Autocrine growth hormone (hGH) and chemotherapeutic drug resistance in mammary carcinoma cells 2

2.11.4 p53 and tumorigenesis……… 69 2.11.5 The Bax family and tumorigenesis……….71 2.12 Telomerase……….73 2.12.1 Telomerase and tumorigenesis………73 2.12.2 Regulation of telomerase………..74

To my dearest grandfather, parents, aunties and uncles,

To Professor KO Lee and Peter Lobie,

To my friends

To my colleagues in IMCB and NUH

Table of Contents

Acknowledgements……… ……i

Table of contents……….……… ii

Summary……… ……… vi

List of publications……… ….…….ix

List of figures……….… x

Abbreviations… ……… ……… xi

Chapter 1 Introduction……… 1

1.1 Hormones and breast cancer………1

1.2 Growth hormone and breast cancer………2

1.3 Possible mechanisms involved in resistance to breast cancer treatment ……4

1.4 Direction of the study………5

1.5 Objectives……… 6

Chapter 2 Literature review……… 9

2.1 Growth hormone………9

2.1.1 Growth hormone structure……….9

2.2 Pituitary regulation and extrapituitary sites of GH expression………… …11

2.2.1 Pituitary regulation of GH expression……….11

2.2.2 Extrapituitary sites of GH expression……… 13

2.3 Cellular and transcriptional regulation by GH………16

2.3.1 Cellular effects of GH………16

2.3.2 Transcriptional regulation by GH……… 18

2.3.3 Other effects of GH……… 19

2.4 GH dependent intracellular signalling……… 21

2.4.1 GH dependent intracellular signalling………21

2.4.2 GH -mediated activation of mitogen-activated protein kinase (MAPK) pathway ……… 22

2.5 GH and mammary gland……… 26

2.5.1 GH regulation of mammary gland development……….26

2.5.2 Effect of GH on the stromal-epithelial compartment……… 28

2.6 GH and mammary carcinoma………31

2.6.1 GH/IGF-1 axis and mammary carcinoma……….31

2.6.2 IGF-independent effect of GH in mammary carcinoma……….32

2.7 Balance between oxidants and antioxidants……… 35

2.7.1 Oxidative stress………35

2.7.2 Antioxidant pathways……… 37

2.7.3 Reactive oxygen species (ROS) and human disease……… 38

2.8 Antioxidant enzymes………40

2.8.1 Catalase………41

2.8.1.1 Regulation of catalase……….42

2.8.2 Superoxide dismutase (SOD)……….43

2.8.3 Glutathione peroxidase (GPX)……… 45

2.8.4 Glutamylcysteine synthetase (GCS)……… 46

2.9 Role of ROS in breast cancer……… 49

2.9.1 DNA damage……….49

2.9.2 Activation of growth-promoting signalling pathways……… 50

2.9.3 Increased blood supply………50

2.9.4 Metastasis……….……….51

2.9.5 Increased resistance to therapy ……… 51

2.10 Apoptosis……….54

2.10.1 Caspase……… ………54

2.10.2 Apoptosis pathways……… 56

2.10.2.1 Death receptor pathway……….57

2.10.2.2 Mitochondria pathway……… 58

2.10.2.3 Other pathways……….………… 59

2.11 Pro- and anti-apoptotic proteins……… …………62

2.11.1 Bcl-2 family……….……… 62

2.11.2 Bcl-2 family and tumorigenesis……… 65

2.11.3 Apoptotic activities of p53……… 66

2.11.4 p53 and tumorigenesis……… 69

2.11.5 The Bax family and tumorigenesis……….71

2.12 Telomerase……….73

2.12.1 Telomerase and tumorigenesis………73

2.12.2 Regulation of telomerase……… 74

Chapter 3 Materials and methods………78

3.1 p44/42 MAP kinase dependent regulation of catalase by autocrine human growth hormone protects human mammary carcinoma cells from oxidative stress induced apoptosis………78

3.2 Autocrine hGH protects mammary carcinoma cells from chemotherapeutic drug induced cell death…….………96

3.3 Regulation of telomerase activity by stabilization of hTERT mRNA…….…99

Chapter 4 Results……….104

4.1 p44/42 MAP kinase dependent regulation of catalase by autocrine human growth hormone protects human mammary carcinoma cells from oxidative stress induced apoptosis……… 104

