland development and human breast cancer.

From transcript profile experiments previously carried out in our lab (Harris et al. 2006), I have chosen to investigate two putative Prl-regulated genes, which were identified as displaying decreased expression in Prlr-/- mammary epithelium, and increased expression in response to Prl in a cell culture model. One of these genes is the Ets transcription factor, Elf5, which has become the focus of a large body of work within our laboratory. Elf5 is required by the epithelium for full lobuloalveolar development (Zhou et al. 2005), and work conducted in our lab has since demonstrated that (1) Re-expression of Elf5 in Prlr-/- MECs can rescue failed alveolar development, (2) Elf5-/- mice fail to lactate during lactation, and (3) Elf5 overexpression in an inducible transgenic model caused precocious development and milk secretion in virgin mice (Harris et al. 2006; Oakes et al. 2008). These observations, amongst others, illustrate a critical role for Elf5 in the Prl-induced differentiation of the mammary gland.

The work in our laboratory has so far revolved around the investigation of Elf5 function in the normal development of the murine mammary gland. In this thesis, I have investigated the role of Elf5 in human breast cancer and normal mammary epithelial cell lines, in order to determine whether this essential function of Elf5 in the mouse can be translated to the human, and importantly, whether this has any implications in the progression of mammary carcinogenesis. In Chapter Three, we examine the role of Elf5 in the immortal human mammary epithelial cell line, MCF-10A. When cultured on basement membrane, this cell line forms spherical acini and is useful for modelling of normal in vivo mammary development. We investigated the effect of overexpression of Elf5 in MCF-10A cells, and the mechanism involved. In Chapter Four, we examine the role of Elf5 in a human breast cancer cell line, T47D. This steroid receptor-positive cell line has high levels of endogenous Elf5 expression, and we utilise it to further characterise the hormonal regulation of

Elf5, and the effect of suppressing Elf5 expression by RNA interference technology.

Another specific aim of this thesis was to characterise a completely novel gene identified from the transcript profile screen. The second molecule I chose to investigate was the EST, AI850846. This EST corresponded to a protein that was later identified and named KIBRA. In Chapter Five, we describe the generation and construction of reagents, which we subsequently used to characterise the expression of KIBRA in the mammary gland and mammary cell lines. In this chapter, we then go on to investigate the effect of hormonal stimulation on KIBRA expression, and the effect of KIBRA overexpression on the growth of normal human mammary cells and breast cancer cell lines. Following on from this work, the aim of Chapter Six was to investigate the function of KIBRA in the mammary gland. We did this by identifying putative interacting partners of KIBRA using a bioinformatic approach, followed by validation of these interactions and further characterisation of the functional consequences. Overall, the work presented in this thesis aimed to further characterise the role and function of two genes downstream of Prl-signalling in the mammary gland, in order to improve our understanding of the mechanisms behind the hormonal influences of normal development and potential tumorigenesis of the mammary gland.

Chapter 2: Materials and Methods

DNA Methods

Expression vector construction

The human KIBRA cDNA was cloned from HEK-293 cells by PCR into the Gateway entry vector pDONR221 (Invitrogen Life Technologies, Carlsbad, CA) using Forward Primer 5’-GGGG ACA AGT TTG TAC AAA AAA GCA GGC TGG AAG ATG CCC CGG CCG GAG C-3’ and Reverse Primer 5’-GGGG AC CAC TTT GTA CAA GAA AGC TGG GTA GAC GTC ATC TGC AGA GAG AGC TGG -3’. Similarly, two overlapping fragments (WW and C2) of the KIBRA cDNA were cloned into pDONR221 by PCR. Fragment 1, containing the two WW domains, was amplified using the Forward Primer described above, and 5’- GGGG AC CAC TTT GTA CAA GAA AGC TGG GTA TTA CGA TAC GGC GGC TGA GAC ACA GG-3’. Fragment 2, containing the C2 domain, was amplified using 5’- GGGG ACA AGT TTG TAC AAA AAA GCA GGC TTC ACC ATG GAG AGG GAC CGG CTG ATC CTT ATC-3’ and the Reverse Primer described above. The KIBRA cDNA and WW and C2 fragments were then cloned into the mammalian Gateway expression vector pcDNA-DEST40 (CMV/C-terminal V5-6xHis) by recombination (Invitrogen). Human KIBRA cDNA was also subcloned into the NotI site of the untagged expression plasmid pcDNA3.1(+) via the pGEM-T Easy Vector System 1 (Promega) using primers KIBRA5’Forward (GG AAG ATG CCC CGG CCG GAG C) and KIBRA3’Reverse (C TTT TCT GGC GAT TAG ACG TCA TCT GC).

The retroviral vector pMIG has an LTR promoter and expresses a bi-cistronic messenger RNA encoding both the gene of interest and green fluorescent protein (GFP), using an IRES (internal ribosome entry site) (Van Parijs et al. 1999). The Elf5 gene was amplified by PCR with a C-terminal V5 tag using primers hElf5-V5 F (GGA TCC ATG TTG GAC TCG GTG ACA CAC) and hElf5-V5 R (GAA TTC TCA CGT AGA ATC GAG ACC GAG) and was cloned into the pMIG

vector. The KIBRA cDNA was also cloned by recombination (Invitrogen) into the retroviral vector, pMIG, which was converted into a Gateway expression construct by Dr Liz Caldon (Cancer Program, Garvan Institute). A retroviral single vector system that enables Doxycycline regulated RNAi (pHUSH) or transgene (ProEx) expression was obtained from Genentech Inc., San Francisco, USA (Gray et al. 2007). A fellow student in our lab, Maria Kalyuga, generated the inducible transgenic Elf5-V5-ProEx and shRNA Elf5-pHUSH constructs according to the methods detailed in Gray et al., 2007.

All new constructs generated were verified by sequencing (Australian Genome Research Facility, University of Queensland, St Lucia, Qld, Australia). The pRK5-DDR1b expression plasmid was kindly provided by Dr A. Ullrich, Max Planck Institute, Germany. The pRcCMV-PKC expression plasmid was kindly provided by Dr T. Biden, Garvan Institute, Australia. The hPRB-1, pMSGluc and pRcCMV-SRC-1 vectors were kindly provided by A. Russell, Garvan Institute, Australia, and pCMV was a kind gift from Dr T. Brummer, Garvan Institute, Australia.

Site-directed mutagenesis

The DDR1 mutants were constructed by site-directed mutagenesis with the pRK5-DDR1b plasmid as a template using the QuikChange II Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA, USA) according to the manufacturer’s instructions. For each mutant, two complementary synthetic oligonucleotides were designed of which one is indicated: AAPY mutant, 5’-GGT CCT AGA GAG GCA GCC CCG TAC CAG GAG-3’, AAAA mutant, 5’-GA GAG GCA GCC GCG GCC CAG GAG CCC CGG-3’ and PPPF mutant, 5’GGT CCT AGA GAG CCA CCC CCG TTC CAG GAG-3’. The mutated plasmids were transformed into E. coli XL1 Blue Supercompetent cells (Stratagene), and the mutants were verified by sequencing of the plasmid DNA.

Cell Culture Methods

Cell lines

HEK-293 cells

HEK-293 (human embryonic kidney) cells were maintained in Minimum Essential Media (MEM) supplemented with 10% foetal bovine serum (FBS) (15- 010-0500V, Thermotrace, Melbourne, Australia) and 0.5 g of sodium bicarbonate per litre.

MCF-10A cells

MCF-10A cells and MCF-10A-EcoR cells (expressing the ecotropic retroviral receptor; a generous gift of Drs. Danielle Lynch and Joan Brugge) were maintained in monolayer cultures, as previously described (Debnath et al. 2003). The primary culture medium consisted of DMEM/F12 (Invitrogen) with 5% horse serum (Invitrogen), 20 ng/mL recombinant human EGF (236-EG; R&D Systems, Minneapolis, MN, USA), 0.5 g/mL hydrocortisone (H-0888; Sigma, Castle Hill, NSW, Australia), 100 ng/mL cholera toxin (C-8052; Sigma), 10 g/mL bovine insulin (I-1882; Sigma), 50 units/mL penicillin G (Invitrogen), and 50 g/mL streptomycin sulfate (Invitrogen).

T47D and BT474 cells

T47D cells were cultured in RPMI 1640 medium supplemented with 10% fetal calf serum (Thermotrace, Noble Park, Vic., Australia) and insulin (10 mg/mL, Actrapid, Novo Nordisk, Baulkham Hills, NSW, Australia). T47D-EcoR cells for retroviral infection were generated as described in (Musgrove et al. 2001).

Other cell lines

A panel of normal and immortalised breast epithelial cell lines and breast cancer cell lines were cultured and maintained within the Cancer Research Program, Garvan Institute of Medical Research.

Transient transfections

Semiconfluent HEK-293 cells were transiently transfected using FuGENE reagent (Roche Diagnostics, Castle Hill, NSW, Australia) according to the manufacturer’s instructions. After 48 h cells were harvested for western blot analysis or immunoprecipitation experiments, as appropriate.

Production of retrovirus

Sixty micrograms of Elf5-V5-pMIG or control pMIG plasmid DNA was transfected into PlatE-EcoR cells, an ecotropic packaging cell line (Morita et al. 2000), using Fugene reagent. The cells were incubated at 37°C and the medium changed 24 h post-transfection. Retroviral supernatants were harvested 24 h later, passed through a 0.45 m filter, and either used for infection immediately or snap frozen and stored at -80°C for later use.

Infection of MCF-10A cells

Low passage MCF-10A/EcoR cells were plated at low density (5 x 105 cells per 10 cm dish) one day prior to infection. The next day the medium was removed, and Polybrene (8 g/mL; Sigma, Castle Hill, NSW, Australia) was added to a 1:3 dilution of the virus, which was added to the cells. The infection was repeated 24 h later. These cells were then expanded and expression confirmed by Western blotting. Cells infected with the pMIG, Elf5-V5-pMIG,

KIBRA-pMIG virus were sterile sorted by flow cytometry to obtain a GFP positive population, and these populations were subsequently sorted into low, medium and high expressing populations. Cells infected with the Elf5-V5-ProEx virus were maintained in culture a further 48 h before 7 days of selection using 2 μg/mL puromycin. Efficiency of selection was tested by Western blotting of protein lysates, which were harvested from cells that had been treated with Doxycycline (Clontech, Palo Alto, CA, USA).

Three-dimensional (3D) culture

The establishment of 3D cultures of MCF-10A cells was carried out as described in Debnath et al. 2003. Pre-chilled chamber slides (BD Falcon, Castle Hill, NSW, Australia) were thinly coated with Growth Factor Reduced Matrigel (Product #354230; BD Biosciences, North Ryde, NSW, Australia). MCF-10A cells were resuspended in Assay Medium (the same as the primary culture medium previously described, but with 5 ng/mL EGF and 2% Matrigel). This cell resuspension was syringed and overlaid at a density of 3000 cells per well of an 8-well chamber slide. This time point was taken as Day 0, and the medium was changed every 3-4 days.

siRNA duplexes and transfection

For knockdown of mKibra in HC11 cells an siRNA duplex was designed using the algorithm as described in Reynolds et al, 2004 (Reynolds et al. 2004). Double-stranded RNA oligonucleotides against the target sequence GCUUCACUGACCUCUAUUA were synthesised by Dharmacon Research Inc. (Layfayette, Colorado, USA). For knockdown of hKibra in MCF-10A and T47D cells, four siRNA duplexes (KIBRA siRNA #1; D-014058-1, KIBRA siRNA #2; D- 014058-02, KIBRA siRNA #3; D-014058-03 and KIBRA siRNA #4; D-014058- 04) were purchased from Dharmacon. For knockdown of hElf5 in T47D cells, four siRNA duplexes (Elf5 siRNA #1; D-011265-1, Elf5 siRNA #2; D-011265-

02, Elf5 siRNA #3; D-011265-03 and Elf5 siRNA #4; D-011265-04) were also purchased from Dharmacon. Control siRNAs used were siGFP and RISC-Free siRNA (Dharmacon).

The day before transfection cells were plated in 6-well plates at approximately 1.5 x 105 cells per well. siRNA duplexes were transiently transfected using Lipofectamine 2000 (Invitrogen), according to the manufacturer’s instructions. Cells were harvested at 2 days post-transfection to check for mRNA and/or protein levels, unless otherwise specified.

Ligand stimulation of cells

Progesterone stimulation experiments involved treating T47D cells with 10 nM ORG2058 (16a-ethoxy-21-hydroxy-19-norpregn-4-en-3,20-dione), which was obtained from Amersham Biosciences Rydelmere, NSW, Australia) or 100 nM RU486 [17 -hydroxy-11-beta-(4-methylaminophenyl)-17--(1-propynyl)- estra-4,9-diene-3-one-6-7], which was generously provided by Dr J-P Raynaud of Roussel-Uclaf, Romainville, France. Estradiol (3,17-Dihydroxy-1,3,5(10)- estratriene) was obtained from Sigma. Human growth hormone was kindly provided by Prof Ken Ho (Garvan Institute of Medical Research). For collagen treatment, HEK-293 and MCF-10A cells were serum starved overnight, then treated with 10 μg/mL of collagen type IV (human placenta; Sigma, Castle Hill, NSW, Australia) or collagen type I (rat tail; Sigma) for 90 min, unless otherwise specified.

Progesterone receptor transactivation assay

HEK-293 cells were plated in six-well plates (2 x 105 cells/well) and 24 h later were transfected with Fugene with 90 ng of hPRB-1, 450 ng of pMSGluc, and 1.2 g of KIBRApcDNA3.1, pcDNA3.1 or SRC-1, and 200 ng of pCMV. The following day cells were treated with 10 nM ORG2058 and harvested 48 h later in 50 L -galactosidase lysis buffer (Galacto-Star, Tropix, Bedford, MA, USA)

and assayed for luciferase activity (LucLite reagent, Packard Bioscience, Meriden, CT), and corrected for transfection efficiency by carrying out - galactosidase assays (Galacto-Star, Tropix), according to the manufacturers' instructions.

Cell proliferation assays

Growth curve analysis

Cells were plated at low density in 12-well or 6-well plates and trypsinised at the timepoints indicated. After neutralisation in harvest medium, the cells were washed with PBS and resuspended in 1 mL of harvest medium (or as appropriate). Cell number was quantitated using haemocytometer counting. Relative proliferation of cultures was analysed by plotting cell number on a log scale.

MTT assays

Cell proliferation was determined by the MTT CellTiter96 Assay (Promega Corporation, Madison, WI, USA) following the manufacturer’s instructions. Cells were plated in 96-well plates at 5000 cells per well with 6 replicates per sample. Proliferation was assessed by measuring the absorbance of cells relative to that of control wells (without cells) using 490-nm wavelength on a spectrophotometer.

Flow Cytometry

Cells grown in monolayers were trypsanised and then neutralised with harvest medium. Cells that were to be sorted for GFP expression levels were analysed immediately at the Flow Cytometry Core Facility (Garvan Institute) on a BD FACS AriaTM Cell Sorter (BD Biosciences). Cells that were to be analysed for

DNA content were collected by centrifugation at 250 g (5 min at room temperature) and resupended in 1 mL of cold PBS. The resuspended cells were added to 9 mL of 70% ethanol for fixation and then incubated at 4oC at least overnight prior to FACS analysis. On the day of analysis, cells were spun down (250 g; 5 min) and the ethanol was aspirated so as to leave 1 mL remaining. Cells were resuspended in this 1 mL of ethanol and counted using a haemocytometer. Approximately 7 x 105 cells of each sample were aliquotted into a fresh tube and collected by centrifugation, as before, and the ethanol aspirated. Cells were resuspended in 500 L cold PBS/1% Tween while gently vortexing, followed by staining with approximately 5 L of 1 mg/mL propidium iodide (PI) in PBS. Twenty-five microlitres of Ribonuclease A (Sigma) was also added, to a final concentration of 500 g/mL, and gently vortexed to mix. Samples were incubated in the dark at room temperature for up to 5 hours. Samples were syringed gently but thoroughly and then analysed using the FL- 2 channel on a FACSCalibur (BD Biosciences) using CELLQuest 2.0 software. The proportion of cells in the G1, S and G2/M phases of the cell cycle were calculated from the resulting DNA histograms using ModFit LT analysis software (Verity Software House, Inc., Topsham, ME, USA).

RNA Methods

Extraction, quantitation and analysis

Total RNA was extracted using the RNeasy Minikit (Qiagen, Doncaster, Vic., Australia) as described in the manufacturer’s instructions. For Affymetrix GeneChip analysis, RNA quality was assessed by RNA Nano LabChip analysis on an Agilent Bioanalyzer 2100 (Agilent Technologies, Palo Alto, CA, USA) and taken to the Ramaciotti Centre for Gene Function Analysis (UNSW) for Affymetrix GeneChip Array processing. For real-time quantitative PCR, single- stranded cDNA was produced by reverse transcription using 1 μg of RNA in a 20 L reaction using the Reverse Transcription System (Promega, Annadale, NSW, Australia) according to manufacturer’s instructions. cDNA was diluted to

o 1:5 with H20 and stored at -20 C. Quantitative PCR was performed using the TaqMan probe-based system on the ABI Prism 7900HT Sequence Detection System (Applied Biosystems, Scoresby, Vic., Australia). All reagents and Gene Expression Assays (Table 2.1) were purchased from Applied Biosystems.

Figure 2.1 Gene expression assays Gene Gene Expression Assay Elf5 (human) Hs00154971_m1 Elf5 (mouse) Mm00468732_m1 Fatty acid synthase (human) Hs00188012_m1 Desmoplakin (human) Hs00189422_m1 KIBRA (human) Hs00392086_m1 GAPDH (mouse) 4352339E

Protein Methods

Antibody preparation

Anti-KIBRA antiserum was prepared in rabbit (Invitrogen) using the synthetic peptide sequence, SAQERYRLEEPGTEGKQ, derived from the human KIBRA central portion, conjugated to keyhole limpet haemocyanin (KLH). The KIBRA IgG polyclonal antibody was purified on a peptide affinity column.

Cell lysate preparation and protein extraction

From monolayer cultures

Protein samples were prepared from cell lines lysed in ice-cold normal lysis buffer (NLB; 50 mM HEPES pH 7.5, 150 mM NaCl, 10% glycerol, 1% Triton X-

100, 1.5 mM MgCl2, 1 mM EDTA, 10 mM pyrophosphate, 100 mM NaF) containing protease and phosphatase inhibitor cocktail tablets (Roche).

Samples were incubated in lysis buffer on ice for 20 min, before being briefly vortexed and centrifuged at 17,000 g at 4oC. Supernatants were stored at - 20oC or -80oC. For analysis of KIBRA phosphorylation T47D lysates were incubated with lambda protein phosphatase (New England Biolabs) according to the manufacturer’s instructions.

From 3D cultures

To recover whole cell lysates from acini cultured in Matrigel, media was gently aspirated from culture slides, and 300 mL of BD Cell Recovery Solution (BD Biosciences) was added per well of a 4-well chamber slide and incubated for 75 min at 4°C to depolymerise the Matrigel. The cell/Matrigel suspension was then resuspended and transferred to a fresh tube, and each well washed with 200 mL cold PBS to collect any remaining cells. The suspension was centrifuged at 400 g for 10 min at 4°C and the supernatant sucked off. Residual Matrigel was removed by washing the pellet with cold PBS and centrifuging again at 400 g for 10 min at 4°C. Cell pellets were resuspended in an appropriate volume of normal lysis buffer and prepared as described above for monolayer culture protein isolation.

From mammary glands

The fourth inguinal mammary glands from Elf5-/- mice was frozen in liquid nitrogen before storage at -80°C prior to use (Oakes et al. 2008). Total RNA and DNA was extracted using TRIZOL Reagent (Gibco/Invitrogen Vic, Australia), following which protein was isolated from the phenol-ethanol supernate obtained, according to the manufacturers instructions. Protein lysates extracted using all methods were quantitated using Bradford Protein Assay Reagent (Bio-Rad), according to the manufacturer’s instructions.

Immunoprecipitation

One milligram of protein from cell lysates in a total volume of 1 mL of NLB was pre-cleared by rotating at 4oC for 1 h with 40 L of Protein-G Sepharose beads/PBS (1:1; Zymed, San Francisco, CA, USA). The beads were then removed by centrifugation (4oC, 1200 g, 10 min) and each lysate transferred to a fresh tube and incubated with antibodies (approximately 10 L) overnight at 4oC. The following morning Protein-G Sepharose beads/PBS (1:1) were added to the lysates and incubated for 1 h at 4oC. The resulting immunoprecipitates were collected by centrifugation and washed four times with NLB and 1 M NaCl, and resuspended in buffers as described below.

Western blot analysis

Immunoprecipitates and lysates (10 – 40 g) were prepared in NuPAGE LDS Sample Buffer and NuPAGE Sample Reducing Agent (Invitrogen) and separated on NuPAGE Bis-Tris acrylamide gels run in MOPS buffer (Invitrogen). Precision Plus Protein (Dual Colour) (Bio-Rad Laboratories, Hercules, CA, USA) standards were used as a molecular marker. Gels were then transferred to polyvinylidene difluoride (PVDF) membranes (Bio-Rad). The membranes were incubated overnight with the primary antibodies listed in Table 2.2. The secondary antibodies used were horseradish peroxidase-conjugated sheep anti-mouse, donkey anti-rabbit (Amersham Biosciences), donkey anti-goat (Santa Cruz) and goat anti-rat (Pierce Biotechnology, Rockford, IL, USA). Proteins were visualised by chemiluminescence (Perkin-Elmer, Rowville, Vic., Australia). Densitometry was performed using IPLab Gel software (Scanalytics Inc., Fairfax, VA, USA).

Antibodies

The following table lists all primary antibodies used, for Western analysis (WB) and immunofluorescence (IF), as well as their dilution and source. Name Source Application Dilution Elf5 Santa Cruz Biotechnology WB; IF 1:100 (Santa Cruz, CA, USA) (WB); 1:50 V5 (R960-25) Invitrogen WB; IF 1:5,000 (WB) 1:150 (IF) GFP (ab6658) Abcam (Cambridge, UK) WB 1:1,000 Laminin V (MAb Chemicon (Temecula, CA, IF 1:100 19562) USA) Bcl-xL BD PharMingen WB 1:1,000 Bcl-2 (124) DakoCytomation WB 1:100 (Carpinteria, CA. USA) Activated Cell Signaling Technologies WB 1:750 caspase-3 (Beverly, MA, USA) Bim Calbiochem (La Jolla, CA, WB 1:100 USA) EGFR (1005): Santa Cruz WB 1:1,000 sc-03 Phospho-ERK Cell Signaling Technologies WB 1:1,000 Total-ERK Cell Signaling Technologies WB 1:1,000 KIBRA N/A WB; IF; IHC 1:500 DDR1 (C-20) Santa Cruz WB 1:500 nPKC (C-20) Santa Cruz WB 1:1,000 Cyclin-D1 (clone Novacastra Laboratories WB 1:100 DCS6) Ltd., United Kingdom -actin (AC-15) Sigma WB 1:40,000

Progesterone Kindly donated by Dean WB 1:1,000 receptor (MAb Edwards, Houston, TX, 1294) USA Phospho-Tyr Upstate Biotechnology WB 1:1,000 (4G10) (Lake Placid, NY, USA)

Immunohistochemistry

4 μm tissue sections were mounted on Superfrost Plus adhesion slides (Lomb Scientific, Sydney, Australia) and heated in a convection oven at 75°C for 2 h to promote adherence. Sections were de-waxed and re-hydrated according to standard protocols. Antigen retrieval was performed using citrate EDTA buffer boiled under pressure (human cells) or Proteinase K enzymatic digestion (human and mouse tissue). Endogenous peroxidase activity was inhibited with

3% H2O2. Sections were incubated with the anti-KIBRA primary antibody for 60 min (1:200, human tissue and cells) or 30 min (1:150, mouse tissue) and bound antibody was detected using LINK/LABEL and 3,3'-diaminobenzidine Plus as substrate. With each run, diluent and rabbit IgG isotype control were the negative technical controls. Counterstaining was performed with haematoxylin and 1% acid alcohol. Cell lines were processed at the same time to enable comparison of signal strength. All immunohistochemistry reagents were obtained from DAKO Corporation (Carpinteria, CA) unless otherwise specified.

