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DISCERNING THE ROLE OF FOXA1 IN MAMMARY GLAND

DEVELOPMENT AND BREAST

by

GINA MARIE BERNARDO

Submitted in partial fulfillment of the requirements

for the degree of Doctor of Philosophy

Dissertation Adviser: Dr. Ruth A. Keri

Department of Pharmacology

CASE WESTERN RESERVE UNIVERSITY

January, 2012 CASE WESTERN RESERVE UNIVERSITY

SCHOOL OF GRADUATE STUDIES

We hereby approve the thesis/dissertation of

Gina M. Bernardo ______

Ph.D. candidate for the ______degree *.

Monica Montano, Ph.D. (signed)______(chair of the committee)

Richard Hanson, Ph.D. ______

Mark Jackson, Ph.D. ______

Noa Noy, Ph.D. ______

Ruth Keri, Ph.D. ______

______

July 29, 2011 (date) ______

*We also certify that written approval has been obtained for any proprietary material contained therein.

DEDICATION

To my parents, I will forever be indebted.

iii TABLE OF CONTENTS

Signature Page ii

Dedication iii

Table of Contents iv

List of Tables vii

List of Figures ix

Acknowledgements xi

List of Abbreviations xiii

Abstract 1

Chapter 1 Introduction 3

1.1 The FOXA family of factors 3

1.2 The nuclear superfamily 6

1.2.1 The

1.2.2 The receptor

1.3 FOXA1 in development 13

1.3.1 and

1.3.2

1.3.3

1.3.4

1.3.5

1.3.6

1.4 The mammary gland 24

1.4.1 Stages of mammary gland development

iv 1.4.2 Mammary epithelial hierarchy

1.4.3 The in mammary gland development

1.5 FOXA1 in cancer 32

1.5.1 FOXA1 in other than breast and prostate

1.5.2 FOXA1 in

1.6 FOXA1 in 42

1.6.1 The molecular subtypes of breast cancer

1.6.2 FOXA1 expression in breast cancer

1.6.3 FOXA1 and the estrogen receptor

1.6.4 Additional roles of FOXA1 in breast cancer

1.7 Statement of Purpose 58

Chapter 2 FOXA1 is an Essential Determinant of ERα Expression 67

and Mammary Ductal

2.1 Abstract 67

2.2 Introduction 69

2.3 Materials and methods 72

2.4 Results 78

2.5 Discussion 87

2.6 Acknowledgements 92

Chapter 3 FOXA1 Represses Basal Breast Cancer Characteristics 115

3.1 Abstract 115

3.2 Introduction 117

3.3 Materials and methods 121

v 3.4 Results 126

3.5 Discussion 134

3.6 Acknowledgments 140

Chapter 4 Summary and Future Directions 188

4.1 Summary 188

4.2 Is FOXA1 expression necessary for maintaining the 191

mammary epithelial lineage?

4.3 Does manipulation of FOXA1 alter breast cancer subtype 195

tumor progression?

4.4 How does FOXA1 repress basal breast cancer expression? 200

4.5 How is FOXA1 differentially regulated in breast cancer? 204

4.6 Concluding Remarks 210

Bibliography 213

vi LIST OF TABLES

Table 1.1 Overview of Foxa1 mouse models of development 59

Table 1.2 Overview of FOXA1 in cancer 60

Table 3.1 commonly decreased upon knockdown of FOXA1 in 141

MCF7, T47D, MB-453 and SKBR3 cells (p<0.001).

Table 3.2 Genes commonly increased upon knockdown of FOXA1 in 147

MCF7, T47D, MB-453 and SKBR3 cells (p<0.001).

Table 3.3 Basal A, basal B and luminal classifier genes (RN) whose 155

expression is changed upon knockdown of FOXA1 in

MCF7, T47D, MB-453 and SKBR3 cells (p<0.05).

Table 3.4 Classifier gene lists (RN) used to discriminate the luminal, 156

basal A, and basal B molecular subtypes for GSEA.

Table 3.5 Lum(B)-ECJ classifier gene list used for GSEA. 157

Table 3.6 Bas-ECJ classifier gene list used for GSEA. 158

Table 3.7 Lum(M)-ECJ classifier gene list used for GSEA. 159

Table 3.8 Mes-ECJ classifier gene list used for GSEA. 160

Table 3.9 GSEA of classifier gene lists that are discriminatory 161

of luminal v. basal breast cancer molecular subtypes.

Table 3.10 Potential binding sites in basal and luminal signature 162

genes regulated by FOXA1.

Table 3.11 Gene order on Luminal (B)-ECJ heatmap in Figure 3.7. 163

Table 3.12 Gene order on Basal-ECJ heatmap in Figure 3.7. 165

vii Table 3.13 Primers used for PCR amplification of DNA 167

that has been subject to FOXA1 ChIP.

viii LIST OF FIGURES

Figure 1.1 Mammary gland terminal end bud (TEB) formation and 61

ductal invasion

Figure 1.2 FOXA1/AR signaling in prostate cancer 63

Figure 1.3 FOXA1/ERα signaling in breast cancer 65

Figure 2.1 FOXA1 is expressed in the developing mammary gland in 93

conjunction with ERα.

Figure 2.2 FOXA1 is not necessary for of 95

the mammary gland.

Figure 2.3 FOXA1 is required for mammary ductal outgrowth in an 97

orthotopic transplantation model.

Figure 2.4 FOXA1 is required for TEB formation and ductal invasion. 99

Figure 2.5 FOXA1 is not required for luminal or basal/myoepithelial 101

lineage specification.

Figure 2.6 Pubertal mice heterozygous for the Foxa1 null allele display 103

decreased mammary ductal invasion.

Figure 2.7 FOXA1 is not required for alveolar differentiation during 105

.

Figure 2.8 FOXA1 is required for expression of ERα in the normal 107

mammary gland.

Figure 2.9 FOXA1 regulates transcription of ESR1. 109

Figure 2.10 FOXA1 regulates transcription of ESR1 in T47D cells. 111

ix Figure 2.11 Schematic of the mammary epithelial hierarchy 113

Figure 3.1 FOXA1 is expressed in the absence of ERα in breast 168

tumors and luminal cell lines.

Figure 3.2 FOXA1 expression correlates with the luminal subtype 170

in breast cancer cell lines.

Figure 3.3 Loss of FOXA1 increases migration and invasion of 172

luminal breast cancer cells.

Figure 3.4 Identification of a FOXA1-dependent luminal transcriptome. 174

Figure 3.5 Loss of FOXA1 decreases enrichment for luminal genes, 176

while increasing enrichment for basal genes.

Figure 3.6 Loss of FOXA1 induces changes in RN classifier 178

.

Figure 3.7 Loss of FOXA1 induces changes in ECJ classifier 180

gene expression.

Figure 3.8 GSEA enrichment plots for BasB-RN, BasAB-RN, 182

Lum(M)-ECJ and Mes-ECJ classifier lists.

Figure 3.9 Loss of FOXA1 induces basal mRNA and expression. 184

Figure 3.10 FOXA1 binds to luminal and basal genes in 186

luminal breast cancer cells.

Figure 4.1 FOXA1 is required for expression of cytokeratin 5/6 211

in the normal mammary gland.

x ACKNOWLEDGEMENTS

My journey to the Ph.D. has been influenced by so many. I must first thank all my professors from Washington & Jefferson College, who thoroughly prepared me for what was to follow. Vinnedge Lawrence, thank you for pushing me to my limits and helping me see my potential. Roy Ickes, for always being so insightful as an advisor. Steve

Malinak, for making chemistry tolerable. Candy DeBerry and Alice Lee, thank you for the constant support and providing me with the opportunity to assist in your laboratory classes. Dennis Trelka, I will forever owe you for directing me towards UCLA. I must also thank my roommate from W&J, Kisa Lape, for your friendship over the years and pulling so many all nighters with me!

Second, I was incredibly fortunate to have had the opportunity to work at UCLA before coming to CWRU. Dennis Slamon, thank you for exposing me to translational breast cancer research. Cindy Wilson, there are no words to express my gratitude for your patience, guidance and friendship. You are an inspiration. Raul Ayala, thank you for safely introducing me to mouse work and East LA cuisine! Chuck Ginther and Lee

Anderson, you have taught me so much about science and life. Especially the joys of food and wine! Thank you for accepting me into your family and for the countless hours of insightful conversation. I especially want to thank you for being such great collaborators over the past couple years. It was so nice working together! Lisa Pinelli, thank you for being the best roommate and friend, and for putting up with so many evenings of science babble.

xi My appreciation to all the members of the Keri lab, you were always there to provide advice and technical help. Marjorie Montañez-Wiscovich and Jonathan Mosley, thank you for your friendship and guidance, especially through my early years in the lab. Ruth, you have been a great mentor! Thank you so much for allowing me to be independent in the lab, but then always being there when I needed you (which we both know was quite often). I am much obliged to my committee members and the entire Department of

Pharmacology. Also, to my dearest Pharmacology friends, Payal Gandhi, Andrea

Moomaw, Elizabeth Sabens and Tara Ellison, thanks for all your advice and help with my research, but most of all, for the great times shared outside of the lab!

Of course, I owe all the thanks in the world to my family. Mom and dad, you have put up with so much over the years! Thank you for never giving up on me, even when I seemed impossible to deal with, and for sacrificing so much so that I could take advantage of every opportunity thrown my way. Dad, thank you for bringing me up with strength and perseverance, Mom, for teaching me that is it important to counterbalance strength with grace and for always listening when I needed to vent, Dan, for keeping the world in perspective. Thank you grandparents, aunts and uncles for your unwavering love and support. Aunt Rosie, Aunt Karen and Aunt Diann, for being such outstanding role models as professional women. I love you all so much. Lastly, Steve and Elvis, the two loves of my life (man and canine, respectively), for making every day so special and entertaining.

Steve, I would never have made it through these past six years without you. You are everything to me, and I am beyond excited to continue our journey in life and science together.

xii ABBREVIATIONS

AML Acute myeloid leukemia

α-SMA α-smooth muscle

AR Androgen receptor

ARE Androgen

AI inhibitor

BRCA Breast cancer susceptibility protein

CBP CREB-binding protein

ChIP immunoprecipitation

CK Cytokeratin

DHT

DBD DNA binding domain

E Embryonic day

E2 17β-

EGF Epidermal

EGFR receptor

ERα Estrogen receptor-α

ERE Estrogen response element

ERKO Estrogen receptor knockout

ERK Extracellular regulated kinase

FACS Fluorescence activated cell sorting

FAIRE Formaldehyde-assisted isolation of regulatory elements

xiii FOX Forkhead box

GSEA Gene set enrichment analysis

GR receptor

HCC

HDAC deacetylase

HER2 Human epidermal growth factor receptor-2

HNF3 Hepatocyte nuclear factor-3

H3K4 Histone H3, 4

IGF -like growth factor

IHC Immunohistochemistry

KDM lysine(K)-specific histone

LBD binding domain

MaSC Mammary stem cell

MAPK Mitogen activated protein kinase

MMTV Mouse mammary tumor virus

NE Neuroendocrine

P Postnatal day

PR receptor

PSA Prostate specific

PyMT Polyoma middle T antigen

SARM Selective androgen receptor modulator

SERM Selective estrogen receptor modulator

SERD Selective estrogen receptor down-regulator

xiv SHH

SOX Sex determining region Y-box

SRC receptor coactivator

TEB Terminal end bud

TGFβ Transforming growth factor-β

TMA Tissue microarray

TFF1 Trefoil factor-1

xv Discerning the Role of FOXA1 in Mammary Gland Development

and Breast Cancer

Abstract

by

GINA MARIE BERNARDO

Breast cancer is a heterogeneous disease with distinct subtypes that are predictive of patient prognosis. Luminal subtype tumors confer the most favorable prognosis due to

ERα-positivity and response to endocrine therapies. In contrast, basal-like subtype tumors confer the worst prognosis due to intrinsic resistance to and the lack of targeted therapies. The forkhead box , FOXA1, is found exclusively in luminal tumors, and positively correlates with ERα and survival. Prior to our study,

FOXA1 was known to mediate the development of many tissues and be required for ERα transcriptional activity in breast cancer cells. Given these findings, along with the requirement for ERα in mammary morphogenesis, we hypothesized that FOXA1 would be similarly necessary in this process. Using Foxa1 null mice, we found FOXA1 is required for -induced ductal invasion. Mammary glands null for Foxa1 lack epithelial ERα. The regulation of ERα by FOXA1 is similarly observed in breast cancer cells. These results revealed that FOXA1 is not only required for ERα activity, but also for its expression, and provided a mechanism for the ductal in Foxa1 knockout glands.

1 The positive correlation between FOXA1 and ERα in breast tumors is significant, but not perfect. FOXA1 is also expressed in luminal breast tumors and cell lines in the absence of

ERα prompting us to investigate an ERα-independent role for FOXA1 in maintaining the luminal subtype. FOXA1 silencing in ERα-positive and ERα-negative luminal breast cancer cells decreases luminal gene expression, and concomitantly increases basal gene expression. These cells are also more aggressive in vitro, indicating a shift toward the basal phenotype. FOXA1 binds to a subset of basal genes, and thus, is likely orchestrating repression of these genes at the transcriptional level. These data implicate

FOXA1 as a mediator of breast cellular plasticity, and suggest that therapeutically reducing FOXA1 in luminal tumors will increase disease aggressiveness. Combined, this dissertation research has detailed the role for FOXA1 in the normal mammary gland and its importance in luminal breast cancer. Our results have led to numerous testable hypotheses that will continue to advance our understanding of FOXA1 in breast cancer.

2 CHAPTER 1

Introduction

Adapted from “FOXA1: a Transcription Factor with Parallel Functions in Development

and Cancer” Bioscience Reports (in press)

by

Gina M. Bernardo & Ruth A. Keri

1.1 THE FOXA FAMILY OF TRANSCRIPTION FACTORS

FOXA1 is the founding member of the forkhead box (FOX) family of transcription factors that is comprised of at least 40 members (reviewed in (1)). FOXA1/HNF3α,

FOXA2/HNF3β and FOXA3/HNF3γ constitute the FOXA subfamily, which were originally named hepatocyte nuclear factor-3 (HNF3) due to the transcriptional regulation of liver-specific gene expression such as (Ttr) and α1-antitrypsin (Serpina1)

(2). Since this seminal study, FOXAs have been found to regulate many genes involved in developmental specification of not just hepatic, but several other tissues [reviewed herein (Section 1.3) and in (3)].

FOXAs contain an ~100 DNA binding domain or forkhead box/winged helix domain that is highly conserved (at least 92%) within the FOXA family (4), and shares extremely high (90%) with that of its namesake, the homolog fork head (fkh) (4, 5). FOXAs also contain conserved nuclear localization sequences and homology in the N-terminal (IV) and C-terminal (II, III) domains (4, 6, 7).

3 The requirement for these domains in transcriptional activation was demonstrated specifically for FOXA2 (6, 7), but can be inferred for the other family members. FOXAs bind as monomers (8) to the consensus element A(A/T)TRTT(G/T)RYTY (9) and crystallization of the DNA binding domain of FOXA3 revealed a “winged helix” structure bound to DNA in a manner similar to that of linker (8). Importantly, unlike linker histones, FOXAs lack the basic amino acids required for chromatin compaction (10). Thus, FOXA binding to nucleosomes induces an open chromatin configuration (10-12) enabling transcriptional activation that may occur either by direct recruitment of transcriptional initiation machinery, or through indirect recruitment of other transcriptional modulators such as members of the nuclear superfamily. Although FOXA1 likely functions independently of co-factor recruitment in initiating transcription, this has not been specifically demonstrated experimentally.

FOXA1 involvement in estrogen receptor (ERα) and androgen receptor (AR) transcriptional activity will be discussed below. This function has led to FOXAs being coined as “pioneering” or “licensing” factors. Recently, the mechanism by which FOXA1 remodels chromatin has been further described, where FOXA1 binding to DNA precedes the loss of cytosine and the dimethylation of histone H3, lysine K4 (H3K4) during the differentiation of pluripotent P19 cells (13). These results place FOXA1 upstream DNA hypomethylation and active histone modifications resulting in an open chromatin configuration and transcriptional activation at early stages of developmental specification.

4 The ability of FOXAs to remodel provides a mechanistic basis for how these factors initiate transcriptional cascades involved in both development and cancer as will be discussed in detail for FOXA1 in Sections 1.3, 1.5, 1.6 and Chapter 2. Since the role for FOXA1 in both these processes is intimately linked to ERα and AR, a brief discussion of the superfamily is warranted.

5 1.2 THE NUCLEAR RECEPTOR SUPERFAMILY

The superfamily of nuclear receptors is comprised of 48 members, and can be subdivided into those with known ligands and those with unknown ligands, which are otherwise referred to as orphan receptors (reviewed in (14)). Nuclear receptors mediate transcriptional regulation involved in development, morphogenesis, , and endocrine signaling, to name a few, and have pathological implications in a multitude of diseases such as in cancer. Detailing nuclear receptor function is beyond the scope of this introduction, but a brief overview, specifically focused on ER and AR, is necessary to supplement the material that follows outlining FOXA1-mediated ERα and AR transcriptional regulation in development and cancer of the breast and prostate, respectively. For more detailed general reviews on nuclear receptors, please see (14-17).

Nuclear receptors are modular with distinct domains (18): (1) a variable amino- terminal region containing a ligand-independent (AF-1), (2) a highly conserved DNA-binding domain (DBD) containing two finger motifs, (3) a linker hinge region often containing a nuclear localization signal (19), and (4) a conserved carboxy-terminal ligand binding domain (LBD) containing a ligand-dependent transactivation domain (AF-2). The LBD is composed of 12 α -helices, where ligand binding induces a conformational shift in the location of helix 12 (20, 21). The result of this structural change is two-fold: the nuclear receptor is released from bound co- , and is capable of binding to DNA as a monomer, homo- or hetero-dimer where it associates with co-activators to induce transcription. This description is a general

6 rule, but nuclear receptors are also known to recruit co-repressors while bound to DNA resulting in transcriptional repression, and can be active under both ligand-dependent and ligand-independent conditions as will be discussed below for ER and AR. Whereas the steroid receptors [AR, ER, estrogen-related receptors (ERRs),

(GR), (PR), and mineralocorticoid receptor (MR)] generally homo- dimerize, hormone receptors (TRs), retinoic acid receptors (RARs), the (VDR), proliferator-activated receptors (PPARs) and some orphan receptors preferentially hetero-dimerize with the (RXR). Nuclear receptors bind to a hexameric DNA consensus element, which is oriented as a monomer, a direct repeat, or an inverted repeat (palindromic) (14, 16). The DNA consensus elements recognized by nuclear receptors are referred to as hormone response elements

(HREs).

An important level of regulation of nuclear receptor transcriptional activity is via co- and co- recruitment (22). Both co-activators and co-repressors contain conserved protein motifs: LxxLL in co-activators (23) and Lxxx I/H I xxx I/L, also known as the CoRNR box, in co-repressors (16, 24). These motifs interact with the hydrophobic groove in the LBD, which is revealed after ligand binding and the rearrangement of helix 12 (23). Co-activators induce transcription through multiple mechanisms including, but not limited to ATP-dependent chromatin opening (e.g. SWI-

SNF), histone acetylation [e.g. CREB-binding protein (CBP)/p300], histone methylation

[e.g. lysine (K)-specific histone demethylase-1 (KDM1)] and recruitment of the general transcriptional machinery (e.g. TATA-box binding protein (TBP), RNA polymerase II).

7 Co-repressor binding to nuclear receptors results in a structural configuration different from that bound to co-activators (25), ultimately leading to a closed chromatin state through modifications such as histone deacetylation [e.g. silencing mediator for retinoid and (SMRT)]. Nuclear receptor activity is also controlled at the level of post-translational modification, where the , acetylation, sumoylation, ubiquitination and/or status of the nuclear receptor can enhance or diminish its function (reviewed in (26)).

1.2.1 The androgen receptor

AR is essential for normal development of male sex organs and the maintenance of male secondary sex characteristics. In the absence of ligand, AR is predominantly cytoplasmic or perinuclear (27). and the testosterone derivative dihydrotestosterone

(DHT) are the endogenous ligands for AR. The expression of each is regulated by the hypothalamic-pituitary-gonadal endocrine axis (28). The binding of endogenous androgens to AR increases stability of this receptor (29), induces its homo-dimerization

(30), translocation to the nucleus (31), and binding to the conserved androgen response element (ARE) in the DNA sequence GGTACAnnnTGTTCT (32). Two isoforms of AR,

AR-A and AR-B, have been identified in normal human (33, 34). AR-B is full-length and is the predominant isoform expressed, while AR-A is an amino-terminal truncation.

Upon ligand binding, AR undergoes a conformational change resulting in dissociation from heat shock proteins (35). AR is also known to interact with the cytoskeleton (36).

8 As with other nuclear receptors, the addition of ligand rearranges helix 12 revealing co- activator binding motifs. AR associates with co-activators such as steroid receptor co- activator (SRC-1) (37, 38) and co-repressors such as SMRT (39), among others

(reviewed in (40)). Interestingly, unlike in other nuclear receptors, the interaction with

SRC-1 occurs via both AF-1 and AF-2 transactivation motifs (38). AR also has non- genomic functions, such as facilitating phosphorylation of the c-Src/Raf-1/extracellular regulated kinase (ERK)-2 (41) and phosphatidylinositol-3-kinase (PI3K)/AKT (42) signaling cascades. Similar to other nuclear receptors, AR is phosphorylated, acetylated, ubiquitylated and sumoylated (26). Phosphorylation of AR occurs in the presence and absence of androgens, where androgen stimulation increases the level of phosphorylation

(43). Phosphorylation is necessary for optimal ligand binding and the transcriptional activation of the well-described AR-target gene, prostate specific antigen (PSA/KLK3)

(44). AR can also be activated in an androgen-independent manner by HER2 (45), insulin-like growth factors (IGF-I), growth factor (KGF) and epidermal growth factor (EGF) (46).

Other than its role in prostate cancer, AR is also pathogenic in male pattern baldness, androgen insensitivity syndrome (AIS), hypogonadism and benign prostate hyperplasia

(reviewed in (40)). Its participation in disease is often due to a change in the expression level and/or transcriptional activity of the receptor, which are altered by gene amplification, , and the presence or absence of amino-terminal CAG- polyglutamine repeats (47). Several hundred of AR have been identified and are cataloged online (http://androgendb.mcgill.ca/; (48)). Testosterone supplementation

9 and treatment with tissue selective androgen agonists, or selective androgen receptor modulators (SARMs), are used therapeutically to combat diseases with decreased AR function (49). Pharmacologic targeting in diseases such as prostate cancer with increased

AR activity is primarily accomplished by use of AR antagonists, or anti-androgens, such as flutamide, bicalutamide, nilutamide and cyproterone acetate (50).

1.2.2 The estrogen receptor

Paralleling the role for AR in male reproductive biology, transcriptional regulation by ER is essential for female reproduction and mammary gland development as will be discussed below (Section 1.4.3). ER also functions in male reproduction, and is active in both sexes in the skeletal system, the cardiovascular system and in (51). The endogenous ligand for ER is 17β-estradiol (E2), a cholesterol derivative governed by the hypothalamic-pituitary-gonadal endocrine axis (28). Ligand-bound ER undergoes homo- dimerization and mediates transcriptional regulation through the recruitment of co- regulators at conserved estrogen response elements (EREs) harboring the DNA sequence

AGGTCAnnnTGACCT (32). There are two known ERs, ERα and ERβ, each with differing tissue localization, affinities to E2 and distinct biological function (52, 53). The studies described herein evaluate FOXA1 cooperation with ERα. Of note, FOXA1 is not known to mediate ERβ expression and/or activity.

Ligand binding to ER, as with all nuclear receptors, induces a conformational change that reveals binding sites for interaction with co-regulators (54). ER complexes with countless co-regulators bearing histone modifying and chromatin remodeling capabilities (reviewed

10 in (22)). For example, CBP, p300 and SRC-1 are known co-activators of ER, where they function synergistically to induce E2-mediated transcription of ER target genes (55, 56).

ER also interacts with the human homolog of SWI/SNF (57, 58). The temporal recruitment of co-regulators in ER transcriptional regulation of the gene encoding pS2, also known as trefoil factor-1 (TTF1), has been described in detail (59). In addition to regulating transcription directly, ER also functions non-genomically, where it is involved in mediating the activation of signaling cascades in the . Localization of a proportion of ER to the plasma membrane via palmitoylation (60) results in activation of signaling pathways such as c-Src, and subsequent phosphorylation of ERK-1 and ERK-2

(61). ER can also interact indirectly with DNA through other transcription factors such as the AP-1 complex, a Jun/Fos heterodimer, where AP-1 binds to ERE half sites dictating transcriptional activation (62). Lastly, ER can also mediate transcription in a ligand- independent manner, such as through interaction with (63). As mentioned, nuclear receptors are subject to post-translational modification. ER is no exception being phosphorylated, acetylated, ubiquitylated and sumoylated (26). In particular, ER is phosphorylated via EGF and IGF mediated mitogen-activated protein kinase (MAPK) signaling (64). This modification is required for ER activation in both E2-dependent (65) and E2-independent (66) conditions. c-Src family kinases, protein kinase A (PKA) and protein kinase C (PKC) are also known to mediate ER phosphorylation (67, 68).

Drugs modifying ER activity are in use for the treatment of osteoporosis, and breast cancer (as discussed further in Section 1.6.1) (69). Three drug classes utilize distinct mechanisms to alter ER transcriptional function: (1) selective estrogen

11 receptor modulators (SERMs) block estrogen from binding to ER, (2) aromatase inhibitors (AI) inhibit synthesis of , and (3) selective estrogen receptor down- regulators (SERDs) decrease expression of ER through down-regulation of the receptor.

Tamoxifen, , and are examples of SERMs that function as agonists or antagonists, depending on tissue specificity (70). For example, both and raloxifene are antagonists in breast tissue and agonists in the bone.

However, in the endometrium, tamoxifen mediates agonistic activity, whereas raloxifene is antagonistic. Thus, SERMs can be efficacious in treating breast cancer and osteoporosis, but can inadvertently promote endometrial cancer. The differing pharmacologic activity between tamoxifen and raloxifene in the endometrium is ascribed to structural differences in these molecules (71). The antagonistic property of tamoxifen in the breast is attributed to the conformational change its binding induces in ER leading to the recruitment of co-repressor instead of co-activator complexes. In fact, tamoxifen- bound ER mimics ER-bound to co-repressor complexes (72). Acting via a different mechanism, AIs inhibit the aromatase, which catalyzes the conversion of testosterone to estradiol, and andostenedione to . When compared to tamoxifen,

AIs are more associated with decreased bone density and musculoskeletal events (73). In addition to treating breast cancer, AIs are being tested in the treatment of endometriosis

(74) and in growth disorders to improve height (75). The last class of endocrine therapeutics is SERDs. These are pure estrogen antagonists that induce down-regulation of ER. The prototypical SERD is (ICI 182,780; Faslodex) (76). The efficacy and use of endocrine therapy in breast cancer treatment will be discussed in Section

1.6.1.

12 1.3 FOXA1 IN DEVELOPMENT

Processes of development are often also involved in disease as just mentioned with AR and ER. FOXA1 is no exception. The original identification of FOXAs as transcriptional regulators of hepatic specification (2) led multiple groups to perform expression pattern analyses for these factors from early development throughout adulthood to gain insight into their potential roles during this process. Foxa2 mRNA is the first to be expressed during embryogenesis, and is observed during in the anterior primitive streak and node with subsequent expression in the notochord, floor plate and gut (77-79). Foxa1 becomes detectable at the late primitive streak stage in the midline of mouse , followed by expression in the ventral floorplate, notochord and gut (77-79).

Foxa3 is the last to be activated, being expressed during hindgut differentiation (78). In the adult mouse, expression of FOXA1 and FOXA2 is observed within endoderm, , and ectoderm-derived tissues (80, 81). Foxa3 mRNA is less restricted in adult tissues, being present within the , , , and testis, in addition to endoderm-derived liver and gastrointestinal tissues (80).

These data, in combination with the chromatin-remodeling function observed for FOXA, provided evidence that FOXAs may function in developmental specification. Through both germ-line and conditional knockout approaches, the functions of FOXA1, FOXA2 and FOXA3, have been investigated both independently, and in the case of FOXA1 and

FOXA2 in combination, and each has been proven to be required during varying aspects of development (reviewed in (1, 3, 82)). Briefly, the loss of Foxa2 is embryonic lethal

13 due to failed node and notochord development (83). Foxa1 null mice survive through embryogenesis, but are postnatally lethal due to severe hypoglycemia and dehydration as described in detail below (84, 85). In contrast, Foxa3 knockout mice are normal, developing without morphological defects through adulthood (86), but are hypoglycemic in response to fasting (87) and the males are subfertile (88). The severity of these respective parallels the onset of expression of Foxa2, then Foxa1, then

Foxa3, during embyogenesis, and suggest compensatory roles for FOXA2 in the Foxa1 knockout mice, and for both FOXA2 and FOXA1 in Foxa3 knockout mice. The involvement of FOXA1, specifically, will be discussed herein and is overviewed in Table

1.1.

1.3.1 Pancreas and Kidney

Mice that are homozygous null for Foxa1 lack any overt morphological abnormalities

(84, 85). However, they are postnatally lethal due to a combined phenotype of severe hypoglycemia and dehydration. Interestingly, loss of Foxa1 also dramatically decreases circulating levels. This is paradoxical to the observed hypoglycemic state in these null mice because the pancreas normally responds to hypoglycemia by releasing glucagon, which then induces conversion of glycogen stores to glucose in the liver.

Further investigation revealed that FOXA1 is normally expressed in glucagon expressing pancreatic α-islet cells where it is necessary to transcriptionally activate the proglucagon (Gcg) (84). Loss of Foxa1 reduces glucagon transcription, subsequently decreasing glucagon secretion into the circulation. These results confirm a role for

FOXA1 in regulating glucose homeostasis by dictating α-islet cell function. In addition to

14 the loss of proglucagon gene expression, Foxa1 null mice also fail to secrete insulin in response to glucose administration (85). This defect is attributed to the upregulation of mitochondrial 2 (Ucp2) that occurs in the pancreatic β-cells of Foxa1 null mice (89). Increased UCP2 expression uncouples oxidative phosphorylation, which decreases glucose-induced ATP synthesis and insulin secretion from β -islet cells.

Together, these studies revealed that FOXA1 modulates glucose homeostasis through multiple mechanisms: transcriptional activation of the proglucagon gene and repression of Ucp2 expression.

To investigate the role of FOXA1 during pancreatic development without the physiological complexity of a global knockout, mice were generated with loxP sites flanking 2 of Foxa1. These mice were then crossed with Pdx1-CreEarly transgenics

(90). PDX1 (pancreas/duodenum protein 1) expressing cells generate each cell type (exocrine, endocrine and duct) of the pancreas allowing for pancreas-selective expression of Cre DNA recombinase and disruption of floxed (91).

Surprisingly, Pdx1-CreEarly; Foxa1loxP/loxP mice are viable and fertile indicating that the postnatal lethality observed in the germ-line knockouts (84) was not solely due to an isolated defect in the pancreas, but likely also involved severe dehydration (90).

Compound conditional studies subsequently revealed that FOXA1 and

FOXA2 cooperatively control pancreatic acinar and islet morphogenesis (90). The presence of at least one wild type allele of Foxa2 can compensate for complete loss of

Foxa1 in the pancreas, resulting in viable mice. Interestingly, the absence of one or two wild type alleles of Foxa1 dictates the level of specification of exocrine and endocrine

15 cell lineages in the pancreas of mice completely lacking Foxa2 (90, 92). In other words, mice that are wild type, heterozygous null and homozygous null for Foxa1 in the pancreas have the most normal, intermediary and severe disruption, respectively, of pancreatic lineage specification when combined with the homozygous null Foxa2 allele.

For a detailed description of FOXA1/2 compound that have been generated to investigate their functions in the pancreas and other tissues, see (3, 82). Notably, mice null for Foxa3 have hypoglycemia when fasted due to decreased expression of the GLUT2 (87), and thus each FOXA family member is necessary for maintaining glucose homeostasis.

In addition to the deregulation of pancreatic proglucagon and Ucp2 expression, pups lacking FOXA1 are severely dehydrated (84), prompting studies to investigate FOXA1 function in the kidney. Foxa1 null mice develop nephrogenic diabetes insipidus as evidenced by the inability to respond to arginine- (antidiuretic hormone) (93).

Interestingly, the development of this disease could not be explained by a decrease in the expression of genes encoding vasopressin 2 receptor, aquaporins, or other proteins involved in water reabsorption. Although the precise mechanism by which FOXA1 modulates kidney function remains to be determined, it has been postulated that the lethality of the Foxa1 null occurs as a result of the combined impact of pancreatic insufficiency and kidney defects. This is supported by the viability of mice with a pancreatic specific of Foxa1 (Pdx1-CreEarly; Foxa1loxP/loxP) as discussed above. Although it is unclear whether the kidney defect in Foxa1 null mice is due to a

16 direct effect on this organ, or changes that occur in response to an abnormal developmental program.

1.3.2 Liver

Given the role for FOXA proteins in regulating liver-specific gene transcription (2), it was hypothesized that FOXA1 would be necessary for normal liver development.

However, while Foxa1 null mice die postnatally (84), they have a morphologically normal liver. Similar to the maintenance of normal pancreatic morphology in Foxa1 knockouts due to FOXA2 compensation, FOXA2 also offsets loss of Foxa1 in the liver.

When investigated combinatorially, genomic loss of Foxa1, within the context of an endoderm-specific conditional deletion of Foxa2 (Foxa3-Cre; Foxa2loxP/loxP), completely blocked the onset of liver specification whereas conditional loss of Foxa2 alone had no effect (94). The endodermal disruption of Foxa2 occurs at embryonic day 8.5 (E8.5) when using Foxa3-Cre to induce recombination, and this precedes liver specification. To investigate liver development after the onset of specification, AlfpCre; Foxa1loxp/loxp;

Foxa2loxp/loxp mice were generated (95). In these mice, Cre expression occurs at E10.5 in the liver primordium due to the use of the albumin promoter in conjunction with the α- fetoprotein for directing expression of Cre, but complete loss of Foxa1/2 was not observed until postnatal day 2 (P2) (96). These mice undergo normal hepatocyte differentiation, but during adulthood develop bile duct hyperplasia and fibrosis as a result of increased -6 (IL6) expression. Under normal conditions, FOXA1/2 and the glucocorticoid receptor (GR) cooperate to repress Il6 transcription. In the absence of

FOXA1/2, this repression is ablated, allowing NFκB-induced upregulation of Il6. The

17 excessive IL6 then induces proliferation of bile duct epithelial cells (cholangiocytes) (95).

These results demonstrate the combined importance of FOXA1 and FOXA2 in liver cell specification and differentiation, and suggest a potential role for FOXA1/2 in human diseases of the liver. Along these lines, FOXA2 expression is decreased in the of with cholestatic disease (97), but a role for FOXA1 in biliary diseases remains to be established.

1.3.3 Lung

FOXA1 is observed in the lung bud at E10.5, and is maintained in mature conducting airway epithelial and secretory alveolar type II cells (81). Mice null for Foxa1 have delayed lung development (98). Normally, the fetal lung develops through three distinct morphological phases: pseudoglandular, canalicular and saccular. While of wild type mice reach the saccular stage by E16.5, those lacking FOXA1 are delayed in the canalicular stage until E17.5. Similarly, lung septation in postnatal Foxa1 knockout mice

(P5) was significantly delayed. Loss of Foxa1 also decreased the expression of lung differentiation markers [e.g. clara cell secretory protein (CCSP), pro-surfactant proteins-

B (SP-B) and -C (SP-C)] during embryogenesis. This is at least partly due to the dependency of both CCSP (Scbg1a1) (99) and SP-B (Sftpb) (100, 101) promoters on

FOXA1 for activation in lung . How FOXA1 modulates SP-C expression is not known. Although FOXA1 deficiency causes a developmental delay during embryogenesis, of the knockout lungs was indistinguishable from controls by

P13. That said, cuboidal type II cells had higher glycogen levels, decreased number of lamellar bodies and less surfactant than those in wild type lungs. As in other tissues (e.g.

18 pancreas, liver), FOXA2 eventually compensates for loss of Foxa1 in the lung. Complete loss of Foxa1, combined with doxycycline-induced deletion of Foxa2 driven by Cre expression under control of the SP-C lung-specific promoter (Foxa1-/-/SPC-rtTA-

/tg -/tg loxP/loxP /(tetO)7Cre /Foxa2 ) during embryogenesis, drastically impairs lung branching morphogenesis and epithelial differentiation (102). Similar to Foxa1 null lungs, CCSP expression is absent in the compound mutants. In addition, loss of Foxa1 and Foxa2 decreases mRNA expression of sonic hedgehog (Shh), a necessary activator of lung epithelial specification (103, 104). FOXA1 expression is increased in a rat model of acute lung injury (ALI), where it is suggested to function in alveolar type II epithelial cell (105), and is similarly necessary for induced apoptosis in

A549 cells (106). Combined, these data imply FOXA1 may participate in the onset or progression of lung diseases not limited to cancer as discussed in Section 1.5.1.

