TRANSCRIPTIONAL REPRESSION OF THE NUCLEAR COACTIVATOR AIB1 BY FOXG1 LEADS TO APOPTOSIS IN BREAST CANCER CELLS

A Dissertation submitted to the Faculty of the Graduate School of Arts and Sciences of Georgetown University in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Pharmacology

By

Jordan Victoria Li, M.S.

Washington, DC April 21, 2013

TRANSCRIPTIONAL REPRESSION OF THE COACTIVATOR AIB1 BY FOXG1 LEADS TO APOPTOSIS IN BREAST CANCER CELLS

Jordan Victoria Li, M.S.

Thesis Advisor: Anna Tate Riegel, Ph.D.

ABSTRACT

The oncogene nuclear receptor coactivator amplified in breast cancer 1 (AIB1) is a transcriptional coactivator that is overexpressed in various types of human cancers. However, the molecular mechanisms controlling AIB1 expression in the majority of cancers remain unclear. In this study, we identified a novel interacting of AIB1, forkhead-box protein G1 (FoxG1), which is an evolutionarily-conserved forkhead-box transcriptional corepressor. We show that

FoxG1 expression is low in breast cancer cell lines, and that low levels of FoxG1 are correlated with a worse prognosis in breast cancer. We also demonstrate that transient overexpression of

FoxG1 can suppress endogenous levels of AIB1 mRNA and protein in MCF-7 breast cancer cells. Exogenously expressed FoxG1 in MCF-7 cells also leads to apoptosis that can be rescued in part by AIB1 overexpression. Using immunoprecipitation (ChIP), we determined that FoxG1 is recruited to a region of the AIB1 promoter previously characterized to be responsible for AIB1-induced, positive auto-regulation of transcription through the recruitment of an activating, multiprotein complex, involving AIB1, and Sp1. Increased FoxG1 expression significantly reduces the recruitment of AIB1, E2F1 and p300 to this region of the endogenous AIB1 gene promoter. Our data imply that FoxG1 can function as a pro-apoptotic factor in part through suppression of AIB1 coactivator transcription complex formation, thereby reducing the expression of the AIB1 oncogene.

ii DEDICATION

I dedicate this work to

Sharon Edwards, Liu Hongjun (刘鸿君) and Li Jiali (李加利)

for giving me the physical strength and mental capacity to achieve my goals;

for teaching me to be humble but also to always take pride in what I do;

and most importantly,

for their unrelenting love, without which nothing would have been possible in this life.

iii ACKNOWLEDGEMENTS

I would like to first and foremost thank my mentor, Dr. Anna T. Riegel, whose critical insights and constant encouragement have guided me through this difficult process. I would also like to thank my “co-mentor” Dr. Anton Wellstein whose wisdom and enthusiasm for science have made this work possible. I would like to thank both Dr. Riegel and Dr. Wellstein for giving me the opportunity to be a part of their lab, for teaching me to approach and solve problems logically and scientifically; and, most importantly, for training me to be a good scientist and thinker not only for my prospective discipline but also for life.

I was also extremely lucky to have had the additional “mentorship” from Dr. Christopher

Chien whose expertise in molecular biology and enthusiasm in teaching have helped me tremendously in developing and completing this project. I would also like to thank Dr. Jason

Garee and Dr. Virginie Ory for their friendship, great sense of humor, and all the fun science and non-science related discussions. I thank all the members of the Riegel and Wellstein labs for yours support and interest in my research.

Finally, I thank Dr. Christopher Albanese, Dr. Jeffrey Toretsky and Dr. Barry Wolfe for serving on my committee. Your insights, suggestions and guidance for experiments have been crucial in bringing this project to completion.

iv TABLE OF CONTENTS Page

I. Introduction 1

A. Hormone Signaling: A Brief History 2

B. Nuclear Receptors 4

C. The p160 steroid receptor coactivators 5

1. Steroid Receptor Coactivators Superfamily 5

2. Structural and functional Domains of SRCs 6

D. Steroid Receptor Coactivator AIB1 10

1. The Role of AIB1 in Normal Physiology 10

2. AIB1, the Master Gene Regulator 12

3. AIB1 is an oncogene and its overexpression fuels tumorigenesis 14

4. Regulation of AIB1 Expression and Activity 17

E. Transcriptional Repression of AIB1 by Foxg1 19

II. Material and Methods 22

A. Plasmids 23

B. Cell Lines and Transient Transfection 23

C. Western Blot, Nuclear Extraction and Immunoprecipitation 23

D. Kaplan-Meier (KM) Analysis 24

E. Annexin V Apoptosis Assay and Flow Cytometry 25

F. ChIP and ChIP-reChIP 25

G. Luciferase Reporter Assay 29

H. RNA Extraction and Real Time PCR 29

III. Results 31

v A. AIB1 Interacts With The Transcriptional Corepressor FoxG1 32

1. Confirmation of the interaction between AIB1 and FoxG1 32

2. AIB1Δ4 also binds to FoxG1 34

3. FoxG1 transcript levels in breast cancer cell lines 36

4. Prognostic significance of FoxG1 in human breast cancer 37

B. FoxG1 Induces Apoptosis in MCF-7 Cells and Represses AIB1 Expression 40

1. Induction of apoptosis in MCF-7 cells by FoxG1 overexpression 40

2. Foxg1 overexpression reduces both AIB1 mRNA and protein levels 43

3. FoxG1-mediated apoptosis in MCF-7 cells is due to AIB1 downregulation 46

C. FoxG1 represses AIB1 promoter activity 49

1. A previously established model on the regulation of AIB1 promoter activity 49

2. FoxG1 represses AIB1 promoter activtity 51

3. The Sp1-binding element is required for FoxG1 recruitment to the transfected AIB1 promoter construct 54

D. FoxG1 Forms a Complex with AIB1 and E2F1 on the Endogenous AIB1 Gene Promoter 58

1. FoxG1 is recruited to the endogenous AIB1 gene promoter 58

2. The AIB1-E2F1 complex is recruited to the endogenous AIB1 gene promoter 60

3. FoxG1 is part of the AIB1-E2F1 complex present at the endogenous AIB1 gene promoter 63

E. FoxG1 compromises the integrity of the activating complex on the AIB1 gene promoter 66

1. FoxG1 overexpression destabilizes the Sp1-assicated transcription complex on the AIB1 gene promoter 66

vi 2. Overexpressing FoxG1 compromises the integrity of the transcriptional complex present at the endogenous AIB1 gene promoter 69

3. FoxG1 overexpression causes decreased co-occupancy of p300-AIB1 complex at the AIB1 gene promoter 71

F. FoxG1 disrupts AIB1’s coactivator function 74

1. The effect of FoxG1 on steroid-dependent and -independent promoters 74

2. FoxG1 has no effect on the expression of E2F1-responsive 77

IV. Discussion 79

A. FoxG1 Downregulates AIB1 Expression by Repressing Transcription of the AIB1 Gene 80

B. Homo- and Heterodimerization of FoxG1 may be Required for the Repression of the AIB1 83

C. FoxG1 May Act as a Short-Range Repressor at the AIB1 Gene Promoter 84

D. FoxG1 Repression of AIB1 Transcription and Coactivation is Not Universal 85

E. Downregulation of AIB1-regulated Pathways and Inhibition of TGF-β Signaling are Likely Involved in the FoxG1-indcued apoptosis in MCF-7 cells 85

F. FoxG1 can be Both Pro- and Anti-oncogenic 86

G. FoxG1 Repression of AIB1 Expression: Its Pharmacological Impacts 87

V. Appendix 90

A. Design of AIB1Δ4 Scorpion Primers - a quantitative bioassay for the high-efficient, low-cost screening of AIB1Δ4 in human breast and pancreatic cancers. 91

I. Introduction 92

1. AIB1Δ4, the Splice Variant of AIB1 93

2. AIB1Δ4 is a More Potent Coactivator Due to Its Nuclear Function 96

vii

II. Results 97

1. Development of the AIB1Δ4 Scorpion Primer 98

2. Validation of the AIB1Δ4 Scorpion Primer 102

3. Higher AIB1Δ4 Levels Are Correlated With Increased 104 Invasiveness and Metastatic Potential in Pancreatic and Breast Cancer Cells Lines

VI. References 109

viii LIST OF FIGURES Pages

Figure 1. Functional domains and sequence identity of p160 steroid receptor family 8

Figure 2. Model of steroid receptor coactivators (SRCs) function 9

Figure 3. Confirmation of the interaction between AIB1 and Foxg1 33

Figure 4. FoxG1 also interacts with AIB1Δ4 35

Figure 5. FoxG1 mRNA levels in breast cancer cell lines 36

Figure 6. Prognostic significance of FoxG1 in human breast cancer 38

Figure 7. Prognostic significance of FoxG1 in human breast cancer 39

Figure 8. FoxG1 overexpression leads to apoptosis in MCF-7 cells 41

Figure 9. FoxG1 overexpression suppresses endogenous AIB1 expression in MCF-7 44

Figure 10. The Effect of FoxG1 overexpression on other SRC family proteins 45

Figure 11. AIB1 rescues MCF-7 cells from FoxG1-induced apoptosis 47

Figure 12. Model of the AIB1 gene promoter 50

Figure 13. FoxG1 represses the activity of the AIB1 promoter reporter 52

Figure 14. Schematic of ChIP procedure 55

Figure 15. Protein association to the Sp1 in the transfected AIB1 gene promoter 56

Figure 16. FoxG1 is recruited to the endogenous AIB1 promoter 59

Figure 17. ChIP-reChIP procedure 61

Figure 18. AIB1 and E2F1 co-occupancy at the endogenous AIB1 gene promoter 62

Figure 19. FoxG1, AIB1 and E2F1 co-occupancy at the AIB1 gene promoter 64

Figure 20. FoxG1 overexpression leads to decreased recruitment of the members of the transcriptional complex to the endogenous AIB1 promoter 67

Figure 21. FoxG1 compromises the integrity of the protein complex 70

ix

Figure 22. Overexpressing FoxG1 causes reduction in p300-AIB1 co-occupancy at the AIB1 promoter 72

Figure 23. FoxG1 interferes with the interaction between AIB1 and p300 73

Figure 24. FoxG1’s effect on steroid-dependent and -independent promoters 75

Figure 25. FoxG1’s effect on estrogen-stimulated transcription 76

Figure 26. FoxG1 has no effect on E2F1-regulated gene expression 78

Figure 27. A proposed model for the role of FoxG1 in regulating AIB1 gene expression 82

Figure 28. The CBP/p300 binding domain is not responsible for AIB1 and FoxG1 interaction 89

Figure 29. mRNA and protein structure of AIB1 and AIB1Δ4 94

Figure 30. Making of the AIB1 and AIB1Δ4 scorpion primers 100

Figure 31. Validation of the AIB1 and AIB1Δ4 scorpion primers 103

Figure 32. AIB1Δ4 transcript levels are higher in metastatic pancreatic cancer cells 105

Figure 33. AIB1Δ4 expression correlates with increased metastatic potential in breast Cancer cells 107

x

I. INTRODUCTION

1 A. Hormone Signaling: A Brief History

When Starling and Bayliss first coined the word “hormone” in 1905, virtually nothing was known about the nature of these substances, except that they were “signal-bearing” pancreatic secretions which seemingly had the ability to communicate important biological information between organs (86). Little did they know, the research from Starling and Bayliss’ laboratory laid the first founding brick for the school of endocrinology and set the ground for the explosive advancement in several disciplines such as physiology, chemistry and biochemistry.

Today, the term “hormone” is used to define a class of chemical messengers secreted from any endocrine glands and released into the bloodstream, through which they are able to travel to, and affect tissues at distant sites with target-precision and high tissue-specificity. The overall purpose of hormones is to coordinate and integrate the activities of metabolic and developmental process in diverse target cells in response to environmental stimuli.

In the 1930s and 1940s, the development of the protein purification technique and the increasing availability of purified made possible the investigation of hormone- direct interaction. The popular assumption at the time was that all hormones function in a unified, common mode of action where they must have been acting on the enzyme systems.

Hormones were thought to interact with enzymes and induce allosteric or conformational changes in these proteins, which subsequently switched them from an inactive to an active state.

Such a hypothesis did not come as a surprise since this was the great era of enzymology.

Nevertheless, it quickly lost its footing due to the fact that a direct effect was only obtained by administering a high concentration of hormones, and the effects of hormones were only evident under pharmacological and toxic conditions. These findings contradicted the high degree of

2 tissue-specificity exhibited by hormones, and failed to explain the important growth and developmental actions of hormones. In the next 20 years, cyclic AMP was shown to be a crucial mediator of peptide hormone signaling (83), and glucocorticoids were known to regulate the protein synthesis of liver enzyme tyrosine aminotransferase (41). However, whether this regulation takes place on the transcriptional or translational level remained a subject of debate.

The discovery of receptors in the 1960s, and the availability of transcription inhibitors, actinomycin D and α-amanitin, led to the complete abandonment of the direct hormone-enzyme interaction theory, and shifted the focus of research on hormone mechanism of action to the control of transcription. Jenson and colleague in 1961 demonstrated that the tissues from the reproductive tract of female rats took up and retained radioactively-labeled hormone estradiol-

17β, which was found primarily in the nucleus of the cells from these tissues, complexed with a binding substance without chemical charge. Later, it was shown that this binding substance was actually the (ER). In the past 50 years, more physical and functional characterizations of hormone receptors have been uncovered, and hormone receptors can be classified in two categories based on their cellular locations: those found on the plasma membrane usually interact with peptide and protein hormones (membrane receptors), and those located in the nucleus have steroid hormone ligands that are lipid-soluble (nuclear receptors).

The estrogen receptor is a member of the nuclear receptors.

Elwood Jenson’s revolutionary work was of crucial importance in that, not only was ER the first steroid to be recognized, but Jenson also put forward the two-step model delineating the mechanism of estrogen-mediated activation of gene transcription which became our current paradigm of hormone action in general. In his model, the binding of

3 hormones with corresponding nuclear receptors causes a conformational change in the receptors leading to disassociation of the ligand-bound receptors from their steroid-binding proteins

(which render nuclear receptors inactive in the cytoplasm in the absence of ligands), followed by the subsequent translocation of the receptors into the nucleus. The ligand-associated nuclear receptors then dimerize and directly bind to specific sequences in the genomic chromatin, resulting in gene activation. This theory outlines the fundamental mechanism by which nuclear receptors exert their action. Nonetheless there are additional levels of complexity to the modulation of gene transcription due to hormone stimulation.

B. Nuclear Receptors

There are currently more than 20 nuclear receptors (NR) which have been discovered and cloned in humans. Those whose ligand have yet to be identified are referred to as “orphan receptors,” suggesting that novel hormone ligands might exist (86). NRs are essentially ligand- inducible transcriptional factors that bind to DNA directly and recruit the basal transcriptional machinery to control gene expression. Typically, NRs regulate hormone-responsive genes that mediate important biological functions in cellular development, homeostasis and metabolism.

Structurally, all NRs share common domains. The central DNA-binding domain (DBD) comprised of two evolutionarily conserved zinc fingers is a unique molecular signature for NRs and distinguishes NRs from other transcription factors. The DBD also enables NRs to interact with specific DNA sequences known as hormone response elements (HRE) in the genomic

DNA. In addition, NRs contain an amino-terminal activation function domain (AF-1), a carboxyl-terminal AF-2 domain and ligand-binding domain (LBD). The LBD allows precise

4 ligand recognition by NRs and ensures both specificity and selectivity of the subsequent physiological response warranted by the hormone-bound receptors (64).