Immunofluorescence and microscopy

Fixation and staining protocol of 2D cultures

Cells were plated onto chamber slides, washed with PBS once prior to fixation, and then fixed in 4% paraformaldehyde/PBS for 15 min at room temperature.

The slides were then rinsed in PBS, permeablised in 0.2% Triton X-100, rinsed again in PBS and blocked with 1% BSA in PBS for at least one hour. Slides were then incubated in primary antibody diluted in 1% BSA/PBS overnight at 4oC. The following morning the slides were rinsed in PBS 2-3 times, cells were stained using the appropriate Cy2- or Cy3-labelled secondary antibodies (Jackson ImmunoResearch Laboratories, Pennsylvania, USA). Where required cells were counterstained with either FITC-phalloidin or TRITC-phalloidin (Sigma) as an actin counterstain, and TOPRO-3 (Molecular Probes OR, USA) as a DNA counterstain. Following 2-3 washes with PBS, the slides were then mounted in 90% glycerol/PBS ready for visualisation.

Fixation and staining protocol of 3D cultures

Cells cultured on Matrigel were rinsed with PBS once, then fixed with 2% paraformaldehyde/PBS (pH 7.4) for 20 min at room temperature. Slides were then permeabilised in 0.5% Triton X-100/PBS for 10 min at 4oC, followed by rinsing 3 times in 100 mM glycine/PBS (10-15 min per wash at room temperature). Slides were then blocked in IF Buffer (7.7 mM azide, 0.1% BSA, 0.2% Triton X-100, 0.05% Tween-20 in PBS) plus 10% goat serum for 1-1.5 h at room temperature. Following this, slides were incubated with primary antibody, where appropriate, diluted in IF Buffer plus 10% goat serum at 4oC overnight. The next morning slides were rinsed with 3 washes of IF Buffer then stained with secondary antibodies and counterstains diluted in IF Buffer plus 10% goat serum for approximately 1 h at room temperature. Following 2 rinses with IF Buffer and 1 rinse with PBS (5 min), slides were mounted with Prolong Antifade Reagent (Molecular Probes, Invitrogen) and allowed to dry overnight at room temperature. Slides were visualised the following day.

Microscopy

Immunofluorescent microscopy was carried out on a Leica DMRBE upright microscope. Imaging of cells grown as monolayers was performed using either

a 63x or 100x PL APO oil objective. Imaging of cells grown in 3D was performed using a 40x HCX PL APO oil or immersion objective.

Bioinformatic Methods

Build 33 of the human proteome, containing 37490 sequences, was downloaded from the NCBI (http://www.ncbi.nlm.nih.gov) and the protein regular expression program 'Preg' (Rice et al. 2000) was used to identify all sequences in the proteome with the PPxY and RxPPxY motifs (where P is proline, R is arginine, Y is tyrosine and x is any amino acid). The GI accession numbers from the matched sequences were used to extract annotations from the 15 June 2003 build of the Affymetrix HGU-133A annotation file. The annotations were then extended by searching batch SOURCE (Diehn et al. 2003) with the LocusLink identity numbers derived from the matched Affymetrix HGU-133A annotations. Protein expression in the mammary gland was determined from the NCBI Unigene database (http://www.ncbi.nlm.nih.gov/unigene).

Statistical analysis

Statistical analyses were performed using StatView 4.5 (Abacus Concepts Inc., Berkely, CA, USA). Differences between groups were evaluated by Fisher’s protected least significant difference test after ANOVA analysis where appropriate.

Microarray analysis

Quality of the arrays was assessed by the Ramaciotti Centre for Gene Function Analysis by measuring 5 different parameters, including the mean of the raw intensity for all of the probes on the array, the mean probeset signal for all

probesets analysed, and the mean over all probesets analysed of the absolute log expression value relative to all other arrays). The arrays were found to be of similar high quality. Normalization of the arrays was performed using the Probe Logarithmic Intensity ERror (PLIER) algorithm as implemented in the Affymetrix Power Tools environment. CEL files from all nine arrays were normalized together. All statistical analyses were carried out in the R statistical programming language (R website; www.R-package.org). The data was analysed for differential expression using LIMMA (Smyth 2005), comparing Pg- treated and Baseline cells, Elf5-KD and Baseline cells, and finally Elf5-KD and Pg-treated cells. The p-values obtained for each comparison were adjusted for multiple testing using the Benjamini-Yekutieli (BY) correction, implemented in the multtest package in R (Benjamini and Yekutieli 2001; Pollard et al. 2005).

Functional Annotation Analysis

Functional annotation analysis of sets of selected genes was carried out using the gene-enrichment annotation analysis tools within DAVID version 2008 (DAVID website; http://david.abcc.ncifcrf.gov/home.jsp). Categories analyzed included GO categories (Biological Process, Molecular Function, and Cellular Component), protein domain categories (InterPro Name, Superfamily Name, SMART Name), pathways databases (BBID, BioCarta, KEGG Pathways), functional categories (COG/KOG Ontology, Sp Pir Keywords, Up Seq Feature) and a disease category (Genetic Association Database). The BY correction for multiple testing was applied to the EASE scores, and the significance threshold set at adjusted P0.05.

Chapter 3: Effect of Elf5 Expression on MCF-10A Cells in 3D Culture

Introduction

The genes that are important for the control of mammary cell proliferation and differentiation during normal mammary gland development are also targets for mutation and dysregulated expression that can result in hyperplasia and cancer. New techniques that define this genomic regulatory network offer an unprecedented opportunity to understand the processes that build a mammary gland, and which go awry in cancer.

To investigate what genes are important in the control of mammary cell proliferation and differentiation, our lab transcript profiled mammary gland epithelial cells from the prolactin receptor knockout (Prlr-/-) mouse (Ormandy et al. 2003). In addition to a number of reproductive defects such as sterility, irregular cycles and failed blastocyst implantation, these mice display a lack of alveolar differentiation and milk secretion (Ormandy et al. 1997a). For this study, Prlr-/- and wildtype (Prlr+/+) mammary epithelia were transplanted into the cleared mammary fat pads of 4-week-old immunocompromised Rag1-/- animals. We transcript profiled the Prlr+/+ and Prlr-/- mammary epithelial transplants during early time points of pregnancy to overcome reduced epithelial cell numbers observed in Prlr-/- mammary glands at later time points and to focus our attention on early events (Harris et al. 2006). To exclude gene expression contributed by the fat pad we also transcript profiled Rag1-/- mammary fat pads cleared of endogenous epithelium. In addition, we profiled prolactin treated and control SCp2 cells, a murine mammary epithelial cell line, that secretes -casein when cultured on extracellular matrix (ECM) and treated with lactogenic hormones. A combined analysis of these different models identified genes that showed decreased expression in the Prlr-/- epithelium compared to wild type during early pregnancy and showed upregulation by prolactin treatment of SCp2 cells (Harris et al. 2006). Many of these genes were known to be important for mammary development, but we also identified

a number of novel epithelial-specific prolactin-regulated genes and expressed sequence tags (ESTs). One of these molecules was Elf5.

Elf5 is an epithelial-specific member of the large Ets transcription factor family characterised by a highly conserved DNA binding domain, the Ets domain, which binds to sites containing a central GGA motif (Sharrocks et al. 1997). Elf5 (e74-like factor 5 or ESE-2) was originally described by Zhou et al. (1998) as being isolated from an adult mouse lung cDNA library following screening with a probe containing the Ets domain of Elf3 (e74-like factor 3 or ESE-1), thus it is most closely related to Elf3. In fact, the divergent Ets domain of Elf5 shares 67% amino acid identity with that of Elf3 (Zhou et al. 1998), and is also 65% similar to ESE-3/EHF, indicating that these three Ets family members form a separate subfamily of genes (Kas et al. 2000). Mouse Elf5 and human ESE-2 are highly conserved, sharing approximately 95% identity, suggesting conservation of function (Zhou et al. 1998).

Elf5 is expressed predominantly in secretory epithelium of organs such as the mammary gland and salivary gland, as well as displaying moderate levels in the prostate, lung, and kidney (Oettgen et al. 1999). In addition, work in our laboratory has shown that in the mammary gland Elf5 expression is restricted to the luminal epithelial cells, and its expression increases dramatically during pregnancy to far higher levels than observed in other tissues (Harris et al. 2006). Elf5 has subsequently been shown to be important in mammary gland development by a number of experiments. Firstly, Elf5 heterozygote (Elf5+/-) mice show defective lobuloalveolar development and decreased milk protein gene expression during pregnancy (Zhou et al. 2005). Secondly, retroviral re- expression of Elf5 in Prlr-/- mammary glands rescued failed alveolar development (Harris et al. 2006). Elf5 can induce transcription of the milk protein gene whey acidic protein (Wap) by binding to its promoter (Thomas et al. 2000). Finally, in Elf5 knockout (Elf5-/-) mice, lobuloalveolar development is severely impaired and milk production is lost. Conversely, forced Elf5 expression in an inducible transgenic model resulted in precocious milk

secretion during pregnancy (Oakes et al. 2008). These data all lend support to the critical role of Elf5 in the differentiation of the alveolar epithelium during pregnancy.

The use of three-dimensional (3D) cell culture models are useful tools for studying the biochemical and cellular processes which occur in mammary epithelial cell morphogenesis in vitro, and which recapitulates numerous features of breast epithelium in vivo (Petersen et al. 1992; Debnath and Brugge 2005). These cell culture systems also allow for the identification and investigation of potential oncogenes and pathways in a biologically relevant context. MCF-10A cells are a widely used cell line for this in vitro modelling of developmental pathways. When grown in 3D culture, these cells form acini-like spheroids of a single outer layer of cells surrounding a hollow lumen, with apicobasal polarisation of cells and deposition of basement membrane components (Debnath et al. 2003). A schematic diagram of the biological processes that contribute to this morphogenesis is depicted in Figure 3.1. The work in our laboratory so far has focussed on investigating the role of Elf5 in normal development of the mouse mammary gland, and so the aim of the work presented here was to examine the role of Elf5 expression in human mammary epithelial cells, and to identify any potential role in carcinogenesis. For clarity, human ESE-2 will be referred to as Elf5 throughout this chapter.

The specific aims of this chapter are to: (1) Generate constitutive and inducible Elf5 overexpressing human mammary cell lines, (2) Investigate the effect of Elf5 overexpression on acinar morphogenesis in three-dimensional culture, and (3) Investigate the mechanism of Elf5 action in MCF-10A cells.

proliferation

polarisation of outer cells

survival signalling in outer cells

luminal cell death

Figure 3.1 Schematic diagram of MCF-10A acinar morphogenesis. The process is depicted over 20 days, and shows the stages of proliferation, polarisation and luminal apoptosis (Figure adapted from Debnath et al. 2003).

Results

Generation of constitutive Elf5 overexpressing stable cell lines

To investigate the effect of Elf5 overexpression in human mammary cells, we used the non-tumorigenic human breast epithelial cell line, MCF-10A, which retains many of the characteristics of normal breast epithelium and fails to exhibit growth in immune-deficient mice (Lane et al. 1999). Together with fellow PhD student, Maria Kalyuga, retroviral vectors expressing Elf5 were constructed and used to infect the MCF-10A-EcoR cell line (which stably expressed the ecotropic retroviral receptor, EcoR). This particular construct consisted of a V5-tagged Elf5 cDNA in the pMIG-IRES-GFP vector (Van Parijs et al. 1999) for constitutive Elf5 expression (Figure 3.2).

Figure 3.2 Schematic representations of Elf5-V5-pMIG vector for constitutive Elf5-V5 overexpression. LTR = long terminal repeat; IRES = internal ribsome entry site; GFP = green fluorescent protein.

The presence of the internal ribosome entry site (IRES) in the pMIG vector enables the expression of both Elf5 and GFP (Green Fluorescent Protein) controlled by one promoter, and this GFP tag allowed us to sort by FACS (fluorescence activated cell sorting) for GFP-positive populations into high, medium and low expressing populations. This corresponded to the level of Elf5 expression, as shown by the relative Elf5 mRNA levels in these sorted cell populations (Figure 3.3A). It should be noted that MCF-10A cells do not express any endogenous Elf5. Immunofluorescence and fluorescence analysis of -Elf5 and GFP respectively, showed that Elf5-V5-pMIG infected cells, which displayed higher levels of GFP did indeed express higher levels of Elf5, demonstrating the direct correlation between GFP and Elf5 levels (Figure 3.3B).

B GFP -Elf5 TOPRO overlay

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Figure 3.3 A Relative levels of Elf5 mRNA transcripts in sorted Elf5-overexpressing MCF- 10A populations B Immunofluorescent images of MCF-10A cells infected with Elf5-V5-pMIG (a – c) or control (d) retrovirus, showing GFP fluorescence and staining for Elf5 (red) and TOPRO (nucleus; blue) as labelled. Scale bars represent 40 m. 78

Investigation of the effect of Elf5 expression on the proliferation of MCF-10A cells in two-dimensional culture

To determine whether Elf5 overexpression had any effect on the growth of MCF-10A cells in 2D culture, we carried out a number of proliferation assays on cells that had been sorted for high Elf5 expression levels. These assays included growth curves, MTT assays and FACS analysis of the proportion of cells in the proliferative S phase. As illustrated in Figure 3.4, Elf5 expression had no significant effect on proliferation in the assays performed. However, as the three-dimensional culture of this cell line on a reconstituted basement membrane is commonly used for in vitro modelling of several aspects of glandular architecture in vivo, we were interested in determining whether Elf5 expression had any effect on acinar morphogenesis in these cells.

Figure 3.4 Elf5 overexpression has no significant effect on the proliferation of MCF- 10A cells in 2D culture. A Growth curve B MTT assay C Percentage of cells in proliferative S- phase as determined by FACS analysis. Error bars represent mean±SE of at least 2 independent experiments, each which consisted of 3 replicates.

Investigation of the effect of Elf5 expression on acinar morphogenesis in three-dimensional culture

The MCF-10A cells which had been infected with Elf5-V5-pMIG retrovirus (Elf5) or corresponding control virus (pMIG) were plated onto Matrigel, a commercially available reconstituted basement membrane, to examine the effect of Elf5 overexpression on 3D culture. As MCF-10A cells normally do not express any endogenous Elf5, the first thing we examined was the expression of Elf5 over the twenty-day time course of the experiment. Figure 3.5 shows that Elf5 levels were repressed over time to low levels. Expression levels of V5 and GFP also decreased accordingly. This phenomenon was due specifically to the expression of Elf5, as levels of GFP in the pMIG control cells remained high throughout the experiment (Figure 3.5A). Figure 3.5B depicts V5 protein levels in the Elf5-overexpressing cells normalised to -actin levels, as measured by densitometric analysis. Repression of expression from transgenic cassettes via methylation and other epigenetic mechanisms acting on cassette promoter regions is an established phenomenon (Gebara et al. 1987). This could occur globally to reduce Elf5 expression, or could confer a growth advantage on a subset of cells expressing low Elf5 levels. This suppression of Elf5 expression over time was a 3D-culture specific phenomenon, as when we maintained Elf5- V5-pMIG MCF-10A cells which had been sorted for high expression in normal 2D culture over a similar time course, there was no decrease in V5 expression in these cells (Figure 3.5C).

A Day 0 Day 7 Day 10 Day 15 Day 20 P E P E P E P E P E

-Elf5

-V5

-GFP

--actin

C Day 0 Day 7 Day 12 Day 20

P E P E P E P E

-V5

Figure 3.5 Elf5 expression levels decreased over time in 3D culture, but not 2D culture. A Elf5 and pMIG control MCF-10A cells were cultured in 3D and collected at 7, 10, 15 and 20 days. Lysates were probed with antibodies to Elf5, V5 and GFP. -actin immunoblotting was performed to indicate relative loading. P = pMIG; E = Elf5. B Quantitation of levels of V5 expression (from Western blot in A) normalised to -actin in Elf5-overexpressing cells. C Protein lysates from cells grown in 2D culture were harvested at time intervals over 20 days and immunoblotted with -V5 antibody.

It is interesting to note that when we probed these lysates with the -V5 antibody, we observed two bands, whereas when we used the -Elf5 antibody we observed one band only (Figure 3.5A). The two bands observed could be due to an alternate start site downstream of the first in the Elf5 cDNA sequence, and a second start site does indeed lie 20 amino acids downstream of the first (Oettgen et al. 1999). As the V5 tag is situated at the C-terminal of the Elf5-V5 fusion protein, the -V5 antibody could therefore detect both isoforms, whereas the -Elf5 antibody, which detects an epitope at the N- terminal of the protein, would only detect a single, larger isoform.

Because of our observation of the marked decrease in Elf5 expression over time, we used populations of MCF-10A cells sorted for high Elf5 expression in these 3D culture experiments, unless otherwise specified. Upon immunofluorescence analysis of these acini growing in 3D culture, the Elf5 expressing acini appeared morphologically similar to controls at early time points (Day 7), with a polarised outer layer and normal deposition of a laminin V basement membrane (Figures 3.6 and 3.7). Therefore, the introduction of Elf5 into MCF-10A cells did not significantly alter the morphology of acini following 7 days of 3D culture.

By Days 15 and 20 of the experiment, the most striking observation was the increased proportion of irregular acini and failure to form a normal hollow lumen in the Elf5 overexpressing cells, compared to controls. Figures 3.8 and 3.9 show a selection of representative GFP fluorescent images of acini at Day 15 and Day 20, respectively. Also interesting was the appearance of a dense mass of cells in the centre of some Elf5-expressing acini, indicated in Figure 3.8. Elf5 was able to exert these effects despite the down-regulation of Elf5 expression that was observed with time (Fig 3.5).

GFP TOPRO Phalloidin Overlay

Figure 3.6 Elf5 expression had no significant effect on morphology of acini after 7 days of culture compared to controls. Immunofluorescent images of pMIG control (A) and Elf5 (B) acini counterstained with TOPRO (nucleus; blue) and Phalloidin (actin; red). Scale bars represent 40 m.

GFP Laminin V Overlay

Figure 3.7 Elf5 expression had no effect on basement membrane deposition of acini. Immunofluorescent images of pMIG control (A) and Elf5-expressing (B) acini after 7 days in 3D culture and immunostained for laminin V. Scale bars represent 40 m.

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Figure 3.8 Elf5-expressing acini exhibited delayed lumen formation following 15 days of culture. GFP fluorescent images of pMIG control (A) and Elf5-expressing (B) acini at high (a) and low (b) magnification. Scale bars represent 40 m.

Figure 3.9 Elf5-expressing acini have a defect in normal hollow lumen formation. GFP fluorescent images of pMIG control (A) and Elf5-expressing (B) acini following 20 days of 3D culture. White arrows indicate appearance of dense mass of cells in the centre of some acini. Scale bars represent 80 m.

We also carried out these 3D acinar morphogenesis assays with MCF-10A cells infected with Elf5-V5 virus and pMIG control virus, but which had not been sorted for positive GFP-expressing populations. Using these unsorted cell populations allowed for the presence of infected and uninfected acini within the same culture, providing an internal control. Again, the Elf5-expressing acini displayed a defect in normal hollow lumen formation, and these Elf5- expressing acini with filled lumens formed alongside hollow uninfected control acini, as indicated by arrows in Figure 3.10.

GFP Phalloidin TOPRO Overlay

Figure 3.10 Immunofluorescent images of unsorted MCF-10A cells infected with Elf5 retrovirus after 20 days of culture. Acini are counterstained with Phalloidin (actin; red) and TOPRO (nucleus; blue). White arrows indicate GFP-negative hollow acini. Scale bars represent 80 m.

To determine the proportion of Elf5-expressing acini which did not form a normal hollow lumen, we counted over 200 acini of Elf5-expressing acini and pMIG control acini each from two independent experiments, and tallied the number of acini with hollow lumen, partially hollow lumen, or filled lumen. After 20 days of culture, approximately 90% of Elf5-expressing acini displayed filled lumens compared to 14% of pMIG controls, whereas only 6% of Elf5- expressing acini formed a normal hollow lumen compared to 78% of controls (Figure 3.11). This data demonstrates that Elf5 expression has a significant effect on the morphology of MCF-10A acini, and causes a defect in normal hollow lumen formation.

Figure 3.11 Altered lumen formation of Elf5-overexpressing MCF-10A cells in three- dimensional culture. Acini with hollow lumen, partially hollow lumen or filled lumen were counted and plotted as a percentage of total counted. Data is based on tallying 267 pMIG control acini and 211 Elf5 acini from 2 independent experiments.

Mechanism of Elf5 action on lumen formation in 3D culture

Apoptosis is essential in forming and maintaining a hollow lumen in MCF-10A acini (Debnath et al. 2002), therefore in order to investigate the mechanism of how expression of Elf5 decreased the rate of luminal clearance, we looked at

the expression level of markers of apoptosis in protein lysates which had been collected throughout the time course. Figure 3.12 shows levels of the anti- apoptotic proteins Bcl-xL and Bcl-2, and the pro-apoptotic proteins activated caspase 3 and Bim. As reported previously, by Day 8 of culture the centrally located non-polarised cells begin to die by apoptosis (Debnath et al. 2002), and this coincided with repression of Bcl-2, and induction of activated caspase 3 and Bim (Figure 3.12). Compared to controls Elf5-expressing acini showed a significantly attenuated increase in Bim expression at Day 10. Bim is a critical regulator of luminal apoptosis and lumen formation during morphogenesis of MCF-10A acini (Reginato et al. 2005).

37 kDa

20 kDa

25 kDa

Figure 3.12 Western blot analysis of apoptotic markers in acini over 20 day time course. Protein lysates from each cell line were immunoblotted with -Bcl-xL, -Bcl-2, - activated caspase-3, -Bim, -Elf5 and --actin. P = pMIG; E = Elf5.