1.3.4 Brain

FOXA1 and FOXA2 are expressed in the notochord and floorplate of the fetal brain (77-

79) and Foxa2 null mice are embryonic lethal as a result of failed node and notochord development (83, 107). Although Foxa1 null pups do not exhibit overt CNS architectural defects (84, 85), they have delayed maturation of mature midbrain dopaminergic (mDA) neurons (108). Conditional loss of Foxa2 using a Nestin-Cre dose-dependently exacerbates the Foxa1 null phenotype, indicating that even one copy of Foxa2 compensates for Foxa1 loss, and vice versa. The phenotypic response to FOXA loss has been attributed to the ability of these proteins to control expression of neurogenin 2,

Nurr1 and 1 (EN1), three factors essential for neuronal differentiation (109-

19 111). Complimenting these studies, conditional loss of both Foxa1 and Foxa2, through breeding with En1-klCre transgenic mice, led to disruption of each FOXA protein earlier in development (E9.75) (112) compared with Nestin-Cre (E10.5) mediated disruption

(108). Through these two approaches, Lin and colleagues (2009) showed that FOXA1/2 positively stimulate expression of the LIM homeodomain transcription factors Lmx1a/1b and repress the morphogenically-associated homeodomain protein Nkx2.2 earlier in development (112). Intriguingly, FOXA1 and FOXA2 are redundantly necessary for the initiation of SHH expression in the midbrain (112, 113), paralleling the regulation of Shh by FOXA1 observed in the lung that was discussed previously. Further demonstrating the importance of FOXA in SHH signaling, at least in this context, FOXA1/2 are also responsible for restricting the expression of Gli1 and Gli2, transcriptional mediators of

SHH signaling, and patched-1, the receptor for SHH, in the ventral midbrain (113).

1.3.5 Gastrointestinal Tract

FOXA1/2 are also expressed in the epithelia of the gastrointestinal tract (81). To investigate how the combined loss of Foxa1 and Foxa2 specifically affects intestinal development, floxed alleles were conditionally disrupted using the villin promoter to direct expression of Cre (Vil-Cre) to the intestinal epithelium (114). The compound conditional mutants are viable, but grow slower, and have decreased body weight due to a reduction in lean muscle mass and less body fat. Although overall intestinal morphology is normal, the number, secretory capacity, and differentiation of goblet cells is reduced in the combined mutants. FOXA1, but not FOXA2, was found to bind to the promoter of the

Muc2 gene, which encodes 2. These data compliment previous in vitro studies

20 where FOXA1 and FOXA2 can both transactivate the Muc2 promoter, with FOXA1 being more efficient (115). In addition to diminished formation, loss of

Foxa1/2 leads to decreased differentiation of L- and D-cell enteroendocrine lineages

(115), which produce involved in intestinal function. Paralleling in vitro regulation of the proglucagon promoter by FOXA1/2 (84, 116), intestines of the combined conditional mutants lack D-cells, as evidenced by loss of cells expressing glucagon-like peptides (GLP)-1 and -2. In addition, cell populations that express somatostatin and peptide YY (PYY) (D- and L-cells, respectively) were decreased.

Furthermore, expression of Islet-1 and Pax6, two transcription factors involved in enteroendocrine cell differentiation, are decreased providing evidence that FOXA1/2 precede Islet-1 and Pax6 expression in the enteroendocrine transcriptional hierarchy.

These data indicate that FOXA proteins are essential for development of both mucin- producing goblet and enteroendocrine cellular populations. The loss of these cell types likely results in insufficient nutrient absorption required for normal growth, thus providing an explanation for the decreased growth rate of these mice during early life.

1.3.6 Prostate

Morphogenesis of the prostate is dependent on stromal AR expression, whereas prostatic secretory function relies on epithelial AR (117, 118). FOXA1 is an established regulator of AR transcriptional activity in prostate cancer cells (discussed below) leading to an investigation of FOXA1 during prostate development. Expression analyses revealed that

FOXA1 is present in prostate tissue from the onset of development at E18 in the urogenital sinus epithelium throughout adulthood, with no detectable expression in the

21 stroma (119-121). Considering the postnatal lethality of the Foxa1 null mice, Matusik and colleagues (2005) investigated prostate morphogenesis in the absence of FOXA1 through utilization of two distinct rescue strategies: renal capsule grafting and tissue recombination (122). While loss of Foxa1 does not alter rudiment formation, luminal epithelial lineage differentiation is blocked as evidenced by luminal cytokeratin 8 (CK8) staining being present only in concert with the basal epithelial lineage marker p63.

Furthermore, cytokeratin 5 (CK5) positive basal cell and α-smooth muscle actin (α-SMA) positive smooth muscle cell populations were aberrantly expanded. The epithelium within the Foxa1 null prostate also fails to polarize and undergo proper lumen formation, and as expected, results in a secretion defect. While prostatic tissue lacking epithelial AR has a similar secretory phenotype (118), Foxa1 null maintain epithelial AR expression. Since FOXA1 facilitates AR transcriptional activity as discussed below, it is possible that while FOXA1 is not required for AR expression, it is necessary for AR function in the developing prostate, thus resulting in the observed secretory defect. This is supported by the loss of probasin (Pbsn) and spermine binding protein (Sbp) mRNA, which are normally stimulated by activated AR, in the Foxa1 null prostate glands (122).

Interestingly, FOXA2 is aberrantly up-regulated in Foxa1 knockout prostate epithelium

(122). Normally, FOXA2 is only found in prostatic epithelia during embryogenesis, and in a small population of mature basal epithelial cells which co-express synaptophysin, a neuroendocrine (NE) marker (121, 123). Thus, unlike in other tissues (e.g. lung, liver, pancreas), FOXA2 does not compensate for FOXA1 loss in the prostate. As discussed below with regard to prostate cancer, the upregulation of FOXA2 in the Foxa1 null

22 prostates may enable AR function in an androgen-independent manner, resulting in the observed hyper-proliferation of basal epithelial cells. Of note, loss of Foxa1 also decreases expression of the homeobox gene Nkx3.1, a putative tumor suppressor whose deficiency also leads to prostatic hyperplasia (124). Prostates lacking FOXA1 also have increased SHH, an established contributor to prostate (125-127).

Interestingly, the negative regulation of SHH by FOXA1 in the prostate differs from the positive regulation observed in lung and brain. This may be due to the absence of

FOXA1/FOXA2 collaboration in the prostate, but this possibility has not yet been tested.

23 1.4 THE MAMMARY GLAND

At the onset of this doctoral work, a role for FOXA1 in mammary gland development was unknown. However, the studies just discussed revealing FOXA1 as a necessary developmental mediator in multiple tissue types, especially prostate, in combination with its role in mediating the hormone responsiveness of ERα and AR in cancer (Sections

1.5.2 and 1.6.3), led us to hypothesize that FOXA1 would be necessary for morphogenesis of the mammary gland. This is further supported by the pronounced similarity between the prostate and mammary gland, which are both hormonally controlled during development and normal adulthood. These tissues are also structurally similar, both having stromal and epithelial (luminal and basal) populations and secretory function. Additionally, both succumb to hormone driven tumorigenesis. Given that several of the processes involved in prostatic and mammary morphogenesis (e.g. hormone response, proliferation, tissue remodeling, apoptosis) are also involved in cancer, investigation of how FOXA1 functions in development will provide a greater understanding of its role in cancer. The following overview of mammary gland development provides background to our analysis of FOXA1 in the mammary gland as described in Chapter 2.

1.4.1 Stages of mammary gland development

Mammary gland development is initiated during embryogenesis, but unlike with most tissues, the majority of its morphogenesis occurs after birth. Mouse mammary gland development commences at approximately E10 where the thickening of two lines of

24 endoderm occurs on the ventral surface of the mouse in a bilateral, craniocaudal fashion.

These lines of endoderm are termed milk lines, and are replaced at E11.5 by five pairs of anlagen, or placodes, that become larger and invaginate into the dermis throughout E12-

13. At E15.5, the mammary epithelium begins to invade into the underlying that will ultimately become the fat pad. At birth, each gland is observed as a minimally arborized rudimentary ductal tree surrounded by a small mammary fat pad. An example of a normal P1 mammary gland is shown in Figure 2.2A. Embryonic mammogenesis is coordinated by a large number of signaling pathways including Wnts and growth factors (FGFs) as major players. For detailed reviews of these processes, please see (128, 129).

After birth, the mammary epithelium and fat pad grow isometrically with the until (3-5 weeks depending on genetic strain), upon which ovarian production of E2 and progesterone stimulates ductal invasion and side-branching, respectively.

Particularly, estrogen signaling through ERα is required for the formation of terminal end buds (TEBs), which are the catalytic structures responsible for ductal invasion throughout the fat pad (130, 131) (Figure 1.1A). Interaction of the mammary epithelium with the stromal compartment is critical in mammary gland development as described below for

ERα. Structurally, TEBs contain an outer layer of cap cells, a highly proliferative population thought to be comprised of stem cells, and an inner layer of body cells, also known as luminal progenitor cells (132) (Figure 1.1C). As the TEB elongates the duct into the fat pad, the body cells undergo apoptosis to form a ductal lumen (133). The mature duct is comprised of two distinct epithelial populations: luminal epithelium and

25 basal/myoepithelium. Luminal cells line the lumen of both ducts and alveolar buds, representing ductal and alveolar luminal populations, respectively. The basal cells line the basement membrane, and are responsible for ductal contractility and milk secretion out of the nipple (134). The basal/myoepithelium also secretes proteins and proteoglycans to form the basement membrane (135). In addition, the mammary gland contains several non-epithelial cell types. It is highly vascularized and is composed of , fibroblasts, neuronal cells and inflammatory cells.

Upon completion of puberty, which in the mouse is approximately 10-12 weeks of age, the mammary epithelium has completely invaded the fat pad. TEBs have now regressed, and are replaced by terminal ducts (Figure 1.1B). Progesterone signaling through the progesterone receptor (PR) throughout puberty results in a mature mammary gland that is highly arborized with extensive ductal side-branching and alveolar bud formation (136).

The number of alveolar buds fluctuates throughout phases of the estrous . During pregnancy, alveoli terminally differentiate in response to increasing levels of progesterone and prolactin to form milk-producing lobulo-alveoli, which are the functional units of (137, 138). After pups are weaned, the mammary gland undergoes involution, or massive programmed cell death. Involution occurs through two main phases: a reversible phase for the first 72 hours, and an irreversible phase that takes at least 8-10 days to complete (139, 140). After involution, the mammary gland is structurally similar to a virgin gland that is poised for another round of pregnancy- induced lobulo-alveolarization, lactation and involution.

26 1.4.2 Mammary epithelial hierarchy

As just discussed, there are two distinct epithelial populations in the mammary gland, luminal and basal/myoepithelial cells. Investigation of a large cohort of human breast cancers revealed that there are multiple subtypes of breast cancer, a number of which are phenotypically similar to the luminal or basal populations of the normal gland that are called luminal (A, B, C) and basal-like, respectively (141) (The molecular subtypes of breast cancer are reviewed in detail in Section 1.6.1.). These data led to the hypothesis that subtypes of breast cancer are dependent on their “cell of origin”, or in other words, tumors of the luminal subtypes may arise in luminal cells, whereas tumors of the basal subtype may arise in basal cells. Importantly, the breast cancer molecular phenotype is predictive of patient prognosis, where luminal tumors have a favorable prognosis, while basal tumor have a poor prognosis (142). A great deal of investigation has been done over the last decade to define those factors involved in the differentiation of luminal and basal lineages. These factors will likely also be critical for maintaining the phenotype of luminal and basal breast cancers, and thus, the aggressiveness of the tumor.

To begin to test the cell of origin hypothesis, it was necessary to distinguish the different epithelial populations of the mammary gland. The luminal and basal mammary epithelial populations can be visualized by performing immunohistochemistry (IHC) for markers of each lineage. Several cytokeratins (CK8, CK18, CK19) and E-cadherin are common markers of the luminal lineage, whereas CK14, p63 and α-SMA are examples of markers of the basal lineage. A means to isolate the distinct populations of the mammary epithelium, including the mammary stem cell (MaSC) population, is through fluorescence

27 activated cell sorting (FACS). Upon enzymatic dissociation of the mammary gland, and removal of non-mammary, or lineage negative (Lin-) populations (e.g. hematopoietic, endothelial), the mammary epithelium can be sorted into four distinct populations based on expression of CD29 (β1-) and CD24 (heat stable antigen): CD29lo/CD24-,

CD29hi/CD24-, CD29lo/CD24+, and CD29hi/CD24+ (143). The Lin-/CD29hi/CD24+ population constitutes MaSCs, where one cell is capable of generating a functional mammary gland upon injection into the cleared fat pad of a mouse (143). Lin-

/CD29lo/CD24+ isolates a population of luminal cells that can be further separated through an additional round of FASC with an to CD61 (β3-intergrin) that distinguishes mature (CD61-) and progenitor (CD61+) luminal populations (144). CD49f

(α6-integrin) can be similarly used in place of CD29 to segregate these populations (145).

Evaluating the mammary epithelial lineages in genetically engineered mice has provided insights into the roles for these distinct populations in development and cancer. For example, mice conditionally null for GATA3 in the mammary gland have an increase in the number of luminal progenitors, suggesting a block in luminal lineage maturation

(144). GATA3 is expressed specifically in the luminal subtype of breast cancer (141), and it was subsequently shown that GATA3 is required for maintenance of the luminal tumor phenotype and disease dissemination (146). Furthermore, mice that over-express the Wnt1 ligand specifically in the mammary gland due to mouse mammary tumor virus

(MMTV) induced expression (MMTV-Wnt1) develop mammary tumors recapitulating the basal phenotype (147), and have an increase in the percentage of MaSCs compared to wild type controls (143). These data suggest that the MaSCs are the cell of origin of this

28 particular tumor model. Similar techniques have been used on normal human tissue revealing a similar mammary epithelial hierarchy (148). Interestingly, this strategy has provided evidence that human basal-like breast cancers likely arise in the luminal progenitor, not in the MaSC population (149, 150). Overall, these analyses exemplify the importance of investigating development in order to gain insights into cancer initiation, progression and .

1.4.3 The estrogen receptor in mammary gland development

Estrogen signaling through the steroid nuclear receptor ERα is essential for normal mammary gland development. In contrast, ERβ is not required for ductal growth (151), but it involved in terminal differentiation of the gland (152). ERα is expressed in both mammary luminal epithelial and stromal populations as shown in Figure 2.1 (153, 154), and is responsible for inducing expression of PR in these cells (155). A number of different experimental approaches have been performed to delineate both epithelial and stromal ERα function. In 1993, Kenneth Korach and colleagues generated a mouse genomic knockout of ERα (ERKO) through disruption of exon 2 of Esr1 (156). These mice exhibit estrogen insensitivity, and are completely infertile with both uterine and ovarian developmental defects. ERKO females were subsequently described to have failed mammary ductal invasion with glands resembling a pre-pubertal rudimentary structure (157).

The function of ERα in both stromal and epithelial compartments has also been described using tissue recombination followed by transplantation into either the renal capsule of

29 athymic nude mice (158), or by orthotopic transplantation into cleared mammary fat pads

(159). In both studies, recombination of ERKO and wild type (wt) stroma (S) and epithelium (E) was performed in every possible combination: (1) E-wt, S-wt, (2) E-wt, S-

ERKO, (3) E-ERKO, S-wt, and (4) E-ERKO, S-ERKO. Experiments by Cunha et al.

(1997) (158) were executed on isolated tissue from postnatal mammary glands and revealed that ERα expression in the stroma is required for ductal outgrowth, whereas epithelial ERα expression was dispensable (i.e. E-wt, S-ERKO did not grow, but E-

ERKO, S-wt did grow). In contrast, orthotopic transplant experiments performed by

Mueller et al. (2002) (159) were done with epithelium from mature females. In these studies, both epithelial and stromal ERα was required for ductal outgrowth.

The ERKO mice (156) used in each of the studies just described were subsequently identified to express a truncated form of ERα that maintains some transactivation capacity (160, 161). Thus, an ERKO mouse was engineered lacking exon 3 of

Esr1 (Ex3αERKO) (162). The mammary glands of these mice do not form TEBs, and do not develop past a rudimentary ductal structure similar to that seen in previous ERKO females (163). Orthotopic transplantation studies were similarly completed on this genetic model by recombining stroma and epithelium from wild type and Ex3αERKO mice, and provided evidence that only epithelial ERα is necessary for mammary ductal outgrowth, whereas stromal ERα is not required (163). The importance of ERα in the epithelium has been further confirmed through mice engineered to lack ERα specifically in the mammary epithelium (MMTV-Cre; Esr1LoxP/LoxP) (164). Lastly, given that the

Ex3αERKO mice have overt reproductive abnormalities (162), investigation of

30 pregnancy and lactational competence is impossible in this model system. To circumvent this limitation, mice were generated to delete the floxed ERα allele after virgin development using the whey acidic protein (WAP)-Cre promoter (164). Both lobulo- alveologenesis and the ability to feed pups are deficient in these mice, suggesting that

ERα is critical in multiple stages of mammary gland development.

Overall, the importance of ERα in the development of the mammary gland is well established. Since FOXA1 is necessary for proper ERα activity in cancer cells as will be discussed in Section 1.6.3, it will also likely be involved in normal development. For this reason, our experimental approach to evaluating FOXA1 has been two pronged: to determine how it functions in normal mammary gland development (Chapter 2) and in breast cancer (Chapter 3).

31 1.5 FOXA1 IN CANCER

1.5.1 FOXA1 in cancers other than breast and prostate

Over the past decade, FOXA1 expression has been examined in several human cancers, with proposed oncogenic and tumor suppressive roles depending on the type, and in some cases, cancer subtype (Table 1.2). Our knowledge of FOXA1’s participation in four cancer types (acute myeloid leukemia (AML), esophageal, lung, pancreatic and thyroid) is very much in its infancy with only one or two reports evaluating a role for FOXA1 in each of these diseases. Briefly, FOXA1 is increased in AML patient samples that are chromosomally normal, but harbor oncogenic mutations in fms-related kinase 3

(FLT3) and neuroblastoma RAS viral homolog (NRAS) (165). Similarly, the

FOXA1 gene is amplified in anaplastic thyroid carcinomas (166), and in a subset of esophageal and lung cancers (167). These data suggest that aberrant FOXA1 transcriptional activity may promote tumorigenesis or the level of aggressiveness in these tumors. Further supporting this possibility, FOXA1 expression is correlated with lymph node metastases in esophageal squamous cell carcinoma (ESCC) and loss of FOXA1 in vitro decreases cellular invasion and migration in an esophageal cancer cell line (168). In stark contrast, loss of either FOXA1 or FOXA2 in pancreatic cancer cells induces epithelial-to-mesenchymal (EMT) transition implying a metastasis suppressive role in this disease (169). The combined knockdown of FOXA1/2 in pancreatic cancer cells leads to an even greater reduction in epithelial-specific gene expression and increased cell motility than silencing either factor alone, suggesting that these genes may cooperate to inhibit metastatic progression. Given the compensatory effect of FOXA2 observed in the

32 pancreas-specific knockout of Foxa1 during development, it is not surprising that the two

FOXA factors have similar functions in cancer cells derived from this tissue.

As indicated above, loss of both Foxa1 and Foxa2 induces bile duct hyperplasia in the murine liver (95). These results suggest that FOXA proteins may reduce tumor susceptibility, but interestingly, expression of both FOXA1 and FOXA2 is maintained in chemically induced mouse liver cancer models (170, 171). Although counterintuitive, these results may indicate that FOXA1 is differentially expressed in a less aggressive subtype of hepatocellular carcinoma (HCC) possibly represented in these mouse models.

This is supported by data suggesting both FOXA1 and FOXA2 positively regulate mir122, a microRNA expressed in a subset of HCC that is correlated with favorable prognosis (172), and would be similar to the differential expression of FOXA1 seen in the varying subtypes of breast cancer as discussed below. The bile duct hyperplasia observed in the Foxa1/2 null liver stems from diminished FOXA1/2 cooperation with GR in the repression of Il6 (95). Importantly, as with ERα and AR, FOXAs are also implicated in GR-mediated transactivation of numerous other target genes (173-181), and

GR signaling is anti-apoptotic in hepatoma cells (182). Thus, the potential for pro- tumorigenic FOXA/GR signaling in HCC requires further evaluation.

It is critical to note that while the studies just discussed are suggestive that FOXA1 functions in a pro-tumorigenic or anti-tumorigenic capacity, the rigorous approaches necessary to define FOXA1 as an oncogene (over-expression causes cancer), tumor suppressor (loss causes cancer), or metastasis suppressor (over-expression blocks

33 metastasis) have not been performed for any cancer type. That said, the role for FOXA1 in prostate and breast cancer has been studied to a much greater degree than any other cancer. In these cancers, disease aggressiveness and associated patient prognosis is complicated by multiple cancer subtypes and states of hormone dependence versus independence. The known participation of FOXA1 in prostate and breast cancer is detailed next.

1.5.2 FOXA1 in prostate cancer

As previously discussed, FOXA1 is critical for prostate development and loss of Foxa1 causes basal epithelial hyper-proliferation, activation of oncogenic SHH and decreased expression of the tumor suppressor Nkx3.1 (122). These results suggest a tumor suppressive role for FOXA1 in prostate epithelia, and pose several questions regarding if, and how, FOXA1 mediates prostate tumor initiation, progression and/or metastasis.

While Foxa1 null prostates maintain epithelial AR expression, they lack AR-dependent secretory differentiation, implying that FOXA1 may modulate AR activation of this process (122). Given the majority of prostate cancers are driven by aberrant AR signaling, much work has been done to define a role for FOXA1 in this process.

FOXA1 expression in prostate cancer

The LPB-Tag LADY mouse model of prostate cancer expresses SV40 large T antigen

(Tag) under control of a large (12kb) fragment of the prostate-specific probasin promoter

(LPB), giving rise to prostate tumors that recapitulate human disease (183). LADY tumors have increased nuclear expression of FOXA1 and AR that accompanies Tag

34 (123). Interestingly, FOXA1 is retained in both androgen-dependent (12T-7f) and androgen-independent (12T-10) models. Furthermore, 12T-10 associated liver metastases maintain FOXA1. Together, these data argue against a tumor suppressive role for

FOXA1 and suggest that it may function independent of androgens. FOXA1 has also been examined in the transgenic adenocarcinoma of mouse prostate (TRAMP) model, which differs from LADY by expressing both SV40 large T and small t downstream of a minimal probasin promoter (184, 185). Chiaverotti and colleagues

(2008) examined murine strain-specific differences on TRAMP-induced tumor formation and found that expression of the transgene in the FVB/N background decreases survival and increases the incidence of neuroendocrine (NE) carcinomas compared to TRAMP-

C57BL/6 animals (186). NE carcinoma, although rare in humans, is unresponsive to androgen ablation therapy (187). Interestingly, TRAMP-induced NE carcinomas co- express FOXA1 and FOXA2. Normal prostate epithelia and atypical hyperplasia have high levels of FOXA1 and AR, whereas FOXA2 is only expressed within a small percentage of mature prostate basal epithelial cells (121, 123). This, along with co- expression of epithelial (E-cadherin) and NE (synaptophysin) lineage markers, support the possibility that NE carcinomas initiate within a bipotential epithelial population that is more susceptible to tumorigenic processes in FVB/N versus C57BL/6 mice. The combined expression of FOXA1 and FOXA2 in NE tumors may provide a growth advantage in the absence of androgens. Indeed, in vitro analyses revealed that FOXA2 activates prostate specific antigen (PSA) promoter activity in an androgen-independent fashion, and suggested that the conversion from FOXA2-negativity to FOXA2-positivity

35 may be responsible for the transition from androgen-dependence to androgen- independence (123).

Although FOXA1 is functionally significant in androgen-independent cells, FOXA1 mRNA positively correlates with human prostate cancer cell line and xenograft models that have been defined as androgen dependent (188). Examination of FOXA1 and

FOXA2 expression in a small cohort of human prostate adenocarcinomas indicated that

FOXA1 is present in all samples independent of Gleason score, with the level of expression being indistinguishable from benign tissue (123). In contrast, FOXA2 is undetectable in low-grade prostate adenocarcinomas, but is found in NE small cell carcinomas similar to the TRAMP mouse model just discussed. The expression of

FOXA1 was not reported for these human NE samples (123). A more recent study investigating FOXA1 protein levels in primary and metastatic human prostate tumors, revealed high FOXA1 expression positively correlates with metastatic disease (189). In addition, an amplicon at 14q21, which includes FOXA1, has been identified in metastatic prostate tumors (190). Overall, these studies confirm that FOXA1 expression is maintained in mouse models of prostate cancer as well as human tumors, but more robust expression analyses in human prostate tumors are necessary to delineate its specific association with patient outcomes and anti-androgen treatment response.

FOXA1 and the androgen receptor

Most men diagnosed with prostate cancer have AR-positive, hormone dependent disease that is initially responsive to androgen-ablation therapy. Unfortunately, the majority of

36 these cases become resistant to this therapeutic approach due to acquisition of androgen- independence (191). To better understand AR signaling within both androgen-dependent and -independent contexts, the cooperation of FOXA1 with AR transactivation has been investigated, albeit with some discrepancies in the resulting data (123, 192-195). Initial reports indicated that FOXA1 is necessary for AR activation of target gene expression

(192). These studies involved blockade of FOXA1 binding to the probasin and PSA promoters by mutating the FOXA1 consensus sequences within the promoters. Such disruption blocked androgen (R1881) induced promoter activity (192), suggesting that

FOXA1 is necessary for androgen activation of gene expression (Figure 1.2A). In contrast to these results, over-expression of FOXA1 in other studies inhibited dihydrotestosterone (DHT)-induced PSA promoter activity (123) and AR activation of transcription (195). While the over-expression of FOXA1 in vitro could non-specifically squelch the transcriptional machinery, and explain these discrepant results, additional studies have shown that reducing FOXA1 expression does not alter DHT-induced expression of PSA/KLK3 (193, 194), TMPRSS2 or PDE9A (193), nor is FOXA1 necessary for androgen-induced progression (193). Interestingly, Jia and colleagues (2008) did observe a reduction in DHT-induced expression of TMPRSS2 following FOXA1 silencing in an LNCaP variant (C4-2B) that is AR-positive, but androgen-independent (Figure 1.2B) (194), suggesting that FOXA1 facilitates AR- stimulated transcription of at least some genes. Supporting this finding, FOXA1 is also necessary for expression of -conjugating enzyme E2C (UBE2C) in an AR- positive, androgen-independent LNCaP variant (), but not in parental cells (196).

FOXA1 also binds to, and may regulate the homeobox transcription factor Hoxb13 (197),

37 which is an androgen-independent gene (198). Overall, these data suggest that FOXA1 modulates AR target gene transcriptional activity, but possibly through varying mechanisms that depend on the cell or gene’s level of responsiveness to androgens. They also indicate that FOXA1 can modulate ligand-dependent and -independent actions of

AR in prostate cancer cells.

Analyses of the binding of FOXA1 to AR target genes and its role in AR transactivation have suggested varied functions for FOXA1 that are gene-specific. FOXA1 binds adjacent to androgen response elements (AREs) in the probasin, PSA (192, 199) and spermine binding protein (199) promoters similarly in the presence and absence of DHT.

Others have also reported that FOXA1 binds to AR bound regions, but recruitment of

FOXA1 to these sites is further enhanced by DHT (193, 194). Importantly, while FOXA1 was necessary for androgen-induced transcriptional activity of the probasin and PSA promoters, blocking direct binding of FOXA1 to these promoters did not impact AR binding (192), suggesting that FOXA1 is not necessary for recruitment of AR. In contrast, AR binding to enhancer regions of several other genes (UBE2C, CDK1,

CDC20) was reduced in response to FOXA1 silencing in parental (LNCaP) and androgen-independent (LNCaP-abl) prostate cancer cell lines (196). Hence, the dependency of AR binding on preoccupancy of FOXA1 at adjacent sites appears to be highly dependent upon gene context (196). Supporting the requirement for FOXA1 binding as an essential modulator of AR activity, a germ line single polymorphism (SNP) identified in prostate cancer patients within the risk 8q24 increases its affinity for FOXA1 binding and potentiates the ability of this chromosomal

38 region to mediate androgen responsiveness of a heterologous promoter compared to the wild type sequence (200). Although these results suggest a tight link between FOXA1 binding and androgen responsiveness, the authors did not directly investigate whether

FOXA1 is necessary for either AR binding or enhancer activity in its native chromosomal context. As a mechanism for how FOXA1 dictates AR binding at specific genes, studies in both prostate and breast cancer cells revealed that H3K4 dimethylation occurs proximal to FOXA1 binding sites, and can precede FOXA1 binding to DNA (201). The over-expression of the histone demethylase KDM1 reduced AR binding, likely due to loss of bound FOXA1 (196). The presence of FOXA1 in LNCaP cells is also associated with DNA hypomethylation (13), and an open chromatin state as measured by formaldehyde-assisted isolation of regulatory elements (FAIRE), where high FAIRE

FOXA1 binding sites are more likely to bind AR (202). Overall, it is quite evident that

FOXA1 participates in AR target gene recognition, but it seems likely that this regulation is dependent on additional co-factors, androgen dependence and/or the distance from the transcriptional start site (i.e. promoter versus enhancer).

As another mode of regulation, the winged helix/forkhead domain of FOXA1 has been found to complex directly with the DNA-binding domain of AR (192, 203). This complex permits AR binding to promoter DNA even in the absence of AR consensus elements and vice versa for FOXA1 binding (Figure 1.2C,D) (203). Formation of a

FOXA1/AR multimeric transcriptional complex that requires only one of the two protein’s consensus elements could explain why mutagenesis of FOXA1 binding sites does not alter AR binding (192), whereas decreasing FOXA1 expression does as

39 discussed above (196). The FOXA1/AR complex may also be necessary for AR nuclear localization. In this case, decreasing FOXA1 expression would greatly impede the access of AR to its target genes, whereas mutating the FOXA1 consensus element would only directly affect FOXA1 binding.

Additional roles of FOXA1 in prostate cancer

Although the majority of work investigating FOXA1 in prostate cancer has been aimed at understanding how it is involved in modulating AR transcriptional activity, FOXA1 has also been implicated in other signaling pathways. FOXA1/2 positively regulates transcription of the gene encoding anterior gradient protein 2 (AGR2), whose expression increases aggressiveness of prostate cancer cells. This regulation can be abrogated by the presence of ERBB3 binding protein 1 (EBP1) (204). AGR2 is an established metastasis inducer (205), and its activation by FOXA1 was first discovered in goblet cells (206).

Placement of FOXA1 in the EBP1/FOXA1/AGR2 pathway indicates that it may play an important modulatory role in metastatic progression. Interestingly, EBP1 has also been shown to negatively regulate AR mediated transcription (207). While not specifically tested, it is possible that EBP1 represses AR transactivation by inhibiting FOXA1 function in a similar manner as seen for the transcriptional regulation of AGR2. In contrast to its pro-metastatic positive regulation of AGR2, expression of FOXA1 is also down-regulated by SOX4 (sex determining region Y-box 4), a prostate cancer oncogene

(208). This contradiction exemplifies the broad spectrum of activity for FOXA1 in the cancerous prostate, and at least partially explains how loss of Foxa1 in the normal

40 prostate could lead to both tumorigenic (e.g. increased SHH) and anti-tumorigenic (e.g. loss of AR function) phenotypes.

41 1.6 FOXA1 IN BREAST CANCER

In the western world, breast cancer is the most commonly diagnosed cancer in women, and is second only behind cancers of the lung and for cancer-related deaths,

(American Cancer Society, 2011). There are a number of risk factors for developing breast cancer including sex, age, family history, BRCA1/2 mutation, personal history, high breast density, atypical hyperplasia, and lifetime exposure to ovarian hormones.

Upon diagnosis, a patient’s treatment strategy is dependent on a number of factors such as tumor size, tumor grade, lymph node infiltration and metastatic spread. The tumor is also investigated for proliferation rate (Ki67 positivity) and the expression of ERα, PR and human epidermal growth factor receptor 2 (HER2), also known as ERBB2.

Depending on pathological and clinical stage, the patient will undergo a combination of surgery, radiation and/or systemic therapy. The term “systemic therapy” broadly encompasses all biologics, endocrine (anti-hormone) and chemotherapeutics provided in neo-adjuvant and/or adjuvant settings. Unlike the non-selective mechanism of action of chemotherapeutics, biologics (targeted) and endocrine therapies are much less toxic since they are designed to block a specific tumorigenic pathway.

1.6.1 The molecular subtypes of breast cancer

Over a decade ago, a seminal study revealed that breast cancer is not a single disease, but is actually composed of several distinct subtypes: luminal A, luminal B, luminal C,

HER2, basal-like and normal-like (141). These classifications have been repeatedly observed (209), and further investigation revealed that these molecular subtypes are

42 predictive of patient survival (142). The subtypes were identified via unsupervised hierarchical clustering of cDNA microarray data from a cohort of human breast tumors, where each subtype was distinguished by the differential expression of numerous genes.

While the six distinct subtypes can only be classified by genome-wide expression profiling (142), they are often identified using surrogate markers (210, 211): ERα, PR,

HER2, epidermial growth factor receptor (EGFR) and cytokeratin 5/6 (CK5/6). Luminal

A tumors are ERα+/PR+/HER2-. Luminal B tumors are ERα+/PR+/HER2+ or

ERα+/PR+/HER2- with a high proliferation index. HER2 tumors are ERα-/PR-/HER2+.

Basal-like tumors are often referred to as triple negative (ERα-/PR-/HER2-), but the added classification of EGFR and/or CK5/6 positivity is more accurate (212-215).

Importantly, not all basal-like tumors are triple negative, and not all triple negative tumors are basal-like (216). Lastly, normal-like tumors are often negative for all five markers and are fairly uncommon (211). These tumors are more likely to be diagnosed in post-menopausal women, are usually small and confer a more favorable prognosis than

HER2 and basal-like tumors (142, 217).

The luminal subtype of breast cancer

Luminal A subtype tumors comprise ~40-75% of breast cancers (211, 218-220). When compared to the other breast cancer subtypes, luminal A tumors are associated with decreased tumor size, decreased tumor grade, and have no or little lymph node involvement (221). These disease characteristics, in association with the hormone receptor positivity of these tumors, results in luminal A tumors having the most favorable prognostic outcome of all the subtypes (142). This outcome is at least partially due to the

43 high efficacy of endocrine (anti-hormone) therapy (222). Luminal B and C tumors are less common, and confer a poorer survival compared to luminal A tumors (142, 223).

The most striking difference between luminal B and C tumors with luminal A tumors is decreased ERα expression (142). This distinction likely correlates with decreased response to ERα-targeted therapies, and thus the observed reduction in survival.

There are three classes of endocrine therapeutics in use clinically: selective estrogen receptor modulators (SERMs), aromatase inhibitors (AIs) and selective estrogen receptor down-regulators (SERDs). As discussed in Section 1.2.2, endocrine therapy decreases the transcriptional activity of ERα either through altering the recruitment of co-regulators via

SERM action, blocking the production of estrogens in the body via AIs, or down- regulating ERα via SERDs. Currently, the standard endocrine therapy for localized hormone receptor positive disease in premenopausal women is five years of tamoxifen; in postmenopausal women, both tamoxifen and AIs are considered (224). Tamoxifen is efficacious in preventing both recurrence and mortality in women with hormone receptor positive disease (222). Although highly effective, many women do not respond to tamoxifen treatment due to de novo resistance and many eventually relapse due to acquired resistance. Mechanisms of resistance to endocrine therapy include altered ERα activity (e.g. methylation of ESR1, ERα phosphorylation site mutants, changes in co- regulator expression), growth factor receptor signaling (e.g. increased EGFR or HER2 activity), and cell cycle/apoptotic mechanisms (e.g. changes in expression of cyclin and

Bcl-2 family members) (reviewed in (225)). Fortunately, second line AI and SERD treatment is capable of slowing disease progression in some patients (226, 227), likely

44 those with altered ER activity. Additionally, tamoxifen is associated with unwanted side effects such hot flashes, thromboembolism and endometrial cancer (73). Comparison between tamoxifen and third generation AIs as first line therapy has been investigated in post-menopausal woman with early breast cancer. These studies suggest that letrozole

(228, 229), exemestane (230) and (231, 232) are more effective at preventing recurrence versus tamoxifen. Since AIs also have unwanted side effects such as osteoporosis and musculoskeletal disease, due to complete blockade of estrogen signaling, additional studies are warranted to determine the most effective, least adverse adjuvant endocrine therapy.

Even accounting for resistance and off target effects, adjuvant endocrine therapy in women with localized, hormone receptor positive, lymph node negative breast cancer has been so successful that there is debate whether combination endocrine therapy plus chemotherapy provides any survival benefit for these patients versus endocrine therapy alone. Given the adverse side effects associated with chemotherapy, diagnostic tools such as the Oncotype DX (233) are now in use to aid in decision-making with regard to adding chemotherapy to the patient’s treatment regimen. The Oncotype DX qualified the expression of 16 genes (plus 5 reference genes) within ERα-positive tumors, and provides a recurrence score that has prognostic power. Clinical trials have shown that women with hormone receptor positive disease and a low Oncotype DX recurrence score do not benefit from adding chemotherapy to tamoxifen in either node-negative (234) or node- positive (235) disease.

45 The HER2 subtype of breast cancer

The second largest breast cancer subtype (~8-30% of patients) (211, 218-220) comprises tumors over-expressing HER2 most often as a result of gene amplification (236). HER2 positivity predicts poor prognosis mostly due to association with lymph node positivity, high tumor grade, recurrence, and metastatic spread (221, 236). Additionally, HER2- positive disease is disproportionately diagnosed in younger women (237). Patients with this subtype of breast cancer are treated adjuvantly with chemotherapy plus trastuzumab

(Herceptin), which is a humanized directed towards the HER2 receptor. This treatment strategy reduces both recurrence and disease progression, although it is associated with cardiotoxicity (238, 239). Trastuzumab is also in clinical trials to determine its effectiveness in the neo-adjuvant setting (240).

As with many treatment options for breast cancer, patients receiving trastuzumab will eventually develop resistance to the drug. Mechanisms of resistance include increased insulin-like growth factor-I receptor (IGF-IR), decreased p27kip1, expression of truncation forms of HER2 (241), and increased c-Src signaling (242). Second line therapies, such as lapatinib (Tykerb), a dual HER2/EGFR tyrosine kinase inhibitor, are currently in use in combination with chemotherapy to treat patients with HER2-positive disease that failed prior trastuzumab and chemotherapeutic regimens (243, 244). There are also many drugs still in clinical trials including trastuzumab-DM1, which is trastuzumab covalently bound to a derivative of maytansine 1, a destabilizing agent (245, 246). Other agents being tested for efficacy in HER2 positive disease include neratinib, a pan-ERBB

46 inhibitor, and pertuzumab, a humanized monoclonal antibody to

HER2 that blocks dimerization with other ERBB family members (247, 248).

