VITAMIN D SIGNALING PATHWAY AND BREAST CANCER Doctoral Thesis by Lei Sheng Vitamin D Signaling Pathway and Breast Cancer Lei Sheng M.Med., B.Med. This thesis is submitted in fulfilment of the requirements for the Doctor of Philosophy Adelaide Medical School The University of Adelaide Adelaide SA, Australia March 2017 Table of Contents Overview i Publications v Acknowledgement vii Declaration ix CHAPTER I 1 INTRODUCTION: VITAMIN D SIGNALING PATHWAY AND BREAST CANCER 1 1.1 Introduction 2 1.2 Vitamin D metabolism 2 1.2.1 The endocrine paradigm of vitamin D metabolism 2 1.2.2 The paracrine/autocrine paradigm of vitamin D metabolism 4 1.2.3 The paracrine/autocrine paradigm of vitamin D metabolism in the breast 5 1.3 Biological function of Vitamin D 6 1.3.1 The effect of vitamin D in bone 7 1.3.2 The effect of vitamin D in the murine mammary gland 8 1.3.3 The effect of vitamin D in cancer, particularly in breast cancer 10 1.4 Vitamin D and breast cancer risk 18 1.5 Vitamin D and the clinical outcome of breast cancer 20 1.6 Target genes of VDR signaling pathway 21 1.7 Conclusion 23 1.8 References 29 CHAPTER II 47 THE EFFECT OF VITAMIN D SUPPLEMENTATION ON THE RISK OF BREAST CANCER: A TRIAL SEQUENTIAL META-ANALYSIS 47 2.1 Prelude 48 2.2 Abstract 49 2.3 Introduction 51 2.4 Methods 53 2.4.1 Search strategy and eligibility criteria 53 2.4.2 Data collection 53 2.4.3 Data analysis 53 2.4.4 Trial sequential analysis 54 2.5 Results 55 2.5.1 Results of database search 55 2.5.2 Quality assessment of included trials 55 2.5.3 Effects of intervention 56 2.5.4 Trial sequential analysis 56 2.5.5 Publication bias 57 2.6 Discussion 58 2.7 Conclusions 61 2.8 References 71 CHAPTER III 76 IDENTIFICATION OF VITAMIN D3 TARGET GENES IN HUMAN BREAST CANCER TISSUE 76 3.1 Prelude 77 3.2 Abstract 80 3.3 Introduction 81 3.4 Material and Methods 83 3.4.1 Patient explant tissue culture 83 3.4.2 RNA-Seq 83 3.4.3 Pathway analysis and statistics 84 3.4.4 Cell culture 84 3.4.5 Quantitative RT-PCR analysis 85 3.4.6 Data analysis and statistical methods 86 3.5 Results 86 3.5.1 Genome-wide transcriptional profiling of 1,25D regulated genes in human breast explants 86 3.5.2 GoSeq KEGG pathway and ontology analysis 87 3.5.3 Confirmation of differentially expressed genes by qRT-PCR 88 3.5.4 Ketoconazole potentiates the effect of 1,25D 89 3.6 Discussion 90 3.7 Conclusions 94 3.8 References 144 CHAPTER IV 151 VITAMIN D3 SIGNALING AND MAMMARY CANCER: INSIGHTS FROM TRANSGENIC MOUSE MODELS 151 4.1 Prelude 152 4.2 Abstract 154 4.3 Introduction 155 4.4 Metabolism of vitamin D3 155 4.5 Vitamin D3 and breast cancer 156 4.6 Vitamin D3 binding protein knockout mice 158 4.7 Cyp2r1 enzyme activity and mammary cancer 159 4.8 Vdr knockout mice and mammary gland development 161 4.9 Vdr knockout mice and mammary cancer 162 4.10 Cyp27b1 knockout mice and mammary gland development 164 4.11 Cyp27b1 knockout mice and mammary cancer 164 4.12 Cyp24a1 knockout mice and mammary gland development 166 4.13 CYP24A1 and human breast cancer 167 4.14 Conclusion 170 4.15 References 171 CHAPTER V 179 CONDITIONAL INACTIVATION OF THE 25-HYDROXYVITAMIN D-24- HYDROXYLASE (CYP24A1) IN THE MOUSE MAMMARY EPITHELIUM ALTERS MAMMARY GLAND DEVELOPMENT 179 5.1 Prelude 180 5.2 Abstract 183 5.3 Introduction 184 5.4 Methods 185 5.4.1 Mice 185 5.4.2 Whole mount preparation 186 5.4.3 Indirect immunofluorescence 186 5.4.4 Isolation of primary mammary epithelial cells 187 5.4.5 Cell labelling, flow-cytometric analysis, and fluorescence activated cell sorting 188 5.4.6 Generation of cDNA by direct reverse transcription and qPCR analysis 189 5.4.7 In vitro culture and cell viability assay 189 5.4.8 Statistical analysis 190 5.5 Results 191 5.5.1 Conditional deletion of the Cyp24a1 gene in mouse mammary epithelium 191 5.5.2 Cyp24a1 ablation does not alter normal growth of mammary gland fat pad, body weight, pup survival, and circulating 1,25(OH)2D levels 193 5.5.3 Cyp24a1 knockout female displays impaired mammary gland ductal morphogenesis 195 5.5.4 The ablation of Cyp24a1 activity reduces proliferation of mammary epithelial cells 198 5.5.5 Ablation of Cyp24a1 activity sensitizes luminal cells to low level of exogenous 1,25(OH)2D treatment 201 5.6 Discussion 204 5.7 References 211 CHAPTER VI 214 EGF-INDUCED CONFLUENCE-DEPENDENT EXPRESSION OF RANKL IN MCF10A CELLS IS ASSOCIATED WITH EPITHELIAL -MESENCHYMAL TRANSITION 214 6.1 Prelude 215 6.2 Abstract 217 6.3 Introduction 218 6.4 Methods 221 6.4.1 Cell culture 221 6.4.2 Patient samples 221 6.4.3 Quantitative RT-PCR analysis 222 6.4.4 Western blot 222 6.4.5 Data analysis and statistical methods 222 6.5 Results 223 6.5.1 Increased RANKL mRNA levels and RANKL/OPG mRNA ratio