Anoikis is a form of programmed cell death that epithelial cells undergo upon detachment from the ECM, resulting in cells dying that lack direct contact with the basement membrane, and thus forming a hollow lumen in the MCF-10A acini (Frisch and Francis 1994; Debnath and Brugge 2005). Bim expression has been shown to be up-regulated after loss of attachment to the ECM and contribute to anoikis in MCF-10A cells (Reginato et al. 2003). While expression of Bim is induced, levels of EGFR are down-regulated during anoikis, and overexpression of EGFR was demonstrated to block Bim induction during anoikis, and consequently inhibit apoptosis (Reginato et al. 2003). To determine whether Elf5 played a role in the regulation of EGFR following loss of ECM attachment, we analysed the levels of EGFR in protein lysates, which had been collected throughout the time course. Figure 3.13 demonstrates that there was an up-regulation of EGFR at Day 10 and Day 15 in Elf5-expressing acini. This corresponded to when Bim levels were repressed in Elf5-expressing acini (Figure 3.12). Densitometry analysis was performed on Western blots probed with -EGFR and -Bim antibodies from two independent 3D culture experiments, showing that these results are reproducible (Figure 3.14). Therefore, expression of Elf5 in MCF-10A acini caused up-regulation of EGFR from Day 10 when anoikis began to occur, and subsequently caused repression of Bim expression and inhibition of lumen formation.

Figure 3.13 Western blot analysis of EGFR expression in MCF-10A acinar morphorgenesis.

Figure 3.14 Relative protein expression levels of EGFR and Bim in Elf5 and control MCF-10A cells cultured in 3D as determined by densitometric analysis. A Relative expression levels of EGFR in pMIG and Elf5-expressing MCF-10A cells harvested at day 10 and day 15, as indicated. B Relative expression levels of Bim in cells harvested at day 10. Data generated from densitometry of Western blots. Data was normalised to -actin and error bars represent range of mean of two independent experiments.

Generation of inducible Elf5 overexpressing stable cell lines

To determine the time period during which MCF-10A cells were sensitive to Elf5 we constructed a second retroviral vector that enabled inducible expression of Elf5-V5, and infected the MCF-10A-EcoR cell line. This construct consisted of a V5-tagged Elf5 cDNA in the pHUSH-ProEx vector, a single retroviral plasmid tetracycline-inducible expression system constructed at Genentech (Gray et al. 2007) (Figure 3.15). This vector consisted of the Elf5-V5 cDNA downstream of a CMV promoter and Tet operon (CMV-TO), and the Tet repressor (TetR) which is constitutively expressed from the human -actin promoter, thereby repressing Elf5-V5 expression by binding the TO. Addition of Doxycycline (Dox), a derivative of tetracycline, results in dissociation of TetR and induction of Elf5-V5 expression.

Figure 3.15 Schematic representations of Elf5-V5-ProEx construct for Dox-inducible Elf5-V5 overexpression. LTR = long terminal repeat; IRES = internal ribosome entry site; TetRopt = codon-optimised tetracycline repressor; PURO = puromycin selection marker.

MCF-10A cells, which had been infected with the inducible Elf5-V5-ProEx construct, were selected in the presence of puromycin (2 g/mL) and pooled. To analyse levels of Elf5-V5 expression in this inducible system, we performed Western analysis of protein lysates collected from a time course of Dox treatment with two different concentrations of Dox. Figure 3.16 shows that there was no expression of Elf5-V5 when cells had not been stimulated with Dox, and that there was a marked induction of Elf5-V5 expression following 24 hours of Dox treatment. This induction decreased slowly over time when a

single dose of Dox was given (Figure 3.16A), but was maintained when Dox was added every 24 hours (Figure 3.16B). There was no significant difference in levels of induction when 0.1 g/mL or 1 g/mL Dox was used. Immunofluorescent staining of these cells also confirmed that induction of Elf5 expression was achieved with Dox treatment, as depicted in Figure 3.17. Immunostaining for both -V5 (Figure 3.17A) and -Elf5 (Figure 3.17B) displayed nuclear expression of Elf5-V5 following Dox treatment. This Elf5-V5- ProEx construct did not have a GFP tag and so these cells could not be sorted into high or low expressing populations, thus these cells expressed varying levels of Elf5-V5.

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20 kDa

Figure 3.16 Induction of Elf5-V5 expression with Dox treatment in MCF-10A cells grown in 2D and infected with Elf5-V5-ProEx retrovirus. A V5 expression over time course following treatment with a single dose of Dox. B V5 expression over time course following treatment with Dox every 24 hours.

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Figure 3.17 Immunofluorescence analysis of inducible Elf5-V5-ProEx MCF-10As. Cells were grown in 2D and stained with -V5 (Green- Panel A) or -Elf5 (Red - Panel B) antibody and counterstained with Phalloidin (actin). Cells were treated without (a) or with (b – f) Dox. Scale bars are 40 m in all images except (b) where it is 20 m. 95

Effect of inducible Elf5 overexpression on levels of EGFR and Bim expression

To investigate the effect of the inducible Elf5-V5-ProEx construct on the levels of EGFR and Bim expression in MCF-10A cells in 3D culture, we cultured these cells on Matrigel and induced Elf5 expression with Dox treatment at different time points. Following our observation of Elf5 expression being repressed over time in the constitutive overexpression system (Figure 3.5), we were interested in seeing whether repression of Elf5 expression in 3D culture also occurred in this inducible system. Figure 3.18 shows that this effect was recapitulated, even though the cells were treated with Dox every 24 hours once Elf5 expression had been induced. Thus despite continuous stimulation of the cells to express Elf5, expression levels were still suppressed over time, providing further support that Elf5 is specifically selected for down-regulation over time when cultured on a basement membrane.

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Figure 3.18 Inducible Elf5 expression levels decreased over time in 3D culture. Elf5 MCF-10A cells were cultured in 3D and stimulated with 1 g/mL Dox every 24 hours from Day 0. Lysates were collected at Days 7, 10, 15 and 20, and probed with the V5 antibody. -actin immunoblotting was performed to indicate relative loading.

In these experiments, we induced Elf5 expression with Dox treatment at Day 0, Day 4 and Day 7, and once induced, the cells were treated with Dox every 24 hours following until harvest. To determine whether this inducible Elf5- expression system also produced the same phenotype as constitutive Elf5 expression did, we analysed expression levels of EGFR and Bim at Day 10, the time point which showed the most significant change in expression of these two proteins in the previous experiments (Figures 3.12 and 3.13). Figure 3.19 shows that when Dox had been added at Day 0 or Day 4 onwards, there was an increase in EGFR levels and decrease in Bim levels in the Elf5-expressing acini, as observed in the constitutive expressing Elf5-V5-pMIG system. It is by Day 7-8 of culture that cells begin to die by apoptosis (Debnath et al. 2002), and so it is possible that Elf5 expression is required prior to this time in order to cause downstream repression of Bim protein levels.

Figure 3.19 Induction of Elf5 expression from Day 0 or Day 4 increases EGFR and decreases Bim protein levels. Protein lysates from Day 10 of 3D culture that had been treated with 0.1 g/mL Dox were immunoblotted with -EGFR, -Bim, and -V5.

Role of the ERK MAPK pathway in repression of Bim expression by Elf5 in acinar morphogenesis

In two-dimensional culture of MCF-10As, loss of ECM attachment has previously been shown to cause a down-regulation of EGFR, leading to induction of Bim through loss of signalling via the ERK MAPK pathway (Reginato et al. 2003). This pathway has also been identified as a key regulator of Bim in a range of different cell types (Luciano et al. 2003; Weston et al. 2003; Ley et al. 2004). As we observed an increase in EGFR and decrease in Bim during anoikis of Elf5-expressing MCF-10A cells in three- dimensional culture, we were interested in determining whether there was an increase in ERK MAPK signalling in the Elf5-expressing acini. In the constitutive Elf5-V5-pMIG expression system, there was a modest increase in ERK activation in Elf5-expressing acini at Days 10 and 15 of culture (Figure 3.20). The focus of our laboratory is now to determine the precise link between Elf5 and EGFR expression, and the role of the ERK MAPK pathway in Elf5- expressing MCF-10A acini in 3D culture.

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Figure 3.20 Western analysis of activation of ERK MAPK in MCF-10A 3D morphogenesis. Protein lysates from constitutive Elf5-expressing and control acini were harvested at Days 7, 10 and 15 and immunoblotted with -phospho ERK and -total ERK. P = pMIG; E = Elf5.

Discussion

To date, Elf5 has been shown to have a crucial role in mediating prolactin- driven mammary gland development and differentiation (Zhou et al. 2005; Harris et al. 2006; Oakes et al. 2008). This data was collected from work carried out in mouse models, and so far the role of Elf5 in the human remains to be established. The aim of this chapter was therefore to investigate Elf5 function in the context of the human mammary gland, and to study the cellular processes and signalling pathways involved in the mechanism of Elf5 action using a human mammary cell culture model.

Despite having no effect on the growth or proliferation of MCF-10A cells in 2D culture, Elf5 expression in MCF-10A cells grown in 3D culture had a profound effect, including repression of Elf5 expression over time, and a defect in lumen formation. This defect was associated with up-regulation of EGFR in Elf5- expressing acini, and a failure to induce Bim, a pro-apoptotic factor required for lumen formation (Reginato et al. 2005). This phenotype that we observed was quite distinct from that induced by oncogenes such as active ErbB2, which in addition to luminal filling, elicits multiacinar structures with excess proliferation (Muthuswamy et al. 2001). The vast majority (90%) of Elf5- expressing acini did not form a hollow lumen even after 20 days of culture, despite the fact these acini were expressing very low levels of Elf5 by this time. This is quite remarkable considering that inhibiting apoptosis by either ectopic expression of anti-apoptotic proteins like Bcl-2 or Bcl-xL, or conversely inhibiting Bim expression by siRNA, delayed but did not prevent luminal apoptosis (Debnath et al. 2002; Reginato et al. 2005).

Although there was a significant decrease in Bim expression in Elf5-expressing acini, there was only a modest decrease in the pro-apoptotic activated caspase-3, and surprisingly, a decrease in Bcl-2 expression (Figure 3.11). This is comparable to the scenario where progestins stimulate proliferation and

survival in breast cancer cells, and although stimulation of Bcl-xL was observed, so was the unexpected inhibition of Bcl-2 expression (Moore et al. 2000). Interestingly, Ets2 has been shown to up-regulate Bcl-xL expression, possibly via the Ets binding site in the Bcl-xL promoter (Sevilla et al. 1999), and Prl has also been demonstrated to promote survival by up-regulation of Bcl-xL (Fujinaka et al. 2007). However we did not observe any increase in Bcl- xL expression in Elf5-expressing acini (Figure 3.11). It must be noted that Bcl- 2 belongs to a large family of apoptosis regulators, including both pro- apoptotic (eg. Bim and Bax) and anti-apoptotic (eg. Bcl-2 and Bcl-xL) proteins, and it is the ratio of these proteins that is important in determining how the cell will respond to apoptotic stimuli (Kroemer 1997). Therefore one possibility to explain our seemingly paradoxical observation might be that it is the total balance of these apoptotic regulators that is important within the Elf5- expressing cells. A second possibility is that down-regulation of Bim expression alone may be sufficient to inhibit luminal clearance in Elf5-expressing acini.

Having demonstrated that Elf5 expression in MCF-10A cells is associated with increased expression of EGFR, it will now be necessary to determine how this occurs, and whether it is through a direct or indirect mechanism. Human Elf5 interacts with DNA sequences that contain a GGAA core (Oettgen et al. 1999), and the human EGFR promoter does indeed contain a number of these motifs, as analysed from The Eukaryotic Promoter Database (http://www.epd.isb- sib.ch/). At present we are unable to test Elf5 recruitment to the EGFR promoter by chromatin immunoprecipitation (ChIP) due to the unavailability of a ChIP-grade Elf5 antibody, and so this will most likely have to be investigated by luciferase reporter assays. Although we demonstrated up-regulation of EGFR in Elf5-expressing MCF-10A acini, the regulation of cellular processes in 2D culture compared to 3D culture can be markedly different. Preliminary data has shown that Elf5 expression alone in MCF-10A cells does not induce EGFR expression, and so the role of Elf5 in anoikis inhibition in monolayer culture of MCF-10A cells will need to be further investigated. It should be noted that an

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example of where luminal filling of MCF-10A acini corresponds to inhibition of anoikis in monolayer culture has recently been reported (Danes et al. 2008).

Overexpression of EGFR in MCF-10A cells grown in 3D culture has recently been demonstrated to increase the proportion of irregular acini with partially filled lumens (Dimri et al. 2007), although not to the extent that we observed with Elf5 expression. Interestingly, when EGFR was co-overexpressed with c- Src, there was a dramatic shift towards irregular and hyperproliferative acini with a marked loss in polarity and some branching morphogenesis (Dimri et al. 2007). This suggests that the expression of particular molecules can biologically cooperate to promote early oncogenic phenotypes in MCF-10A cells. Therefore, as Elf5 expression up-regulates EGFR expression in 3D culture, it would be interesting to investigate the effect of co-expression of Elf5 and c-Src on MCF-10A acinar morphogenesis to determine whether, in an appropriate context, Elf5 overexpression is capable of initiating oncogenic transformation. The potential role of Elf5 in mammary carcinogenesis will be discussed further in Chapter Seven.

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Chapter 4: Investigation of the Role of Elf5 in the Human Breast Cancer Cell Line, T47D

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Introduction

The Ets transcription factors are a large family that play key roles in development, differentiation and apoptosis, and have long been implicated in the control of cellular proliferation and tumorigenesis (Macleod et al. 1992; Seth et al. 1992; Wasylyk et al. 1993; Graves and Petersen 1998). The Ets family may play an important role in breast cancer, as suggested by several reports demonstrating aberrant expression of Ets transcription factors in mammary tumors and breast cancer cell lines. However, it is unclear how this altered expression is correlated with carcinogenesis due to reports of conflicting data. For example, the closely related epithelial-specific Ets family member, Pdef, has been reported to be both up-regulated (He et al. 2007) and down-regulated (Feldman et al. 2003) in human breast cancer cell lines. Elf5, together with the closely related Ets factors, Elf3 and EHF, has been shown to be expressed at higher levels in breast cancer cell lines compared with the nominally “normal” epithelial cell lines, however, the comparison here is also between luminal and basal phenotypes. Elf5 is most abundantly expressed by the T47D breast cancer cell line (Harris 2004; He et al. 2007). Most informatively, this sub-family of Ets transcription factors have also been shown to be down-regulated during breast cancer progression. One laser capture microdissection study reported that Elf5 mRNA expression by epithelial cells is much reduced in breast cancers compared to adjacent normal tissue, and was one of the most consistently down regulated genes in the genome (Ma et al. 2003). This important result suggests that a loss in Elf5 expression may be required for carcinogenesis, either by down regulation of Elf5 levels or by the selection of highly proliferative and undifferentiated low Elf5 expressing cells. Another study demonstrated that EHF expression was restricted to normal epithelium and clearly excluded from transformed epithelium in breast ductal carcinoma samples (Tugores et al. 2001). It remains to be defined whether expression of epithelial Ets factors is lost during carcinogenesis, or whether these cells do not participate in this process.

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As reported by Harris et al. (2006), pregnancy induces a massive increase of Elf5 expression in the mammary gland. This expression peaks at day 2 of lactation, remains high throughout lactation, and decreases during involution (Harris et al. 2006). These changes in expression are presumably triggered by the changing levels of reproductive hormones throughout pregnancy and lactation. As described in Chapter One, ductal morphogenesis is primarily regulated by estrogen and growth hormone, lobuloalveolar morphogenesis requires prolactin and progesterone and lactation is induced by the loss of progesterone and a further increase in prolactin (Neville et al. 2002). Although the effects of prolactin on Elf5 levels are defined (Harris et al. 2006) the potential role of estrogen and progesterone are unknown. It is not yet understood whether aberrant expression of Elf5 is a risk factor for human breast cancer, whether expression of Elf5 can be correlated with disease progression or outcome and how these potential actions may interact with the hormonal environment. Therefore, given the involvement of the Ets family in oncogenesis and the pivotal role of Elf5 in normal mammary development, it is of importance to investigate the role of Elf5 in models of hormone-sensitive breast cancer.

The specific aims of this chapter are to: (1) Characterise the regulation of Elf5 by the other key hormones involved in lobuloalveolar development and to determine to what extent Elf5 may mediate their effects. (2) Investigate the effect of suppression of Elf5 expression in T47D cells on endpoints such as proliferation and differentiation

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Results

Hormonal regulation of Elf5

As described in detail in the Introduction, Elf5 is a key mediator of the structural and functional development of the milk secreting alveolar structures, a process which is dependant on a synergy between Prl and Pg. Work performed in our lab has previously shown that Elf5 is a Prl-regulated gene (Harris et al. 2006) and shows high levels of expression in several steroid receptor-positive cell lines (Harris 2004), and so we were interested in determining whether Elf5 was also regulated by steroid hormones.

Regulation of Elf5 expression by sex-steroid hormones in the mouse mammary gland

To investigate the hormonal regulation of Elf5 in vivo, we obtained mouse mammary gland tissue from Assoc Prof Liz Musgrove (Garvan Institute of Medical Research, Australia) and extracted the RNA. These wildtype mice had been subjected to short-term hormonal treatment whereby one progesterone pellet or one estrogen pellet, or a pellet of each hormone together, was implanted into the mouse for 6 days, after which the mammary glands were harvested. As shown in Figure 4.1, mice that had progesterone pellet implants showed a 9-10 fold increase in the number of Elf5 transcripts in the mammary gland, compared to placebo controls. Estrogen treatment had no effect on the number of Elf5 transcripts, and interestingly, combined estrogen and progesterone treatment also had no effect on Elf5 levels, indicating that estrogen treatment inhibited the increased transcription of Elf5 in response to progesterone.

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Figure 4.1 Elf5 is up-regulated by progesterone treatment in vivo. Relative numbers of transcript numbers of Elf5 following short-term hormonal stimulation by pellet implanting in the mouse mammary gland. Each bar represents one mouse. (Plac = placebo; Pg = progesterone; E = estrogen; P + E = progesterone plus estrogen.)

Regulation of human Elf5 expression by progestins in breast cancer cells

To determine whether this progesterone up-regulation of Elf5 in the mouse could be extended into a human context, we treated the PgR-positive T47D mammary carcinoma cell line with the progestin ORG2058, a synthetic analogue of progesterone. As shown in Figure 4.2A, Elf5 mRNA levels were increased with prolonged ORG2058 exposure. This up-regulation was initially observed following 16 hours of treatment, and maintained up until at least 6 days of treatment. This pattern of up-regulation was also observed at the protein level (Figure 4.2B). This progestin-induced up-regulation of Elf5 was also observed in BT474 cells, a second PgR-positive human breast cancer cell line. Figure 4.3A shows the levels of Elf5 mRNA transcripts over a 2-day time course following ORG2058 treatment. To determine whether this increase in Elf5 expression was occurring in a PgR-specific manner, we also co-treated T47D cells with ORG2058 and the progestin antagonist, RU486, for 16 and 24 hours. Figure 4.3B shows that co-treatment inhibited the increase in Elf5 expression, thus demonstrating that Elf5 expression is up-regulated by Pg via the PgR.

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Figure 4.2 Elf5 expression is strongly up-regulated in T47Ds by progestin treatment. A Relative number of Elf5 transcripts in T47Ds over 6 day time course following treatment with ORG2058 (each error bar is mean±SE, and is representative of an independent experiment which was repeated at least 3 times). Chart is representative of one experiment with 3 replicates, and which was repeated at least 3 times. B Western blot of Elf5 protein expression in T47Ds following treatment with ORG2058.

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Figure 4.3. A Relative number of Elf5 transcripts in BT474 cells over time course following treatment with ORG2058 (each error bar is mean±SE). B Western blot of Elf5 protein levels in T47D cells following treatment with 10 nM ORG2058 alone, and co-treatment with ORG2058 and 100 nM RU486.

Progestin treatment of breast cancer cells in culture elicits a biphasic response, whereby there is initially a proliferative burst (approximately 12 hrs), followed by onset of differentiation and long-term inhibition of proliferation (following approximately 24 hrs of treatment) (Musgrove et al. 1991; Sutherland et al. 1998). This suggests that Elf5 expression is up-regulated during the differentiation phase of T47D cells, consistent with our knowledge of Elf5’s role in the differentiation of mouse mammary epithelial cells.

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Regulation of Elf5 expression by co-treatment of T47D breast cancer cells with estrogen and progestin

Having demonstrated that Elf5 was up-regulated by progesterone and progestin treatment in vivo and in vitro, we were interested in determining whether combined treatment of T47D cells with estrogen and Pg counteracted the increase in Elf5 expression as it had in vivo (Figure 4.1). Evidence of Pg reversing the effects of E in vitro has previously been reported (Kester et al. 1997), however the mechanism of this action is unclear. Cells were cultured in phenol red-free media and stimulated with 10 nM ORG2058 or 100 nM estradiol, or both, for 48 hours. As depicted in Figure 4.4, the number Elf5 transcripts were greatly increased upon Pg treatment, as well as co-treatment with E. Therefore the repression of Pg-induced Elf5 expression observed in vivo is not recapitulated in T47D cells in vitro. This suggests that the repression of Pg-induced Elf5 expression in vivo may not be a direct transcriptional event, but rather a result of the action of complex hormonal and genomic signalling networks at play in vivo. Furthermore, T-47D cells are responsive to estrogen, but less so than MCF-7 cells (Keydar et al. 1979).

Figure 4.4 Regulation of Elf5 expression in T47D breast cancer cells with treatment of estradiol (E2), progestin (Pg), or both.

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Regulation of Elf5 expression by growth hormone in T47D cells

In addition to Prl and Pg, T47D cells are also responsive to human growth hormone (hGH), a lactogenic hormone closely related to Prl but far more stable in solution and less prone to aggregation, which binds the Prlr and the GHR. Also similarly to Prl and Pg, hGH induces lipid biosynthesis and differentiation in T47D cells (Shiu and Paterson 1984b; Chambon et al. 1989). To determine whether hGH regulated Elf5 we treated T47D cells with hGH (500 ng/mL) for 24 h and 48 h, in addition to co-treating the cells with hGH and ORG2058 (10 nM). Elf5 protein levels were indeed up-regulated following 24 h and 48 h hGH treatment (Figure 4.5). Interestingly this up-regulation was increased additively upon co-treatment with ORG2058, suggesting that there may be cross-talk between the progestin and growth hormone downstream signalling pathways. This is consistent with the literature where cross-talk between progestin and GH signalling in human breast cancer has previously been described (Milewicz et al. 2005). Furthermore, it must be noted that in this case hGH may be acting via Prlr, and working synergistically with Pg signalling, as described in Chapterhapter One.One. A

Figure 4.5 Elf5 expression is up-regulated in T47Ds by hGH treatment. A Western

blot of Elf5 protein expression in T47Ds following treatment with ORG2058, hGH, or both. B Corresponding densitometric analysis depicting relative levels of Elf5 following hormone treatment for 24 hours (red) or 48 hours (blue). 110

Effects of down-regulation of Elf5 expression in T47D breast cancer cells by siRNA

Elf5 is a known target of Prl, and the data just presented indicate that Elf5 is markedly up-regulated by progestin treatment. Prl, GH and Pg regulate differentiation in the mammary gland (reviewed in (Neville et al. 2002), and in human breast cancer cells (Shiu and Paterson 1984; Chambon et al. 1989). Since Elf5 is a transcription factor with a known role in differentiation in the mouse, we hypothesised that up-regulation of Elf5 in T47D cells may contribute to the differentiative effects of these hormones. To investigate this idea we established a system whereby we transiently knocked down Elf5 expression in the presence and absence of progestins. We screened four Elf5 small interfering RNA (siRNA) oligoduplexes for their efficiency in the T47D cells, which express very high levels of endogenous Elf5. All four siRNAs tested reduced Elf5 mRNA levels (Figure 4.6). Elf5 siRNAs #3 and #4 consistently achieved the highest level of knockdown by 50% or more, and were chosen for use in subsequent experiments.

