i Acknowledgements 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 ii Table of Contents Acknowledgements…………………………………………………………… ……i Table of contents………………………………………………………….……… ii Summary…………… …………………………………………………………… vi List of publications……………………………………………………… ….…….ix List of figures…………………………………………………………………….… x Abbreviations… …………………………………………… ………………… xi Chapter Introduction…………………………………………………………… 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……………………………………………………………………… Chapter Literature review……………………………………………………… 2.1 Growth hormone…………………………………………………………………9 2.1.1 Growth hormone structure……………………………………………….9 2.2 Pituitary regulation and extrapituitary sites of GH expression………… …11 iii 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 iv 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 v 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 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 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 Discussion…………………………………………………………… 177 Chapter General Discussion……………………………………………………192 Chapter Reference.…………………………………………………………….199 vi Summary 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 vii 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 viii 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 of targets on which to base biological therapies and therefore shed light on the improvement of breast cancer prognosis ix Publications 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 III Regulation of Telomerase Activity by Poly(C)-rich Segment Binding Protein Mediated 3’-UTR Stabilization of hTERT mRNA Manuscript submitted 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 x 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 xi 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 xii Abbreviations 3’UTR 3-AT 5-FU αCPs AAPH ABTS AIF APC Bcl-2 bGH BH BSA CAT CARD CDK CHO CKI C/EBPs DED DDs DISC DMSO DRs ECL EC-SOD Egr-1 Endo G Elk-1 ER ERKs ERK1/2 FA gel FADD GHRH GCS GCSh GCSl GH GH-N GHRH 3’ untranslated region amino-1, 2, 4-triazole 5-fluorouracil α-globin mRNA poly(C)-rich 2, 2’-Azobis-(2-amidinopropane) dihydrochloride 2, 2' azino-bis-[3-ethylbenz-thiazoline-6-sulfonic acid] Apoptosis-inducing factor Adenomatous polyposis coli B-cell lymphoma Bovine growth hormone Bcl-2 homology Bovine serum albumin Catalase Caspase activation and recruitment domain Cyclin-dependent kinase Chinese hamster ovary cells Cyclin kinase inhibitor CCAAT/enhancer binding proteins Death effector domain Death domains Death-inducing signaling complex Dimethyl Sulfoxide Death receptors Enhanced chemiluminescence Extracellular SOD Early growth response protein Endonuclease G ETS-domain protein-1 Estrogen receptor Extracellular signal-regulated kinases Extracellular signal-regulated protein kinase-1 and -2 Formaldehyde agarose gel Fas-associated protein with death domain Growth hormone releasing hormone Glutamylcysteine synthetase Glutamylcysteine synthetase heavy subunit Glutamylcysteine synthetase light subunit Growth hormone Normal growth hormone Growth hormone-releasing hormone xiii GPX Glutathione peroxidase GSH Glutathione Hydrogen peroxide H2O2 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 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 TRAIL TNF-α-related apoptosis-inducing ligand VEGF Vascular endothelial growth factor ... 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... 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... p44/ 42 MAP kinase dependent regulation of catalase by autocrine human growth hormone protects human mammary carcinoma cells from oxidative stress induced apoptosis Oncogene 20 05 May 26 ;24 (23 ):3774-85