THE MECHANISM OF PPARN3 MEDIATED DOWN REGULATION OF SODIUM HYDROGEN EXCHANGER 1 (NHE1) GENE EPXRESSION AND ITS INHIBITION BY ESTROGEN RECEPTOR n1 1

82 3A1.5 Pharmacologcial PPARγ antagonist abrogates the effect of PPARγ ligand on NHE1 gene expression.. 88 3A2.1 Transcription-defective PPARγ abrogates the effect of PPARγ ligand on NH

THE MECHANISM OF PPARγ-MEDIATED REGULATION OF SODIUM HYDROGEN EXCHANGER 1 (NHE1) GENE EXPRESSION AND ITS INHIBITION BY

DOWN-ESTROGEN RECEPTOR α

Zhou Ting (BSc (Hons.), NUS)

A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY

NUS GRADUATE SCHOOL FOR INTEGRATIVE

SCIENCES AND ENGINEERING

NATIONAL UNIVERSITY OF SINGAPORE

2012

ACKNOWLEDGEMENTS

I wish to express my heartfelt gratitude for my supervisor Professor Shazib Pervaiz I want to thank him for taking me as an honours student during undergraduate years, and allowing me to continue my graduate study in this lab I am grateful for all the guidance and encouragement that he has offered during all these years

To Dr Alan Prem Kumar, thank you for giving me the initial guidance when I first entered the field of research, and throughout the course of my study Your advice and help will always be remembered

I would also like to thank my TAC members, Prof Marie-Veronique Clement and Prof Edison Liu for there invaluable input into this project To Marie, thank for your constructive comments during all the meeting sessions I am also grateful for the opportunity Prof Liu offered to let me do some of the crucial experiments in his lab and for all the resources he provided

My warmest thanks to all the colleagues I have worked with and learned from throughout all these years Special thanks to Ms Kong Say Li for teaching me the ChIP techniques, and Ms Quak Ai Li for helping me establish the initial direction of the project To NUMI girls and Team Xtream, thank you for making my PhD years enjoyable and for being great friends

Finally, I would express my deepest gratitude for my parents Though you are not physically with me during all these years in Singapore, your encouragement and support has always been the source of my strength at every step of my endeavors

TABLE OF CONTENTS

ACKNOWLEDGEMENTS II SUMMARY X LIST OF FIGURES XII LIST OF TABLES XV LIST OF ABBREVIATIONS XVI LIST OF PUBLICATIONS XXII

1 INTRODUCTION 1

1.1 PEROXISOME PROLIFERATOR-ACTIVATED RECEPTORS (PPARS) 1

1.1.1 Identification of PPARs 1

1.1.2 Structural domains of PPARs 2

1.1.3 Mechanism of action of PPARs 3

1.1.4 Subtypes of PPARs 5

1.1.5 Ligands and physiological functions of PPARα and PPARδ 7

1.1.6 Ligands of PPARγ 8

1.1.7 PPARγ and adipogenesis 10

1.1.8 PPARγ and insulin sensitization 12

1.1.9 PPARs and cancer 13

1.1.10 PPARγ and breast cancer 15

1.2 ESTROGEN RECEPTORS (ERS) 18

1.2.1 Identification and structures of ERs 18

1.2.2 Mechanism of action of ERs 20

1.2.3 Estrogen and ERs in human breast cancer 23

1.2.4 Ligands of ERs 26

1.2.5 ERs cross talk with each other and with other signaling pathways… 29

1.3 SODIUM-HYDROGEN EXCHANGER 1 (NHE1) 32

1.3.1 Intracellular pH regulation 32

1.3.2 Mammalian Na+/H+ exchanger 32

1.3.3 NHE1 and cell volume 34

1.3.4 NHE1 and cell proliferation and differentiation 34

1.3.5 NHE1 and cell migration 35

1.3.6 NHE1 and heart disease 35

1.3.7 NHE1 and cancer 36

1.3.8 Regulation of NHE1 activity 39

1.3.9 Regulation of NHE1 transcription 41

1.4 REACTIVE OXYGEN/NITROGEN SPECIES (ROS/RNS) 43

1.4.1 ROS/RNS species 43

1.4.2 Intracellular sites of ROS production 43

1.4.3 NO production and its derivatives 45

1.4.4 The antioxidant system 47

1.4.5 ROS/RNS-mediated cell death 48

1.5 AIM OF STUDY 49

2 MATERIALS AND METHODS 51

2.1 MATERIALS 51

2.1.1 Chemicals and reagents 51

2.1.2 Cell lines and cell culture 52

2.1.3 Antibodies 53

2.1.4 Plasmids and siRNAs 54

2.1.5 Primers and Oligonucleotides 55

2.2 METHODS 56

2.2.1 Crystal violet cell viability assay 56

2.2.2 Colony forming assays 57

2.2.3 Immunofluorescence 57

2.2.4 Western Blot analysis 58

2.2.5 Nuclear-cytoplasmic fractionation 59

2.2.6 Reverse Transcription-Polymerase Chain Reaction (RT-PCR) 60

2.2.7 Transient Transfection 61

2.2.8 Luciferase gene reporter assay 61

2.2.9 Chloramphenicol Acetyl Transferase (CAT) assays 62

2.2.10 Measurement of Reactive Oxygen Species (ROS) 62

2.2.11 Noshift Transcription Factor Assay 63

2.2.12 Chromatin immunoprecipitation assay 63

2.2.13 Coimmunoprecipitation 65

2.2.14 Morphology studies 66

2.2.15 Protein determination 66

2.2.16 Statistical analysis 66

3 RESULTS 68

3A PPARγ-MEDIATED REGULATION OF NHE1 68

3A.1 PPARγ AND THE EXPRESSION OF NHE1 68

3A1.1 Identification of putative PPRE on NHE1 promoter 68

3A1.2 Down-regulation of NHE1 by PPARγ ligands 73

3A1.3 Down-regulation of NHE1 by PPARγ ligands is PPARγ-dependent… 80

3A1.4 Silencing PPARγ abrogates the effect of PPARγ ligand on NHE1…… 82

3A1.5 Pharmacologcial PPARγ antagonist abrogates the effect of PPARγ ligand on NHE1 gene expression 84