Figure 4.6 Repression of Elf5 expression in T47D cells by RNAi. Transfection of T47D cells with a panel of four Elf5 siRNAs (10 nM) results in reduction in Elf5 transcript levels after 48 hrs by all siRNAs.

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Changes in cellular morphology in T47D cells with suppressed Elf5 expression

When we reduced Elf5 expression in T47D breast cancer cells, the first observation we made was that it altered the morphology of these cells (Figure 4.7). At higher magnifications, a number of Elf5 siRNA-transfected cells appeared more enlarged and flattened, compared to the normal cobblestone appearance of control cells. In addition, mock-transfected and control RISCfree siRNA-transfected cells grew in packed colonies, whereas Elf5 siRNA- transfected cells grew sparsely with a scattered morphology and minimal cell colony formation (Figure 4.7B).

Effect of suppression of Elf5 expression on cell cycle progression in T47D cells

FACS analysis of T47D cells transfected with control and Elf5 siRNAs revealed that suppression of Elf5 led to cell cycle changes. We observed a decrease in the proportion of cells in the proliferative S phase in Elf5 siRNA-transfected cells (14%), compared with mock-transfected (21%) and RISCfree control siRNA-transfected (23%) cells. We also observed an increase in the proportion of cells in the G1/G0 phase in Elf5 siRNA-transfected cells (67%), compared with mock-transfected (62%) and RISCfree siRNA-transfected cells (61%). Figure 4.8 depicts DNA histograms obtained from FACS analysis used to quantitate the proportion of cells in S phase.

We also performed Western analysis on protein lysates harvested 48 hours after transfection of Elf5 and control siRNAs. We transfected two different Elf5 siRNAs, as well as a pool of these two duplexes, and observed that, compared to controls, the suppression of Elf5 expression suppressed the activation of the ERK MAPK pathway and expression of the cell cycle protein cyclin D1, both molecules which promote growth and proliferation (Figure 4.9). In addition, we

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observed down-regulation of the progesterone receptor, which corresponds with a decrease in proliferation in T47D cells (Botella et al. 1994).

A (a) (b) (c)

(d) (e) (f)

Mock RISCfree Elf5 siRNA siRNA #4

Figure 4.7 Suppression of Elf5 expression in T47D cells alters cellular morphology. A Phase contrast images of T47D cells transfected with (a) Mock, (b) RISCfree siRNA, (c) GFP siRNA, (d) Elf5 siRNA #3, (e) Elf5 siRNA #4 and (f) Elf5 siRNA #3 and #4. B Immunofluorescent images of T47D cells transfected as labelled and counterstained with Phalloidin (actin; red) and TOPRO (nucleus; blue). Scale bars represent 80 m.

Figure 4.8 (over page) Suppression of Elf5 expression in T47D cells decreases the proportion of cells in the proliferative S phase. The proportion of cells in S phase was determined by quantitating DNA histograms using flow cytometry. The percentage of cells in S phase and the coefficient of variation (CV) are as labelled.

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Mock S phase = 21% CV = 3.57%

0 20 40 60 80 100 120 Channels

RISCfree siRNA S phase = 23% CV = 4.31%

0 20 40 60 80 100 120 Channels

Elf5 siRNA #4 S phase = 14% CV = 4.40%

0 20 40 60 80 100 120 Channels

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Figure 4.9 Suppression of Elf5 expression in T47D cells decreases expression of growth-promoting proteins. A Western blot of duplicate samples of protein lysates harvested from cells transfected with Elf5 and control siRNA duplexes and immunoblotted with -phospho ERK, -total ERK, -cyclin D1, -PR and -Elf5 antibodies. Levels of --actin are shown to indicate relative loading. B Relative expression of ERK phosphorylation normalised to --actin levels as determined by densitometry analysis.

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Comparison with in vivo mouse model

We examined the question in a different model to determine whether this effect could be reproduced in vivo. Our lab has generated Elf5 knockout (Elf5-/-) embryos and serially transplanted Elf5-/- mammary epithelium from this mouse into wild-type immune-compromised hosts. Upon collection of these mammary transplants it was observed there was a reduction in the number of proliferating Ki67-positive cells during early time points pregnancy {Oakes et al., 2008; see Appendix III}. This prompted us to investigate whether activation of the ERK MAPK pathway was involved in the mechanism of reduced proliferation in Elf5-/- mammary epithelium. Figure 4.10 demonstrates that there was indeed a significant reduction of ERK phosphorylation in Elf5-/- transplants, compared with the corresponding wildtype mammary epithelium from the same mouse. Therefore, Elf5 expression has downstream effects on activation of the ERK MAPK pathway in vivo, and provides supporting evidence to confirm our observation of the effect of suppression of Elf5 expression in T47D breast cancer cells in vitro.

Figure 4.10 Deletion of Elf5 in the mouse inhibits proliferation in the mammary gland. Western blot of protein lysates from wild-type (WT) and Elf5-/- mammary transplants at 4.5 dpc. Each pair of lanes represents a single mouse. Lysates were immunoblotted with -phospho ERK MAPK, -total ERK MAPK and --actin.

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Role of Elf5 in progestin-induced differentiation in T47Ds

Since progestin treatment caused an up regulation of Elf5 expression we wondered to what extent the transcriptional activity of Elf5 contributed to progestin action. To suppress Elf5 expression in the presence of progestins, we transfected T47D cells with Elf5 siRNA duplexes, and 24 hours later we stimulated the cells with 10 M ORG2058 for at least 48 hours before harvesting lysates. As depicted in Figure 4.11, Elf5 mRNA levels were markedly up-regulated by progestin stimulation in controls, however this induction was reduced or abolished in Elf5 siRNA-transfected cells depending on the siRNA used. This model system allowed us to study the relevance of Elf5 expression for progestin-induced differentiation in T47D cells.

Figure 4.11 Repression of Elf5 expression in the presence of progestins. T47D cells were transiently transfected with Elf5 siRNAs followed by stimulation with ORG2058 (10 nM; 48 hrs).

Progestins induce lipogenesis in T47D cells (Chambon et al. 1989). To determine whether Elf5 expression was important for this lipid synthesis, we transiently transfected T47D cells with siRNA duplexes and cultured these cells for 5 days in the presence of ORG2058. We chose to look at levels of fatty acid synthase (FASN) as an endpoint, as this enzyme indicates activation of the

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lipid synthesis pathway (Horton et al. 2002), and is a known target of progestins (Chambon et al. 1989). Our results show that Elf5 transcript numbers increased with progestin stimulation, and this induction was suppressed in Elf5 siRNA-transfected cells (Figure 4.12A). Levels of FASN also increased upon progestin treatment, however, we observed no consistent decrease in FASN induction when Elf5 expression was suppressed (Figure 4.12B).) A

Figureure 4.124 12 ReducedReduced Elf5Elf5 expressionexpression hashas nono significantsignificant effectseffects onon thethe Pg-mediatedPg mediated induction of FASN. Numbers of Elf5 (A) and FASN (B) transcripts in T47D cells transfected with Elf5 siRNA #4 and control duplexes, and cultured in the presence of 10 nM ORG2058 (Pg) for 5 days.

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We also repeated this experiment looking at the transcript levels of a second molecule, desmoplakin (DSP), a known marker of progestin-induced differentiation in T47Ds (Kester et al. 1997). Our results demonstrate that we did not observe any significant changes in progestin-induced DSP expression levels in Elf5 siRNA-transfected cells (Figure 4.13).

Figure 4.13 Reduced Elf5 expression has no significant effects on the Pg-mediated induction of DSP. Numbers of Desmoplakin transcripts in T47D cells transfected with control and two Elf5 siRNA duplexes, and cultured in the presence of 10 nM ORG2058 (Pg) for 5 days.

Identification of potential downstream targets of Elf5 in T47Ds

The data just presented showed that increased Elf5 expression was not essential in the up-regulation of the differentiation markers, FASN and DSP, prompting us to take a genome-wide screen to better investigate and identify potential Elf5-mediated targets downstream of progesterone action. To do this, we utilised a stable inducible Elf5 shRNA T47D cell line which had been recently generated by fellow student, Maria Kalyuga, in our laboratory. These cells were made by inserting an shRNA (designed to match the Elf5 siRNA #4; Figure 4.6) into the pHUSH vector system created by Genentech (described in

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Chapter Three; (Gray et al. 2007)). Upon treatment with Doxycycline, the expression of endogenous Elf5 is repressed long-term, both in the presence and absence of progestin treatment (Figure 4.14; as performed by Maria Kalyuga). For this experiment, we transcript profiled these cells under three conditions: (1) Treatment with ethanol vehicle only (Baseline), (2) Plus ORG2058 treatment for 4 days (to identify Pg targets in these cells), and (3) Plus Dox and ORG2058 treatment for 4 days (to knockdown the progestin- induction of Elf5 expression to identify genes induced by progestin via Elf5).

Figure 4.14 Stable repression of Elf5 expression induced by Dox treatment. T47D breast cancer cells retrovirally infected with a Elf5 shRNA-pHUSH vector were induced to suppress Elf5 expression with 1 g/mL Dox. Twenty-four hours later the cells were stimulated with or without 10-8M ORG2058 for 48 hours and protein lysates were harvested.

We chose 4 days of ORG2058 treatment as our time point as the cells have undergone growth arrest and are differentiating at this time (Musgrove et al. 1991; Caldon et al. 2008). RNA harvested from samples of these cells under each condition was hybridised to Affymetrix Human Gene 1.0 ST Gene Arrays at the Ramaciotti Centre for Gene Function Analysis at the University of New South Wales. Each condition was carried out in triplicate, and each replicate was a pool of three wells, to minimise experimental variation. Quality control analysis as reported by the Ramaciotti Centre using Affymetrix Expression ConsoleTM software revealed the quality of the arrays was high.

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To get an overview of the proportion of genes that displayed differential expression, Figure 4.15 below depicts the total numbers of genes which changed when comparisons were made between the various experimental conditions. For example, it illustrates that 2707 genes were differentially expressed in Pg-treated Elf5 knockdown (KD) cells compared with Baseline cells, and that 1486 of these were up-regulated, and 1221 were down- regulated.

Figure 4.15 Schematic diagram of total differential gene expression from microarray analysis. The number of genes up-regulated ()ordown-regulated() are shown and labelled with the table number in which those genes are listed further on in this chapter.

We first identified progestin-regulated genes. Searching for genes that differed significantly (p<0.05; for details see Materials and Methods) between the Baseline samples and the Pg-treated samples identified 2649 genes that showed altered expression in response to progestin treatment. Functional annotation analysis indicated a number of categories relating to the immune

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response were enriched in the genes up-regulated in Pg-treated cells vs Baseline cells, and a large number of categories relating to the cell cycle were enriched in the genes down-regulated in this comparison, reflecting the inhibition of proliferation induced by long-term progestin treatment. In addition, a number of known progesterone targets showed significant differential expression, validating the response to ORG2058 treatment in the cells (Table 4.1) (Chambon et al. 1989; Kester et al. 1997; Lange et al. 1998).

Table 4.1 Foldchange in expression of known progesterone targets. Foldchange Gene Symbol // Gene Name FASN // fatty acid synthase 4.6 DSP // desmoplakin 1.5 STAT5A // signal transducer and activator of transcription 5A 16.8 ErbB2 // v-erb-b2 erythroblastic leukemia viral oncogene homolog 2 2.4

We next sought to identify genes regulated by knockdown of Elf5 but which were insensitive to progesterone treatment. These comparisons revealed that 64 probesets that were differentially regulated in Pg-treated Elf5 KD vs Baseline cells, but showed no change in the Pg-treated vs Baseline cells. These probesets were potential targets of Elf5, which were not regulated by progestin treatment. Of these, 54 genes were up-regulated by Elf5 KD (un-adjusted p<0.005; foldchange > 1; Table 4.2). These genes are thus repressed by Elf5, and included a number of proteins involved in chromatin remodelling, for example Snf-2 related CREBBP activator protein; (Ruhl et al. 2006)). There were also 10 targets down-regulated by Elf5 KD (un-adjusted p<0.005; foldchange > 1; Table 4.3). These included Cyclin E1, a protein essential for

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cell cycle progression, which is consistent with our observations of the requirement of Elf5 expression for normal cell proliferation described earlier in this chapter.

Table 4.2 Potential genes repressed by Elf5 and not regulated by progestin. Genes unchanged in Pg-treated vs baseline (unadjusted p>0.5) and up-regulated in Pg-treated Elf5 KD vs Pg-treated cells (unadjusted p<0.005). Genes labelled with “---“ represent unknown genes, which displayed differential expression. Pg-treated Elf5 KD Pg-treated Elf5 KD vs Baseline vs Pg-treated Pg vs. Baseline BY Un- Unadjusted p- adjusted Fold- adjusted Fold- Gene Name value p-value change p-value change Hect (homologous to the E6-AP (UBE3A) carboxyl terminus) domain and RCC1 (CHC1)-like domain (RLD) 1 0.616 0.024 1.513 0.0002 1.469 PI-3-kinase-related kinase SMG-1 0.559 0.060 1.409 0.0003 1.459 KIAA1217 0.621 0.087 1.362 0.0005 1.402 --- 0.942 0.064 1238.89 0.0005 1354.44 DENN/MADD domain containing 4C 0.564 0.039 1.335 0.0005 1.300 --- 0.664 0.087 2.124 0.0005 2.258 Chondroitin sulfate GalNAcT-2 0.558 0.041 1.353 0.0005 1.315 Snf2-related CREBBP activator protein 0.704 0.054 1.282 0.0006 1.261 Islet amyloid polypeptide 0.701 0.133 1.510 0.0009 1.560 --- 0.802 0.085 1.443 0.0010 1.418 E1A binding protein p300 0.971 0.102 1.414 0.0010 1.411 Ash1 (absent, small, or homeotic)-like (Drosophila) 0.779 0.135 1.355 0.0010 1.379 LPS-responsive vesicle trafficking, beach and anchor containing 0.812 0.137 1.267 0.0011 1.282 KIAA2018 0.899 0.140 1.334 0.0013 1.345 Chromosome 10 open reading frame 118 0.588 0.081 1.567 0.0013 1.498 Desmoglein 2 0.988 0.130 1.282 0.0013 1.281 Nuclear factor I/B 0.642 0.215 1.220 0.0015 1.246 RAB GTPase activating protein 1-like 0.709 0.108 1.272 0.0016 1.250 Utrophin 0.757 0.121 1.426 0.0017 1.395 Fermitin family homolog 2 (Drosophila) 0.982 0.157 1.290 0.0018 1.288

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--- 0.838 0.137 1.992 0.0018 1.934 --- 0.532 0.309 2.164 0.0019 2.447 Protein phosphatase 1, regulatory (inhibitor) subunit 9A 0.754 0.139 1.611 0.0021 1.562 Inter-alpha (globulin) inhibitor H4 (plasma Kallikrein-sensitive glycoprotein) 0.786 0.246 1.416 0.0022 1.449 Rho-associated, coiled- coil containing protein kinase 1 0.858 0.233 1.333 0.0023 1.349 Senataxin 0.960 0.201 1.284 0.0026 1.281 Jumonji domain containing 1C 0.767 0.165 1.321 0.0027 1.297 Bromodomain adjacent to zinc finger domain, 2A 0.731 0.309 1.239 0.0027 1.262 Sp1 transcription factor 0.823 0.281 1.187 0.0028 1.198 Neuregulin 4 0.957 0.240 1.380 0.0028 1.386 Phospholipase D1, phosphatidylcholine- specific 0.555 0.416 1.255 0.0029 1.302 SET binding factor 2 0.615 0.146 1.267 0.0029 1.236 Zinc finger with KRAB and SCAN domains 5 0.717 0.333 1.314 0.0029 1.348 Coiled-coil domain containing 131 0.942 0.220 1.320 0.0030 1.314 Tetratricopeptide repeat domain 14 0.689 0.162 1.352 0.0030 1.318 --- 0.587 0.145 2.196 0.0030 2.007 FERM domain containing 4A 0.647 0.384 1.244 0.0031 1.278 --- 0.776 0.333 1.836 0.0032 1.919 Sarcolemma associated protein 0.888 0.291 1.195 0.0033 1.202 --- 0.697 0.178 1.318 0.0033 1.288 Family with sequence similarity 59, member A 0.598 0.165 1.257 0.0036 1.224 Zinc finger protein 644 0.983 0.268 1.444 0.0036 1.441 ATPase, class V, type 10D 0.683 0.423 1.219 0.0037 1.247 Bromodomain and WD repeat domain containing 1 0.973 0.294 1.316 0.0038 1.319 --- 0.502 0.152 1.515 0.0039 1.427 --- 0.502 0.152 1.515 0.0039 1.427 Serine/arginine repetitive matrix 2 0.553 0.540 1.375 0.0039 1.454 Early endosome antigen 1 0.701 0.209 1.268 0.0041 1.242 Inhibitor of Bruton agammaglobulinemia tyrosine kinase 0.958 0.322 1.187 0.0042 1.189 --- 0.828 0.384 1.852 0.0042 1.920

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F-box and leucine-rich repeat protein 11 0.597 0.186 1.241 0.0043 1.210 Son of sevenless homolog 1 (Drosophila) 0.999 0.335 1.256 0.0047 1.256 --- 0.551 0.632 1.379 0.0048 1.463 KIAA0368 0.563 0.197 1.267 0.0050 1.228

Table 4.3 Potential genes induced by Elf5 and not regulated by progestin. Genes unchanged in Pg-treated vs baseline (unadjusted p>0.5) and down-regulated in Pg-treated Elf5 KD vs Pg-treated cells (unadjusted p<0.005). Pg-treated Elf5 KD Pg-treated Elf5 KD vs Baseline vs Pg-treated Pg vs. Baseline BY Un- Unadjusted p- adjusted Fold- adjusted Fold- Gene Name value p-value change p-value change --- 0.528 0.051 0.575 0.0008 0.610 CDC42 effector protein (Rho GTPase binding) 2 0.800 0.126 0.780 0.0009 0.770 --- 0.614 0.259 0.550 0.0018 0.511 Cyclin E1 0.519 0.350 0.799 0.0022 0.769 Hypothetical LOC646471 0.592 0.415 0.814 0.0030 0.790 --- 0.836 0.289 0.714 0.0031 0.702 --- 0.775 0.322 0.432 0.0031 0.406 Phospholipid scramblase 4 0.672 0.382 0.687 0.0032 0.659 Klotho 0.612 0.525 0.850 0.0043 0.830 Solute carrier family 17 (anion/sugar transporter), member 5 0.859 0.382 0.757 0.0044 0.747

Finally we sought to determine the role of Elf5 in progestin-regulated gene expression. There was a high degree of similarity between the number and types of genes identified which displayed differential expression in Pg-treated vs Baseline cells, and in Pg-treated Elf5 KD vs Baseline cells. This can be seen by comparing the gene ontology tables of gene categories which were over- represented in the genes which changed in Pg-treated vs Baseline cells (Tables 4.4 and 4.5) with those which changed in Pg-treated Elf5 KD vs Baseline cells (Tables 4.6 and 4.7). Similarly to the Pg-treated cells, immune response categories were over-represented in the genes that were up-regulated, and many cell cycle categories were over-represented in the list of down-regulated genes.

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Table 4.4 Functional annotation categories enriched with BY p<0.05 in genes up- regulated in Pg-treated vs Baseline cells.

BY p- Category Term Count p-Value value GOTERM_BP_ALL GO:0002376~immune system process 87 1.49E-09 4.03E-05 GOTERM_BP_ALL GO:0006955~immune response 66 8.44E-08 0.00114 GOTERM_BP_ALL GO:0009615~response to virus 20 3.80E-07 0.00343 GO:0042612~MHC class I protein GOTERM_CC_ALL complex 11 5.90E-07 0.00399 GO:0002474~antigen processing and presentation of peptide antigen via MHC GOTERM_BP_ALL class I 10 9.40E-07 0.00509 GO:0019882~antigen processing and GOTERM_BP_ALL presentation 15 3.21E-06 0.0145 SP_PIR_KEYWORDS antiviral defense 12 6.19E-06 0.0209 GOTERM_CC_ALL GO:0005737~cytoplasm 379 6.87E-06 0.0209 SP_PIR_KEYWORDS microsome 19 7.08E-06 0.0209 GO:0048002~antigen processing and GOTERM_BP_ALL presentation of peptide antigen 10 7.72E-06 0.0209 GOTERM_MF_ALL GO:0003824~catalytic activity 326 9.22E-06 0.0227 IPR001039:MHC class I, alpha chain, INTERPRO alpha1 and alpha2 9 1.11E-05 0.0253 UP_SEQ_FEATURE domain:Ig-like C1-type 9 1.35E-05 0.0281 GOTERM_CC_ALL GO:0042611~MHC protein complex 11 1.47E-05 0.0284 SP_PIR_KEYWORDS mhc i 6 2.63E-05 0.0445 GO:0009607~response to biotic GOTERM_BP_ALL stimulus 33 2.63E-05 0.0445

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Table 4.5 The top 10 functional annotation categories most over-represented with BY p<0.05 in genes down-regulated in Pg-treated vs Baseline cells.

BY p- Category Term Count p-Value value GOTERM_CC_ALL GO:0005694~chromosome 116 1.27E-48 4.15E-44 GOTERM_BP_ALL GO:0000278~mitotic cell cycle 111 3.42E-47 5.10E-43 GOTERM_BP_ALL GO:0007049~cell cycle 191 4.68E-47 5.10E-43 SP_PIR_KEYWORDS cell cycle 122 4.88E-46 3.99E-42 GOTERM_BP_ALL GO:0022403~cell cycle phase 115 9.92E-46 6.49E-42 GOTERM_BP_ALL GO:0006259~DNA metabolic process 170 2.34E-45 1.28E-41 GOTERM_CC_ALL GO:0044427~chromosomal part 102 1.80E-44 8.41E-41 SP_PIR_KEYWORDS phosphoprotein 531 2.82E-44 1.15E-40 GOTERM_BP_ALL GO:0000279~M phase 101 4.89E-44 1.78E-40 GO:0000087~M phase of mitotic cell GOTERM_BP_ALL cycle 90 1.91E-43 6.25E-40

Table 4.6 Functional annotation categories enriched with BY p<0.05 in genes up- regulated in Pg-treated Elf5 KD vs Baseline cells.