in breast cancer as compared with matched adjacent normal tissue 223 6.5.2 Restoration of RANKL protein expression in hormone receptor negative MCF10A cells 223 6.5.3 RANKL expression is associated with induction of two RANKL cleavage enzymes and suppression of OPG and RUNX2 mRNA 224 6.5.4 EGF-induced confluence-dependent expression of RANKL in MCF10A was associated with epithelial-mesenchymal transition (EMT) 225 6.6 Discussion 227 6.7 References 235 FINAL CONCLUSION 239 Overview Although the mortality rate of breast cancer has continued to decrease over the past several decades, it remains the most frequently diagnosed cancer and the leading cause of cancer death in women worldwide. Accumulating epidemiological and clinical evidence suggest that vitamin D insufficiency is associated with increased breast cancer incidence and poor clinical outcomes in patients with breast cancer, which makes vitamin D supplementation a potential preventive or therapeutic option for the management of breast cancer. However, the detailed mechanisms on how vitamin D protects against breast cancer remains largely unknown. This thesis mainly investigates the role of vitamin D signaling pathway in the prevention of breast cancer. There are two major themes. The first theme focuses on identifying target genes and molecular pathways of vitamin D in human breast cancer, while the second theme aims to characterize the role of the vitamin D inactivating enzyme Cyp24a1 in the development of mouse mammary gland morphogenesis. This thesis is in the publication format with Chapter III as a published article, Chapter IV, V, and VI as manuscripts in preparation, while the remaining parts of this thesis (Chapter I and II) are regarded as an introduction. Theme 1: Identification of vitamin D3 target genes in human breast cancer tissue i Chapter I summarizes the basic knowledge of vitamin D metabolism in breast tissue, our current understandings of the anti-tumor effects of vitamin D, and the role of vitamin D in breast cancer prevention based on epidemiological and clinical studies. Target genes of the vitamin D signaling pathway mainly generated from investigation of breast cancer cell lines are also reviewed. Chapter II presents the evidence from randomized clinical trials that vitamin D, plus or minus calcium supplementation, has the potential to prevent breast cancer. Our analyses indicate that the available data are insufficient to confirm any protective effect of vitamin D supplementation, with or without calcium, on the risk of breast cancer. More participants in future trials are required to make a reliable and conclusive assessment. Chapter III, published as an original research article in the Journal of Steroid Biochemistry and Molecular Biology, determines the target genes and molecular pathways influenced by 1,25(OH)2D in normal human breast and cancer tissues in an ex vivo explant system. RNA-Seq revealed 523 genes that were differentially expressed in breast cancer tissues in response to 1,25(OH)2D treatment, and 127 genes with altered expression in normal breast tissues. The major finding of the present study is that exposure of both normal and malignant breast tissue to 1,25(OH)2D results in changes in cellular adhesion, metabolic pathways and tumor suppressor-like ii pathways, which support epidemiological data suggesting that adequate vitamin D3 levels may improve breast cancer outcome. Theme 2: Characterization of the vitamin D-inactivating enzyme Cyp24a1 in pubertal mouse mammary gland morphogenesis. Chapter IV, presented as a manuscript in preparation, summarizes our current understanding from transgenic mouse models of the vitamin D receptor (Vdr) and vitamin D associated metabolic enzymes in mammary gland development, cancer initiation, and progression and discusses the implications of these findings for human breast cancer. An improved understanding of the mechanisms of action of vitamin D signaling pathway derived from these mouse models provides support that activation of vitamin D signaling pathway is a potential approach for human breast cancer prevention. Chapter V, also presented as a manuscript in preparation, characterizes the role of vitamin D-inactivating enzyme Cyp24a1 in mammary gland development. A novel mouse model was available with conditional knockout of the Cyp24a1 gene specifically in the mammary epithelium (MMTV-Cre x Cyp24a1lox/lox). Our major finding is that mammary glands from virgin Cyp24a1 knockout females display impaired ductal morphogenesis compared with age- and weight-matched wild-type mice, which is due iii to reduced proliferation of mammary epithelial cells with the ablation of Cyp24a1 activity.