3A.2 THE MECHANISM OF PPARγ-MEDIATED DOWN-REGULATION OF NHE1 88

3A2.1 Transcription-defective PPARγ abrogates the effect of PPARγ ligand on NHE1 gene expression 88

3A2.2 Activated PPARγ binds to the identified PPRE on NHE1 promoter… 92

3A.3 THE MECHANISM OF ROS/RNS-MEDIATED

DOWN-REGULATION OF NHE1 95

3A3.1 Production of ROS/RNS by PPARγ ligands in breast cancer

cells……… 963A3.2 ONOO– is the main RNS species produced by PPARγ ligands

in breast cancer cells 973A3.3 ONOO– is partially responsible for PPARγ ligand-mediated

down-regulation of NHE1 expression 102

3A.4 THE ANTI-TUMOR EFFECTS OF PPARγ LIGANDS 109

3A4.1 PPARγ ligands induce loss of cell viability in breast cancer

cells……… 1093A4.2 PPARγ ligands inhibit colony formation by breast cancer cells 1113A4.3 Anti-tumor effect of 15d-PGJ2 is PPARγ-dependent 1143A4.4 Anti-tumor effects of 15d-PGJ2 is partially ROS/RNS-

dependent 1193A4.5 Reduced NHE1 expression is responsible for PPARγ-mediated

anti-tumor effects 119

REGULATION 121 3B.1 ESTROGEN BLOCKS THE EFFECT OF PPARγ ON NHE1 121

3B1.1 Regular serum versus dextran stripped serum condition 1213B1.2 Estrogen blocks PPARγ-mediated down-regulation of NHE1 in

CS serum condition 126

3B.2 ACTIVE ERα BLOCKS EFFECT OF PPARγ ON NHE1 129

3B2.1 Re-expression of ERα in ER negative MDA-MB-231 cells

restores its response to E2 on inhibiting PPARγ-mediated down-regulation

of NHE1… 1293B2.2 Transient silencing of ERα in ER positive MCF-7 cells

abrogates the inhibitory effect of E2 on PPARγ-mediated down-regulation

of NHE1… 133

3B2.3 Depletion of active ERα in ER positive MCF-7 cells enhances

PPARγ-mediated down-regulation of NHE1 136

3B2.4 Re-expression of ERα in ER negative MDA-MB-231 cells blocks PPARγ-mediated down-regulationof NHE1 141

3B.3 TRANSCRIPTIONALLY ACTIVE ERα BLOCKS THE EFFECT OF PPARγ ON NHE1 EXPRESSION 145

3B3.1 ERα antagonist enhances the PPARγ-mediated NHE1 repression… 145

3B3.2 ERα defective in DNA binding enhances the effect of PPARγ ligand on NHE1 down-regulation 149

3B.4 THE MECHANISM BY WHICH ERα BLOCKS THE EFFECT OF PPARγ ON NHE1 EXPRESSION 153

3B4.1 ERα does not bind to the putative ERE on NHE1 promoter 153

3B4.2 ERα suppresses binding of PPARγ to NHE1 promoter 155

3B4.3 ERα inhibits transactivation of PPARγ 159

3B4.4 PPARγ inhibits binding of activated ERα to ERE 162

3B4.5 PPARγ physically interacts with ERα 163

3B4.6 Growth inhibitory effect by PPARγ ligand combined with ERα antagonists 167

4 DISCUSSION 169

4.1 ESTABLISHING THE RELATIONSHIP BETWEEN PPARγ ACTIVATION AND NHE1 EXPRESSION 169

4.1.1 Identification of NHE1 gene as a transcriptional target of PPARγ…… 169

4.1.2 The mechanism of PPARγ-mediated repression of NHE1 gene 171

4.1.3 Production of ROS/RNS by PPARγ ligands in breast cancer cells………… 174

4.1.4 The mechanism of ROS/RNS-mediated repression of NHE1 gene……… 177

4.2 ANTI-CANCER EFFECTS OF PPARγ LIGANDS 179

4.2.1 PPARγ-dependent anti-cancer effects of PPARγ agonists 179

4.2.2 PPARγ-independent anti-cancer effects of PPARγ agonists 1824.2.3 Repression of NHE1 is involved in anti-tumor effect of PPARγ

ligand…… 186

DOWN-REGULATION OF NHE1 189

4.3.1 ERα negatively interferes with PPARγ-mediated

down-regulation of NHE1 gene expression 1894.3.2 Unravelling the mechanism of how ERα inhibits PPARγ-

mediated down-regulation of NHE1 gene expression 1924.3.3 Signal cross talk between ERα and PPARγ in breast cancer

cells……… 195

4.4 CLINICAL SIGNIFICANCE OF PPARγ-MEDIATED BREAST

CANCER THERAPY AND ITS POSSIBLE MODULATION BY

ER SIGNALLING PATHWAY 199 4.5 CONCLUSION 201 REFERENCES 205

SUMMARY

In addition to its role in lipid and glucose metabolism, peroxisome activated receptor gamma (PPAR) has been associated with the process of carcinogenesis, which therefore presents a promising target for cancer treatment Having identified a Peroxisome Proliferator Response Element (PPRE) in the

proliferator-promoter region of the pH regulator, Na+/H+ exchanger 1 (NHE1), we recently