Category Term Count p-Value BY p-value GOTERM_BP_ALL GO:0002376~immune system process 104 3.13E-09 0.000108 IPR004827:Basic-leucine zipper (bZIP) INTERPRO transcription factor 17 1.14E-07 0.00197 GOTERM_BP_ALL GO:0007243~protein kinase cascade 56 3.49E-07 0.00362 SMART SM00338:BRLZ 17 4.19E-07 0.00362 GOTERM_BP_ALL GO:0006955~immune response 76 1.02E-06 0.00538 SP_PIR_KEYWORDS cytoplasm 231 1.07E-06 0.00538 SP_PIR_KEYWORDS phosphoprotein 399 1.16E-06 0.00538 UP_SEQ_FEATURE domain:Leucine-zipper 22 1.39E-06 0.00538 GOTERM_CC_ALL GO:0005737~cytoplasm 486 1.40E-06 0.00538 GOTERM_MF_ALL GO:0005515~protein binding 543 3.16E-06 0.0109 GOTERM_BP_ALL GO:0009615~response to virus 21 4.85E-06 0.0153 GO:0045637~regulation of myeloid cell GOTERM_BP_ALL differentiation 13 5.96E-06 0.0166 GOTERM_CC_ALL GO:0042612~MHC class I protein complex 11 6.24E-06 0.0166 GO:0050793~regulation of developmental GOTERM_BP_ALL process 39 7.59E-06 0.0188 GO:0002474~antigen processing and presentation of peptide antigen via MHC class GOTERM_BP_ALL I 10 8.35E-06 0.0193 GOTERM_MF_ALL GO:0008134~transcription factor binding 54 1.16E-05 0.0251 SP_PIR_KEYWORDS antiviral defense 13 1.36E-05 0.0277

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Table 4.7 The top 10 functional annotation categories most over-represented with BY p<0.05 in genes down-regulated in Pg-treated Elf5 KD cells vs Baseline cells.

BY p- Category Term Count p-Value value GOTERM_BP_ALL GO:0007049~cell cycle 181 7.94E-54 2.10E-49 GOTERM_BP_ALL GO:0022403~cell cycle phase 111 6.12E-51 8.10E-47 GOTERM_CC_ALL GO:0005694~chromosome 107 5.57E-49 4.91E-45 GOTERM_BP_ALL GO:0000278~mitotic cell cycle 103 3.03E-48 2.01E-44 GOTERM_BP_ALL GO:0000279~M phase 97 5.01E-48 2.65E-44 SP_PIR_KEYWORDS cell cycle 114 7.50E-48 3.31E-44 GOTERM_BP_ALL GO:0022402~cell cycle process 157 1.03E-46 3.90E-43 GOTERM_CC_ALL GO:0044427~chromosomal part 96 2.04E-46 6.75E-43 GO:0000087~M phase of mitotic cell GOTERM_BP_ALL cycle 84 2.35E-44 6.91E-41 GOTERM_BP_ALL GO:0006259~DNA metabolic process 151 4.53E-44 1.20E-40

To discern the effects of the knockdown of Elf5 on Pg target molecules, we searched for genes that changed expression in response to progestin, but not when Elf5 was knocked down as well. When we did this comparison, no genes passed the adjusted P threshold of Benjamini-Yekutieli (BY) p<0.05 for differential expression, including Elf5, which demonstrated that we had set too-stringent cutoffs. We therefore investigated probesets with an un-adjusted (or “raw”) p<0.001, which identified 167 probesets that now included Elf5. Of these, 134 were up-regulated by Elf5 KD. Several functional annotation categories relating to the Spectrin sequence repeat were over-represented with BY adjusted p<0.05 in this gene list (Table 4.8). In the 33 probesets down-regulated by Elf5 KD, some cell-division categories were over- represented, but did not pass the significance threshold of BY adjusted p<0.05 (Table 4.9). However, as cell-division genes were down-regulated when Elf5 expression was repressed, this trend again lends support to the observation of Elf5 having a role in proliferation described earlier in this chapter.

To better identify potential Pg-targets mediated by Elf5, we selected probesets differentially expressed with BY adjusted p<0.05 in the Pg-treated vs Baseline experiment and with an un-adjusted p<0.001 in the Pg-treated Elf5 KD vs Pg-

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treated experiment. There were 61 probesets which fulfilled these criteria. There were 7 targets that displayed up-regulated expression upon Pg- treatment, and were down-regulated in Pg-treated Elf5 KD cells. That is, these are Pg-targets that are potentially positively regulated by Elf5. The most significantly changing gene of these was Elf5 itself, which displayed an approximate 2-fold increase in expression upon Pg treatment, and 2-fold decrease in Pg-treated Elf5 KD cells compared to Pg-treated cells (Table 4.10). This was confirmed at the level of protein expression (Figure 4.16) and demonstrates that knockdown of Elf5 expression in the presence of Pg was successful. The second most significantly changing gene was glycerol-3- phosphate dehydrogenase 1 (GPD1), which is an enzyme important in lipid metabolism (Park et al. 2006). This is in line with our hypothesis, however we did not identify any other lipogenic enzymes, as expected.

Table 4.8 Functional annotation categories over-represented with BY-adjusted p<0.05 in genes up-regulated in Pg-treated Elf5 KD cells vs Pg-treated cells.

BY adj Category Term Count P-Value P-values SP_PIR_KEYWORDS phosphoprotein 54 3.80E-11 1.85E-07 UP_SEQ_FEATURE repeat:Spectrin 2 5 4.59E-06 0.007449 UP_SEQ_FEATURE repeat:Spectrin 1 5 4.59E-06 0.007449 SP_PIR_KEYWORDS Coiled coil 23 8.46E-06 0.010297 INTERPRO IPR002017:Spectrin repeat 5 2.43E-05 0.015823 UP_SEQ_FEATURE repeat:Spectrin 9 4 3.25E-05 0.015823 UP_SEQ_FEATURE repeat:Spectrin 8 4 3.25E-05 0.015823 UP_SEQ_FEATURE repeat:Spectrin 7 4 3.25E-05 0.015823 UP_SEQ_FEATURE repeat:Spectrin 6 4 3.25E-05 0.015823 UP_SEQ_FEATURE repeat:Spectrin 5 4 3.25E-05 0.015823 GO:0005515~protein GOTERM_MF_ALL binding 50 4.10E-05 0.018146 GO:0016043~cellular component organization GOTERM_BP_ALL and biogenesis 29 4.55E-05 0.01846 SMART SM00150:SPEC 5 5.51E-05 0.020635 UP_SEQ_FEATURE repeat:Spectrin 4 4 1.26E-04 0.040896 UP_SEQ_FEATURE repeat:Spectrin 3 4 1.26E-04 0.040896

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Table 4.9 Functional annotation categories over-represented with un-adjusted (raw) p<0.05 in genes down-regulated in Pg-treated Elf5 KD cells vs Pg-treated cells. Category Term Count P-Value BY adj P SP_PIR_KEYWORDS cell division 3 0.017451 1 GOTERM_BP_ALL GO:0051301~cell division 3 0.032793 1 GOTERM_BP_ALL GO:0000279~M phase 3 0.044854 1

Table 4.10 Potential Elf5-mediated Pg targets. Genes up-regulated in Pg-treated vs Baseline experiment (BY adj p<0.05) and down-regulated in Pg-treated Elf5 KD vs Pg-treated experiment (unadjusted p<0.001). Pg-treated vs Pg-treated Elf5 KD vs Baseline Pg-treated Probeset BY p- Foldchange Raw p Foldchange ID Gene Symbol // Gene Name values no filter ELF5 // E74-like factor 5 (ets 7947481 domain transcription factor) 0.0010 2.158 <0.0001 0.548 7924967 --- 0.0203 2.239 <0.0001 0.313 GPD1 // glycerol-3-phosphate 7955348 dehydrogenase 1 (soluble) 0.0023 2.086 0.0002 0.657 GUCY2D // guanylate cyclase 8004763 2D, membrane (retina-specific) 0.0116 1.459 0.0005 0.770 ATP2A3 // ATPase, Ca++ 8011516 transporting, ubiquitous 0.0070 1.646 0.0006 0.740 RARRES1 // retinoic acid receptor responder (tazarotene 8091723 induced) 1 0.0226 1.378 0.0008 0.788 8021047 SETBP1 // SET binding protein 1 0.0115 1.447 0.0009 0.792

Figure 4.16 Western analysis of Elf5 protein levels in Baseline, Pg-treated and Pg- treated Elf5 KD cells.

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There were 21 targets which displayed up-regulated expression upon Pg treatment, and were further up-regulated in Pg-treated Elf5 KD cells (Table 4.11). That is, these are Pg targets that are potentially negatively regulated by Elf5. Surprisingly, desmoplakin was identified in this list, despite us detecting no change in desmoplakin expression when we transiently transfected T47D cells with Elf5 siRNA duplexes (Figure 4.13). This would suggest that Elf5 may be involved in repressing the expression of desmoplakin, which is contrary to our hypothesis given that desmoplakin is a marker of progestin-induced differentiation in T47Ds (Kester et al. 1997). However, in addition to differentiation, desmoplakin is also important in other cellular processes, for example, cell-cell adhesion and proliferation. Desmoplakin is a component of desmosomes at cell junctions, and interestingly, so is desmoglein 2, which was identified as a potential target repressed by Elf5 (Table 4.6) (Burdett 1998). Furthermore, down-regulation of desmoplakin can enhance cell growth and activation of ERK MAPK (Wan et al. 2007), and so Elf5 may play a role in repressing these functions of desmoplakin, rather than differentiation.

Progestin treatment also down-regulated a large number of genes. There were 29 targets, which displayed down-regulated expression upon Pg treatment, that was relieved in Pg-treated Elf5 KD cells. That is, these are genes that are repressed by Pg, and de-repressed by Elf5 (Table 4.12). Finally, 4 probesets were identified as being down-regulated upon Pg treatment, and further down- regulated in Pg-treated Elf5 KD cells (Table 4.13). Interestingly, three of these genes (cell division cycle associated 5, SPC25, NDC80 kinetochore complex component and CDC28 protein kinase regulatory subunit 2) are involved in promoting mitosis or cell cycle progression (Bharadwaj et al. 2004; Rankin 2005; Lan et al. 2008). This suggests that Elf5 may be required for the regulation of expression of these two genes, and thus the normal proliferation of these cells. Furthermore, four functional annotation categories were over- represented in these genes with an adjusted p-value passing the significance threshold of BY adjusted p<0.05 (Table 4.14). These categories were related

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to the cell cycle and cell division, lending further support to the hypothesis that Elf5 expression is required for normal proliferation.

In summary, from these transcript profiling experiments we identified a large number of genes which displayed differential expression in response to long- term progestin treatment. Induction of Elf5 expression by Pg only mediated a very small fraction of Pg action. This was depicted in Figure 4.15 where it can be seen that of the 2649 genes that changed following Pg treatment, only 61 of these displayed differential expression when Elf5 levels were repressed. This equates to approximately 2.3% of Pg-regulated genes. Within the Pg-target genes that did show altered expression when Elf5 levels were decreased, was a gene involved in lipogenesis and two genes that promote cell cycle progression, which is consistent with our current understanding of Elf5 function. It is important to note that a large proportion of the genes identified from these transcript profiles are of unknown or undefined function, for example, CAPRIN2 and RFX3 (Table 4.11), and FIGN and CCDC41 (Table 4.12). Therefore, at this time we cannot understand the functional relevance of the identification of all of the genes identified from this microarray analysis. However, as these signalling pathways and networks continue to be defined, the significance of a number of these genes may later be revealed.

Overall, the data obtained from this microarray analysis has suggested that in T47D cells specifically, the role played by Elf5 in the Pg signalling pathway is relatively minor. It is possible that Pg modulates Elf5 to provide a way of potentiating the activity of related pathways, such as growth factor receptor or hormone receptor pathways. For example, A kinase anchor protein 13 (identified in Table 4.11) has been reported to bind the ER and modulate ER signalling (Rubino et al. 1998), and microtubule-associated protein 2 (also identified in Table 4.11) has been shown to interact with Src and Grb2 (Lim and Halpain 2000), both of which are members downstream of the Prlr signal transduction pathway, as described in Chapter One. Therefore, rather than being a critical transcription factor downstream of Pg action, Elf5 expression

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may be more important in participating cross-talk with other signal transduction cascades, such as the Prl signalling pathway, which is consistent with the synergistic action of Prl and Pg in mammary gland development.

Table 4.11 Potential Elf5-mediated Pg targets. Genes up-regulated in Pg-treated vs Baseline experiment (BY adj p<0.05) and up-regulated in Pg-treated Elf5 KD vs Pg-treated experiment (unadjusted p<0.001).

Pg-treated Elf5 KD vs Pg- Pg-treated vs Baseline treated Un- Probeset Gene Symbol // BY p- Foldchange adjusted Foldchange ID Gene Name values p value 8116780 DSP // desmoplakin 0.028 1.498 0.000 1.682 7963410 KRT6C // keratin 6C 0.000 18.583 0.000 2.765 AKAP13 // A kinase (PRKA) anchor protein 7985695 13 0.003 1.807 0.000 1.478 7904482 --- 0.011 1.387 0.000 1.314 CAPRIN2 // caprin 7962112 family member 2 0.005 1.661 0.000 1.437 RFX3 // regulatory factor X, 3 (influences HLA class II 8159876 expression) 0.027 1.421 0.000 1.420 MAP2 // microtubule- 8047926 associated protein 2 0.018 2.086 0.000 1.946 SRGAP2 // SLIT- ROBO Rho GTPase 7909175 activating protein 2 0.002 1.946 0.000 1.403 TRIM29 // tripartite 7952290 motif-containing 29 0.000 6.048 0.000 1.300 LPP // LIM domain containing preferred translocation partner 8084742 in lipoma 0.036 1.522 0.000 1.464 7927091 --- 0.043 1.400 0.000 1.375 MYCBP2 // MYC 7972069 binding protein 2 0.047 1.525 0.001 1.477 LOC440973 // similar to Nuclear transcription factor Y subunit beta (Nuclear transcription factor Y subunit B) (NF-YB) (CAAT-box DNA- binding protein 8089459 subunit B) 0.044 1.599 0.001 1.514 8155849 ANXA1 // annexin A1 0.000 5.641 0.001 1.372

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MAFB // v-maf musculoaponeurotic fibrosarcoma oncogene homolog B 8066266 (avian) 0.000 21.121 0.001 1.467 AHNAK // AHNAK 7948667 nucleoprotein 0.004 2.016 0.001 1.433 ZCCHC6 // zinc finger, CCHC domain 8162147 containing 6 0.007 1.556 0.001 1.288 TM4SF1 // transmembrane 4 L 8091411 six family member 1 0.003 2.461 0.001 1.538 7904469 --- 0.038 1.331 0.001 1.262 PCLO // piccolo (presynaptic 8140620 cytomatrix protein) 0.026 1.886 0.001 1.605 TMEM164 // transmembrane 8169365 protein 164 0.000 4.019 0.001 1.424

Table 4.12 Potential Elf5-repressed Pg-repressed targets. Genes down-regulated in Pg- treated vs Baseline experiment (BY adj p<0.05) and up-regulated in Pg-treated Elf5 KD vs Pg- treated experiment (unadjusted p<0.001). Pg-treated Elf5 KD vs Pg- Pg-treated vs Baseline treated Un- Probeset Gene Symbol // BY p- Foldchange adjusted p Foldchange ID Gene name values value

7896578 --- 0.0007 0.001 <0.0001 3858.684 8042462 --- 0.0026 0.000 <0.0001 1403.737 7895623 --- 0.0073 0.265 <0.0001 5.277 7893995 --- 0.0009 0.286 <0.0001 3.773 7975309 --- 0.0121 0.504 <0.0001 2.027 7892616 --- 0.0201 0.063 0.0001 20.760 8053797 --- 0.0196 0.394 0.0001 2.659 C21orf15 // chromosome 21 open reading frame 8069505 15 0.0098 0.413 0.0002 1.935 C9orf165 // chromosome 9 open 8160816 reading frame 165 0.0043 0.411 0.0002 1.785 NAP1L3 // nucleosome assembly protein 1- 8173917 like 3 0.0287 0.568 0.0002 1.736 KIAA1641 // 8053834 KIAA1641 0.0150 0.595 0.0002 1.585

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AHNAK2 // AHNAK 7981514 nucleoprotein 2 0.0454 0.674 0.0002 1.548 7893340 --- 0.0036 0.613 0.0002 1.370 8056323 FIGN // fidgetin 0.0049 0.627 0.0002 1.363 ATP11C // ATPase, 8175492 class VI, type 11C 0.0010 0.553 0.0002 1.313 7893780 † --- 0.0114 0.001 0.0003 172.675 SNORD22 // small nucleolar RNA, C/D 7948896 box 22 0.0185 0.491 0.0004 1.752 8009351 --- 0.0288 0.550 0.0004 1.687 HSP90AA5P // heat shock protein 90kDa alpha (cytosolic), class A member 5 8084299 (pseudogene) 0.0250 0.592 0.0005 1.533 TMEM46 // transmembrane 7970676 protein 46 0.0337 0.648 0.0005 1.465 7896647 --- 0.0130 0.655 0.0005 1.350 ARFGEF1 // ADP- ribosylation factor guanine nucleotide- exchange factor 1(brefeldin A- 8151149 inhibited) 0.0074 0.640 0.0005 1.322 LAMC1 // laminin, gamma 1 (formerly 7908041 LAMB2) 0.0060 0.563 0.0006 1.398 TPR // translocated promoter region (to activated MET 7922912 oncogene) 0.0408 0.737 0.0007 1.300 INTS6 // integrator 7971692 complex subunit 6 0.0245 0.712 0.0007 1.300 CCDC41 // coiled-coil domain containing 7965486 41 0.0128 0.576 0.0007 1.441 NPY1R // neuropeptide Y 8103494 receptor Y1 0.0003 0.104 0.0008 1.897 LOC220594 // TL132 8005689 protein 0.0145 0.593 0.0009 1.415 TOP2B // topoisomerase (DNA) 8085815 II beta 180kDa 0.0033 0.626 0.0009 1.251

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Table 4.13 Potential Elf5-induced Pg-repressed target genes. Genes down-regulated in Pg-treated vs Baseline experiment (BY adj p<0.05) and down-regulated in Pg-treated Elf5 KD vs Pg-treated experiment (unadjusted p<0.001).

Pg-treated vs Pg-treated Elf5 KD vs Baseline Pg-treated Un- Probeset BY p- Foldchange adjusted Foldchange ID Gene Symbol // Gene Name values p value CDCA5 // cell division cycle 7949364 associated 5 0.0003 0.392 0.0003 0.746 SPC25 // SPC25, NDC80 kinetochore complex component, homolog (S. 8056572 cerevisiae) 0.0001 0.379 0.0004 0.780 CKS2 // CDC28 protein kinase 8156290 regulatory subunit 2 0.0009 0.449 0.0007 0.740 LOC201164 // similar to 8013068 CG12314 gene product 0.0220 0.703 0.0010 0.779

Table 4.14 Functional annotation categories over-represented with un-adjusted p<0.05 in potential Elf5-induced Pg-repressed targets. These genes were down-regulated in Pg-treated vs Baseline experiment (BY adj p<0.05) and down-regulated in Pg-treated Elf5 KD vs Pg-treated experiment (unadjusted p<0.001).

BY P- Category Term Count p-Value values SP_PIR_KEYWORDS cell division 3 1.64E-04 0.022 SP_PIR_KEYWORDS cell cycle 3 7.06E-04 0.043 GOTERM_BP_ALL GO:0051301~cell division 3 9.56E-04 0.043 GOTERM_BP_ALL GO:0000279~M phase 3 0.00135 0.045

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Discussion

Elf5 is a prolactin-regulated Ets transcription factor critical in the differentiation of the mammary gland in vivo, and in this chapter we have demonstrated that Elf5 is also regulated by Pg and human growth hormone. The induction of Elf5 expression by progesterone in vivo was antagonized by co-treatment with estrogen. Induction of Elf5 expression by Pg was observed in both the mouse mammary gland in vivo and in human breast cancer cells in vitro, suggesting functional similarities between the mouse and human Elf5 regulation. As T47D breast cancer cells express high levels of endogenous Elf5, we have investigated the effects of repressing Elf5 expression in these cells. We observed that Elf5 expression is required for normal proliferation, similar to what occurs in vivo. Finally, when Elf5 expression was repressed in the presence of progestin treatment in T47D cells, we determined that Elf5 expression mediated only a very small fraction of progestin action.

Analysis of the effects of Elf5 expression on proliferation of T47D cells was achieved by transiently transfecting the cells with Elf5 siRNA, after which we observed altered cellular morphology and a lack cell colony formation. In addition, suppression of Elf5 expression reduced the proportion of cells in the proliferative S phase, and decreased the expression of growth-promoting signalling molecules, such as phospho-ERK and cyclin D1. These growth inhibitory observations suggest a requirement of Elf5 in the normal proliferation of T47D cells. To support our data demonstrating Elf5 expression is required for normal levels of ERK activation, we investigated this hypothesis in a second model. Elf5 has previously been demonstrated to have a role in proliferation in vivo. Compared to wild-type mammary epithelium, the number of Ki67-positive cells in Elf5-/- mammary transplants was reduced, as was ERK phosphorylation (Figure 4.11 and Oakes et al. 2008; See Appendix III). This effect is comparable to the significant, although not dramatic, effect that we observed when we knocked down Elf5 expression in T47D cells. Furthermore,

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we identified a number of genes and functional annotation categories involved in cell division and the promotion of proliferation, which were down-regulated in Pg-treated Elf5 KD cells. Together, these findings demonstrate Elf5 expression is required for normal proliferation in mammary epithelial cells.

Knowing that long-term Pg treatment induces differentiation in T47D breast cancer cells, and that Elf5 is a transcription factor important in the differentiation of mammary epithelial cells and is up-regulated with long-term Pg treatment, we hypothesised that Elf5 expression is required to induce the transcription of some Pg target genes involved in differentiation. We investigated this hypothesis using two different methods. Firstly, we transiently transfected T47D cells with Elf5 siRNA duplexes and measured the levels of two specific Pg target genes involved in differentiation. We also transcript profiled T47D cells which can be induced to stably repress Elf5 expression. From these analyses we discovered that induction of Elf5 expression by Pg did not play a major role in mediating Pg action. Pg treatment of T47D cells modulated large subsets of genes (approximately 2,700 probesets), however reduced Elf5 expression influenced only 2.3% of these particular probesets.