Generally, hormone-activated NRs alone are sufficient to turn on gene transcription.

However, their actions are greatly enhanced by interacting with transcriptional coactivators which catalyze the rate of gene transcription and facilitate amplified gene expression. Common

NR-interacting coactivators include histone modifiers such as CPB/p300 histone (19) and the NSD (nuclear receptor-binding SET domain protein) methyltransferases (57), as well as the p160 steroid receptor family proteins.

C. The p160 steroid receptor coactivators

1. Steroid Receptor Coactivators Superfamily

Gene-specific activation and repression of transcription play a central role in gene regulation. The coregulator family proteins of corepressors and coactivators, which dampen and potentiate gene expression, respectively, work together in competition to oversee and control the highly complex process of gene transcription.

Coactivators interact with and enhance the transcriptional activity of NRs and other transcription factors. There are currently three known members of the steroid receptor coactivator (SRC) family proteins: SRC-1 (NCOA-1), TIF2 (SRC-2, GRIP1, NCOA-2) and

AIB1 (SRC-3, NCOA-3, ACTR, p/CIP, RAC3, TRAM-1). The first of its kind, SRC-1, was identified by O’Malley and colleagues in 1995, and was shown to directly interact with and increase the human transcriptional activity (72). In this study SRC-1 was reported to associate with and enhance ERα, (GR), thyroid hormone

5 receptor (TR), and (RXR) transcriptional activity through their cognate DNA response elements in the presence of ligands. The agonist-activated association between SRC-1,

ERα and PR was blocked by the use of antagonists, indicating that proper conformational change induced by an agonist is necessary for the receptor to interact with SRC-1. In the following year, two groups independently cloned transcriptional mediators/intermediary factor 2, TIF2/SRC-2, and its mouse homolog glucocorticoid receptor-interacting protein 1, GRIP1 (32, 93), and observed ligand-dependent TIF2 coactivation on ER, PR, RXR, TR, GR, vitamin D3 receptor

(VDR) and (AR). Finally, the third SRC family member was isolated in 1997 as p/CIP in mice (91), and the human homolog amplified in breast cancer 1, AIB1/SRC-3 was cloned independently by several groups (19, 52, 84).

2. Structural and functional Domains of SRCs

The SRC members have a molecular weight of about 160 kDa and approximately 40% identity in their sequence. All SRC family members contain an amino-terminal basic helix-loop-helix (bHLH), Per Arnt Sim (PAS) A and B domain, nuclear receptor interaction domain (RID), a polyglutamine region, and two activation domains in the carboxyl-terminal

(Figure 1). The bHLH domain is the most conserved region and typically functions for DNA- binding in transcriptional factors. However, as of today, none of the SRC family proteins has been shown to bind DNA directly. The PAS domain is a region of homology shared with the period circadian (Per), aryl hydrocarbon receptor nuclear translocator (ARNT), and single minded (Sim) proteins, and is mainly responsible for mediating protein-protein interaction, especially homo- and hetero-dimerization of PAS domain-containing proteins. The RID domain

6 facilitates the binding of SRCs with ligand-bound nuclear receptors through three leucine-rich motifs with the amino acid consensus sequence LXXLL.

At the carboxyl-end of the SRCs there are two transcriptional activation domains, AD1 and AD2 that interact with two major classes of coactivators. AD1 contains the critical binding element (CID) allowing SRCs to directly associate with CBP/p300 histone and p300/CBP-asspicated factor (p/CAF) (19, 91, 109). The AD2 domain interacts with methyltransferases, coactivator-associated arginine methyltransferase-1 (CARM1) (18) and protein arginine methyltransferase-1 (PRMT1) (42). These proteins are chromatin modifiers that, once ligand-activated nuclear receptors translocate to the nucleus and recruit SRCs, they bind to

SRCs and further recruit basal transcription machinery to DNA. Their general purpose is to enhance transcription by local remodeling of the chromatin to a more relaxed conformation, consequently permitting RNA polymerase II access to DNA and transcription initiation (Figure

2).

7

Figure 1. Functional domains and sequence identity of p160 steroid receptor family proteins

Domains: basic helix-loop-helix (bHLH)/Per/Arnt/Sim (PAS), receptor interaction domain (RID), CBP/p300 interaction domain (CID), glutamine-rich region (Q-rich)/histone acetyltransferase domain (HAT). NLS, nuclear localization signal; LXXLL (L = leucine, X = any amino acids).

8

Figure 2. Model of steroid receptor coactivators (SRCs) function

SRCs bind to ligand-bound nuclear receptors (NR) and recruit coactivating partners such as CBP/p300 and CARM1. These proteins are chromatin modifiers capable of acetylating histones, which lead to a relaxed state and allow RNA Pol II to access DNA and initiate transcription.

9 D. Steroid Receptor Coactivator AIB1

1. The Role of AIB1 in Normal Physiology

The nomenclature Amplified In Breast Cancer 1 (AIB1) can be somewhat misleading as the name explicitly implicates AIB1 as a protein relating solely to cancer etiology and pathology.

However, as numerous studies have shown, AIB1 also plays an important role in mediating normal physiological functions such as the development of functional reproductive system, maintenance of lipid and energy homeostasis, control of wound healing and angiogenesis, and suppression of cytokine inflammatory response.

It is been shown in AIB1 (p/CIP) -/- knockout mice that disruption of the AIB1 gene leads to postnatal somatic growth retardation, resulting in an average 15 to 20% weight loss in AIB1 null animals as compared to wild-type (WT) littermates (94, 103). Female AIB1 null mice exhibit delayed onset of puberty, poorly developed mammary gland structure with minimal ductal branching, and abnormal reproductive function manifesting as decreased ovulation, lower pregnancy frequency, small litter size, and longer estrous cycling time (103). In addition to having an effect on reproduction, AIB1 (p/CIP), and SRC-1 in mice have been implicated in the regulation of energy homeostasis and adipogenesis by controlling the expression of proliferator-activated receptor-gamma (PPARγ) target genes involved in lipid metabolism (60,

95). Wang et al. observed that the combined loss of AIB1 and SRC-1 function in AIB1-/-/SRC-1-

/- double knockout mice leads to reduced brown fat development and near 50% weight loss as compared to WT animals due to diminished lipid storage in the brown adipose tissue. These animals also show increased metabolic rates but are defective in adaptive thermogenesis as they appear to respond poorly to temperature changes (from high to low) in comparison to WT mice

10 (95). Interestingly, Louet et al. further demonstrate that AIB1 is recruited to PPARγ gene promoter and the decreased expression of PPARγ and its target genes in AIB1-/-!mice are linked to impaired white adipocyte development, which can be restored by ectopic re-expression of

AIB1 in these animals (60).

In terms of AIB1’s role in wound healing and angiogenesis, endothelial cells form the barrier layer between the bloodstream and the parenchyma, and we have previously reported that

AIB1 depletion in AIB1 homozygous and null mice significantly impairs endothelial cells to sprout and form tubes and monolayers in vitro (3). We have also shown that the loss of a single

AIB allele in mice reduces the angiogenic response by 70 to 90% in animals with subcutaneous wounds inflected by the matrigel injection. Additionally, the loss of AIB1 leads to a slower tissue repair process as shown by incomplete healing of punctured skin lesions due to poorer wound contraction, significantly delayed lesion closure and fibrin breakdown, and very little collagen deposition (all of which are normal healing features) in AIB1+/- mice as compared to

WT animals (3). AIB1 also has an anti-inflammatory function. Excessive cytokine production by macrophages in response to infection, as a result of unrestrained transcription or translation, is detrimental to the host and must be under precise regulation. In a recent report presented by

O’Malley and colleague, AIB1-/- mice are shown to be highly susceptible to lipopolysaccharides

(LPS) - induced endotoxic shock due to increased production of serum proinflammatory cytokines such as TNF-α, IL-1b, and IL-6 as compared to the WT animals (111). They show that this increase in cytokine production is a result of enhanced translation of the cytokines due to decreased AIB1 levels in the null animals. This is because under normal physiological condition

(i.e. in WT mice), AIB1 directly binds to, and cooperates with translational repressors TIA-1 and

11 TIAR to suppress TNF-α mRNA translation, consequently leading to repression of excessive and uncontrolled inflammatory response (111). Taken together, it is evident that AIB1 plays a distinct role in diverse biological processes.

2. AIB1, the Master Gene Regulator

The concept of “master genes” was first proposed by Britten and Davidson in 1969 to describe a subgroup of genes in animal genomes that function to implement the coordinated expression of subservient genes termed as “producer genes.” More specifically, a true master gene is able to integrate the transcription of many producer genes at multiple cellular levels and of various biological and physiological functions, in response to a single molecular cue.

AIB1 fits the mold predicted for master genes. Although initially identified as a nuclear hormone receptor coactivator that promotes and is the rate-limiting step required for maximal transcriptional activation of multiple steroid receptors such as the ER, PR, TR and RXR (4, 19,

82), we now know that AIB1 coactivator function extends well beyond the simple role of one- dimensional executer of hormone-induced transcriptional events. In fact, AIB meets the criteria framed by Britten and Davidson as master genes because it has the ability to bind across unrelated families of transcription factors and coordinately regulate the expression of multiple genes required for complex physiological goals.

First, AIB1 controls different growth factor activated signaling pathways as shown by

O’Malley and colleague that disruption of AIB1 gene in AIB1-/- knockout mice results in a diverse phenotype of dwarfism, delayed puberty, abnormal reproductive function, and mammary gland growth retardation, suggesting that AIB1 could play a role in growth factor singling (103).

12 Wang et al. went further to demonstrate that AIB1 deletion in these mice caused a dramatic decrease in circulating serum IGF-1 (insulin-like growth factor 1) levels and a reduction in overall size (94). Since then, several studies show that AIB1 is a major mediator in IGF-1 signaling. Our lab has reported that the gene silencing, and the resultant reduction, of endogenous AIB1 levels by small interfering RNA in MCF-7 breast cancer cells prevents IGF-1 mediated anchorage-independent growth by reducing IGF-1 dependent anti-anoikis (71). Also, in contrast to the AIB1-/- knockout animals, transgenic mice overexpressing human AIB1 cDNA gene exhibit increased serum IGF-1 levels and activation of the IGF/IR/P13K/AKT pathway in mammary epithelial cells, as well as increased size and accelerated differentiation of murine mammary gland (92). Furthermore, there is emerging evidence that AIB1 could also be functionally involved in modulating the activity of the human epidermal growth factor (HER) signaling pathways. AIB1 and epidermal growth factor receptor (EGFR) are frequently overexpressed in epithelial tumors and their expression is associated with poor prognosis. We have previously shown that AIB1 knockdown in breast, lung and pancreatic cancer cell lines leads to decreased EGFR tyrosine phosphorylation, signaling, and proliferation in these cells

(45). We have also demonstrated that AIB1 is required for HER2/neu (Human Epidermal

Growth Factor Receptor 2) oncogenic activity and for the phosphorylation and activation of the

HER2/neu receptor. In mice with deletion of one copy of AIB1, we show a reduction in HER2- induced mammary gland hyperplasia and delayed tumor formation and development in animals with decreased AIB1 levels (24).

Second, AIB1 interacts with nonreceptor transcription factors such as E2F1, nuclear factor-κB (NF-κB), activating protein-1 (AP-1) and STAT6, down stream of growth factor

13 receptors (5, 62, 96, 107). AIB1 controls cell cycle gene expression by coactivating E2F1- dependent transcription. AIB1 is shown to interact with E2F1 directly and binds to the promoters of E2F1 responsive genes such as Cdk2, cyclin D1, cyclin E and E2F1 (61, 62). Yan et al. also reported that AIB1 is recruited to the IRS-2 (insulin receptor substrate 2) and IGF-1 gene promoters and enhances AP-1 induction of both promoter activities (107). In addition, AIB1 coactivates NF-κB and STAT6-dependent transcriptional activity (5, 98). By inhibiting caspases and coactivating these transcription factors, AIB1 participates in, and promotes, pro-proliferation and anti-apoptotic activities in cancer cells (11, 21). The upregulation of anti-apoptotic/pro- growth signaling represents an important alternate mechanism by which AIB1 contributes to hormone-independent tumor growth (46).

In summary, AIB1 has an effect on transcription initiation, elongation, receptor and coregulator turnover and mRNA translation. Such a capacity to mediate diverse physiological and molecular functions suggests that AIB1 is a true master gene regulator.

3. AIB1 is an oncogene and its overexpression fuels tumorigenesis

Gene amplification is a frequent mechanism of increased gene expression in human cancers. From 20q microdissection, a known region with increased DNA copy number shown by cytogenetic analysis of breast cancers, Anzick et al. reveals that AIB1 is a gene ubiquitously expressed in normal human tissues but amplified in breast cancer (4). In this study, the levels of AIB1 expression are detected in ER-positive (ER+) and ER-negative (ER-) breast and ovarian cancer cell lines by Fluorescent In Situ Hybridization (FISH), and high levels of

AIB1 amplification of greater than 20 fold are found in 4 out of 5 ER+ cell lines, including BT-

14 474, MCF-7, ZR75-1 and BG-1. To exclude the possibility of AIB1 amplification as a result of cell culture selection, unselected breast cancer specimens from tumor biopsies are also tested for

AIB1 gene amplification. Out of 105 samples, FISH analysis indicates that 10% of the primary tumors have AIB1 amplification although the levels are not as high as in the cell lines.

Additionally, AIB1 expression is also examined by mRNA in situ hybridization in the primary breast tumors previously tested by FISH where, in samples without AIB1 gene amplification,

64% of tumors still show increased AIB1 mRNA expression in comparison to normal mammary epithelium, while the transcript levels of the other SRC members remain constant (4). This suggests that AIB1 transcript levels can be increased in breast tumors by mechanisms other than amplification of the gene.

An oncogene by definition is a gene that contributes to the production and growth of cancer and is capable, when activated, of transforming cells. AIB1 was first identified as an oncogene based on the work conducted by Myles Brown and colleague, in which a transgenic mouse model (AIB1-tg) that expresses human AIB1 cDNA under the transcriptional control of the mouse mammary tumor virus (MMTV) promoter was generated to study the effect of higher

AIB1 levels in vivo. They reported that AIB1 overexpression caused mammary gland hyperplasia, delays in mammary gland involution and spontaneous development of various types and tissue-specific malignant tumors. In these mice, while mammary gland adenocarcinomas, pituitary adenomas, uterine leiomyosarcomas, and lung adenocarcinomas occurred in large numbers and most frequently, there were also tumors forming at other organs. Additionally, they observed that serum IGF-1 levels were significantly elevated (>3 fold) in the AIB1-tg animals accompanied by increased activation of IGF-1 signaling pathway, relative to the WT mice. This

15 study is of crucial importance because it shows that AIB1 overexpression is not only the causative agent for mammary gland tumorigenesis but also, given the multitude of diverse tumor types driven by AIB1 expression, it may also be involved in a much wider range of human tumors than previously assumed. Indeed, over the years AIB1 gene amplification or overexpression has been detected in colorectal (48, 101), endometrial (7), esophageal squamous cell carcinoma (102), gastric cancer (76), meningioma (16), non small cell lung cancer

(14, 29), ovarian cancer (10, 85), pancreatic cancer (30), and prostate cancer (112). Clinically, the AIB1 gene is amplified in 5 to 10% of human breast cancers and the overexpression of AIB1 mRNA and protein has been observed in ~30% of human breast tumors (105). AIB1 has been strongly implicated in the development of hormone-responsive and nonresponsive cancers (35,

104), and a strong correlation exists between high levels of AIB1 and high HER2 levels, larger tumor size, higher tumor grade, increased cancer reoccurrence and worse prognoses. Overall, the potent oncogenic potential of AIB1 is attributed to its ability to coactivate steroid hormone receptors as well as a plethora of growth factor induced transcription.