showed that exposure of ER-negative breast cancer cells to PPAR ligands repressed NHE1 expression, which could be inhibited by the PPAR antagonist, GW9662 Moreover, inhibition of NHE1 expression either by direct silencing or pre-incubation with PPAR ligands in breast cancer cells increased their sensitivity to doxorubicin and paclitaxel However, recent evidence of cross talks between nuclear receptors including PPAR and the estrogen receptor (ER) pathways has been demonstrated Here we investigated the effect of PPAR activation on NHE1 gene repression in the presence of 17-estradiol (E2) using ER-positive MCF-7 breast cancer cells Results show that E2 prevented the strong inhibition of NHE1 expression by PPAR ligands (natural or synthetic) On the contrary, E2 was unable to

prevent the inhibition of NHE1 expression by PPAR ligands in the ER -negative

breast cancer cell line, MDA-MB-231 or in MCF-7 cells where ER was silenced by specific ER siRNA This result suggested that a functional activated ER is necessary to prevent PPAR-dependent down-regulation of NHE1 by E2 Indeed, a putative ER binding site (ERE) in close proximity and upstream of the PPRE was identified; however, ER did not bind to the putative ERE ER was found to physically associate with PPAR at the PPRE and functionally interfered with PPAR binding efficiency to NHE1 promoter Disruption of ER-PPAR complex by anti-estrogens led to increased efficacy of the anti-tumor activity of PPAR and its

repressive effect on NHE1 gene expression in vivo, which could be a potential

combination therapy for enhanced efficacy of anti-estrogen therapy in breast cancer patients

LIST OF FIGURES

Figures INTRODUCTION

Figure A: Chemical structures of PPARγ agonists and antagonist

Figure B: Chemical structures of ER agonist and antagonists

RESULTS

Figure 1: Sequence Analysis of NHE1 Promoter

Figure 2: PPARγ ligands down-regulate NHE1 protein levels in human

breast cancer cells

Figure 3: PPARγ ligands down-regulate NHE1 mRNA levels in human

breast cancer cells

Figure 4: Overexpression of PPARγ enhances the inhibition of 15d-PGJ2

on NHE1 expressions

Figure 5: Silencing PPARγ attenuates the inhibition of 15d-PGJ2 on

NHE1 expression

Figure 6: PPARγ inhibitor abrogates the effects of 15d-PGJ2 on PPARγ

activity and on NHE1 expression

Figure 7: Transcription-defective PPARγ abrogates the effect of PPARγ

ligand on NHE1 gene expression

Figure 8: PPARγ binds to NHE1 promoter upon 15d-PGJ2 treatment Figure 9: PPARγ ligands produce ROS/RNS in breast cancer cells

Figure 10: PPARγ ligands produce ONOO- in breast cancer cells

Figure 11: ROS/RNS contributes to down-regulation of NHE1

Figure 12: ROS/RNS is partially responsible for 15d-PGJ2-mediated

down-regulation of NHE1

Figure 13: Effects of PPARγ ligands on breast cancer cell morphology and

cell viability

Figure 14: PPARγ agonists reduce colony-forming ability of breast cancer

cells

Figure 15: PPARγ antagonist rescues the loss of cologenic ability induced

by PPARγ ligand in breast cancer cells

Figure 16: FeTPPS rescues the loss of cell viability and colonogenic

ability induced by PPARγ ligand in breast cancer cells

Figure 17: Regular serum versus charcoal/dextran-treated serum

Figure 18: Estrogen blocks PPARγ-mediated down-regulation of NHE1

expression

Figure 29: Re-expression of ERα blocks PPARγ-mediated

down-regulation of NHE1 expression in MDA-MB-231 cells

Figure 20: Silencing ERα attenuates the inhibitory effect of E2 on

PPARγ-mediated down-regulation of NHE1

Figure 21: Reduced ERα level enhances PPARγ-mediated

down-regulation of NHE1 in regular serum condition

Figure 22: Re-expression of ERα blocks PPARγ-mediated

down-regulation of NHE1 in MDA-MB-231 cells kept in regular serum condition

Figure 23: ERα antagonists enhance PPARγ-mediated down-regulation of

NHE1 in regular serum condition

Figure 24: Transfection of DNA-binding defective ERα enhances

PPARγ-mediated down-regulation of NHE1 in regular serum condition Figure 25: Identification of putative ERE on NHE1 promoter

Figure 26: Binding of PPARγ to PPRE is blocked in the presence of

Figure 29: Physical interaction between PPARγ and ERα

Figure 30: ERα antagonists enhanced the effect of PPARγ ligand on

colony-forming ability in MCF-7 cells

Figure 31: The mechanism of PPARγ-mediated down-regulation of NHE1

and its inhibiton by ERα

AluRRE Alu Receptor Response Element

ATP 2-adenosine 5’-triphosphate

Bcl-2 B-cell lymphoma protein 2

BME Beta-mercaptoethanol

bp Base pair

Caspase Cysteine-dependent aspartate-specific protease CAII Carbonic anhydrase II

CaM Calmodulin

CAT Chloramphenicol acetyl transferase

CBP/p400 CREB-binding protein/E1 A binding protein P400

CDK2 Cyclin dependent kinase

cDNA Complementary DNA

ChIP Chromatin Immunoprecipitation

CM-H2DCFDA 5-(and-6)-chloromethyl-2’,7’-dichlorofluorescin

diacetate-dichlorofluorescein diacetate COX Cyclooxygenase

COMT Catehol O-methyltransferase

Cosi siRNA control

COUP-TF Chicken ovalbumin upstream

promoter-transcription factor

CS Charcoal-stripped Cu/Zn SOD Copper/zinc superoxide dismutase

DAF-FM

4-Amino-5-Methylamino-2',7'-Difluorofluorescein Diacetate DBD DNA binding domain

EDTA Ethylenediaminetetraacetic acid

EFR-1 Early growth response-1

ERE Estrogen response element

ERK Extracellular regulated kinase

ETC Electron transport chain

FACs Fluorescence activated cell sorter

FATP Fatty acid transport protein

FeTPPS

5,10,15,20-Tetrakis(4-sulfonatophenul)porpyrinato Iron(III), Chloride GAPDH Glyceraldehyde-3-phosphate dehydrogenase