This suggests that, at least in this experimental model system, the induction of Elf5 expression by Pg is utilised more to influence other signal transduction cascades, such as the Prl pathway. Cross-talk between Pg and various other signalling pathways has previously been proposed to account for seemingly paradoxical effects of Pg on cell proliferation (Lange et al. 1998; Richer et al. 1998; Lange et al. 1999). For example, perhaps Pg-induced up-regulation of Elf5 is required for Pg to promote differentiative rather than proliferative effects, via Elf5 acting in concert with Prl signalling. If a similar experiment was performed to compare microarray analyses of Prl-treated cells with Prl- treated Elf5 KD cells, we would expect large subsets of genes to display differential expression in this comparison, as Elf5 expression is sufficient to compensate for loss of Prl signalling (Harris et al. 2006). We hypothesise that

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Elf5 is primarily a major transcription factor downstream of Prl, and that this action is enhanced by Pg. Evidence of the overlap between Pg and Prl signalling has previously been demonstrated at the level of other molecules, for example, Stat5 (Gouilleux et al. 1994; Richer et al. 1998) and the c- Src/Ras/ERK MAPK pathway (Acosta et al. 2003; Lange 2008). Data from this chapter suggests that Elf5 also participates in cross-talk between the Pg and Prl signalling cascades. Furthermore, as Prl and Pg have both been implicated in mammary carcinogenesis (see Chapter One), dysregulation of this cross-talk mechanism may contribute to breast cancer biology.

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Chapter 5: Expression Profile and Hormonal Regulation of KIBRA

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Introduction

From the study that compared opposing models of prolactin action outlined in Chapter Three, a second candidate gene was chosen for further investigation. The murine EST AI850846 was identified as a mammary epithelium specific transcript that was decreased in Prlr-/- mammary epithelium compared to Prlr+/+ mammary epithelium, and increased in response to prolactin treatment in the SCp2 cell model system. We named this molecule GOBLIN and performed BLAST homology searches of the GenBank database to determine the full-length nucleotide sequence of GOBLIN in the mouse, and its orthologue in the human. This approach identified a human sequence that lacked the 5’ region of the mouse coding sequence. Work undertaken by Dr Prudence Stanford in the laboratory using rapid amplification of cDNA (RACE) from the 5’ end of this human mRNA identified an additional 566 nucleotides in the human mRNA, which shared high sequence homology to the mouse sequence. This allowed the identification of the full-length human GOBLIN nucleotide sequence. When the full-length mouse and human protein sequences were aligned, it was revealed there was conservation of two WW domains and a C2 domain, and the mouse and human orthologues shared 91% overall amino acid identity. Subsequent sequence database searches identified that GOBLIN corresponded to a protein which has since been named KIBRA/WWC1 (GenBank accession no. NM_170779 [mouse]; NM_015238 [human]). We selected KIBRA for further study as its function had not been previously described, and it had two functional domains (the C2 phospholipid binding domain and two WW motifs), suggesting it may play a structural or signalling role.

As published in Hilton et al. (2008) (see Appendix II), absolute quantitative PCR undertaken by Dr Jessica Harris in our laboratory demonstrated that KIBRA transcripts were present in Prlr+/+ mammary glands during early pregnancy, were decreased in numbers in Prlr-/- mammary glands at the same time points, and were only just detectable in Prlr+/+ fat pads cleared of

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epithelium, confirming the transcript profiling data. It was also demonstrated that KIBRA transcripts were detected at all stages of development in the normal murine mammary gland, and that its expression increased during mid- pregnancy (day 12) to a peak at late pregnancy (day 18) and early lactation (day 2), followed by a decrease at later time points (day 9). At involution of the mammary gland (day 1), KIBRA transcript levels increased again to levels observed at day 18 of pregnancy. This expression profile is characteristic of a gene that is involved in tissue remodelling during lobuloalveolar development and involution. KIBRA transcript numbers were also examined in a panel of primary human breast cancer cell lines, as well as normal and immortal basal epithelial cell lines. KIBRA was expressed in all the cell lines analysed, and no correlation with expression of the Prlr or steroid hormones receptors was apparent, as had been observed for Elf5. KIBRA expression was low in the normal cell lines (which have a finite lifespan), higher in the immortal mammary cell lines, and showed variable expression in the breast cancer cell lines. At the same time we began investigating the function of the KIBRA protein, it was published as being identified from a yeast two-hybrid screen of a human brain cDNA library as a protein which interacts with the postsynaptic protein Dendrin (Kremerskothen et al. 2003). It was shown to contain two N- terminal WW domains and a C2 domain, and to be expressed predominantly in the kidney and brain (Kremerskothen et al. 2003). We were therefore interested in investigating the role of KIBRA in the mammary gland.

Specifically, the aims of this chapter are to: (1) Describe the generation and construction of reagents used for the investigation of KIBRA, (2) Characterise the expression of KIBRA protein in the mammary gland and mammary cell lines, (3) Investigate the effect of hormonal stimulation on KIBRA expression, and (4) Investigate the effect of KIBRA overexpression on the growth of normal human mammary cells and breast cancer cell lines.

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Results

Generation of reagents

Cloning of KIBRA

In order to begin investigating the expression and function of a novel protein like KIBRA, we needed to generate reagents such as expression constructs and antibodies. Figure 5.1 depicts the constructs made, and illustrates their sizes, tags and the location of the functional WW and C2 domains. Details of primers, vectors and restriction sites used are described in Chapter Two. We produced two overlapping truncated V5-His-tagged constructs – one without the WW domains, and one without the C2 domain. These fusion constructs would allow an analysis of the requirement of each of these functional domains in later experiments.

Figure 5.1 Schematic diagram of KIBRA constructs generated. i. KIBRApcDNA3.1 ii. KIBRA-V5 iii. WW-V5 iv.C2-V5.

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Validation of KIBRA antibody

There are currently no commercial KIBRA antibodies available, so in order to perform a comprehensive analysis of KIBRA protein expression in mouse and human tissue and cell lines, we generated an affinity purified anti-KIBRA antibody. The location of the peptide sequence used to raise the antibody is shown in Figure 5.1, and this sequence is conserved between human and mouse. To determine the specificity of our antibody, we transfected HEK-293 cells with the constructs described in Figure 5.1. Western blot analysis of protein lysates from these cells showed that our KIBRA antibody recognises all four constructs (Figure 5.2). This antibody is highly specific for KIBRA, as there is little cross-reactivity to other proteins, and the bands detected are almost identical to those detected by the monoclonal V5 antibody.

Figure 5.2 Validation of KIBRA antibody as shown by Western blot. Lysates (20 g) of HEK-293 cells transfected with constructs as labelled, and immunoblotted with -KIBRA and - V5 antibodies. Reference molecular weight markers (kDa) are indicated.

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Downregulation of KIBRA expression by RNA interference

To provide additional validation of our KIBRA antibody, and to test whether we could knock down KIBRA expression for further experimentation, four KIBRA siRNAs (Dharmacon; see Chapter Two) were screened for their efficiency in MCF-10A normal human mammary cells, and T47D human breast cancer cells. The siRNAs were transfected at concentrations of 50, 25 and 10 nM for 48 hours, followed by western blotting for KIBRA (Figure 5.3). All siRNAs tested reduced KIBRA protein expression compared to controls, with KIBRA siRNAs #1 - #3 being the most effective, particularly in the MCF-10A cell line. The concentration of the siRNA used did not appear to have a major effect, and so a low concentration (10 nM) of siRNA duplexes could therefore be used for later functional experiments.

Figure 5.3 Knockdown of KIBRA expression in human mammary cells. Transfection of MCF-10A and T47D cells with a panel of four KIBRA siRNAs results in reduction in KIBRA protein after 48 hrs by all siRNAs at 10-50 nM concentrations. Expression of -actin is shown as a loading control.

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KIBRA expression in mammary gland and breast

Expression of KIBRA protein in human breast cancer cell lines

Figure 5.4 shows two Western blots of KIBRA expression in various mammary cell lines and demonstrates that we are able to detect endogenous KIBRA expression in both human and mouse cell lines. Lysates of HC11 (normal murine mammary epithelial cells) transfected with KIBRA siRNA, and HEK-293 cells transfected with KIBRApcDNA3.1, provide further validation of the specificity of our KIBRA antibody. We observed specific bands of the KIBRA protein running below the 150 kDa size marker and often as a doublet. When T47D breast cancer cell lysates were treated with lambda protein phosphatase only the lower band of the doublet remained, indicating that the upper band is due to post-translational phosphorylation. Whether KIBRA is present predominantly as the phosphorylated form or non-phosphorylated form depends on the cell line, for example in mouse HC11 cells KIBRA is observed as the phosphorylated form only, and in the human breast cancer cell line MDA-MB-231 KIBRA is observed only as the non-phosphorylated form.

Localisation of KIBRA as visualised by immunofluorescence

KIBRA shows a predominantly cytoplasmic localisation (Kremerskothen et al. 2003), however a significant amount can also be seen in the nucleus of a subset of cells, as shown by immunofluorescence microscopy of endogenous levels of KIBRA in T47D cells (Figure 5.5). KIBRA has previously been reported to have a nuclear localisation signal in the protein sequence (Rayala et al. 2006), and this interesting expression pattern suggests a potential nuclear function of KIBRA. The cellular distribution of KIBRA in transfected BT20 breast

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cancer cells and HeLa cells is also predominantly cytoplasmic with some perinuclear enrichment, as well as showing staining at membranous regions, particularly in membrane ruffle structures (Figure 5.6).

Figure 5.4 KIBRA protein expression in mouse and human cells. A Lysates (20 g) were loaded as labelled. Lysate of HEK-293 transfected with KIBRApcDNA3.1 is shown at shorter exposure due to over-expression. B Lysates (20 g) were loaded as labelled. Expression of - actin is shown as a loading control.

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Figure 5.5 Localisation of KIBRA as examined by immunofluorescence. A – C Localisation of endogenous KIBRA is shown by staining T47D human breast cancer cells with - KIBRA antibody, shown in green (i). Overlay with actin staining (red) and the nucleus (blue) is shown in (ii). Scale bars represent 20 m.

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Figure 5.6 Localisation of exogenous KIBRA as examined by immunofluorescence. A-B BT20 breast cancer cells transfected with KIBRApcDNA3.1. C Immunofluorescence of HeLa cells transfected with KIBRApcDNA3.1. Cells were stained with -KIBRA antibody, shown in green (i). Overlay with actin staining (red) and the nucleus (blue) is shown in (ii). Scale bars represent 10 m.

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Localisation of KIBRA as visualised by immunohistochemistry

The KIBRA transcript levels measured in a panel of breast cancer cell lines by quantitative RT-PCR were reproduced by immunohistochemical signal strength as shown by BT474 cells (Figure 5.7A), BT20 cells (Figure 5.7B) and MDA-MB- 157 cells (Figure 5.7C). In human breast tissue, KIBRA protein is present in the luminal epithelium surrounding the ducts in the normal breast, but is not expressed in the myoepithelium, stromal adipocytes or fibroblasts (Figure 5.7D). KIBRA protein was also detected in cancerous tissue and Figure 5.7E-F shows heterogeneous expression of KIBRA in invasive ductal carcinoma (IDC). Shown also is the expression of KIBRA protein in mouse mammary tissue throughout development (Figure 5.7G-L). It is observed as surrounding the alveoli structures and is predominantly cytoplasmic, as seen at time points of 18.5 days post coitus (Figure 5.7K) and 1 day post partum (Figure 5.7L).

Figure 5.7 (over page) KIBRA protein expression in mouse and human cells as examined by immunohistochemistry. A BT474 cells B BT20 cells C MDA-MB-157 cells D Normal human breast tissue. Blue arrow indicates positive KIBRA staining of luminal epithelial cells. Black arrow shows negative KIBRA staining of surrounding myoepithelial cells. E and F High grade IDC. Blue arrows indicate KIBRA staining at membranous regions. Black arrow indicates cytoplasmic staining of KIBRA G – L Immunohistochemistry analysis of KIBRA protein expression in mouse mammary tissue throughout development (G 12 week virgin, H 2.5 days post coitus, I 6.5 days post-coitus, J 12.5 days post coitus, K 18.5 days post coitus, L 1 day post partum). Scale bars represent 20 m.

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Effect of hormonal stimulation on KIBRA gene expression

As we identified KIBRA as being a gene showing decreased expression in Prlr-/- mammary epithelium and increased expression by MAS analysis in two SCp2 cell experiments following treatment with prolactin, we were also interested in investigating the effect of other hormones that are required for alveolar morphogenesis. Like for Elf5 (see Chapter Four), when we treated T47D cells with ORG2058, we observed a strong up-regulation of KIBRA expression. Figure 5.8A shows KIBRA transcript levels following ORG2058 treatment over a 48-hour time course, and Figure 5.8B shows levels of KIBRA protein expression over a five day time course. Up-regulation of KIBRA expression is observed from about 8 hours of treatment and was maintained to at least 5 days. Figure 5.8C depicts the relative increase in KIBRA expression from densitometry analysis of five independent experiments following 24 hours of ORG2058 treatment.

This progestin-induced up-regulation of KIBRA was also observed in BT474 cells, which is a second Pgr-positive human breast cancer cell line. Figures 5.9A and 5.9B depict the levels of KIBRA transcripts and protein expression, respectively, over a time course following ORG2058 treatment. We also co- treated T47D cells with ORG2058 and the progestin antagonist RU486 for 16 and 24 hours, and this inhibited the increase in KIBRA expression, indicating this up-regulation occurs in a Pgr-specific manner (Figure 5.9C). Together these experiments demonstrate that KIBRA expression is regulated by a second hormone that drives alveolar morphogenesis.

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C p<0.0001

Figure 5.8 KIBRA expression is strongly up-regulated in T47D cells by progestin treatment. A Relative number of KIBRA transcripts in T47D cells over time course following treatment with ORG2058 (each bar is mean±SE). B Western blot of time course showing KIBRA protein expression in T47D cells following treatment with ORG2058 for between 4 h and 5 days. C Data generated from densitometry of 5 separate Western blots.

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Figure 5.9 KIBRA is up-regulated by progestin treatment in a Pgr-specific manner. A Relative number of KIBRA transcripts in BT474 cells over time course following treatment with ORG2058 (each bar is mean±SE). B Western blot of time course showing KIBRA protein expression in BT474 cells following treatment with ORG2058 for between 16 h and 3 days. C KIBRA protein levels in T47D cells following treatment with 10 nM ORG2058 alone, and co-treatment with ORG2058 and 100 nM RU486.

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KIBRA is not a co-activator of the progesterone receptor

KIBRA is regulated by progestin treatment and localises to the nucleus (Figure 5.5). KIBRA has also previously been shown to be a co-activator of the estrogen receptor (Rayala et al. 2006), therefore we were interested in determining whether KIBRA could also regulate the function of the progesterone receptor. In order to determine whether KIBRA could enhance transcriptional activation by Pgr, we transiently transfected HEK-293 cells, which lack endogenous Pgr, with a Pgr expression vector (hPRB-1), and the progestin-responsive MMTV-luciferase reporter construct (pMSGluc) together with KIBRA, pcDNA3.1 (empty vector) or SRC-1 (as a positive control). KIBRA had virtually no effect on progestin (ORG2058)-induced luciferase activity compared to empty vector control (Figure 5.10), and is therefore not a co- activator of the Pgr.

Figure 5.10 KIBRA does not enhance transcriptional activation by PgR. Luciferase reporter assays were carried out using HEK-293 cells transfected with KIBRA, SRC-1 or empty vector. Cells were harvested for luciferase activity assays following 48 h of treatment with either 10 nM ORG2058 or ethanol vehicle.

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Effect of KIBRA overexpression levels on cell growth

As mentioned in Chapter Four, progestins have a biphasic effect on the proliferation of T47D breast cancer cells, whereby they initially accelerate the cells through the first cell cycle (up to 12 h following ORG2058 treatment), followed by cell cycle arrest and inhibition of growth (Musgrove et al. 1991). Previously we observed that KIBRA protein expression was strongly up- regulated with long-term progestin treatment, we were therefore interested in determining whether the level of KIBRA expression had any effect on cell growth or proliferation. In order to study this, we transiently transfected T47D breast cancer cells with KIBRApcDNA3.1, confirmed KIBRA overexpression, and performed growth curves and MTT assays. Figure 5.11 shows that overexpression of KIBRA had no effect on the proliferation of T47D breast cancer cells.

Having carried out proliferation assays using cells that were transiently transfected, we wanted to create cell lines that stably overexpressed KIBRA. We attempted this by transfecting the cells followed by antibiotic selection, however we were unsuccessful in creating stable clones or pools of cells with significant KIBRA overexpression in T47D, MCF-10A and MCF-12A cell lines. An alternative method for creating stable cell lines is by retroviral infection, and we employed this method in MCF-10A-EcoR cells by infecting them with a KIBRA-IRES-GFP retrovirus (KIBRA) or empty vector control virus (pMIG), similar to as what was performed with Elf5 (see Chapter Three). Using this approach we were successful in making stable MCF-10A cells, which normally only expresses relatively moderate amounts of the protein, with high expression of KIBRA.

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pcDNA3.1 KIBRA

Figure 5.11 KIBRA overexpression in T47D breast cancer cells has no effect on proliferation. A Representative growth curve showing the proliferation of T47D cells transfected with pcDNA3.1 and KIBRApcDNA3.1 over time. Each point is mean±SE. B Absorbances as measured by MTT assay of T47D cells transfected with pcDNA3.1 and KIBRApcDNA3.1. Inset: Western blot showing level of KIBRA overexpression of cells 72 hours post-transfection ie. Day 2 of the MTT assay.

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To determine whether KIBRA overexpression had an effect in the three- dimensional culture system, we plated an unsorted population of these KIBRA overexpressing MCF-10A cells onto Matrigel. As shown in Figure 5.12A, there was large overexpression of KIBRA at Day 0 when the cells were plated. Interestingly, by Day 6 of culture this overexpression had been completely lost to basal expression levels. This was specifically due to the overexpression of KIBRA, as GFP expression was maintained in the pMIG control acini over the duration of the experiment. Also, there is no suppression of KIBRA overexpression when maintained in normal 2D culture, as shown by Western analysis of lysates taken over 4 consecutive days (Figure 5.12B). There appears to be more than one isoform or splice variant of KIBRA that is overexpressed in these cells, however it is the larger, more dominant form which corresponds to that which is expressed endogenously in MCF-10A cells (Figure 5.12B).

Because KIBRA expression was completely suppressed over a short time in three-dimensional culture, this was not a suitable system to study the function of KIBRA, so we investigated the effect of KIBRA overexpression on proliferation in two-dimensional culture. We took these MCF-10A-KIBRA cells and selected high and low GFP-expressing populations by FACS (Figure 5.13A). Interestingly, the high KIBRA-expressing population did not grow in culture (data not shown), and so the low KIBRA-expressing population was used for proliferation assays. The representative growth curve in Figure 5.13B shows that there was no difference in the rate of growth of KIBRA-expressing cells compared to controls.

The data presented here suggests that high levels of KIBRA overexpression in MCF-10A cells are selectively silenced, or that high levels of expression stops the growth of MCF-10A cells, when cultured on Matrigel. These results also demonstrate that high KIBRA expression also inhibits the growth of these cells when maintained in normal two-dimensional culture. It cannot be concluded whether this is a specific effect of KIBRA overexpression, or whether it is an

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artefact of such high expression of KIBRA. To circumvent this effect we selected a population of MCF-10A cells expressing low levels of KIBRA, but saw no difference in growth rate of the cells.

B Day 1 Day 2 Day 3 Day 4 P K P K P K P K

-KIBRA

-actin

Figure 5.12 KIBRA overexpression in MCF-10A cells. A Transfected KIBRA expression is completely lost following 6 days of three-dimensional culture. The Western blot of KIBRA expression is shown at both long and short exposure to show the levels of endogenous KIBRA expression in these cells. B KIBRA overexpression is maintained over time in two-dimensional culture. P = pMIG; K = KIBRA.

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Figure 5.13 MCF-10A-KIBRA cells sorted for level of expression. A FACS plots of MCF-10A cells expressing KIBRApMIG showing populations selected for low and high expression B Growth curve of MCF-10A cells expressing low levels of KIBRA versus control cells.

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Discussion

Comparison of the SCp2 transcript profiles with transcript profiles of Prlr-/- mammary epithelium during early pregnancy identified KIBRA as a novel gene putatively regulated by prolactin in the mammary epithelium during pregnancy. KIBRA encodes a large protein of just under 150 kDa, and includes two conserved WW domains and a C2 phospholipid-binding domain. The WW domain is a protein interaction module that recognises specific proline- containing peptide sequences (Otte et al. 2003). They are found in a variety of proteins involved in diverse cellular processes including protein degradation, transcription regulation and differentiation, and are also associated with a number of human diseases including muscular dystrophy, Alzheimer’s disease and cancer (Sudol et al. 2001). The C2 domain is a conserved membrane- targeting motif that mediates an array of intracellular processes, the majority of which involve signal transduction and membrane trafficking (Nalefski and Falke 1996). The presence of these domains suggested a scaffold protein role for KIBRA in the formation of a signalling complex.

Results presented in this chapter demonstrate how we utilised our own affinity purified anti-KIBRA antibody to examine the expression of KIBRA protein in both human and mouse mammary tissue and cell lines, using immunofluorescence, immunohistochemistry and Western analysis techniques. We have shown that KIBRA was expressed in all cell lines tested, although with variable expression. In agreement with Kremerskothen et al., (2003), we also show that KIBRA protein is expressed predominantly in the cytoplasm in mammary cells, however we also show that in T47D breast cancer cells there are a subset of cells which show nuclear localisation of KIBRA. This nuclear localisation has also been demonstrated in other labs (Rayala et al. 2006). In addition, we have shown that KIBRA expression is epithelial-specific, and that it is expressed in the luminal epithelial cell population of the normal human breast and breast cancer cell lines.

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As well as showing decreased expression in Prlr-/- mice, we have demonstrated that KIBRA is markedly up-regulated with progesterone treatment. It is interesting to note that a number of other genes were identified in the original study which, similarly to KIBRA, displayed decreased expression levels in Prlr-/- mammary epithelial cells, and are positively regulated by progesterone (Harris et al. 2006). In addition to Prlr itself, these genes included Elf5, amphiregulin, calcitonin and Wnt-4 (see Chapter Four;(Das et al. 1995; Ormandy et al. 1997b; Brisken et al. 2000; Ismail et al. 2004). Both the prolactin and progesterone signalling pathways are critical in mammary development, and they work synergistically in driving alveolar morphogenesis. This has been demonstrated in mammary cells where prolactin causes up-regulation of the progesterone receptor, and conversely progesterone up-regulates the prolactin receptor (Ormandy et al. 1997b). This may provide an additional explanation for our detection of KIBRA as differentially regulated in Prlr-/- epithelium compared to wild type.