The basal-like/triple negative subtype of breast cancer

The subtype of breast cancer associated with the highest patient mortality is the basal-like or triple negative subtype (249). Patients with basal-like and/or triple negative breast cancer make up ~12-20% of all patients (211, 218-220). In addition to lacking ERα, PR and HER2, these tumors are also further delineated through positive expression of EGFR and/or CK5/6 (210). This disease is associated with age and ethnicity, where young (<50 years of age) and African American women are more likely to have basal-like or triple negative breast cancer (210, 217) (220, 250). These tumors are high grade (221), are more likely to lead to visceral metastasis (251), and harbor a high risk of recurrence and death within five years of diagnosis, but heightened risk declines after this point (252).

Given the absence of the targetable receptors (e.g. ERα and HER2) that are expressed in other subtypes of breast cancer, patients with basal-like/triple negative tumors are treated with surgery and standard chemotherapy regimens. Tumors are fairly responsive to treatment, but overall survival rates are always low (253). This dichotomy suggests a high level of intrinsic resistance, and a great need for targeted therapeutics.

The absence of targeted therapies for treating basal-like/triple negative breast cancer has spawned extensive programs aimed at identifying putative therapeutic strategies for this subtype (251). A number of subtype-specific markers are now known, including -1, fascin, , p-cadherin, and α,β - (254). Additionally, this

47 subtype exhibits aberrant expression of a number of tumor suppressors such as mutations in TP53 (255), or loss of protein (pRb) concomitant with a gain in p16

(256, 257). Furthermore, women with mutations in the breast cancer susceptibility protein type 1 (BRCA1) tend to develop basal-like breast cancer (223, 254, 258), suggesting an association with BRCA1 function and subtype-specific disease. Since , pRb and

BRCA1 are all involved in the cell cycle and the DNA damage response, the high proliferation rates of these tumors are most likely a result of these processes gone awry.

For this reason, agents targeting poly(adenosine diphosphate [ADP]-ribose) polymerase

(PARP), a mediator of single strand DNA break repair, are being tested for their efficacy in treating tumors with BRCA1/2 mutation. Two PARP inhibitors (olaparib and iniparib) have exhibited promising results regarding clinical benefit in phase I and phase II trials respectively, with iniparib increasing patient survival when combined with chemotherapy

(259, 260). Other drugs in pre-clinical and clinical testing for this subtype of breast cancer are malate, a non-selective tyrosine kinase inhibitor, and dasatinib, a c-

Src inhibitor (251, 261, 262).

1.6.2 FOXA1 expression in breast cancer

Since being identified as a novel luminal subtype gene, the clinical significance of

FOXA1 protein expression in breast tumors has been investigated by multiple groups.

Wolf and colleagues (2006) were the first to investigate a breast tumor tissue microarray

(TMA) for FOXA1 expression, revealing that FOXA1 is associated with low tumor grade, and not surprisingly, ERα expression (263). A number of publications soon followed with varying TMA compositions, and when combined, provide expression data

48 for FOXA1 on over 5000 human breast cancers (264-269). FOXA1 significantly associates with ERα expression in every study. Moreover, FOXA1 positively correlates with the luminal subtype as defined by ERα and/or PR positivity, HER2 negativity (264,

265), and luminal cytokeratin expression (264-266). Negative correlations have been observed for FOXA1 and the basal subtype when defined as ERα-negative, HER2- negative with CK5/6 and/or EGFR positivity (265, 269), or by basal cytokeratin expression (265, 266). Importantly, several groups have proposed utilizing FOXA1 as a prognostic tool to stratify patients within the ERα-positive, luminal subtype (264, 265,

267). Data from these studies suggest that FOXA1 can predict survival within this population, and thus aid in therapeutic decision-making. However, TMA data generated by one group argues that FOXA1 is unable to further stratify prognosis within the subgroup of patients with ERα-positive disease (266). To directly test the independent prognostic and predictive ability of FOXA1, Ademuyiwa and colleagues (2010) investigated the correlation between FOXA1 expression and Oncotype DX recurrence scores in patients with ERα-positive, node negative disease (268). In this study, FOXA1 negatively correlated with recurrence score leading the authors to suggest FOXA1 immunostaining could function as a more cost-effective pathological marker than the

Oncotype DX. Of note, FOXA1 is not a component of Oncotype DX. Lastly, increased copy number of chromosomal locus 14q13, where FOXA1 is located, is associated with the ERα, PR-positive breast cancer subtype (270), providing further evidence that

FOXA1 may promote tumor differentiation.

49 Although FOXA1 statistically correlates with ERα, it has been noted that some ERα- negative tumors also have high FOXA1 expression (263-267, 269). A subset of ERα, PR- negative tumors is molecularly more similar to ERα, PR-positive than to triple negative tumors, and FOXA1 was identified as one of the differentially expressed genes within this “ERα-positive-like” subgroup (271). These tumors are more likely to possess apocrine features, express AR, have an androgen responsive molecular signature, and a breast cancer cell line (MDA-MB-453) that recapitulates this phenotype is growth stimulated by androgens and inhibited by anti-androgens (272). Given the role of FOXA1 in modulating AR transactivation in prostate cancer, it is possible that the presence of

FOXA1 in this subset of ERα-negative tumors is required for androgen-dependent responsiveness mediated by AR. Supporting this hypothesis, testosterone increases

FOXA1 mRNA expression in another AR-positive breast cancer cell line (SUM190)

(273). In fact, a recent study provided evidence that FOXA1 mediates AR transcriptional regulation in the MDA-MB-453 cells (274).

1.6.3 FOXA1 and the estrogen receptor

Functioning as a chromatin remodeler, FOXA1 was discovered to cooperate with ERα in activating the liver-specific vitellogenin promoter (275), and had been shown to regulate the well-described ERα target, TFF1 (pS2) (276). In 2005, two groups independently extended these findings to breast cancer, demonstrating that FOXA1 was necessary for estrogen-induced ERα binding to specific genes, and the upregulation of those genes, including TFF1 (277, 278). Carroll and colleagues (2005) identified forkhead consensus motifs in close proximity to roughly half of the ERα binding sites spanning

50 21 and 22 of a breast cancer cell line (277). Likewise, Laganière et al. (2005) analyzed

ERα binding sites via a human promoter microarray, and found 12% of ERα-bound promoters also contained forkhead motifs (278). Several genome-wide analyses have confirmed these data (201, 279-282), although one group has reported much less (<10%) commonality in the location of forkhead and ERα consensus motifs in the genome (283).

That said, considerable between FOXA1 and ERα has been repeatedly observed in comparisons of full genome ERα and FOXA1 ChIP-chip (201) and ChIP-seq

(282) data, showing that over half of ERα binding sites are also occupied by FOXA1.

This cooperation is observed for both ERα up-regulated and down-regulated genes (201,

282) (Figure 1.3A). For example, FOXA1 is necessary for ERα repression of Reprimo

(RPRM) transcription (284). FOXA1 and ERα can also have opposing roles in regulating gene expression as demonstrated for BASE (breast cancer and salivary gland expression):

FOXA1 is required for BASE expression, whereas ERα represses transcription of this gene (285).

Functionally, FOXA1 is necessary for (286) and specifically for estrogen- mediated cell cycle progression of luminal breast cancer cells (278, 287). These results are at least partly explained by the requirement for FOXA1 to mediate ERα stimulation of CCND1, the gene encoding cyclin D1 through a downstream enhancer (287). The presence of FOXA1 correlates with bound RNA polymerase II, active chromatin and histone H4 acetylation (287) at ERα-activated genes, where each is a hallmark of proliferating cells. Similar to its role in prostate cancer cells, FOXA1 binding is associated with an open chromatin state as measured by FAIRE, where loss of FOXA1

51 decreases the level of open chromatin on a global scale (202, 282). Specifically, FOXA1 binding at high FAIRE regions is accompanied by increased histone H3, lysine 9 (H3K9) acetylation, decreased H3K9 mono- and di-methylation, and increased H3K4 dimethylation (202). The presence of FOXA1 is also associated with DNA hypomethylation (13). Together, these data support a pioneering function for FOXA1 in modulating chromatin structure for subsequent ERα binding and activity (Figure 1.3A), similar to that predicted for AR, although the specific timing of events is not well established in cancer cell lines.

FOXA1 is preferentially bound to intergenic enhancers and , mirroring that of ERα

(277). It is recruited to DNA in the presence and absence of estradiol, but unlike with androgen stimulation, binding of FOXA1 generally decreases with estrogen treatment

(277, 279). A direct comparison of FOXA1 in ERα and AR signaling using breast and prostate cells, respectively, revealed that while there are common binding sites, FOXA1 has a distinct binding profile depending on whether it is co-expressed with ERα or AR in these distinct cell types (201). This tissue specificity is dependent on differential recruitment of FOXA1 to ERα versus AR target genes due to lineage-specific H3K4 dimethylation (201). Moreover, the presence of FOXA1 at the TFF1 gene enhancer is also dependent on binding of the histone variant, H2A.Z, by p400 (288), providing further evidence that tissue-specific FOXA1 activity is dictated by histone modifications.

Another factor has recently been identified that also delineates FOXA1-specific recruitment and subsequent ERα transcriptional regulation. CCCTC-binding factor

(CTCF) is an insulator binding protein (289, 290) whose loss was shown to impede

52 FOXA1 binding to known target genes (e.g. TFF1, PGR), while loss of FOXA1 had no impact on CTCF binding (290). In contrast, Hurtado et al. (2010) found that loss of

CTCF increases FOXA1 binding capacity (282). Despite the discrepancy between these two reports, it is apparent that CTCF can modulate FOXA1 binding. The context dependency of this interaction must be further explored to unravel the molecular mechanisms by which CTCF enhances or impedes binding of FOXA1 to DNA.

The interplay between FOXA1 and ERα may extend beyond the ability of FOXA1 to control binding activity of ERα. Using a different experimental approach than Carroll et al. (2005) (277) who identified FOXA1 binding sites in ERα target genes through motif analysis, Laganière and colleagues (2005) first identified FOXA1 as an estrogen-induced

ERα target gene and then subsequently observed FOXA1 binding sites within ERα responsive genes (278). Providing support for FOXA1 being an ERα target, a more recent analysis of the ERα chromatin interaction network revealed that ERα binding sites bracket the FOXA1 gene (291). In contrast, others have reported that FOXA1 expression is decreased with estrogen treatment (263, 292-294) (Figure 1.3B). These disparate observations are further complicated by the varying roles of FOXA1 in mediating ERα signaling in tissues other than the breast (282, 295, 296). Osteosarcoma cells that have been engineered to express ERα (U2OS-ER) have undetectable levels of FOXA1 protein, suggesting ERα is insufficient to induce expression of FOXA1 in these cells. Given the absence of FOXA1 in U2OS-ER cells, it is not surprising that forkhead consensus motifs do not correlate with the ERα cistrome, nor is FOXA1 required for ERα-regulated transcription in this context (296). These results suggest that the role of FOXA1 in

53 modulating ERα activity is cell-context dependent. In support of an indispensable role for

FOXA1 in ERα activity in breast epithelial-specific gene regulation, exogenous expression of FOXA1 in U2OS-ER cells induces ERα binding to otherwise breast- specific ERα targets (e.g. TFF1, XBP1) that is accompanied by increased expression of these genes (282). Furthermore, FOXA1 over-expressing U2OS-ER cells also become sensitive to the growth inhibitory effects of tamoxifen. In contrast, FOXA1 is endogenously expressed in, and is necessary for, -induced ERα transactivation of bone morphogenetic protein-2 (BMP2) gene expression in the cell line,

MC3T3-E1 (295). It is possible that the distinct observations seen with FOXA1 and ERα in these studies can be attributed to the differences between the U2OS cells, which were derived from a human osteosarcoma versus the non-transformed MC3T3-E1 cells, which were isolated from newborn mouse calvaria (297).

FOXA1 has been proposed as a putative breast cancer therapeutic target based on the concept that ERα dependency on FOXA1 will translate to an increase in the efficacy of tamoxifen in ERα-dependent breast cancer (298). Functioning as a SERM, tamoxifen has antagonistic properties in the breast, but is agonistic in the bone, where it maintains estrogen signaling and is protective against osteoporosis. The differing roles of FOXA1 in cancerous and normal bone tissue should be further defined before considering targeting FOXA1 in breast cancer patients. If FOXA1 is expressed in normal bone, as observed in the MC3T3-E1 cells, but loss of FOXA1 correlates with tumorigenic progression, as observed in tumorigenic U2OS cells, then the systemic reduction of

FOXA1 expression may inadvertently promote formation of bone tumors.

54 1.6.4 Additional roles of FOXA1 in breast cancer

In addition to the well-described ability of FOXA1 to control ERα activity, there have been other facets of FOXA1 described in breast cancer. One of these is a possible role in

HER2 signaling (273, 286). Treatment of the breast cancer cell line SUM190 with heregulin, an indirect activator of HER2, induces FOXA1 expression (273). This suggests that FOXA1 may be downstream of HER2 signaling and mediate some of its actions.

Indeed, FOXA1 silencing potentiates the cellular toxicity of Herceptin (286). While the specific mechanisms remain unknown, these data support a role for FOXA1 in HER2- induced cell survival. However, it is important to note that the presence or absence of

FOXA1 does not correlate with clinical outcome of patients with HER2-positive disease

(299), indicating that FOXA1 may not play a critical role in HER2-induced signaling in vivo.

In addition to many other genes, FOXA1 binds to, and activates transcription of the gene encoding 72 (HSPA1A), the stress-inducible cytosolic form of

(300). HSP70 independently associates with poorer outcome in breast cancer patients with node-negative disease (301). Thus, it is possible that FOXA1 and HSP72 cooperatively dictate the aggressive behaviors of these cancers, but this has not been tested. As mentioned earlier, FOXA1 also participates in ERα-dependent transactivation of CCND1 (cyclin D1) (287). Intriguingly, FOXA1, ERα and cyclin D1 are coordinately up-regulated in tumors arising in mice that are bitransgenic for mammary gland-specific expression of integrin-linked kinase (ILK) and Wnt1 (MMTV-Wnt1/Ilk) (302). Forced co- expression of ILK and Wnt1 leads to increased epithelial proliferation, a greater

55 percentage of luminal progenitor cells, and increased tumor formation versus expression of either alone. The authors of this work propose that the convergence of Wnt1 and ILK on β-catenin activation may result in upregulation of FOXA1, which is downstream of

SOX17/β-catenin during endodermal development in Xenopus (303). Whether FOXA1 is necessary for expansion of luminal progenitors and the formation of mammary tumors, or is simply expressed as a bystander in this bi-transgenic, or in other mouse models of breast cancer, remains to be seen.

Given its correlation with the less aggressive luminal subtype of breast cancer, it is not surprising that FOXA1 activates the transcription of genes (e.g. CDH1, CDKN1B) implicated in decreased breast cancer tumorigenicity independently of ERα (304, 305).

Exogenous over-expression of FOXA1 in a triple negative, basal breast cancer cell line

(MDA-MB-231) induces expression of the cell- molecule, E-cadherin (306).

In this context, FOXA1 directly stimulates transcription of the E-cadherin gene (CDH1), and the associated induction of E-cadherin expression decreases the migratory capacity of these cells, suggesting that FOXA1 may promote their differentiation. Activation of

CDH1 occurs in the absence of ERα, supporting the notion that FOXA1 has ERα- independent roles in dictating a more differentiated luminal cell phenotype. Another example of FOXA1 functioning in a tumor suppressive role is its regulation of the cyclin dependent kinase inhibitor p27Kip1 (307). FOXA1 binds to the promoter of p27Kip1

(CDKN1B) in breast cancer cells and synergizes with BRCA1 to induce its transactivation in a colon cancer cell line. In addition, breast cancer cells over-expressing

FOXA1 have increased p27Kip1 promoter activity and decreased cell number (263). When

56 combined, these studies paint a complicated picture of FOXA1 as a participant in multiple signaling pathways in breast cancer, that are both oncogenic and tumor suppressive. It is likely that the specific molecular functions of FOXA1 are dictated by many components, including, but not limited to, the epithelial origin of the tumor initiating cell, and the co-expression of ERα, HER2 and other regulatory factors that have not yet been identified.

57 1.7 STATEMENT OF PURPOSE

Prior to the onset of this doctoral work, the participation of FOXA1 in mediating ERα and AR transcriptional regulation was well described. FOXA1 had also been demonstrated as a critical mediator in the normal development of several organs, including the hormonally regulated prostate gland. Given additional evidence that

FOXA1 and ERα are positively correlated in breast cancer, and that ERα is necessary for mammary gland morphogenesis, we hypothesized that FOXA1 would also be indispensable for the normal development of the mammary gland. Our findings that

FOXA1 is required for the mammary epithelial ERα expression and hormone induced ductal invasion is described in Chapter 2. These results reveal that in addition to being required for ERα transcriptional activity, FOXA1 is also required for its expression.

Interestingly, although FOXA1 positively correlates with ERα in breast cancer, this correlation is not perfect. FOXA1 expression in ERα-negative tumors has also been described. Since FOXA1 independently correlates with the luminal subtype of breast cancer and a more favorable patient prognosis, we hypothesized that in addition to its known co-modulatory role in ERα signaling, FOXA1 also functions in an ERα- independent manner to promote the differentiated phenotype of luminal subtype tumors.

Studies identifying a role for FOXA1 in repressing the basal breast cancer molecular phenotype are described in Chapter 3. Combined, these data provide novel insight into

FOXA1 as an orchestrator of mammary ductal development and the differentiation status of breast cancers.

58 Table 1.1

Overview of Foxa1 mouse models of development

Foxa2 Tissue Origin Genotype Viability Phenotype Ref(s) Redundancy Lethal, Insulin secretion defect; Hypoglycemia; 84, 85, Pancres Endoderm Foxa1-/- No P2-12/14 Hypotriglyceridemia 89 Pdx1-CreE/Foxa1loxP/loxP Adulthood No major defects Yes 90 Pdx1-CreE/Foxa1loxP/loxP/Foxa2loxP/+ Adulthood No major defects Yes 90 Pdx1-CreE/Foxa1loxP/+/Foxa2loxP/loxP Lethal, P5 Hypoplasia n/a 90 Failed endocrine and exocrine Pdx1-CreE/Foxa1loxP/loxP/Foxa2loxP/loxP Lethal, P2 n/a 90 differentiation; Hypoplasia

Lethal, Dehydration; Nephrogenic diabetes Kidney Mesoderm Foxa1-/- No 84, 93 P2-12 insipidus

Lethal, Liver Endoderm Foxa1-/-/Foxa3Cre/Foxa2loxP/loxP Failed fetal liver development n/a 94 E10 Alf-pCre/Foxa1loxP/loxP/Foxa2loxP/loxP Adulthood Bile duct hyperplasia; Fibrosis n/a 95

Lethal, Delayed alveolarization; Surfactant Lung Endoderm Foxa1-/- Yes 98 P2-13 secretion defect Foxa1-/-/SPC-rtTA-/tg/(tetO) Cre-/tg/ At least 7 Impaired branching morphogenesis n/a 102 Foxa2loxP/loxP E18.5

Lethal, Delayed dopaminergic neuron Brain Ectoderm Foxa1-/- Yes 108 P2-12 maturation At least *Foxa1-/-/Nestin-Cre/Foxa2loxP/loxP Failed dopaminergic neuron maturation n/a 108 E18.5 At least En1-klCre/Foxa1loxP/loxP/Foxa2loxP/loxP Failed dopaminergic neuron maturation n/a 112 E11.5 At least Failed ventral midbrain progenitor Foxa1-/-/Wnt1-Cre/Foxa2loxP/loxP n/a 113 E18.5 specification

Impaired goblet & enterendocrine cell GI Tract Endoderm Villin-Cre/Foxa1loxP/loxP/Foxa2loxP/loxP Adulthood n/a 114 maturation; Mucin secretion defect

Failed luminal lineage differentiation; Prostate Endoderm Foxa1-/- Rescued Epithelial hyperproliferation; Secretion No 122 defect

*Investigated dose-dependent effect of each allele of FOXA1/A2. For simplicity, these combinations were not reported here. n/a = not-applicable; in these models Foxa2 was deleted in combination with Foxa1 P = Post-natal day E = Embryonic day

59 Table 1.2

Overview of FOXA1 in human cancer

Cancer Type FOXA1 Expression Relative to Normal Predicted Activity Ref(s) AML Increased Oncogenic 165

Breast No difference Unclear 263-269 Amplified in ER+/PR+ tumors Oncogenic 270

Esophageal Amplified/Overexpressed Oncogenic 167, 168

HCC Expressed in Favorable Prognosis Tumors Tumor suppressive 172

Lung Amplified/Overexpressed Oncogenic 167

Pancreatic Decreased in poorly differentiated disease Tumor suppressive 169

Prostate No difference Unclear 123 Increased in associated metastases Oncogenic 189 Amplified in associated metastases Oncogenic 190

Thyroid Amplified/Overexpressed Oncogenic 166

60 Figure 1.1

Mammary gland terminal end bud (TEB) formation and ductal invasion.

(A) During puberty, ovarian hormones induce terminal end bud (TEB) formation (arrow).

These structures are responsible for ductal invasion throughout the mammary fat pad. (B)

After puberty, TEBs regress and are replaced by terminal ducts (arrow). (C) A cartoon diagram of the TEB.

61 Figure 1.1

62 Figure 1.2

FOXA1/AR signaling in prostate cancer.

(A-B) Lineage-specific FOXA1 binding sites are marked by histone H3, lysine 4 dimethylation (H3K4me2). Under both (A) androgen-dependent and (B) androgen- independent conditions, FOXA1 mediates AR transactivation of varied responsive genes.

FOXA1 over-expression has been shown to repress androgen-induced PSA promoter activity, mRNA and protein expression, but due to the limited number of reports and the unclear mechanism involved, this is not included in the figure. (C-D) FOXA1 complexes with AR: (C) FOXA1 binds to ARE containing DNA in the presence of AR, and (D) AR binds to FOXA consensus motif containing DNA in the presence of FOXA1. These complexes can also form in the absence of DNA. It is unknown whether FOXA1/AR can co-transactivate in the absence of one of the DNA binding motifs, and whether these processes are dependent on androgen.

63 Figure 1.2

64 Figure 1.3

FOXA1/ERα signaling in breast cancer.

(A) Lineage-specific FOXA1 binding sites are marked by histone H3, lysine 4 dimethylation (H3K4me2). FOXA1 mediates both estrogen-induced gene transactivation and repression. (B) Expression of ERα has been proposed to modulate FOXA1 expression in an estrogen-dependent manner.

65 Figure 1.3

66 CHAPTER 2

FOXA1 is an Essential Determinant of ERα Expression and Mammary Ductal

Morphogenesis

Adapted from Development 137, 2045-2054 (2010)

by

Gina M. Bernardo, Kristen L. Lozada, John D. Miedler, Gwyndolen Harburg, Sylvia C.

Hewitt, Jonathan D. Mosley, Andrew K. Godwin, Kenneth S. Korach, Jane E. Visvader,

Klaus H. Kaestner, Fadi W. Abdul-Karim, Monica M. Montano & Ruth A. Keri

2.1 ABSTRACT

FOXA1, estrogen receptor-α (ERα) and GATA-3 independently predict favorable outcome in breast cancer patients, and their expression correlates with a differentiated, luminal tumor subtype. As transcription factors, each functions in the morphogenesis of various organs, with ERα and GATA-3 being established regulators of mammary gland development. Interdependency between these three factors in breast cancer and normal mammary development has been suggested, but the specific role for FOXA1 is not known. Herein, we report that Foxa1 deficiency causes a defect in hormone-induced mammary ductal invasion associated with a loss of terminal end bud formation and ERα expression. In contrast, Foxa1 null glands maintain GATA-3 expression. Unlike ERα and

GATA-3 deficiency, Foxa1 null glands form milk-producing alveoli indicating that the defect is restricted to expansion of the ductal epithelium, further emphasizing the novel role for FOXA1 in mammary morphogenesis. Using breast cancer cell lines, we also demonstrate that FOXA1 regulates ERα expression, but not GATA-3. These data reveal

67 that FOXA1 is necessary for hormonal responsiveness in the developing mammary gland and ERα-positive breast cancers, at least in part, due to its control of ERα expression.

68 2.2 INTRODUCTION

The epithelium of the mammary gland is composed of luminal and basal/myoepithelial cell lineages (308). Luminal cells line the ductal lumen and secrete milk upon terminal differentiation into lobulo-alveolar cells. Basal/myoepithelial cells reside between the luminal cells and the basement membrane and are necessary for ductal contractility.

Breast cancer subtypes (luminal v. basal) have been defined by patterns of gene expression that reflect these lineages (223). Luminal subtype tumors maintain a more differentiated state and are less aggressive than basal subtype cancers. Processes of normal postnatal mammary gland development directly mirror those of tumorigenesis

(e.g. invasion, proliferation, angiogenic remodeling and apoptotic resistance) (309).

Hence, determining how cell fate is regulated during normal mammary gland development should facilitate identifying the mechanistic basis for phenotypic differences between luminal and basal breast cancers, and should advance the development of subtype-specific therapeutics.

Expression of the transcription factors ERα, GATA-3 and FOXA1 strongly correlates with the luminal subtype of breast cancer and favorable patient prognosis (223, 264, 266,

310). Estrogenic signaling through ERα, a member of the nuclear receptor superfamily, is a primary determinant of luminal tumor biology, and patients with luminal tumors have a better prognosis, in part, due to estrogen-targeted therapies (222). The consistent concomitant expression of FOXA1, ERα and GATA-3 in this subtype is suggestive of a co-modulatory loop that may be responsible for maintaining the luminal phenotype. In

69 breast cancer cells, FOXA1 facilitates estrogen responsiveness by modulating ERα binding to a subset of target gene promoters (277, 278). For example, FOXA1 is specifically required for ERα-induced transcription of CCND1 (cyclin D1) (287), an established oncogene in breast cancer (287, 311). In contrast to the role of FOXA1 in

ERα activity, ERα and GATA-3 have been suggested to function in a positive feedback loop, where expression of ERα is required for the transcription of GATA-3 and vice versa

(312). These data imply an interdependence of FOXA1, ERα and GATA-3 in the maintenance of luminal breast cancer. Further defining this collaboration should provide insight into how ERα-positive tumors become resistant to anti-hormone therapies as well as reveal the function of FOXA1 that occurs in tumors in the absence of ERα (266).

The ability of FOXA1, ERα and GATA-3 to form a regulatory network in luminal breast cancer cells suggests that they may also co-modulate normal mammary gland morphogenesis, a process that requires ERα and GATA-3. ERα is expressed within a subset of the normal luminal epithelial and stromal populations of the mammary gland

(313). Disruption of Esr1 blocks development at a rudimentary ductal structure, and signaling from estradiol through ERα during puberty is required for mammary epithelial proliferation, ductal elongation, bifurcation, and invasion throughout the mammary fat pad (159, 163, 164). GATA-3 is also necessary for mammary gland development (144,

314). Specifically, Gata-3 deficiency leads to expansion of the CD61-positive luminal progenitor population indicating that GATA-3 is necessary for terminal differentiation of the luminal lineage (144). Further investigation revealed that forced expression of

GATA-3 induces tumor differentiation and inhibits metastatic progression (146). In

70 support of a transcriptional interdependence between ERα, GATA-3 and FOXA1, loss of

Gata-3 in the normal mammary gland decreases the ERα-expressing luminal population

(144, 314), and over-expression of GATA-3 in murine mammary tumors (146) and a human embryonal kidney epithelial cell line increases FOXA1 mRNA (315). Moreover, chromatin immunoprecipitation (ChIP) in primary mammary cells revealed that GATA-3 binds, and can potentially regulate, transcription of Foxa1 (314), although this has not been directly demonstrated.

Although expression of FOXA1, GATA-3 and ERα are positively correlated in breast tumors and form a co-regulatory network in breast cancer cell lines, the functional relationship between these three factors during development and tumor initiation has not been fully explored. In particular, the role of FOXA1 in mammary morphogenesis remains unknown. Herein, we report that Foxa1 deficiency in the mammary gland results in loss of ERα, a block in terminal end bud formation and an inability of the ducts to properly invade the mammary fat pad in response to pubertal or pregnancy hormones. In contrast, Gata-3 expression and formation of lobulo-alveoli are independent of FOXA1.

These data provide the first direct evidence that FOXA1 is critical for mammary gland morphogenesis and maintenance of ERα expression.

71 2.3 MATERIALS AND METHODS

Immunohistochemistry and Immunofluorescence. For analysis of proliferation, mice were injected i.p. with 10 mg/g BrdU (Sigma) 2 hours prior to sacrifice. Glands were fixed in 4% paraformaldehyde for 4 hours, transferred to 1XPBS, paraffin embedded and sectioned (5 µm). Sections were re-hydrated, and antigen retrieval performed using 10 mM sodium citrate (pH = 6) in a pressure cooker (125˚C for 10 minutes; 90˚C for 2 minutes) or for milk antibody, incubated in 10 ug/ml pepsin in 0.01 N HCl for 15 minutes at room temperature. Sections were blocked with peroxidase blocking reagent (DAKO) and incubated with primary antibody overnight at 4°C [FOXA1, Santa Cruz; ERα, Santa

Cruz; E-cadherin, ; CK8 (TROMA-1), Developmental Studies Hybridoma

Bank, University of Iowa; α-SMA, Sigma; BrdU, Becton-Dickinson) or at room temperature for 1 hour (Milk, Nordic Immunology; PR, DAKO]. Secondary detection of

FOXA1, ERα, E-cadherin, Milk and PR was performed using the appropriate Vectastain

Elite ABC Kit (Vector Laboratories) as per manufacturer’s recommendations. CK8, α-

SMA and BrdU were detected using the EnVision+System-HRP for mouse

(DAKO) as recommended. Secondary conjugates were detected using 3,3’- diaminobenzidine (DAKO). Sections were counterstained with Gill’s #3 Hematoxylin

(Fisher), dehydrated and mounted. TUNEL was performed as per manufacturer’s recommendations except using Gill’s #3 Hematoxylin as a counterstain (ApopTag

Peroxidase In Situ Apoptosis Detection Kit, Chemicon). Alexafluor-596 (anti-goat) and

Alexafluor-488 (anti-rabbit) secondary antibodies (Invitrogen) were used for detection of

FOXA1 and ERα by IF. IF and IHC was quantified by counting the percentage of

72 positive ductal epithelium in at least 2-5 fields per section per mouse. H&E staining was performed by the Case Western Reserve University Tissue Procurement and Histology

Core Facility.

Animal Breeding. All animal procedures, except production of Ex3αERKO and MMTV-

Cre;Gata-3f/f mice, were approved by the Case Western Reserve University IACUC.

Ex3αERKO mice were generated under an approved protocol at the National Institute of

Environmental Health Sciences/NIH, and a contract to Xenogen Inc. (Caliper Life

Sciences) using a strategy similar to that described previously (162). This resulted in an

Esr1 gene with exon 3 flanked by loxP sites. Exon3 was deleted by crossing mice carrying the floxed exon 3 Esr1 to a global -Cre mouse line (Tg(Sox2-cre)1Amc/J;

Jackson Labs). DNA was evaluated by PCR using P1 and P3 primers as described (162).

The MMTV-Cre;Gata-3f/f mice were generated at the Walter and Eliza Hall Institute of

Medical Research as described (144). Foxa1+/- males (84) were bred with wild type

C57BL/6 females generating Foxa1+/- progeny that were intercrossed to generate Foxa1-/- progeny. Transgenic mice were identified by PCR using primers as described (84).

Mammary anlagen transplantation. Transplantation of mammary anlagen into recipient mice has been described (316). Briefly, the mammary anlagen of embryonic day 14 (e14) female mouse embryos were dissected and cultured at 37°C/5% CO2 in DMEM/F12

(supplemented with 10% FBS, 1% Pen-Strep, 2 mM L- and 0.75 ug/ml

Fungizone) until the genotypes were determined. Three-week old recipient C57BL/6 females were anesthetized with 2.5% avertin, and inguinal fat pads were cleared of

73 endogenous epithelium. The cleared fat pad was examined by whole mount to verify successful clearing. Foxa1+/+ anlage was inserted into the #4 cleared fat pad, and Foxa1-/- anlage was inserted into the #9 cleared fat pad of the same mouse. The incision was sutured and infiltrated with marcaine (0.25%). Recipient mice were aged 5 or 8 weeks, and the transplanted glands were collected and whole mounts examined. Alternatively, recipient mice were aged 8 weeks, mated with C57BL/6 males, and the transplanted glands collected and whole mounts assessed at 18.5 days post-coitum (dpc).

Renal Capsule Grafting. Tissue grafting into the renal capsule has been described (317).

Briefly, inguinal fat pads of postnatal day 1 female pups were removed, and incubated at

4°C in DMEM/F12 culture media as described above until the genotypes were determined. Recipient C57BL/6 females were anesthetized with 2.5% avertin, and a kidney exteriorized. A small incision was made to separate the kidney capsule from the parenchyma. A polished glass pipette was used to create a pocket between the kidney capsule and parenchyma. The inguinal fat pad from a Foxa1+/+ pup was grafted into the pocket, and the kidney placed back into the body cavity. The same procedure was used to graft the inguinal fat pad from a Foxa1-/- pup on to the contralateral kidney. The incisions were closed using wound clips, and the wound infiltrated with marcaine (0.25%).

Recipient mice were aged either 2 or 4-5 weeks, and the glands harvested for further analysis. Alternatively, recipients were aged 4-5 weeks, mated, and the glands were harvested at 18.5 dpc.

74 Mammary Gland Whole Mounts. Glands were fixed in Kahle’s fixative for at least 4 hours, washed in 70% ethanol, gradually rehydrated to 100% water, stained with carmine alum, dehydrated, cleared in xylenes, and mounted as previously described (318). Ductal area was obtained by taking the area of a box drawn around the gland, including the nipple. Ductal length was obtained by measuring from the farthest edge of the lymph node from the nipple to the end of the longest duct. When the duct did not reach the lymph node, the distance from the end of the duct to the farthest edge of the lymph node from the nipple was measured and given a negative value.

Mammary Cell Preparation and FACS. Inguinal mammary glands from 10 (per experiment) wild type FVB/N virgin females (~8 weeks of age) were isolated and prepared as described (143). Experiments #1 and #2 were sorted by fluorescence- activated cell sorting (FACS) using antibodies against CD24, CD29 and CD61 as described (144). Experiment #2 was sorted twice to enhance purity.

Quantitative Real-time PCR. Total RNA was isolated using TRIzol Reagent

(Invitrogen), treated with DNAse I (DNA-free, Ambion), and cDNA produced using

SuperScript II Reverse Transcriptase (Invitrogen). Real-time PCR was performed using

Applied Biosystems TaqMan Gene Expression Assays ( IDs = Foxa1,

Mm00484713_m1; Pgr, Mm00435625_m1; Gata-3 Mm00484683_m1; CK8,

Mm00835759_m1; ESR1 Hs01046817_m1; GAPDH, Hs99999905_m1) or SYBR-green primers (Foxa1-Foward: GGATCCCCGCTACTCCTTTA; Foxa1-Reverse:

AGCACGGGTCTGGAATACAC).

75 Cell Culture and RNA Interference. All cell lines were obtained from ATCC. MCF7 cells were grown in DMEM (Mediatech); T47D cells in RPMI 1640 (Gibco). Media was supplemented with 10% FBS and 1% Penicillin-Streptamycin (Invitrogen). Cells were seeded in 100 mm dishes to be 30-50% confluent upon . siRNA targeting firefly luciferase mRNA (siCONTROL Non-targeting siRNA #2, Dharmacon) or human

FOXA1 mRNA (siGENOME M-010319-01 and -04, Dharmacon) were transfected in

OPTI-MEM media (Invitrogen) using Lipfectamine 2000 (Invitrogen) to a final concentration of 100 nM. Culture media was changed to complete growth medium after

16-24 (MCF7) or 24 (T47D) hours. Cells were harvested 36 (MCF7) or 72 (T47D) hours post-transfection.

Immunoblots. Cells were lysed (50 mM Tris-HCl, pH7.4; 100 mM NaCl; 1 mM EDTA;

1 mM EGTA; 1 mM NaF; 0.1% SDS; 0.5% Sodium Deoxycholate; 1% Triton-X-100;

10% Glycerol; 2 mM Sodium Orthovanadate; Protease Inhibitor Cocktail (Sigma)), and protein levels quantified (Bradford Assay, Biorad). Protein lysate was resolved using

SDS-PAGE, and transferred to PVDF membrane (BioRad). Blots were blocked (5%- milk-1XPBST) and incubated overnight at 4°C with primary antibody (FOXA1, Santa

Cruz; ERα, Santa Cruz; GATA-3, Santa Cruz; β-actin, Sigma) diluted in 5%-BSA-

1XPBST. Blots were incubated with an HRP-conjugated secondary antibody (Santa

Cruz) diluted in 5%-milk-1XPBST and developed using ECL reagent (Amersham).

Quantification of protein levels was determined using Image J (319).

76 Chromatin Immunoprecipitation. ChIP was performed as previously described (320).

For the FOXA1 ChIP analysis, MCF7 cells were treated with vehicle or 17β-estradiol

(10-7 M) for 45 minutes. Cleared lysate was incubated with either normal goat IgG or

FOXA1 antibody (Abcam, Ab5089). For RNA Polymerase II ChIP analysis, MCF7 cells were transfected with a non-targeting siRNA or FOXA1 siRNA as described above. Cells were harvested 36 hours post-transfection. Cleared lysate was incubated with either normal mouse IgG or RNA Polymerase II antibody (Covance, 8WG16). Binding of

FOXA1 and RNA Polymerase II to the ESR1 proximal promoter was detected using the following primers: 5’-AGGAGGGGGAATCAAACAGA-3’ and 5’-

TTTACTTGTCGTCGCTGCTG-3’. Quantification of precipitated DNA relative to input was accomplished using Image J (319).