GSH Glutathione

GSSG Glutathione disulfide

Gpx Glutathione peroxidase

HEPES 4-(2-hydroyethyl)-1-piperazineethanesulfonic acid

HER-2 Human epidermal growth factor receptor 2

HMG-CoA 3-hydroxy-3-methyl-glutaryl-CoA

L-NMMA L-NG-monomethyl Arginine citrate

MnSOD Manganese superoxide dismutase

MOMP Mitochondrial outer membrane permeabilization

MPT Mitochondrial Permeability Transition

MTT 3-(4,5-dimethylthiazol- 2-yl)-2,5

diphenyltetrazolium bromide MYC v-myc myelocytomatosis viral oncogene

homolog

NAC N-Acetyl cysteine

NCoR Nuclear receptor corepressor

NFкB Nuclear factor of kappa light

NHE Na+/H+ exchanger

PARP Poly(ADP-ribose) polymerase

PEPCK Phosphoenolpyruvate carboxy kinase

PI3K Phosphatidylinositol-3-kinase

PIP2 Phosphatidylinositol diphosphate

PIP3 Phosphatidylinositol triphosphate

PMSF Phenylmethylsuphonyl fluoride

PP1 Phosphatase protein phosphatase 1

PPAR Peroxisome proliferator-activated receptor PPRE Peroxisome proliferator response element

PR Progesterone receptor

PTEN Phosphatase and tensin homolog located on

chromosome ten RAR-1 Retinoic acid receptor α-1

RNS Reactive nitrogen species

RPMI 1640 Rosewell Park Memorial Institute 1640

RT-PCR Reverse transcription-polymerase chain reaction

SDS-PAGE SDS-polyacrylamide gel electrophoresis

SERM Selective estrogen receptor modulators

SMRT Silencing mediator for retinoid and thyroid

TRAIL TNF-related apoptosis inducing factor

LIST OF PUBLICATIONS

Kumar, A P., Quake, A L., Chang, M K., Zhou, T., Lim, K S., Singh, R., Hewitt, R E., Salto-Tellez, Pervaiz,S.Clement, M Repression of NHE1 Expression by PPARγ Activation Is a Potential New Approach for Specific

inhibition of the Growth of Tumor Cells In vitro and In vivo Cancer Research

69(22): 8636-8644 (2009)

CONFERENCE PAPERS

Ting Zhou, Alan Prem Kumar, Marie Veronique Clement, Shazib Pervaiz Moleular Mechanism by which estrogen prevents PPARã-mediated transrepression of the Na+/H+ Exchanger-1 (NHE1) gene in ERá-positive human breast cancer cells American Association for cancer research (2008) Ting Zhou, Alan Prem Kumar, Marie Veronique Clement, Shazib Pervaiz.Estrogen receptor- inhibits PPAR-induced repression of Na+/H+ Exchanger-1 expression and sensitivity to apoptosis via direct interaction with PPAR Nuclear Receptors Keystone Symposium (2009)

Ting Zhou, Alan Prem Kumar, Marie Veronique Clement, Shazib Pervaiz Estrogen receptor- inhibits PPAR-induced repression of Na+/H+

Exchanger-1 expression Nuclear Receptors European Molecular Biology Organization (2011)

of hydrogen peroxide, process of transaminations, purine and polyamine catabolism and alcohol oxidation (Tolbert, 1981)

Peroxisome proliferators (PP) are a class of structurally distinct compounds that was classified based on their ability to upregulate the number and size of hepatic peroxisomes in rodents (Kliewer et al., 1994) Such compounds include the phthalate plasticizers, herbicides and fibrate class of hypolipidemic agents (Reddy

et al., 1982) Chlofibrate was the first member of PP to be identified by its ability

to induce hepatomegaly in rats and increase peroxisomes in hepatocytes (Hess et al., 1965) Later, peroxisomes were also reported to be induced by high-fat diet and cold acclimatization (Kliewer et al., 1994)

Although the PPs were characterized early in 1960, the molecular mechanism of how they regulate a panel of genes involved in peroxisomal β-oxidation of long-

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chain fatty acids remained unclear In 1990, a mammalian transcription factor which is activated by diverse class of PPs was successfully cloned (Issemann and Green, 1990) This receptor was classified as a new member of steroid hormone receptor superfamily, which include members such as thyroid, retinoid acid, viatamin D, estrogen and glucocorticoid receptors (Wahli et al., 1995) Due to its ability to be activated by peroxisome proliferators, the transcription factor was termed peroxisome proliferator-activated receptor-α (PPARα) Later on, PPARγ and PPARδ were cloned and characterized to be the isoforms of PPARα (Dreyer

et al., 1992; Kliewer et al., 1994)

1.1.2 Structural domains of PPARs

As typical transcription factor of nuclear receptor superfamily, PPARs consist of four functional domains The N-terminal domain (A/B domain) is responsible for ligand-indepednet transactivation function (AF-1) and is subjected to MAPKs-mediated phosphorylation (Werman et al., 1997) The C region is the DNA-binding domain consisting of two zinc fingers, and it is the most conserved region across all three PPAR isoforms (Berger and Moller, 2002) The D domain is involved in interaction with various cofactors crucial for its transcriptional activity, and the E/F domain represents the ligand-binding domain (LBD) with its ligand-dependent activation domain, AF-2 detected on the C terminus (Berger and Moller, 2002) In comparison to DBD, LBD is relatively less conserved among the three receptor isoforms The LBD is composed of 13 α-helices and 4-stranded β-sheet that assume a conformation with a hydrophobic ligand-binding cavity; the