We have demonstrated here that transient overexpression of KIBRA does not have any significant effect on the growth of the cell lines tested. A previous study reported that they had been successful in generating stable KIBRA- overexpressing MCF7 human breast cancer cells, and have shown that KIBRA enhanced basal and estrogen-induced levels of Cyclin D1 (Rayala et al. 2006). Although they did not perform any proliferation assays, this might suggest that KIBRA confers pro-proliferative effects in this cell line, however from the data presented it was difficult to determine the level of KIBRA overexpression that was obtained. It could be speculated that the effect on cell proliferation is dependent on the level of KIBRA overexpression, and that very high levels of KIBRA expression inhibit cell growth, which would provide a possible explanation as to why the high KIBRA-expressing MCF-10A cell population did not grow. As we experienced difficulty in establishing cell lines stably overexpressing KIBRA, we undertook an alternative approach to investigate the function of KIBRA by using bioinformatics to identify potential interacting partners. This is discussed in detail and forms the focus of Chapter Six.

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Chapter 6: Identification and Characterisation of KIBRA Interaction with DDR1

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Introduction

As described in Chapter Five, KIBRA consists of two N-terminal WW domains and a C2 domain. The WW domain is a major protein-protein interaction module and is so named for its highly conserved two tryptophan (W) residues spaced 20-22 amino acids apart. They are widely distributed in all biological systems, often localised in the cytoplasm as well as in the nucleus, and have been implicated in a diverse range of processes, such as protein degradation, transcription, and RNA splicing. Examples of WW domain containing proteins include the transcriptional co-activator TAZ (Kanai et al. 2000), the scaffolding protein MAGI-2 (Wu et al. 2000), and the tumor suppressor WWOX (Aqeilan and Croce 2007). WW domains recognise specific proline-containing peptides, in particular the sequence motif, PPxY (where P is proline, Y is tyrosine, and x is any amino acid) (Sudol and Hunter 2000). The core of the structure also contains conserved aromatic residues, and it is these conserved residues which are key to the specificity of WW domain interactions with its proline-containing ligands (Ilsley et al. 2002).

The C2 domain was first discovered in protein kinase C as a calcium-binding domain that is approximately 120 amino acids in length (Newton and Johnson 1998). Upon calcium binding, the C2 domain can bind to phospholipids, inositol polyphosphates, and some proteins. They are often involved in targeting proteins to cell membranes, and are found in many cellular proteins involved in signal transduction or membrane trafficking, for example, the synaptic plasticity protein SynGAP (Pena et al. 2008), the tumor suppressor lipid phosphatase PTEN (Lee et al. 1999), and lactadherin (a membrane-associated human milk glycoprotein) (Taylor et al. 1997). Similarly to KIBRA, the Nedd4 family of E3 ubiquitin ligases contain both WW domains and a C2 domain, in which these domains are important in regulating cellular localisation and substrate selection (Ingham et al. 2004).

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In Chapter Five we examined the expression profile and hormonal regulation of KIBRA in human mammary epithelial cells. To further investigate the function of KIBRA, we were interested in identifying novel KIBRA interacting partners. Since this work began, KIBRA has been demonstrated to bind a number of proteins. These include the postsynaptic protein Dendrin (Kremerskothen et al. 2003), the signalling enzyme protein kinase C zeta (PKC) (Buther et al. 2004), the cytoplasmic component dynein light chain 1 (DLC1) (Rayala et al. 2006), and the endosomal sorting molecule sorting nexin-4 (SNX4) (Traer et al. 2007). Common methods of identifying novel protein interacting partners are yeast two-hybrid analysis, pull-down assays, co-immunoprecipitation and mass spectrometry. Using the fact that WW domains bind proteins with PPxY motifs, we were interested in looking at possible targets that contained this motif. We did this by using bioinformatics to identify a list of candidate proteins which contained this peptide sequence, as well as being expressed in the mammary gland, and to see whether we could demonstrate an interaction with KIBRA. This would thus aid in elucidating more about the cellular function of KIBRA.

The specific aims of this chapter are to: (1) Use bioinformatics to identify a list of candidate KIBRA interacting proteins, (2) Confirm the interaction of KIBRA with one or more of these candidates, (3) Characterise the domains and regions required for this interaction to occur, and (4) Define a signalling pathway endpoint that responds to manipulation of KIBRA expression.

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Results

Use of bioinformatic approach to identify KIBRA interacting partners

In collaboration with Dr Warren Kaplan from the Peter Wills Bioinformatics Centre at the Garvan Institute, we undertook a bioinformatic approach to identify potential KIBRA interacting proteins. Using the fact that WW domains recognise specific proline-containing peptides, we mined the human proteome for proteins that contained the PPxY motif using the protein regular expression program 'Preg' (Rice et al. 2000). This list was refined to 105 candidates by selecting proteins that contained the motif RxPPxY (where R is arginine), the preferred putative consensus recognition motif for KIBRA, as identified in Kremerskothen et al., 2003. By discounting candidates which were “hypothetical” proteins or “similar to hypothetical” proteins, and only selecting those which were expressed in the mammary gland (determined from the NCBI Unigene database), this list was further refined to 29 candidates (Table 6.1). From this list of potential binding partners we selected DDR1, a collagen activated tyrosine kinase receptor, as our top candidate due to its essential role in mammary gland development (Vogel et al. 2001) and its involvement in signalling pathways, as KIBRA was proposed to act as a potential signalling scaffold molecule due to the presence of the WW and C2 domains.

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TABLE 6.1. List of candidate KIBRA interacting proteins which contain a RxPPxY motif and are expressed in mammary gland.

Sequence RxPPxY Sequence Derived Gene From Gene Title Symbol Function

NM_003035 TAL1 (SCL) interrupting locus SIL cell proliferation RQPPAY

NM_001447 FAT tumor suppressor homolog 2 (Drosophila) FAT2 cell proliferation and adhesion RVPPNY

NM_023038 a disintegrin and metalloproteinase domain 19 ADAM19 cell-cell and cell-matrix interactions RPPPDY

NM_005401 protein tyrosine phosphatase, non-receptor type 14 PTPN14 signal transduction RPPPPY

NM_005529 heparan sulfate proteoglycan 2 HSPG2 basement membrane molecule RCPPGY

NM_007039 protein tyrosine phosphatase, non-receptor type 21 PTPN21 signal transduction RPPPPY

NM_007286 synaptopodin SYNPO cytoskeletal molecule RSPPSY

NM_014945 actin binding LIM protein family member 3 ABLIM3 cytoskeletal molecule RKPPIY

NM_006720 actin binding LIM protein 1 ABLIM1 cytoskeletal molecule RKPPIY

NM_004393 dystroglycan 1 DAG1 muscle contraction RSPPPY

NM_003494 dysferlin, limb girdle muscular dystrophy 2B DYSF muscle contraction RPPPHY

NM_001954 discoidin domain receptor 1 DDR1 protein tyrosine kinase receptor REPPPY

NM_001982 v-erb-b2 erythroblastic leukemia viral oncogene homolog 3 ERBB3 protein tyrosine kinase receptor RDPPRY

NM_005157 v-abl Abelson murine leukemia viral oncogene homolog 1 ABL1 protein tyrosine kinase activity REPPFY

NM_005642 TAF7 RNA polymerase II TAF7 RNA polymerase II transcription factor RLPPEY

NM_005933 myeloid/lymphoid or mixed-lineage leukemia MLL RNA polymerase II transcription factor RQPPEY

NM_014757 mastermind-like 1 (Drosophila) MAML1 transcriptional co-activator RPPPQY

NM_012245 SKI-interacting protein SKIIP transcription co-activator REPPPY

NM_012406 PR domain containing 4 PRDM4 transcription factor RPPPQY

NM_014345 zinc finger protein 318 ZNF318 RNA binding RIPPNY

NM_012091 adenosine deaminase, tRNA-specific 1 ADAT1 RNA binding and processing RNPPDY

NM_133330 Wolf-Hirschhorn syndrome candidate 1 WHSC1 embryogenesis and morphogenesis RKPPPY

NM_022902 solute carrier family 30 (zinc transporter) SLC30A5 zinc transporter RLPPEY

NM_003730 ribonuclease 6 precursor RNASE6PL ribonuclease activity RDPPDY

NM_018641 chondroitin 4-O-sulfotransferase 2 C4S-2 transferase activity RFPPSY

NM_014262 leprecan-like 2 protein LEPREL2 protein metabolism REPPAY

NM_014427 copine VII CPNE7 lipid metabolism RIPPKY

NM_001649 apical protein-like (Xenopus laevis) APXL sodium channel activity RHPPLY NM_013423 Rho GTPase activating protein 6 ARHGAP6 Rho GTPase activator activity RPPPPYY

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KIBRA interacts with DDR1 in a collagen-regulated manner

We co-expressed full-length cDNAs of KIBRA and DDR1 in HEK-293 cells, and observed co-immunoprecipitation of KIBRA and DDR1 (Figure 6.1A). In addition, when we treated the cells with collagen type IV, a ligand of DDR1, we observed that more DDR1 co-immunoprecipitated with KIBRA in the absence of collagen than in the presence of this ligand (Figure 6.1A). We confirmed this interaction was collagen-regulated by performing the reciprocal immunoprecipitation, where we observed KIBRA immunoprecipitation with DDR1 antibodies, but not in the presence of collagen IV (Figure 6.1B). Furthermore, although the sensitivity of the KIBRA antibody precluded detection of an association between endogenous KIBRA and endogenous DDR1, we could detect recruitment of exogenous KIBRA to endogenous DDR1 (Figure 6.1C).

To further investigate the collagen inhibition of the KIBRA-DDR1 interaction, we performed a kinetic study of DDR1 activation over a four-hour time course and observed a gradual dissociation of KIBRA from DDR1 over time (Figure 6.2A). Exposure to collagen causes tyrosine phosphorylation of DDR1 (Vogel et al. 1997), and particularly interesting was the observation that the time point at which DDR1 was first shown to be tyrosine phosphorylated upon collagen treatment (t = 1 hr), was also the first time point at which less DDR1 was observed to co-immunoprecipitate with KIBRA (Figure 6.2A). This displacement also occurred in a dose-responsive manner upon collagen treatment (Figure 6.2B). Furthermore, we also have demonstrated that this collagen inhibition of the KIBRA-DDR1 interaction was not specific to collagen type IV, as when we used a second DDR1 ligand, collagen type I, we also observed that less DDR1 co-immunoprecipitated in the presence of this ligand (Figure 6.2C). Together, these data show that KIBRA interacts with DDR1 and that this interaction is regulated by collagens that bind and activate DDR1.

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150 kDa

100 kDa

Figure 6.1 KIBRA interacts with DDR1. A KIBRA immunopreciptates from HEK-293 cells transiently co-transfected with KIBRA and DDR1 cDNAs showing that DDR1 co-preciptates with KIBRA. B DDR1 immunoprecipitates from HEK-293 cells +/- collagen showing that KIBRA co- precipitates with DDR1 in the absence of collagen IV. C Endogenous DDR1 immunoprecipitates from T47D cells transiently transfected with KIBRA showing an interaction between KIBRA and endogenous DDR1. IP = immunoprecipitation; WB = Western blot; NLB = normal lysis buffer control.

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_ _

Figure 6.2 KIBRA interacts with DDR1 in a collagen-dependent manner. A Co- immunoprecipitation of KIBRA and DDR1 over a 4 hr time course of collagen IV treatment. Levels of total and phosphorylated DDR1 in whole cell lysates are shown. B Co- immunoprecipitation of KIBRA and DDR1 in response to increasing concentrations of collagen IV. C Co-immunoprecipitation of KIBRA and DDR1 +/- collagen type I.

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The WW domains of KIBRA bind the PPxY motif in DDR1

To determine whether the KIBRA-DDR1 interaction was occurring via the PPxY motif of DDR1, we performed site-directed mutagenesis on this motif. Three DDR1 mutants were made where the target motif (PPPY) was mutated to AAAA, AAPY and PPPF (where A is alanine, P is proline, Y is tyrosine and F is phenylalanine). Full-length KIBRA was co-expressed with either wild-type DDR1 (DDR1-wt) or the DDR1 mutants. Only DDR1-wt, and not DDR1-AAAA, DDR1-AAPY or DDR1-PPPF, co-immunoprecipitated with KIBRA (Figure 6.3). Similar expression in the protein lysates of DDR1-wt and the mutant constructs is demonstrated in the bottom panel of Figure 6.3. Therefore, the loss of the two proline residues or the tyrosine residues in the PPxY sequence abolishes binding of DDR1 to KIBRA, confirming that integrity of this consensus motif is critical for the interaction to occur.

Figure 6.3 KIBRA interacts with wildtype DDR1 only. Co-immunoprecipitation of KIBRA and DDR1 wt or DDR1 mutants (AAPY, AAAA or PPPF).

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PPxY motifs bind to WW domains of their target proteins. To determine whether the KIBRA and DDR1 interaction was mediated by the two WW domains of KIBRA, we constructed 2 two overlapping V5-tagged fusion proteins, as described in Chapter Four. One included the WW domains of KIBRA, and the other included the C2 domain (Figure 5.1). We co-expressed DDR1 with either WW-V5, C2-V5 or full-length KIBRA-V5. When we immunoprecipitated with the KIBRA antibody, DDR1 was pulled down with full- length KIBRA-V5 and WW-V5, but not with the C2-V5 fusion protein (Figure 6.4A). These data indicate that KIBRA and DDR1 interact via a fragment of KIBRA containing the WW domains and the PPPY motif of DDR1.

KIBRA interacts with DDR1 and PKC in a trimeric complex

KIBRA has been reported to interact with protein kinase C (PKC) (Buther et al. 2004). We confirmed this by immunoblotting and showed that endogenous PKC co-immunoprecipitates with KIBRA in HEK-293 cells (Figure 6.4A). PKC is pulled down with C2-V5 KIBRA, which is in agreement with Buther et al. (2004) who mapped the KIBRA binding site to a short 44 amino acid fragment downstream of the C2 domain (Buther et al. 2004). We observed that both DDR1 and endogenous PKC co-precipitate with full-length KIBRA, but not the C2 domain or WW domain fragments of KIBRA, suggesting that KIBRA, DDR1 and PKC interact in a complex. When we co-expressed KIBRA, DDR1 and PKC in HEK-293 cells we observed co-immunoprecipitation of all three proteins using DDR1 antibodies (Figure 6.4B). The presence of collagen reduced the amount of KIBRA co-precipitating with DDR1, and the amount of associated PKC decreased in proportion (Figure 6.4B). In addition, collagen treatment has no effect on the KIBRA-PKC interaction. Although the levels of DDR1 co-immunoprecipitated with KIBRA decrease over a time course of collagen treatment, the levels of PKC remain the same (Figure 6.4C). These

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data indicate that KIBRA, DDR1 and PKC interact in a tripartite complex, but predominantly in the absence of collagen.

Modulation of KIBRA expression has downstream effects on the collagen-stimulated activation of ERK MAPK cascade

Having demonstrated that KIBRA interacts in a complex with DDR1 and PKC,, we performed literature searches to identify common downstream targets of both DDR1 and PKC that may provide clues as to the functional role of KIBRA. We identified the ERK MAPK cascade, a ubiquitous cytoplasmic signalling pathway with a critical role in fundamental biological processes such as cell proliferation, differentiation and apoptosis, as being a downstream target of both DDR1 and PKC. To investigate whether KIBRA may also be involved in this pathway we transfected HEK-293 cells with KIBRA (KIBRApcDNA3.1) or vector (pcDNA3.1) and stimulated with collagen type IV. As shown in Figure 6.5A, KIBRA-transfected cells showed increased levels of phospho-ERK when exposed to collagen, relative to vector-only cells. No changes were observed in total ERK levels. Figure 6.5B shows densitometric analysis of a representative Western blot, revealing an approximate three to four-fold increase in levels of phospho-ERK in cells transfected with KIBRA in the presence of collagen, compared to empty vector controls. To confirm KIBRA was involved in the collagen-regulated stimulation of the MAPK pathway we used the normal human mammary epithelial cell line, MCF-10A, to determine whether there was any effect on the activation of this signal transduction cascade when endogenous KIBRA expression was knocked down by siRNA. Figure 6.6A shows that ERK was activated upon short-term stimulation with collagen in mock and RISCfree siRNA controls, but that this activation was suppressed when KIBRA expression is knocked down using 2 different siRNA duplexes. Figure 6.6B depicts the corresponding densitometric analysis. These data demonstrate that KIBRA plays a positive role in the collagen-stimulated activation of the ERK MAPK cascade.

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Figure 6.4 KIBRA, DDR1 and PKC interact in a trimeric complex. A KIBRA immunoprecipitates from HEK-293 cells transfected with DDR1 and each of WW-V5, C2-V5 or KIBRA-V5 cDNAs. Co-immunoprecipitates of endogenous PKC are also shown. B DDR1 immunoprecipitates from HEK-293 cells (+/- collagen) transiently transfected with DDR1, KIBRA and PKC. C KIBRA immunoprecipitates over a time-course of collagen treatment.

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Figure 6.5 KIBRA overexpression in the presence of collagen activates the ERK MAPK cascade. A Representative Western blots of lysates from HEK-293 cells transiently transfected with KIBRApcDNA3.1 or pcDNA3.1 and treated +/- collagen for 20 min. B Corresponding graph depicting fold increase in ERK phosphorylation in the presence of collagen.

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Figure 6.6 KIBRA expression is required for collagen activation of the ERK MAPK cascade in MCF-10A cells. A Western blot of lysates from MCF-10A cells transfected with KIBRA siRNA duplexes and treated +/- collagen for 20 min. B Corresponding graph depicting fold increase in ERK phosphorylation as determined by densitometric analysis.

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Discussion

An important step in elucidating the function of a novel protein, such as KIBRA, is the identification of its protein interaction partners. Here we have used a bioinformatic approach to mine the human proteome to isolate proteins containing the PPxY motif and which thus potentially interact with the WW domains of KIBRA. We have shown that KIBRA interacts in a collagen- regulated manner with DDR1, a protein known to have an important role in mammary development (Vogel et al. 2001), and that this interaction occurs via the PPxY motif of DDR1. Furthermore, we have demonstrated that KIBRA interacts simultaneously with DDR1 and PKC, and that KIBRA is involved in the collagen-regulated stimulation of the MAPK cascade.

DDR1, a collagen-activated receptor tyrosine kinase, shows similar expression patterns to KIBRA in that it is epithelial-specific, shows an increase in expression in the mouse mammary gland during pregnancy, and is also overexpressed in several primary breast tumors (Barker et al. 1995). Female DDR1 knockout mice show defects in blastocyst implantation together with hyperproliferation and abnormal branching of the mammary ducts which result in a lactational defect (Vogel et al. 2001). DDR1 mutant mice also show an increased amount of collagenous extracellular matrix surrounding the mammary epithelium (Vogel et al. 2001). This observation suggests a role for DDR1 in mediating extracellular matrix signalling within the mammary gland. The interaction between the mammary epithelium and the extracellular matrix is essential for complete alveolar morphogenesis and in the regulation of processes such as cell motility and adhesion (Fata et al. 2004), which when dysregulated can drive tumor progression by causing enhanced migration, invasion or metastasis. A number of studies have detected an overexpression of DDR1 in human tumours, particularly in primary breast cancer (Barker et al. 1995), and pediatric brain cancer (Weiner et al. 1996). It has also been identified as a dysregulated gene in epithelial ovarian cancer (Heinzelmann- Schwarz et al. 2004), and more recently as a novel marker for differentiation

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of invasive ductal and lobular breast carcinomas (Turashvili et al. 2007). Since KIBRA binds DDR1 and regulates ERK MAPK activation in response to the DDR1 ligand collagen, it will be important to determine KIBRA expression in breast and other cancers.

Our data demonstrate that the KIBRA-DDR1 complex dissociates upon activation of DDR1 by collagen, suggesting a role for KIBRA in the downstream signalling pathways induced by the extracellular matrix. It is therefore interesting to note that forced expression and activation of DDR1 with collagen in mouse mammary epithelial HC11 cells resulted in increased activation of Stat5, a downstream target of Prlr, as well as increased -casein gene expression (Faraci-Orf et al. 2006). This suggests that the collagenous extracellular matrix and DDR1 signalling pathway work in conjunction with the prolactin pathway in order to maintain efficient lactogenesis. As KIBRA expression is regulated by two hormones required for alveolar morphogenesis, we postulate a role for the KIBRA-DDR1 interaction in integrating hormone- stimulated and matrix-derived signals essential in maintaining normal mammary gland function.

Other interacting proteins for both KIBRA and DDR1 have previously been identified. KIBRA has been reported to be a novel substrate for protein kinase C (PKC) (Buther et al. 2004), a member of the family of atypical PKCs, which are characterised by the presence of only one zinc finger module, and do not bind or respond to phorbol esters and diacylglycerol (Moscat and Diaz-Meco 2000). PKC is important in regulating many cell processes, such as proliferation and differentiation, and when stably over expressed in a murine mammary epithelial cell line has been shown to modulate cell proliferation, adhesion and migration, via activation of the ERK MAPK pathway (Urtrerger et al. 2005). PKC isozymes have long been implicated in carcinogenesis, and specifically, PKC shows increasing expression correlating with increasing tumor grade in urinary bladder cancer (Varga et al. 2004), is involved in

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cancer cell motility in pancreatic adenocarcinoma cells (Laudanna et al. 2003), and has been shown to be overexpressed in breast carcinomas (Schondorf et al. 2001). With respect to DDR1, other interacting proteins have previously been identified which bind this receptor in a phosphotyrosine-dependent manner. One study reported an interaction between the neuronal phosphoprotein DARPP-32 and DDR1, which similarly to KIBRA was shown to be stronger in the absence of collagen, and their interaction was shown to inhibit the migration of human breast cancer cells (Hansen et al. 2006). In contrast, previous studies determined that several other cytoplasmic signalling proteins, including Shc, Nck2 and Shp-2, bind DDR1 but only following activation by collagen (Vogel et al. 1997; Koo et al. 2006).

In addition to the PKC pathway, ERK MAPK has also previously been shown to be a downstream effector in the DDR1 pathway (Curat and Vogel 2002; Ongusaha et al. 2003), and so our data demonstrating an interaction between KIBRA, PKC and DDR1 in a complex, suggests a potential role for KIBRA in activation of the ERK MAPK cascade. This is further supported by our data showing that overexpression of KIBRA in the presence of collagen causes an increase in ERK phosphorylation, and conversely that knockdown of KIBRA expression inhibits ERK MAPK activation upon collagen treatment. It is interesting to note that while unstimulated DDR1 has been shown to suppress ERK MAPK phosphorylation, collagen-stimulated DDR1 induces ERK MAPK activation (Curat and Vogel 2002). As collagen treatment is also required for ERK phosphorylation when KIBRA is over-expressed, and collagen inhibits the KIBRA-DDR1 interaction, we propose a model whereby these proteins must dissociate in order for activation of the ERK MAPK cascade to occur, and that unstimulated DDR1 potentially interacts with KIBRA to suppress ERK signalling. One possibility of how this activation occurs is either via downstream signalling by the KIBRA-PKC complex, or by KIBRA dissociation allowing access to the collagen-activated receptor for molecules that participate in Ras/ERK signalling, such as Shc. These models are not mutually exclusive. In

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conclusion, in this chapter we have identified DDR1 as a novel binding partner of KIBRA and shown that this interaction occurs in a collagen-regulated manner. Subsequently we have demonstrated that KIBRA expression plays a role in downstream signalling of the extracellular matrix.