Statistical Methods. Significance was determined by Student’s t-test assuming a two- tailed distribution and equal variance among sample populations.

77 2.4 RESULTS

FOXA1 is expressed in the developing mammary gland in conjunction with ERα

The consistent expression of FOXA1 in luminal breast cancers led us to postulate that

FOXA1 may also regulate luminal epithelial cells in the normal breast. To begin to address this possibility, we examined the pattern of FOXA1 expression throughout various stages of murine mammary gland development. Given the ability of FOXA1 to regulate ERα activity at numerous target genes in breast cancer, we also assessed the pattern of ERα expression. FOXA1 is expressed in the majority of body cells (i.e. luminal progenitors), but is absent from cap cells (i.e. myoepithelial progenitors) within the terminal end bud (TEB) (Figure 2.1A). TEBs appear at the duct’s leading edge during puberty, and are the highly proliferative structures required for ductal elongation and branching of the mammary epithelium throughout its associated fat pad (308). The expression pattern for FOXA1 was similar to that observed for ERα in the pubertal gland

(313). Both FOXA1 and ERα are maintained in the ductal epithelium of post-pubertal virgin mammary glands, but the subset of cells that are positive for either protein decreases within the virgin alveolar population and is further reduced during pregnancy, where only a few positive cells are present per field. Importantly, lobulo-alveoli do not express either FOXA1 or ERα. Detection of the cell population that expresses FOXA1 and ERα is restored as the mammary gland undergoes involution. These data indicate that

FOXA1 is present within the structures that are necessary for puberty-associated mammary morphogenesis (i.e. TEBs) and in the same developmental stages as ERα. To define whether FOXA1 and ERα are co-expressed within the same cells, we performed

78 dual-immunofluorescence (IF) within the adult virgin gland (Figure 2.1B). At this stage, approximately 30% of luminal epithelial cells express both FOXA1 and ERα, while a subset of cells express FOXA1 alone, or to a lesser degree, ERα alone. In addition, while

ERα is present within the stroma, FOXA1 expression is undetectable (data not shown).

FOXA1 is essential for mammary ductal invasion

The pattern of FOXA1 expression in the TEB (Figure 2.1A) suggests that it may contribute to mammary morphogenesis. To determine if loss of Foxa1 disrupts embryonic development of the mammary primordium, we analyzed mammary rudiments from Foxa1+/+ (wild type) and Foxa1-/- (null) mice (84) on postnatal day 1 (Figure 2.2A).

Using whole mount analysis, we observed no difference between Foxa1+/+ and Foxa1-/- rudiments in the number of terminal ducts or area occupied by ducts (Figure 2.2B).

These results indicate that FOXA1 expression is not required for embryonic development of the mammary ductal rudiment.

Other than early rudiment formation, most mammary gland development occurs postnatally with the onset of puberty. Thus, we next examined the impact of FOXA1 on postnatal mammary morphogenesis in Foxa1-/- mice. These mice exhibit postnatal lethality due to severe hypoglycemia and dehydration (84). Thus, we implemented two well-established rescue strategies to investigate postnatal mammary gland development: orthotopic transplantation and renal capsule grafting (316, 317). The orthotopic transplantation paradigm examines if epithelia are capable of growing and invading the wild type stroma in response to the pubertal and post-pubertal hormonal milieu of the

79 recipient. Mammary anlagen were collected from E14 Foxa1+/+ or Foxa1-/- embryos and inserted into the cleared fat pads of wild type syngeneic recipient females (Figure 2.3).

Transplanted glands were retrieved from recipient mice 5 and 8 weeks later (Figure

2.3A,B). In addition, a subset of the 8-week recipients was mated, and transplanted glands were collected from mice with a verified pregnancy (Figure 2.3C). At 5 weeks post-transplant, 50% (3/6) of the wild type anlagen formed mammary ductal outgrowths.

This take-rate is consistent with previous reports using this approach (321, 322). In contrast to the wild type donor glands, no detectable outgrowths occurred from Foxa1-/- anlagen (0/5). At 8 weeks post-transplant, 59% (10/17) of the wild type versus 0% (0/9) of the Foxa1-/- anlagen formed mammary ductal outgrowths. Even when exposed to pregnancy-associated hormones, all of the Foxa1-/- mammary anlagen failed (0/9) to develop while 71% (5/7) of the wild type anlagen formed outgrowths with extensive alveoli. Although FOXA1 is not necessary for formation of the primordial ductal tree at postnatal day 1 (Figure 2.2), these results reveal that the presence of FOXA1 in the mammary epithelium is essential for the ductal outgrowth that occurs with puberty.

The complete absence of ductal outgrowth in transplanted glands from Foxa1-/- mice could be due either to an invasion defect of the developing gland, or a loss of epithelial cells from the transplanted embryonic anlagen. To test this directly, we utilized renal capsule grafting of postnatal day 1 glands (317). This approach maintains the endogenous epithelial-stromal architecture, rather than requiring the formation of a tissue recombinant. In the case of Foxa1-/- glands, the intact mammary rudiment/fat pad serves as an ideal source of donor tissue because the glands have a normal rudimentary ductal

80 structure postnatally (Figure 2.2). The grafts were harvested 2 weeks after transplantation, and whole mount analysis revealed numerous TEBs actively invading the fat pad within wild type glands (Figure 2.4A). In contrast, TEBs are not present in Foxa1 null glands. Rather, the rudimentary ducts have not extended into the fat pad. Hence, loss of Foxa1 leads to failed development of TEBs and a subsequent inability to invade the mammary fat pad in response to an adult milieu of mammogenic hormones. Whole mount analysis at 4-5 weeks post-transplant demonstrates that even after wild type glands have completed invasion of the fat pad, Foxa1-/- glands remain severely dysmorphic

(Figure 2.4B).

The TEB contains a high frequency of luminal progenitors (323) and FOXA1 is expressed in a subset of the body cells in this structure (Figure 2.1A). To determine if

Foxa1 is expressed specifically within luminal progenitors, we compared expression levels between sorted epithelial cell populations enriched for normal mammary stem cell

(MaSC), CD61-positive luminal progenitor, and mature luminal epithelial populations

(Figure 2.4C) (144). When compared to the MaSC-enriched population, Foxa1 mRNA expression is increased in the luminal progenitor population, and is further increased in mature luminal cells. These results, in combination with the pattern of expression of

FOXA1 in the TEB, suggest that FOXA1 may contribute to specification of the luminal lineage. To test this directly, we analyzed the expression of proteins that distinguish luminal [E-cadherin and cytokeratin-8 (CK8)] and basal/myoepithelial [α-smooth muscle actin (α-SMA)] lineages (Figure 2.5). Expression and localization of CK8, E-cadherin and α-SMA are unaltered in renal grafts of Foxa1-/- glands harvested at 4-5 weeks post-

81 transplantation. Both lineages were also observed in Foxa1-/- renal transplanted glands harvested at 2 weeks post-transplant and during pregnancy (data not shown). Hence,

FOXA1 is not necessary for lineage specification, but is essential for expansion and invasion of ductal cells.

Due to the blockade of ductal invasion observed in the complete absence of Foxa1, we also investigated whether Foxa1 displays . Unlike Foxa1-/- mice,

Foxa1+/- mice are viable precluding the need for additional transplantation. Mammary gland development was analyzed at both mid-puberty (5 weeks) and late-puberty (7 weeks) and a significant decrease in ductal invasion was observed at both time points

(Figure 2.6A-D). Ovariectomy and estradiol plus progesterone (E+P) replacement did not rescue this defect (Figure 2.6E,F), indicating that it is not due to an ovarian steroid deficiency. Foxa1+/- mice are capable of lactating (data not shown), thus loss of a single allele delays, but does not prevent, mammary gland development. Growth inhibition was associated with an increase in epithelial apoptosis without a change in proliferation

(Figure 2.6G-J). These data suggest that the expression level of FOXA1 may be critical for ductal expansion and invasion due to its regulation of ductal cell survival.

Alveologenesis is independent of FOXA1

The mammary luminal lineage terminally differentiates into secretory lobulo-alveolar cells during pregnancy and lactation. To determine if FOXA1 is necessary for alveolar differentiation, recipients of renal capsule grafts were mated at 4-5 weeks post- transplantation, and transplanted glands were harvested at 18.5 dpc. Alveoli fill the fat

82 pads of wild type glands, whereas Foxa1-/- glands remain severely hampered from invading the surrounding stroma (Figure 2.7A). However, histological evaluation of epithelium in Foxa1-/- glands revealed the presence of alveoli immediately surrounding the truncated ducts that contained lipid droplets indistinguishable from wild type controls

(Figure 2.7A). Expression of milk protein was confirmed in both wild type and Foxa1-/- glands indicating that the stunted, non-invaded Foxa1-/- glands can undergo terminal differentiation (Figure 2.7B). Consistent with these data, the E+P treated Foxa1+/- mammary glands have increased alveoli when compared to Foxa1+/+ controls (Figure

2.6E,F), suggesting that suppression of FOXA1 may promote alveologenesis.

FOXA1 is required for ERα expression in the mammary epithelium

The inability of the Foxa1-/- glands to properly invade the mammary fat pad is a phenocopy of the ERα-knockout (ERKO) mouse (144, 159, 163, 164, 314) and FOXA1 expression co-localizes with ERα in ~30% of luminal cells, suggesting that Foxa1 may be epistatic with Esr1 within luminal cells. Analysis of ERα expression in renal transplanted mammary glands revealed that ERα is undetectable within the epithelium of

Foxa1-/- glands (Figure 2.8A). In contrast, ERα is highly expressed in the epithelium of wild type transplanted controls and in the stromal population of both wild type and

Foxa1-/- glands. To confirm that ERα activity is lost in Foxa1-/- glands, we assessed expression of the progesterone receptor (PR), an established transcriptional target of ERα

(324). Similar to ERα, PR expression is undetectable in Foxa1-/- epithelium, while being maintained in wild type glands and Foxa1-/- stroma (Figure 2.8A). PR mRNA (Pgr) is similarly decreased (Figure 2.8B). These data indicate that FOXA1 is necessary for ERα

83 and PR expression and that this requirement is epithelial-specific. The percentage of epithelial cells expressing ERα and PR was unchanged in pubertal Foxa1+/- versus

Foxa1+/+ control glands, (51 ± 6% v. 51 ± 2%, 48 ± 3% v. 47 ± 2%, respectively; n = 3-4 per group), indicating that retention of one FOXA1 allele is sufficient to maintain the percentage of cells expressing these receptors.

To verify that FOXA1 is upstream of ERα during normal mammary gland development, we analyzed FOXA1 expression in ERα knockout (Ex3αERKO) mice. These mice are devoid of all ERα transcriptional activity as a result of genomic deletion of exon 3, the coding region for the DNA binding domain, in Esr1. FOXA1 mRNA and protein levels are maintained in Ex3αERKO mammary glands compared to wild type controls (Figure

2.8C,D). Combined, these results indicate that FOXA1 functions upstream, and is necessary for, ERα expression in the normal mammary gland.

FOXA1, ERα and GATA-3 have been proposed to collaborate during mammary morphogenesis (314), thus we also evaluated Gata-3 expression in the absence of

FOXA1. We found no significant change in Gata-3 mRNA in Foxa1-/- glands (Figure

2.8B) indicating that FOXA1 is not required for Gata-3 transcription in the mammary gland. In addition, the presence of Gata-3, but absence of ERα in Foxa1-/- epithelium suggests that, in contrast to breast cancer cells (312), transcription of Gata-3 in normal mammary epithelium may be independent of ERα. This dichotomy was further confirmed by the sustained expression of Gata-3 mRNA in Ex3αERKO mammary glands (Figure

2.8E). We also evaluated whether GATA-3 regulates expression of FOXA1 using

84 mammary glands deficient for Gata-3 (MMTV-Cre;Gata-3f/f) (144). FOXA1 expressing cells in MMTV-Cre;Gata-3f/f (null) versus Gata-3+/f (intact) controls were indistinguishable (Figure 2.8F).

FOXA1 regulates transcription of ESR1

Loss of ERα in Foxa1 null mammary glands could be due either to a loss of FOXA1/ERα expressing cells, or to a requirement for FOXA1 to induce expression of ERα. To determine if FOXA1 regulates expression of ERα, we silenced FOXA1 expression and assessed the impact on ERα mRNA and protein expression in MCF7 (Figure 2.9A-C) and T47D (Figure 2.10) breast cancer cell lines, both of which endogenously express

FOXA1 and ERα (307). Transient knockdown of FOXA1 resulted in a significant reduction in ERα protein levels in both cell lines (Figure 2.9A,B; Figure 2.10A,B), recapitulating the loss of ERα in Foxa1 null mammary glands. ESR1 mRNA levels were also significantly decreased, suggesting that FOXA1 may regulate its transcription

(Figure 2.9C; Figure 2.10C). Importantly, knockdown of FOXA1 in MCF7 cells did not affect GATA-3 mRNA or protein levels (data not shown and Figure 2.9A) providing additional evidence that FOXA1 is not required for GATA-3 expression.

To investigate the mechanism underlying regulation of ERα by FOXA1, we queried a publicly available dataset of genome-wide FOXA1 binding sites in MCF7 cells (201).

This dataset indicates that FOXA1 binds to ten distinct regions of the ESR1 gene, with five sites in the promoter and five in intragenic regions (Figure 2.9D). We then confirmed FOXA1 binding to one of these predicted regions within the ESR1 proximal

85 promoter through chromatin immunoprecipitation (ChIP) followed by site-directed PCR.

FOXA1 binds to this region independently of estradiol treatment (Figure 2.9E). To ascertain whether FOXA1 regulates transcription of ESR1, we examined binding of RNA polymerase II to the ESR1 proximal promoter following transient knockdown of FOXA1

(Figure 2.9F). Silencing FOXA1 reduces RNA polymerase II binding by ~50%, which is comparable to the reduction in ESR1 mRNA levels after FOXA1 knockdown (Figure

2.9C). Combined, these data reveal a previously unrecognized requirement for FOXA1 in regulating ERα expression, suggesting that FOXA1 may directly regulate ESR1 although these experiments do not rule out an indirect effect of FOXA1 on ESR1 transcription.

86 2.5 DISCUSSION

FOXA1 is critical for both ERα expression and functional activity

Previous studies using breast cancer cells revealed that FOXA1 is required for ERα binding to target gene promoters, and subsequent estrogen responsiveness (277, 278). We predicted that FOXA1 may function similarly during mammary morphogenesis. We found that FOXA1 and ERα follow identical expression patterns throughout normal development, and are co-expressed in luminal epithelial cells. Our studies also revealed that FOXA1 is unnecessary for embryonic development of the mammary rudiment, but is required for mammary ductal invasion in three different models: orthotopic and renal capsule transplantation, and Foxa1 heterozygous null mice. The absence of TEBs in renal transplanted Foxa1 null glands, along with the presence of Foxa1 in the luminal progenitor population, indicates that FOXA1 is essential for ductal lineage expansion and morphogenesis (see model, Figure 2.11). The loss of epithelial ERα in Foxa1 null glands provides a specific mechanism for this phenotype because ERα is also essential for TEB development and ductal morphogenesis (159, 163, 164).

Depletion of epithelial ERα with deficiency of Foxa1 could have been due to regulation of ERα expression by FOXA1 or a loss of differentiated cells that can express ERα, as seen in GATA-3 depleted mammary glands (144, 314). Complimenting our observations in the developing mammary gland, transient suppression of FOXA1 results in decreased transcription of ESR1 and protein expression of ERα in breast cancer cells. Hence,

FOXA1 not only mediates ERα activity as has been described (277, 278), but is also

87 essential for sustained ERα expression. These data reveal that FOXA1 tightly regulates

ERα activity through two distinct mechanisms, i.e. basal expression and functional activity.

Previous reports examining a role for FOXA1 in mediating ERα binding to target gene promoters did not observe a decrease in ERα expression upon transient knockdown of

FOXA1 (277, 278, 287). The disparity between these results may be explained by variation in experimental conditions. For the studies reported herein, changes in ERα in response to transient knockdown of FOXA1 were observed using media containing hormone-replete serum. In contrast, previous studies where sustained ERα occurred following FOXA1 silencing were performed under hormone-deprivation. The presence of estradiol substantially decreases the stability of ERα mRNA and protein (325). Thus, the experimental paradigm used herein likely maintains a higher turnover rate of ERα mRNA and protein, and thus, are permissive to detecting changes in expression as a result of

FOXA1 silencing.

Expression of GATA-3 is independent of FOXA1 and ERα

FOXA1, ERα and GATA-3 are positively correlated in breast cancer, and ERα appears necessary for GATA-3 expression in breast cancer cell lines (312). However, Gata-3 expression is sustained in the Foxa1 null mammary glands that also lack detectable ERα.

In addition, Gata-3 mRNA is maintained in mammary glands that lack functional ERα providing further evidence that ERα is not necessary for Gata-3 expression in normal mammary epithelium. These data reveal that expression of GATA-3 occurs

88 independently of FOXA1 and ERα during lineage specification. We confirmed these data by silencing FOXA1 in vitro and found that GATA-3 remains constant even with a reduction in ERα. These results contrast with previous analyses of breast cancer cell lines

(312). To reconcile these data, we propose that while ERα may not be required for

GATA-3 expression under normal conditions, it may become necessary during tumorigenesis. It is also important to note that the FOXA1 knockdown in breast cancer cells presented herein resulted in only a 50% reduction in ERα expression, which may be sufficient to sustain GATA-3.

It has also been suggested that GATA-3 regulates ERα expression in breast cancer cell lines (312). While our results do not refute this conclusion, they do indicate that GATA-3 alone is insufficient to maintain ERα in the absence of FOXA1. This conclusion stems from the loss of ERα, but not GATA-3 that occurs both in Foxa1 null glands and with transient silencing in breast cancer cells. Lastly, GATA-3 was previously reported to bind to the Foxa1 promoter in primary mammary cells (314) and induce expression of FOXA1 in mammary tumors (146) and a kidney cell line (315). We found that FOXA1 expression is maintained in glands deficient for Gata-3, indicating that GATA-3 is not necessary for

FOXA1 expression during normal development.

Development of the mammary ductal, but not alveolar lineage is dependent on FOXA1

Both orthotopic and renal transplantation models used herein revealed that Foxa1 null glands were unable to invade the mammary fat pad in response to pregnancy-associated hormones. However, the rudimentary ductal epithelium that was grafted into the renal

89 capsule developed differentiated alveoli in response to pregnancy. While these alveoli arose from a rudimentary duct and were substantially fewer in number, they were otherwise indistinguishable from wild type glands. These data reveal that Foxa1 is unnecessary for lobulo-alveolar lineage specification (see model, Figure 2.11) and provide additional evidence that ductal expansion and alveolar lineage specification are independent processes. A similar phenotype has been observed in murine mammary glands lacking amphiregulin (326), ErbB3 (327) and FGFR2b (328), or in glands exposed to exogenous TGF-β1 (329, 330). Interestingly, TEB development is also disrupted in all of these models. Thus, it is possible that FOXA1 participates in a signaling network that includes one or more of these mediators of and cancer progression

(331-333).

Previous studies have shown that ERα and PR are independently required for alveologenesis (137, 163, 164). Thus, the loss of ERα and PR in Foxa1 null glands along with the sustained ability to form alveoli was unanticipated. A trivial explanation for these data is that although we cannot detect ERα and PR by IHC, low levels still occur and are sufficiently functional. Supporting this notion, Pgr mRNA is still present, albeit only at ~10% of normal levels. Like FOXA1, ERα is not expressed in lobulo-alveoli.

Thus it is not clear whether ERα acts in a cell-autonomous manner to regulate alveologenesis, or if intercellular communication or lineage progression involving ERα silencing is involved (broken arrow in Figure 2.11). It is also possible that FOXA1 maintains the ductal epithelium in an undifferentiated state, thus inhibiting alveologenesis. The loss of FOXA1 could then induce alveolar differentiation in response

90 to pregnancy-associated hormones even in the absence of ERα. This hypothesis is supported by the enhanced alveologenesis observed in Foxa1 heterozygotes when treated with pregnancy-level hormones. Notably, both orthotopic and renal capsule transplantation models preclude investigating lactational differentiation in detail because the transplanted glands undergo involution post-partum due to the lack of suckling (334).

Hence, conditional knockout of Foxa1 is necessary to directly examine the function of

FOXA1 in lactation and involution and these studies are currently underway.

Conclusions and Implications

FOXA1 is critical for development and specification of cell fate in the prostate, liver, kidney, pancreas and lung (84, 85, 93, 98, 122). We now describe an indispensable role for FOXA1 in mammary ductal morphogenesis (Figure 2.11). Our studies also reveal that FOXA1 is necessary for expression of ERα in the normal mammary epithelium, and modulates transcription of ESR1 in vitro. Approximately 75% of breast cancers are ERα- positive, hence these findings have implications in hormone receptor positive disease because FOXA1 expression occurs in most, if not all ERα-positive breast cancers. It is likely that the positive correlation seen between FOXA1 and the differentiated luminal breast tumor subtype stems from this previously undefined role of FOXA1 in regulating the differentiation of the mammary ductal lineage and controlling ESR1 transcription. We also suggest that FOXA1 may also modulate other well-known pathways of tumorigenesis (e.g. amphiregulin-EGFR, heregulin-ErbB3, TGF-β1) providing a possible explanation and function for FOXA1 in breast tumors lacking ERα.

91 2.6 ACKNOWLEDGMENTS

We thank Kay-Uwe Wagner and Jennifer Yori for instruction in performing orthotopic transplants, Marie-Liesse Asselin-Labat for assistance with Gata-3 deficient mice and

Yanduan Hu for technical support for the ChIP analyses. Paraffin embedding and sectioning was performed by the Case Western Reserve University Tissue Procurement and Histology Core Facility. The CK8 antibody was developed by Philippe Brulet and

Rolf Kemler, and obtained from the Developmental Studies Hybridoma Bank formed under the auspices of the NICHD and maintained by the University of Iowa, Department of Biological Sciences, Iowa City, IA 52242. G.M.B. is supported through a pre-doctoral fellowship from the Department of Defense (DoD) (W81XWH-06-1-0712). This work was also supported by the Case Western Reserve University Comprehensive Cancer

Center (P30 CA043703), a University Hospitals of Cleveland Research

Associates Grant (F.W.A. and R.A.K.), the Division of Intramural Research of the

National Institute of Environmental Health Sciences/NIH (S.C.H and K.S.K), an Ohio

Innovation Incentive Fellowship (J.D.Mosley.), the NIH (T32-GM0720, J.D.Mosley; P01

DK049210, K.H.K.; R01 CA090398, R.A.K), a Fox Chase Cancer Center Support Grant

(P30 CA006927, A.K.G.), the National Health and Medical Research Council of

Australia and the DoD (W81XWH-08-1-0347, R.A.K.).

92 Figure 2.1

FOXA1 is expressed in the developing mammary gland in conjunction with ERα.

(A) Representative images of FOXA1 and ERα IHC in virgin TEBs (5 week old), virgin ductal epithelium (8 week old), virgin alveoli (20 week old), pregnant alveoli (day 18), lactating alveoli (day 2) and an involuting gland (day 5). Within the TEB, arrows mark the luminal progenitor cells and arrowheads mark the basal/myoepithelial progenitors.

Empty arrowheads indicate expressing cells in the pregnant alveoli and involuting gland.

FOXA1 and ERα expression (brown nuclei) is counterstained with hematoxylin. Scale bars = 20 µm. (B) Representative image of dual-IF for FOXA1 and ERα in virgin ductal epithelium (n = 4). The luminal epithelium consists of four populations: cells co- expressing FOXA1 and ERα (31.8% ± 4.4) (yellow cells in “Merge”), expressing

FOXA1 (12.1% ± 5.0) or ERα (3.8% ± 0.6) alone (arrowheads), or expressing neither

(52.3% ± 6.8). Scale bars = 20 µm.

93 Figure 2.1

94 Figure 2.2

FOXA1 is not necessary for embryonic development of the mammary gland.

(A) Representative whole mounts of postnatal day 1 Foxa1+/+ and Foxa1-/- mammary glands (LN = Lymph Node). Scale bars = 0.5 mm. (B) Quantification of the number of terminal ducts and total ductal area. Values represent the average ± S.D. (+/+, n = 4; -/-, n

= 3) (NS = not significant).

95 Figure 2.2

96 Figure 2.3

FOXA1 is required for mammary ductal outgrowth in an orthotopic transplantation model.

(A-C) Representative whole mounts of ductal outgrowths arising from mammary anlagen collected from E14 Foxa1+/+ and Foxa1-/- mice and transplanted into cleared fat pads of

3-4 week old syngeneic C57BL/6 recipients. (A) Recipients aged 5 weeks post- transplant. (B) Recipients aged 8 weeks post-transplant. (C) Recipients aged 8 weeks post-transplant with subsequent pregnancy (18.5 dpc). Epidermal cysts (*) form as a result of co-transplantation of hair follicles along with the mammary gland. Scale bars =

2 mm. The number and percentage of mammary outgrowths for each donor genotype is indicated.

97 Figure 2.3

98 Figure 2.4

FOXA1 is required for TEB formation and ductal invasion.

(A-B) Representative whole mounts of renal grafts of Foxa1+/+ and Foxa1-/- mammary glands (into wild type C57BL/6 recipients) harvested at (A) 2 weeks (+/+, n = 4; -/-, n =

3) and (B) 4-5 weeks post-transplantation (+/+, n = 4; -/-, n = 4). Dashed lines outline the mammary fat pad. Scale bars = 1 mm. (C) Quantitative real-time PCR of Foxa1 mRNA levels in the MaSC-enriched population (CD24+/CD29hi), the luminal progenitor population (CD24+/CD29lo/CD61+), and the mature luminal population

(CD24+/CD29lo/CD61-) isolated from wild type FVB/N inguinal mammary glands (n =

10 per independent experiment). The results of two independent cell sorting experiments are shown. Values were normalized to 18S rRNA (Exp#1) or Gapdh mRNA (Exp#2) and then expressed relative to the values obtained with the mature luminal population.

99 Figure 2.4

100 Figure 2.5

FOXA1 is not required for luminal or basal/myoepithelial lineage specification.

Representative images of FOXA1, E-cadherin, CK8 and α-SMA IHC (brown) in renal grafts from Foxa1+/+ and Foxa1-/- mammary glands harvested 4-5 weeks post- transplantation (+/+, n = 3; -/-, n = 3). All sections were counterstained with hematoxylin.

Scale bars = 20 µm.

101 Figure 2.5

102 Figure 2.6

Pubertal mice heterozygous for the Foxa1 null allele display decreased mammary ductal invasion.

(A) Representative whole mounts of mammary glands from 5-week old Foxa1+/+ and

Foxa1+/- mice. Scale bars = 2 mm. (B) Glands from 5-week old Foxa1+/- mice have significantly decreased ductal length compared to wild type glands. Values represent the average ± S.D. (+/+, n = 6; +/-, n = 6; *p<0.01). (C) Representative whole mounts of mammary glands from 7-week old Foxa1+/+ and Foxa1+/- mice. Scale bars = 2 mm. (D)

Glands from 7-week old Foxa1+/- mice have significantly decreased ductal length. Values represent the average ± S.D. (+/+, n = 8; +/-, n = 10; *p<0.05). (E) Representative whole mounts of mammary glands from Foxa1+/+ and Foxa1+/- mice ovariectomized at 4 weeks of age and treated s.c. with 17β-estradiol (1 µg) and progesterone (500 µg) daily for 3 weeks. Scale bars = 2 mm. (F) Treated Foxa1+/- glands have significantly decreased ductal length. Values represent the average ± S.D. (+/+, n = 4; +/-, n = 4; *p<0.05). (G)

Representative sections of glands from 5-week old mice analyzed by TUNEL (brown).

Scale bars = 20 µm. (J) Mammary glands from 5-week old Foxa1+/- mice have significantly increased TUNEL positivity. Values represent the average ± S.D. (+/+, n =

4; +/-, n = 3; *p<0.01). (I) Representative sections of glands from 5-week old mice stained for BrdU (brown). Scale bars = 20 µm. (J) Quantification of BrdU positivity.

Values represent the average ± S.D. (+/+, n = 4; +/-, n = 3; NS = not significant).

103 Figure 2.6

104 Figure 2.7

FOXA1 is not required for alveolar differentiation during pregnancy.

(A) Representative whole mounts and H&E stained sections (+/+, n = 5; -/-, n = 3; scale bars = 0.5 mm) and (B) images of milk protein IHC (brown) (+/+, n = 3; -/-, n = 3; scale bars = 20 µm) in renal grafts from Foxa1+/+ and Foxa1-/- mammary glands harvested 4-5 weeks after transplantation and during late pregnancy (18.5 dpc). Sections were counterstained with hematoxylin.

105 Figure 2.7

106 Figure 2.8

FOXA1 is required for expression of ERα in the normal mammary gland.

(A) Representative images of ERα and PR IHC (brown nuclei) in renal grafts from

Foxa1+/+ and Foxa1-/- mammary glands harvested 4-5 weeks post-transplantation (+/+, n

= 3; -/-, n = 3). ERα and PR are maintained in the stroma of Foxa1-/- glands (arrows). (B)

Foxa1, Pgr and Gata-3 mRNA levels in renal transplanted Foxa1+/+ and Foxa1-/- mammary glands. Values represent the average ± S.D. and are relative to CK8 mRNA

(+/+, n = 3; -/-, n = 3; *p<0.01; NS = Not significant). (C) Quantitation of Foxa1 mRNA and (D) representative images of FOXA1 IHC (brown) in wild type and Ex3αERKO mammary glands. Values represent the average ± S.D. and are relative to CK8 mRNA

(Wild type, n = 3; Ex3αERKO, n = 3; NS = not significant). (E) Gata-3 mRNA levels in wild type and Ex3αERKO mammary glands. Values represent the average ± S.D. and are relative to CK8 mRNA (Wild type, n = 3; Ex3αERKO, n = 3; NS = not significant). (F)

Representative images of FOXA1 IHC in Gata-3+/f and MMTV-cre;Gata-3f/f mammary glands (+/f, n = 3; f/f, n = 3). IHC quantification is depicted in the bottom right corner of each image. All sections were counterstained with hematoxylin. Scale bars = 20 µm.

107 Figure 2.8

108 Figure 2.9

FOXA1 regulates transcription of ESR1.

(A-C) MCF7 cells were transiently transfected with non-targeting (NT) or two different siRNAs targeting FOXA1 (si#1 and si#4). (A) Representative immunoblots of FOXA1,

ERα and GATA-3 (n = 3) (* = mutant form of GATA-3 (315)). (B) Quantitation of ERα protein levels. Bars represent the mean of three experiments ± S.D. (*p<0.01;

**p<0.005). (C) Quantitation of ESR1 mRNA levels. Bars represent the mean of three experiments ± S.D. relative to GAPDH mRNA (*p<0.005). (D) ESR1 is comprised of eight exons and at least seven promoters (only A and F are shown) (325). Regions previously identified to bind FOXA1 by ChIP-chip are indicated by black boxes (201).

(E) Representative (n = 3) FOXA1 ChIP of the ESR1 promoter using primers amplifying a predicted (* in D). MCF7 cells were treated with and without 17β-estradiol

(E2). (F) MCF7 cells were transiently transfected with NT or FOXA1 si#1. Quantification of RNA Polymerase II ChIP of the ESR1 promoter (n = 3). Bars represent the average fold change relative to input and normalized relative to NT ± S.D. (*p<0.0005).

109 Figure 2.9

110 Figure 2.10

FOXA1 regulates transcription of ESR1 in T47D cells.

(A-C) T47D cells were transiently transfected with non-targeting (NT) or siRNA targeting FOXA1 (si#4). (A) Representative immunoblots of FOXA1 and ERα protein levels (n = 3). (B) Quantitation of ERα protein levels represented as the average of three experiments ± S.D. (*p<0.05). (C) Quantitative real-time PCR of ESR1 mRNA levels represented as the average of three experiments ± S.D. relative to GAPDH mRNA

(*p<0.001).

111 Figure 2.10

112 Figure 2.11

Schematic of the mammary epithelial cell hierarchy

FOXA1 is expressed in and required for ductal development. GATA-3 is expressed in and required for both ductal and alveolar development and is independent of FOXA1 expression. ERα is required for ductal and alveolar development, but is only expressed in ductal cells. This supports intercellular communication and/or lineage progression from

ERα-positive ductal to ERα-negative alveolar cells (broken arrow).

113 Figure 2.11

114 CHAPTER 3

FOXA1 Actively Represses the Molecular Phenotype of Basal Breast Cancers

Adapted from manuscript in submission

by

Gina M. Bernardo, Gurkan Bebek, Charles L. Ginther, Steven T. Sizemore, Kristen L.

Lozada, John D. Miedler, Lee A. Anderson, Andrew K. Godwin, Fadi W. Abdul-Karim,

Dennis J. Slamon & Ruth A. Keri

3.1 ABSTRACT

Breast cancer is a heterogeneous disease comprised of at least five major subtypes.

Luminal subtype tumors confer a more favorable patient prognosis, which is in part, attributed to the estrogen receptor-α (ERα) positivity and anti-hormone responsiveness of these tumors. Expression of the forkhead box transcription factor, FOXA1, also correlates with the luminal subtype and patient survival, but is present in a subset of ERα-negative tumors. Similarly, FOXA1 is consistently expressed in luminal breast cancer cell lines even in the absence of ERα. In contrast, basal breast cancer cell lines do not express

FOXA1, and loss of FOXA1 in luminal cells increases migration and invasion, characteristics of the basal subtype. To delineate an ERα-independent role for FOXA1 in maintaining the luminal phenotype, and hence a more favorable prognosis, we performed cDNA microarray analyses on FOXA1-positive, ERα-positive (MCF7, T47D) and

FOXA1-positive, ERα-negative (MDA-MB-453, SKBR3) cell lines in the presence or absence of transient FOXA1 silencing. This resulted in three FOXA1 transcriptomes: (1) a luminal-signature (consistent across cell lines), (2) an ERα-positive signature (restricted

115 to MCF7 and T47D) and (3) an ERα-negative signature (restricted to MDA-MB-453 and

SKBR3). Use of Gene Set Enrichment Analyses (GSEA) as a phenotyping tool revealed that FOXA1 silencing resulted in a transcriptome shift from luminal to basal gene expression signatures. FOXA1 binds to both luminal and basal genes within luminal breast cancer cells, suggesting that it not only transactivates luminal genes, but also represses basal-associated genes. From these results we conclude that FOXA1 controls plasticity between basal and luminal cells, playing a dominant role in repressing the basal phenotype, and thus tumor aggressiveness, in luminal breast cancer cells. Although it has been proposed that FOXA1-targeting agents may be useful for treating luminal tumors, these data suggest that this approach may promote transitions toward a more aggressive cancer.

116 3.2 INTRODUCTION

Breast cancer is a heterogeneous disease, which includes at least six major molecular subtypes: luminal A, luminal B, luminal C, HER2, basal-like and normal-like. These subtypes were identified through unsupervised hierarchical clustering of human tumor gene expression data, and are predictive of patient prognosis (141, 142). Specifically, tumors of the luminal A subtype are estrogen receptor-α (ERα) positive, and patients with this disease have a more favorable prognosis, which is at least partially due the efficacy of anti-hormone therapies (222). Conversely, patients with tumors of the basal subtype have a poorer prognosis due to the intrinsic resistance of these tumors to standard chemotherapeutics and the lack of targeted therapies (249). These discrete subtypes of breast cancer were named luminal versus basal based on the similarity of the tumor gene expression profile with that of the normal luminal and basal epithelial populations of the breast (141). It has been proposed that these different signatures are either reflective of the cell of origin of the individual tumor types, or that oncogenic events induce transcriptomes that mimic those of distinct cell lineages (335). While basal tumors were originally postulated to arise from the basal lineage, more recent evidence suggests that basal breast cancer cells are actually more similar to the normal luminal progenitor population (149, 150). These findings suggest that either basal tumors initiate within luminal progenitor cells, or that a cancerous population of luminal progenitor cells is capable of de-differentiating resulting in a basal-like phenotype. Moreover, these data raise the possibility that exists between both normal and tumorigenic basal, luminal progenitor and mature luminal populations. Of note, the molecular phenotype of breast cancer cell lines has also been characterized using gene

117 expression analyses, resulting in the generation of classifier gene lists for luminal and basal subtypes (336, 337), which provide useful tools for examining the extent of plasticity that may exist in breast cancer subtypes.

In addition to ESR1, the mRNA encoding ERα, FOXA1 is specifically expressed in luminal subtype tumors (141, 142). Tissue microarray studies have revealed that FOXA1 protein levels also associate with breast cancer patient survival and are correlated with

ERα expression (263-267, 269). Furthermore, FOXA1 expression correlates with the luminal subtype as defined by ERα and/or PR positivity, HER2 negativity, or luminal- specific markers (e.g. E-cadherin or cytokeratin 18) (264-267, 269). The co-expression of

FOXA1 and ERα in human tumors spawned in vitro analyses investigating the putative functional cooperation of these transcriptional regulators in breast cancer cell lines. These initial studies revealed that regions of genes containing estrogen response elements

(EREs) are enriched for forkhead DNA consensus motifs, and that FOXA1 is necessary for estrogen-induced ERα binding to target genes and subsequent transcriptional activation (277, 278). Given the previously described role for FOXA1 as a chromatin- remodeling factor (10-12), it has been proposed that FOXA1 is required to prime chromatin for subsequent ERα binding. Functioning in this context, FOXA1 modulates both estrogen-induced ERα transcriptional activation and repression (201, 282). In addition to being necessary for ERα activity, FOXA1 also regulates the expression of

ERα mRNA and protein in breast cancer cell lines (338). Supporting these in vitro data,

Foxa1 null mammary glands fail to express epithelial ERα and do not invade the mammary fat pad in response to pubertal hormones (338), a phenotype reminiscent of

118 that observed in mice lacking ERα (159, 163, 164). Further supporting their cooperation,

FOXA1 and ERα share an overlapping pattern of expression throughout normal mammary morphogenesis (338).