3

larger than normal ligand-binding pocket may contribute to its ability to bind to diverse class of structurally distinct compounds (Nolte et al., 1998) As a typical ligand-activated transcription factor, upon ligand-binding at the LBD, the AF-2 domain undergoes conformational change to facilitate its interaction with the heterodimeric partner, retinoid X receptor (RXR) for transduction of the hormonal signal into transcriptional activity (Gearing et al., 1993; Nolte et al., 1998)

1.1.3 Mechanism of action of PPARs

Unlike other steroid hormone receptors which function as homodimers, PPAR strictly requires its heterodimeric partner RXR for its functional activity (Miyata

et al., 1994) Three subtypes of RXR have been identified, namely, RXRα, β and

γ Common ligands include vitamin A derivatives and 9-cis-retinoic acid and all three isoforms bind to PPARs upon ligand activation (Mangelsdorf et al., 1992) Among them, RXRγ is the most potent in promoting DNA binding of PPARs upon dimerization, while RXRα induces binding of PPAR to weak response elements (Desvergne and Wahli, 1999)

Interestingly, activated RXR was shown to promote heterodimeric formation and exert anti-diabetic effects in a similar way as activation by PPARγ ligands (Mukherjee et al., 1997) Furthermore, it was reported that co-activation of PPARγ and RXR produce synergistic transcriptional effect (Schulman et al., 1998) Other than binding to PPARs, RXRs are common heterodimeric partners for other nuclear receptors, such as thyroid receptors (TRs), retinoic acid receptors (RARs) and vitamin D receptors (VDR) (Gronemeyer et al., 2004) The

to the 5’ hexameric repeat while RXR interacts with the 3’ core motif (A et al., 1997) The first functional PPRE was identified on the enhancer region of acyl-CoA oxidase gene (ACO) (Tugwood et al., 1992) Subsequently, more DR1 PPREs were discovered mainly on promoters of genes involved in adipocyte differentiation and lipid metabolism (Wahli et al., 1995) However, more recently, direct repeat of core motif separated by 2 nucleotides (DR2) has been reported to respond to PPARγ activation by serving as a functional PPRE (Gervois et al., 1999; Fontaine et al., 2003; Kumar et al., 2004) Hence, PPARs can bind to DR1

as well as DR2 for its transcriptional activity On the other hand, other members

of the nuclear receptors such as vitamin D receptor, thyroid hormone receptor and retinoic acid receptor recognize direct repeats separated by 3, 4 and 5 nucleotides respectively (Lemberger et al., 1996) Other than the number of interspaced nucleotides between repeat sequences, 5’ flanking nucleotide sequences of PPRE have been shown to be important in determining the binding affinity of PPAR to its response element (Hsu et al., 1998) However, PPARγ shows higher binding affinity to majority of PPREs than PPARα and PPARδ, making it less dependent

on the 5’ flanking sequence (Desvergne and Wahli, 1999)

by activated PPAR:RXR complex on PPRE for chromatin decondensation (Zhu et al., 1996; Torchia et al., 1997) The transcription is initiated by the recruitment of second group of coactivators, such as PPAR binding protein (PBP)/TRAP220 to functionally link the basic transcription initiation machinery to activated nuclear receptors (Zhu et al., 1997) Similar to other nuclear receptors which recruit different groups of cofactors depending on the binding ligands, the pattern of association to coactivators also vary in PPAR stimulated by different ligands (Oberfield et al., 1999) As a result, the selective transactivation of target genes by different agonist in distinct cell types is achieved through the altered binding patterns between coactivators and PPARs

1.1.4 Subtypes of PPARs

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The three isoforms of PPARs have different yet overlapping biological functions Their physiological roles and tissue distribution also vary significantly PPARα is mainly found in heart, kidney, large intestines and skeletal muscles in human tissue (Auboeuf et al., 1997) These are the sites where high rate of fatty acid oxidation occurs for primary energy source Insulin is shown to down-regulate PPARα (Steineger et al., 1994) and glucocorticoids are able to upregulate its expression (Lemberger et al., 1994) In contrast, PPARδ is ubiquitously expressed

in a wide range of tissues, with higher expression in large intestine and placenta (Rodie et al., 2005)

For PPARγ, two isoforms, PPARγ-1 and 2 have been identified in both mouse and human They arise from alternative splicing using different promoters of the single PPARγ gene (Elbrecht et al., 1996) PPARγ-1 protein is encoded by three mRNA transcript variants, namely PPARγ1, γ2, γ3 mRNA, but PPARγ-2 is translated from a single PPARγ2 mRNA transcript (Rumi et al., 2004) The two isoforms differ in 30 extra amino acids in the N-terminus of PPARγ-2 (Fajas et al., 1997) Compared to PPARα and δ, PPARγ is predominantly expressed in the adipose tissue, as it is mainly involved in functions like adipogenesis and fatty acid storage (Tontonoz et al., 1994) Insulin-responsive tissues, such as skeletal muscles, heart and liver also express low levels of PPARγ-1 protein (Mukherjee

et al., 1997) In mouse, PPARγ is also detected in spleen and T lymphocytes, whereas the same tissue in human does not express the receptor except in several transformed B lymphocytes and myeloid cell lines (Greene et al., 1995) PPARγ expression was reported to be upregualted by co-administration of corticosteroids

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and insulin in human adipocytes (Vidal-Puig et al., 1997), and down-regulated by tumor necrosis factor-α (TNFα) (Xing et al., 1997)