4. Regulation of AIB1 Expression and Activity

Total AIB1 cellular expression can be regulated at three levels: gene transcription, RNA translation, and degradation, as well as protein stability and turnover (46).

Hossain et al. reported that the translation of AIB1 mRNA can be regulated by microRNAs, which inhibit translation by binding to the 3’-untranlated regions of target mRNAs.

They show that levels of AIB1 mRNA can be reduced post-transcriptionally by mir-17-5p in

16 MCF-7 breast cancer cells, and expression of this particular microRNA is low in breast cancer cell lines with high levels of AIB1 protein (33).

A number of proteasomal degradation pathways have been implicated for the regulation of AIB1 protein levels. For example, we have previously reported an interaction between AIB1 and the E3- E6 associated protein (E6-AP) in MCF-7 cells, in which the latter may target AIB1 for proteasomal degradation (65). Other groups have shown that phosphorylation of AIB1 at serines 505 and 509 by glycogen synthase kinase 3 (GSK3) marks

AIB1 for SCFFbw7a -dependent ubiquitination of lysine residues 723 and 786, which leads to increased AIB1 protein turnover (99). Additionally, AIB1 is also a known substrate for Speckle- type POZ protein (SPOP)-mediated ubiquitination and proteolysis (50).

In terms of AIB1 gene transcription, we have previously reported that all-trans retinoic acid, antiestrogens ICI 182,780 and tamoxifen, and transforming growth factor β (TGF-β) can upregulate AIB1 transcripts, whereas estrogen can suppress AIB1 gene expression (49). In addition, a recent study demonstrates that transcription of the AIB1 gene is controlled by regulatory sequences within the bp -250 to +350 region of its promoter which enable AIB1 to auto-regulate and enhance the expression of its own gene (62, 67). In these studies, an Sp1- binding site down-stream of exon 1 was described within the -250/+350 region that also recruited

E2F1. This enables AIB1 to complex with E2F1, and this Sp1-associated transcription complex significantly increases the coactivation of the AIB1 gene (67).

AIB1 function can be controlled by post-translational modifications (PTM) such as methylation (23, 68), sumoylation (97) and (19), However, the most studied modification of AIB1 is phosphorylation. Mitogen activated protein kinase (MAPK) has been

17 shown to primarily phosphorylate AIB1 on serine residues which increase its ability to recruit p300/CBP and its associated HAT activtity (25). In addition, AIB1 can be phosphorylated by differenat kinases (ERK, JNK, p38 MAPK, GSK3, CK1δ and PKA) on multiple serine (505,

543, 857, 860, 867) and threonine (24) sites, and phosphorylations of AIB1 are required for optimum interactions with transcriptional activators and other coactivators such as NF-κB, p300/CBP and CARM 1 (100). Both estrogen and tumor necrosis factor-α (TNF-α) can increase the phosphorylation of AIB1 (98, 100). TNF-α stimulation is shown to activate IκB kinase (IKK) which leads to phosphorylation of both IκB and AIB1. As a result, phospho-IκB is rapidly degraded, resulting in activation, and nuclear translocation, of its binding protein NF-κB, where in parallel phosphorylation of AIB1 by IKK promotes cytoplasmic to nuclear transport of AIB1

(98). Finally, our lab has reported that AIB1 is phosphorylated at tyrosine 1357 by V-abl

Abelson murine leukemia viral oncogene homolog 1 (abl) kinase in response to estrogen, EGF and IGF, and loss of posphorylation at this site in AIB1 leads to decreased interaction with p300 and ERα (70). In summary, AIB1 phosphreylation is required for binding to other transcriptional cofactors, nuclear translocation and for its abilty to fully function as a transcritpional coactivator in the tumorigensis (46).

E. Transcriptional Repression of AIB1 by Foxg1

AIB1 is known to directly bind to other coactivators such as histone acetyltransferase p300/CBP, p300/CBP-associated p/CAF, and arginine methyltransferase CARM 1, and enhance transcriptional activation by bringing these potent cofactors capable of modifying chromatin organization to the target gene promoter (19, 79, 88). The ability to interact with a

18 wide range of transcriptional coactivators enables AIB1’s potent coactivating function. In contrast, only a few transcriptional repressors that interact with the SRC are also known to interact with AIB1, such as nuclear receptor corepressor 1 (N-CoR) and silencing mediator for retinoid or thyroid-hormone receptors (SMRT/N-CoR2) (39, 56). However, little is known about other factors that might serve to squelch AIB1 function.

To uncover potential repressors of AIB1 function, we have undertaken broad screens of

AIB1-interacting proteins using mass spectrometry (MS) to detect low abundance AIB1 binding partners (36). From the MS results, we focused on AIB1-interacting proteins that segregated under the category of “transcriptional repressors,” which could potentially negatively regulate the AIB1 gene expression. Here we demonstrate that the winged-helix, DNA-binding transcriptional corepressor Foxg1 (also known as brain factor 1, BF1), which we identified as an

AIB1 interacting protein, down-regulates AIB1 promoter activity and suppresses both AIB1 transcript and protein expression in MCF-7 cells. Foxg1 is a member of the forkhead-box (FOX) protein family of transcriptional regulators. The FOX proteins have been shown to have diverse functions for , proliferation, differentiation and longevity, many of which are also important for embryonic development (15). Although named after and categorized by the evolutionarily conserved, defining feature of the forkhead-box (FOX) DNA binding motif, there is actually very little structural similarity amongst the members of the FOX protein family. As mentioned before, the forkhead motif, also known as the “winged helix” domain, is a sequence of 80 to 100 amino acids that is highly conserved across species, and is responsible for mediating

Fox protein binding to DNA. Structurally, the forkhead domain encompasses a helix-turn-helix

19 core of three α-helices, which is also flanked by two loops. The 3D structure of this domain resembles the shape of a butterfly, giving rise to the nomenclature “winged-helix” (15).

In particular, FoxG1 on 14q12, is a protein mainly expressed in the brain and testis in human (69, 106), and is shown to control the development of the telencephalon and cerebral corticogenesis (106). While Foxg1 knockout mice develop cerebral hypoplasia and die at birth, human patients with Foxg1 haploinsufficiency suffer from severe mental retardation and microcephaly (28, 81, 106). The prominent developmental phenotype associated with Foxg1 pathology has focused most investigations of Foxg1 function in the brain and on neurogenesis.

Therefore, very little is known about Foxg1’s role in cancer and the molecular mechanism underlying Foxg1 function. However, studies have shown that Foxg1 interacts with global transcriptional corepressors and histone deacetylases to potentiate transcriptional repression

(108). Foxg1 is also known to directly interact with androgen receptor (AR) and suppress AR- mediated transactivation (69).

Our data indicate that Foxg1 is recruited to the AIB1 promoter directly. We report a mechanism in which Foxg1 overexpression compromises the integrity of an Sp-1-associated activating transcriptional complex which is required for the upregulation of AIB1 gene expression, resulting in reduced recruitment, disassembly and detachment of the activating complex from the AIB1 promoter. Our data indicate that FoxG1 is directly recruited to the AIB1 promoter. We report a mechanism in which FoxG1 overexpression compromises the integrity of an Sp-1-associated activating transcriptional complex which is required for the upregulation of

AIB1 gene expression, resulting in reduced recruitment, disassembly and detachment of the

20 activating complex from the AIB1 promoter. We also show that FoxG1 downregulates AIB1 expression which leads to apoptosis in human breast cancer cells.

21

II. MATERIAL AND METHODS

22 A. Plasmids

HA-p300, E2F1 and IRES-FoxG1 constructs were kindly provided by Dr. Maria L.

Avantaggiati (Georgetown University), Dr. Hongwu Chen (University of California, Davis) and from Dr. Joan Massague (Sloan-Kettering Institute), respectively. FLAG-AIB1 plasmid was previously described (70, 74). HA-FoxG1 was generated first by restriction enzyme digestion of the IRES-FoxG1 construct with EcoRI and BamHI, followed by insertion of the excised FoxG1 coding region into phCMV2.

B. Cell Lines and Transient Transfection

HEK293T and MCF-7 were obtained from the Tissue Culture Shared Resource at

Georgetown University. HEK293T and MCF-7 were grown in Dulbecco modified Eagle’s medium (DMEM, Invitrogen, Carlsbad, CA, USA) and phenol red-free Iscove’s modified

Eagle’s medium (IMEM, Invitrogen), respectively, and both were supplemented with 10% FBS.

The human mammary epithelial cells (HMEC) were purchased and cultured in commercially supplied medium (BulletKit, Lonza, Walkersville, MD, USA). Transient transfection was performed in HEK293T and MCF-7 with FuGENE 6 (Roche, Indianapolis, IN, USA) and

FuGENE HD (Promega, Madison, WI, USA), respectively.

C. Western Blot, Nuclear Extraction and Immunoprecipitation

(i) For interaction of AIB1 with FoxG1 in HEK293T, cells were transfected with 8 µg of either FLAG-AIB1, HA-FoxG1, or FLAG-AIB1 and HA-FoxG1 together. 48 hours post transfection, cells were washed with cold 1X PBS and lysed in 1% Nonidet P-40 lysis buffer

23 containing 1mM NaO3VO4 and 1X Complete protease inhibitor tablet (Roche). Whole cell lysates were subjected to IP with anti-FLAG M2 affinity gel (Sigma, St Louis, MO, USA) as described previously (45) and samples were subjected to SDS-PAGE. WB was probed with antibodies against FLAG (M2, Sigma) or HA (Y-11, Santa Cruz Biotechnology, Santa Cruz, CA,

USA).

(ii) For the endogenous interaction of AIB1 with FoxG1, MCF-7 cells were plated in 15- cm dishes and nuclear lysates were prepared from cells as per the protocol recommended by the

CelLytic NuCLEAR Extraction kit (NXTRACT, Sigma). 2 mg of nuclear lysates were used to immunoprecipitate AIB1 with anti-AIB1 antibody (611105, BD Biosciences, San Jose, CA,

USA). The amount of FoxG1 associated with AIB1 was detected with a FoxG1 antibody

(ab3394, Abcam, Cambridge, MA, USA), and the precipitated AIB1 was probed using FLAG

M2 antibody (Sigma).

(iii) For protein expression levels in MCF-7 cells overexpressing FoxG1, cells were transfected with an empty vector (EV) control or FoxG1 constructs for 24 hours. Whole cell lysates were prepared as indicated in (i), and relative protein levels were assessed with the following antibodies: AIB1 (5E11, Cell Signaling Technology Inc, Danvers, MA); E2F1 (KH95,

Santa Cruz Biotechnology); Sp1 (PEP2, Santa Cruz Biotechnology); p300 (C-20, Santa Cruz

Biotechnology); FoxG1 (ab3394, Abcam) and human actin (C4, Millipore, Billerica, MA, USA).

D. Kaplan-Meier (KM) Analysis

KM survival curves are generated from Kaplan-Meier Plotter (http://kmplot.com) (27).

Analysis parameters used to generate FoxG1 and AIB1 mRNA KM plots are the following:

24 Affymetrix IDs of “206018_at” and “209062_x_at” were used for FoxG1 and RAC3/AIB1 respectively; data were plotted for relapse free survival (RFS) at 15 years follow-up threshold; patient data were split and analyzed by median using all probe sets per gene; database “n=2361” was used to generate all the KM plots in this study.

E. Annexin V Apoptosis Assay and Flow Cytometry

MCF-7 cells were grown in 10-cm dishes with IMEM containing 10% FBS and transfected with an EV control or FoxG1-expressing constructs. After 24 hours, cells were trypsinized and stained with FITC-conjugated Annexin V and propidium iodide (PI) as per the protocol recommended by the TACS Annexin V kit (Trevigen Inc, Gaithersburg, MD, USA).

The percentage of cells in early and late apoptosis was determined using fluorescence-activated cell sorting (FACS) on a Facstar-Plus Dual Laser flow cytometer (Becton Dickinson, Franklin

Lakes, NJ, USA), provided by the Flow Cytometry Shared Resource at Georgetown University.

Duplicate samples from each transfection condition were subjected to FACS analysis for apoptotic index.

F. ChIP and ChIP-reChIP

For ChIP assays using transfected HEK293T, cells in 15-cm dishes were transfected with

12 µg of either wild-type AIB1(-250/+350) or mutant AIB1(-250/+350)-Sp1-del promoter reporter constructs. 6 hours after transfection, cells were fixed with 1% formaldehyde (3.7% formaldehyde, 100 mM NaCl, 50 mM Tris/HCl, pH 8.0, 1 mM EDTA, 0.5 mM EGTA) for 10 min at 37 ºC and the reaction was stopped by 0.125 M of glycine solution for 5 min at room

25 temperature. Cells were washed three times with 1X PBS and resuspended in SDS lysis buffer

(50 mM Tris, pH 8.0, 10 mM, EDTA, pH 8.0, 1% SDS). Cells were then sonicated and resuspended in ChIP dilution buffer (20 mM Tris, pH 8.0, 2 mM EDTA, pH 8.0, 150 mM NaCl,

1% Triton X-100) and pre-cleared with 70 µl of protein G-agarose/salmon sperm DNA

(Millipore) for 1 hr. 1 mg of total protein was immunoprecipitated with 5 µg of antibodies against either FoxG1 (ab18259, Abcam), AIB1/NCoA3 (C-20, Santa Cruz Biotechnology), E2F1

(1:1 mixture of C-20 & KH95, Santa Cruz Biotechnology), Sp1 (PEP2x, Santa Cruz

Biotechnology) or IgG (as negative control) for 16 hr and immunoprecipitated with 60 µl of protein G-agarose/salmon sperm DNA for 4 hr. Agarose was washed with once with low salt buffer (20 mM Tris, pH 8.0, 2 mM EDTA, pH 8.0, NaCl 150 mM, 0.1% SDS, 1% Triton X-100), twice with high salt buffer (20 mM Tris, pH 8.0, 2 mM EDTA, pH 8.0, 500 mM NaCl, 0.1%

SDS, 1% Triton X-100), once with LiCl salt buffer (10 mM Tris, pH 8.0, 1 mM EDTA, pH 8.0,

250 mM LiCl, 1% sodium deoxycholate, 1% Nonidet P-40), and twice with TE buffer (10 mM

Tris, pH 8.0, 1 mM EDTA, pH 8.0). Samples were eluted with elution buffer (1% SDS, 0.1 M

NaHCO3) for 15 min on a rotator and 10 min on a vortexer. Cross-links were removed with 200 mM NaCl overnight at 65 °C and proteins digested with 1 µg of proteinase K for 1 hr at 45 °C.

DNA was purified using GENECLEAN Turbo kit (Obiogene Inc, Carlsbad, CA, USA). Real time PCR (RT-PCR) (iCycler, Bio-Rad, Hercules, CA, USA) was performed in triplicates using

IQ SYBR Green Supermix (Bio-Rad) and 2µl purified ChIP DNA to examine protein recruitment to the WT and mutant reporter constructs with the following primers: 5’-

GCGAGTTTCCGATTTAAAGC (complementary to the 5’AIB1 promoter sequence) and 5’-

CTTTATGTTTTTGGCGTCTTCCA (complimentary to the 5’ reporter sequence) (67).