While the positive correlation and functional collaboration of FOXA1 and ERα in human tumors is well documented, several groups have reported tumors expressing FOXA1 in the absence of ERα (263-267, 269). These data are recapitulated in the normal mammary gland where co-localization studies revealed that a sub-population of mature luminal epithelial cells exists that expresses FOXA1 in the absence of ERα co-expression (338).

Interestingly, decreased FOXA1 expression associates with the survival of ERα-negative patients (267). Moreover, FOXA1 is one of several genes differentially expressed in an

ERα-positive-like, androgen responsive subgroup of breast cancers that lack ERα and

Progesterone Receptor (PR) (271). FOXA1 is required for AR binding to its target genes and promotes the expression of the apocrine signature in these breast cancer cells (274).

FOXA1 has also been implicated in other signaling pathways including HER2 (273, 286) and BRCA1 (307). Combined, these data suggest that in addition to its well-known role as a co-modulator of estrogen-induced transcription, FOXA1 may also maintain the luminal phenotype of breast cancer through ERα-independent mechanisms.

Herein, we confirm the presence of FOXA1 in a subset of ERα-negative breast tumors, and in all cell lines that have been classified as luminal, even in cells that lack ERα.

Utilizing transient siRNA knockdown of FOXA1 in both ERα-positive and ERα-negative luminal cell lines, we define three FOXA1 transcriptional signatures: ERα-positive, ERα-

119 negative and luminal. Within the luminal signature, FOXA1 is not only necessary for the maintenance of luminal-specific gene expression, but also for the repression of genes that are specific to basal breast cancer cells. FOXA1 binds adjacent to, or within a representative subset of both luminal and basal classifier genes. In support of FOXA1 actively repressing basal gene expression, loss of FOXA1 induces the migratory and invasive capacity of luminal cells. Thus, for the first time, we reveal an ERα-independent, luminal-specific function for FOXA1 in maintaining the highly differentiated characteristics of luminal breast cancer cells through transcriptional regulation of both luminal and basal genes.

120 3.3 MATERIALS AND METHODS

Immunohistochemistry. De-identified human breast tumor samples were obtained through the Biosample Repository at Fox Chase Cancer Center, where they were processed and grouped by grade (high vs. low), ERα, PR and HER2 status. Sections were re-hydrated, and antigen retrieval performed using 10 mM sodium citrate (pH = 6) in a pressure cooker (125˚C for 10 minutes; 90˚C for 2 minutes). Sections were blocked with peroxidase blocking reagent (DAKO) and incubated with primary antibody (FOXA1,

Santa Cruz) overnight at 4°C. Secondary detection was performed using the Goat IgG

Vectastain Elite ABC Kit (Vector Laboratories) per manufacturer’s recommendations.

Secondary conjugates were detected using 3,3’-diaminobenzidine (DAKO). Sections were counterstained with Gill’s #3 Hematoxylin (Fisher), dehydrated and mounted.

Scores for FOXA1 IHC were determined by multiplying signal intensity (1 = lowest; 3 = highest) by the percentage of positive cells (1=10%, 2=20%, etc.) as previously described

(264).

Quantitative Real-time PCR. Total RNA was isolated using TRIzol Reagent

(Invitrogen), treated with DNAse I (DNA-free, Ambion), and cDNA produced using

SuperScript II Reverse Transcriptase (Invitrogen). Real-time PCR was performed using

Applied Biosystems TaqMan Gene Expression Assays: ANXA1 = Hs00167549_m1;

CD58 = Hs00156385_m1; ERBB3 = Hs00176538_m1; FNDC3B = Hs00224289_m1;

FOXA1 = Hs00270129_m1; GAPDH = Hs99999905_m1; JAG1 = Hs01070036_m1;

PRNP = Hs00175591_m1; SPDEF = Hs01026048_m1; XBP1 = Hs00231936_m1.

121 Cell Culture and RNA Interference. All cell lines were obtained from ATCC. MCF7 and

MDA-MB-453 cells were grown in DMEM (Mediatech); T47D cells in RPMI 1640

(Gibco); SKBR3 cells in McCoy’s 5A (Sigma). Media was supplemented with 10% FBS and 1% Penicillin-Streptamycin (Invitrogen). Cells were seeded in 100 mm dishes to be

30-50% confluent upon transfection. siRNA targeting firefly luciferase mRNA

(siCONTROL Non-targeting siRNA #2, Dharmacon, herein described as NT) or human

FOXA1 mRNA (siGENOME M-010319-01 and -04, Dharmacon) were transfected in

OPTI-MEM media (Invitrogen) using Lipfectamine 2000 (Invitrogen) to a final concentration of 100 nM. Culture media was changed to complete growth medium after

24 hours. Cells were harvested at varying time points post-transfection as described below for protein or mRNA isolation.

Immunoblots. Cells were lysed (50 mM Tris-HCl, pH7.4; 100 mM NaCl; 1 mM EDTA;

1 mM EGTA; 1 mM NaF; 0.1% SDS; 0.5% Sodium Deoxycholate; 1% Triton-X-100;

10% Glycerol; 2 mM Sodium Orthovanadate; Protease Inhibitor Cocktail (Sigma)), and protein levels quantified (Bradford Assay, Biorad). Protein lysate was resolved using

SDS-PAGE, and transferred to PVDF membrane (BioRad). Blots were blocked (5%- milk-1XPBST) and incubated overnight at 4°C with primary antibody (FOXA1, Santa

Cruz; ERα, Santa Cruz; β-actin, Sigma; Annexin 1, BD Biosciences) diluted in 5%-BSA-

1XPBST. Blots were incubated with an HRP-conjugated secondary antibody (Santa

Cruz) diluted in 5%-milk-1XPBST and developed using ECL western blotting substrate

(Pierce). Quantification of protein levels was determined using Image J (319).

122 Migration and Invasion. MCF7 and MDA-MB-453 cells were transfected with siRNA as described. At 48 hours post-transfection, cells were trypsinized and 50,000 cells were seeded into the top of uncoated transwell inserts (Corning) or matrigel coated invasion chambers (BD Biosciences) in serum free media. Cells were allowed to migrate/invade towards complete serum containing media (10% FBS) for 24 (migration) or 48 (invasion) hours. Migrated/invaded cells were fixed and stained using a DiffQuik staining procedure. Three technical replicates were performed per experiment, where 5-10X fields were quantified per replicate.

cDNA microarrays. MCF7, T47D, SKBR3 and MB-453 cells were harvested at 36 or 72 hours post-transfection with siRNA. Three biological replicates were performed per cell line at the 72-hour time point. Total RNA was isolated using TRIzol Reagent (Invitrogen) and treated with DNAse I (DNA-free, Ambion). RNA quantity was measured using a

NanoDrop ND-1000 Spectrophotometer (NanoDrop Technologies) and by separation via capillary electrophoresis using the Agilent 2000 Bioanalyzer (Agilent Technologies).

Characterization of individual transcripts was performed by comparison of control (non- targeting siRNA) with FOXA1 knockdown (siRNA to FOXA1) samples labeled with

Cy5-UTP or Cy3-UTP using the Agilent Quick Amp Labeling Kit (Agilent

Technologies). Equal amounts of labeled treated and untreated RNA were mixed and hybridized to Agilent Human 44K microarray slides at 65°C for 24 hours. After washing, microarray slides were read using an Agilent Scanner, and Agilent Feature Extraction software v7.5 was used to calculate gene expression values. The feature extracted files were imported into the Rosetta Resolver® system v7.1 for gene expression data analysis

123 (Rosetta Biosoftware). The intensity ratios between the cell line sample and mixed reference calculated for each sequence were computed according to the Agilent error model. A particular sequence was considered differentially expressed if the calculated p- value of change was p≤0.05. Biological replicates were further combined using a ratio- error weighted ANOVA. Venn diagrams were used to compare common gene expression changes between the four cell lines at p≤0.001.

Gene Set Enrichment Analysis (GSEA). The Agilent Human 44K microarray reported log-ratio values of non-targeting versus FOXA1 knockdown expression data. On the other hand, GSEA requires individual data for each phenotype (339). Hence, log- intensities of the array measurements instead of the log-ratios were calculated by individual channel analysis of the two-color Agilent microarrays using R-package Limma

(340). An average microarray measurement across all the cell lines (MCF7, T47D, MB-

453, SKBR3) was generated using Limma. Background correction and normalization was done on the two-color microarray data, and a linear model was fit to the data to acquire average values for each biological replicate. Averages were determined for three biological replicates plus a technical dye reversal replicate for each cell line. Then, the cell line averages were combined to obtain a common average value. The GSEA was conducted on a subset of the classifier genes that were identified in Neve et al. (2006)

(RN) (336) and Charafe-Jauffret et al. (2006) (ECJ) (337) (Tables 3.4 through 3.8).

Enrichment of each classifier gene list was then determined.

124 Chromatin Immunoprecipitation. Identification of DNA regions bound by FOXA1 was completed using the cross-linking (X)-ChIP protocol as detailed by Abcam. Briefly,

MCF7 and MB-453 cells were fixed with formaldehyde and sonicated using a Branson

Digital Sonifier (Model#150). Sonicated lysate was rotated overnight at 4°C with either normal goat IgG (Santa Cruz) or FOXA1 antibody (Abcam, Ab5089), and ChIP-Grade

Protein G Agarose Beads (Cell Signaling). The beads were washed, DNA was eluted and crosslinks reversed overnight at 65°C. Purified DNA was then analyzed by PCR, where binding of FOXA1 was detected using the gene specific primers listed in Table 3.13.

Quantification of precipitated DNA relative to the normal goat IgG control was accomplished using Image J (319).

125 3.4 RESULTS

FOXA1 is expressed in the absence of ERα and correlates with the luminal tumor subtype

Although previous tissue microarray analyses revealed a statistical correlation between

FOXA1 and ERα expression, several groups have also reported that FOXA1 can be expressed in a subset of tumors defined as ERα-negative (263-267, 269). To confirm these results in an independent cohort, we investigated FOXA1 expression in breast cancer specimens using immunohistochemistry (IHC). While FOXA1 is expressed in

100% of ERα-positive tumors, it is also expressed in ~50% of ERα-negative tumors, albeit at statistically decreased levels compared to the ERα-positive cohort (Figure

3.1A,B). We further determined if FOXA1 expression correlates with mammary epithelial phenotype using a panel of breast cancer cell lines previously classified as luminal, basal A or basal B using gene expression signatures (336). Of note, basal A cell lines are more characteristic of human basal-like breast cancers, while basal lines are more mesenchymal and display features of stem cells (336, 337). Similar to breast tumors, FOXA1 is expressed in all luminal cell lines examined. In contrast, cells that are defined by their transcriptome as luminal can lack ERα [MDA-MB-453 (hereafter referred to as MB-453) and SKBR3] (Figure 3.1C,D). These results suggest that FOXA1 more precisely correlates with the luminal subtype than ERα, and may be independently required for maintenance of the luminal tumor phenotype.

The expression pattern we identified for FOXA1 and ERα protein in this subset of cell lines mirrors publically available cDNA microarray expression data for a larger group of

126 breast cancer cell lines (Figure 3.2) (336). These data revealed additional lines (AU565,

HCC202, HCC2185, SUM185PE) (Figure 3.2) that are defined as luminal, but have

ESR1 mRNA levels similar to that of SKBR3 and MB-453 cells, which do not have detectable ERα protein expression (Figure 3.1D). In contrast, none of the luminal cell lines examined to date fail to express FOXA1, further suggesting that FOXA1 may be a critical driver of the luminal phenotype. Notably, of those cell lines that express FOXA1 in the absence of ESR1, the MB-453, SKBR3, HCC202 and AU565 cell lines over- express HER2 (336), a proto-oncogene amplified in ~25-30% of breast cancers. In contrast, the luminal HCC2185, SUM185PE and 600MPE cells do not over-express

HER2 (336), indicating that the presence of FOXA1 in ERα deficient cells is not dependent on constitutively active HER2 signaling. The precise characteristics of

FOXA1 and HER2 co-expressing cells may diverge to some extent from luminal cell lines that fail to over-express HER2, however, all of these cell lines express the luminal signature genes including FOXA1.

FOXA1 represses luminal breast cancer cell aggressiveness

FOXA1 positively correlates with favorable patient prognosis and the less aggressive luminal tumor subtype (263-267, 269). To investigate whether FOXA1 can maintain the less aggressive phenotype of luminal breast cancer cells independently of ERα, we reduced FOXA1 expression in both ERα-positive (MCF7) and ERα-negative (MB-453) cells by transient transfection of non-targeting or FOXA1-targeted siRNAs. We then assessed the impact of FOXA1 silencing on migration and invasion, two hallmarks of highly aggressive breast cancer cells. Both MCF7 and MB-453 cells exhibit increased

127 migratory capacity following FOXA1 silencing compared to the non-targeting control

(Figure 3.3A). Cellular invasion was also increased in MCF7 cells (Figure 3.3B). These results were not secondary to an increase in cell number (Figure 3.3C). Indeed, FOXA1 silencing resulted in a significant decrease in MCF7 cell number, which is consistent with previous reports (278, 286, 287). Overall, these results indicate that FOXA1 actively represses in vitro cellular aggressiveness independently of ERα expression.

Generation of the FOXA1-dependent luminal transcriptome

Studies examining the role of FOXA1 in luminal breast cancer have been primarily restricted to analyzing its participation in the regulation of ERα expression and activity.

To identify the ERα-independent function(s) of FOXA1 in regulating luminal characteristics we utilized genome-wide expression analysis. We silenced FOXA1 in both ERα-positive (MCF7, T47D) and ERα-negative (MB-453, SKBR3) FOXA1- expressing luminal cells, and performed genome-wide microarray analysis at 36 or 72 hours post-transfection with FOXA1 or non-targeting siRNA. After confirming decreased

FOXA1 protein levels, which was on average between 60%-87% reduced (Figure 3.4A), gene expression changes were quantified with Agilent Human 44K microarrays. Three biological replicates were performed at the 72-hour time point, and the expression data from these three replicates was combined through error-weighted ANOVA (schematic in

Figure 3.4B) Gene expression changes (p<0.001) within the combined datasets for each cell line were then compared to generate three distinct FOXA1 transcriptomes (Figure

3.4C): (1) changes specific to the ERα-positive MCF7 and T47D cells, (2) changes specific to the ERα-negative MB-453 and SKBR3 cells, and (3) changes observed in all

128 four luminal lines, which are autonomous to ERα expression (referred to as the “luminal signature”). Gene lists for the luminal signature, both decreased and increased upon

FOXA1 knockdown, are provided (Table 3.1 and 3.2).

Loss of FOXA1 induces a phenotypic shift from luminal to basal transcriptomes

Differentially expressed genes in luminal versus basal breast cancers (141, 142) and cell lines (336, 337) have previously been reported. Our analysis of the FOXA1-associated transcriptomes revealed that loss of FOXA1 in both ERα-positive (MCF7, T47D) and

ERα-negative (MB-453, SKBR3) luminal breast cancer cell lines (i.e. the luminal signature) significantly (p<0.05) induced the expression of a number of previously defined basal A (16.3%) and basal B (10.1%) genes, while concomitantly reducing the expression of a number of luminal (21%) genes (Table 3.3) (336). Notably, no basal A classifying genes were significantly decreased and only ~9% of the luminal signature genes were increased upon FOXA1 knockdown consistently in all four cell lines (data not shown). Ordered heat maps depicting the change in expression of genes comprising classifiers for luminal and basal cells are shown in Figure 3.5 [Luminal and Basal A (top

50 genes) classifiers from Neve, et al. (2006)], Figure 3.6 [Basal A (entire gene set) and

Basal B from Neve, et al. (2006)] and Figure 3.7 [Luminal (B) and Basal classifiers from

Charafe-Jauffret et al. (2006)]. The classifier genes are listed in order of subtype classification power, where more predictive genes are at the top of the heat map, and the less predictive genes are at the bottom.

129 Silencing of FOXA1 decreases the expression of a subset of luminal genes and increases the expression of a subset of basal genes in all four luminal cell lines. To determine if there was a statistically significant shift from luminal to basal gene classifier expression we utilized Gene Set Enrichment Analysis (GSEA). Expression data was compiled from each cell line for GSEA as described in the methods. We used a subset of the classifier lists generated by Neve et al. (2006) (336) to discriminate between the basal A, basal B and luminal subtypes (Table 3.4). As shown in Table 3.3, loss of FOXA1 leads to a decreased enrichment of luminal genes, with a concomitant increase in the enrichment of basal A genes [False Discovery Rate (FDR) q<0.05; Nominal (NOM) p<0.05]. In contrast, there is no change in enrichment of the basal B genes. The combined group of basal A and B genes is also significantly enriched (FDR q<0.05; NOM p<0.05), indicating the core enrichment power of genes encompassing the basal A list. These data confirmed our initial observations that loss of FOXA1 increases the expression of genes associated with the basal A phenotype (16.3%) more so than basal B genes (10.1%)

(Table 3.3). To confirm these findings, we performed a second GSEA using a subset of the classifier lists defined by Charafe-Jauffret et al. (2006) (337) (Tables 3.5-3.8). These genes discriminate between luminal and basal subtypes, without further subdivision of the basal subtype into basal A and basal B. A separate gene set delineates luminal and mesenchymal subtypes. The luminal classifier that discriminates between the luminal and basal subtypes is referred to as luminal (B), whereas the luminal classifier that discriminates between the luminal and mesenchymal subtypes is referred to as luminal

(M). Of note, the mesenchymal gene set is descriptive of a phenotype similar to that of the Neve et al. (2006) basal B gene set (341). Confirming the previous analysis, loss of

130 FOXA1 similarly led to a decrease in the enrichment of the luminal (B) genes, and an increase in the enrichment of the basal genes (FDR q<0.05; NOM p<0.001). Similar to the Neve et al. (2006) basal B gene set, there was no change in the enrichment of the luminal (M) or mesenchymal gene sets (Table 3.9). Enrichment plots for the subtype- defining gene sets are shown in Figures 3.5 and 3.8.

To confirm changes in gene expression observed via microarray analysis, mRNA expression of a representative subset of luminal and basal genes was quantified using

Real-Time RT-PCR (qRT-PCR). The subset of luminal and basal genes selected for confirmation are present in the Neve et al. (2006) luminal or basal A, and Charafe-

Jauffret et al. (2006) luminal (B) or basal classifying lists, respectively. Upon knockdown of FOXA1, expression of the basal genes is significantly increased in each luminal cell line (Figure 3.9). Of note, expression of the selected luminal genes was decreased to a greater extent in ERα-positive cell lines (MCF7, T47D). These genes have previously been shown to be responsive to estradiol (292, 342). To confirm that changes in basal classifier gene activity in response to FOXA1 resulted in increased protein levels, we evaluated the expression of Annexin 1, which was similarly increased in each cell line with FOXA1 silencing (Figure 3.9).

FOXA1 binds to luminal and basal genes in luminal breast cancer cells

FOXA1 binds to DNA, can regulate transcription through chromatin remodeling (10-12), and more recently has been associated with localized DNA hypomethylation (13). Hence, we hypothesized that in addition to its known ability to facilitate estrogen-regulated ERα

131 transactivation and transrepression in breast cancer (201, 277, 278, 282), FOXA1 may also repress basal gene expression through direct DNA binding independent of ERα function. To begin to test this, we utilized a publically available dataset of FOXA1 chromosomal binding locations in MCF7 cells (201). Each basal A gene that was induced and luminal gene that was repressed within the luminal signature (Table 3.3) was investigated in silico for potential FOXA1 binding sites upstream (50kb), intragenically, or downstream (50kb). This analysis revealed binding sites for FOXA1 either within, or adjacent to 53% of basal A and 94% of luminal genes within an ERα-positive breast cancer cell line (Table 3.10). For those genes not bound to FOXA1 within this ChIP-chip dataset, we interrogated the proximal promoter (-1000bp) sequence for FOXA1 consensus elements using the Transcription Element Search System (TESS) (343). To directly assess whether FOXA1 could bind to a subgroup of basal A genes and to three representative luminal genes (ERBB3, SPDEF, XBP1) in FOXA1-positive, ERα-positive

(MCF7) and FOXA1-positive, ERα-negative (MB-453) luminal breast cancer cells, gene- specific ChIP was performed using PCR primers surrounding the proposed binding sites

(Figure 3.10). These experiments revealed that FOXA1 binds to both luminal and basal

A genes independently of ERα co-expression. More specifically, FOXA1 binds to all of the basal A genes tested in MCF7 cells, and to FNDC3B, PAM and TRIM2 in MB-453 cells. The ANXA1 proximal promoter has two predicted FOXA1 consensus motifs,

FOXA1 only binds to one of these regions in MCF7 cells, while binding is minimal to either sequence in the MB-453 cells. These studies also revealed that the relative level of

FOXA1 binding is similar between luminal and basal genes, suggesting that FOXA1 has

132 a direct role in repressing basal signature genes as well as inducing luminal gene expression.

FOXA1 binding to gene enhancer regions was recently shown to correlate with DNA hypomethylation (13). Hence, we postulated that the loss of FOXA1 could alter the methylation status of the basal gene promoters, leading to an increase in the transcription of these genes. To test this, methylated DNA was immunoprecipated from cells following

FOXA1 silencing and analyzed on a methylation-specific promoter microarray (Agilent

Human CpG Island Array #G4492A). These experiments were done in duplicate in ERα- positive (MCF7) and ERα-negative (SKBR3) cells, and cells were harvested at 72 hours post-transfection. Knockdown of FOXA1 did not affect either genome wide promoter methylation, or the methylation status of the basal gene promoters bound by FOXA1 in

Figure 3.10 (data not shown). These data indicate that while FOXA1 represses expression of a subset of basal genes, it does not alter the DNA methylation status of their promoters. Further studies are necessary to determine if FOXA1 regulates the methylation status of putative enhancer regions surrounding the basal genes.

133 3.5 DISCUSSION

FOXA1 actively represses basal breast cancer genes

It is well established that FOXA1 is necessary for estrogen-induced ERα binding and subsequent transcriptional activation of luminal genes in breast cancer cells (201, 277,

278, 282, 287). These analyses were performed in ERα-positive cell lines in the presence or absence of estrogens. This co-modulatory role between FOXA1 and ERα has also been reported for estrogen-mediated transcriptional repression (201, 282), and has been specifically demonstrated for the repression of RPRM, the gene encoding Reprimo (284).

In this context, FOXA1 and ERα form a tripartite complex with Histone Deacetylase 7

(HDAC7) that silences RPRM transcription independently of histone deacetylase activity.

Interestingly, FOXA1 does not bind to the RPRM promoter when ERα is silenced in

MCF7 cells, but does bind to HDAC7 in an estrogen-independent fashion.

FOXA1/ERα/HDAC7 are likely required for the estrogen-induced repression of additional target genes, but it is unknown whether FOXA1 can interact with HDAC7 to repress transcription in cells lacking ERα. Herein, we report that FOXA1 is necessary for sustained repression of a subset of basal breast cancer classifier genes. Loss of FOXA1 increases these genes in both ERα-positive and ERα-negative luminal breast cancer cells and FOXA1 binds to these genes. FOXA1 is not known to have intrinsic repressor activity. Hence, it likely recruits repressors to these chromosomal locations. HDAC7 is a possible candidate given its known role with FOXA1 in MCF7 cells (284). Our data indicate that FOXA1 represses basal gene expression in an ERα-independent fashion, and thus, FOXA1 and HDAC7 may similarly participate in gene repression in the absence of

ERα. It is also possible that FOXA1 represses basal genes in complex with HDAC7 only

134 in ERα-positive lines, and interacts with other co-repressors in ERα-negative cells. Based on an in silico evaluation of a publically available ChIP-chip dataset defined for MCF7 cells (201), ERα does bind (<50kb upstream, intragenically, and/or <50kb downstream) to five of the basal genes (CD58, LYN, MGP, TF, TRIM2) that are consistently induced in all four luminal cell lines following FOXA1 knockdown. Thus, it is possible that in ERα- positive cell lines, ERα also participates in the repression of a subset of basal genes.

Additional repressor complexes may also be involved in mediating FOXA1 repression of basal gene expression. Loss of BRCA1 in T47D cells induces transcription of KRT5,

KRT16 and CDH3, three genes associated with the basal subtype, and a BRCA1/c- complex binds to the respective promoters to mediate repression (344). FOXA1 can synergize with BRCA1 in the regulation of the p27Kip1 promoter (307), thus, it tempting to speculate that FOXA1 and BRCA1 cooperate to repress basal gene expression.

While no additional studies have investigated FOXA1 repression of gene expression in breast cancer, FOXA1 can silence liver-specific genes through recruitment of Groucho 3

(Grg3) (345). In this context, binding of FOXA1 and Grg3 leads to a condensed chromatin state that is resistant to DNAse I treatment, and is not bound by transcriptional activators (e.g. NF-1, TBP or Pol II). The Groucho family has been implicated in the tumorigenesis of tissues other than breast (346), and Groucho 1 (TLE1) functions in ERα transactivation in a manner similar to FOXA1 in breast cancer cell lines (347). However, a functional role for Grg3 in breast cancer remains to be determined. Another putative mode of FOXA1 repression can be inferred from the C. elegans homolog of FOXA,

PHA-4. PHA-4 interacts with TAM-1/TRIM, a Nucleosome Remodeling and histone

135 Deacetylase (NuRD) complex, and represses expression of genes involved in embryonic cell fate (348). Several components of NuRD also participate in transcriptional repression in the breast (349), but no studies to date have investigated a connection between FOXA1 and NuRD in mammalian cells. To begin to define the mechanism by which FOXA1 represses basal breast cancer genes, the potential cooperation of FOXA1 with HDAC7,

Grg3 and components of the NuRD complex should be examined in both ERα-positive and ERα-negative breast cancer cell lines. The identification of factors that are capable of repressing basal gene expression will have profound implications in the development of targeted therapies to induce differentiation and hormone responsiveness to tumors of the highly aggressive basal subtype.

Although loss of FOXA1 induces the expression of a subset of basal genes, and this induction is statistically significant as measured by GSEA, not all the basal genes are increased upon FOXA1 silencing. Similarly, not all luminal signature genes are decreased. The simplest explanation for these results is that FOXA1 expression was transiently suppressed. The expression of basal genes that are not directly regulated by

FOXA1, or those that mRNAs with long half-lives may not have changed by 72 hours after transfection with the FOXA1 siRNA. FOXA1 knockdown was also not 100% effective, and this may have given rise to an incomplete phenotypic change. Other than technical limitations, it is also possible that FOXA1 represses only a subset of the basal genes, and additional factors control other basal gene subsets. It is also likely that a group of transcriptional activators are necessary to express the full complement of basal genes, but reducing FOXA1 may not be adequate to induce their expression. Even if transient

136 loss of FOXA1 induced expression of these activators, the time course of this study may have been insufficient to detect alterations in the expression of their gene targets. Stable knockdown of FOXA1 will be necessary to ultimately determine if FOXA1 can repress the entire basal gene set, and hence, cause a complete conversion of luminal to basal cells.

FOXA1 maintains a less aggressive phenotype in breast cancer cells

We provide evidence that transient knockdown of FOXA1 in luminal breast cancer cells results in a switch in the molecular phenotype of these cells. Breast cancer phenotypes have been proposed to reflect the lineage progression of normal mammary epithelial cells, with basal tumors arising from luminal progenitors and luminal tumors arising from mature luminal epithelium (149, 150). The degree of plasticity between these states is not yet known, however, our studies suggest that they may be highly dynamic wherein more differentiated breast cancer cells can acquire phenotypic characteristics of less differentiated, or basal cells. In the case of the results presented herein, FOXA1 plays a critical role in maintaining the luminal state and preventing expression of genes associated with basal cancers. It will be important to determine whether FOXA1 functions similarly during developmental specification of the mammary epithelial hierarchy. If so, this would indicate that a major function of FOXA1 in breast development and cancer is the regulation of lineage commitment.

Another transcription factor, GATA3, has been proposed to play a similar role as

FOXA1. It is also necessary for normal mammary morphogenesis (144, 314), and its

137 forced expression in tumors induces differentiation and prevents disease dissemination in mice (146). However, the impact of GATA3 on the expression of basal signature genes has not yet been reported. We have found previously that FOXA1 and GATA3 expression are independent in the normal mammary gland, and that loss of FOXA1 does not alter GATA3 in breast cancer cells (338). Studies examining the GATA3-regulated transcriptome will be necessary to discern the overlapping and distinct functions of

FOXA1 versus GATA3 in maintaining the luminal subtype of breast cancer.

Paralleling the observed molecular switch towards a basal phenotype, transient loss of

FOXA1 similarly increases the aggressiveness of luminal breast cancer cells as measured in vitro through migration and invasion assays. Importantly, the changes in migration and invasion that are observed with FOXA1 silencing are not secondary to an increase in proliferation. In fact, the loss of FOXA1 in an ERα-positive breast cancer cell line

(MCF7) led to a significant decrease in the number of cells at 72 hours post-transfection.

These data are consistent with previous reports that loss of FOXA1 blocks estrogen mediated cell cycle progression in these cells (278, 287). FOXA1 knockdown is similarly reported to reduce cell number under hormone-replete conditions (286). While the combined phenotype of increased aggressiveness and decreased cell number appears counterintuitive, heterogeneity within breast cancer cell lines may explain this dichotomy

(350). Specifically, loss of FOXA1 may lead to growth arrest of the subpopulation of cells that are fully differentiated (i.e. mature luminal). In contrast, the remaining cells may have greater plasticity to de-differentiate towards the basal phenotype. MCF7 cells, in particular, have a side-population with tumor initiating capabilities (350). Loss of

138 FOXA1 may increase the percentage of this population of cells resulting in the observed shift toward the basal subtype.

Targeting FOXA1 as an approach for breast cancer therapy

Although we did not identify any ERα-positive tumors that lack FOXA1 within our tumor cohort, others have reported such tumors (263-267, 269). Since there are currently no breast cancer cell lines that recapitulate the FOXA1-negative, ERα-positive molecular phenotype, future studies should aim to delineate the molecular and phenotypic differences between these and FOXA1-positive, ERα-positive cancers because it will be important to determine if ERα has a distinct function in the absence of FOXA1. Perhaps

ERα transcriptional regulation in these breast tumors recapitulates ERα activity in other cancer types (e.g. osteosarcoma, ovarian) that do not co-express FOXA1 (282, 296).

The observation that FOXA1 controls luminal and basal classifier genes further supports the prognostic value of FOXA1 because its expression level is not only correlative, but is functionally important in maintaining luminal tumors in a differentiated, less aggressive state. It has been suggested that targeting FOXA1 in patients with hormone receptor positive tumors would abrogate ERα signaling and possible aid in the efficacy of tamoxifen (298). Our analyses refute this postulate because loss of FOXA1 may lead to the selection of a more aggressive, basal-like population of tumor cells, or cause de- differentiation of existing cells. For example, we found that loss of FOXA1 substantially induces expression of Annexin 1 (ANXA1), which is required for metastasis of basal breast cancer cells (351). These results, in combination with the increase in in vitro

139 migration and invasion that occurs in response to FOXA1 silencing, indicate that FOXA1 directly maintains the more favorable, luminal phenotype, even in the absence of ERα.

While pharmacologic reduction of FOXA1 in luminal tumors may be contraindicated, elevating expression of FOXA1 in basal breast cancers may prove useful for inducing luminal differentiation and acquisition of hormone responsiveness. Such differentiation approaches may uncover a novel therapeutic approach for treating these recalcitrant tumors.

3.6 ACKNOWLEDGEMENTS

G.M.B is supported through a pre-doctoral fellowship from the Department of Defense

(DoD) (W81XWH-06-1-0712) and a National Institute of Health (NIH) pre-doctoral training grant (T32-HD-07104-33). G.B. is supported by the NIH (UL1-RR024989). STS is supported through a post-doctoral fellowship from the DoD (W81XWH-09-1-0696).

This work was also supported by the Case Western Reserve University Comprehensive

Cancer Center (P30 CA043703; G.B. and R.A.K.), a University Hospitals of Cleveland

Pathology Research Associates Grant (F.W.A. and R.A.K.), the NIH (R01 CA090398,

R.A.K.), a Fox Chase Cancer Center Support Grant (P30 CA006927, A.K.G.) and the

DoD (W81XWH-08-1-0347, R.A.K.).

140 Table 3.1

Genes commonly decreased upon knockdown of FOXA1 in MCF7, T47D, MB-453 and SKBR3 cells (p<0.001).