1.1.5 Ligands and physiological functions of PPARα and PPARδ

PPARα is activated by synthetic hypolipidemic fibrates and natural ligands such

as unsaturated fatty acids and phytanic acids (Issemann and Green, 1990; Desvergne and Wahli, 1999) PPARα is present in human hepatocytes at lower levels compared to rodent hepatocytes, hence PPARα activation does not lead to hepatic peroxisome proliferation, hepatomegaly and hepatocarcinogenesis in human liver (Palmer et al., 1998)

PPARα is primarily involved in cellular uptake and metabolism of fatty acids Its activation enhances cellular uptake of fatty acids by increasing the expression of fatty acid transport proteins, such as fatty acid transport protein (FATP) (Martin et al., 1997) and fatty acid translocase (FAT) (Motojima et al., 1998) PPARα-mediated β-oxidation of fatty acids is through transcriptional induction of acyl-CoA oxidase (ACO) (Schoonjans et al., 1995) and keto-acyl-CoA thiolase enzymes (Zhang et al., 1993) The clinical application of PPARα ligands stems from its ability to reduce triglycerides and increase high density lipoprotein (HDL), thus reducing cardiovascular events or death by 22% in the case of PPARα agonist, emfibrozil (Linton and Fazio, 2000) This effect is also achieved partly by upregulation of lipoprotein lipase (LPL) and down-regulation of hepatic apolipoprotein (apo) C-III (Staels et al., 1995; Schoonjans et al., 1996) This positive outcome from trial results has presented a promising strategy for treating

1.1.6 Ligands of PPARγ

Transcriptional activation of PPARγ/RXR complex is induced upon binding of various ligands including saturated and unsaturated fatty acids, arachidonic acid derivatives and different synthetic compounds Since the discovery of thiazolidinedione (TZD) antidiabetic drugs as PPARγ agonists, more synthetic ligands for PPARγ have been developed (Lehmann et al., 1995; Willson and Wahli, 1997) Figure A presents the chemical structures of various TZD compounds Among the TZDs, their affinity to PPARγ are ranked in the order of rosiglitazone, pioglitazone, troglitazone and ciglitazone (Berger and Moller,

9

2002) Rosiglitazone is available clinically for treatment of type II diabetes mellitus It was found that the antihyperglycemic effect of these drugs is dependent on their activation of PPARγ and subsequent regulation of insulin-responsive genes involved in glucose and fatty acid metabolism (Berger et al., 1996) However, troglitazone was later withdrawn from market due to its hepatotoxicity Rosiglitazone was also reported to increase myocardial infarction and risk of death from cardiovascular events (Nissen and Wolski, 2007) Since then, the use of TZD compounds for treating diabetes has been under controversy Other than the synthetic ligands, PPARγ is also activated by endogenous ligands, such as polyunsaturated fatty acids, arachidonic acid and eicosapentaenoic acid (Berger and Moller, 2002) Metabolic intermediates of linoleic acid, 9-HODE and 13-HODE are able to function as PPARγ ligands (Nagy et al., 1998) 15-deoxy-

12, 14-PGJ2 (15d-PGJ2), a metabolite of the eicosanoid prostaglandin J2, is reported to be the most potent natural ligand of PPARγ, with Kds varying from 325nM to 2.5M (Boitier et al., 2003) Prostaglandins are metabolites of arachidonic acid generated via the cyclooxygenase pathway (Scher and Pillinger, 2005) Prostaglandin J2 and prostaglandin A2 are produced from the non-enzymatic dehydration of prostaglandin D2 and prostaglandin E2, respectively (Fukushima, 1990; Fukushima et al., 1992; Noyori and Suzuki, 1993) It was shown that 15d-PGJ2, the endogenous PPARγ ligand, is present at low concentration of picomolar in the medium of 3T3-L1 preadipocytes (Bell-Parikh

et al., 2003), although this endogenous PPARγ ligand concentration has not been proven to lead to functional PPARγ activation Given the central role of COX in

10

pathway of 15d-PGJ2 production, its activity is shown to be important in regulating the endogenous level of PPARγ ligand and the basal level of receptor activation (Birnbaum et al., 2005; Yano et al., 2007)

Apart from PPARγ agonists, Leesnitzer et al identified a selective PPARγ antagonist in 2002 after screening through various PPARγ ligands This compound, GW9662 elicits its antagonistic function by covalently modifying a cysteine residue in the LBD, and its antagonistic property was validated by inhibiting adipocyte differentiation (Leesnitzer et al., 2002)

Figure A: Chemical structures of PPARγ agonists and antagonists

1.1.7 PPARγ and adipogenesis

The findings that most of PPARγ response genes bearing functional PPRE are involved in adipocyte differentiation incited subsequent studies to confirm the role of PPARγ in promoting adipocyte differentiation Successful differentiation

of NIH-3T3 fibroblasts to adipocytes was demonstrated by overexpression of

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PPARγ with administration of PPARγ ligands (Tontonoz et al., 1994) Later on, the role of PPARγ in adipocyte differentiation was further confirmed in PPARγ heterozygous null mice which displayed reduced adipose tissue (Rosen et al., 1999) The conclusive evidence came from a study in PPARγ-/- mice, which were completely devoid of adipose tissue (Kubota et al., 1999; Rosen et al., 1999; Miles et al., 2000)