26 For the association of FoxG1 with the endogenous AIB1 promoter, MCF-7 cells were plated in 15-cm dishes and grown until 70-80% confluence. Cells were cross-linked and fragmented for ChIP assays as described above. The Protein-DNA complexes were immunoprecipitated with 5 µg of negative control rabbit IgG (Santa Cruz Biotechnology) or anti-

FoxG1 antibody. Purified DNA was analyzed by RT-PCR with a pair of AIB1 promoter-specific primers: 5’- GCGAGTTTCCGATTTAAAGC and 5’-GCCTTGGCAGATCTGAAG (67). To further verify the specificity of FoxG1 binding to the AIB1 promoter, ChIP DNA samples were also analyzed by RT-PCR with primers amplifying an region in exon 4 of the AIB1 gene (5’-

AGACGGGAGCAGGAAAGTAA and 5’-CGCACATTTATCTGGTTTGACATTG) or primers that detect the albumin promoter (5’-TGGGGTTGACAGAAGAGAAAAGC and 5’-

TACATTGACAAGGTCTTGTGGAG) (67). To investigate protein recruitment to the AIB1 promoter in MCF-7 cells overexpressing FoxG1, cells were transfected with either an EV control or FoxG1 constructs. 24 hours after transfection, cells were collected, sonicated and crude chromatin solution was diluted and incubated overnight at 4ºC with specific antibodies against

FoxG1, AIB1, E2F1, Sp1, p300 (C-20x, Santa Cruz Biotechnology), Pol II (C-21x, Santa Cruz

Biotechnology) and IgG as negative control. Purified DNA was analyzed by RT-PCR using the

AIB1 promoter-specific primers.

The two-step ChIP-reChIP (reChIP) experiments were performed in MCF-7 cells.

Chromatin was pre-cleared with 20 µl Magna ChIP Protein A+G Magnetic Beads (16663,

Millipore) and 2 mg chromatin DNA was immunoprecipitated with 40 µl beads and 5 µg of either AIB1, E2F1 or FoxG1 antibodies in the first round of ChIPs. The ChIP precipitates were gently washed as in the usual ChIP assay and the chromatin-protein complexes were eluted from

27 the beads in 75 µl TE buffer (10mM Tris, pH 8.0, 1mM EDTA, pH 8.0) containing 1X Complete protease inhibitor (Roche) and 10 mM DTT for 30 min at 37 ºC. After centrifugation, the supernatant from each sample was diluted with 1.5 ml ChIP dilution buffer (20 mM Tris, pH 8.0,

2 mM EDTA, pH 8.0, 150 mM NaCl, 1% Triton X-100) containing 1X Complete protease inhibitor and subjected to the second round of ChIP with 40 µl beads and 5 µg antibodies specifically against either E2F1, AIB1, or FoxG1. All first-round ChIPs were also followed by an IgG ChIP as a negative control. Cross-links were reversed in the precipitated complexes with

200 mM NaCl for 16 hours at 65 ºC and proteins digested with 1 µg of proteinase K for 1 hour at

45 ºC. For reChIP assays performed in MCF-7 cells with exogenously expressed FoxG1, sonicated chromatin was prepared from cells transfected with either an EV control or FoxG1 vectors, and subjected to reChIP procedures as described above with the exception of first-round immunoprecipitations using anti-E2F1 antibody, followed by second-round ChIPs with either

IgG (negative control), Sp1, AIB1 or FoxG1 antibodies. Recovered ChIP DNA was purified and analyzed by RT-PCR with the AIB1 promoter-specific primers.

Data (Ct values obtained from RT-PCR) collected from all ChIP and reChIP experiments in this study were first calculated as percentage of their respective inputs. The IgG-ChIPs and - reChIPs were then arbitrarily set as 1 and all the samples were analyzed and plotted in reference to IgG.

G. Luciferase Reporter Assay

Cells were plated in triplicates for all the luciferase reporter assays. 50,000 MCF-7 cells per well in a 24-well dish were transfected in serum-free IMEM with 0.5 µg WT AIB1(-

28 250/+350) promoter reporter construct alone, or together with 0.25 µg E2F1, with or without cotransfection of 0.25 µg FoxG1. 24 hours post transfection, cells were lysed in 100 µl 1X passive lysis buffer (Promega) and incubated at room temperature for 30 min on a rocker.

Luciferase values were measured using the luciferase reporter assay kit (Promega). Protein concentration for each sample was determined using the BCA protein assay (Pierce, Rockford,

IL, USA) and luciferase values were normalized with their protein concentrations. Reporter assays using HEK293T cells were performed as descried above. Cells were transfected in

DMEM without serum with AIB1 expression plasmids and either 0.2 µg multimerized AP-1 or

NF-κB reporter constructs (Stratagene, Santa Clara, CA, USA), in the presence or absence of 0.5

µg FoxG1. 25 ng of c-fos and c-jun expression vectors were also cotransfected with the AP-1 reporter. Hormone-stripped HEK293T cells used for estrogen-responsive promoter (ERE) reporter assays were transfected with AIB1- (0.5 µg), ERα (20 ng) constructs, and ERE luciferase vector (0.2 µg), with or without 0.5 µg FoxG1 for 24 hours. Cells were then treated with hormone for 24 hours before assessing for luciferase activity.

H. RNA Extraction and Real Time PCR

Total RNA was harvested using RNeasy mini kit (Qiagen, Valencia, CA, USA) and reverse-transcribed with iScript can synthesis kit (Bio-Rad) using 1 µg of total RNA. cDNA fragments were amplified in triplicates by RT-qPCR (iCycler, Bio-Rad) of 45 cycles with primers listed in Table 1 (61, 62). For AIB1 and FoxG1 gene expression in the 7 tested cell lines, frozen cell pellets of BT-483, T47D, BT-549, HCC-1937 and BT-20 cell lines were obtained from the Tissue Culture Shared Resource at Georgetown University. HMEC and MCF-7 cells

29 were plated in 10-cm dishes and grown in culture until 70-80% confluence. The fold change in

AIB1 and FoxG1 gene expression was normalized first to the human actin gene, then calculated

by the comparative Ct method, with relative transcript levels determined as y = 2^-ΔCT. AIB1

and FoxG1 mRNA expression of the 6 cancer cell lines was further normalized to the normal

HMEC (set as 1), which expresses the lowest levels of AIB1 in the group. For E2F1-regulated

gene expression in MCF-7 cells overexpressing FoxG1, cells were transfected with an EV

control or FoxG1 constructs. After 24 hours of transfection, the ∆Ct values of E2F1-regulated

genes were obtained by normalizing to actin.

TABLE 1. RT-PCR PRIMERS USED IN THIS STUDY

PRIMERS FORWARD SEQUENCE REVERSE SEQUENCE

AIB1 AGACGGGAGCAGGAAAGTAA CGCACATTTATCTGGTTTGACATTG Foxg1 AGAAGAACGGCAAGTACGAGA TGTTGAGGGACAGATTGTGGC CDK2 TTTGCTGAGATGGTGACTCGC CACTGGAGGAGGGGTGAGATTAG CDC25a TGAAGAATGAGGAGGAGACCCC CTGATGTTTCCCAGCAACTGTATG MCM7 AAGCCAGGAGTGCCAAACCAAC GCAGCAGTGCCTTCTTCACATC E2F1 CGCATCTATGACATCACCAACG GAAAGTTCTCCGAAGAGTCCACG CDC6 AAAGAGAATGGTCCCCCTCACTC AGTTTTTCCAGTTCCAGGAGCAC

Actin CCTGGCACCCAGCACAAT GCCGATCCACACGGAGTACT

30

III. RESULTS

31 A. AIB1 Interacts With The Transcriptional Corepressor FoxG1

1. Confirmation of the interaction between AIB1 and FoxG1

In order to identify proteins that interact with AIB1, we performed AIB1-specific immunoprecipitations (IP) of lysates from HEK293T cells transfected with a FLAG-tagged

AIB1 construct. After IP with a FLAG antibody, we isolated FLAG-associated immunocomplexes by denaturing gel electrophoresis, followed by Coomassie Blue staining.

Twelve visible bands were excised, and extracted proteins were subjected to mass spectrometry

(MS) analysis to identify potential interacting partners of AIB1 (described in (36)). FoxG1 was identified through our MS evaluation as a candidate AIB1-interacting protein. It was of interest for further investigation since it is known to function in certain contexts as a transcriptional repressor (108) and could potentially regulate AIB1 function and gene expression. To verify the interaction of AIB1 with FoxG1, we performed coimmunoprecipitation (co-IP) experiments.

FLAG-AIB1 was expressed by transient transfection together with HA-FoxG1 in HEK293T cells and HA-FoxG1 was detected in the immunoprecipitates of FLAG-AIB1 from whole cell lysates

(Figure 3a). We also confirmed the interaction of endogenous AIB1 and FoxG1 in MCF-7 breast cancer cells which harbor the 20q AIB1 gene amplicon, express high levels of AIB1 protein and a detectable amount of FoxG1 (Figure 3b). These IP results confirm the MS data and together demonstrate that FoxG1 is present in complexes that co-immunoprecipitate with AIB1.

32 a

Input IP: FLAG

FLAG-AIB1 HA-FoxG1 WB: HA

WB: FLAG

b

Input IP: IgG IP: AIB1

FoxG1 WB: FoxG1

WB: AIB1

Figure 3. Confirmation of the interaction between AIB1 and Foxg1

(a) AIB1 interacts with FoxG1 in HEK293T cells. FLAG-AIB1 was cotransfected with

HA-FoxG1 constructs. 48 hours post transient transfection, whole cell lysates were collected and used for immunoprecipitation (IP) and western blot (WB) analysis with anti-FLAG and anti-

HA antibodies as indicated. (b) Interaction of endogenous AIB1 with FoxG1 in MCF-7 breast cancer cells. Nuclear lysates were prepared from MCF-7 cells and immunoprecipitated with an

AIB1 antibody or control IgG. FoxG1 protein associated with AIB1 in the IP was detected by

WB as indicated.

33 2. AIB1Δ4 also binds to FoxG1

We had previously identified a splice variant of AIB1, where exon 4 was spliced from the mature mRNA and the resulting N-terminally truncated isoform was named AIB1Δ4 (74).

AIB1Δ4 protein was found to be a more potent coactivator of hormone-dependent transcription in comparison to the full-length AIB1 protein (1, 2). AIB1Δ4 transcript levels were increased in breast tumor tissue relative to normal breast tissue (74). We have shown that AIB1Δ4 increases the efficacy of estrogenic compounds and the agonist effects of the selective estrogen receptor modulator tamoxifen in breast and endometrial tumor cells (74, 75). Overexpressing AIB1Δ4 in mice leads to ductal ectasia in the mammary gland with an increased expression of proliferative markers such as proliferating cell nuclear antigen, phospho-histone H3, and cyclin D1 (90). More recently, AIB1Δ4 was shown to act as a molecular bridge between epidermal growth factor receptor (EGFR) and focal adhesion kinase (FAK) in the cytoplasm, and its overexpression is correlated with increased invasiveness and metastatic capability of human cancer cell lines (20,

59).

To test whether AIB1Δ4 also interacts with FoxG1, we transfected FLAG-tagged

AIB1Δ4 and the N-terminal fragment AIB1 1-198 (that’s truncated from AIB1Δ4) in HEK293T cells. Endogenous FoxG1 was immunoprecipitated and its associated immunocomplexes were probed with an FLAG antibody. Interestingly, we found that while AIB1Δ4 co- immunoprecipitated FoxG1, the N-terminal 1-198 fragment was not detected in the FoxG1 immunoprecipitates (Figure 4). This suggests that the interaction of FoxG1 with AIB1 is more likely mediated through the C-terminus of AIB1. It is also possible that FoxG1 binding to AIB1 requires both intact N-terminus and C-terminus.

34

INPUT IP: FoxG1

FLAG-AIB1)ODJ$,%¨Flag-AIB1(1-198)empty vector FLAG-AIB1)ODJ$,%¨Flag-AIB1(1-198)empty vector

WB: FLAG

WB: FoxG1

Figure 4. FoxG1 also interacts with AIB1Δ4

FLAG tagged AIB1, AIB1Δ4 and the N-terminal fragment AIB1 1-198 were transfected into HEK293T cells. 24 hours post transient transfection, whole cell lysates were collected and used for IP and WB analysis with anti-FoxG1 and anti-FLAG antibodies as indicated.

35 3. FoxG1 transcript levels in breast cancer cell lines

FoxG1 is predominantly expressed in the brain and its non-neuronal expression in normal tissues is low (69, 106). However, the expression of FoxG1 in human cancer has not been widely reported. In a comparison of normal breast cells and breast cancer cell lines, we found that the mRNA expression level of FoxG1 was significantly lower in breast cancer cell lines, irrespective of their estrogen receptor (ER) status, as compared to FoxG1 expression in the normal human mammary epithelial cells (HMEC) (Figure 5). These data suggest a loss of FoxG1 expression from normal to cancerous transition (Figure 5) and indicate that FoxG1 expression might have prognostic significance in human breast cancer.

AIB1 and FoxG1 mRNA Figure 5. FoxG1 mRNA levels 8 AIB1 in breast cancer cell lines 4 FoxG1 4 AIB1 and FoxG1 mRNA expression levels in breast cancer 3 cell lines. Total RNA was 2 Fold Change harvested from breast cancer cell 1 lines to determine the relative 0 gene expression for AIB1 and

T47D FoxG1. HMEC BT-20 MCF-7 BT-483 BT-549 HCC1937

36 4. Prognostic significance of FoxG1 in human breast cancer

To examine the prognostic significance of FoxG1 in human breast cancer we used a gene expression microarray provided by Kaplan-Meier (KM) Plotter (http://kmplot.com) (27) and determined that higher FoxG1 mRNA levels, in 2241 breast cancer samples, were predictive of increased survival rate and better overall clinical outcome (Figure 6a) (KM analysis parameters are described in Materials and Methods). In contrast, in the same data set, elevated AIB1 transcript levels corresponded to reduced rate of relapse-free survival (RFS) (Figure 6b), which is consistent with previous reports on AIB1 prognostic significance in human breast cancer

(reviewed in 46).

Our analysis of the microarray data generated from breast cancer population indicates that lower levels of FoxG1 segregate with worse clinical outcome and as expected, higher AIB1 expression correlates with shorter disease-free interval in the same population. Since we have previously shown that estrogen can suppress AIB1 transcript and protein levels in MCF-7 breast cancer cells (49), it is possible that ER status may contribute to the better prognosis associated with higher FoxG1 levels in patients. However, population analysis shows that the risk of relapse with low Foxg1 is the same in both ER-positive (ER+) and -negative (ER-) patients, suggesting that ER-mediated repression of AIB1 does not play a role in FoxG1 function, at least in breast cancers (Figure 7a and b).

37

a b Low High Low High 1138 1103 1260 981 FoxG1 mRNA AIB1 mRNA 1.0 1.0 Low 0.8 0.8 High 0.6 0.6 RFS

RFS 0.4 0.4 0.2 Low Low 0.2 High High 0.0 p<0.001 p<0.001 0.0 0 5 10 15 0 5 10 15 Years Years

Figure 6. Prognostic significance of FoxG1 in human breast cancer

Analysis of the levels of AIB1 and FoxG1 mRNA on a gene expression microarray of breast cancer samples from patients with known overall relapse-free survival (RFS) times provided by Kaplan-Meier Plotter (http://www.kmplot.com) (KM analysis parameters are described in Material and Methods) (27) .