141 Table 3.1

Sequence Name Sequence Code Sequence Name Sequence Code SEPT7 A_24_P291973 ARHGEF16 A_23_P114670 SEPT7 A_23_P8482 ASAP2 A_24_P362540 SEPT7 A_32_P79584 ASH2L A_23_P134827 SEPT11 A_23_P306964 ASPSCR1 A_23_P32320 SEPT13 A_24_P855187 ATAD2 A_24_P59596 A_23_P51966 A_23_P51966 ATP2C2 A_23_P117992 A_24_P109962 A_24_P109962 ATP8B2 A_32_P185229 A_24_P118813 A_24_P118813 ATP8B2 A_24_P117896 A_24_P169976 A_24_P169976 BAZ1B A_23_P215449 A_24_P24230 A_24_P24230 BC007568 A_24_P145122 A_24_P307075 A_24_P307075 BC029473 A_32_P12065 A_24_P307306 A_24_P307306 BCL9 A_23_P382602 A_24_P323916 A_24_P323916 BDH2 A_23_P92490 A_24_P341489 A_24_P341489 BG576864 A_32_P186348 A_24_P349869 A_24_P349869 BLVRA A_23_P71148 A_24_P409471 A_24_P409471 BOD1 A_24_P117964 A_24_P409824 A_24_P409824 BPTF A_24_P392475 A_24_P41530 A_24_P41530 C10orf81 A_24_P286951 A_24_P480464 A_24_P480464 C10orf97 A_23_P23983 A_24_P560332 A_24_P560332 C14orf1 A_23_P25935 A_24_P632230 A_24_P632230 C16orf14 A_23_P3663 A_24_P651129 A_24_P651129 C18orf1 A_24_P130363 A_24_P739075 A_24_P739075 C19orf52 A_23_P310532 A_24_P752362 A_24_P752362 C1orf210 A_32_P42946 A_24_P780609 A_24_P780609 C1orf53 A_32_P210572 A_24_P929650 A_24_P929650 C20orf20 A_23_P17307 A_32_P222030 A_32_P222030 C21orf33 A_23_P109333 A_32_P4608 A_32_P4608 C3orf52 A_23_P58009 A_32_P96036 A_32_P96036 C4orf34 A_23_P112634 ABCG1 A_23_P166297 C5orf30 A_23_P122007 ABHD10 A_24_P308590 C5orf32 A_23_P259506 ABHD10 A_23_P92213 C9orf23 A_23_P169409 ABI2 A_24_P933418 C9orf75 A_23_P323836 ACOT7 A_24_P205589 CACNA1D A_24_P880000 ACTB A_23_P135769 CASP6 A_23_P500799 AF15Q14 A_23_P100127 CBX3 A_23_P31315 AFF1 A_24_P414332 CCBL2 A_24_P824592 AFG3L1 A_23_P355289 CCDC115 A_24_P54485 AK027315 A_23_P102071 CCDC115 A_23_P17074 AK096500 A_32_P155035 CCDC117 A_23_P432034 AK124080 A_32_P80610 CCDC134 A_23_P155106 AL359585 A_32_P157385 CCDC28B A_23_P62764 ALDH6A1 A_23_P128967 CCNA2 A_23_P58321 ALKBH3 A_23_P35989 CCND1 A_24_P193011 ALS2CR4 A_23_P370097 CCND1 A_23_P202837 AMD1 A_23_P214121 CCND1 A_24_P124550 ANG A_23_P428738 CCNF A_24_P193592 ANKRD20A1 A_32_P168326 CCNF A_23_P37954 ANKRD39 A_23_P95027 CCT7 A_23_P102404 ANXA2P1 A_24_P204244 CD302 A_23_P131435 ANXA6 A_23_P357104 CDC20 A_23_P149200 AP1M1 A_23_P55802 CDC26 A_32_P228501 APH1B A_23_P205997 CDC42EP2 A_23_P1602 APOA1BP A_23_P126448 CDC45L A_23_P57379 ARHGAP19 A_23_P1387 CDC6 A_23_P49972

142 Table 3.1 continued

Sequence Name Sequence Code Sequence Name Sequence Code CDT1 A_23_P37704 ELK1 A_23_P171054 CDT1 A_24_P176374 ENST00000239730 A_24_P66398 CENPH A_23_P110802 ENST00000259575 A_24_P289043 CEP55 A_23_P115872 ENST00000297984 A_24_P409115 CEP68 A_23_P210323 ENST00000305536 A_24_P143785 CHD1L A_23_P45831 ENST00000316369 A_24_P212596 CHEK1 A_23_P116123 ENST00000318909 A_24_P409521 CHML A_24_P712350 ENST00000325023 A_24_P289834 CHML A_23_P46118 ENST00000327086 A_24_P75879 CHMP7 A_23_P303203 ENST00000327893 A_24_P375510 CHPT1 A_23_P105571 ENST00000330752 A_24_P41309 CHRAC1 A_23_P123544 EPHB4 A_23_P168443 CHST14 A_23_P106532 ERCC6L A_23_P96325 CLPP A_23_P4754 ERLIN1 A_23_P202029 CMBL A_23_P144668 ERLIN1 A_23_P52219 COL24A1 A_23_P74701 ESD A_24_P737416 CORO2A A_23_P135061 ESRP2 A_23_P100220 CPT1A A_24_P347065 EXOSC3 A_23_P123905 CPT1A A_23_P104563 F12 A_23_P167674 CREB3 A_23_P423389 FAM120AOS A_24_P832156 CREBBP A_23_P163850 FAM131A A_24_P243086 CTDSP1 A_23_P28263 FAM13B A_24_P20700 CTDSP2 A_23_P81880 FAM48A A_23_P140050 CTNNAL1 A_23_P157795 FAM64A A_23_P49878 CXCR4 A_23_P102000 FANCD2 A_32_P24165 CXorf40A A_23_P306919 FARP1 A_23_P308763 CXorf40A A_23_P316239 FBL A_23_P78892 CYTSB A_23_P38567 FBXL6 A_23_P43296 DCP1A A_24_P576174 FBXO45 A_23_P400794 DCP1A A_23_P166826 FKBP5 A_23_P111206 DCUN1D5 A_23_P127533 FLJ20618 A_23_P68868 DEK A_23_P254702 FLJ36840 A_24_P59471 DENND4B A_23_P774 FOXK2 A_23_P397969 DENND5A A_23_P321201 FOXM1 A_23_P151150 DKFZp434N035 A_23_P166336 FOXP1 A_23_P155257 DKFZp434P055 A_24_P157156 FREQ A_23_P217049 DKFZP547L112 A_24_P171043 FUCA2 A_23_P145408 DLX4 A_23_P164196 GALNT10 A_23_P19102 DMAP1 A_23_P586 GAPDHL7 A_24_P662086 DNAJA4 A_23_P206140 GAS8 A_23_P206435 DNAJC9 A_23_P104372 GINS1 A_23_P210853 DRG2 A_23_P141656 GIYD2 A_23_P501451 DSTYK A_23_P23894 GLT25D1 A_23_P209183 DTX3 A_23_P53247 GNAS A_24_P168574 DULLARD A_24_P142473 GNB1 A_24_P71021 DUSP16 A_23_P308924 GNB1L A_23_P218751 DUSP16 A_24_P189739 GNPTAB A_24_P281975 DUSP16 A_23_P353905 GNPTAB A_23_P204380 A_23_P385034 GNPTAB A_24_P288979 EBF4 A_23_P109122 GOLT1A A_24_P280846 EBP A_23_P171077 GPD2 A_23_P119812 EFHD1 A_24_P234391 GPR160 A_23_P167005 EIF1AD A_23_P127467 GPX8 A_23_P122052 EIF2C1 A_23_P11764 GRB10 A_23_P122863 EIF2C2 A_23_P112159 GTF2I A_24_P95007

143 Table 3.1 continued

Sequence Name Sequence Code Sequence Name Sequence Code GTF2I A_32_P88791 MCM6 A_23_P90612 GTF2IRD2 A_24_P876862 MESDC1 A_23_P99891 GTPBP3 A_23_P28068 MEX3A A_24_P857404 H1FX A_23_P96087 MLLT6 A_32_P98683 H2AFV A_23_P316487 MLX A_24_P13991 HBS1L A_23_P254379 MMAB A_24_P364381 HDGF A_24_P376707 MPDU1 A_24_P198844 HDGFRP3 A_23_P344451 MPI A_23_P60579 HES4 A_23_P149448 MRPL18 A_23_P8339 HIPK2 A_23_P169756 MRPL19 A_23_P102262 HIPK2 A_24_P500621 MRPL30 A_24_P302695 HIST2H3C A_23_P435029 MRPL41 A_32_P115130 HLTF A_24_P277155 MSH6 A_23_P102202 HMGB1 A_24_P169148 MTDH A_24_P760130 HMGB3 A_23_P217236 MTMR1 A_23_P73530 HMGN1 A_32_P78250 MTMR2 A_23_P64019 HNRNPA1 A_32_P168202 MUTED A_24_P77870 HOOK3 A_23_P216080 MYL3 A_23_P155638 HSD17B12 A_23_P47377 MYL6B A_23_P2223 HSPC159 A_23_P430818 NAB2 A_23_P368187 HYAL2 A_23_P57927 NAB2 A_23_P76151 IGFBP5 A_23_P154115 NAPG A_24_P185314 IGFBP5 A_23_P383009 NAT10 A_23_P87329 IGHMBP2 A_23_P393713 NCAPH2 A_23_P80398 IKBKE A_23_P887 NDE1 A_24_P210675 IMPDH1 A_24_P89708 NDE1 A_23_P206901 INPPL1 A_23_P36322 NDUFC1 A_23_P92362 ISOC1 A_23_P250982 NEDD1 A_24_P198355 ITGB1BP1 A_23_P154507 NEK2 A_23_P35219 ITPA A_23_P68529 NGFRAP1 A_23_P45524 KAZALD1 A_23_P421011 NHP2L1 A_24_P382765 KBTBD2 A_23_P70951 NHP2L1 A_23_P80362 KDELR2 A_23_P19938 NLE1 A_23_P141315 KIAA0317 A_23_P369987 NMB A_23_P88522 KIAA0391 A_23_P14340 NMU A_23_P69537 KIF14 A_23_P149668 NOL9 A_23_P126727 LARP2 A_23_P7353 NOP56 A_23_P79927 LASS6 A_24_P289366 NRAS A_23_P63190 LIG1 A_23_P39116 NRGN A_23_P116264 LMCD1 A_23_P6771 NSFL1C A_24_P134340 LOC145837 A_32_P46594 NT5C3L A_23_P49924 LOC344967 A_24_P698141 NUCKS1 A_24_P216964 LOC387922 A_32_P201677 NUCKS1 A_24_P216968 LOC401057 A_24_P684119 NUDT16L1 A_24_P399065 LOC402152 A_24_P409697 NUDT16L1 A_23_P49429 LOC440309 A_24_P523061 NUF2 A_23_P74349 LOXL1 A_23_P124084 OBSCN A_23_P63128 LRFN4 A_23_P63980 ODF2 A_23_P60488 LRRC45 A_23_P381461 OLA1 A_23_P28420 MACC1 A_32_P131031 OPN3 A_23_P74391 MAGED2 A_23_P33894 ORC1L A_23_P45799 MAP3K6 A_32_P223017 PACSIN1 A_23_P258088 MAPK1 A_24_P237265 PAPOLA A_23_P419051 MAPKAPK2 A_23_P201483 PCGF5 A_23_P202117 MAX A_23_P205549 PECI A_23_P156852

144 Table 3.1 continued

Sequence Name Sequence Code Sequence Name Sequence Code PEF1 A_23_P313 SCAMP1 A_24_P42527 PEX19 A_23_P160188 SCD A_23_P63618 PFDN5 A_24_P181120 SEC13L1 A_24_P214506 PHF17 A_23_P167263 SEC13L1 A_23_P29555 PI4KB A_24_P222860 SEPHS1 A_23_P150092 PIK3CB A_23_P346969 SEPHS1 A_24_P118452 PLAC1 A_23_P148609 SGSH A_23_P254254 PLCG1 A_23_P254801 SLC24A3 A_23_P79978 PLDN A_24_P27373 SLC25A17 A_23_P57547 PLEKHG3 A_32_P311737 SLC2A4RG A_23_P102571 PLEKHH3 A_32_P172188 SMEK1 A_23_P218158 POLA2 A_23_P161615 SMUG1 A_24_P75072 POLB A_32_P34552 SNX21 A_23_P154585 PPIL4 A_23_P42507 SNX24 A_23_P19095 PPP1R13B A_23_P205273 SOAT1 A_24_P216654 PPP2R5E A_23_P3042 SOAT1 A_23_P63319 PREB A_23_P91114 SOCS3 A_23_P207058 PRELID2 A_23_P110585 SORD A_32_P89691 PREX1 A_23_P413641 SORD A_23_P77103 PRO2463 A_23_P390190 SPDEF A_23_P111194 PROCR A_23_P80040 SPTLC1 A_23_P311192 PRR7 A_23_P30464 SRCAP A_23_P394917 PSEN2 A_23_P96985 SRRD A_24_P288954 PSEN2 A_23_P103398 SRRD A_32_P135385 PSEN2 A_24_P62521 SSH1 A_24_P332647 PSIP1 A_32_P22702 SSNA1 A_23_P159476 PSIP1 A_24_P98371 SSNA1 A_24_P303915 RAB35 A_23_P204484 ST6GALNAC2 A_32_P9597 RAD17 A_24_P97836 STAU2 A_32_P649 RAD23A A_24_P22887 STIL A_24_P214231 RALGDS A_23_P135184 STK38 A_24_P63727 RALGDS A_23_P135190 STT3A A_23_P104734 RANBP9 A_23_P424513 SUB1 A_32_P2333 RAPH1 A_24_P924862 SURF4 A_23_P32052 RAPH1 A_24_P929570 SURF4 A_24_P89971 RBM41 A_32_P135091 SUV39H2 A_23_P202392 RBMX2 A_24_P322966 SYNJ2 A_23_P316974 RCN2 A_32_P175539 TACC3 A_23_P212844 REPS1 A_23_P70748 TALDO1 A_24_P81740 RFT1 A_23_P155441 TALDO1 A_32_P18258 RHOH A_23_P58132 TCEA2 A_23_P147641 RNF114 A_24_P82948 TCEAL1 A_23_P73801 RNF8 A_23_P70384 TCF3 A_24_P365365 RNPS1 A_23_P152272 TCOF1 A_23_P310317 RNPS1 A_24_P725630 TERT A_23_P110851 ROBO3 A_23_P356581 TFAM A_23_P202258 RPL7L1 A_24_P332971 TGIF2 A_23_P79794 RPL7L1 A_23_P42163 THC1804460 A_24_P896373 RPS2 A_32_P14544 THC1809100 A_32_P40673 RPS2P32 A_24_P84045 THC1838438 A_32_P214054 RRP1B A_23_P256297 THC1859012 A_32_P224157 RTF1 A_23_P77321 THC1862126 A_24_P732106 RTKN2 A_23_P52410 THC1924749 A_24_P32920 SALL2 A_23_P48585 THC1956189 A_32_P514599 SAP30 A_23_P121602 THC1977331 A_32_P145790

145 Table 3.1 continued

Sequence Name Sequence Code Sequence Name Sequence Code THOC6 A_23_P37949 ZWILCH A_23_P112673 THUMPD1 A_24_P99371 ZWINT A_23_P63789 TIGD7 A_23_P129577 TK1 A_23_P107421 TM6SF1 A_24_P35935 TMEM106C A_23_P48175 TMEM109 A_23_P203364 TMEM149 A_23_P142424 TMEM199 A_23_P27048 TMEM199 A_24_P56884 TMEM93 A_23_P78134 TNFRSF10A A_23_P255653 TNRC6C A_24_P718833 TOR1A A_24_P269687 TRAF5 A_23_P201731 TRAIP A_23_P170491 TRIB3 A_24_P305541 TRIB3 A_23_P210690 TRIM14 A_23_P216655 TRIM14 A_24_P197964 TRIM32 A_24_P29966 TRIM32 A_23_P112311 TSEN15 A_23_P104022 TSEN15 A_23_P104025 TSGA14 A_23_P215070 TSPAN13 A_23_P168610 TTC3 A_24_P84984 TUBA1C A_23_P128161 TUBB A_32_P78528 TUBB A_23_P81912 TYMS A_23_P50096 UBE2S A_32_P171328 UBE2Z A_23_P83438 UBE2Z A_24_P378506 UGT2B10 A_23_P7342 UGT2B11 A_23_P212968 UROS A_23_P35609 USMG5 A_23_P127095 VMA21 A_32_P151621 WDFY2 A_23_P218108 WDYHV1 A_23_P71415 WIZ A_23_P405531 XBP1 A_24_P100228 XM_087553 A_32_P228331 XYLT2 A_23_P15582 ZBED1 A_32_P86623 ZBED3 A_23_P93032 ZBTB4 A_23_P100654 ZBTB8 A_23_P352365 ZDHHC17 A_23_P314191 ZFHX3 A_23_P118266 ZHX2 A_32_P227657 ZNF428 A_23_P311468 ZNF639 A_24_P123155 ZNRF1 A_23_P163858

146 Table 3.2

Genes commonly increased upon knockdown of FOXA1 in MCF7, T47D, MB-453 and SKBR3 cells (p<0.001).

147 Table 3.2

Sequence Name Sequence Code Sequence Name Sequence Code A_23_P103951 A_23_P103951 ANXA8L2 A_32_P105549 A_23_P129405 A_23_P129405 ARAP2 A_32_P83784 A_23_P21234 A_23_P21234 ARFIP1 A_23_P95050 A_24_P178444 A_24_P178444 ARFIP1 A_24_P166094 A_24_P212764 A_24_P212764 ARHGAP18 A_32_P17163 A_24_P675386 A_24_P675386 ARHGAP18 A_32_P162250 A_24_P904723 A_24_P904723 ARHGAP8 A_24_P561713 A_32_P134167 A_32_P134167 ARHGEF15 A_23_P49674 A_32_P149735 A_32_P149735 ARL6IP5 A_23_P166640 A_32_P156017 A_32_P156017 ARL8B A_23_P385217 A_32_P169243 A_32_P169243 ARMC8 A_23_P29784 A_32_P175313 A_32_P175313 ARMETL1 A_24_P93309 A_32_P187875 A_32_P187875 ASS1 A_24_P929818 A_32_P214860 A_32_P214860 ATF6 A_23_P62907 A_32_P23113 A_32_P23113 ATG2B A_23_P88163 A_32_P25972 A_32_P25972 ATG7 A_24_P944827 A_32_P30014 A_32_P30014 ATMIN A_24_P176493 A_32_P30187 A_32_P30187 ATN1 A_24_P24244 A_32_P35303 A_32_P35303 ATP11A A_24_P161973 A_32_P85578 A_32_P85578 ATP6V0E1 A_23_P213840 AASDHPPT A_24_P295620 ATP6V1H A_23_P157478 ACRC A_23_P171237 AU158367 A_24_P933965 ACYP2 A_23_P256988 AUH A_23_P20852 ADAMTS4 A_23_P360754 AVL9 A_24_P55356 ADAT1 A_24_P48139 AW269579 A_32_P146552 ADAT1 A_23_P141100 B3GNT1 A_23_P86900 ADI1 A_32_P52911 BAIAP2L2 A_23_P379034 AEBP2 A_24_P18621 BBS1 A_24_P184305 AGAP4 A_32_P221958 BC039397 A_32_P123168 AGGF1 A_23_P250554 BC040982 A_24_P288116 AGPAT9 A_23_P69810 BE181102 A_32_P184417 AGTPBP1 A_23_P169278 BE881987 A_32_P54987 AHCYL2 A_24_P72518 BI497361 A_32_P220161 AHI1 A_24_P943484 BI820718 A_32_P144629 AK002036 A_24_P839239 BM701156 A_32_P171232 AK023328 A_24_P462725 BM928197 A_24_P893239 AK026295 A_32_P187009 BM930591 A_24_P11587 AK057652 A_24_P892402 BMPR2 A_24_P753161 AK057720 A_24_P759674 BQ188770 A_32_P50670 AK057835 A_24_P931944 BQ276781 A_32_P130290 AK090739 A_32_P201958 BQ717518 A_24_P924681 AK095167 A_24_P931579 BQ926066 A_32_P6682 AK098597 A_32_P153833 BRAF A_23_P42935 AK123264 A_24_P930088 BX436400 A_32_P36075 AK123861 A_32_P158723 BZW2 A_23_P157215 AL137325 A_32_P500996 C10orf2 A_24_P258073 ALG1 A_24_P565556 C10orf57 A_23_P97853 ALG9 A_24_P795594 C12orf66 A_24_P68294 AMFR A_23_P141005 C13orf10 A_24_P919840 AMY1A A_23_P23611 C16orf71 A_23_P414281 ANKRA2 A_23_P159012 C19orf63 A_23_P208674 ANKRA2 A_24_P337397 C1orf102 A_23_P103433 ANKRD46 A_23_P94095 C1orf107 A_23_P34946 ANKRD5 A_23_P256047 C1orf107 A_23_P34953 ANXA1 A_23_P94501 C1orf123 A_23_P23017

148 Table 3.2 continued

Sequence Name Sequence Code Sequence Name Sequence Code C1orf123 A_24_P334005 CLN5 A_23_P117286 C1orf25 A_24_P190877 CLN5 A_24_P270357 C1orf63 A_24_P2584 CLN8 A_23_P407206 C20orf111 A_23_P79818 clone 39-1 A_24_P930647 C20orf12 A_23_P40315 CLYBL A_23_P376591 C20orf177 A_23_P68505 CMTM6 A_23_P57736 C20orf177 A_24_P6428 CMTM6 A_24_P97526 C20orf30 A_23_P17490 CNOT4 A_23_P300781 C22orf23 A_23_P132308 COPZ1 A_23_P116809 C2orf4 A_32_P122925 COX16 A_23_P53957 C3orf50 A_24_P68183 CPSF2 A_23_P99837 C4orf32 A_23_P58390 CRABP2 A_23_P115064 C5orf41 A_23_P404606 CREM A_23_P201979 C5orf53 A_24_P110558 CRYZL1 A_23_P218731 C6orf164 A_23_P418434 CRYZL1 A_24_P408341 C6orf35 A_23_P134026 CTAGE5 A_32_P121362 C6orf70 A_32_P129527 CTNNB1 A_23_P29495 C8G A_23_P20713 CTSB A_24_P397928 C8orf83 A_32_P117313 CX3CR1 A_23_P407565 C9orf41 A_24_P320970 CYB561D1 A_24_P348885 C9orf45 A_23_P216935 CYCL A_24_P403501 C9orf5 A_23_P146417 CYLD A_24_P48078 C9orf95 A_23_P32036 CYP4B1 A_23_P114713 CA438977 A_24_P621023 DDIT4 A_23_P104318 CABC1 A_23_P85598 DDX52 A_23_P118660 CALCOCO1 A_23_P98995 DHRS1 A_24_P133475 CAPZA2 A_32_P224666 DHRS1 A_23_P48747 CASC1 A_23_P95231 DHX57 A_23_P28307 CASC3 A_23_P374288 DIAPH2 A_32_P50834 CATSPERB A_23_P77043 DIDO1 A_24_P944144 CCDC18 A_24_P481375 DIRC1 A_23_P131139 CCDC28A A_23_P31085 DIRC2 A_23_P80778 CCDC57 A_23_P356965 DISP2 A_23_P324340 CCDC6 A_32_P184279 DKFZP586M0622 A_23_P334271 CCDC76 A_23_P51317 DKFZp686N09198 A_24_P333993 CCDC82 A_23_P312246 DKFZp781N1041 A_24_P509948 CCL24 A_23_P215491 DLX2 A_23_P28598 CD104030 A_32_P115749 DMXL1 A_23_P250571 CD164 A_23_P254756 DNAJC16 A_23_P358009 CD2AP A_24_P329152 DNAJC19 A_23_P121396 CD55 A_23_P374862 DNER A_23_P362148 CD55 A_24_P188377 DPY19L1 A_23_P358628 CD59 A_32_P50275 DPY19L1P1 A_24_P652033 CD59 A_23_P75523 DPY19L4 A_32_P196047 CD59 A_24_P639441 DSCR3 A_24_P254346 CDC23 A_23_P133629 DUSP3 A_23_P129956 CDC42 A_23_P200560 DYRK2 A_24_P56270 CDK6 A_24_P166663 DYRK2 A_24_P942786 CDKL3 A_23_P110643 ELF5 A_24_P247820 CDKN1A A_23_P59210 ELK3 A_24_P133171 CG012 A_24_P500771 ENDOD1 A_23_P104624 CLDN12 A_23_P157268 ENOX2 A_23_P258251 CLDN3 A_24_P35109 ENPP4 A_23_P70318 CLDN3 A_23_P71017 ENPP5 A_23_P214244 CLK1 A_23_P16817 ENST00000324709 A_24_P238525

149 Table 3.2 continued

Sequence Name Sequence Code Sequence Name Sequence Code ENST00000327625 A_23_P81484 GPR107 A_24_P295379 ENST00000329768 A_24_P367100 GRAMD4 A_24_P23258 ENST00000329775 A_24_P101742 GRB2 A_23_P77847 ENST00000332402 A_32_P62963 GRIK5 A_24_P576591 ENST00000334072 A_23_P128868 GTDC1 A_23_P153945 ENTPD4 A_24_P269598 GTF2B A_23_P34628 ENTPD7 A_24_P112447 GTF2IRD2 A_23_P59616 EPHX2 A_23_P8834 HAVCR2 A_23_P18903 ERMP1 A_23_P169154 HBP1 A_23_P215787 ERO1L A_24_P407311 HCCS A_23_P436407 ETF1 A_23_P133582 HER2/neu receptor A_24_P933108 EVI5 A_24_P96593 HERC4 A_24_P346807 FAM119B A_23_P320878 HEXIM1 A_24_P356601 FAM155B A_23_P22647 HGSNAT A_23_P112061 FAM160B1 A_32_P26895 HIPK3 A_24_P364066 FAM175B A_23_P364478 HN1L A_23_P434900 FAM178B A_24_P231546 HSPB1 A_24_P86537 FAM73A A_24_P58395 HSPBAP1 A_23_P250118 FAM78A A_23_P314250 HUS1 A_23_P156977 FAM98A A_23_P313728 IDS A_23_P400847 FBXO28 A_24_P310756 IGLC1 A_24_P104980 FBXO32 A_24_P218259 IL11 A_23_P67169 FBXO32 A_23_P82814 IL1RAP A_23_P170857 FBXO32 A_24_P918147 IL20RB A_23_P91850 FBXO36 A_23_P422981 INADL A_23_P321034 FCER1G A_23_P160849 ING4 A_23_P48070 FITM2 A_23_P431638 IPPK A_23_P123582 FLJ00254 A_24_P162145 IQCK A_23_P324523 FLJ21616 A_24_P226700 IQGAP1 A_24_P321919 FLJ22313 A_32_P40615 IRF8 A_23_P332190 FLJ22833 A_23_P329198 ITGAV A_23_P50907 FLJ31131 A_23_P380076 ITGB1 A_23_P104199 FLJ38482 A_23_P253677 ITGB1 A_32_P95397 FLJ38482 A_24_P20524 JARID2 A_23_P214876 FLJ39609 A_24_P940999 JMY A_32_P176550 FLJ44253 A_32_P46171 KDSR A_32_P515088 FLJ45244 A_24_P678418 KIAA0184 A_24_P940499 FMO4 A_23_P160992 KIAA0226 A_23_P304171 FNDC3B A_23_P253434 KIAA0494 A_24_P233917 FNTA A_23_P24926 KIAA1609 A_24_P213924 FNTB A_23_P25835 KIAA1671 A_23_P309619 FRMD8 A_24_P67988 KIAA2018 A_32_P138617 FSTL5 A_24_P251734 KIF1B A_24_P649624 GAB1 A_24_P936319 KLHL24 A_23_P251574 GABARAPL2 A_23_P118313 KLHL7 A_23_P324994 GABPB2 A_23_P73208 LAMB3 A_23_P86012 GALNT3 A_24_P114249 LAMP1 A_23_P162846 GAN A_23_P88873 LAMP2 A_24_P396231 GEMIN5 A_23_P336513 LAP1B A_23_P46660 GLRX5 A_23_P65370 LCE1C A_23_P303891 GLT8D3 A_23_P336796 LCMT1 A_23_P112825 GM2A A_24_P925314 LENG8 A_23_P56288 GM2A A_23_P144872 LEPR A_24_P231104 GNRHR A_23_P155796 LHFP A_23_P88069 GOLT1B A_24_P321511 LMBRD2 A_24_P58147

150 Table 3.2 continued

Sequence Name Sequence Code Sequence Name Sequence Code LOC100131989 A_32_P89310 MPZL2 A_23_P150379 LOC144438 A_23_P98963 MPZL3 A_24_P270033 LOC149705 A_23_P253561 MRAS A_24_P88850 LOC149913 A_32_P154223 MRPS6 A_23_P102890 LOC153546 A_32_P197698 MRS2 A_23_P111373 LOC157562 A_32_P192922 MT3 A_23_P129629 LOC158381 A_24_P358245 MTA3 A_23_P411431 LOC201229 A_23_P392126 MTMR6 A_24_P419211 LOC253039 A_24_P649582 MTMR6 A_23_P25515 LOC283788 A_24_P801197 MYCBP A_23_P86230 LOC283874 A_23_P95599 MYH14 A_23_P78734 LOC284454 A_24_P734060 MYO1E A_23_P37497 LOC339924 A_24_P521994 MYO6 A_23_P255952 LOC388114 A_32_P76992 N4BP2L2 A_23_P65262 LOC389833 A_32_P71183 NARS A_23_P101185 LOC390595 A_24_P481783 NAT12 A_24_P66679 LOC400684 A_24_P59569 NAT12 A_23_P25868 LOC402247 A_24_P152325 NEXN A_24_P409971 LOC440317 A_24_P649327 NFIB A_24_P658427 LOC441104 A_24_P680857 NFIB A_24_P804992 LOC641467 A_32_P23989 NHLRC3 A_32_P129950 LOC644246 A_24_P221327 NIPSNAP3A A_23_P20606 LOC649294 A_24_P401270 NQO1 A_23_P206661 LOC727916 A_32_P120791 NUDT3 A_24_P554156 LOC728537 A_24_P456723 NUDT3 A_23_P93389 LPIN1 A_32_P52609 NUFIP2 A_24_P921144 LRPAP1 A_24_P196568 OCLN A_23_P92672 LRPPRC A_24_P26073 OR1E1 A_23_P101073 LRRC23 A_23_P36689 ORC3L A_23_P42045 LRRC37A3 A_23_P331400 OSBPL2 A_24_P408321 LRRC57 A_24_P219266 OSTM1 A_23_P404785 LRRC68 A_23_P101303 OSTM1 A_23_P31097 LSG1 A_23_P132417 OSTM1 A_32_P154830 LUZPP1 A_23_P254061 OTUD1 A_32_P60459 LYG1 A_23_P165707 OTUD4 A_24_P402898 LYST A_23_P354074 OVOL1 A_23_P202810 LZTFL1 A_23_P41049 P25 A_24_P579482 MACROD2 A_23_P22398 PAFAH1B2 A_23_P370142 MACROD2 A_32_P89352 PAFAH2 A_24_P71153 MAGT1 A_24_P485219 PARD6B A_32_P205637 MAN2A1 A_23_P18931 PARS2 A_24_P54253 MAOB A_23_P85015 PBLD A_23_P149998 MAP1LC3B A_24_P108005 PBLD A_24_P112395 MAP2 A_24_P231483 PCDH1 A_24_P234838 MAP3K7IP2 A_23_P19702 PCMTD1 A_32_P63858 MAPK14 A_23_P426292 PCMTD2 A_23_P210829 MDS025 A_32_P157295 PDXK A_24_P160088 MERTK A_23_P32955 PEX12 A_24_P416411 MFAP3 A_24_P217758 PHF10 A_24_P535219 MFSD1 A_23_P166677 PHF6 A_32_P229299 MFSD11 A_23_P26704 PHIP A_32_P61339 MKRN1 A_23_P59798 PHLDB2 A_24_P240166 MKRN4 A_24_P28208 PIBF1 A_23_P411881 MOBKL1B A_23_P108922 PIGG A_23_P136547 MOV10 A_23_P161125 PIGZ A_23_P143935

151 Table 3.2 continued

Sequence Name Sequence Code Sequence Name Sequence Code PLAC2 A_23_P359666 RNF141 A_24_P372625 PLD1 A_23_P155335 RNF145 A_23_P167559 PLEKHA1 A_24_P269814 RNF187 A_24_P23995 PLEKHB2 A_24_P873414 RNF214 A_24_P32520 PLK3 A_23_P51646 RNMT A_24_P48408 PLXDC2 A_23_P161424 ROCK1 A_24_P538403 PNAS-138 A_32_P189680 RPGRIP1L A_32_P123966 PNPLA4 A_24_P943815 RPGRIP1L A_23_P388489 PPAP2A A_23_P70060 RPIA A_23_P131646 PPAP2B A_23_P201808 RPL35 A_24_P726215 PPAPDC2 A_24_P40907 RPS27L A_23_P14734 PPAT A_24_P123347 RRAGA A_23_P169117 PPAT A_23_P80940 RRAS A_23_P39076 PPL A_23_P106906 RRAS A_23_P39074 PPP1R13L A_23_P119095 RSAD1 A_23_P152807 PPP1R3E A_23_P428640 RSL1D1 A_24_P778836 PPP2R3B A_23_P357504 RSPH3 A_23_P59397 PPP3CA A_24_P414371 S100A11 A_23_P126593 PQLC3 A_23_P131375 S100A9 A_23_P23048 PREPL A_24_P943193 SAMD4A A_23_P335661 PRKCH A_23_P205567 SAP18 A_24_P381555 PRRG1 A_24_P31929 SARDH A_23_P135079 PSKH1 A_23_P390596 SCFD2 A_23_P92543 PSORS1C2 A_23_P81926 SDCBP A_23_P157580 PTGR2 A_23_P48713 SDR39U1 A_23_P163161 PTMS A_23_P128337 SECISBP2 A_23_P84872 PTPN1 A_23_P338890 SELT A_23_P250042 PTPRJ A_23_P405049 SERINC5 A_24_P6381 PUM1 A_23_P10815 SETD7 A_24_P251841 QDPR A_23_P167212 SGPL1 A_24_P940815 QKI A_24_P941322 SH3D19 A_23_P33364 RAB11FIP1 A_23_P31873 SLC1A5 A_24_P409500 RAB15 A_24_P193295 SLC1A5 A_32_P31165 RAB40B A_23_P129801 SLC20A2 A_23_P94921 RABL2A A_24_P88721 SLC35A2 A_24_P310616 RABL3 A_24_P302785 SLC41A2 A_23_P204801 RAC1 A_24_P194845 SLC41A3 A_23_P18317 RAGE A_23_P76731 SLC48A1 A_23_P204511 RAP1B A_23_P2661 SLC6A13 A_24_P923483 RAP2C A_24_P374319 SLK A_24_P146670 RAP2C A_23_P147826 SLPI A_23_P91230 RASSF3 A_23_P117546 SLPI A_24_P190472 RBM47 A_23_P132910 SMAD1 A_23_P212870 RCBTB2 A_23_P14105 SMCR5 A_23_P388798 REPS2 A_32_P100109 SNRK A_23_P211985 REXO2 A_24_P316364 SNRPB2 A_23_P40307 RFK A_23_P216708 SNRPB2 A_24_P724040 RG9MTD2 A_23_P398637 SPTY2D1 A_23_P429082 RHBDD1 A_24_P134834 STAU2 A_24_P374634 RHBDD1 A_23_P147514 STEAP3 A_23_P90601 RHOU A_24_P62530 STX3 A_23_P139143 RMND5A A_23_P257753 SUGT1 A_32_P161455 RMND5A A_23_P257755 SUPT4H1 A_24_P1910 RMND5A A_24_P114334 SYPL1 A_23_P93881 RNF121 A_23_P75973 SYPL1 A_24_P141005

152 Table 3.2 continued

Sequence Name Sequence Code Sequence Name Sequence Code SYTL2 A_23_P53193 THC1931812 A_32_P963 TBC1D14 A_23_P155828 THC1933362 A_32_P115446 TBC1D15 A_23_P139558 THC1933362 A_32_P115451 TBC1D20 A_24_P392958 THC1933926 A_32_P181548 TBC1D22A A_32_P10633 THC1949165 A_32_P9491 TBC1D3F A_32_P24372 THC1952955 A_32_P17635 TBC1D5 A_23_P355455 THC1953000 A_32_P177097 TBC1D5 A_24_P289029 THC1953521 A_32_P94122 TBC1D9 A_23_P41487 THC1953717 A_32_P5168 TBCEL A_23_P410017 THC1953802 A_24_P286845 TBX19 A_23_P137705 THC1954712 A_32_P114235 TCEANC A_23_P320159 THC1957416 A_24_P390583 TCTA A_23_P58002 THC1957642 A_24_P479364 TECPR2 A_23_P394216 THC1958780 A_32_P55438 TF A_23_P212500 THC1965240 A_23_P114582 THAP2 A_24_P350437 THC1965702 A_24_P738130 THC1807966 A_24_P71938 THC1968370 A_32_P135450 THC1808056 A_32_P107219 THC1971019 A_24_P791814 THC1817947 A_32_P377577 THC1977999 A_24_P345993 THC1818158 A_32_P155811 THC1978975 A_24_P194260 THC1818601 A_24_P787947 THC1985992 A_32_P80781 THC1819997 A_32_P182511 THC1990965 A_32_P234996 THC1826099 A_32_P224040 THC1992140 A_32_P154121 THC1831576 A_24_P122403 THC1992648 A_32_P179706 THC1835478 A_24_P364072 THC1999284 A_32_P181061 THC1836416 A_32_P151133 THRSP A_23_P105212 THC1836785 A_32_P221631 TMBIM6 A_24_P355876 THC1840393 A_32_P186038 TMCO1 A_24_P276583 THC1843049 A_32_P35452 TMCO1 A_23_P355067 THC1844946 A_24_P727868 TMEM106B A_23_P8522 THC1844946 A_32_P58999 TMEM106B A_32_P353072 THC1847203 A_24_P744259 TMEM117 A_23_P2573 THC1848213 A_32_P226700 TMEM188 A_23_P397417 THC1848900 A_32_P140220 TMEM38A A_23_P101392 THC1848922 A_32_P190613 TMEM70 A_23_P253963 THC1857560 A_24_P157053 TMEM80 A_24_P90349 THC1857619 A_24_P295709 TMEM86A A_32_P66035 THC1858909 A_32_P119949 TMX4 A_24_P942517 THC1866189 A_24_P932632 TNFRSF10B A_24_P218265 THC1867405 A_24_P343377 TNFRSF1A A_23_P139722 THC1870233 A_32_P108592 TOM1L1 A_23_P118493 THC1877485 A_24_P706752 TOMM20L A_24_P117942 THC1878189 A_32_P96692 TOR2A A_23_P60534 THC1881187 A_23_P82047 TP53INP1 A_23_P168882 THC1881737 A_24_P54847 TPD52L3 A_24_P309216 THC1883248 A_32_P109181 TPK1 A_23_P145957 THC1889620 A_23_P214273 TRA2A A_23_P31389 THC1891147 A_24_P97785 TRAF6 A_32_P192984 THC1891603 A_32_P157481 TRIM2 A_24_P208909 THC1903859 A_32_P48526 TRIM23 A_24_P208998 THC1904048 A_24_P633825 TRIM35 A_23_P502553 THC1911551 A_32_P173177 TSEN2 A_23_P92012 THC1913382 A_24_P285179 TSGA10 A_23_P17103 THC1916767 A_32_P186018 TSN A_24_P242820 THC1924953 A_32_P194563 TSN A_23_P28105

153 Table 3.2 continued

Sequence Name Sequence Code Sequence Name Sequence Code TTC19 A_24_P126393 ZNF565 A_23_P101615 TTC19 A_23_P50008 ZNF586 A_24_P83944 TTC30A A_23_P131611 ZNF597 A_23_P3753 TTC37 A_23_P61854 ZNF624 A_23_P153037 TUBA4A A_23_P154065 ZNF75D A_23_P397635 TUBA4B A_23_P84448 ZNF862 A_24_P333421 TWISTNB A_23_P386561 ZZZ3 A_23_P11507 UBQLN2 A_23_P114164 UBR3 A_23_P5405 UBR3 A_24_P285501 UHMK1 A_32_P84389 unknown A_32_P22571 USP30 A_24_P390055 USP31 A_24_P147263 USPL1 A_24_P338757 USPL1 A_23_P128641 UTP23 A_32_P208733 VAPA A_23_P382199 VAPB A_23_P91293 VAV3 A_23_P201551 VCPIP1 A_23_P256445 VNN3 A_23_P214935 WASF1 A_23_P168306 WDFY1 A_23_P502797 WDR19 A_24_P394368 WDR19 A_24_P394361 WFDC10B A_32_P951 WHAMM A_24_P307827 XM_293276 A_24_P178167 XM_374900 A_24_P571864 YME1L1 A_24_P167052 YPEL5 A_23_P108835 YRDC A_23_P62840 ZBTB1 A_23_P99693 ZBTB20 A_23_P40866 ZBTB41 A_32_P108826 ZBTB47 A_24_P71700 ZBTB5 A_23_P216476 ZCWPW1 A_23_P70897 ZDHHC20 A_23_P341418 ZDHHC24 A_23_P333330 ZF A_23_P203645 ZFAND6 A_32_P220472 ZHX1 A_23_P43150 ZNF12 A_23_P31335 ZNF14 A_23_P101811 ZNF177 A_24_P168398 ZNF177 A_23_P164706 ZNF185 A_23_P11025 ZNF251 A_24_P49517 ZNF268 A_23_P255591 ZNF490 A_24_P49142 ZNF518B A_32_P144421 ZNF554 A_23_P343250 ZNF559 A_24_P284584

154 Table 3.3

Basal A, basal B and luminal classifier genes (RN) whose expression is changed upon knockdown of FOXA1 in MCF7, T47D, MB-453 and SKBR3 cells (p<0.05).

Basal A Basal B Luminal Increased in all four Increased in all four Decreased in all four lines lines lines ANXA1 DSE ABCG1 ARHGEF9 ELK3 ALDH6A1 CD58 FSTL1 CDC20 FNDC3B GLS DUSP4 JAG1 LHFP EFHD1 KRT16 MALT1 EMP2 LYN PALM2 ERBB3 MGP PALM2-AKAP2 GALNT10 PAM 10.1% of total BasB HDGFRP3 PRNP IGFBP5 S100A2 INHBB SLPI PECI SOX9 SPDEF TF SPINT1 TRIM2 TFF3 16.3% of total BasA TSPAN13 XBP1 21.0% of total Lum

155 Table 3.4

Classifier gene lists (RN) used to discriminate the luminal, basal A, and basal B molecular subtypes for GSEA. BasAB-RN = BasA + BasB as listed below.