Microarray analysis on PPARγ ligand-induced differentiated mouse adipocytes revealed a large number of novel genes potentially responsible for adipocyte differentiation One such gene is oxidized low-density lipoprotein receptor 1 (OLR1) OLR1 was shown to be a direct PPARγ target gene which is potently induced by TZDs In addition, TZDs also stimulated the uptake of oxidized low-density lipoprotein (oxLDL) into adipocytes, partly through the upregulated OLR1 As a result, adipocyte cholesterol content and free fatty acid uptake were greatly enhanced (Guan, 2002) Other PPARγ target genes that have been identified to be involved in adipogenesis include adipocyte fatty acid-binding protein aP-2 (Tontonoz et al., 1994), phosphoenolpyruvate carboxy kinase (PEPCK) (Tontonoz et al., 1995), acyl-CoA oxidase (ACO) (Schoonjans et al., 1995), fatty acid transport protein (FATP) (Martin et al., 1997) and malic acid enzyme (ME) (Castelein et al., 1994) All these genes contain experimentally validated PPRE and are involved in critical steps of adipogenesis, such as fatty acid and triglycerides synthesis (ME), glyceroneogenesis (PEPCK), fatty acid esterifcation (ACO) and cellular uptake of fatty acid (FATP, aP2) Due to its role

in promoting lipid accumulation in adipocytes, PPARγ ligands used in treatment

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of diabetes are suspected of increasing cardiovascular risk in diabetic patients by inducing weight gain Hence, a more innovative therapeutic approach is needed for treatment of metabolic diseases, such as hyperglycemia, obesity and dyslipidemia without causing adverse cardiovascular side-effects One possible approach is to combine with PPARα ligands, which promote catabolic oxidation

of fatty acid

1.1.8 PPARγ and insulin sensitization

PPARγ ligands TZDs were initially used for treatment of type II diabetes mellitus without understanding the molecular mechanism of action It was only after discovery of the TZDs as potent PPARγ agonists, was their therapeutic mechanism involving transcriptional regulation of genes associated with insulin action unearthed It was reported that genes important in positive regulation of insulin-signaling cascade, such as c-CBL-associated protein (CAP) (Ribon et al., 1998) and insulin receptor substrate-2 (IRS-2) (Smith et al., 2001) are induced by TZDs in adipocytes Meanwhile, a protein responsible for insulin resistance, 11 β-hydroxysteroid dehydrogenase 1 (11β-HSD-1) is down-regulated by PPARγ

activation (Berger et al., 2001) In vivo data from human subjects receiving

rosiglitazone show increased plasma concentrations of adipocyte-related complement protein (Acrp)30 (Combs et al., 2002), which serves to down-regulate glucose, free fatty acids and triglycerides levels (Berg et al., 2001), suggesting the PPARγ-induced Acrp30 as an important mediator of improved clinical outcomes in diabetic patients receiving TZDs In addition, suppression of tumor necrosis factor-α (TNFα) was demonstrated to contribute to PPARγ-

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mediated insulin sensitization The pro-inflammatory cytokine secreted by adipocytes, TNFα negatively interferes with insulin signaling cascade (Hotamisligil et al., 1994), leading to insulin resistance (Hotamisligil et al., 1993) Activated PPARγ rescues insulin receptor and IRS-1 from TNFα –suppressed phosphorylation (Peraldi and Spiegelman, 1997), and inhibits TNFα-induced lipolysis (Souza et al., 1998) by repressing TNFα expression in white adipose tissue (Hofmann et al., 1994) However, this antagonism between PPARγ and TNFα is bi-directional TNFα has been shown to inhibit PPARγ expression (Zhang et al., 1996) and thus its transcriptional activity (Gao et al., 2006) Overall, the cross talk between PPARγ and TNFα plays an important role in the regulation

of lipid and glucose metabolism and its associated pathogenesis

1.1.9 PPARs and cancer

As described previously, the role of PPARγ in adiocyte differentiation has been clearly established It was also noted that adipocyte differentiation is closely related to cell proliferation Transcription factors important in regulating genes involved in DNA synthesis and cell cycle progression, E2Fs are induced in the initial stage of adipocyte differentiation (Richon et al., 1997) Other cell cycle associated gene, such as c-Myc, cyclin D1 and cyclin E were also shown to be upregulated in the early stage of adipogenesis (Reichert and Eick, 1999) More recently, PPARs are not only implicated in cell cycle progression, but are shown

to regulate cell proliferation, differentiation and cell death (Sertznig et al., 2007)

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Among the three receptor subtypes, PPARγ is the most studied for its anti-tumor effects in various cancer type On the other hand, although PPARα has been reported to have antiangiogenic and tumouricidal properties, its further application in treating cancer is unlikely for two reasons Firstly, PPARα is not overexpressed in cancer cells relative to normal cells, suggesting the use of its ligands may equally affect viability of cancer cells as well as normal cells (Sertznig et al., 2007) Moreover, the rodent model used in PPARα studies is not comparable to human systems, limiting the relevance of data on anti-cancer efficacy and toxicity of PPARα ligands obtained from mice (Palmer et al., 1998)

As for PPARδ, so far the evidence from literature has suggested its role in cancer

as promoting tumor progression rather than inhibiting it It was observed that PPARδ+/- colorectal cancer cell line had enhanced ability to form tumors in nude mice as compared to PPARδ-/- cells (Park et al., 2001), and PPARδ agonist increased small intestine tumorigenesis in APCmin mice (Gupta et al., 2004) Even more is the ability of PPARδ to rescue colorectal cancer cells from PPARγ-induced apoptosis, further confirming its prosurvival role in tumorigenesis (Wang

et al., 2012) Overall, activation of PPARδ seems to promote rather than to block tumor formation

It was widely reported that the PPARγ expression is elevated in tumor cells compared to their normal counterparts, hence its activation using PPARγ agonist for tumor-specific growth inhibition has been proposed as an attractive approach

in cancer therapy PPARγ overexpressing tumors include breast, colorectal, prostate, stomach, cervix, ovary, bladder, salivary gland, testes, lung, leukemia