38

Low High Low High 930 906 208 197

1.0 Foxg1 mRNA 1.0 FoxG1 mRNA

0.8 0.8

0.6 0.6 RFS RFS 0.4 0.4

0.2 Low 0.2 Low High p<0.0001 High p=0.001 0.0 0.0

0 5 10 15 0 5 10 15 Years Years

ER+ Population ER- Population

Figure 7. Prognostic significance of FoxG1 in human breast cancer

Analysis of the levels of FoxG1 mRNA in ER+ and ER- populations on a gene expression microarray of breast cancer samples from patients with known overall relapse-free survival (RFS) times provided by Kaplan-Meier Plotter (http://www.kmplot.com) (KM analysis parameters are described in Material and Methods) (27) .

39 B. FoxG1 Induces Apoptosis in MCF-7 Cells and Represses AIB1 Expression 1. Induction of apoptosis in MCF-7 cells by FoxG1 overexpression

The expression pattern for AIB1 and FoxG1 in MCF-7 cells was of interest because these cells significantly overexpress AIB1 protein. However, they have low levels of FoxG1 mRNA expression compared to HMEC (Figure 1c). We therefore chose to study the phenotypic effect of

FoxG1 overexpression on MCF-7 cells. Twenty four hours after expression vector transfection, overexpressing FoxG1 led to cell detachment from culture dishes, and the induction of apoptosis as determined by annexin V staining (Figure 8, upper panel). The early and total apoptosis average indices for duplicate samples of 14.5 and 28.64 % in FoxG1-expressing cells were significantly elevated compared to MCF-7 cells transfected with the control empty vector (EV) with average apoptosis indices of 1.68 and 9.4 %, respectively (Figure 8, lower panel). Increased expression of FoxG1 in MCF-7 cells was correlated with the apoptotic response.

40 EV FoxG1 Propidium Iodide (PI)

Annexin V-FITC

32 15 *** ** 24 10 16 5 8 % Total Apoptosis % Total % Early Apoptosis % Early 0 0 EV FoxG1 EV FoxG1

41

Figure 8. FoxG1 overexpression leads to apoptosis in MCF-7 cells

MCF-7 cells were transfected with either an empty vector (EV) control or FoxG1 constructs. 24 hours after transfection, cells were subjected to Annexin V apoptosis analysis. The percentages of cells in early- and late apoptosis are represented by bottom right- and top right quadrants of the FACS analysis, respectively. Percent total apoptosis was the total percentage of cells in both early- and late apoptosis. The mean ± SEM values were obtained from duplicate samples from each transfection condition. ***, P<0.001; **, P<0.01 relative to EV. Statistical analysis was done by Student’s t test.

42 2. Foxg1 overexpression reduces both AIB1 mRNA and protein levels

Previous studies reported increased incidence of apoptosis in MCF-7 cells when AIB1 expression is downregulated by small-interfering RNA (siRNA)-directed gene silencing (71).

Thus, since AIB1 and FoxG1 form a complex, we conjectured that a portion of the FoxG1- induced apoptotic effect might be mediated through changes in AIB1 expression. Consistent with this notion, as shown in Figure 9, FoxG1 overexpression caused a twofold decrease in both AIB1 mRNA and protein expression levels (Figure 9).

Since we observed this suppression of endogenous AIB1 expression in MCF-7 cells, we wanted to investigate whether the FoxG1-indcued repression is specific for AIB1 by examining the effect of FoxG1 overexpression on the other SRC family proteins. As Figure 10 demonstrates, increased FoxG1 levels had no effect on the relative mRNA expression of SRC-1.

We did observed a decrease in the expression of SRC-2 transcripts, however, the relative change of reduction in SRC-2 mRNA levels was less than twofold (Figure 10). Taken together, our data suggest that FoxG1 specifically downregulates AIB1 expression (of both mRNA and protein), whereas the other members of the SRC family remain unaffected.

43

a b AIB1 mRNA + - EV - + FoxG1 1 WB: AIB1 1 0.5 0.5 ** Fold Change WB: FoxG1

0.25 WB: Actin EV FoxG1

Figure 9. FoxG1 overexpression suppresses endogenous AIB1 expression in MCF-7 Analysis of endogenous AIB1 expression in MCF-7 cells overexpressing FoxG1. Cells were transfected with EV or FoxG1 as in Figure 6. Total RNA and whole cell lysates were collected to determine the relative levels of mRNA and protein for AIB1. Cells transfected with

EV were arbitrarily set at 1 and cells expressing FoxG1 were analyzed in reference to it.

Student’s t test. **, P<0.01 relative to EV. Relative protein levels were determined by WB with antibodies as indicated.

44

SRC-1 mRNA SRC-2 mRNA 2

ns 1 1 Fold Change Fold Change *** 0.5 0.5 EV FoxG1 EV FoxG1

Figure 10. The Effect of FoxG1 overexpression on other SRC family proteins

MCF-7 cells were transfected with either an empty vector (EV) control or FoxG1 constructs. 24 hours after transfection, total RNA was collected and the relative mRNA expression of SRC-1 and SRC-2 was measured by RT-PCR. ***, P<0.001; ns = not significant relative to EV. Statistical analysis was done by Student’s t test.

45

3. FoxG1-mediated apoptosis in MCF-7 cells is due to AIB1 downregulation

We next asked whether the FoxG1 induction of apoptosis was mediated directly by loss of AIB1 by determining whether exogenously expressed AIB1 was sufficient to rescue these cells from FoxG1-induced apoptosis. Our analysis revealed that AIB1 co-expression with FoxG1 allowed an approximately 50% rescue from apoptosis compared to cells transfected with only

FoxG1 (Figure 11, bottom panel; compare 2nd bar with the 4th bar). Thus, our phenotypic studies argue that a significant portion of the FoxG1 induction of apoptosis in MCF-7 cells is mediated through downregulation of AIB1 expression.

46 AIB1 AIB1+FoxG1 Propidium Iodide (PI)

Annexin V-FITC

18 15 *** 12 9 *** 6

3 % Early Apoptosis % Early 0 EV FoxG1 AIB1 AIB1+ FoxG1

47 Figure 11. AIB1 rescues MCF-7 cells from FoxG1-induced apoptosis

MCF-7 cells were transfected separately with expression vectors for either EV control,

FoxG1, AIB1, or AIB1 and FoxG1 together. 24 hours after transfection, cells were subjected to

Annexin V apoptosis analysis. The percentages of cells in early- and late apoptosis are represented by bottom right- and top right quadrants of the FACS analysis, respectively. The mean ± SEM values were obtained from duplicate samples from each transfection condition.

***, P<0.001, EV vs. FoxG1; or FoxG1 vs. AIB1+FoxG1. One-way ANOVA with Bonferroni post test.

48 C. FoxG1 represses AIB1 promoter activity

1. A previously established model on the regulation of AIB1 promoter activity

Our data demonstrate that FoxG1 overexpression represses the levels of AIB1 transcript, we hypothesized that FoxG1 could directly regulate the AIB1 promoter. Previous studies have identified a region in the AIB1 gene promoter responsible for the positive auto-regulation of transcription through the recruitment of an activating transcriptional protein complex involving

AIB1, E2F1, and Sp1 (62, 67). This critical positive regulatory sequence covers a -250 to +350 span (-250/+350) up and downstream of the transcription start site of the AIB1 gene. An intronic

Sp1 binding site, marked by a GC box at bp +150/+160 is required for the AIB1 promoter activation by E2F1 and AIB1. Moreover, the Sp1 binding sequence serves as a docking site for the recruitment of the AIB1-E2F1 complex, through which AIB1 can act as a coactivator on its own promoter. Since AIB1 is a known transcriptional coactivator for the expression of E2F1- responsive genes, the finding of an E2F1 binding site in the AIB1 promoter suggests that AIB1 can self-regulation. Indeed, Mussi et al show that via binding to Sp1, E2F1 and AIB1 together establish a positive auto-regulatory loop through which the AIB1 gene expression is significantly enhanced (Figure 12) (67).

49

AIB1 RNA Pol II E2F1 Sp1 GC AIB1 -250 +350 Exon 1 +150/+160 +1

Figure 12. Model of the AIB1 gene promoter

Model showing an activating transcriptional complex consisting of AIB1, E2F1 and Sp1, anchored to DNA through a Sp1-binding site, the GC box, which is down stream of exon 1

(black box) in the -250 to +350 region of the AIB1 promoter. The red arrows represent the locations and orientations of the AIB1 promoter-specific primers.

50 2. FoxG1 represses AIB1 promoter activtity

To determine whether FoxG1 impacted AIB1 promoter activity we cotransfected MCF-7 cells with an E2F1 expression vector in the presence or absence of FoxG1 and a wild-type (WT)

AIB1 promoter-luciferase reporter containing the intact positive regulatory sequence of the AIB1 gene promoter. E2F1 significantly enhanced the AIB1 promoter reporter activity, as shown previously (67), whereas the addition of FoxG1 suppressed E2F1-induced AIB1 promoter activation back to the basal level, indicating an inhibitory role for FoxG1 in AIB1 gene expression (Figure 13).

51

AIB1(-250/+350) GC LUC

-250 exon1 +350

8

6 ** 6

4

RLU (x10 ) 2 **

0 AIB1(-250/+350) + + + E2F1 + + FoxG1 +

52 Figure 13. FoxG1 represses the activity of the AIB1 promoter reporter

MCF-7 cells were transfected with WT AIB1(-250/+350) promoter reporter alone, or together with E2F1 in the presence or absence of FoxG1. The assay was plated in triplicate and

24 hours post-transfection, luciferase values were normalized to the protein concentration of each cell lysate. A representative graph is shown from two independent experiments and data were analyzed by one-way ANOVA with Bonferroni post test. **, P<0.01 when E2F1 is compared to promoter alone; or FoxG1 and E2F1 together relative to E2F1.

53 3. The Sp1-binding element is required for FoxG1 recruitment to the transfected AIB1 promoter construct

Because the intronic GC-rich, Sp1 binding sequence is essential for the recruitment of

Sp1, AIB1 and E2F1 (67), we next tested whether FoxG1 is also recruited to the AIB1 gene promoter through this element.

We performed chromatin immunoprecipitation (ChIP) (Figure 14) assays using

HEK293T cells transfected with either the WT luciferase reporter AIB1(-250/+350) or the same reporter with a deleted Sp1 site - AIB1(-250/+350)-Sp1-del (Figure 15a). PCR primers with a forward primer positioned on exon 1 of the AIB1 gene and a reverse primer in the luciferase sequence were used to specifically detect and distinguish the transfected luciferase vectors from the endogenous AIB1 promoter (Figure 15a). Our ChIP analysis showed that FoxG1 is associated with the WT AIB1(-250/+350) reporter but not the mutant reporter AIB1(-250/+350)-

Sp1-del (Figure 15b). Consistent with published literature (67), our data also demonstrated that

AIB1, E2F1 and Sp1 bind to the WT AIB1(-250/+350) reporter, whereas the Sp1 site deletion abolished recruitment of these proteins (Figure 15b). These results indicate that the Sp1 binding site is not only required for Sp1, E2F1 and AIB1 binding to the AIB1 promoter, but it is also critical for the recruitment of FoxG1.

54

Cross-link proteins to DNA in living cells with formaldehyde Figure 14. Schematic of ChIP procedure

A Chromatin Immunoprecipitation DNA (ChIP) technique allows the investigation of

the interaction between proteins and DNA in

add a cell. Beads Procedure: protein is cross-linked to ab to protein A

Beads associated chromatin in living cells by

formaldehyde fixation. Cell are then lysed A and sonicated to produce protein-bound DNA

fragments of 500-800 bp. Protein(s) of

interest are selectively immunoprecipitated

with specific antibodies and agarose beads.

After an incubation period of typically 16

hours, the DNA-immunocomplexes are eluted Elute the immuno-complex off the beads after overnight incubation off the beads and proteins are removed by Decross-link DNA from bound proteins Remove protein by proteinase digestion proteinase digestion. The associated/enriched

DNA fragments are purified and their

sequence is determined by either end-point

PCR or real-time PCR.

Assess enriched DNA products by End-point - or real-time PCR

55 a

AIB1(-250/+350) GC LUC -250 exon 1 +350

AIB1(-250/+350)-Sp1-del LUC -250 exon 1 +350

b

4 *** ** 3 ** 2 *

Relative Units 1

0 IgG IgG Sp1 Sp1 AIB1 AIB1 E2F1 E2F1 FoxG1 GFoxg1

AIB1(-250/+350) AIB1(-250/+350)-Sp1-del

56 Figure 15. Protein association to the Sp1 binding site in the transfected AIB1 gene promoter

(a) HEK293T cells were transfected with either the WT AIB1 reporter or the mutant reporter where the Sp1 binding sequence is deleted. (b) Cells were processed for ChIP 6 hours post transfection. Recruitment of FoxG1, AIB1, E2F1 and Sp1 to both the WT- and mutant promoter reporters was assessed with a pair of primers that specifically detect the transfected reporter DNA, as shown in red arrows in (a). The IgG-ChIP was arbitrarily set as 1 and all the samples were analyzed and plotted in reference to IgG. Data represent two independent experiments and were analyzed by Student’s t test. ***, P<0.001; **, P<0.01; *, P<0.05 compared with IgG control.

57 D. FoxG1 Forms a Complex with AIB1 and E2F1 on the Endogenous AIB1 Gene Promoter

1. FoxG1 is recruited to the endogenous AIB1 gene promoter

We next performed ChIP assays to investigate whether FoxG1 is directly recruited to the

-250/+350 region of the endogenous AIB1 gene promoter in MCF-7 cells. Using a pair of AIB1 promoter-specific primers where the forward primer is positioned on exon 1 of the AIB1 gene and the reverse primer is situated down-stream of the Sp1 binding sequence (Figure 12), we found that an antibody specific to FoxG1, but not the IgG control, successfully immunoprecipitated endogenous FoxG1 on the AIB1 promoter (Figure 16a). As a negative control we demonstrated no specific FoxG1 binding relative to the IgG control to a region in the coding sequence of exon 4 of the AIB1 gene (Figure 16b). We also show that FoxG1 is not recruited specifically to the non-target albumin promoter (Figure 16c). Together, these ChIP data support FoxG1 specific recruitment to the AIB1 promoter.

58 a b c AIB1 promoter AIB1 exon 4 Albumin promoter 4 4 4 *** 3 3 3

2 2 2 Relative Units Relative Units 1 1 Relative Units 1

0 0 0 IgG FoxG1 IgG FoxG1 IgG FoxG1

Figure 16. FoxG1 is recruited to the endogenous AIB1 promoter

ChIP assays were performed in MCF-7 cells, where endogenous FoxG1-DNA complex

was immunoprecipitated with anti-FoxG1 antibody or isotype IgG. Protein-enriched DNA was

analyzed by RT-PCR using (a) AIB1 promoter-specific primers (Figure 9, red arrows) or primers

that will either amplify (b) a region in exon 4 of the AIB1 gene or (c) the albumin promoter.

ChIP results were analyzed by Student’s t test, where ***, P<0.001 relative to IgG.