4.2 Autocrine hGH protects mammary carcinoma cells from chemotherapeutic drug induced cell death…….……… 144

4.3 Regulation of telomerase activity by stabilization of hTERT mRNA ……164

Chapter 5 Discussion……… 177

Chapter 6 General Discussion………192

Chapter 6 Reference.……….199

Breast cancer is the most frequent cancer and the leading cause of cancer related death in women Chemotherapy is usually effective in early stages of breast cancer, but frequently become resistant to the same treatments as the cancer advances Studies indicate the possible involvement of autocrine growth hormone (hGH) in this phenomenon The purpose of my study was to identify possible mechanisms by which autocrine hGH protects mammary carcinoma cells from cell death induced by chemotherapeutic drugs A cellular model using a human mammary carcinoma cell line, MCF-7 cells, stably transfected with hGH gene or a translation-deficient hGH gene, was adopted in this study Results showed that autocrine hGH protected mammary carcinoma cells from different chemotherapeutic drugs

Given that increased cellular oxidative stress is a key effector mechanism of chemotherapeutic agents, I analyzed the effect of autocrine hGH on oxidative stress induced cell death Results showed that autocrine hGH protected mammary carcinoma cells from oxidative stress Increased anti-oxidant status due to autocrine hGH was found in mammary carcinoma cells Further analysis of the expression of anti-oxidant enzymes revealed that autocrine hGH increased both the mRNA and protein levels of catalase, superoxide dismutase 1(SOD1), glutathione peroxidase (GPx) and glutamylcysteine synthetase (GCS) Furthermore, the activity of catalase, one key anti-oxidant enzyme, was induced by autocrine hGH in mammary carcinoma

cells Catalase promoter reporter assay suggested the effect of autocrine hGH on the level of catalase mRNA was exerted at the transcriptional level and this transcriptional upregulation was abolished by p44/42 MAP kinase inhibition p44/42 MAP kinase inhibition also prevented autocrine hGH stimulated increase in catalase protein and activity and abrogated the protective effect of autocrine hGH against oxidative stress induced apoptosis

Secondly, to further elucidate the mechanism of the protective effect of autocrine hGH to chemotherapeutic drugs, the effect of autocrine hGH in mammary carcinoma cells was studied by measuring a variety of proteins involved in the apoptotic process Protein levels of the antiapoptotic protein, Bcl-xl, was dramatically induced by autocrine hGH In addition, although autocrine hGH did not alter the level of Bcl-2, the functional activity of Bcl-2 was decreased by autocrine hGH by inhibiting its phosphorylation

Lastly, given that chemotherapeutic drugs could induce cell death by inducing telomere dysfunction and by telomerase activity inhibition, the effect of autocrine hGH on telomerase activity was investigated The results demonstrated that autocrine hGH increased telomerase activity in human mammary carcinoma cells by increasing hTERT mRNA stability

As such, enhanced resistance to anti-neoplastic agents by induction of antioxidant enzymes and antiapoptotic molecules by autocrine hGH is likely to make a significant

contribution to the mechanisms of chemoresistance Therefore, the understanding of the molecular basis of the protective effect of autocrine hGH obtained in this study may provide us with a number oftargets on which to base biological therapies and therefore shed light on the improvement of breast cancer prognosis

Manuscripts

I p44/42 MAP kinase dependent regulation of catalase by autocrine human

growth hormone protects human mammary carcinoma cells from

oxidative stress induced apoptosis

Oncogene 2005 May 26;24(23):3774-85

II Regulation of Telomerase Activity by Poly(C)-rich Segment Binding

Protein Mediated 3’-UTR Stabilization of hTERT mRNA

Manuscript submitted III Autocrine hGH protects mammary carcinoma cells from

chemotherapeutic drug induced cell death

Manuscript in preparation

Published abstracts:

I Regulation of Telomerase Activity by Poly(C)-rich Segment Binding

Protein Mediated 3’-UTR Stabilization of hTERT mRNA

Yong Chen, Zhe Zhu, Tao Zhu, Kok-Onn Lee, and Peter E Lobie

Presented at Endocrine Society 84th Annual Meeting, San Francisco, California, USA, 2002

II Autocrine hGH protects mammary carcinoma cells from chemotherapeutic drugs induced cell death

Zhe Zhu, Svetlana Mukhina, Kok-Onn Lee and Peter E Lobie

Presented at 4th International Symposium on Hormonal Carcinogenesis, Valencia, Spain, 2003

III p44/42 MAP kinase dependent regulation of catalase by autocrine human

growth hormone protects human mammary carcinoma cells from oxidative stress induced apoptosis

Zhe Zhu, Svetlana Mukhina, Kok-Onn Lee and Peter E Lobie

Presented at Endocrine Society’s 86th Annual Meeting in New Orleans, Louisiana, USA, 2004