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and neuroblastoma (Krishnan et al., 2007) The initial observation of elevated PPARγ expression led to the hypothesis that PPARγ acts as an oncogene in tumor cells However, later it was found out that the higher level in transformed cells may be induced by myc-associated zinc finger protein (MAZ) using a tumor-specific PPARγ promoter in MCF-7 breast cancer cells (Wang et al., 2008) Although PPARγ are present in tumour tissues at higher levels, they are generally inactive unless exogenous PPARγ ligands are introduced (Keshamouni et al., 2004) An alternative explanation for elevated PPARγ expression in tumor cells is its lack of endogenous ligands, which under normal circumstances cause receptor ubiquitination and proteasomal degradation after inducing its transcriptional activity (Hauser et al., 2000) The anti-proliferative effects of PPARγ ligands have been demonstrated in a large variety of tumor cells ranging from colon, breast, pituitary, gastric, bladder, liposarcoma, prostate to neruoblastoma caners, as well

as in melanoma (Sertznig et al., 2007) The ability of PPARγ agonists to promote differentiation (Tontonoz et al., 1997), stall cell cycle (Morrison and Farmer, 1999), and induce apoptosis (Elstner et al., 1998) collectively contributes to its anti-tumor effects in cancer cells

1.1.10 PPARγ and breast cancer

Breast cancer is the most frequent malignancy identified in women Each year, about 212,930 new cases of breast cancer are diagnosed, and 40,840 related deaths are reported in the United Sates alone, presenting a heavy public health burden (Jemal et al., 2005) Over the years, hormonal and cytotoxic therapies

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have been employed in treating breast cancer patients with some but limited success The hormonal therapy works by depleting the systematic estrogen available to estrogen receptor either through reducing the overall estrogen level or

by blocking the estrogen receptor (Yager and Davidson, 2006) Although estrogen therapy has been functional in treating ER positive tumors, it is not effective in ER negative tumors, which are usually more aggressive and metastatic Moreover, ER positive cells sometimes develop anti-estrogen resistance where their dependence on estrogen is attenuated and they no longer respond the hormonal treatment Hence, the limited therapeutic regimes available for ER negative breast cancers and the possibility of developing hormonal resistance by ER positive breast cancers stimulate the search for an alternative therapeutic approach in treating breast cancer patients

anti-The overexpression of PPARγ receptor and its anti-proliferative effect in breast cancer cells makes PPARγ an attractive molecular target in breast cancer therapy The elevated PPARγ level was demonstrated in human breast cancer cell line (MCF-7, MDA-MB-231, T47D and BT474) as well as in primary metastatic breast carcinoma samples (Elstner et al., 1998; Mueller et al., 1998; Clay et al., 1999; Suzuki et al., 2006) Moreover, PPARγ was reported to be an independent prognostic factor from ERα in assessing improved clinical outcome in ER positive breast cancer patients (Suzuki et al., 2006)

Although contradictory data exist, majority of the studies have reported that transactivation of PPARγ by PPARγ agonists lead to remission of breast cancer

In breast caner cell lines treated with PPARγ ligands, inhibition of cell

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proliferation, loss of colonogenic ability, and change in gene signature associated with a less malignant and more differentiated state were observed (Mueller et al.,

1998) In vivo data from mouse models, also demonstrated the ability of PPARγ

synthetic ligand, troglitazone, to reduce the breast tumor size and weight in immunodeficient mice (Elstner et al., 1998) In accordance with the report, another PPARγ activator, GW7875, was shown to inhibit tumorigenesis in chemically induced mouse mammary tumors (Yin et al., 2005)

The anti-tumor effect of PPARγ has been affirmed in breast carcinoma, however, the mechanism of it remains to be clarified It was reported that PPARγ ligands together with retinoid receptor ligands induced a significant decrease in Bcl-2 gene expression, which in turn led to apoptosis in Bcl-2 overexpressing MCF-7, MDA-MB-231 and ZR-75-1 human breast cancer cells (Elstner et al., 2002) On the other hand, cell cycle arrest was caused by upregulated p21 and repressed CDK2, CDK4 and cyclin D1 together with reduced retinoblastoma phosphorylation in breast cancer cell lines exposed to PPARγ ligands (Samid et al., 2000; Yin et al., 2001) However, the genes identified are not direct transcriptional targets of PPARγ as they lack functional PPRE Moreover, their regulation by PPARγ agonist may be through receptor-independent pathway

More recently, PTEN, a well known tumor suppressor was identified as a bona

fide transcriptional target of PPARγ in MCF-7 breast cancer cells and its role in

PPARγ-mediated anti-cancer effect is implicated (Patel et al., 2001) On grounds

of the promising in vitro and in vivo data, further studies need to be conducted in

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search for other direct PPARγ targets that are responsible for the mediated anti-tumor effects in breast cancer

1.2.1 Identification and structures of ERs

Estrogen is a hormone that regulates various cellular processes ranging from growth, development to differentiation Its main physiological role is for development of female reproductive system, central nervous system, cardiovascular systems and bone metabolism (Katzenellenbogen, 1996) The ability of estrogen to regulate transcription was discovered in 1968, when O’Malley observed changes in amount of RNA in the chick oviduct stimulated by estrogen (O'Malley et al., 1968) 3 years later, the estrogen-binding protein was found in breast tumors, and its expression was shown to be important for estrogen-mediated tumor response, demonstrating for the first time how estrogen regulates breast cancer progression (Jensen et al., 1971; McGuire, 1973) Another decade passed, before this estrogen receptor α (ERα) was cloned and its structural domains characterized, suggesting its function as a ligand-dependent transcription factor (Green et al., 1986; Greene and Press, 1986; Kumar et al., 1987) For the following decade, ERα was believed to be the only receptor for estrogen, until in

1996 the second estrogen receptor, ERβ was identified in rat prostate (Kuiper et al., 1996) Following that, ERβ was also reported to be found in human and murine tissue (Mosselman et al., 1996; Tremblay et al., 1997) Human ERα and