59

2. The AIB1-E2F1 complex is recruited to the endogenous AIB1 gene promoter

AIB1 has been shown to directly interact with E2F1 and the AIB1-E2F1 complex is essential for E2F1-regulated gene transcription (62). Individual binding of AIB1 and E2F1 to the

-250/+350 region of the endogenous AIB1 gene promoter is well established (67), and our goal was to determine whether AIB1 and E2F1 are recruited to this region as a complex. We performed reciprocal ChIP-reChIP (reChIP) assays (Figure 17) to address this question.

Chromatin samples were prepared from MCF-7 cells, incubated and immunoprecipitated first with either AIB1 or E2F1 antibodies, followed by subsequent reChIP with antibodies specific to either E2F1 or AIB1, respectively. As a control, isotype IgG was used for the first round of IP followed by E2F1- or AIB1 reChIP. As shown in Figure 18, the two-step reciprocal reChIP successfully precipitated the endogenous AIB1 promoter, indicating that AIB1 and E2F1 form a protein complex at the -250/+350 region of the AIB1 promoter (Figure 18).

60

Beads ab to protein A Figure 17. ChIP-reChIP procedure A B Formaldehyde-crosslinked, DNA protein–DNA complexes from living

Elute the immuno-complex cells are subjected to two sequential off the beads after the 1st round of ChIP ChIPs with antibodies of different

specificity. ReChIP has been used to ab to protein B A address, in a qualitative manner, Beads B whether two proteins can DNA simultaneously co-occupy a stretch of

Elute the immuno-complex DNA in vivo. off the beads after the 2st round of ChIP Procedures for single ChIP are

reviewed in Figure 14. Decross-link DNA from bound proteins Remove protein by proteinase digestion

Assess enriched DNA products by End-point - or real-time PCR

61 4 4 ** ** 3 3

2 2

Relative Units 1 Relative Units 1

0 0

IgG/E2F1AIB1/E2F1 IgG/AIB1E2F1/AIB1

Figure 18. AIB1 and E2F1 co-occupancy at the endogenous AIB1 gene promoter

Two-step ChIP-reChIP assays were performed in MCF-7 cells, and all DNA samples were subjected to two rounds of ChIPs. Sonicated DNA was immunoprecipitated first with AIB1 or E2F1 antibodies, followed by reChIP with antibodies specific to E2F1 or AIB1. As a negative control, isotype IgG was used for the first-round ChIPs followed by reChIP of the respective second-round antibodies. Student’s t test. **, P<0.01; when compared with each respective IgG- reChIP control.

62 3. FoxG1 is part of the AIB1-E2F1 complex present at the endogenous AIB1 gene promoter

We next assayed for endogenous FoxG1 participation in the AIB1-E2F1 complex. In order to determine the presence of FoxG1 in the AIB1-E2F1 transcriptional complex we used reciprocal reChIP assays. MCF-7 cells were cross-linked, sonicated and samples were subjected to ChIP and reChIP with FoxG1, AIB1 and E2F1 antibodies. We found that the endogenous

AIB1 promoter was precipitated from Foxg1/AIB1 and Foxg1/E2F1 reChIP immunoprecipitates

(Figure 19a), as well as AIB1/Foxg1 and E2F1/Foxg1 reChIP immunoprecipitates (Figure 19b).

These data suggest simultaneous chromatin co-occupancy of the three proteins, and provide evidence that endogenous AIB1, E2F1 and FoxG1 can be recruited as a complex to the -

250/+350 regulatory sequence of the AIB1 promoter under steady-state conditions.

63

a 3 3

*** 2 2 *

1 1 Relative Units Relative Units

0 0

IgG/AIB1 IgG/Foxg1 FoxG1/AIB1 AIB1/FoxG1

b 3 4

** *** 3 2

2

1

Relative Units Relative Units 1

0 0

IgG/E2F1 IgG/Foxg1 FoxG1/E2F1 E2F1/FoxG1

64

Figure 19. FoxG1, AIB1 and E2F1 co-occupancy at the AIB1 gene promoter

ReChIP assays were performed in MCF-7 cells. Chromatin samples were immunoprecipitated first with (a) FoxG1 or AIB1-, or (b) FoxG1 or E2F1 antibodies, followed by reChIP with antibodies specific to (a) AIB1 or FoxG1, or (b) E2F1 or FoxG1. The endogenous AIB1 promoter bound to each immunocomplex as indicated in the figure was analyzed by RT-PCR using the AIB1 promoter-specific primers. Student’s t test. ***, P<0.001;

**, P<0.01; *, P<0.05 when compared with each respective IgG-reChIP control.

65 E. FoxG1 compromises the integrity of the activating complex on the AIB1 gene promoter

1. FoxG1 overexpression destabilizes the Sp1-assicated transcription complex on the AIB1 gene promoter

Our data thus far have shown that there is a basal level recruitment of endogenous FoxG1 to the AIB1 gene promoter. We next performed ChIP assays to investigate the effect of overexpressing FoxG1, (at levels that can cause suppression of AIB1 gene expression and apoptosis), on the transcription complex present on the endogenous AIB1 promoter at bp

+150/+160. Cross-linked and fragmented DNA was harvested from MCF-7 cells that had been transfected with either an EV control or a FoxG1-expression vector. The samples were then subjected to ChIP with a panel of antibodies, directed against components of the Sp1-associated transcription complex. We demonstrate that increased expression of FoxG1 resulted in a dramatic decline (>50%) in the promoter-binding activities of AIB1, E2F1, p300 and RNA polymerase II (Pol II), although there was no significant change in Sp1 recruitment at the promoter level (Figure 20a). The loss of promoter binding was not due to FoxG1-induced changes in gene expression since protein levels of these factors were either unchanged or increased (e.g. E2F1) when FoxG1 was overexpressed (Figure 20b). Interestingly, we did not observe increased chromatin occupancy of FoxG1 when it was overexpressed (Figure 20a) suggesting that once cellular expression of FoxG1 is above a threshold level, it can promote rapid disassembly of the Sp1-associated complex without affecting the direct binding of Sp1 to the +150/+160 AIB1 promoter binding element.

66

a 3 EV FoxG1 ** 2 ** *** *** ** ns 1

Relative Units

0 IgG FoxG1 AIB1 E2F1 Sp1 p300 Pol II

b

EV FoxG1 EV FoxG1 WB: AIB1 WB: p300

1 0.47 1 1.06

WB: E2F1 WB: FoxG1

1 1.44 WB: Sp1 WB: Actin 1 0.9

67

Figure 20. FoxG1 overexpression leads to decreased recruitment of the members of the transcriptional complex to the endogenous AIB1 promoter

(a) ChIP assays were performed in MCF-7 cells transfected with EV or FoxG1 vectors.

Protein-bound endogenous AIB1 promoter was enriched with antibodies as indicated. RT-PCR was performed using the AIB promoter-specific primers to amplify AIB1 gene promoter in the

ChIP products. Student’s t test, where ***, P<0.001; **, P<0.01 were FoxG1-expressing cells

(black bars) relative to EV (white bars). (b) MCF-7 cells were transfected with EV or FoxG1 expression vectors for 24 hours, and relative protein levels after FoxG1 transfection are shown by WB and probed with antibodies as indicated.

68

2. Overexpressing FoxG1 compromises the integrity of the transcriptional complex present at the endogenous AIB1 gene promoter

To test whether increased FoxG1 expression has a negative effect on the integrity of the transcriptional protein complex, we performed reChIP experiments. We show that FoxG1 overexpression in MCF-7 cells caused significant reduction in the recruitment of protein complexes comprising E2F1 and Sp1, AIB1 and FoxG1 to the endogenous AIB1 promoter, respectively (Figure 21). Most interestingly, while both E2F1/AIB1 and E2F1/FoxG1 complexes showed more than twofold decrease in their co-recruitment, we observed that overexpressing

FoxG1 led to a near threefold decrease in the co-recruitment of E2F1 complexed with Sp1 to the

AIB1 promoter (Figure 21, compare “E2F1/Sp1” between white bar and black bar).

69

EV 3 *** FoxG1

2 ** *

1 Relative Units

0

E2F1/IgGE2F1/Sp1E2F1/AIB1 E2F1/IgGE2F1/Sp1E2F1/AIB1 E2F1/FoxG1 E2F1/FoxG1

Figure 21. FoxG1 compromises the integrity of the protein complex

Affinity of the immunocomplexes associated with the AIB1 promoter was assessed by

ChIP-reChIP experiments, where chromatin was immunoprecipitated sequentially first with anti-

E2F1 antibody, followed by reChIP with antibodies specific to either Sp1, AIB1, or FoxG1. The

E2F1-ChIP was also followed by a reChIP of IgG as a negative control. ***, P<0.001; **,

P<0.01; *, P<0.05 relative to E2F1/IgG. Student’s t test.

70 3. FoxG1 overexpression causes decreased co-occupancy of p300-AIB1 complex at the AIB1 gene promoter

The recruitment of p300 to the +150/+160 Sp1-associated complex has not been described previously and overexpression of FoxG1 also reduces its association with the complex at the AIB1 promoter (Figure 20a), without a reduction in p300 protein expression (Figure 20b).

The binding of p300 to AIB1 is known to promote and stabilize transcriptional complex formation, and exert a positive effect on gene transcription (19, 43, 91). Therefore, we wanted to determine whether overexpressing FoxG1 had any effect on the recruitment of the AIB1-p300 complex. We assessed co-occupancy of AIB1 and p300 at the AIB1 promoter by reciprocal reChIP, and discovered a five- to eightfold reduction in the recruitment of AIB1-p300 complex to the AIB1 promoter in MCF-7 cells transfected with FoxG1 as compared to control (Figure

22).

It is possible that FoxG1 binding to AIB1 causes a conformational change in AIB1 which may subsequently reduces its affinity to p300. As mentioned before, the p300-AIB1 interaction is critical for the stable formation of a transcriptional complex that is required for activation of gene transcription (19, 43, 91). To test this hypothesis we examined the interaction of AIB1 with p300 by WB analysis in the presence or absence of FoxG1. HEK293T Cell lysates containing

FLAG-AIB1 were mixed with HA-p300 lysates, with or without FoxG1. Samples were then subjected to immunoprecipitation with a HA antibody and FLAG-AIB1 was detect in the HA- p300 immunoprecipitates. We found that in the context of FoxG1, the binding of AIB1 to p300 was reduced by > 50% when compared to their interaction without FoxG1 addition (Figure 23).

71

a b 6 8 * EV * EV FoxG1 FoxG1 6 4

4 2 Relative Units

Relative Units 2

0 0

p300/IgG p300/IgG AIB1/IgG AIB1/IgG p300/AIB1 p300/AIB1 AIB1/p300 AIB1/p300

Figure 22. Overexpressing FoxG1 causes reduction in p300-AIB1 co-occupancy at the AIB1

promoter

MCF-7 cells were transfected with EV or FoxG1 as in (a), and harvested for reChIP

experiments by performing reciprocal and sequential ChIPs using antibodies specific to p300,

followed by AIB1, or to AIB1, followed by p300. The AIB1 promoter-specific primers were

used to assess the relative occupancy of the AIB1-p300 complex at the endogenous AIB1

promoter. *, P<0.05 relative to p300/IgG or AIB1/IgG. Student’s t test.

72

HA-p300: + + + FLAG-AIB1: - + + FoxG1: - - +

WB: HA

IP: HA

WB: FLAG

1 0.43

Figure 23. FoxG1 interferes with the interaction between AIB1 and p300

HEK293T cells were transfected with either p300-HA, FLAG AIB1, or FoxG1 separately. Equal amounts of FLAG AIB1 were incubated with equal amounts of p300-HA, with or without the addition of FoxG1 cell lysates. After immunoprecipitation with HA antibody to pull down p300 and associated proteins, a FLAG WB was performed to determine how much

AIB1 immunoprecipitated with p300.

73 F. FoxG1 disrupts AIB1’s coactivator function

1. The effect of FoxG1 on steroid-dependent and -independent promoters

We next wanted to test if the effect of FoxG1 on AIB1-containing transcription complexes was limited to the Sp1 binding site in the AIB1 promoter or if other promoter elements known to involve AIB1 were also affected. AIB1 is a known coactivator for ER- mediated transactivation and has previously been shown to coactivate NF-κB and AP-1 (96,

107), therefore we tested the impact of FoxG1 on promoters containing these binding sites using gene promoter reporter assays. As reported previously (70), AIB1 overexpression significantly induced transcription from all these reporters (Figure 24 and 25).

Concomitant FoxG1 overexpression caused a dramatic reduction in the AIB1-induced transcription of the AP-1 and NF-κB promoters (Figure 24a and b). We also observed a near- complete reversal of AIB1 coactivation on the estrogen-responsive promoter (ERE) reporter in the presence of FoxG1 and estrogen (Figure 25).

74

a b AP-1 1)ț% 3 12 2

5 6 9 1 0.1 6

RLU (x10 ) RLU (x10 ) 3

0 0 + + :FoxG1 + + :FoxG1 EV AIB1 EV AIB1

Figure 24. FoxG1’s effect on steroid-dependent and -independent promoters

HEK293T cells were transfected with AIB1 expression constructs as indicated with either

(a) a multimerized AP-1 reporter or (b) a multimerized NF-κB reporter, in the presence or absence of FoxG1. c-fos and c-jun expression vectors were also cotransfected with the AP-1 reporter. 24 hours after transfection, cells were lysed to measure luciferase activity.

75

ERE 6

4 3 6

2

RLU (x10 ) 1

0 E2 (10 nM): + + + +

EV EV+ AIB1 AIB1+ Foxg1 Foxg1

Figure 25. FoxG1’s effect on estrogen-stimulated transcription

AIB1 was cotransfected with ERα and estrogen-responsive promoter reporter (ERE) constructs into hormone-stripped HEK293T cells, with or without cotransfection of FoxG1. Cells were treated with ethanol (-) or 10 nM estradiol (E2) (+) for 24 hours and analyzed for reporter activity. Results are expressed as changes in the level of activation compared with EV- transfected cells.

76 2. FoxG1 has no effect on the expression of E2F1-responsive genes

Previous studies have shown that AIB1 directly interacts, through its N-terminal domain, with E2F1, and AIB1 is recruited to not only E2F1, but also E2F1 target gene promoters. AIB1 coactivation is shown to significantly increase the expression of E2F1 responsive genes that are critically involved in cell cycle control and G1/S transition (61, 62).

To demonstrate that the repressive effect of FoxG1 on AIB coactivated gene promoters was not universal we tested the effect of FoxG1 overexpression on the transcription of E2F1- regulated genes (since AIB1 is also a coactivator for E2F1). We found in parallel experiments that FoxG1 overexpression had no impact on endogenous E2F1-regulated genes such as CDK2,

CDC25a, MCM7, E2F1 and CDC6 in MCF-7 cells (Figure 26). These functional data indicate that FoxG1 is important not only for the control of AIB1 promoter, but also for some AIB1 regulated steroid-dependent and -independent transcription, although the impact of FoxG1 is dependent on the promoter context.

77

CDK2 CDC25aMCM7 E2F1 CDC6 0

2

4

¨&W

6

8 EV 10 FoxG1

Figure 26. FoxG1 has no effect on E2F1-regulated gene expression

MCF-7 cells were transfected with EV or FoxG1 and total RNA was harvested from cells to determine the relative gene expression for CDK2, CDC25A, MCM7, E2F1, and CDC6. The

Ct values were normalized to actin expression as control.