Lum-RN BasA-RN BasB-RN SPDEF MSN APOBEC3B EMP3 COL4A2 RHOB KLK6 EN1 PTRF LOXL2 FOXA1 IFITM1 MET AXL SGCB XBP1 PLSCR1 ARHGEF9 FOSL1 TPM2 GATA3 KLK5 S100A2 CAV2 FBN1 TFF1 CRYAB PPM2C ELK3 IMPA1 ERBB3 KRT16 RNF15 NT5E IGFBP6 TOB1 ALDH1A3 PTK7 TGFBR2 DKK3 SLC9A3R1 PDZK1IP1 HLA-F PHLDA1 NMT2 AGR2 ANXA1 MGP VIM CORO1C PBX1 LCN2 LAMP3 ANPEP POLR3G GPD1L KLK8 SOX9 AKR1B1 PVRL3 TFF3 IFITM3 MX1 FSTL1 GNG12 MLPH SLPI ANXA3 GPD1L BIN1 CA12 GABRP JAG1 PLAU MT1E TJP3 RARRES1 TRIM22 SACS SERPINE1 KRT19 PRNP ANXA8 SNAI2 SNX7 ALDH3B2 IFI27 KRT6B TGFBI GJA1 GALNT6 TRIM2 FNDC3B PROCR RAB32 AP1G1 IFITM2 PLS3 MAP4K4 KIAA0182 ST6GALNAC5 CHI3L1 CD44 KRT8 GBP1 CD58 PALM2 NEBL CALB2 SERPINB5 BAG2 EFHD1 CDKN2A SERPINA3 DFNA5 EPN3 IFI44L SRD5A1 TUBB-5 ALDH6A1 NMI PSMB9 PRKCDBP SFRP1 HERC6 UPP1 KRT23 IFIH1 TGFB1I1 LYN HLA-C SUMF2 IFI44 NAB1 FSCN1 PELI1 HHLA3 FLRT2 C3 HLA-B GNG11 DSC3 HLA-G EPHA2 DDX58 VGLL1 VEGFC KRT5 HDGFRP3 HMGA2 KRT17 HLA-A CTNNAL1 TBC1D1 PSPH NNMT FXYD5 SCHIP1 COL4A1 PAM CBS MLLT11 PIM1 MBNL2 MALT1 MTAP BTG3 TGFB1 TF LHFP CALD1 GLS

156 Table 3.5 Lum(B)-ECJ classifier gene list used for GSEA.

PREX1 FLJ38379 TEGT TSGA2 PVRL2 MYCN PRLR ASTN2 LZTR1 LOC284591 LOH3CR2A GGA3 SLC24A3 CRNKL1 SPTLC2 SOX13 CCT6A LASS6 TFF1 KCTD15 GART CAPN9 GARS ZNF398 ABCA3 SLC1A4 KDELR2 IGSF4 LMCD1 KIAA0089 RHPN1 GSPT1 MAPT LASS2 ARID2 AP1G1 FOXA1 ELOVL2 RDH13 ATP6AP1 SLC37A1 LYK5 GATA3 NUCB2 LOC202451 DNALI1 DDAH2 SYCP2 SPDEF EMP2 BAI2 HPX POLE KRT19 XBP1 KIAA0644 BAZ2A USP3 HPN SLC4A8 PLEKHH1 Rab11-FIP3 GTF3C1 PYCR1 TFF3 TM4SF13 RAI17 ACVR1B TCFL1 KIAA1324 CACNA1D SDCCAG3 FKBP4 GGA1 CTNND2 FLJ22184 FLJ36445 WFS1 DNAJA4 TGFB3 CLSTN2 ZNF24 FLJ36032 GALNT6 STARD10 MCCC2 TBC1D16 AD023 INHBB RABEP2 GARNL1 TBL1X MYO6 DAAM1 HMG20B F7 HNRPA2B1 CIRBP ATP8B1 TRIM3 DACH1 MDM4 LOC151963 RNF103 ELL3 DUSP8 USP42 JMJD2B BCAS1 BCOR KIAA0232 SH3GLB2 TTC9 TBX3 RHOB MAGED2 MYEF2 FGFR4 KIAA1211 ZNF444 ASB8 GAMT PTPRF GP1BB KIAA0217 ANXA6 DF GARNL3 FLJ90013 USP7 LLGL2 CYB561 SLC38A1 PCBP2 KIFC2 SHANK2 NET-7 RHBDF1 SLC26A11 EVL FLJ33814 POGZ MGC4368 FLJ20174 ISG20 HIP1R LRP3 GPRC5C GPR160 SCUBE2 SMARCC2 RALGPS1 PRKAG1 SLC16A6 KIAA1543 CREB3L1 LUC7A SLC2A10 TOB1 CHN2 NLK NME3 KIF12 CREB3L4 MGC23280 PACE-1 KIAA0556 FLJ31568 RUSC1 KIAA0241 STRBP CGI-40 PDCL3 FALZ TTC3 SERF2 SLC35A1 LOC284018 LARGE SLC9A3R1 KIAA1244 IVD KIAA1856 MLPH ADCY6 SNX27 CTXN1 MAPK9 ULK1 KIAA0040 ARFIP2 PGGT1B LFNG PBX1 EPN3 DLG3 TOMM70A MYO5B NPDC1 KIAA0226 TRPS1 SRRM2 RAB3D TESK1 EIF3S9 PPP2R2C NACA ARF3 MYB WBSCR21 KIAA1181 ICA1 ENPP1 TLE3 FLJ33674 KIAA0738 DNAJC1 ABCG1 SGEF MARS PPP1R16A LOC338692 MGC3121 NUDT4 KIAA0703 HK2 CA12 MGC3047 RAB40C FZD4 AGR2 KIAA0767 TGIF2 CACNA2D2 PCP4 HMGCS2 ZFYVE16 KIAA1467 PIB5PA FLJ30092 CAP350 RAB17 KIAA1598 CACNB3 MGC9712 CCND1 EGFL5 ESR1 ANKRD30A MYST4 NDUFS8 SOX12 SFMBT2 UAP1L1 CISH ZNF84 UBN1 ASH1L LOC283400 AR DDX42 LOC80298 TJP3 VIPR1 MGRN1 CXXC5 FRS2 PCK2 ERBB3 POMT1 SLC7A8 SECISBP2 LRRN1 CACNG4 SLC25A29 CNNM3 ANXA9 EPS8L1 KLF2 RBAK LOC153561 KIAA0913 ZNF74

157 Table 3.6 Bas-ECJ classifier gene list used for GSEA.

ZDHHC2 MRCL3 C1R MBNL1 CAMTA1 ELK3 LY6K CXCL3 SH3KBP1 SFN SLC6A15 ASAM TNFAIP8 CRIPT IL1RAP SMAD3 STK17A ANXA8 KLK5 ANXA1 MGC10946 ZNF145 RAC2 SERPINB2 IFI44 KLK10 MT1E ARPC2 SAA1 SOAT1 SH3GLB1 VAMP3 RBM7 LOX GBP1 ICAM1 RHBDL2 PTPN2 COL8A1 GALNT2 HIF1A COL4A2 DIA1 ETS2 SP100 KRT6B RALBP1 BIRC3 CASP4 ADM VSNL1 ATP1A1 LAMA3 BTG3 GSTP1 C3 DIRC2 IL7R MPZL1 LAMC2 NMI INHBA MAML2 TNFRSF10D RUNX3 HMGA2 ETF1 ANTXR1 EXT1 CRYAB ADA HIC ARNTL2 NOB1P SFRP1 KRT17 GABRE MDH1 YAP1 GLIPR1 PERP DHRS8 DUOX1 RGS2 APOL6 NACSIN CDC42EP3 SPTBN1 BF CD44 ADORA2B TBPL1 PLA2G4A SLC16A1 LOC137392 ASXL1 TGFBI FLJ23518 HLA-E RRAS2 LRAP FGF2 RGC32 COL4A1 JAG1 NSEP1 KIAA1228 IGFBP6 RIOK3 NCK1 PTK7 ELL2 NUDT15 SERPINE2 LAMC1 ACTN1 PLAT RBM9 LAMB3 ABCD3 SNX7 ITGA3 LOC285812 COTL1 MET EMP1 SRI CD59 ITGA6 CTSC TLE4 PI3 CDH3 IL1A NAV2 MPPE1 GPSM2 PBEF1 URB SLC1A3 MTMR2 NUP50 GTF2B TP73L BNC1 FLJ13391 EPHA2 TWSG1 DGKA ACY1L2 CXCL2 IGFBP7 IL15 DSC3 ADRB2 PPM2C IFI27 CDK6 OGFRL1 FGG FOSL1 TRIM22 ADD3 GNA15 EREG KIAA1212 NFAT5 FLJ22833 BICD2 PPP1R14C CRK AXL FSCN1 CALD1 PSMB8 FHL1 RPL5 CLIC4 IFITM3 LOC51186 BTN3A2 NRP1 BPAG1 MYO1B SERPINB5 DCTD RGS20 PSAT1 B3GNT5 YES1 OSBPL9 GPM6B IL18 ITM2C ETS1 HOXA1 CORO1C RALB PHLDA1 TRIM29 NT5E HOXA3 FMNL2 BIN1 TGFA AKR1C1 UPP1 SCAP2 SLC9A6 LYN CHST3 C6orf67 GM2A TAZ F2RL1 BMP1 EGFR STAT3 SOX7 LIPG TKT PRNP COPS8 NNMT CDH13 GNG12 ZNF258 SVIL IRX1 KIRREL DSG3 OSBPL3 GNAI1 PGM2 GBP3 AGPS TWIST2 PPP4R1 AMD1 ALDH3A2 LOC340061 CSNK2A2 SCHIP1 CD109 LOXL2 DCBLD2 FAP CYBRD1 BTN3A3 TAP2 NAB1 AKR1C3 ATP1B3 AIM1 FKBP1A INPP1 SRPX S100A10 S100A2 MMP14 DCBLD1 B2M SDCCAG8 MT2A CYLD SNAI2 KLF5 IRS2 PTPRM MFGE8 MT1G CDCP1 SPRY2 PKP2 MGC4083 FBLP-1 KRT15 GFOD1 OSMR CAV2 DKFZP566E144 UBE2E3 FLJ43339 FDFT1 NDEL1 SLPI DPYD CSDA NSFL1C SSFA2 CD14 F3 FXYD5 AKR1B10 CFL2 ARHGAP23 RGL1 MAP17 TBC1D1 ALDH1A3 MSN ANXA3 AKT3 ANXA4 NR3C1 CHMP1.5 KRT14 AKR1B1 CGI-100 FST ITGB1 GAS1 EMP3 CD58 KRT16 GART NFE2L2 PLAU PHLDB2 HP1-BP74 PIK3CD TGFBR2 TUBA1 TRIP10 FZD6 PDGFC FSTL1 PTPNS1 CARD6 BJ-TSA-9 KIF1B TNFAIP3 PTGS2 RBMS3 GPX1 IFI16 TIP-1 RGNEF MT1X CFLAR LUZP1 PLS3 PTRF CCNA1 MGC14376 ARHGAP5 FLJ40432 IF FOXQ1 CASP1 CBR1 KPNA1 WDR1 PLSCR1 FLJ32028 PSMB9 LEPREL1 RBMS1 MYO1E ANKH KIAA0746 SCPEP1 ANXA2 KRT5 DSG2 FLJ31951 MAP4K4 HOXA5 HRH1 GJB3 NDFIP2 STAT4 MBNL2 KRT6A DMD PRKCDBP CD97 MME DNAPTP6 TLR2 DNAJB4 C1S

158 Table 3.7 Lum(M)-ECJ classifier gene list used for GSEA.

RAB25 MYB TSGA2 VAV3 MLPH MYO5C SCAMP2 MAL2 SPTLC2 FLJ21749 PIP5K2C FLJ21125 MAP7 ITPK1 FAM31C KIAA1324 ASB8 SMPDL3B SERF2 CACNG4 RHOH F11R GPR160 EPS8L1 HIP1R RAB5B MGC20255 EFHD1 TFCP2L3 FLJ22457 ESR1 CGN ARF3 TOB1 BLVRB EPN3 EPHA1 PLA2G12A KIFC2 OCLN CACNB3 RAB11-FIP4 FLJ36445 MYO5B FLJ22531 RABEP2 EFNA4 SPTBN2 SLC25A29 BSPRY RHPN1 EPHB3 KIAA0767 GRB7 FLJ46072 KIAA0251 TJP3 EPPK1 LOC118430 FKBP4 TRIM3 IGFBP2 ARHGEF5 ERBB3 PKP3 LAD1 TRIB3 ARRDC4 CSAD PERLD1 SPDEF PACE-1 NPDC1 TIGA1 PADI2 ENSA INHBB IRF6 WBSCR21 ARFIP2 LISCH7 PNN SSH-3 BIK PRSS8 TMG4 EPB41L5 APH-1A PLEKHF2 FLJ11506 DKFZP586A0522 KRT19 ELF3 CBLC NME3 ZNF289 TSPAN-1 SYMPK FXYD3 SH3YL1 KIAA0247 RPS6KA5 TLE3 MKNK2 MGC23280 CD24 LLGL2 FLJ22318 TMPRSS2 FGD3 NOL3 SIGIRR SYTL1 CLDN3 S100A14 TRPS1 RAB40C VRK3 BCAS1 MB PVRL4 LOC196264 H2AFJ FLJ22386 PRODH PROM2 IRX5 RAB3D CLDN4 FBP1 JUP TM4SF13 SH2D3A AP1M2 SYCP2 HIG2 EGFL5 KIAA0703 CLN3 PREX1 ZNF385 TDE2L PPP2R5A ST14 NEBL ELMO3 MYO6 ATP6AP1 ALDH6A1 KIAA1543 CLDN7 EMP2 CRIP2 CGI-143 MGMT ARHGAP8 KIAA1522 ABAT CCDC6 WDR23 CGI-119 ALDH3B2 SLC35A1 FLJ90165 MUC1 ZCCHC8 SYNGR1 FLJ20174 RHOB MRPL41 RHBDF1 TMC4 CHN2 SPINT1 CEBPA RUSC1 VPS45A GSTO2 FEM1B TFCP2L2 MGC9712 IBRDC2 ALDH4A1 TRAF4 PPFIBP2 CACNA1D LASS6 CTNND2 DDR1 WFS1 BTBD5 ELL3 SLC9A3R1 SLC7A8 VIPR1 LOC202451 LOC339745 SULT2B1 FOXA1 SPINT2 MGC15416 ANKRD22 AP1G1 CREB3L4 BCR SEPP1 ZNF24 PPP1R16A CDH1 KIAA0040 ENTPD2 ABCA12 SOX13 LOC400451 PCK2 CDS1 ICA1 ZNF165 MYLIP TM7SF2 SSBP2 KIAA0644 FRAT2 ARRDC1 KIAA0089 FLJ33718 PPM1H ABCG1 CRABP2 TUFT1 TMEM16A FUT1 PCDH1 STARD10 HOOK2 ABCC11 LOC124220 LMTK3 CHD2 CYB561 TFF1 GSPT1 RERG CEACAM6 IDH2 GATA3 DNAJA4 SLC37A1 USP18 AIM1L CYP4B1 AGR2 MCF2L PER2 GPR56 FANCF FLJ10980 LIMK2 MGC13102 TFF3 MCCC2 RALGPS1 SEPHS2 ANXA9 TNRC9 SEMA4A CIRBP TNKS1BP1 CRIP1 TTC9 FUK PRKD2 INADL SORL1 HIST2H2AA PRLR DBP TPD52L1 CA12 CADPS2 NR2F6 ENPP5 MPP7 DLG3 DNAJC1 SSR4 S100A8 ADCY6 XBP1 SYT7 FAAH BLOC1S1 EZI MSX2 C4.4A DAAM1 SHANK2 CNNM4 KIAA0556 LOC222171 PIB5PA LOC93622 PEX11B ZNF74 FLJ11017 MYO1D PLXNB1 GALNT6 MYH14 FLJ38379 ZHX2 RAB17 PDCD4 DUSP16 RNF103 COG7 ULK1 OVOL1 LOC146439 ERBB2 PTPN6 GPR157 RAB27B ANK3 ACVR1B SEMA3F TEGT SLC29A2 COMMD3 LOC161291 BC-2 AZGP1 TP53AP1 FA2H GJE1 SLC16A14 JTB CYB5 PWWP2 KRT23 ANKRD30A RAB45 EHF TRPM4 S100P KIAA1244 PPP1R3D

159 Table 3.8 Mes-ECJ classifier gene list used for GSEA.

ACTG1 ATM TFPI DERP6 TAP2 PTPLA FSTL1 NAP1L5 CORO2B MID1 LOC51334 B3GNT5 PLAU LYN FAM20C IFNGR2 RAB8B POPDC3 ZDHHC2 FST TNFAIP3 HIC ELAVL1 CDK6 DZIP1 CDC20 TGFBI SLIT2 CTSC PSMB6 ASB1 FLI1 RAB6IP1 TBC1D1 COL6A2 PTGS2 SFPQ G0S2 ARHGAP21 FZD2 INPP1 TGFBR2 SNRPD1 RAB31 NONO PPM2C ZNF258 AGPS GFPT2 EXTL2 DEPDC1 TTL SERPINB1 CKLFSF3 ILF3 AKT3 ITGAV PAFAH1B1 OK/SW-cl.56 SLC35B4 MALT1 BICD2 PVRL3 PPP4R1 SH3GLB1 LOC400745 EPB41L2 ACTN1 RAI14 GRK5 NT5E NAV1 PTPRM GNG12 TNFRSF10D GALNT2 SHOX2 GLS ENG EIF4A1 RGS2 SACS FSCN1 TGFB1I1 SFRP1 RALBP1 PDE3A NR3C1 ARHGAP23 HRH1 IL6 SERPINE1 CKLFSF7 ACTA2 MMP2 IFI16 TOX IL11 ACTR2 ADAMTS5 CUL4B LDHB LOXL2 IL7R AKR1C3 TEAD1 SSFA2 NR2F1 CFL2 LRRK1 NAB1 NNMT FBN1 CHST2 TAZ MPP1 FHOD3 STK17A PRKCDBP CALD1 ADD3 SLC16A7 ETV5 LAMC1 ANXA5 SDCCAG8 JAG1 SPTBN1 CHSY1 GJA1 EXT1 BACH1 FLJ23518 NUP88 ASAM PAPPA LOC285550 RAB34 ABCC4 ITM2C D2S448 FXYD5 TIMP2 TWIST2 DOCK10 DHRS8 ANTXR1 ANKH NUP155 PHLDA1 TCF8 GNAI1 IL15 PTRF TYMS PMP22 CD99 PDGFC DCBLD2 FOSL1 PIK3CD LOXL1 COL6A3 CD44 CHST3 MGC4083 CAV2 IRAK2 SPRY2 RPL5 DAB2 COL5A1 AK5 FLJ10357 DFNA5 MAPRE1 SPARC TIMP1 MCAM ASXL1 NACSIN ZNF515 DDEF1 RTN4 DNAJB4 NID2 AXL VCL MSN PLP2 LHFP COL6A1 MCFD2 TGFB2 UPP1 WASPIP HAS2 PRG1 PDE7B MGC49942 LOC137392 MAP1B COL4A1 PLK1 PICALM EYA4 DKK3 MAP4K4 KCTD12 EDIL3 CPNE2 BNC2 ANKRD25 SPAG9 CDH11 HMGA2 KIRREL INCENP NOL7 TRAM2 CTNNAL1 DRCTNNB1A FOXQ1 PNMA2 DNM1 PFKP KIAA1033 GPR TRIO GNG11 AKR1B1 ANLN ANTXR2 PSMB2 GPRC5B MYLK LOC143903 SH3KBP1 FOSL2 LOC51186 LEPRE1 ELK3 BASP1 IGFBP6 FLRT2 PYGL EMILIN2 GULP1 DPYD TWSG1 BIN1 SERPINE2 FLJ23323 RGS4 MFGE8 ECHDC1 ADORA2B ETS1 LEPREL1 FZD7 ANXA2 FER SRPX BAG2 EPHA2 FLNC VPS54 AKAP12 SNX7 ZIC2 MAML2 SEMA3A COL4A2 FBLP-1 UBE2S NAV3 SOCS3 BCAT1 TGFB1 RBMS3 CDC27 DHX33 GPR161 PDE4A NMT2 PFAS EMP3 ANKRD28 MET TPM1 SDPR FEZ2 FGG PTX3 CD97 DMD SGCB LTBP2 MRC2 MGC17337 GSTP1 PIM1 STK10 NRP1 FLJ13391 IKIP KIF3C RGS20 CORO1C ITSN1 CRIM1 EGFR CSRP2 GNG2 KIAA1212 COL13A1 LUZP1 BCHE AP1S2 PHLDB2 F2RL2 VIM C1S S100A2 NDEL1 INHBA KIAA1949 OSMR FOXF2 EFEMP2 PRNP SNAI2 CRTAP KIAA0802 PARVA CASP1 PTTG1 SKP2 PABPC4 CXCL2 FLJ37034 BUB1 SDCBP FLJ31951 GADD45A ALS2CR2 EDG2 ARNTL2 NMNAT2 TRAF3 KIAA1754 ANXA1 PSMB9 CDKN2C SV2A MTMR2 KPNB1 BIRC5 PRKCA MGC33424 FOXC1 RAFTLIN BDNF PLAT FMNL2 SIL QKI EIF5A2 CYP26B1 NEXN IGFBP7 LAMB1 FHL1 CYLN2 PSMD1 WNT5B PROS1

160 Table 3.9

GSEA of classifier gene lists that are discriminatory of luminal v. basal breast cancer molecular subtypes.

Lum- BasA- BasB- BasAB- Lum(B) Bas- Lum(M) Mes- RN RN RN RN -ECJ ECJ -ECJ ECJ ES -0.60 0.59 0.28 0.45 -0.36 0.43 0.18 0.3 Normalized ES -1.83 2.26 1.00 1.89 -1.69 1.98 0.84 1.38 NOM p-value 0.002 0 0.452 0 0 0 0.948 0.004 FDR q-value 0.004 <1x10-4 0.497 <1x10-4 0.008 <1x10-4 0.806 0.066 FWER p-value 0.005 0 0.994 0.002 0.025 0.001 1.000 0.375 Classifier gene lists based on Neve et al. (2006) (RN) (336) & Charafe-Jauffret et al. (2006) (ECJ) (337). See Tables 3.4 through 3.8. ES = Enrichment Score, NOM = nominal, FDR = false discovery rate, FWER = familywise-error rate.

161 Table 3.10

Potential binding sites in basal and luminal signature genes regulated by FOXA1.

FOXA1 FOXA1 Basal A Bound* Consensus** ANXA1 2 sites ARHGEF9 0 sites CD58 ✓ FNDC3B ✓ JAG1 ✓ KRT16 1 site LYN ✓ MGP 3 sites PAM ✓ PRNP 1 sites S100A2 0 sites SLPI 1 site SOX9 ✓ TF ✓ TRIM2 ✓ FOXA1 FOXA1 Luminal Bound* Consensus** ABCG1 ✓ ALDH6A1 ✓ CDC20 ✓ DUSP4 ✓ EFHD1 ✓ EMP2 ✓ ERBB3 ✓ GALNT10 1 site HDGFRP3 ✓ IGFBP5 ✓ INHBB ✓ PECI ✓ SPDEF ✓ SPINT1 ✓ TFF3 ✓ TSPAN13 ✓ XBP1 ✓ *FOXA1 is bound <50kb upstream, intragenically, or <50kb downstream according to ChIP-chip data from Lupien et al. (2007) (201). **FOXA1 Consensus motif identified in the proximal (-1000bp) promoter by TESS. Shaded grey = Confirmed by ChIP.

162 Table 3.11 Gene order on Luminal (B)-ECJ heatmap in Figure 3.7.

PREX1 LOC112476 ANXA6 ZNF24 PRLR PH-4 LLGL2 FLJ14360 SLC24A3 PBX1 KIFC2 TBC1D16 TFF1 NPDC1 EVL TBL1X ABCA3 TESK1 ISG20 HNRPA2B1 PIK4CA MYB SCUBE2 MDM4 C17orf28 TLE3 KIAA1543 USP42 RHPN1 SGEF C7orf26 SH3GLB2 FOXA1 NUDT4 CHN2 MYEF2 GATA3 RAB40C KIAA0182 GAMT KIAA0984 KIAA1545 MGC23280 DF SPDEF CACNA2D2 KIAA0241 RND1 XBP1 PIB5PA TTC3 EZI KIAA0476 CACNB3 SLC9A3R1 LNX SLC4A8 ANKRD30A ADCY6 SHANK2 TFF3 UAP1L1 C9orf7 FLJ33814 KIAA1324 LOC283400 KIAA0040 HIP1R CTNND2 VIPR1 EPN3 SMARCC2 TGFB3 ERBB3 FLJ11164 CREB3L1 STARD10 CACNG4 KIAA0226 NLK N2N KLF2 EIF3S9 PACE-1 RABEP2 FLJ38379 WBSCR21 STRBP DKFZP547E1010 ASTN2 FLJ33674 SERF2 HMG20B CRNKL1 MARS KIAA1244 TRIM3 DP1 KIAA0703 SNX27 ELL3 FLJ23825 FRMD4 ARFIP2 BCOR KCTD15 FZD4 DLG3 ENTPD8 SLC1A4 PCP4 TRPS1 RHOB GSPT1 FLJ30092 PPP2R2C ZNF444 KIAA0310 MGC9712 KIAA1181 KIAA0217 C12orf22 MYST4 PLXN3 USP7 ELOVL2 RAB2L KIAA0738 PCBP2 NUCB2 CISH PPP1R16A SLC26A11 EMP2 AR HK2 FLJ20174 C14orf66 KIAA1023 AGR2 GPR160 ZNF278 MGRN1 HMGCS2 C1orf34 KIAA0644 POMT1 CAP350 C9orf91 PLEKHH1 SLC25A29 CCND1 MGC15523 C14orf132 RBAK NDUFS8 LOC112868 TM4SF13 TEGT ZNF84 LAF4 C7orf24 LZTR1 DDX42 SLC16A6 CACNA1D SPTLC2 CXXC5 TOB1 FLJ22184 GART SLC7A8 MGC26885 CLSTN2 KDELR2 CNNM3 CREB3L4 ZNF342 MAPT LOC153561 ZNF325 MCCC2 DEPDC6 LOC149670 RUSC1 GARNL1 RDH13 TSGA2 C21orf5 MGC21874 LOC202451 LOC284591 FALZ F7 BAI2 SOX13 LARGE DACH1 BAZ2A CAPN9 KIAA0934 SIP Rab11-FIP3 IGSF4 MLPH DUSP8 RAI17 LASS2 FLJ13710 KIAA0232 SDCCAG3 ATP6AP1 ULK1 MAGED2 FLJ36445 DNALI1 C14orf83 ASB8 KIAA1718 HPX

163 Table 3.11 continued

USP3 C6orf29 RAB3D GTF3C1 FRS2 ARF3 ACVR1B C20orf22 ENPP1 FLJ14299 SECISBP2 ABCG1 FKBP4 ANXA9 MGC3121 WFS1 KIAA0913 MGC3047 FLJ36032 PVRL2 TGIF2 AD023 KIAA0542 T1 MYO6 LOH3CR2A C10orf82 CIRBP CCT6A KIAA1467 LOC151963 GARS KIAA1598 JMJD2B LMCD1 ESR1 SYNGR2 ARID2 FLJ12650 TTC9 SLC37A1 SFMBT2 FGFR4 FLJ10761 ASH1L FLJ20202 DDAH2 MTAC2D1 SPEC1 POLE TJP3 PTPRF HPN PCK2 GARNL3 PYCR1 LRRN1 CYB561 TCFL1 EPS8L1 NET-7 FLJ00133 ZNF74 POGZ GGA1 MGC39325 LRP3 DNAJA4 MYCN RALGPS1 GALNT6 GGA3 LUC7A INHBB LASS6 NME3 DAAM1 ZNF398 KIAA0556 ATP8B1 KIAA0089 C10orf12 RNF103 FLJ11323 CGI-40 BCAS1 AP1G1 MGC33338 TBX3 LYK5 SLC35A1 KIAA0763 SYCP2 IVD FLJ42654 LOC283232 C7orf32 KIAA1211 KRT19 CTXN1 GP1BB FLJ20847 MGC13010 KIAA0446 PGGT1B FLJ44186 TOMM70A FLJ90013 SRRM2 BLNK NACA MGAT4A ICA1 SLC38A1 DNAJC1 RHBDF1 LOC338692 MGC4368 CA12 FLJ21919 C9orf152 GPRC5C KIAA0767 PRKAG1 ZFYVE16 SLC2A10 RAB17 KIF12 XTP2 FLJ31568 TTC6 PDCL3 EGFL5 LOC284018 SOX12 KIAA1856 DKFZp586I1420 MAPK9 FLJ20274 LFNG UBN1 MYO5B LOC80298 KIAA1608

164 Table 3.12 Gene order on Basal-ECJ heatmap in Figure 3.7.

KRT5 COL4A2 CD44 MGC34923 C2orf25 CAV1 GBP1 DUOX1 OGFRL1 TAZ LEPREL1 ARPC2 KRT17 IGFBP7 ATP10D HBP17 MGC10946 EXT1 BNC1 UPP1 CASP1 CRIPT INHBA PBEF1 FAD104 (FNDC3B) PTRF LY6K KIAA0650 TLE4 TRIM29 IFI16 C1S GSTP1 COTL1 IL18 CARD6 PRKCDBP KBTBD9 PLAT DCTD TGFBR2 GJB3 IMP-3 NCK1 SRPUL KRT16 ANXA2 BIRC3 RGC32 IFITM3 AKR1B1 PSMB9 HIF1A ASXL1 AXL MSN FOXQ1 MT1H BF EREG AKR1B10 PLS3 LOX DHRS8 CDK6 DPYD TNFRSF6 MIRAB13 C21orf96 CXCL2 CAV2 GPX1 MT1E SFRP1 TP73L SPRY2 PTPNS1 ANXA1 ANTXR1 GPSM2 SNAI2 PIK3CD KIAA1959 NMI FLJ35036 S100A2 CD58 TNFAIP8 BTG3 LBH AKR1C3 KRT14 ELK3 RALBP1 CTSC LOXL2 ALDH1A3 DNAJB4 IFIT4 LOC285812 SGK FXYD5 DMD GALNT2 ACTN1 PPP4R1 SLPI HRH1 RBM7 RIOK3 DSG3 OSMR SCPEP1 KLK10 FGF2 KIAA1726 CDCP1 FLJ32028 KLK5 LOC137392 NNMT CYLD IF C6orf145 SPTBN1 EGFR S100A10 LUZP1 ASAM MSCP LYN NAB1 RBMS3 C6orf80 PERP FMNL2 CD109 CHST6 CAMTA1 UMP-CMPK HOXA1 TWIST2 FSTL1 TLR2 NOB1P B3GNT5 KIRREL HP1-BP74 KRT6A ETF1 NRP1 COPS8 EMP3 HOXA5 LAMC2 PSMB8 BMP1 CHMP1.5 KIAA0746 LAMA3 FLJ22833 FLJ20073 C20orf100 PLSCR1 KRT6B FOSL1 SLC9A6 TBC1D1 FLJ40432 COL8A1 DSC3 HOXA3 F3 CFLAR VAMP3 EPHA2 ETS1 NDEL1 PTGS2 IFI44 SLC1A3 PSAT1 GFOD1 PDGFC ANXA8 ANKRD3 BTN3A2 MT1G PHLDB2 SLC6A15 CDH3 AMSH-LP MT2A GAS1 MBNL1 EMP1 CALD1 SRPX NR3C1 DNAPTP6 ANXA2P1 ADPRTL1 TAP2 MAP17 C9orf5 LAMB3 NFAT5 SCHIP1 CD14 C21orf63 ELL2 MGC4655 IMP-2 FDFT1 FLJ21069 JAG1 IFNGR1 AGPS C10orf38 40422 FLJ23518 FGG IRX1 KRT15 MBNL2 ADORA2B PNLIPRP3 PRNP MFGE8 MAP4K4 RGS2 IL15 F2RL1 SDCCAG8 ANKH FLJ11196 FLJ13391 SCAP2 INPP1 WDR1 GABRE URB NT5E PRSS11 ARHGAP5 SART2 PI3 ITM2C BTN3A3 MT1X CRYAB MET RGS20 CSNK2A2 TNFAIP3 MAML2 RBM9 LOC51186 PRKCN FZD6 C3 PTK7 ARHE GBP3 PLAU C20orf42 COL4A1 FSCN1 SVIL C2orf12 CASP4 TGFBI KIAA1212 TKT ITGB1

165 Table 3.12 continued

DKFZp434D0215 KIF1B NDFIP2 C14orf31 ANXA4 TRIP10 DSG2 DIA1 RGL1 NFE2L2 RBMS1 ICAM1 SSFA2 FST CBR1 SAA1 FLJ43339 AKT3 CCNA1 ZNF145 FBLP-1 ARHGAP23 TIP-1 IL1RAP PTPRM NSFL1C BJ-TSA-9 CXCL3 B2M UBE2E3 TUBA1 ZDHHC2 FKBP1A MGC4083 GART CYBRD1 IRS2 CGI-100 LOC340061 DCBLD1 38245 PGM2 AIM1 ESDN ZNF258 FAP ANXA3 LIPG ALDH3A2 CFL2 GM2A FLJ12649 CSDA AKR1C1 GNAI1 DKFZP566E144 PHLDA1 GNG12 PKP2 GPM6B SOX7 KLF5 ITGA1 C6orf67 MMP14 SERPINB5 TGFA ATP1B3 CLIC4 RALB MGC45871 PRKCL2 OSBPL9 DCBLD2 CRK MYO1B C10orf10 GNA15 RPL5 AMD1 IFI27 PPP1R14C OSBPL3 ACY1L2 CGI-09 CDH13 GTF2B FLJ23091 STAT3 MPPE1 ADD3 CHST3 ITGA6 PPM2C BIN1 ITGA3 DGKA CORO1C LAMC1 NUP50 YES1 IGFBP6 NAV2 BPAG1 LRAP CD59 DOCK5 SLC16A1 SNX7 FHL1 CDC42EP3 SERPINE2 BICD2 GLIPR1 KIAA1228 TRIM22 ARNTL2 RRAS2 ADRB2 HMGA2 C20orf129 HRB2 MPZL1 PLA2G4A TWSG1 ATP1A1 NACSIN MTMR2 SP100 YAP1 IL1A PTPN2 HIC SRI SH3GLB1 RUNX3 ABCD3 CEBPD IL7R NUDT15 SERPINB2 AKAP2 ARAP3 STK17A VSNL1 NSEP1 SFN ETS2 HLA-E C1R RHBDL2 TBPL1 MME SOAT1 APOL6 STAT4 RAC2 MDH1 FLJ31951 SMAD3 LOC51240 MYO1E SH3KBP1 ADA KPNA1 MRCL3 TNFRSF10D MGC14376 CD97 DIRC2 RGNEF BBP ADM

166 Table 3.13

Primers used for PCR amplification of DNA that has been subject to FOXA1 ChIP.

Gene Primer Sequence ANXA1-1 F 5'-AAAATTTTGAGCCAATCTGGAA-3' ANXA1-1 R 5'-CCACCATGCCTAGCTGTTTT-3'

ANXA1-2 F 5'-GCCTGGGCAATATAACGAGA-3' ANXA1-2 R 5'-CTGCCCCAATCCTAATACCA-3'

CD58 F 5’-ATGGGCATCTCTCTGCAGTT-3’ CD58 R 5’-GGATTTGCCCTTGATCTTCA-3’

ERBB3 F 5’-TCCCCAGAGGTGTTGTTTGT-3’ ERBB3 R 5’-CGTGCCCATAAGTGTTTGTG-3’

FNDC3B F 5’-TATTTGGCGGGGCCTATTTA-3’ FNDC3B R 5’-TGCTGCATACTGTTCCCAAG-3’

KRT16 F 5'-CGCATGTTTCTTTGTGGCTA-3' KRT16 R 5'-CGGGATCTCCAGAAGTGTGT-3'

PAM F 5’-TAGAAAGGATGGCCACCAAG-3’ PAM R 5’-ACTTGCCGACAAAGCAGAGT-3’

SPDEF F 5’-CGAGTGAATGAGCGAGTGAA-3’ SPDEF R 5’-GCTGGGAGGAAGTCAGACAG-3’

TF F 5’-GCTGCTTCTCAAGGATGACC-3’ TF R 5’-GTGCAGGAAAGCTGGAAAAG-3’

TRIM2 F 5’-GTGTGCCCAGGGTAGTGTTT-3’ TRIM2 R 5’-TGACTGCCACTCCTCAACAG-3’

XBP1 F 5’-TCTCAGTGGGGGAGATCTTG-3’ XBP1 R 5’-GCCTGGTAAACCCCATTTCT-3’

167 Figure 3.1

FOXA1 is expressed in the absence of ERα in breast tumors and luminal cell lines.

(A) Representative FOXA1 IHC of ERα-positive (n = 32) and ERα-negative (n = 13) breast tumor sections. FOXA1 expression (brown) is counterstained with hematoxylin.

Scale bars = 100 µm. (B) FOXA1 is expressed in all ERα-positive and ~50% of ERα- negative tumors. ERα-negative tumors express significantly less FOXA1 than their ERα- positive counterparts as revealed by the IHC score (*p<1x10-6). Scores were computed by multiplying signal intensity (1 = lowest; 3 = highest) by the percentage of positive cells

(1=10%, 2=20%, etc.) (264). (C) Quantitative real-time PCR of FOXA1 mRNA levels in a diverse group of breast cancer cell lines. Bars represent the mean of three experiments

(cells harvested on separate occasions) ± s.e.m. relative to GAPDH. (D) Immunoblot analysis of a cohort of breast cancer cell lines for FOXA1 and ERα (BaA = Basal A; BaB

= Basal B).

168 Figure 3.1

169 Figure 3.2

FOXA1 expression correlates with the luminal subtype in breast cancer cell lines.