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Chapter 7: Discussion

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Overview

Development of the adult female mammary gland occurs through recurrent cycles of growth, differentiation and apoptosis. These processes are under tight control by hormonal signals elicited by the ovaries and pituitary gland. Prolactin, and its co-operation with progesterone signalling, is the major driving force behind the development of the alveoli during pregnancy into the milk secreting units required during lactation. Although the hormonal regulation of these morphological and functional stages has been well described, much remains to be understood about the changes in gene expression that occur during these developmental events.

In order to understand more about the genomic network, which is modulated by Prl and Pg signalling during development, we investigated two genes that had been identified by a transcript profile screen of two contrasting models of Prl action. These two genes were KIBRA, a novel gene of unknown function, and Elf5, which had been previously demonstrated in this laboratory to be critical in mediating Prl-induced mammary development (Harris et al. 2006). The overall aim of this project was to investigate the function of two putative Prl-regulated genes not previously characterised in the human mammary gland.

A Role for Elf5 in Proliferation and Survival of Mammary Epithelial Cells

In the mouse, several in vivo models have demonstrated that Elf5 has an essential role in the proliferation of the mammary epithelium. These include the failed proliferation during mid-pregnancy in Elf5 heterozygous mice (Zhou et al. 2005), rescue of the alveolar proliferation defect during pregnancy by re-

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expression of Elf5 in Prlr knockout mice (Harris et al. 2006), and decreased proliferation during early pregnancy in Elf5 knockout mice (Oakes et al. 2008). The function of Elf5 in the human mammary gland is so far poorly understood, however data presented in this thesis suggests that Elf5 is also important in proliferation in human mammary cells. We have shown suppression of Elf5 expression in T47D breast cancer cells inhibits proliferation and decreases ERK MAPK activation (Chapter Four). This inhibition of ERK MAPK was similar to that which mediated the decreased proliferation observed in Elf5 knockout mice (Figure 4.14 and Oakes et al. 2008). Furthermore, microarray analyses revealed that when Elf5 levels were repressed in the presence of Pg in T47D breast cancer cells, a number of genes involved in the promotion of cell division were down-regulated, providing further evidence that Elf5 expression is required for normal proliferation.

Prolactin is critical in driving mammary epithelial proliferation and differentiation, and this occurs via a number of major downstream signalling pathways, including the ERK MAPK pathway (Das and Vonderhaar 1997). In the human breast cancer cell lines T47D and MCF7 specifically, this mechanism occurs via prolactin activation of c-Src, which mediates the activation of the Fak/Ras/Raf/ERK signalling cascade, providing the signals for cyclin D1 expression required for cell proliferation (Acosta et al. 2003). We observed decreased proliferation and levels of cyclin D1 expression and ERK activation in T47D cells which were transfected with Elf5 siRNAs. Furthermore, as Elf5 is downstream of Prl signalling, we hypothesise that Elf5 expression is, at least in part, required in this pathway for normal proliferation to occur. Deletion or reduction of molecules in this signal transduction cascade, including Prlr (Ormandy et al. 1997a; Binart et al. 2003) and Fak (Nagy et al. 2007), as well as Elf5 itself (Figure 4.14 and (Oakes et al. 2008)), results in decreased ERK MAPK activation and proliferation. Therefore we hypothesise that Elf5 expression is required at a point downstream of Prlr and upstream of ERK, in order for normal proliferation to occur in mammary epithelial cells.

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Our transcript profiling data determined that Elf5 is not a major downstream mediator of Pg action, at least in the T47D cell culture model. We postulate that the induction of Elf5 expression by Pg is utilised more to influence other signal transduction cascades, for example the Prl pathway. Both Prl and Pg fulfill crucial roles during proliferation and differentiation of the mammary gland, however differentiation is most often coupled to inhibition of proliferation and growth arrest. Therefore, depending on the hormonal milieu of the cell, the Prl- and Pg-induced regulation of Elf5 and other factors may cooperate to specify whether the cell will follow a proliferative or differentiative fate. For example, Pg has been shown to promote proliferation of T47D cells via the ERK MAPK pathway through cross-talk with both ER signalling (Migliaccio et al. 1998), and EGF signalling (Lange et al. 1998). One could speculate that upon activation of Prl signalling in this context, Pg may then switch to preferentially synergise with the Prl signal cascade to promote differentiative, rather than proliferative effects.

Data presented in this thesis also proposes a pro-survival function of Elf5 in MCF-10A cells. This was demonstrated by a failure of the inner cell population in Elf5-expressing acini to undergo apoptosis and give rise to a normal hollow lumen when cultured on a basement membrane (Chapter Three). This occurred due to a lack of induction of the pro-apoptotic protein Bim, caused by an apparent up-regulation of EGFR. These observations are similar to reports in the literature where overexpression of EGFR in MCF-10A cells grown in 3D culture resulted in a higher frequency of acini with increased numbers of cells and partially filled lumen (Dimri et al. 2007). Furthermore, inhibition of Bim expression by siRNA transiently prevented luminal apoptosis (Reginato et al. 2005). Notably, our observations with Elf5-expressing acini were more dramatic, where the majority of lumens remained filled for the duration of the experiment, and so additional factors must therefore be at play when Elf5 is overexpressed in MCF-10A acini.

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It is widely recognised that both the negative regulation of Bim by cell adhesion and EGFR signalling play a role in protection from anoikis. However the precise role and mechanism of action of Bim, which is also required for normal lumen formation in mammary ducts and TEBs in vivo (Mailleux et al. 2007), is one of debate. While some report that Bim is a key apoptotic sensor which responds directly to the loss of integrin-mediated signals (Reginato et al. 2003), others report that Bim acts instead as a sensor of loss of EGF- dependent survival signals (Wang et al. 2004). This is most likely explained by the different cell types in which these experiments are performed. In the case of MCF-10A cells, Bim appears to integrate growth factor signals and ECM attachment via the Raf/MEK/ERK pathway (Reginato et al. 2003; Marani et al. 2004).

One of the major questions that remains to be answered is how Elf5 regulates EGFR expression in MCF-10A cells grown in 3D culture. Is Elf5 able to bind the EGFR promoter, similarly to how the closely related Ets member, Elf3, can transactivate the ErbB2 promoter (Chang et al. 1997; Neve et al. 2002)? Indeed, overexpression of at least eight different Ets transcription factors stimulates ErbB2 expression (Scott et al. 2000), and Elf5 has previously been suggested to contribute to ErbB2 expression by binding and transactivating its promoter (Zhou et al. 1998). Whether Elf5 is able to regulate EGFR phosphorylation and activity, as well as total levels of expression, will also need to be addressed. There are examples of cross-talk between Prlr and EGFR/ErbB receptors. The ErbB ligand, amphiregulin, was down-regulated in Prlr-/- epithelium (Harris et al. 2006) suggesting co-operation between Prlr and the ErbB receptors. Another study reported that in T47D cells, Prl caused ERK- dependent phosphorylation of both EGFR and ErbB2, as well as inhibiting EGF- induced down-regulation of EGFR (Huang et al. 2006). Growth hormone has also been demonstrated to induce ERK-dependent activation of EGFR and ErbB2 (Huang et al. 2003). Therefore, as a target of Prl and GH, Elf5 may be important in the regulation of EGFR in these scenarios.

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There is evidence for a role of prolactin in survival of the mammary gland and breast cancer cell lines (Flint and Knight 1997; Perks et al. 2004). This is in agreement with the demonstration of Prl as a potent survival factor in other cell lines, such as Nb2 lymphoma cells (Fernandez et al. 2003), thymocytes (Krishnan et al. 2003) and the PC3 prostate cancer cell line (Ruffion et al. 2003). Furthermore, Stat5 has also been shown to be important in survival in the mammary gland, in addition to its roles in proliferation and differentiation (Cui et al. 2004). Elf5 has very recently been identified as a potential target of DEC1, an important regulator of survival of breast cancer cells (Ehata et al. 2007; Qian et al. 2008). It is interesting to note that DEC1, which is also believed to be involved in the control of proliferation and/or differentiation of various cell types (Shen et al. 1997), also acts downstream of EGFR signalling (Li et al. 2006). These observations place Elf5 downstream of signalling pathways important in survival.

Perhaps we can draw additional clues about the function and mechanism of Elf5 action by other important mediators of Prl signalling in mammary development, for example, RankL. Like Elf5, RankL was identified as displaying decreased expression in Prlr-/- mouse model (Harris et al. 2006) and is also a target of the Pgr signalling pathway important in the proliferation and differentiation of the mammary epithelium during pregnancy (Fata et al. 2000). Treatment of 3D primary mammary epithelial acini with RankL resulted in larger acini with filled lumens, although the molecular mechanism behind this observation was not elucidated (Gonzalez-Suarez et al. 2007). It would be interesting to determine whether EGFR expression was implicated in these findings, as EGFR has very recently been demonstrated to be involved in Rank downstream signalling (Yi et al. 2008). In this study by Yi et al., RankL was shown to transactivate EGFR in osteoclasts and the authors postulate a novel mechanism whereby EGFR expression is important in RankL-induced osteoclast differentiation and survival. I speculate that Elf5 works via a similar mechanism of action, at least in the MCF-10A 3D model.

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The results presented in Chapters Three and Four of this thesis are part of on- going work, and raise some interesting questions which need to be addressed. What precise mechanism is responsible for the Elf5’s regulation of EGFR expression and promotion of survival of the inner cell population in MCF-10A acini? What other factors and molecules play key roles? Once a detailed mechanism has been elucidated, it will be interesting to determine whether this effect is reproduced in 3D morphogenesis of primary mammary epithelial cells. Another major issue that remains to be resolved is the exact relationship between Elf5 and ERK MAPK, and whether Elf5 directly activates ERK MAPK. Overall, the data presented in Chapters Three and Four of this thesis suggest a pro-proliferative and pro-survival role of Elf5 in human mammary epithelial cells, which is concordant with what is known about the role of prolactin signalling in the mammary gland. Figure 7.1 illustrates some of the key ideas addressed in this thesis together with what we already know regarding the role of Elf5 in human mammary epithelial cells.

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Figure 7.1 Hypothetical model of role of Elf5 in signalling pathways in mammary epithelial cells. Prl bound to Prlr dimers activates Jak/Stat pathway to promote differentiation, and the Ras/Mek/ERK pathway to promote proliferation. Pg bound to Pgr dimers also stimulate proliferation via this Ras/Mek/ERK pathway. Elf5 levels are up-regulated by both Pg and Prl, which co-operatively drive proliferation and differentiation. Suppression of Elf5 expression inhibits activation or ERK MAPK and proliferation, and so Elf5 is likely to act at a point downstream of Prlr and upstream of ERK. Pg also potentiates EGF signalling via the Ras/Mek/ERK pathway to promote proliferation (Lange et al. 1998). In addition, Elf5 expression induces up-regulation of EGFR by an unknown mechanism, which represses Bim induction in an ERK MAPK-dependent manner, subsequently inhibiting apoptosis.

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A Potential Role for Elf5 in Breast Cancer?

Prolactin has long been implicated in mammary carcinogenesis (see Chapter One), but so far the involvement of Elf5 in breast cancer has not yet been established. Elf5 was originally identified as a novel gene that was localised to human chromosome 11p13-15, a region that frequently undergoes loss of heterozygosity (LOH) in many cancers such as ductal breast carcinoma, and was tentatively suggested to have tumour suppressor properties (Lichy et al. 1998; Zhou et al. 1998). Indeed, Elf5 mRNA levels in epithelium of pre- malignant, pre-invasive and invasive breast cancers is lower compared to adjacent normal epithelium (Ma et al. 2003). In contrast, other studies have found significantly elevated expression of Elf5 in mammary tumors and breast cancer cell lines, compared to normal epithelial tissue (Galang et al. 2004; He et al. 2007).

A number of Ets transcription factors have been shown to have dysregulated expression in mammary tumors and have been implicated in the control of breast carcinogenesis. Members of the PEA3 subfamily of Ets factors (Trimble et al. 1993; Shepherd et al. 2001), Ets-1 (Span et al. 2002) and PSE (Ghadersohi and Sood 2001) have all been shown to display elevated levels of expression in mammary tumors. Elf3 has also been demonstrated to be overexpressed in breast tumors (Chang et al. 1997), and has been shown to be sufficient to transform normal MCF-12A mammary epithelial cells (Prescott et al. 2004), as well as significantly increase their survival (Eckel et al. 2003). Elf5 has previously been shown to share common functional aspects with its closely related family members, Elf3 and EHF. For example, in a microarray analysis of gene expression in normal terminal duct lobular units (TDLUs) compared to hyperplastic enlarged lobular units (HELUs), Elf5, Elf3 and EHF were identified as being significantly down-regulated in HELUs compared to the more differentiated TDLUs (Lee et al. 2007). Interestingly, changes in ErbB genes were particularly prominent in this study.

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Elf5 and EHF were also identified together in a microarray analysis as overexpressed in non-tumorigenic cells compared to cancer stem cells (which share some attributes of early progenitor cells) in breast tumors of MMTV- Wnt1 mice (Cho et al. 2008). These two observations are in agreement with the role of Elf5 as a critical cell fate determinant, whereby Elf5 drives the differentiation of the luminal progenitor cell population to the secretory alveolar epithelium required for lactation (Oakes et al. 2008). In the studies by Lee et al. (2007) and Cho et al. (2008), Elf5 expression also appears to be required to maintain the more differentiated state of TDLUs and non- tumorigenic stem cells, compared to HELUs and cancer stem cells, respectively. These studies suggest that perhaps Elf3 and EHF also have similar roles in cell fate specification. Indeed, the expression of these two Ets transcription factors are already known to be important in maintaining the differentiated phenotype in epithelial cells (Andreoli et al. 1997; Tugores et al. 2001).

As just mentioned, Elf5 expression is essential in driving the differentiation of luminal progenitor cells. In addition, we have observed that in human breast cancer cell lines, Elf5 expression correlates with Prlr and steroid hormone receptor expression, and was absent in the normal or non-cancerous mammary epithelial cell lines, which are commonly associated with a basal epithelial phenotype (Harris 2004). Although luminal epithelial cells are widely believed to give rise to the majority of human breast cancers, a subset of breast cancers (approximately 15-20%) exhibit a basal phenotype which most likely arise from basal epithelia (Perou et al. 2000; Sorlie et al. 2001). Perhaps the conflicting observations of Elf5 expression in breast tumours reported in the literature are due to the heterogeneity of the breast cancers investigated. For example, Chehani Alles from our laboratory identified Elf5 as displaying very high expression in approximately 50% of basal tumor cell types (Alles, M., personal communication). This subtype of breast cancer is characterised by negative expression of hormone receptors and ErbB2, and is associated with aggressive behaviour and poor prognosis, as recently reviewed in Rakha et al.

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2008. Interestingly, EGFR is also often overexpressed in this subtype of breast cancer (Hoadley et al. 2007). Since Elf5 is not normally expressed in basal epithelial tissue or cell lines, it is possible that inappropriate expression of Elf5 in this cell type promotes carcinogenesis. The MCF-10A cell model resembles a basal epithelial cell type, and so perhaps our demonstration that forced expression of Elf5 in these acini results in luminal filling of acini, a hallmark feature of oncogenes in this cell model, will provide clues as to the correlation between high Elf5 expression in basal tumours in vivo.

In summary, the data presented in Chapters Three and Four of this thesis suggests that in addition to differentiation, Elf5 is also involved in regulating growth and survival of mammary epithelial cells. The major questions that remain to be answered now are to delineate the relationship between Elf5 and EGFR expression, to determine whether this mechanism is utilised in two- dimensional culture or in other cell lines, and to elucidate the association between Elf5 expression and ERK MAPK activation. Continuation of this study will address these questions and others, further elaborating on the details that define the role of Elf5 as a critical transcription factor in normal mammary development and function.

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KIBRA and Modulation of ECM Signalling

In this thesis we have also taken an unknown EST, subsequently named KIBRA/WWC1, which was identified as an epithelial-specific transcript showing decreased expression in the Prlr knockout mammary epithelium, and characterised its expression and hormonal regulation in mammary epithelial cells. In addition to this, we identified a binding partner of KIBRA, and defined a signalling pathway endpoint that responds to manipulation of KIBRA’s expression. Specifically, we have shown that KIBRA is a progesterone- regulated phosphoprotein of approximately 150 kDa, which displayed a predominantly cytoplasmic localisation in human mammary epithelial cells. In human breast tissue, KIBRA was expressed specifically in the luminal epithelium, and displayed heterogeneous expression in cancerous tissue. We have also shown that KIBRA interacts in a collagen-regulated manner with DDR1, a protein known to have a role in mammary development (Vogel et al. 2001), and that this interaction occurs via the PPxY motif of DDR1. Furthermore, we have demonstrated that KIBRA interacts simultaneously with DDR1 and PKC, and that KIBRA is involved in the collagen-regulated stimulation of the ERK MAPK cascade.

Our results demonstrate that the KIBRA-DDR1 complex dissociates upon activation and phosphorylation of DDR1 by collagen. In addition, we have shown that overexpression of KIBRA in the presence of collagen causes an increase in ERK MAPK phosphorylation, and conversely that knock-down of KIBRA expression inhibits ERK MAPK cascade. It has previously been demonstrated that while unstimulated DDR1 has been shown to suppress ERK MAPK phosphorylation, collagen-stimulated DDR1 induces ERK MAPK activation (Curat and Vogel 2002). Using this combined data, we propose a model whereby both proteins must be dissociated from each other in order for activation of the MAPK cascade to occur, and that unstimulated DDR1 potentially interacts with KIBRA to suppress mitogenic signalling. Figure 7.2

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illustrates this hypothetical model whereby in the unstimulated state of the cell KIBRA is bound to DDR1 and PKC in a complex, and upon contact of the cell with collagen, KIBRA and PKC dissociate from DDR1 and the MAPK cascade is activated. Cell adhesion has been reported to signal PKC activation, for example, adhesion of HeLa cells to a collagen substratum induces PKC activity (Chun and Jacobson 1993; Xu and Clark 1997). We have demonstrated that KIBRA and PKC bind in the absence and presence of collagen, and so perhaps PKC activity induced by collagen stimulation is another mechanism through which ERK MAPK is activated.

Figure 7.2 Hypothetical model of regulation of MAPK cascade by the KIBRA/DDR1/PKC complex. In the unstimulated cell there is a pool of PKC and KIBRA which bind in the cytoplasm of the cell, and potentially also in the nucleus. When at the membrane of the cell, the two molecules can bind DDR1 to form a complex. Upon contact of the cell with the extracellular matrix, DDR1 is activated and phosphorylated (depicted by ‘P’) by its ligand collagen, inhibiting the KIBRA-DDR1 interaction. KIBRA and PKC then dissociate from DDR1, and the ERK MAPK cascade is activated. This may occur via activation of PKC, or via downstream signalling of DDR1 or KIBRA, or via a combination of these pathways.

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It has previously been reported that progestins can stimulate proliferation of mammary epithelial cells derived from adult virgin mice in the presence of collagen type IV (Xie and Haslam 1997). One might speculate that this proliferation is a result of activation of the ERK MAPK cascade due to an up- regulation of KIBRA expression by progestin, in the presence of collagen. Using the data presented in this thesis, and the information we know about DDR1 from the literature, we can also hypothesise how the KIBRA-DDR1 interaction may be involved in mammary development in vivo. As demonstrated by Vogel et al. in 2001, DDR1 knockout mice were shown to display aberrant ductal growth, with a higher number of secondary ducts with wider lumens, and significantly larger amounts of ECM being deposited around the ducts. In addition, the epithelium of virgin mice was hyper-proliferative and pregnant females displayed a defect in milk secretion (Vogel et al. 2001). From these observations and others, it has been speculated that DDR1 has regulatory functions in suppressing ECM synthesis, MAPK activation and proliferation in the mammary gland (Vogel et al. 2001; Curat and Vogel 2002). Combined with our data, we hypothesise that during normal mammary development when KIBRA expression levels increase due to hormonal stimulus, DDR1 potentially interacts with KIBRA and sequesters it away from activating the MAPK cascade downstream, in order to regulate proliferation. This is depicted in the schematic diagram in Figure 7.3A. Therefore in the absence of DDR1, KIBRA is up-regulated due to hormonal stimulus and in the presence of excess collagen, causes downstream activation of the MAPK cascade and hyper-proliferation, as exhibited in the DDR1 knockout mouse model (Figure 7.3B). This would most likely resemble the situation of KIBRA overexpression in vivo, and so it would be interesting to determine whether a KIBRA transgenic mouse model exhibited a similar mammary phenotype to that of the DDR1 knockout mouse.

In summary, from the data presented in Chapters Five and Six of this thesis, we hypothesise that in cells which are in contact with the extracellular matrix, the interaction of KIBRA with DDR1, and the modulation of KIBRA expression

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by progesterone and prolactin, is important for the regulation of activation of the ERK MAPK cascade in the mammary gland. Thus the hormonally regulated interactions between KIBRA and DDR1 and the extracellular matrix are necessary for proliferation and tissue remodelling of the mammary gland throughout its cycle of development.

Figure 7.3 Schematic diagram of the KIBRA-DDR1 interaction in the mammary gland. A In the wild-type mammary gland, DDR1 is important in mediating cell-matrix contacts and regulating ECM deposition, as well as suppressing excess proliferation. B In the absence of DDR1, KIBRA is not sequestered and in the presence of excess collagen, causes downstream activation of the MAPK cascade, and subsequent excess proliferation.

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Summary and Conclusions

In summary, in this thesis we have investigated two genes whose function had previously not been characterised in the human mammary gland. Both molecules were identified as hormonally-regulated epithelial-specific mammary gland transcripts that showed decreased expression in the Prlr knockout mammary epithelium. We have subsequently shown they both display increased expression in response to progesterone. We have also demonstrated that Elf5 expression promotes the growth and survival of mammary epithelial cells. Specifically, we have shown that Elf5 expression in normal mammary epithelial MCF-10A cells grown on basement membrane promotes luminal filling via up-regulation of EGFR and inhibition of the pro-apoptotic factor, Bim. Furthermore, suppression of Elf5 expression inhibits proliferation and ERK MAPK activation. We have also investigated a novel EST, KIBRA, and characterised its expression in mouse and human mammary epithelial cells. In addition, we identified DDR1 as an interaction partner of KIBRA, and showed that these molecules interacted in a collagen-dependent manner via the PPxY motif of DDR1. Furthermore, we have demonstrated that KIBRA interacts simultaneously with DDR1 and PKC, and that KIBRA is involved in the collagen-regulated stimulation of the MAPK cascade. In conclusion, this work has contributed to the growing volumes of information which detail the genomic regulatory networks that regulate mammary gland development. Defining these mechanisms in a normal mammary cell context is essential to understand how they may be dysregulated during carcinogenesis.

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