78

IV. DISCUSSION

79 A. FoxG1 Downregulates AIB1 Expression by Repressing Transcription of the AIB1 Gene

This is the first study, to our knowledge, that describes the regulation of the AIB1 gene by a transcriptional repressor. Since its discovery in 1997, the role of AIB1 in breast cancer and tumorigenesis has been extensively researched (4). Its frequent detection and amplification in various types of human cancers have implicated AIB1 as a cancer-overexpressed oncogene (63) .

Although initially established as a potent transcriptional coactivator for nuclear receptor (NR)- mediated transcription, AIB1 also functions to promote the activity of other transcriptional cofactors such as E2F1, AP-1 and NF-κB. The ability to interact with and coactivate a wide range of transducers and activators of transcription enables AIB1 to participate in a number of tumor growth-promoting pathways, and controls cellular motility and proliferation (110).

Considering the pivotal effects AIB1 exerts over cellular metabolism and cancer morphogenesis, regulation of both AIB1 transcript and protein expression has been the focus of intense research.

Total cellular AIB1 levels can be controlled at multiple levels including control of AIB1 levels of gene transcription (67), control of AIB1 mRNA stability (with e.g. miRNA-17-5p (33)) , control of protein modification including phosphorylation (25, 70, 98, 100), acetylation (19) and sumoylation (97) and control of proteasomal degradation of AIB1 protein (65, 68, 99). In the current study we have determined that FoxG1 can control levels of AIB1 mRNA by directly influencing the transcription of the AIB1 gene. Based on our data we propose a model (Figure

27) whereby the Sp1 site at bp +150/+160 of the AIB1 gene promoter is directly repressed by increasing levels of FoxG1. AIB1 can complex with E2F1 and together regulate the activity of its own promoter (34, 61). More specifically E2F1 can regulate AIB1 promoter activity by interacting with Sp1 bound at bp +150/+160 (67) which, via direct binding to DNA, appears to

80 bridge and anchor the E2F1-AIB1 coactivating complex to the AIB1 gene promoter. This allows

AIB1 to coactivate and enhance the transcriptional activity of its own prompter. Our data show a reduction in the recruitment of the “anchorage complex”, E2F1-Sp1, as well as the essential

“coactivating complex”, E2F1-AIB1 to the AIB1 promoter when FoxG1 is overexpressed. Our data also indicate that p300 is recruited as part of the activating complex necessary for high levels of AIB1 gene transcription. CBP/p300 can bind AIB1 directly, promote stable formation of the transcription complex and has strong histone acetylase activity necessary for altering local chromatin structure and activating transcription (19, 91). We show that overexpressing Foxg1 led to a dramatic reduction in the recruitment of p300-AIB1 complex to the AIB1 promoter.

Together, our data indicate that as FoxG1 levels rise in the cell, the Sp1-associated transcription complex is disrupted, causing E2F1, AIB1 and p300 to dissociate from Sp1, thus reducing AIB1 gene transcription (Figure 27). Inhibition of AIB1 gene transcription by FoxG1 requires an intact

Sp1 binding site but no other elements of the AIB1 gene promoter, as we have shown that deletion of the Sp1 binding sequence effectively prevents recruitment of FoxG1, AIB1, E2F1, and Sp1 to the AIB1 promoter reporter.

81 + p300 AIB1

RNA Pol II E2F1 Sp1 GC AIB1 -250 Exon 1 +150/+160 +350 +1 transcription machinery

“ on ” +

Foxg1

Foxg1

p300

Foxg1 AIB1

RNA Pol II E2F1

Sp1

GC + decreased -250 Exon 1 +150/+160 +350 gene expression +1 transcription machinery

“ off ”

Figure 27. A proposed model for the role of FoxG1 in regulating AIB1 gene expression

FoxG1 binds to, and reduces AIB1 binding to the components of the activating transcription complex that is required for the upregulation of AIB1 gene expression. In the presence of increased FoxG1 levels, the activating complex disassembles and disassociates from the AIB1 promoter, leading to reduced AIB1 gene transcription.

82 B. Homo- and Heterodimerization of FoxG1 may be Required for the Repression of the

AIB1 Gene Expression

Previous studies have shown that FoxG1 can cause transcriptional repression by binding

DNA directly (54) and nucleating a repressosome by recruiting histone deacetylase 1 (HDAC1) and TLE family proteins (108). However, a search (http://www.ncbi.nlm.nih.gov/nuccore) of the region flanking the Sp1 site in the AIB1 gene promoter using AIB1 genomic DNA (GenBank accession no. AL353777) shows no match for the consensus FoxG1 binding sequence,

AATGTAAACA, which is evolutionarily conserved in avian, rat and human (53). Furthermore, the interaction of AIB1 with FoxG1 in cell lysates occurs in the absence of tethering DNA. This suggests that in the context of the Sp1 binding site in the AIB1 promoter, FoxG1 inhibits transcription by disrupting an activating transcription complex bearing histone acetylase activity, rather than by forming a de novo repressosome after direct DNA-binding to the AIB1 gene promoter. Also consistent with this paradigm of FoxG1-induced repression mechanism is that, in our ChIP assays in cells overexpressing FoxG1, we did not observe increased FoxG1 binding to the AIB1 promoter in the vicinity of the Sp1 binding site. In fact, as exogenous FoxG1 levels increase in the cell, the amount of FoxG1 present in the Sp1-associated transcription complex decreases along with the loss of AIB1 and E2F1. This suggests that at higher concentrations of

FoxG1, there is an increase in its access or affinity for AIB1 binding, possibly though dimerization, and this in turn would accelerate degeneration and disassembly of the activating transcription complex. Similar models of repression have been seen with Foxp1, a transcriptional repressor of the forkhead protein family which has been shown to be tumor suppressive in several types of cancers (8). Foxp1 can homo- and heterodimerize with Foxp2 and Foxp4, where

83 the specific combination of homo- and heterodimers are required for the interaction with other transcriptional cofactors as well as for executing transcriptional repression (55).

C. FoxG1 May Act as a Short-Range Repressor at the AIB1 Gene Promoter

Our data also suggest that FoxG1 may fall under the category of “short-range repressors,” which generally act within 100 bp of, or adjacent to a transcriptional activator, causing inhibition through “quenching” (6, 9, 26). Short-range repressors may also directly interact with an activating cofactor and interfere with its activity or block its access to the basal transcriptional machinery (51, 77). It is been demonstrated that several members of the short-range repressors mediate transcriptional repression in a repressor concentration-dependent manner, where higher repressor protein levels (as compared to low levels) are sufficient to switch a gene from an active to an inactive state (31, 44, 77). Therefore, it is possible that repression of the AIB1 gene promoter occurs when FoxG1 protein levels rise in a cell. Since high AIB1 expression can lead to uncontrolled cell proliferation and tumorigenesis (92), it is likely that cells employ FoxG1, by altering its cellular concentration gradient, to control and dampen AIB1 transcription. FoxG1 thus may serve as a short-range repressor for the AIB1 promoter, as we have shown that FoxG1 binding to AIB1 disassembles the Sp1-associated transcription complex. In this sense, FoxG1 acts like a tumor suppresser and this in part, may help to explain the progressive loss of FoxG1 expression as cells evolve from normal to cancerous.

D. FoxG1 Repression of AIB1 Transcription and Coactivation is Not Universal

Another intriguing observation from our study is that, despite the disruptive effect FoxG1 exerts on AIB1-mediated coactivation, we found that this is not limited to, or exclusive to the

84 Sp1 regulatory sequence. In fact, we observed dramatic reductions in AIB1 coactivation at NF-

κB and AP-1 regulatory elements. However, it is worth mentioning that not all AIB1-associated promoter elements are influenced by FoxG1, as we have demonstrated that the transcription of a number of E2F1-driven genes was unaffected by increasing amounts of FoxG1 in the cell. This implies that FoxG1 repression of a gene promoter activity is context-specific for AIB1 in a transcription complex. A recent genome-wide location analysis of AIB1 chromatin affinity sites in 17β-estradiol (E2) -treated MCF-7 cells demonstrated a significant overlap of AIB1 with

FoxA1 binding sites in the breast cancer cell DNA (47). FoxA1 is a determining mediator for estrogen receptor function and endocrine response (37). For comparison, it would be interesting to investigate whether the portion of the AIB1 genomic binding sites overlapping with FoxA1 is also engaged by FoxG1, and also to determine whether such a population represent a subset of

FoxG1- regulated genes.

E. Downregulation of AIB1-regulated Pathways and Inhibition of TGF-β Signaling are

Likely Involved in the FoxG1-indcued Apoptosis in MCF-7 cells

Reintroducing AIB1 into FoxG1-induced apoptotic MCF-7 cells was only able to partially restore viability in these cells. This could indicate that FoxG1 induction of apoptosis also involves genes that are not directly regulated by AIB1. However, complete replenishment of endogenous AIB1 levels is difficult to achieve after knockdown and also the temporal response of different AIB1 regulated genes can be variable. Global ChIP assays have revealed that AIB1 is widely distributed in the genome (47) and our data have shown that FoxG1 downregulates AIB1 coactivation of AP-1 and NF-κB transcription. AP-1 is known to promote

85 the expression of genes involved in cell cycle progression (58), and NF-κB-dependent gene transcription is crucial for pro-proliferation and anti-apoptosis signals (11). Thus reduction in

AIB1 levels by FoxG1 repression likely has effects on multiple AIB1-regulated pathways which in combination, could result in apoptosis.

FoxG1 is also known to antagonize TGF-β signaling by binding to, and blocking, the action of SMAD-3/4 proteins, both of which are major signal-transducers of TGF-β (78).

Although TGF-β can be tumor suppressive, there is also evidence to suggest TGF-β involvement in stimulating invasiveness and metastasis of cancer cells (73). Interestingly, AIB1 is one of a number of TGF-β responsive genes in A549 human lung carcinoma cells (2) and TGF-β can significantly upregulate AIB1 gene transcription in MCF-7 cells (49). Thus FoxG1 inhibition of

TGF-β signaling might also be involved in the FoxG1-mediated apoptosis in MCF-7 cells.

F. FoxG1 can be Both Pro- and Anti-oncogenic

AIB1 has been designated an oncogene (92) and its overexpression is associated with worse disease outcome in multiple types of tumors (105). Paradoxically, even though aberrant

AIB1 expression can lead to tumorigenesis (92) , loss of AIB1 can also be pro-oncogenic in certain contexts, as shown by Coste et al. that absence of AIB1 results in B-cell lymphoma (22).

Similarly, although FoxG1 can interact with androgen receptor (AR) directly in vivo and acts as a corepressor to both AR- and PR-mediated transactivation (69), FoxG1 is also shown to be upregulated in ovarian cancer (17), and its gene amplification is associated with the development of bladder cancer and medulloblastoma (1, 40). This suggests that FoxG1 can act as both a tumor

86 suppressor and an oncogene in a cell-type- and tissue-type-specific manner, depending on the cellular environment.

G. FoxG1 Repression of AIB1 Expression: Its Pharmacological Impacts

Our observations in the present study suggest that FoxG1 acts like a tumor suppressor, and downregulation of FoxG1 function could represent an important alternative mechanism to derail the control of cell viability in breast tumors that are dependent on AIB1 for survival and growth.

A question that arises from the proposed schematic in Figure 27 is how does FoxG1 cause destabilization of the Sp1-associaed transcription complex? One possibility is that through binding, FoxG1 induces a conformational change in AIB1 which leads to reduced affinity between AIB1 and the other members of the Sp1 transcription complex. As mentioned before, the interaction of AIB1 with p300/CBP is critical for AIB1’s function as a transcriptional coactivator, and the binding of p300 to AIB1 secures and stabilizes transcription complex formation. We originally hypothesized that FoxG1 binding to AIB1 may interrupts with the p300-AIB1 interaction since both of our ChIP analysis and co-immunoprecipitation experiments show that increasing levels of FoxG1 in cells led to decreased co-recruitment of p300 and AIB1 to the endogenous AIB1 gene promoter, and reduced protein binding of AIB1 to p300. Based on our data it is possible that FoxG1 interferes with the interaction between p300 and AIB1 by directly occupying the p300/CBP-binding domain of AIB1 and subsequently displacing p300.

However, as shown in Figure 28, the FLAG-tagged AIB1 construct with a deleted CBP/p300- binding domain was able to co-immunoprecipitate FoxG1, at levels higher than the full-length

87 AIB1. This suggests that the p300/CBP-binding domain is not likely responsible for the binding of FoxG1 to AIB1, and a conformational change in AIB1 is most likely the cause for p300 disassociation from AIB1.

More experiments will be directed to examine the domains of AIB1 and FoxG1 responsible for complex formation and repression of gene transcription. If FoxG1 binding to

AIB1 could indeed induce a conformational or allosteric change in AIB1 then mimicking FoxG1 binding to AIB1 with small molecule inhibitors may likely be a potential therapeutic target in cells where the AIB1 oncogene is operational.

88

FLAG-AIB1 + - - + -

)/$*$,%¨&%3 - + - - + HA-FoxG1 - - + + +

WB: FLAG

,3)/$*

WB: HA

WB: FLAG Input WB: HA

Figure 28. The CBP/p300 binding domain is not responsible for AIB1 and FoxG1 interaction

FLAG-AIB1 or FLAG-AIB1ΔCBP was cotransfected with HA-FoxG1 constructs in

HEK293T cells. 48 hours post transient transfection, whole cell lysates were collected and used for immunoprecipitation (IP) and western blot (WB) analysis with anti-FLAG and anti-HA antibodies as indicated.

89

V. APPENDIX

90

A. DESIGN OF AIB1Δ4 SCORPION PRIMERS - A QUANTITATIVE BIOASSAY FOR

THE HIGH-EFFICIENT, LOW-COST SCREENING OF AIB1Δ4 IN HUMAN BREAST

AND PANCREATIC CANCERS.

91

I. INTRODUCTION

92 1. AIB1Δ4, the Splice Variant of AIB1

Previously, in an attempt to identify naturally occurring splice variants of AIB1 present in breast cancer cells that might encode proteins with altered function relevant to breast cancer progression, we assembled the exon-intron structure of AIB1 by comparing the published sequence of the cDNA. Using reverse transcription and polymerase chain reaction (PCR), we detected two PCR products that differed in size by 150 base pairs. We discovered that the smaller PCR product was actually an NH2-terminal truncated version of AIB1 whereas exon 3 was spliced from the mature mRNA. The resulting protein lacked the bHLH and PAS A domains and was named AIB1Δ3 at the time (74). More recently, an additional 5’ exon 80 kilobases upstream of the known 5’UTR was identified. Therefore, the deleted exon is now exon 4, and we now refer to the splice variant as AIB1Δ4. The full-length AIB1 protein has a molecular weight of 155 kDa consisting of 1424 amino acids. The AIB1Δ4 transcript lacks exon 4 due to alternative splicing and the resultant protein lacks the N-terminal 223 amino acids. AIB1Δ4 is a protein of 130 kDa and consists of 1201 amino acids (Figure 29).