List of Figures

Fig 2.1 ………25

Fig 2.2 ………55

Fig 2.3 ………60

Fig 2.4 ………63

Fig 4.1.1A ………107

Fig 4.1.1B ………109

Fig 4.1.2 ……….111

Fig 4.1.3 ………113

Fig 4.1.4 ………115

Fig 4.1.5A ………117

Fig 4.1.5B……….119

Fig 4.1.6 ………121

Fig 4.1.7………123

Fig 4.1.8A ………125

Fig 4.1.8B ………126

Fig 4.1.9A……….128

Fig 4.1.9B.………130

Fig 4.1.10A ………133

Fig 4.1.10B……… 135

Fig 4.1.11A……… 138

Fig 4.1.11B ……… 139

Fig 4.1.11C ……….140

Fig 4.1.12 ……….142

Fig 4.2.1A……….147

Fig 4.2.1B……….148

Fig 4.2.1C……….149

Fig 4.2.1D……….150

Fig 4.2.1E……… 151

Fig 4.2.2 ………153

Fig 4.2.3 ……….155

Fig 4.2.4 ……….157

Fig 4.2.5A……….159

Fig 4.2.5B……….160

Fig 4.2.6 ……… 162

Fig 4.3.1 ………167

Fig 4.3.2 ………169

Fig 4.3.3 ……… ………171

Fig 4.3.4……… ……….173

3’UTR 3’ untranslated region

3-AT 3 amino-1, 2, 4-triazole

5-FU 5-fluorouracil

αCPs α-globin mRNA poly(C)-rich

AAPH 2, 2’-Azobis-(2-amidinopropane) dihydrochloride

ABTS 2, 2' azino-bis-[3-ethylbenz-thiazoline-6-sulfonicacid]

AIF Apoptosis-inducing factor

APC Adenomatous polyposis coli

Bcl-2 B-cell lymphoma 2

bGH Bovine growth hormone

BH Bcl-2 homology

BSA Bovine serum albumin

CAT Catalase

CARD Caspase activation and recruitment domain

CDK Cyclin-dependent kinase

CHO Chinese hamster ovary cells

CKI Cyclin kinase inhibitor

C/EBPs CCAAT/enhancer binding proteins

DED Death effector domain

DDs Death domains

DISC Death-inducing signaling complex

DMSO Dimethyl Sulfoxide

DRs Death receptors

ECL Enhanced chemiluminescence

EC-SOD Extracellular SOD

Egr-1 Early growth response protein 1

Endo G Endonuclease G

Elk-1 ETS-domain protein-1

ER Estrogen receptor

ERKs Extracellular signal-regulated kinases

ERK1/2 Extracellular signal-regulated protein kinase-1 and -2

FA gel Formaldehyde agarose gel

FADD Fas-associated protein with death domain

GHRH Growth hormone releasing hormone

GCS Glutamylcysteine synthetase

GCSh Glutamylcysteine synthetase heavy subunit

GCSl Glutamylcysteine synthetase light subunit

GH Growth hormone

GH-N Normal growth hormone

GHRH Growth hormone-releasing hormone

GPX Glutathione peroxidase

GSH Glutathione

H2O2 Hydrogen peroxide

hGH Human growth hormone

hGHR Human growth hormone receptor

HMEC Human mammary epithelial cells

HNF-1α Hepatocyte nuclear factor-1α

Hsp90 Heat shock protein 90

hTERT Human telomerase-specific reverse transcriptase

hTR Human telomerase RNA HIF-1 hypoxia inducible factor-1

IFN-α alpha interferon

IGF-1 Insulin-like growth factor-1

IGFBPs IGF binding proteins

JNK/SAPK c-Jun N terminal protein kinase/Stress activated protein kinase

LAP Liver activating protein

LB Luria-Bertani medium

LIP Liver inhibitory protein

MAPKs Mitogen-activated protein kinases

MAPKK MAPK kinases

MDM2 Mouse double minute 2

MMP-1 Matrix metalloproteinase-1

NOS Nitric oxide synthase

PCBPs Poly(C)-binding proteins

PHGPX Phospholipid hydroperoxide glutathione peroxidase

PI-3 Phosphatidylinositol-3

PL Placental lactogen

PRL Prolactin

PRLR Prolactin receptor

Prop-1 Prophet of Pit-1

PTGF-beta Placenta transforming growth factor-beta

RA Retinoic acid

ROS Reactive oxygen species

SAP kinases Stress-activated protein kinases

SOD Superoxide dismutase

Sos Son-of-sevenless

SRE Serum response element

STAT Signal transducer and activator of transcription

TEB Terminal end bud

TNF Tumor necrosis factor

TNF-α Tumor necrosis factor α

TP1 Telomerase associated protein 1

TRAIL TNF-α-related apoptosis-inducing ligand

VEGF Vascular endothelial growth factor