Publicly available cDNA microarray expression data from Neve et al. (2006) were evaluated for FOXA1 (top) and ESR1 (bottom) mRNA expression (336). Expression measurements are shown in log-scale. Cell lines with mRNA expression greater than the median (black line) for that probe are defined as positive. Cell lines are designated as luminal, basal A or basal B according to Neve, et al. (2006) (336). Immunoblot analysis for FOXA1 and ERα protein expression was performed on a representative subset

(arrows) (see Figure 1C). Luminal cell lines that express FOXA1, but have low levels of

ESR1 mRNA expression, are indicated by arrowheads in the bottom panel.

170 Figure 3.2

171 Figure 3.3

Loss of FOXA1 increases migration and invasion of luminal breast cancer cells.

(A-D) MCF7 and MB-453 cells were transiently transfected with non-targeting (NT) or siRNA targeting FOXA1 (siA1#1). At 48 hours post-transfection, cells were plated in modified Boyden chambers to analyze (A) migration at 24 hours or (B) invasion at 48 hours. (C) Number of viable cells (trypan blue excluded) at 48 hours post-transfection.

Bars in A-C represent the mean of three experiments ± s.e.m. relative to NT (*p<0.05;

**p<0.01). (D) Representative immunoblots confirming knockdown of FOXA1 with siA1#1 at 72 hours post-transfection (MCF7, n = 3; MB-453, n = 2).

172 Figure 3.3

173 Figure 3.4

Identification of a FOXA1-dependent luminal transcriptome.

(A) Representative immunoblots confirming FOXA1 knockdown with siA1#4 at 36 (n =

1) and 72 hours (n = 3) post-transfection. NT = non-targeting siRNA. (B) Schematic of the experimental design for each cell line. Technical replicates from each experiment were performed in triplicate and were processed for microarray analysis. Biological replicates were combined via error-weighted ANOVA. (C) Venn diagrams of commonly changed genes at 72 hours post-transfection (p<0.001).

174 Figure 3.4

175 Figure 3.5

Loss of FOXA1 decreases enrichment for luminal genes, while increasing enrichment for basal genes.

(A) Heat maps depicting expression changes of the genes comprising the Neve et al.

(2006) (RN) (336) luminal and basal A classifier lists upon knockdown of FOXA1

(siA1#4) at 72 hours post-transfection. Genes are ordered from highest to lowest classification power. A propensity of red or green is indicative of a directional shift in global expression of the gene classifier. (B) GSEA enrichment plots utilizing a subset of the luminal and basal discriminatory gene sets generated by Neve et al. (2006) (RN)

(336) and Charafe-Jauffret et al. (2006) (ECJ) (337). Vertical lines represent individual genes of the respective classifier that contribute to the enrichment score. Genes are ranked by signal to noise ratio: left (most positive) to right (most negative). Values below

0 indicate reduced enrichment of a signature gene set, while values above 0 indicate a gain in enrichment.

176 Figure 3.5

177 Figure 3.6

Loss of FOXA1 induces changes in RN classifier gene expression.

Heat maps depicting expression changes of the genes comprising the Neve et al. (2006)

(RN) (336) basal A and B classifier lists upon FOXA1 silencing (siA1#4) at 72 hours post-transfection. Genes are ordered from highest to lowest classification power. A propensity of red or green is indicative of a directional shift in global expression of the gene classifier.

178 Figure 3.6

179 Figure 3.7

Loss of FOXA1 induces changes in ECJ classifier gene expression.

Heat maps depicting expression changes of the genes comprising the Charafe-Jauffret et al. (2006) (ECJ) (337) luminal (B) and basal classifier lists upon FOXA1 silencing

(siA1#4) at 72 hours post-transfection. Genes are ordered from highest to lowest classification power. The genes are listed in the order shown in Table 3.11 and Table

3.12. A propensity of red or green is indicative of a directional shift in global expression of the gene classifier.

180 Figure 3.7

181 Figure 3.8

GSEA enrichment plots for BasB-RN, BasAB-RN, Lum(M)-ECJ and Mes-ECJ classifier lists.

GSEA enrichment plots were constructed using a subset of luminal and basal discriminatory gene sets generated by Neve et al. (2006) (RN) (336) and Charafe-Jauffret et al. (2006) (ECJ) (337). Vertical lines represent individual genes of the respective classifier that contribute to the enrichment score. Genes are ranked by signal to noise ratio: left (most positive) to right (most negative). Values below 0 indicate reduced enrichment of a signature gene set while values above 0 indicate a gain in enrichment.

182 Figure 3.8

183 Figure 3.9

Loss of FOXA1 induces basal mRNA and protein expression.

(A) MCF7, (B) T47D, (C) MB-453, and (D) SKBR3 cells were transiently transfected with non-targeting (NT) or siRNA targeting FOXA1 (siA1#4). (Left) Quantification of mRNA levels for a subset of luminal and basal classifying genes at 72 hours post- transfection. Bars represent the mean of three experiments ± s.e.m. relative to GAPDH mRNA (*p<0.05). (Right) Representative immunoblots at 72 hours post-transfection showing the induction of the basal protein, Annexin 1, in response to FOXA1 silencing (n

= 3).

184 Figure 3.9

185 Figure 3.10

FOXA1 binds to luminal and basal genes in luminal breast cancer cells. (A)

Representative (n = 3) FOXA1 ChIP of a subset of basal and luminal genes in MCF7 and

MB-453 cells. (B-C) Quantification of FOXA1 ChIP in MCF7 (B) and MB-453 (C) cells.

Bars represent % binding relative to input ± s.e.m..

186 Figure 3.10

187 CHAPTER 4

Summary and Future Directions

4.1 SUMMARY

Prior to our investigation, the understanding of the role for FOXA1 in breast cancer was primarily limited to its selective expression in the luminal subtype of breast cancer, its correlation with a more favorable patient prognosis, and its requirement in ERα target gene regulation. Based on this prior knowledge, we sought to define (1) whether the requirement for FOXA1 in ERα activity was similarly present in the normal mammary epithelium, and if so, whether this co-modulation was required for normal mammary gland development (Chapter 2), and (2) whether FOXA1 expression was required to maintain the luminal phenotype of breast cancer cells (Chapter 3).

To analyze mammary gland development in the absence of FOXA1, we utilized Foxa1 null mice. Investigation of the mammary glands from mice heterozygous for Foxa1 and those rescued from postnatally lethal Foxa1 null pups revealed that FOXA1 is essential for mammary ductal invasion in response to pubertal or pregnancy hormones. The absence of epithelial ERα expression in the Foxa1 null mammary glands provides a mechanism for this observed phenotype, which mimics that of the ERKO mouse. The requirement for FOXA1 in ERα expression was investigated further in breast cancer cell lines revealing that FOXA1 binds to the ESR1 proximal promoter, and that FOXA1 silencing in vitro reduces ERα mRNA and protein expression. These studies also defined

188 the transcriptional hierarchy of FOXA1, ERα and GATA3, another breast cancer luminal- subtype specific transcription factor, in the normal mammary gland. This was accomplished by analyzing expression of FOXA1, ERα and GATA3 in Foxa1 null,

Ex3αERKO and MMTV-Cre; GATA3LoxP/LoxP tissues. These studies demonstrated that loss of FOXA1 decreases ERα, but not GATA3 expression; loss of ERα does not affect either FOXA1 or GATA3; and conditional loss of GATA3 decreases ERα, but not

FOXA1. Additionally, unlike ERα and GATA3, FOXA1 is not required for terminal differentiation of the ductal epithelium into lobulo-alveoli. Combined, our investigation revealed a function for FOXA1 independent of ERα and GATA3 in mammary gland development, and for the first time, described FOXA1 as a necessary enhancer of ERα expression in addition to its activity. Future studies are needed to determine how loss of

FOXA1 affects mammary epithelial lineage differentiation and luminal tumorigenesis.

These are discussed in Section 4.2 and Section 4.3, respectively.

The observation that FOXA1 was consistently expressed in luminal breast tumors and cell lines led us to hypothesize that FOXA1 was necessary for maintaining the molecular phenotype of these less aggressive tumors. Moreover, although we described an indispensable role in for FOXA1 in ERα expression (Chapter 2), FOXA1 can be expressed in normal luminal epithelium, luminal tumors, and luminal cell lines in the absence of ERα. To address whether FOXA1 is independently necessary for maintenance of the luminal phenotype, we transiently silenced FOXA1 in luminal ERα-positive and luminal ERα-negative breast cancer cells, and monitored changes in the molecular phenotype. Loss of FOXA1 induces migration and invasion of these cells, which are in

189 vitro characteristics of the more aggressive, basal breast cancer subtype. Transcriptional profiling followed by GSEA also revealed a selective decrease in the expression of genes associated with the luminal subtype, concomitant with an enrichment of genes associated with the basal subtype. Combined, these data reveal that FOXA1 is not just a marker of the luminal subtype, but that it is functionally necessary for maintaining this phenotype.

FOXA1 actively represses basal associated gene expression, and data showing that

FOXA1 binds directly to a subset of the basal genes suggests that it does so at the level of transcription. Future studies investigating how FOXA1 mediates repression of these genes will be discussed in Section 4.4. Most importantly, our studies exhibit a requirement for FOXA1 in maintaining the less aggressive nature of luminal breast cancer. Others have proposed therapeutically reducing FOXA1 in luminal breast cancers as a means to increase the efficacy of tamoxifen, but our data suggests that decreasing

FOXA1 will inadvertently increase the aggressiveness of these cancers. We suggest developing a method to induce FOXA1 in basal breast cancers in order to induce differentiation and hormone responsiveness to these essentially untreatable tumors.

Future analyses that involve over-expressing FOXA1 in basal cell lines and mouse models are discussed in Section 4.3, and those defining the mechanisms that regulate

FOXA1 expression in breast cancer are discussed in Section 4.5.

190 4.2 IS FOXA1 EXPRESSION NECESSARY FOR MAINTAINING THE

MAMMARY EPITHELIAL LINEAGE?

In Chapter 2, we describe an indispensable role for FOXA1 in mammary gland morphogenesis. FOXA1 is necessary for ductal invasion throughout the mammary fat pad in response to pubertal and pregnancy hormones, and this phenotype is attributed to the loss of epithelial ERα expression, a known mediator of this developmental process.

Although the Foxa1 null mammary glands are severely stunted, there is no apparent change in the expression of known markers of the luminal (E-cadherin, CK8) or basal (α-

SMA) lineages at 4-5 weeks post-transplantation suggesting that loss of FOXA1 is not affecting epithelial lineage maturation (Figure 2.5). However, we have found that the

Foxa1 knockout glands exhibit loss of CK5/6, a historically basal marker that is also expressed in the luminal progenitor lineage (149) (Figure 4.1). These results indicate that glands lacking FOXA1 may have an aberrant shift in the normal distribution of the mammary epithelial populations. In addition to being highly expressed in the mature luminal epithelium, FOXA1 is expressed in the luminal progenitor population, as seen by positive immunostaining of the body cells in the TEB (Figure 2.1A) and by FACS analysis (Figure 2.4C). Combined, these data imply that FOXA1 expression in luminal progenitor cells may be necessary to maintain the proper distribution of the mammary epithelial lineages.

We can test this directly by analyzing the percentage of mammary stem cell (MaSC), luminal progenitor and mature luminal populations present in wild type and Foxa1 null

191 glands through marker assisted FACS as described in Section 2.3 and in (144). This technique requires a large amount of starting tissue, or approximately 10 inguinal glands from adult virgin animals. The experiments examining loss of FOXA1 in mammary tissue discussed in Chapter 2 utilized a renal capsule transplantation approach to rescue the postnatally lethal Foxa1 null glands. Although this model has many advantages, the surface area of the resulting mammary gland is no larger than the size of the kidney it was transplanted into, and thus, is only a fraction of the size of a mammary gland from an adult mouse. This limitation led us to transition our studies into a mammary-specific

Foxa1 knockout mouse model to bypass the postnatal lethality of these mice. Mice harboring LoxP sites flanking exon 2 of the Foxa1 allele (Foxa1loxP) were generated previously by Klaus Kaestner’s research group (90), and are currently being crossed with

MMTV-Cre transgenics in our laboratory. Of note, the MMTV promoter is well described to induce/delete expression in tissues such as the testis, salivary glands, pancreas, etc. (352). Crossing the Foxa1loxP mice with the MMTV-Cre Line F mice (353) resulted in an absence of MMTV-Cre; Foxa1LoxP/LoxP progeny, and thus, this promoter was not sufficiently selective to bypass the hypoglycemic/dehydration defect that causes postnatal lethality in global Foxa1 null mice. We have obtained a different line of

MMTV-Cre mice (Line D), and are in the process of testing the efficacy of this promoter on mammary-specific deletion of Foxa1.

Upon successful genotyping of conditionally null animals, mammary glands will be harvested from wild type, heterozygous and null females at mid- (7-8 weeks of age) and post-puberty (10-12 weeks of age) to confirm a dose dependent reduction in FOXA1

192 mRNA and protein expression. An inguinal gland from the same mouse will also be removed for whole mount analysis to determine if Foxa1 loss alters mammary gland ductal invasion as seen with the rescued Foxa1 null glands in Chapter 2. Differences in the epithelial subpopulations of wild type versus Foxa1 conditionally null mammary glands will be assessed by performing IHC and FACS for lineage specific markers. The efficacy of the FACS experiment will also require confirmation by analyzing the in vivo repopulating capacity of the sorted cells, and by examining each population for expression of subpopulation-specific markers.

Given that Foxa1 null mammary epithelia have greatly reduced CK5/6 expression, and that FOXA1 is expressed in the luminal progenitor population, we predict that loss of

FOXA1 will reduce the percentage of luminal progenitor cells compared to wild type tissue. It will be interesting to determine if this loss results in a concomitant gain in either the MaSC or mature luminal populations. Since loss of FOXA1 has no effect on lobulo- alveologenesis and milk secretion (Figure 2.7), it is possible that there will be an increase in the percentage of mature luminal epithelial cells. This result would indicate that the conditionally null Foxa1 glands may be entirely terminally differentiated, and may not be capable of repopulating the tissue after pregnancy. Performing the same analysis just described on females after one or two rounds of pregnancy will answer this question. It is also possible that loss of FOXA1 will increase the percentage of MaSCs, which have characteristics recapitulating the basal breast cancer subtype (354). As discussed in

Section 1.4.2, the cell of origin of basal-like breast cancers has been hypothesized as the

MaSC (355), but more recently evidence suggests it is the luminal progenitor cell (149,

193 150). Given that loss of FOXA1 in human breast cancer cell lines induces a shift towards the basal molecular phenotype (Chapter 3), we expect that loss of FOXA1 under normal conditions will lead to an increase in the epithelial population representative of the cell of origin of the basal subtype of breast cancer. Thus, these studies should provide novel insight into the initiation of basal-like breast cancers. To determine if FOXA1 is a breast tumor suppressor, these mice should be also be aged to investigate whether loss of

FOXA1 alone is sufficient to induce tumorigenesis. Lastly, the experiments described in this section will be essential to establish a foundation for interpreting how FOXA1 mediates tumor progression in mouse models of luminal and basal breast cancer as described in the next section.

194 4.3 DOES MANIPULATION OF FOXA1 ALTER BREAST CANCER SUBTYPE

TUMOR PROGRESSION?

FOXA1 actively represses the basal breast cancer subtype in human breast cancer cell lines (Chapter 3). These results were obtained by transiently silencing FOXA1 in luminal breast cancer cells, and thus, only investigated phenotypic changes that occurred over the course of three days. Future studies stably reducing FOXA1 in luminal breast cancer cells will be necessary to conduct long-term experiments testing whether loss of

FOXA1 increases anchorage independent growth, in vivo tumor formation, and metastatic progression. In parallel, stable over-expression of FOXA1 in basal A breast cancer cell lines should induce the more differentiated luminal phenotype, and reduce the tumorigenicity of these cells. We have attempted to stably knockdown FOXA1 in MCF7 cells, but due to the requirement of FOXA1 for ERα expression (Chapter 2), and the requirement for estrogen-induced ERα activity for survival of these cells (356), we were unable to generate stable populations with successful knockdown. Performing stable knockdown of FOXA1 in luminal, ERα-negative cells (MB-453, SKBR3) should overcome this limitation. Additionally, FOXA1 stably over-expressed in a basal B cell line (MB-231) induces the up-regulation of CDH1 mRNA (encodes E-cadherin), an epithelial-like morphology and a decrease in migration (306). These results support our observation that FOXA1 mediates breast cancer aggressiveness, but we found FOXA1 preferentially represses basal A genes, not basal B genes (Chapter 3). Furthermore, basal

A cell lines more accurately reflect the human basal-like subtype (336, 337). Stable over-

195 expression of FOXA1 in basal A cells (HCC1187, HCC1143) should determine if enforced FOXA1 expression causes a loss of the basal phenotype.

In addition to being necessary for maintaining the luminal breast cancer subtype in human cancer cell lines, our data is also suggestive that loss of FOXA1 in a mouse model of luminal breast cancer will result in a more basal-like tumor. To test this, we will cross the conditionally null Foxa1 mice (MMTV-Cre; Foxa1LoxP/LoxP) described in the previous section with MMTV-PyMT mice, which develop luminal tumors (147). These mice express the polyoma virus middle T antigen downstream of the MMTV promoter, and while tumor latency is dependent on strain, the line (634) we are currently maintaining in the laboratory develops palpable tumors around 5 weeks of age, and most develop lung metastasis at three months of age (357). Tumor latency, tumor size and extent of lung metastases will be compared between mice with two conditionally null alleles of Foxa1 in the presence and absence of the PyMT transgene. These data will reveal whether loss of FOXA1 affects several aspects of tumorigenesis. We anticipate that an increase in tumor aggressiveness (e.g. decreased tumor latency, increased tumor size, increased number of metastases) will be a result of these tumors being phenotypically more basal.

To test this directly, we will examine the tumors for loss of luminal-associated markers concomitant with the gain of basal-associated markers by qRT-PCR analysis and IHC.

Similar results have been reported for MMTV-PyMT mice heterozygous for Adipoq, the gene encoding adiponectin (358). Importantly, the initiation of MMTV-PyMT tumors is estrogen-dependent (359, 360), and thus it is possible that loss of FOXA1 via MMTV-

Cre-mediated excision will reduce tumor formation due to secondary loss of ERα. These

196 data would not negate our in vivo findings that loss of FOXA1 increases breast cancer aggressiveness, but would further support a requirement for FOXA1/ERα signaling in the early stages of MMTV-PyMT tumorigenesis. To investigate how loss of FOXA1 alters the tumor phenotype after tumor initiation, we will excise established MMTV-PyMT tumors, dissociate the cells, and stably transfect with an shRNA to FOXA1 ex vivo. After confirming FOXA1 knockdown, the cells will be injected into the cleared fat pads of three week old syngeneic recipients as described for the orthotopic transplants in

Chapter 2. This strategy has been successfully used in the stable reduction of GATA3 in the MMTV-PyMT tumors (146). A second, but much more labor-intensive method is to generate a mammary specific doxycyline-inducible Cre mouse model to selectively delete

FOXA1 in the mammary gland (MMTV-tTA; tetO-Cre; FOXA1LoxP/LoxP) after tumor initiation, however this requires expression of MMTV-PyMT and MMTV-tTA in the same cells and this is not guaranteed. Providing evidence that loss of FOXA1 induces a more basal-like phenotype of breast tumors will confirm a role for FOXA1 in maintaining the luminal lineage and further support the plasticity of breast cancers as described in Chapter 3. Importantly, if the MMTV-Cre; Foxa1LoxP/LoxP mice are found to develop mammary tumors, it will be interesting to investigate the molecular subtype of these tumors, which we predict will be representative of the basal-like subtype.

In parallel to testing how FOXA1 over-expression alters basal A breast cancer cell characteristics in vitro as already discussed, it is also necessary to determine if forced over-expression of FOXA1 decreases tumor formation in mouse models of basal breast cancer. If so, these data would strongly support the development of therapeutic strategies

197 to induce FOXA1 expression in women with basal-like breast cancer. To begin to test this, mice exogenously expressing Foxa1 downstream the MMTV promoter will be engineered and characterized. Lines with high levels of FOXA1 in the mammary gland will then be crossed with MMTV-Wnt1 mice. MMTV-Wnt1 mice develop basal-like mammary tumors (147) likely due to induction of Wnt1 signaling in the basal cell population (361) and increased MaSC number (143). Importantly, FOXA1 is undetectable these tumors (302). Changes in tumor latency, volume and metastatic progression will be determined for MMTV-Wnt1 mice with and without MMTV-Foxa1 over-expression. The resulting tumors will be tested for mammary epithelial lineage composition as described previously. We predict that FOXA1 over-expression will reduce the MaSC number, induce differentiation, and reduce the tumor aggressiveness of these basal-like tumors. If substantiated, these results would strongly support targeting

FOXA1 therapeutically in basal breast cancers. Although both transgenes are driven by the same MMTV promoter, it is possible that FOXA1 and Wnt1 will not be induced in the same cells, and thus Wnt1-induced tumorigenesis will occur in the absence of

FOXA1 co-expression. It will be necessary to confirm both FOXA1 and Wnt1 tumor expression to verify transgene activity. Furthermore, the MMTV-Wnt1 promoter enhances Wnt1 secretion from the luminal cells, resulting in paracrine signaling to the basal/myoepithelium, which are the hypothesized cell of origin of these tumors. Although we predict that FOXA1 over-expression should induce differentiation and decrease the number of resident MaSC, there is a chance that even a reduction in the number of MaSC will not be enough to decrease tumorigenicity in these mice. An alternative approach is to

198 induce FOXA1 over-expression in the basal/myoepithelium directly using a K14 promoter.

199 4.4 HOW DOES FOXA1 REPRESS BASAL BREAST CANCER GENE

EXPRESSION?

In Chapter 3, we provide evidence that FOXA1 represses the basal breast cancer phenotype, where loss of FOXA1 induces basal-associated gene expression and breast cancer aggressiveness as measured in vitro. ChIP studies revealed that FOXA1 likely mediates repression through direct association with the basal genes (Figure 3.10). As mentioned in the discussion section of Chapter 3, FOXA1 does not have known intrinsic repressor activity, but it has been reported to participate in gene repression through interaction with HDAC7 in breast cancer cells (284), Groucho 3 (Grg3) in hepatocytes

(345) and with a NuRD complex in C. elegans (348). To begin to define the mechanism by which FOXA1 represses basal breast cancer genes, the potential cooperation of

FOXA1 with HDAC7, Grg3 and the components of the NuRD complex should be tested in both ERα-positive and ERα-negative breast cancer cell lines to define an ERα- independent, luminal-dependent role for FOXA1 in this process. Initial experiments should examine if knockdown of the potential co-repressor(s) recapitulates the luminal to basal transcriptional shift observed with knockdown of FOXA1 in these cells. These experiments will require verifying successful knockdown of the co-repressor at the mRNA and protein level, and should also determine if reducing co-repressor expression causes a change in FOXA1 levels. These studies will reveal which, if any, potential co- repressor complex warrants further investigation. It is possible that knockdown of one or more of the co-repressors will induce basal gene expression exclusively in ERα-positive or ERα-negative cells. This is certainly possible for HDAC7, which has been shown to

200 complex with both FOXA1 and ERα in mediating repression of RPRM in MCF7 cells

(284). Upon identifying a putative complex, additional studies should determine if the co- repressor(s) bind to the same regions of the basal genes that bind FOXA1 (Figure 3.10).

These studies should also be done in the presence and absence of FOXA1 knockdown to verify that loss of FOXA1 alleviates binding of the co-repressor(s) to the target genes.

Finally, we predict that FOXA1 directly complexes with the co-repressor(s), and this can be tested through co-immunoprecipitation analysis. Identifying a FOXA1-directed repressor complex in luminal breast cancer will reveal putative therapeutic targets to induce repression of the basal breast cancer transcriptome in these essentially non- targetable cancers.

Another mode by which FOXA1 may be repressing basal gene expression is by competitively inhibiting another transcription factor from binding and inducing transactivation. FOXA1 is known to function in this context by antagonizing FOXA2 transcriptional activation in the endoderm (362). While FOXA2 is not expressed in the breast, expression of another forkhead box transcription factor, FOXC1, is inversely correlated with FOXA1 in breast tumors and cell lines ((188); data not shown). In this context, FOXA1 is expressed in luminal breast cancers, while FOXC1 is expressed in basal breast cancers. It is possible that the relative levels of the two transcription factors dictates a subtype-specific set of gene transcription: high levels of FOXA1 leading to luminal gene expression, and high levels of FOXC1 leading to basal gene expression. In this case, loss of FOXA1 would permit FOXC1 binding to high affinity consensus sites resulting in transcriptional activation. Although FOXC1 is undetectable in these cells,

201 low expression of this factor may be sufficient to cause activation of the basal signature genes. This could explain why loss of FOXA1 only elevates expression of a subset of the basal genes. The recruitment of FOXC1 to these genes upon knockdown of FOXA1 would substantiate this hypothesis. If true, then knockdown of FOXA1 in combination with FOXC1 over-expression should completely shift luminal breast cancer cells to a basal phenotype. In parallel, FOXC1 silencing in the presence of FOXA1 over- expression should induce basal breast cancer cells to become luminal. The combined manipulation of these two FOX family members could be more efficacious than therapeutically inducing FOXA1 expression in basal breast cancers alone.

The induction of FOXA1 expression is also known to delineate an epigenetic program during retinoic acid differentiation of P19 embryonal carcinoma cells (13). In these cells, retinoic acid treatment induces FOXA1 and its subsequent binding to enhancer regions.

Importantly, FOXA1 binding occurs prior to H3K4 mono- and di-methylation, and DNA hypomethylation, and therefore functions in establishing competency within these enhancer regions. FOXA1 is similarly correlated with DNA hypomethylation and H3K4 dimethylation in breast and prostate cancer cells (13, 201). These data suggest that loss of

FOXA1 could subsequently cause an alteration in histone and/or DNA methylation patterns of basal genes resulting in transcriptional activation. We have investigated changes in promoter methylation through methylated DNA immunoprecipitation followed by microarray analysis (MeDIP) as briefly described in Chapter 3, but found no difference in the CpG methylation status of the basal gene promoters between cells transfected with the non-targeting siRNA versus the siRNA targeting FOXA1. However,

202 there were two major limitations with this study. First, the microarrays were designed to test CpG islands within proximal promoter regions, but FOXA1 is known to mediating changes at distal enhancers as has been previously described (201). These experiments should be repeated examining immunoprecipitated methylated DNA after FOXA1 knockdown on full genome arrays, through MeDIP followed by next generation sequencing, or through whole-genome shotgun bisulphate sequencing. Another limitation of the study reported in Chapter 3 was the transient time frame of FOXA1 silencing.

Analyzing DNA methylation upon stable knockdown of FOXA1 (discussed in Section

4.3) will permit analyzing DNA methylation after several rounds of DNA replication.

Changes in histone methylation at the basal genes should also be investigated with and without FOXA1 silencing. ChIP with antibodies specific for the varying methylation states of active (H3K4-me1, me2, me3), and repressive (H3K9-me1, me2, me3) histone configurations should be performed (363).

203 4.5 HOW IS FOXA1 DIFFERENTIALLY REGULATED IN BREAST CANCER?

FOXA1 is expressed specifically in the luminal subtype of breast cancer, and based on our data its expression in this subtype is necessary for repression of genes associated with the more aggressive, basal phenotype (Chapter 3). Those factors that positively regulate expression of FOXA1 in luminal cells, or negatively regulate FOXA1 in basal cells are putative therapeutic targets to enforce expression of FOXA1 in basal breast cancers.

Interestingly, while transcriptional regulation by FOXA1 has been studied for decades with countless target genes being identified during developmental progression and in cancer as discussed in Chapter 1, the mechanism(s) regulating the expression of FOXA1 are less defined, with a smattering of publications in a wide variety of biological systems and tissue types. If we are to pharmacologically induce FOXA1 in basal breast cancers, we need to further investigate how FOXA1 is repressed in these cancers, as well as how it is activated in luminal breast cancers.

Outside of the breast, FOXA1 is positively regulated by dexamethasone in the rat liver

(364), TGFβ in mouse hepatocytes (365), SOX17/β-catenin in Xenopus embryos (303),

SOX4 in the human prostate (208), and C/EBPβ in mouse adipocytes (366). Retinoic acid treatment induces FOXA1 expression in mouse and human embryonal carcinoma cells

(13, 367-370), where FOXA1 is a primary target of RAR mediated transcriptional activity (370). In addition, FOXA1 has been shown to regulate it own promoter suggesting positive feedback (119). The promoter is also positively regulated by thryroid transcription factor-1 (TTF), and is repressed by UF1-H3β and SP1 (119). Each of these

204 pathways participates in carcinogenesis, and thus, it is worthwhile directing future studies to investigate each pathway’s ability to stimulate FOXA1 transcription in breast cancer cells as described below.

The mechanisms underlying the induction of FOXA1 expression specifically in breast luminal epithelium have not been described, although several groups have placed FOXA1 downstream a handful of signaling pathways in breast cancer cell lines and mouse models. First, several studies have suggested that FOXA1 may be an estrogen-responsive gene, being both up-regulated (278, 291) and down-regulated (263, 292-294) with E2 treatment in MCF7 cells. However, our data revealed that mice lacking ERα maintain

FOXA1 expression in the mammary epithelium suggesting that ERα is not required for

FOXA1 expression in normal cells, in vivo (Figure 2.8). Others have shown that treatment of breast cancer cells with either heregulin or testosterone induces FOXA1 expression (273). In MCF7 cells, the transcription factor AP-2γ (TFAP2C) binds to the

FOXA1 promoter and is necessary for maintaining FOXA1 expression (371). Lastly, mice over-expressing Wnt1 in combination with integrin-linked kinase (ILK) in the mammary gland (MMTV-Wnt1/Ilk) have increased expression of FOXA1 in tumors arising in the mammary glands of these mice, although the precise mechanism behind this induction has not been defined (302).

In total, these studies are suggestive that transcription of FOXA1 in breast cancer cells may be responsive to direct binding and regulation by SOX17/β-catenin, SOX4, C/EBPβ,

TTF, UF1-H3β, SP1 and/or AP-2γ. FOXA1 may also be downstream of TGFβ signaling

205 through SMADs, heregulin signaling through the EGFR family and/or retinoic acid signaling through RAR. Interestingly, FOXA1 gene regulation via dexamethasone through GR, estradiol through ERα, and testosterone through AR in combination with the requirement for FOXA1 in mediating GR, ERα and AR transcriptional regulation as described in Chapter 1, suggest a cross-modulatory feedback loop between FOXA1 and these nuclear receptors. This has been proposed for FOXA1 and ERα previously (278,

291). Consensus elements for C/EBPβ, SP1, GR, RAR and ERα are present in the human

FOXA1 proximal promoter (-2000bp) as determined in silico by the online Transcription

Element Search System (TESS), but there are no putative sites for SOX17, SOX4, UF1,

TFF1, AP-2γ or SMADs (data not shown). To begin to identify which of these transcription factors mediates FOXA1 gene expression in luminal breast cancer, activity of the human FOXA1 promoter should be investigated in both ERα-positive and ERα- negative luminal breast cancer cells. Constructs containing portions of the promoter at varying lengths will define particular regions critical for FOXA1 transcription within the different cell types. Narrowing down the regions of the promoter that are necessary for transcriptional activity should reveal the transcription factor(s) necessary for expression.

Upon identification, expression of these factor(s) can be silenced to confirm their role in potentiating FOXA1 mRNA and protein expression. Furthermore, the direct binding of these factors to the FOXA1 gene can be assessed by ChIP at predicted consensus sites.

Over-expression of these factors may induce expression of FOXA1 in basal breast cancer cells, depending on the chromosomal configuration of FOXA1 in these cells. If the signaling pathway is ligand-mediated (e.g. retinoic acid, estradiol), then promoter

206 activity, FOXA1 mRNA and protein expression should be also determined with and without ligand treatment.

Performing these experiments in both ERα-positive and ERα-negative luminal breast cancer cells will delineate whether the identified regulatory mechanism underlies FOXA1 expression in the luminal subtype of breast cancer. Of the five (C/EBPβ, SP1, GR, RAR and ERα) putative transcription factors listed above, only ERα is expressed specifically in the luminal subtype, but as discussed in Chapter 3, is not expressed in all luminal tumors/cell lines. A factor conferring subtype specific expression of FOXA1, that is itself not subtype-specific would imply the involvement of additional co-factors that form a unique higher-order complex that induces FOXA1 expression. It is also possible that a multimeric complex controls transcription of FOXA1 in breast cancer cells where only one component of the complex is subtype-specific. Lastly, rather than requiring a unique activator, the expression of FOXA1 in luminal cells may be due to absence of expression of a basal subtype-specific repressor. Hence, it will also be necessary to investigate the

FOXA1 promoter in a basal cell line that lacks FOXA1 expression. Deletion of repressive

DNA sequences and silencing of putative repressors will reveal which is necessary for inhibiting FOXA1 expression in this subtype.

Epigenetic control of the FOXA1 gene has been described previously in basal B cell lines

(MB-231, MB-435). These studies revealed that treatment with DNA methyltransferase

(5-Aza-2’-deoxycytidine) or histone deacetylase (HDAC) [trichostatin A (TSA); valproic acid] inhibitors increases FOXA1 expression in these cells (306, 372). These results imply

207 that the FOXA1 may not be transcribed in these cells as a consequence of promoter hypermethylation and/or histone deacetylation. Alternatively, a factor necessary for

FOXA1 transactivation could be itself regulated epigenetically. Bisulfite sequencing of the FOXA1 promoter in both luminal and basal cells will reveal if DNA methylation is a subtype-specific mechanism of transcriptional control. The histone acetylation status of the promoter should also be determined.

In addition to being regulated at the level of transcription, expression of FOXA1 in breast cancer may also be controlled by post-transcriptional or post-translational mechanisms.

Our preliminary studies have revealed that treatment of MCF7 and SKBR3 cells with

TSA reduces FOXA1 expression to nearly undetectable levels (data not shown).

Interestingly, these data are in complete contrast to that seen in the basal B cells lines just discussed, where treatment with TSA induces expression of FOXA1. A similar dichotomy has been described for the regulation of ESR1 where TSA indirectly reduces

ESR1 mRNA in MCF7 cells by altering the subcellular localization of HuR, an RNA binding protein responsible for maintaining ESR1 mRNA stability (373). FOXA1 mRNA stability in the presence and absence of TSA should be tested in MCF7 and SKBR3 cells by inhibiting nascent RNA synthesis with actinomycin D. HuR binds to the 3’-UTR of mRNA, and thus the removal of the 3’-UTR of the FOXA1 transcript should alleviate the decrease in FOXA1 mRNA observed with TSA treatment. Lastly, the altered subcellular localization of HuR in response to TSA treatment should be confirmed by performing westerns for both nuclear and cytoplasmic fractions, and through immunofluorescence of

208 HuR in ERα-positive and ERα-negative breast cancer cell lines to determine if this is a generalized effect.

In addition to directly inhibiting histone deacetylases, HDACs are also known to deacetylate non-histone substrates, such as transcription factors (374). Interestingly,

FOXA1 is acetylated, and its acetylation status regulates binding to DNA (375). These data, in combination with the auto-regulation of FOXA1 described previously (119), suggest that the observed decrease in FOXA1 mRNA expression with TSA treatment may be due to the FOXA1 protein becoming fully acetylated. This would displace FOXA1 from chromatin and block its ability to induce its own expression. To test this, the acetylation status of FOXA1 upon TSA treatment can be determined by immunoprecipitating FOXA1, followed by western analysis using an antibody that recognizes acetylated lysine residues. The absence of bound FOXA1 to its promoter would further support this hypothesis. Of note, FOXA1 binds ~7000bp upstream of its own transcriptional start site in MCF7 cells as determined by ChIP-chip (201). Lastly, previous analysis has revealed BRCA1 expression increases FOXA1 expression by increasing its protein stability (307). BRCA1 related cancers are characterized as basal- like (376). These data suggest that the loss of BRCA1 expression may be directly responsible for the absence of FOXA1 expression in the basal subtype. The protein stability studies performed by Williamson et al. (2006) were performed in a colon cancer cell line, and thus, it is necessary to repeat these experiments in breast cancer cell lines.

The protein half-life of FOXA1 with and without stable over-expression of BRCA1 should be determined by inhibiting with cycloheximide. Another approach is

209 to transiently silence BRCA1, and determine the protein stability of FOXA1 by pulse- chase with [35S]-methionine.

4.6 CONCLUDING REMARKS

Since first described as a liver-specific transcription factor in 1989 (2), FOXA1 has been linked to the development and differentiation of multiple tissues. FOXA1 has roles in the pancreas, kidney, liver, lung, gastro-intestinal tract, brain, prostate, and based on our studies, is also required for normal mammary gland development. Commonly, FOXA1 is critical in mediating lineage specification and has a propensity to cooperate with nuclear hormone receptors (e.g. ERα and AR). We show for the first time, that in addition to being necessary for ERα activity, FOXA1 is also necessary for its expression under both normal and cancerous conditions. We have also revealed that FOXA1 correlates with the luminal subtype of breast cancer, even more so than ERα, and that its expression is functionally required for maintenance of the luminal molecular phenotype by actively repressing basal associated gene expression. Our studies have led to the generation of many testable hypotheses that will further define the luminal-specific expression of

FOXA1 in breast cancer, and possible identify a means to induce FOXA1 expression in basal-like breast cancer.

210 Figure 4.1

FOXA1 is required for expression of cytokeratin 5/6 in the normal mammary gland.

Representative images of CK5/6 IHC (brown nuclei) in renal grafts from (A) Foxa1+/+ and (B) Foxa1-/- mammary glands harvested 4-5 weeks post-transplantation (+/+, n = 3; -

/-, n = 3). All sections were counterstained with hematoxylin. Scale bars = 20 µm.

211 Figure 4.1

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