93

AIB1

Exon: 1 2 3 4 5 6 ORF 23

NLS CBP/p300 HAT

1 A B 1424

bLHL PAS RID NES

$,%¨

Exon: 1 2 3 5 6 ORF 23 4

CBP/p300 HAT

224 B 1424

PAS RID NES

Figure 29. mRNA and protein structure of AIB1 and AIB1Δ4

The full-length AIB1 mRNA has 23 exons which make a 155 kDa protein of 1424 amino acids. Exon 4 is lost in AIB1Δ4 mRNA due to alternative splicing and the resultant protein lacks the N-terminal 223 amino acids. AIB1Δ4 has a molecular weight of 130 kDa and is a protein of

1201 amino acids. The PAS B, RID, CBP/p300 binding domains as well as the nuclear export sequence (NES) are retained in both the full-length AIB1 and AIB1Δ4 proteins. Splicing of exon

4 results in the loss of nuclear localization signal (NLS), bHLH and PAS A domain in AIB1Δ4.

94 In a series of studies, we found that AIB1Δ4 mRNA expression was significantly increased in breast cancer cells in comparison to normal or non-transformed breast epithelial cells. AIB1Δ4 transcript levels were elevated in breast tumor tissue relative to normal breast tissue. Interestingly, we found that AIB1Δ4 was much more effective and potent in coactivating estrogen, progesterone and EGF singling than the wild type AIB1 (74), and the overexpression of

AIB1Δ4 significantly increased the efficacy of estrogenic compounds and the agonist effects of the selective estrogen receptor modulator tamoxifen at both estrogen receptor-α (ER- α) and ER-

β in breast, ovarian and endometrial cancer cell lines (75). We also made transgenic mice expressing human AIB1Δ4 driven by the CMV promoter to determine the role of AIB1Δ4 in vivo. These mice had significant increases in mammary epithelial cell proliferation, mammary gland mass and the expression of IGF-1 receptor protein. At 13 months of age, mammary ductal ectasia was found in the AIB1Δ4 mice, and increased expression of proliferative markers such as proliferating cell nuclear antigen (PCNA), phospho-histone H3, and cyclin D1 were also observed in these animals (90).

In a recent study, AIB1Δ4 was shown to act as a molecular bridge and facilitate the interaction of EGFR and focal adhesion kinase (FAK) in the cytoplasm. The RID domain of

AIB1Δ4 was identified to be responsible for the interaction with FAK though the N-terminus of

FAK, whereas the N-terminus of AIB1Δ4 was defined as the EGFR interaction domain. In this study, it was reported that, upon EGF stimulation, p21 activated kinase (PAK1) (a known mediator of EGF signaling) was able to phosphorylate AIB1Δ4 at T56, S659 and S676, which in turn promoted AIB1Δ4 localization to plasma membrane (i.e. fliopodia), the interaction of

AIB1Δ4 with FAK and EGFR and, ultimately, cell migration. Using the metastatic breast cancer

95 cells MDA-MB-231, this study found that stable AIB1Δ4 overexpression, without affecting cell or tumor growth rate, significantly increased MDA-MB-231 cell migration and invasion, as well as mammary tumor metastasis to the lymph node and lung in the mouse model. In addition, the number of circulating tumor cells was also elevated with AIB1Δ4 overexpression (59).

2. AIB1Δ4 is a More Potent Coactivator Due to Its Nuclear Function

The full-length human AIB1 transcript contains 23 exons, and the nuclear localization signal (NLS) sequence of AIB1 is located in the bHLH domain, which is lacking in AIB1Δ4 due to the loss of exon 3 and the subsequent N-terminal truncation. Therefore, any function of this protein in cancer to date has been attributed predominantly to its role in cytoplasm. However, in a recent study, we showed that AIB1Δ4, like full-length AIB1, could also enter the nucleus by interacting with other NLS containing proteins such as p300/CBP. Dimerization with full-length

AIB1 was also able to facilitate the nuclear transport of AIB1Δ4. We demonstrated that endogenously and exogenously expressed AIB1Δ4 was recruited as efficiently as full-length

AIB1 to estrogen-response elements of genes, and it enhanced estrogen-dependent transcription more effectively than AIB1. We showed that AIB1Δ4 was a more potent nuclear coactivator than full-length AIB1 - most likely due to its loss of the N-terminus, which possibly contains an inhibitory domain that normally serves to squelch the transcriptional activity of full-length AIB1

(20).

96

II. RESULTS

97 1. Development of the AIB1Δ4 Scorpion Primer

We have shown previously that the expression of AIB1Δ4 in breast cancer cell lines and tumor tissues is significantly higher than normal cell lines or breast tissues (74). A recent publication has also reported the correlation of higher AIB1Δ4 expression with increased metastatic potential of the beast cancer cells MDA-MB-231 (59). However, detection of the levels of AIB1Δ4 expression in these studies was conducted by Western blotting and IHC, both of which are semi-quantitative. Also, even though we have recently developed the affinity purified AIB1Δ4 antibody, only “relative” Δ4 protein expression levels can be obtained via the use of this antibody. Therefore, our goal was to design a quantitative and high throughput bioassay that was able to determine AIB1Δ4 expression in cell lines and tissues.

Initially, we designed Taqman and CYBR green based primers specifically for full-length

AIB1 and the splice variant Δ4 to be used in real-time PCR reactions. However, since both transcripts differ only by the lack of exon 4, it was extremely difficult to distinguish AIB1Δ4 independently from the full-length AIB1 in real-time PCR experiments using both techniques.

After a few failed attempts to redesign primers for both Taqman and CYBR green based PCR reactions, the “Scorpion Primer and Probe” technique was our final strategy. The Scorpion primer came to our attention because it had been used successfully to distinguish single nucleotide polymorphisms as well as for identifying splice variants of gene products (87, 89).

Typically, a scorpion consists of a specific probe sequence that is complementary to a sequence of the gene of interest, and is held in a hairpin loop configuration by complementary stem sequences (stem) on the 5’ and 3’ sides of the probe. There is a fluorophore (reporter) attached to the 5’-end of the loop (at the end of the 5’-stem), and its activity is quenched by a moiety

98 (quencher) joined to the 3’-end of the loop (at the end of the 3’-stem). The hairpin loop is linked to the 5’-end of a forward primer via a PCR stopper. During PCR amplification, after a few cycles of extension with the scorpion probe conjunct forward primer and a reverse primer, the specific probe sequence will bind to its complement within the same strand of DNA (if the target is indeed present in the reaction). This hybridization event will open the hairpin loop which is held closed by the stem sequences, consequently physically separating the fluorophore from the quencher so that fluorescence is no longer quenched and an increase in signal can be detected.

The function of the PCR stopper is to prevent opening of the hairpin loop and non-specific read- through in the absence of target DNA sequence. As shown in Figure 30a, the probe sequence of the AIB1Δ4 scorpion covers the unique “junction” between exon 3 and 5, where the first half of the probe contains the last 8 bp of exon 3 (shown in red), and the last half comprises the first 16 bp of exon 5 (shown in green). In contrast, the probe sequence of full-length AIB1 scorpion is complementary to a sequence in exon 4 (shown in light blue), which is absent in the splice variant Δ4 (Figure 30).

99 a AIB1 Scorpion Primer

5’ 3’ 3 4 5 3 4 5

$,%¨6FRUSLRQ3ULPHU

5’ 3’ 3 5 3 5

Figure 30. Making of the AIB1 and AIB1Δ4 scorpion primers

(a) Scorpion primers were designed to quantitatively and independently detect and distinguish AIB1Δ4 from the full-length AIB1. Both scorpion primers consist of forward primer

(black half arrow), stopper (black jagged line), quencher (purple circle), probe (light blue - exon

4, red – exon 3, green – exon 5) and reporter (dark pink and yellow circles). Colored boxes: numbered exons. In a real-time PCR reaction, the stem region (black cross bars) of the scorpion primer will only disassociate when the probe binds to its specific DNA target. (b) Design and sequences of AIB1 and AIB1Δ4 scorpion primers.

100 B AIB1 Scorpion

Target Sequence: 5- TCTTACCTGCAGTGGTGAAAAAC -3 (Top Strand)

Probe Sequence: 3- AGAATGGACGTCACCACTTTTTG -5 (Bottom Strand) 5- GTTTTTCACCACTGCAGGTAAGA –3 (23bp, Tm=56°C, GC% = 44)

Stem sequence: CCCGCGC______GCGCGGG

Scorpion Primer for full-length AIB1 (Amplicon =181):

5-HEX CCCGCGC GTTTTTCACCACTGCAGGTAAGA GCGCGGG BHQ1 HEG GCCATGTGATACTCCAGGA -3 (Forward Primer: 19bp, Tm= 53, 53%GC)

Reverse Primer: 5- ACGTATCTGTCTTACTGTTTCC -3 (22bp, Tm=53, 41% GC)

HEX: fluorophore BHQ1: quencher HEG: stopper

$,%¨6FRUSLRQ

Target Sequence: 5- GGACAAGGGAAAAACTATTTCCAA-3

Probe’ Sequence: 3- CCTGTTCCCTTTTTGATAAAGGTT-5 5-TTGGAAATAGTTTTTCCCTTGTCC-3 (24bp, Tm=54.1ºC, GC% 38%)

Stem sequence: CCCGCGC______GCGCGGG

6FRUSLRQ3ULPHUIRU$,%¨ $PSOLFRQ 

5- FAM CCCGCGC TTGGAAATAGTTTTTCCCTTGTCC GCGCGGG BHQ1 HEG CGCAAATTGCCATGTGATAC -3 (Tm=52.6, 45%GC, 20bp)

Reverse Primer: 5- CCATCCAATGCCTGAAGTAA -3 (Tm=52.5, 45%GC, 20bp)

FAM: fluorophore BHQ1: quencher HEG: stopper

101 2. Validation of the AIB1Δ4 Scorpion Primer

We then tested the specificity of both AIB1 and AIB1Δ4 scorpion primers against expression plasmids that contain cDNA for either full-length AIB1 or AIB1Δ4. For AIB1 scorpion primer, we observed increased fluorescence activity in real-time PCR (RT-PCR) reactions only when AIB1 plasmid was present (Figure 31a). For AIB1Δ4 scorpion primer, increased fluorescence was detected in the presence of AIB1Δ4 plasmid and not full-length AIB1 cDNA (Figure 31b). This suggests that the scorpion primers are specific, and we can now quantitatively and independently detect and distinguish AIB1Δ4 from the full-length AIB1.

102

a b AIB1 Scorpion Primer $,%¨6FRUSLRQ3ULPHU 350 800 AIB1 AIB1 250 $,%¨ $,%¨ 600

150 400 Fluorescence 50 Fluorescence

200 0 10 20 30 40 50 60 10 20 30 40 50 60

Figure 31. Validation of the AIB1 and AIB1Δ4 scorpion primers

(a) Real-time PCR reaction containing both AIB1 and AIB1Δ4 scorpion primers but

AIB1 expression plasmids only. (b) Real-time PCR reaction containing both AIB1 and AIB1Δ4

scorpion primers but AIB1Δ4 expression vectors only. Cycling conditions for AIB1 and AIB1Δ4

scorpion primers consisted of an initial denaturing step at 94 °C (2 min) and 60 cycles (20s at

94°C, 15s at 55.5°C, and 20s at 72°C). Unlike SYBR Green real time PCR analysis where data

are collected during the extension step, data for the Scorpion primer reactions were collected

during the 55.5 °C annealing step.

103 3. Higher AIB1Δ4 Levels Are Correlated With Increased Invasiveness and Metastatic

Potential in Pancreatic and Breast Cancer Cells Lines

With the development of the AIB1Δ4 scorpion primers, we wanted to determine whether

AIB1Δ4 expression levels correlated with invasiveness and metastatic potential. We investigated

AIB1Δ4 mRNA levels in the human pancreatic cancer cell line COLO 357 and its two metastatic variants, COLO PL (pancreas to liver) and COLO SL (spleen to liver). These cell lines were generated by injecting COLO 357 cells to the pancreas or spleen of nude mice. Liver metastases formed shortly after injection in these mice, and were harvested and re-injected into the spleen or pancreas. After several cycles of in vivo selection, the metastatic variants COLO PL and SL were obtained from the parental 357 cells (13). Phenotypically, the variant cells had increased expression of pro-angiogenic markers such as vascular endothelial growth factor (VEGF) and interleukin-8 (IL-8), and produced a higher incidence and number of lymph node and liver metastases than the parental cells. The metastatic variants also had significantly shorter doubling time, and exhibited increased motility and invasiveness in comparison to the parental COLO 357 cells(13). We decided to use the AIB1Δ4 scorpion primer to determine if AIB1Δ4 was correlated with the metastatic phenotype of these cells. We found that there was a near-threefold increase in the levels of AIB1Δ4 transcript in both of the metastatic variant cell lines COLO PL and SL as compared to the parental 357 cells, indicating a correlation of AIB1Δ4 expression with metastatic capability (Figure 32).

104 COLO 357 3

2

1

$,%¨P51$ Fold change

0

COLO PL COLO SL COLO 357

Figure 32. AIB1Δ4 transcript levels are higher in metastatic pancreatic cancer cells

COLO 357, PL and SL cells were grown until 70 ~ 80 % confluence. Total mRNA was harvested and reverse transcribed. Relative AIB1Δ4 transcript levels were determined by using

AIB1Δ4 scorpion primers. Actin mRNA levels were assessed with a pair of CYBR green primers. All Ct values were normalized first to actin, then to the value of the parental COLO 357 cells which was arbitrarily set to 1. Cycling conditions for the human actin primers include a denaturing step at 94 °C (2 min) and 45 cycles (20 s at 94 °C, 30 s at 58 °C, and 40 s at 72 °C).

105 We next tested AIB1Δ4 expression in the highly metastatic human breast cancer adenocarcinoma MDA-MB-231 cells. The cell line MDA-MB-231 was originally derived from the pleural effusion of a breast cancer patient suffering from widespread metastasis years after removal of her primary tumor. Since then, three metastatic sub lines of MDA-MB-231 were made by injecting the parental cells into the arterial circulation, and the resulting bone (SCP2), brain (BrM2) and lung (4175) homing variants were harvested and characterized (12, 38, 66).

We compared the levels of AIB1Δ4 mRNA between the parental MDA-MB-231 cells and the three metastatic variants, as well as the normal, non-tumorgenic HMEC cells. We found that

HMEC had the lowest relative AIB1Δ4 expression compared to the MDA 231 parental and the variant cells. In addition, all of the in vivo selected tissue-specific variants had significantly higher AIB1Δ4 transcript expression (seven- to tenfold increase) than the parental cells (Figure

33). Taken together, our data suggest that there is indeed a strong correlation between AIB1Δ4 expression and increased metastatic potential.

106 MDA-MB-231 15

10

5 $,%¨P51$ )ROGFKDQJH

0

HMEC

MDA-MB-231

0'$ OXQJ 0'$%U0 EUDLQ 0'$6&3 ERQH

Figure 33. AIB1Δ4 expression correlates with increased metastatic potential in breast cancer cells

HMEC, MDA-MB-231, MDA231-BrM2, MDA231-SCP2 and MDA231-4175 cells were grown until 70 ~ 80 % confluence. Total mRNA was collected from each cell line and reverse transcribed. Relative AIB1Δ4 transcript levels were determined by using AIB1Δ4 scorpion primers. Actin mRNA expression was assessed with a pair of CYBR green primers. All values were normalized first to actin, then to the value of HMEC cells which was arbitrarily set to 1.

107

Abbreviations

SCFFBW7α, a SKP1-cullin-1-F-box complex that contains FBW7 as the F-box protein; ERK, extracellular-signal-regulated kinases; JNK, c-Jun N-terminal kinases; CK1δ, casein kinase 1 delta; PKA, protein kinase A

108

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